Curcumin Triggers S-Phase Accumulation and p53-Bax/Bcl-2-Mediated Intrinsic Apoptosis in HCT-116 Colorectal Cancer Cells

Curcumin Triggers S-Phase Accumulation and p53-Bax/Bcl-2-Mediated Intrinsic Apoptosis in HCT-116 Colorectal Cancer Cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Curcumin Triggers S-Phase Accumulation and p53-Bax/Bcl-2-Mediated Intrinsic Apoptosis in HCT-116 Colorectal Cancer Cells Ahmed Abualshroud, Faris Abdon, Abdalla Elamin, Abdalla ElGenaidi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8432900/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract Background Curcumin has been widely investigated as a bioactive phytochemical with anticancer potential, but its coupled effects on apoptosis-related transcriptional programs and cell-cycle distribution in colorectal cancer models remain incompletely characterized. Methods HCT-116 colorectal carcinoma cells were exposed to curcumin (0-100 µg/mL) for 24–72 h, and viability was quantified by MTT to derive IC₅₀ values. Cell death morphology was assessed by acridine orange/ethidium bromide staining at a cytotoxic exposure. TP53, BAX, and BCL2 mRNA expression was quantified by SYBR Green RT-qPCR, and DNA-content profiling was performed by propidium iodide staining and flow cytometry. Results Curcumin reduced HCT-116 viability with increasing exposure, with IC₅₀ values of 48.87 µg/mL (24 h), 31.0 µg/mL (48 h), and 17.5 µg/mL (72 h). At 35 µg/mL for 48 h, AO/EtBr staining showed 40% viable cells, 10% early apoptosis, 32% late apoptosis, and 18% necrosis. Curcumin increased TP53 (2.74-fold) and BAX (1.70-fold) and decreased BCL2 (0.37-fold) versus control (all P < 0.0001). Flow cytometry demonstrated a shift from G0/G1 (71.58% to 60.26%) toward S phase (7.50% to 18.69%), with minimal change in G2/M. Conclusion In HCT-116 cells, free curcumin is associated with S-phase accumulation and a pro-apoptotic transcriptional shift characterized by increased TP53/BAX and reduced BCL2, accompanied by predominantly apoptotic cell death. These findings support further evaluation of curcumin-based strategies (particularly optimized formulations and rational combinations) in colorectal cancer models. curcumin colorectal cancer HCT-116 apoptosis cell cycle p53 Bax Bcl-2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Colorectal cancer remains a major contributor to global cancer morbidity and mortality, and clinical outcomes are strongly influenced by stage at diagnosis and therapeutic resistance [ 1 , 2 ]. While advances in screening and systemic therapy have improved survival in some settings, recurrence and treatment-limiting toxicity remain persistent barriers [ 2 , 3 ]. These challenges continue to motivate evaluation of low-toxicity agents that can modulate proliferation and cell-death programs and potentially complement established regimens [ 4 – 6 ]. Current CRC management combines surgery and, where indicated, radiotherapy with systemic therapy built around fluoropyrimidines, oxaliplatin, and irinotecan, with targeted agents or immunotherapy used in selected patients [ 5 , 7 ]. Although these approaches improve outcomes (particularly when disease is detected early), durable control in locally advanced and metastatic CRC is frequently constrained by treatment resistance, cumulative toxicity, and recurrence after apparently successful therapy [ 8 – 10 ]. Relapse is often attributed to residual tumor subpopulations that survive initial therapy and later repopulate the tumor. One explanatory framework is the cancer stem cell (CSC) model, in which a minority of self-renewing cells contributes to tumour maintenance, dissemination, and therapeutic resistance [ 11 , 12 ]. CRC models enriched for CSC-associated markers (e.g., CD44, CD133, CD166, and ALDH) show enhanced invasion, sphere formation, and regrowth after FOLFOX or FOLFIRI exposure and can be difficult to eradicate with conventional agents alone [ 12 – 14 ]. Accordingly, combination strategies that add targeted or dietary agents to standard chemotherapy have been explored as a means to reduce CSC-associated phenotypes and potentially improve long-term control [ 9 , 12 , 15 ]. Curcumin, the principal curcuminoid of Curcuma longa , is widely studied as a pleiotropic modulator of cancer-associated signalling and is frequently described as having a favourable toxicity profile in non-malignant tissues [ 16 – 18 ]. In experimental colon carcinogenesis (including azoxymethane-induced and inflammation-driven models) dietary curcumin reduced tumour incidence and severity [ 19 , 20 ]. In established CRC cell lines such as HCT-116, HT-29, SW480, and SW620, curcumin and its derivatives suppress proliferation, migration, and invasion, decrease clonogenic and sphere-forming capacity, and enhance chemosensitivity [ 15 , 21 – 23 ]. Mechanistically, curcumin can modulate transcription factors (e.g., NF-κB, STAT3), growth-factor signalling, inflammatory mediators, redox balance, and mitochondrial apoptotic signalling [ 17 , 18 , 24 , 25 ]. In addition, curcumin can reverse multidrug resistance (MDR) in colon cancer models by decreasing P-glycoprotein and survivin, increasing intracellular Rhodamine 123 accumulation, and sensitising vincristine-resistant cells to multiple chemotherapeutic agents [ 26 ]. Apoptosis in CRC is tightly regulated by p53 and the Bcl-2 family. Foundational studies demonstrated that Bcl-2 can heterodimerise with Bax and that the relative balance of anti- and pro-apoptotic members functions as a molecular rheostat governing commitment to programmed cell death following cellular stress [ 27 , 28 ]. p53 integrates DNA damage and other stress signals and can shift this balance by transcriptionally up-regulating pro-apoptotic genes, such as BAX, and down-regulating anti-apoptotic genes, such as BCL2 [ 25 , 29 ]. More recent work emphasises that p53 sits within a broader stress-response network in which phosphorylation and acetylation events, protein-protein interactions, and subcellular localisation collectively influence whether cells undergo transient arrest, senescence, or apoptosis [ 30 , 31 ]. In colon cancer models, curcumin has been reported to promote apoptosis with increased p53 and Bax and decreased Bcl-2, along with caspase activation and characteristic apoptotic morphology; however, the magnitude and timing of these changes vary by cell line, dose, and exposure duration [ 21 , 23 , 32 , 33 ]. Curcumin also interferes with cell-cycle progression in CRC. Depending on the cell line, dose, and formulation, curcumin has been reported to cause arrest in G₀/G₁, S, and/or G₂/M phases, associated with modulation of cyclin-dependent kinases (CDKs), p21, and the retinoblastoma (Rb) pathway, and in some settings with evidence of DNA damage and impaired DNA repair [ 17 , 21 , 34 , 35 ]. In HCT-116 cells, curcumin and curcumin-based nanoprodrugs can induce cell-cycle alterations, reduce clonogenic growth, and downregulate regulators such as PLK1 and several CDKs, with promising effects in xenograft and colitis-associated CRC models [ 23 , 34 , 36 ]. Delivery approaches, including dendrosomal and phytosomal formulations, can improve solubility and bioavailability and frequently display enhanced antiproliferative activity and multi-phase cell-cycle effects compared with free curcumin [ 34 , 37 , 38 ]. Despite this growing literature, important gaps remain. Many studies use heterogeneous curcumin preparations or nano-formulations, focus on a single read-out, or examine cell lines with mutant p53, making it difficult to link curcumin-induced growth inhibition to apoptosis-related transcriptional changes and cell-cycle redistribution in a defined genetic context [ 9 , 15 , 35 ]. HCT-116 cells, which harbour wild-type TP53 and mutant KRAS, are widely used in vitro CRC models and are particularly suitable for dissecting p53-dependent responses [ 39 , 40 ]. However, there are limited data that simultaneously quantify, in the same experimental system, the time- and concentration-dependent cytotoxicity of free curcumin, the distribution of viable/apoptotic/necrotic cells, transcriptional regulation of TP53, BAX, and BCL2, and PI-based cell-cycle distribution at a defined cytotoxic concentration in this line [ 9 , 15 , 35 ]. Accordingly, we profiled the effects of free curcumin on viability, cell death morphology, TP53/BAX/BCL2 expression, and PI-based cell-cycle distribution in HCT-116 cells to link cytotoxicity with apoptosis-related transcriptional changes and cell-cycle perturbations. Materials and Methods Chemicals and reagents Dimethyl sulfoxide (DMSO), curcumin (≥ 96% purity), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), fetal bovine serum (FBS), and trypan blue were purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640 medium, HEPES buffer solution, L-glutamine, gentamicin, and 0.25% trypsin-EDTA were obtained from Biowest (Nuaillé, France). Acridine orange and ethidium bromide were purchased from Merck KGaA (Darmstadt, Germany). Total RNA was isolated using TransZol reagent (TransGen Biotech, China). First-strand cDNA was synthesized with the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). Quantitative real-time PCR (qPCR) was performed using Maxima SYBR Green qPCR Master Mix (Thermo Scientific). Propidium iodide (PI), RNase A, binding buffer, and the CycleTEST™ PLUS DNA reagent kit were obtained from BD (Franklin Lakes, NJ, USA). All other reagents were of analytical grade. Cell line and culture conditions The human colorectal carcinoma cell line HCT-116 (epithelial morphology; large intestine/colon carcinoma) was used as an in vitro model of colorectal cancer. This cell line was originally derived from the colon of an adult male with colon cancer and harbours a mutation in codon 13 of the RAS proto-oncogene. Cells were maintained in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated FBS, 1% penicillin/streptomycin (100 U/mL penicillin and 100 µg/mL streptomycin), 2 mM L-glutamine, and 10 mM HEPES. Cultures were incubated at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. The culture medium was replaced every 2–3 days, and cells were sub-cultured at 70–90% confluence using 0.25% trypsin-EDTA. Cell number and viability were routinely assessed by trypan blue exclusion. All in vitro experiments (MTT assay, AO/EtBr staining, qPCR, and cell-cycle analysis) were performed in at least three independent experiments unless otherwise stated. Preparation of curcumin stock solution and treatment Curcumin was dissolved in DMSO to prepare a 10 mg/mL stock solution, which was aliquoted and stored at -20°C in the dark until use. Immediately before each experiment, the stock solution was diluted in complete RPMI-1640 medium to obtain the desired working concentrations (0, 5, 10, 20, 40, 50, and 100 µg/mL) [ 18 ]. The final DMSO concentration in all wells, including vehicle controls, did not exceed 0.5% (v/v). For treatments, HCT-116 cells were seeded and allowed to attach for 24 h, then exposed to curcumin at the indicated concentrations for 24, 48, or 72 h. Untreated cells and DMSO vehicle controls (0.5% DMSO) were included in each experiment [ 41 ]. Cell viability assay (MTT) Curcumin-induced cytotoxicity was quantified using the MTT assay, which measures the reduction of MTT to insoluble formazan by mitochondrial dehydrogenases in viable cells. HCT-116 cells were suspended in complete medium and seeded into flat-bottom 96-well plates at 5 × 10⁴ cells/well in 100 µL. Plates were incubated for 24 h at 37°C to allow cell attachment. The culture medium was then carefully aspirated and replaced with fresh medium containing curcumin at final concentrations of 0, 5, 10, 20, 40, 50, or 100 µg/mL, or vehicle control (0.5% DMSO). Each concentration was tested in triplicate wells in each independent experiment. Plates were incubated for 24, 48, or 72 h. At each time point, the medium was gently removed and replaced with 90 µL fresh RPMI-1640 and 10 µL MTT stock solution (5 mg/mL in PBS; final MTT concentration 0.5 mg/mL) per well. Plates were incubated for 4 h at 37°C to allow viable cells to form purple formazan crystals. The supernatant was then discarded, and the formazan crystals were solubilised in 50 µL DMSO per well, followed by gentle mixing on a plate shaker for 5 min. The optical density (OD) was measured at an appropriate wavelength range (530–630 nm) using a microplate reader. Cell viability (%) was calculated as: Viability (%) = (OD t /OD c )×100. Where ODt is the mean optical density of wells treated with the tested sample, and ODc is the mean optical density of untreated cells. Dose-response curves were plotted, and half-maximal inhibitory concentration (IC₅₀) values at 24, 48, and 72 h were estimated by non-linear regression of the dose-response data using GraphPad Prism (GraphPad Software, San Diego, CA, USA), according to the general principles described by Mosmann [ 42 ]. Analysis of apoptosis and necrosis by acridine orange/ethidium bromide staining The mode of cell death (viable, early apoptotic, late apoptotic, and necrotic) induced by curcumin was evaluated using acridine orange/ethidium bromide (AO/EtBr) dual staining. HCT-116 cells were cultured on 8-well chamber slides (SPL, Seoul, South Korea) at a density of 1 × 10⁴ cells/well and allowed to adhere for 24 h. Cells were then treated with curcumin at 35 µg/mL (approximately the 48-h IC₅₀ concentration) for 48 h; untreated cells (0 µg/mL) served as controls. At the end of treatment, the medium was aspirated, and cells were gently washed with PBS. A freshly prepared AO/EtBr staining solution (100 µg/mL acridine orange and 100 µg/mL ethidium bromide in PBS, mixed 1:1) was added to each well. Stained cells were immediately examined under a fluorescence microscope (Axio Imager Z2, Zeiss, Jena, Germany). Several random fields were analysed per condition. Cells with uniformly green nuclei were scored as viable; cells with green/yellow fragmented or condensed nuclei as early apoptotic; cells with bright orange/red condensed or fragmented nuclei as late apoptotic; and cells with uniformly red nuclei without chromatin condensation as necrotic. The percentage of cells in each category was calculated. RNA extraction and quantitative real-time PCR All solutions were prepared using sterile RNase-free water, and all tubes and pipette tips were handled with appropriate precautions to minimise RNase contamination. HCT-116 cells were treated with curcumin at 35 µg/mL or vehicle control for 48 h, then harvested for RNA extraction. Total RNA was extracted using TransZol reagent according to the manufacturer’s protocol. Briefly, 1 mL cold TransZol was added to each sample, mixed thoroughly, and incubated for 5 min at room temperature. Chloroform (0.2 mL per 1 mL TransZol) was added, samples were vigorously shaken for 30 s, incubated for 3 min, and centrifuged at 10,000 × g for 15 min at 4°C. The aqueous phase was transferred to a new tube, mixed with 0.5 mL isopropanol per 1 mL TransZol, incubated for 10 min, and centrifuged at 10,000 × g for 10 min at 4°C. The RNA pellet was washed with 1 mL 75% ethanol, centrifuged at 7,500 × g for 5 min at 4°C, air-dried for approximately 15 min, and dissolved in 50 µL RNase-free water. RNA concentration and purity (A₂₆₀/A₂₈₀) were determined using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). RNA samples were stored at -80°C until use. First-strand cDNA was synthesised from 1 µg total RNA using the RevertAid First Strand cDNA Synthesis Kit according to the manufacturer’s instructions. Gene expression of TP53 (p53), BAX, and BCL-2 was quantified by SYBR Green-based qPCR using Maxima SYBR Green qPCR Master Mix in a real-time PCR system (DTlite, DNA-Technology). GAPDH was used as the endogenous control. Each 25 µL qPCR reaction contained 12.5 µL 2× SYBR Green master mix, 1 µL of each forward and reverse primer, cDNA template (≤ 100 ng), and nuclease-free water. All reactions were run in technical triplicate for each biological replicate. Relative mRNA expression was calculated using the 2⁻ΔΔCq method, normalising target genes to GAPDH and comparing curcumin-treated samples to untreated controls. Cell cycle analysis by flow cytometry Cell-cycle distribution and the hypodiploid (sub-G1) population were analysed by propidium iodide staining and flow cytometry. HCT-116 cells were cultured in flasks until approximately 70–80% confluence, then treated with curcumin at the 48-h IC₅₀ concentration (≈ 35 µg/mL) for 48 h or left untreated (vehicle only) as controls. At the end of the incubation period, both floating and adherent cells were collected; adherent cells were detached by trypsinisation and pooled with floating cells, pelleted by centrifugation, and washed twice with cold PBS. Cell pellets were gently resuspended in PBS, and ice-cold 70% ethanol was added dropwise while vortexing to achieve fixation. Samples were fixed at -20°C for at least 1 h. Fixed cells were then centrifuged to remove ethanol, washed with PBS, and resuspended in staining solution containing PI and RNase A using the CycleTEST™ PLUS DNA reagent kit (BD Biosciences) according to the manufacturer’s instructions. Samples were incubated in the dark at room temperature for 30–60 min before acquisition. PI fluorescence histograms were generated, and the proportions of cells with hypodiploid DNA content (sub-G1 population, used as an index of late apoptosis) and with diploid/tetraploid DNA content corresponding to G0/G1, S, and G2/M phases were determined using the instrument’s analysis software [ 43 ]. Treated and control samples were compared to evaluate curcumin-induced alterations in cell-cycle distribution and in the sub-G1 fraction. Statistical data analysis Data are presented as mean ± standard deviation (SD) from at least three independent experiments. Dose-response curves for cell viability were fitted using non-linear regression with a five-parameter logistic model in GraphPad Prism version 10.3.1 (GraphPad Software, San Diego, CA, USA), and IC₅₀ values were obtained from the fitted curves. For other endpoints, comparisons between control and curcumin-treated groups were performed using two-tailed unpaired Student’s t-tests when data were approximately normally distributed and the Wilcoxon rank-sum test when distributional assumptions were not met. A P value < 0.05 was considered statistically significant. Results Curcumin reduces viability of HCT-116 cells in a time- and concentration-dependent manner The effect of curcumin on the viability of HCT-116 colorectal carcinoma cells was evaluated by MTT assay after 24, 48, and 72 h of exposure to 0-100 µg/mL curcumin (Fig. 1 , Table 1 ). At 24 h, cell viability remained above 80% at 5–20 µg/mL (81.03 ± 2.23%, 99.73 ± 11.14%, and 83.75 ± 12.31%, respectively) but decreased to 64.06 ± 14.76% at 40 µg/mL, 48.87 ± 2.68% at 50 µg/mL, and 39.44 ± 5.88% at 100 µg/mL (P = 0.0058 for overall effect vs control). The estimated IC₅₀ at 24 h was 48.87 µg/mL. At 48 h, curcumin produced a more marked reduction in viability (P = 0.001). Viability declined from 100.00 ± 7.56% in untreated cells to 97.00 ± 14.19% at 5 µg/mL, 86.61 ± 18.33% at 10 µg/mL, 85.95 ± 9.77% at 20 µg/mL, and sharply to 18.15 ± 11.13%, 17.38 ± 6.70%, and 14.89 ± 4.43% at 40, 50, and 100 µg/mL, respectively. The corresponding 48-h IC₅₀ was 31.0 µg/mL. At 72 h, curcumin markedly suppressed cell viability (P = 0.0015). Viability remained relatively high at 5–10 µg/mL (81.24 ± 17.25% and 78.13 ± 19.00%), but decreased to 38.33 ± 14.36% at 20 µg/mL and to 6.31 ± 2.92%, 5.77 ± 0.85%, and 5.41 ± 2.23% at 40, 50, and 100 µg/mL, respectively. The 72-h IC₅₀ was 17.5 µg/mL. Curcumin reduced HCT-116 cell viability in a time- and concentration-dependent manner, with IC₅₀ values of 48.87 µg/mL at 24 h, 31.0 µg/mL at 48 h, and 17.5 µg/mL at 72 h (Fig. 1 , Table 1 ). Curcumin at 35 µg/mL induces apoptosis and necrosis in HCT-116 cells To characterize the mode of cell death at a cytotoxic concentration, HCT-116 cells were exposed to 35 µg/mL curcumin (approximately the 48-h IC₅₀) for 48 h and stained with acridine orange/ethidium bromide. Representative fluorescence micrographs are shown in Fig. 2 , and quantitative data are summarized in Table 2 . In untreated controls, the majority of cells were viable (95%), with a small fraction (5%) exhibiting features of early apoptosis; late apoptotic and necrotic cells were not observed (0% each). After treatment with 35 µg/mL curcumin, the proportion of viable cells decreased to 40%, while 10% of cells were in early apoptosis, 32% in late apoptosis, and 18% were necrotic (Table 2 ). Curcumin-treated cells exhibited morphological hallmarks of apoptosis, including cell shrinkage, membrane blebbing, and chromatin condensation/fragmentation, together with a subset of cells showing necrotic morphology (Fig. 2 ). Curcumin at 35 µg/mL modulates p53, BAX, and BCL-2 expression The impact of curcumin on apoptosis-related genes was assessed by SYBR Green qPCR after 48 h exposure of HCT-116 cells to 35 µg/mL curcumin. Expression levels were normalized to GAPDH and calculated using the 2⁻ΔΔCq method, with untreated cells as calibrator (RQ = 1.0). Detailed Ct, ΔCt, ΔΔCt, and RQ values are provided in Supplementary Table 1. Curcumin significantly increased BAX mRNA expression compared with control cells, with a mean fold change (RQ) of 1.70 (P < 0.0001) (Table 3 , Fig. 3 A). The tumor suppressor TP53 (p53) was also significantly upregulated, with a mean fold change of 2.74 (P < 0.0001) (Table 3 , Fig. 3 B). In contrast, curcumin treatment reduced the expression of the anti-apoptotic gene BCL-2, with a mean relative expression of 0.37 compared with control (≈ 63% decrease; P < 0.0001) (Table 3 , Fig. 3 C). At 35 µg/mL, curcumin increased p53 and BAX mRNA levels and decreased BCL-2 expression in HCT-116 cells (Table 3 , Fig. 3 ; Supplementary Table 1). Curcumin at 35 µg/mL induces S-phase accumulation in HCT-116 cells To examine the effect of curcumin on cell-cycle distribution, HCT-116 cells were treated with 35 µg/mL curcumin for 48 h, stained with propidium iodide, and analyzed by flow cytometry (Fig. 4 , Table 4 ). In untreated control cells, 71.58% were in G1 phase, 7.50% in S phase, 0.53% in G2/M, and 19.33% in the sub-G1 (hypodiploid) population, which was used as an index of late apoptosis (Table 4 ). After treatment with curcumin, the fraction of cells in G1 decreased to 60.26%, whereas the proportion in S phase increased to 18.69%. The G2/M fraction remained low (0.64%), and the sub-G1 population showed a slight increase to 20.33% (Table 4 , Fig. 4 ). At 35 µg/mL, 48-h curcumin exposure was associated with a redistribution of HCT-116 cells from G1 into S phase, i.e., S-phase accumulation, with minimal change in the G2/M fraction (Table 4 , Fig. 4 ). Table 1 Time- and concentration-dependent effects of curcumin on viability of HCT-116 colorectal carcinoma cells (MTT assay) Curcumin (µg/mL) 24 h viability % (mean ± SD) 48 h viability % (mean ± SD) 72 h viability % (mean ± SD) 0 (control) 100.00 ± 6.96 100.00 ± 7.56 100.00 ± 6.37 5 81.03 ± 2.23 97.00 ± 14.19 81.24 ± 17.25 10 99.73 ± 11.14 86.61 ± 18.33 78.13 ± 19.00 20 83.75 ± 12.31 85.95 ± 9.77 38.33 ± 14.36 40 64.06 ± 14.76 18.15 ± 11.13 6.31 ± 2.92 50 48.87 ± 2.68 17.38 ± 6.70 5.77 ± 0.85 100 39.44 ± 5.88 14.89 ± 4.43 5.41 ± 2.23 Footnote. Values are expressed as mean ± standard deviation (SD) of percentage viability relative to untreated control (100%) from three independent experiments (each performed in triplicate). Curcumin significantly reduced HCT-116 cell viability in a concentration-dependent manner at all time points (overall effect vs control: P = 0.0058 at 24 h, P = 0.001 at 48 h, and P = 0.0015 at 72 h). The corresponding IC₅₀ values, estimated by non-linear regression of the dose–response curves, were 48.87 µg/mL (24 h), 31.0 µg/mL (48 h), and 17.5 µg/mL (72 h). Table 2 Distribution of viable, apoptotic, and necrotic HCT-116 cells after 48 h of curcumin treatment was assessed by acridine orange/ethidium bromide staining Condition Curcumin (µg/mL) Viable cells (%) Early apoptosis (%) Late apoptosis (%) Necrosis (%) Control 0 95 5 0 0 Curcumin-treated 35 40 10 32 18 Footnote. Percentages represent the proportion of cells in each death category after 48 h exposure of HCT-116 cells to curcumin at a concentration close to the 48-h IC₅₀ (≈ 35 µg/mL), compared with untreated controls, as determined by AO/EtBr staining and morphological assessment under a fluorescence microscope. Table 3 Relative expression of apoptosis-related genes in HCT-116 cells after 48 h of curcumin treatment (qPCR) Gene Curcumin (µg/mL) Fold change vs untreated control (RQ) Regulation vs control P value BAX 35 1.70 Upregulated < 0.0001 TP53 35 2.74 Upregulated < 0.0001 BCL-2 35 0.37 Downregulated < 0.0001 Footnote. Gene expression was quantified by SYBR Green qPCR after 48 h exposure of HCT-116 cells to curcumin at a concentration close to the 48-h IC₅₀ (35 µg/mL or ≈ 35 µg/mL, depending on the notation you choose to standardize). Expression levels were normalized to GAPDH and calculated using the 2⁻ΔΔCq method, with untreated cells as the calibrator (RQ = 1.0). The RQ values shown correspond to the mean relative quantities derived from the underlying calculations (BAX control RQ ≈ 1.01, treated 1.70; TP53 control RQ ≈ 1.00, treated 2.74; BCL-2 control RQ ≈ 1.00, treated 0.37), which are presented in detail in Supplementary Table 1. All genes showed significant differences compared with the control (P < 0.0001). Table 4 Cell-cycle distribution of HCT-116 cells after 48 h of curcumin treatment at the 48-h IC₅₀ Cell-cycle phase Control (0 µg/mL), n (%) Curcumin (≈ 35 µg/mL), n (%) Sub-G1 19.33 20.33 G1 71.58 60.26 S 7.50 18.69 G2/M 0.53 0.64 Footnote. HCT-116 cells were treated for 48 h with curcumin at a concentration approximating the 48-h IC₅₀ (≈ 35 µg/mL) or left untreated. DNA content was assessed by propidium iodide staining and flow cytometry. Sub-G1 represents the hypodiploid/apoptotic fraction. Cells in G0 and G1 phases cannot be distinguished by DNA content alone and are therefore reported together as G0/G1 Discussion This in vitro study examined the effects of free curcumin on cell viability, cell death morphology, apoptosis-related gene expression, and cell cycle distribution in HCT-116 colorectal carcinoma cells. Curcumin reduced viability in a clear time- and concentration-dependent manner, with IC₅₀ values decreasing from 48.87 µg/mL at 24 h to 31.0 µg/mL at 48 h and 17.5 µg/mL at 72 h. At a cytotoxic concentration close to the 48-h IC₅₀ (35 µg/mL), curcumin induced predominantly apoptotic cell death with a smaller necrotic component, increased TP53 and BAX mRNA levels, decreased BCL2 mRNA, and produced S-phase accumulation with minimal change in G₂/M. Collectively, these findings support a model in which curcumin imposes replication-associated stress and engages a p53-centred programme that shifts the Bax/Bcl-2 balance toward mitochondrial apoptosis in this p53-competent CRC line. The progressive leftward shift in IC₅₀ values over 72 h indicates that both exposure time and concentration are critical determinants of curcumin cytotoxicity in this system. At 5–10 µg/mL, viability remained relatively preserved, particularly at earlier time points, whereas at ≥ 40 µg/mL, viability declined sharply by 48–72 h. This profile is more consistent with a compound that perturbs multiple cellular networks over time than with an immediate membrane toxin. In colorectal cancer models, curcumin and related formulations typically suppress viability and clonogenic growth over tens of micromolar concentrations and multi-day exposures, and these effects are linked to modulation of NF-κB, STAT3, PI3K/Akt, AMPK-COX-2, and redox homeostasis rather than a single dominant target [ 15 , 23 , 24 , 35 ]. Consistent with this, in HCT-116 cells, nanocurcumin and other advanced formulations often achieve comparable or stronger effects at lower nominal doses due to improved solubility and uptake [ 34 , 37 ]. Under the relatively stringent conditions used here (free curcumin in standard medium), the marked loss of viability by 48–72 h supports a genuine antiproliferative and cytotoxic effect. The dose-response pattern (modest effects at 5–10 µg/mL with a steep decline above ~ 40 µg/mL at later time points) suggests threshold behaviour. Below a certain intracellular burden, cells appear able to accommodate curcumin-induced stress; above that threshold, damage accumulates and commitment to death increases. Mechanistically, curcumin is known to disrupt cellular redox balance and increase reactive oxygen species (ROS) in colon cancer cells, with superoxide and other ROS contributing to mitochondrial dysfunction and apoptosis [ 17 , 33 ]. Curcumin can also interfere with survival signalling (including NF-κB, STAT3, and PI3K/Akt) and sensitize CRC cells to agents such as 5-fluorouracil and oxaliplatin [ 23 , 24 , 35 , 44 ]. The temporal IC₅₀ shift observed here is compatible with progressive amplification of stress signalling and cumulative disruption of adaptive responses. At 35 µg/mL, AO/EtBr staining demonstrated that apoptosis predominated: the viable fraction decreased from 95% to 40%, late apoptotic cells accounted for 32%, early apoptotic cells for 10%, and necrotic cells for 18%. The morphological hallmarks (cell shrinkage, membrane blebbing, chromatin condensation, and nuclear fragmentation) support activation of regulated apoptosis rather than purely accidental necrosis. Similar patterns have been reported in HT-29 and other CRC models in which curcumin increases annexin V-positive fractions, induces DNA fragmentation, and activates caspase-3 and caspase-9, often accompanied by changes in p53, Bax, and Bcl-2 [ 21 , 23 , 32 , 33 ]. The necrotic fraction observed here is plausible at a clearly cytotoxic dose: some cells may undergo primary necrosis when injury is too severe, while others enter apoptosis but progress to secondary necrosis in 2D culture in the absence of phagocytic clearance. Although we did not directly assess caspase activity or mitochondrial membrane potential, the combined morphology and transcriptional profile (below) support apoptosis as the dominant mode of death under these conditions. The transcriptional response to curcumin (TP53 up 2.74-fold; BAX up 1.70-fold; BCL2 reduced to 0.37-fold of control) is consistent with engagement of the p53–Bax/Bcl-2 axis. Foundational work established that anti-apoptotic Bcl-2 can heterodimerise with pro-apoptotic Bax, and that the relative abundance of these proteins functions as a rheostat influencing susceptibility to mitochondrial apoptosis after stress [ 27 , 28 , 45 ]. p53 integrates DNA damage and broader stress signals and can transcriptionally up-regulate BAX and other pro-apoptotic mediators while down-regulating BCL2 or functionally antagonising Bcl-2, thereby lowering the threshold for intrinsic apoptosis [ 25 , 27 – 29 , 40 ]. Importantly, p53 outcomes are controlled by a complex network of post-translational modifications, protein-protein interactions, and changes in subcellular localisation rather than a single linear pathway [ 30 , 31 ]. Within this framework, the coordinated increase in TP53 and BAX alongside suppression of BCL2 matches the direction of change expected to favour mitochondrial apoptosis. Prior CRC studies have reported comparable shifts in the pathway at the protein level. In HT-29 cells, for example, curcumin increased phospho-p53 (Ser15), up-regulated Bax, down-regulated Bcl-2, and triggered caspase activation, leading to apoptotic morphology [ 32 ]. In HCT-116 and related CRC systems, curcumin and curcumin-based formulations have also been reported to increase p53 and Bax while decreasing Bcl-2, with associated shifts in Bax/Bcl-2 balance and enhanced chemosensitivity [ 23 , 35 , 39 ]. Our data extend these observations by showing a coordinated transcriptional engagement of this axis under a defined exposure condition in wild-type p53 HCT-116 cells, consistent with the observed apoptotic phenotype. Curcumin altered the cell-cycle distribution in a pattern more compatible with S-phase accumulation than with a classic G₂/M block. After 48 h at 35 µg/mL, G1 decreased from 71.58% to 60.26% while S phase increased from 7.50% to 18.69%; G₂/M remained low (~ 0.6%), and the sub-G1 fraction rose only slightly (19.33% to 20.33%). This redistribution suggests that curcumin-treated cells enter S phase but fail to complete DNA replication efficiently, consistent with replication stress and/or DNA damage signalling. Across CRC models, curcumin and its analogues have been reported to induce arrest at G₀/G₁, S, and/or G₂/M depending on the cell line, dose, exposure time, and formulation [ 17 , 21 , 34 , 35 ]. In HCT-116, curcumin has been linked to S-phase accumulation associated with DNA damage and impaired repair, whereas other settings report G₀/G₁ or G₂/M effects mediated via p21-Rb signalling and CDK modulation [ 17 , 21 , 23 ]. Curcumin-based nano-prodrugs and phytosomal or dendrosomal formulations can induce multi-phase arrest and suppress CDKs and PLK1 with improved potency relative to free curcumin [ 17 , 34 ]. In this context, our data indicate that free curcumin at 35 µg/mL over 48 h primarily delays S-phase progression in HCT-116 cells. The modest rise in sub-G1 by PI staining, despite substantial apoptosis on microscopy, is compatible with a dynamic state in which many cells have initiated apoptosis but have not yet progressed to extensive DNA fragmentation sufficient to be scored as hypodiploid. Mechanistically, the combination of S-phase accumulation and increased TP53 expression is consistent with replication stress engaging p53-dependent checkpoints, followed by Bax/Bcl-2 reprogramming and commitment to intrinsic apoptosis. Although we did not directly assess cancer stem cell (CSC) markers, multidrug resistance (MDR) transporters, or drug combinations, the pattern observed here is relevant to these areas. CSC-enriched CRC models (often CD133- and ALDH-positive) exhibit increased resistance to 5-FU and oxaliplatin and are implicated in relapse after standard therapy [ 24 , 35 ]. Curcumin analogues, such as GO-Y030, inhibit STAT3, reduce ALDH⁺/CD133⁺ colon CSC populations, suppress tumor sphere formation, and inhibit tumor growth in vivo [ 46 ]. Curcumin has also been reported to modulate MDR mechanisms: in vincristine-resistant colon cancer cells, curcumin decreased P-glycoprotein and survivin, increased intracellular rhodamine-123 accumulation, and resensitized cells to multiple cytotoxics [ 26 ]. In HCT-116, curcumin enhanced 5-FU activity against viability, invasion, and migration [ 23 ], and in inflammation-driven CRC models, phytosomal curcumin reduced tumor burden and modulated inflammatory and proliferative markers [ 19 , 47 ]. Taken together, our results in parental HCT-116 cells provide a mechanistic baseline for these observations: an S-phase-linked stress response, with activation of p53-Bax/Bcl-2 signalling, would be expected to lower the apoptotic threshold in proliferating cells, potentially increasing vulnerability to antimetabolites and DNA-damaging agents in combination settings. Several limitations should be considered. First, all experiments were performed in a single cell line (HCT-116; TP53 wild-type, KRAS mutant), and responses may differ in TP53-mutant CRC lines or in non-malignant colon epithelial cells [ 15 , 39 ]. Second, mechanistic readouts (AO/EtBr, qPCR, and cell-cycle analysis) were conducted at a single curcumin concentration (35 µg/mL) and a single time point (48 h), which does not define dose-response relationships or early versus late dynamics. Third, TP53, BAX, and BCL2 were measured only at the mRNA level; because both p53 and Bcl-2 family proteins are strongly regulated post-translationally, protein-level and functional confirmation (p53 stabilisation/phosphorylation, Bax/Bcl-2 protein ratio, mitochondrial membrane potential, caspase activation) is needed to strengthen causal inference [ 28 , 30 , 31 , 48 ], Fourth, AO/EtBr staining is informative but cannot distinguish primary from secondary necrosis and is less quantitative than annexin V/PI flow cytometry. Finally, the work was conducted in 2D monolayers; organoids, CSC-enriched systems, and in vivo models may yield different sensitivities and microenvironment-dependent effects [ 9 , 24 , 35 ]. Future work should extend these findings in several directions. Time-course and multi-dose experiments would better map the temporal sequence among S-phase perturbation, p53 activation, Bax/Bcl-2 remodelling, and apoptosis execution, alongside upstream readouts such as ROS and DNA damage (e.g., γH2AX) [ 21 , 33 , 49 ]. Studies in additional CRC lines with differing TP53 and KRAS status, together with normal colon epithelial models, would clarify genotype-dependent and selective effects [ 15 , 39 ]. Combination studies with 5-FU, oxaliplatin, or irinotecan (particularly in CSC-enriched and drug-resistant settings) could test whether curcumin-associated S-phase stress and apoptotic priming translate into additive or synergistic effects [ 23 , 35 , 44 ]. Finally, direct comparisons of free curcumin with nano-curcumin, dendrosomal, or phytosomal formulations under the same endpoints would clarify how improved delivery influences the S-phase-linked and p53–Bax/Bcl-2-centred mechanisms identified here [ 17 , 34 , 37 , 38 ]. Conclusion This study shows that in HCT-116 colorectal carcinoma cells, free curcumin reduces viability in a clear time- and concentration-dependent manner and, at cytotoxic doses, induces predominantly apoptotic cell death with a smaller necrotic component. At 35 µg/mL, curcumin is associated with S-phase accumulation and coordinated modulation of the p53-Bax/Bcl-2 axis, characterised by up-regulation of TP53 and BAX and down-regulation of BCL2, consistent with activation of intrinsic, mitochondria-mediated apoptosis. These findings support the view that curcumin is not simply a non-specific cytotoxin but a multi-target agent that couples cell-cycle perturbation with intrinsic apoptotic signalling in CRC cells. In a KRAS-mutant, p53-wild-type background, this mechanistic profile provides a rational basis for further evaluation of curcumin and more bioavailable derivatives as adjuncts to standard colorectal cancer therapies, particularly in strategies aimed at lowering the apoptotic threshold and overcoming treatment resistance. Future work in additional genetic backgrounds, CSC-enriched and drug-resistant models, in vivo systems, and in combination with conventional chemotherapeutics and optimised formulations will be essential to define the translational potential of these observations. Abbreviations AO/EtBr acridine orange/ethidium bromide CRC colorectal cancer Ct cycle threshold DMSO dimethyl sulfoxide FBS fetal bovine serum GAPDH glyceraldehyde 3-phosphate dehydrogenase IC50 half-maximal inhibitory concentration MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide PI propidium iodide qPCR quantitative polymerase chain reaction RT-qPCR reverse transcription quantitative polymerase chain reaction SD standard deviation. Declarations Ethics approval and consent to participate Not applicable (in vitro study using an established human cell line). Consent for publication Not applicable. Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary information files. Competing interests The authors declare that they have no competing interests. Funding No specific funding was received for this work. Authors’ contributions Ahmed Mahamed Abualshroud: Conceptualization; Methodology; Investigation (cell culture experiments, MTT assay, AO/EtBr staining, flow cytometry, RT-qPCR); Data curation; Formal analysis; Writing original draft. Faris Abdon: Conceptualization; Methodology; Writing original draft; Writing review & editing; Critical revision for intellectual content; Corresponding author responsibilities. Abdalla Ahmed Eldaw Elamin: Methodology; Investigation; Data curation; Writing review & editing. Abdalla Ramadan ElGenaidi: Investigation; Validation; Data curation; Writing review & editing. Ayman Balla Mustafa: Methodology; Formal analysis; Visualization; Writing review & editing. Manal M Sami: Supervision; Methodology; Resources; Writing review & editing. All authors contributed substantially to the work, reviewed and approved the final manuscript, and agree to be accountable for all aspects of the work. References Siegel RL, Kratzer TB, Giaquinto AN, Sung H, Jemal A. Cancer statistics, 2025. CA Cancer J Clin. 2025;75(1):10–45. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209–49. Aggarwal BB, Sundaram C, Malani N, Ichikawa H. Curcumin: the Indian solid gold. Adv Exp Med Biol. 2007;595:1–75. Jokhadze N, Das A, Dizon DS. Global cancer statistics: A healthy population relies on population health. CA Cancer J Clin. 2024;74(3):224–6. Siegel RL, Wagle NS, Cercek A, Smith RA, Jemal A. Colorectal cancer statistics, 2023. CA Cancer J Clin. 2023;73(3):233–54. Islami F, Goding Sauer A, Miller KD, Siegel RL, Fedewa SA, Jacobs EJ, et al. Proportion and number of cancer cases and deaths attributable to potentially modifiable risk factors in the United States. CA Cancer J Clin. 2018;68(1):31–54. Dienstmann R, Salazar R, Tabernero J. Personalizing colon cancer adjuvant therapy: selecting optimal treatments for individual patients. J Clin Oncol. 2015;33(16):1787–96. Siegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. CA Cancer J Clin. 2024;74(1):12–49. de Porras VR, Layos L, Martínez-Balibrea E, editors. Curcumin: A therapeutic strategy for colorectal cancer? Seminars in cancer biology. Elsevier; 2021. Bracci L, Fabbri A, Del Corno M, Conti L. Dietary polyphenols: promising adjuvants for colorectal cancer therapies. Cancers. 2021;13(18):4499. Kunnumakkara AB, Bordoloi D, Padmavathi G, Monisha J, Roy NK, Prasad S, et al. Curcumin, the golden nutraceutical: multitargeting for multiple chronic diseases. Br J Pharmacol. 2017;174(11):1325–48. Nautiyal J, Kanwar SS, Yu Y, Majumdar AP. Combination of dasatinib and curcumin eliminates chemo-resistant colon cancer cells. J Mol Signal. 2011;6(1):1–11. Lane DP. p53, guardian of the genome. Nature. 1992;358(6381). Hewlings SJ, Kalman DS, Curcumin. A review of its effects on human health. Foods. 2017;6(10):92. Weng W, Goel A, editors. Curcumin and colorectal cancer: An update and current perspective on this natural medicine. Seminars in cancer biology. Elsevier; 2022. Sharma RA, Gescher AJ, Steward WP. Curcumin: the story so far. Eur J Cancer. 2005;41(13):1955–68. Selvam C, Prabu SL, Jordan BC, Purushothaman Y, Umamaheswari A, Zare MSH, et al. Molecular mechanisms of curcumin and its analogs in colon cancer prevention and treatment. Life Sci. 2019;239:117032. Shanmugam MK, Rane G, Kanchi MM, Arfuso F, Chinnathambi A, Zayed M, et al. The multifaceted role of curcumin in cancer prevention and treatment. Molecules. 2015;20(2):2728–69. Villegas I, Sánchez-Fidalgo S, de la Alarcon C. New mechanisms and therapeutic potential of curcumin for colorectal cancer. Mol Nutr Food Res. 2008;52(9):1040–61. Villegas I, Sánchez-Fidalgo S, de la Lastra CA. Chemopreventive effect of dietary curcumin on inflammation‐induced colorectal carcinogenesis in mice. Mol Nutr Food Res. 2011;55(2):259–67. Li P, Pu S, Lin C, He L, Zhao H, Yang C, et al. Curcumin selectively induces colon cancer cell apoptosis and S cell cycle arrest by regulates Rb/E2F/p53 pathway. J Mol Struct. 2022;1263:133180. Yu H, Wu H, Zhao Q, Zhao R, Liu J, Yang Z, et al. Curcumin suppresses colorectal cancer by inhibiting TRIM2 and mTOR signaling. Translational Oncol. 2025;61:102517. Xiang L, He B, Liu Q, Hu D, Liao W, Li R, et al. Antitumor effects of curcumin on the proliferation, migration and apoptosis of human colorectal carcinoma HCT–116 cells. Oncol Rep. 2020;44(5):1997–2008. Li S, Zhu X-R, Qin B-C, Chen M-B. Curcumin in colorectal cancer: mechanistic insights, pharmacological limitations, and translational perspectives. Front Pharmacol. 2025;16:1667731. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646–74. Lu W-D, Qin Y, Yang C, Li L. Effect of curcumin on human colon cancer multidrug resistance in vitro and in vivo. Clinics. 2013;68:694–701. Oltval ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programed cell death. Cell. 1993;74(4):609–19. Youle RJ, Strasser A. The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol. 2008;9(1):47–59. Vousden KH, Lane DP. p53 in health and disease. Nat Rev Mol Cell Biol. 2007;8(4):275–83. Lakin ND, Jackson SP. Regulation of p53 in response to DNA damage. Oncogene. 1999;18(53):7644–55. Prives C, Hall PA. The p53 pathway. J Pathol. 1999;187(1):112–26. Song G, Mao Y, Cai Q, Yao L, Ouyang G, Bao S. Curcumin induces human HT-29 colon adenocarcinoma cell apoptosis by activating p53 and regulating apoptosis-related protein expression. Braz J Med Biol Res. 2005;38:1791–8. Watson JL, Hill R, Yaffe PB, Greenshields A, Walsh M, Lee PW, et al. Curcumin causes superoxide anion production and p53-independent apoptosis in human colon cancer cells. Cancer Lett. 2010;297(1):1–8. Xu D, Feng X, Wan Y, Yang L, Gao Q, Yang Z, et al. Curcumin nano-prodrug induces multi-phase cell cycle arrest in colorectal cancer through suppression of CDKs and specific down-regulation of PLK1. Smart Mater Med. 2023;4:648–60. Pricci M, Girardi B, Giorgio F, Losurdo G, Ierardi E, Di Leo A. Curcumin and colorectal cancer: from basic to clinical evidences. Int J Mol Sci. 2020;21(7):2364. Chen J. The cell-cycle arrest and apoptotic functions of p53 in tumor initiation and progression. Cold Spring Harbor Perspect Med. 2016;6(3):a026104. Shehata M, Rafaat Y, Gad NA, Gaafar HF. Selective Cytotoxic and Chemoprotective effect of nanocurcumin against human HCT-116 cell line. Biochem Lett. 2018;14(1):85–93. Gabr SA, Elsaed WM, Eladl MA, El-Sherbiny M, Ebrahim HA, Asseri SM, et al. Curcumin modulates oxidative stress, fibrosis, and apoptosis in drug-resistant cancer cell lines. Life. 2022;12(9):1427. Sritharan S, Sivalingam N. Curcumin induced apoptosis is mediated through oxidative stress in mutated p53 and wild type p53 colon adenocarcinoma cell lines. J Biochem Mol Toxicol. 2021;35(1):e22616. Kinzler KW, Vogelstein B. Lessons from hereditary colorectal cancer. Cell. 1996;87(2):159–70. Ciuca MD, Racovita RC, Curcumin. Overview of extraction methods, health benefits, and encapsulation and delivery using microemulsions and nanoemulsions. Int J Mol Sci. 2023;24(10):8874. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65(1–2):55–63. Leif RC. Practical Flow Cytometry, 3rd Edition, by, Howard M, Shapiro MD, Wiley-Liss. Inc., New York, 1995, 542 pages, $ 79.95. Cytometry. 1995;19(4):376-. Shakibaei M, Mobasheri A, Lueders C, Busch F, Shayan P, Goel A. Curcumin enhances the effect of chemotherapy against colorectal cancer cells by inhibition of NF-κB and Src protein kinase signaling pathways. PLoS ONE. 2013;8(2):e57218. Dean RT, Fu S, Stocker R, Davies MJ. Biochemistry and pathology of radical-mediated protein oxidation. Biochem J. 1997;324(1):1–18. Lin L, Liu Y, Li H, Li P, Fuchs J, Shibata H, et al. Targeting colon cancer stem cells using a new curcumin analogue, GO-Y030. Br J Cancer. 2011;105(2):212–20. Marjaneh RM, Rahmani F, Hassanian SM, Rezaei N, Hashemzehi M, Bahrami A, et al. RETRACTED: Phytosomal curcumin inhibits tumor growth in colitis-associated colorectal cancer. J Cell Physiol. 2018;233(10):6785–98. Kvansakul M, Hinds MG. The Bcl-2 family: structures, interactions and targets for drug discovery. Apoptosis. 2015;20(2):136–50. Lu J-J, Cai Y-J, Ding J. Curcumin induces DNA damage and caffeine-insensitive cell cycle arrest in colorectal carcinoma HCT116 cells. Mol Cell Biochem. 2011;354(1):247–52. Additional Declarations No competing interests reported. Supplementary Files SuppTable1.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 17 Feb, 2026 Reviews received at journal 09 Feb, 2026 Reviews received at journal 07 Feb, 2026 Reviewers agreed at journal 06 Feb, 2026 Reviews received at journal 02 Feb, 2026 Reviewers agreed at journal 02 Feb, 2026 Reviewers agreed at journal 31 Jan, 2026 Reviewers agreed at journal 29 Jan, 2026 Reviewers invited by journal 29 Jan, 2026 Editor invited by journal 09 Jan, 2026 Editor assigned by journal 25 Dec, 2025 Submission checks completed at journal 25 Dec, 2025 First submitted to journal 23 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8432900","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":583638935,"identity":"2674aa2a-a455-4d31-822f-44ee5231349f","order_by":0,"name":"Ahmed Abualshroud","email":"","orcid":"","institution":"Misurata University","correspondingAuthor":false,"prefix":"","firstName":"Ahmed","middleName":"","lastName":"Abualshroud","suffix":""},{"id":583638936,"identity":"fb0e03e3-25bf-4210-b4bd-9260d2f52df7","order_by":1,"name":"Faris Abdon","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIie2QMQuCQBiGv+PgXE5dk+o/GEKj/ZVCsEVanKLBoLX+S9M3K0Iu1Ry4ZEG7Q5BbpwTRorYF3TN893J8D9x7ABLJD6IsAUJxjsUkZxG41qTw8K1Qs7xgbRR4KaxTpmZFOWRR4dkzLdns5nfP7jGg2eVUp/CpGavo+Mb+6KZ9dMTDmGV5NcoIXIgJ0sn25A1TA6lQOOvWKVy/QVRgUCm+gUELpeNCqGJcKSTHuI1yA9ElEV32Vpdgwhlt6MJ1l+YFLsSPrQdlGOnKKrvWKR9QXs226yXk8c22RCKR/A1PWvdJdEQBxa4AAAAASUVORK5CYII=","orcid":"","institution":"Orotta College of Medicine and Health Sciences","correspondingAuthor":true,"prefix":"","firstName":"Faris","middleName":"","lastName":"Abdon","suffix":""},{"id":583638940,"identity":"761cefe1-5e18-48ce-a1b9-79737d7a2fa6","order_by":2,"name":"Abdalla Elamin","email":"","orcid":"","institution":"Ras al-Khaimah Medical and Health Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Abdalla","middleName":"","lastName":"Elamin","suffix":""},{"id":583638942,"identity":"80ea13a0-90bd-4b1e-a60f-b82f1978a6a7","order_by":3,"name":"Abdalla ElGenaidi","email":"","orcid":"","institution":"Misurata University","correspondingAuthor":false,"prefix":"","firstName":"Abdalla","middleName":"","lastName":"ElGenaidi","suffix":""},{"id":583638945,"identity":"af791637-b586-42a2-b85f-91ed9e2b050d","order_by":4,"name":"Ayman Mustafa","email":"","orcid":"","institution":"Misurata University","correspondingAuthor":false,"prefix":"","firstName":"Ayman","middleName":"","lastName":"Mustafa","suffix":""},{"id":583638946,"identity":"b404eedc-cb39-4215-ab37-e4f234c7cdb9","order_by":5,"name":"Manal Sami","email":"","orcid":"","institution":"Ras al-Khaimah Medical and Health Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Manal","middleName":"","lastName":"Sami","suffix":""}],"badges":[],"createdAt":"2025-12-23 11:23:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8432900/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8432900/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101658887,"identity":"13551d2b-1568-44b2-b8f1-a519ee0b62a9","added_by":"auto","created_at":"2026-02-02 10:28:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":35686,"visible":true,"origin":"","legend":"\u003cp\u003eTime- and concentration-dependent inhibition of HCT-116 cell viability by curcumin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLegend\u003c/strong\u003e. HCT-116 colorectal carcinoma cells were treated with curcumin (0-100 μg/mL) for (A) 24 h, (B) 48 h, or (C) 72 h. Cell viability was assessed by MTT assay and expressed as a percentage of untreated controls. Data are presented as mean ± SD of three independent experiments, each performed in triplicate. Dose-response curves were fitted by non-linear regression using a five-parameter logistic model, and IC₅₀ values were estimated from the fitted curves (48.87 μg/mL at 24 h; 31.0 μg/mL at 48 h; 17.5 μg/mL at 72 h). Statistical comparisons between each treated group and the corresponding untreated control were performed in GraphPad Prism (unpaired t-test for normally distributed data or Wilcoxon test for non-normal data); *P \u0026lt; 0.05 versus control.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8432900/v1/4d359e6cda25c21dfd1de4b1.png"},{"id":101658893,"identity":"fa9f43a6-2d2a-4793-a768-e517d7578506","added_by":"auto","created_at":"2026-02-02 10:28:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":389249,"visible":true,"origin":"","legend":"\u003cp\u003eCurcumin induces apoptosis and necrosis in HCT-116 cells (AO/EtBr staining).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLegend\u003c/strong\u003e. Representative acridine orange/ethidium bromide (AO/EtBr) fluorescence micrographs of HCT-116 cells (A) untreated (control) and (B) treated with 35 μg/mL curcumin for 48 h. Viable cells exhibit uniform green nuclei (AO-positive, EtBr-negative), early apoptotic cells show bright green condensed or fragmented nuclei, late apoptotic cells display orange-red condensed/fragmented nuclei, and necrotic cells show uniformly orange-red nuclei with loss of membrane integrity. Curcumin treatment increased apoptotic and necrotic fractions compared with control (quantified in Table 2). Scale bar = 50 μm.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8432900/v1/428ad7f9b369ee9e50957fee.png"},{"id":101658888,"identity":"5355ed95-a90d-48be-b7e1-5a404f273af6","added_by":"auto","created_at":"2026-02-02 10:28:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":37996,"visible":true,"origin":"","legend":"\u003cp\u003eCurcumin modulates TP53, BAX, and BCL2 mRNA expression in HCT-116 cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLegend.\u003c/strong\u003eHCT-116 cells were treated with 35 μg/mL curcumin for 48 h, and mRNA levels of (A) BAX, (B) TP53, and (C) BCL2 were measured by quantitative real-time PCR. Expression was normalised to GAPDH and calculated using the 2⁻ΔΔCq method, with untreated cells set to 1.0 (dashed horizontal line). Bars show mean ± SD of three independent experiments. Curcumin significantly increased TP53 (2.74-fold) and BAX (1.70-fold) expression and decreased BCL2 expression (0.37-fold) compared with control (P \u0026lt; 0.0001 for all comparisons, unpaired t-test).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8432900/v1/f99d7ee65659e2fc764b5d8f.png"},{"id":101658891,"identity":"974385d0-acbb-4696-a36f-82ee92f50c6c","added_by":"auto","created_at":"2026-02-02 10:28:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":121469,"visible":true,"origin":"","legend":"\u003cp\u003eCurcumin induces S-phase accumulation in HCT-116 cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLegend\u003c/strong\u003e. HCT-116 cells were left untreated (control) or treated with 35 μg/mL curcumin for 48 h, stained with propidium iodide, and analysed by flow cytometry. (A) Representative DNA-content histograms showing Sub-G1, G0/G1, S, and G2/M peaks in control and curcumin-treated cells. (B) Quantitative distribution of cells in Sub-G1 (hypodiploid/apoptotic), G0/G1, S, and G2/M phases. Curcumin treatment reduced the G0/G1 fraction (from 71.58% to 60.26%) and increased the S-phase fraction (from 7.50% to 18.69%), with minimal change in G2/M and a slight increase in Sub-G1 (from 19.33% to 20.33%). Bars represent mean ± SD of three independent experiments. Percentages were derived from gating on DNA-content histograms using the instrument’s analysis software.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8432900/v1/462e55f7140e87cd034f3670.png"},{"id":101658967,"identity":"827f1156-dada-4960-b17e-cffd2ade2f75","added_by":"auto","created_at":"2026-02-02 10:28:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1654206,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8432900/v1/4ee62382-1a7a-4edf-b422-5192a853e0c3.pdf"},{"id":101658844,"identity":"e57dc3c5-2e70-49d9-aea3-4ab9e104dceb","added_by":"auto","created_at":"2026-02-02 10:28:27","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":19367,"visible":true,"origin":"","legend":"","description":"","filename":"SuppTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8432900/v1/dff516d9792f7e4485a2504b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Curcumin Triggers S-Phase Accumulation and p53-Bax/Bcl-2-Mediated Intrinsic Apoptosis in HCT-116 Colorectal Cancer Cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer remains a major contributor to global cancer morbidity and mortality, and clinical outcomes are strongly influenced by stage at diagnosis and therapeutic resistance [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. While advances in screening and systemic therapy have improved survival in some settings, recurrence and treatment-limiting toxicity remain persistent barriers [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These challenges continue to motivate evaluation of low-toxicity agents that can modulate proliferation and cell-death programs and potentially complement established regimens [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrent CRC management combines surgery and, where indicated, radiotherapy with systemic therapy built around fluoropyrimidines, oxaliplatin, and irinotecan, with targeted agents or immunotherapy used in selected patients [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Although these approaches improve outcomes (particularly when disease is detected early), durable control in locally advanced and metastatic CRC is frequently constrained by treatment resistance, cumulative toxicity, and recurrence after apparently successful therapy [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Relapse is often attributed to residual tumor subpopulations that survive initial therapy and later repopulate the tumor.\u003c/p\u003e \u003cp\u003eOne explanatory framework is the cancer stem cell (CSC) model, in which a minority of self-renewing cells contributes to tumour maintenance, dissemination, and therapeutic resistance [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. CRC models enriched for CSC-associated markers (e.g., CD44, CD133, CD166, and ALDH) show enhanced invasion, sphere formation, and regrowth after FOLFOX or FOLFIRI exposure and can be difficult to eradicate with conventional agents alone [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Accordingly, combination strategies that add targeted or dietary agents to standard chemotherapy have been explored as a means to reduce CSC-associated phenotypes and potentially improve long-term control [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurcumin, the principal curcuminoid of \u003cem\u003eCurcuma longa\u003c/em\u003e, is widely studied as a pleiotropic modulator of cancer-associated signalling and is frequently described as having a favourable toxicity profile in non-malignant tissues [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In experimental colon carcinogenesis (including azoxymethane-induced and inflammation-driven models) dietary curcumin reduced tumour incidence and severity [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In established CRC cell lines such as HCT-116, HT-29, SW480, and SW620, curcumin and its derivatives suppress proliferation, migration, and invasion, decrease clonogenic and sphere-forming capacity, and enhance chemosensitivity [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Mechanistically, curcumin can modulate transcription factors (e.g., NF-κB, STAT3), growth-factor signalling, inflammatory mediators, redox balance, and mitochondrial apoptotic signalling [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In addition, curcumin can reverse multidrug resistance (MDR) in colon cancer models by decreasing P-glycoprotein and survivin, increasing intracellular Rhodamine 123 accumulation, and sensitising vincristine-resistant cells to multiple chemotherapeutic agents [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eApoptosis in CRC is tightly regulated by p53 and the Bcl-2 family. Foundational studies demonstrated that Bcl-2 can heterodimerise with Bax and that the relative balance of anti- and pro-apoptotic members functions as a molecular rheostat governing commitment to programmed cell death following cellular stress [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. p53 integrates DNA damage and other stress signals and can shift this balance by transcriptionally up-regulating pro-apoptotic genes, such as BAX, and down-regulating anti-apoptotic genes, such as BCL2 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. More recent work emphasises that p53 sits within a broader stress-response network in which phosphorylation and acetylation events, protein-protein interactions, and subcellular localisation collectively influence whether cells undergo transient arrest, senescence, or apoptosis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In colon cancer models, curcumin has been reported to promote apoptosis with increased p53 and Bax and decreased Bcl-2, along with caspase activation and characteristic apoptotic morphology; however, the magnitude and timing of these changes vary by cell line, dose, and exposure duration [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurcumin also interferes with cell-cycle progression in CRC. Depending on the cell line, dose, and formulation, curcumin has been reported to cause arrest in G₀/G₁, S, and/or G₂/M phases, associated with modulation of cyclin-dependent kinases (CDKs), p21, and the retinoblastoma (Rb) pathway, and in some settings with evidence of DNA damage and impaired DNA repair [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In HCT-116 cells, curcumin and curcumin-based nanoprodrugs can induce cell-cycle alterations, reduce clonogenic growth, and downregulate regulators such as PLK1 and several CDKs, with promising effects in xenograft and colitis-associated CRC models [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Delivery approaches, including dendrosomal and phytosomal formulations, can improve solubility and bioavailability and frequently display enhanced antiproliferative activity and multi-phase cell-cycle effects compared with free curcumin [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite this growing literature, important gaps remain. Many studies use heterogeneous curcumin preparations or nano-formulations, focus on a single read-out, or examine cell lines with mutant p53, making it difficult to link curcumin-induced growth inhibition to apoptosis-related transcriptional changes and cell-cycle redistribution in a defined genetic context [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. HCT-116 cells, which harbour wild-type TP53 and mutant KRAS, are widely used in vitro CRC models and are particularly suitable for dissecting p53-dependent responses [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. However, there are limited data that simultaneously quantify, in the same experimental system, the time- and concentration-dependent cytotoxicity of free curcumin, the distribution of viable/apoptotic/necrotic cells, transcriptional regulation of TP53, BAX, and BCL2, and PI-based cell-cycle distribution at a defined cytotoxic concentration in this line [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccordingly, we profiled the effects of free curcumin on viability, cell death morphology, TP53/BAX/BCL2 expression, and PI-based cell-cycle distribution in HCT-116 cells to link cytotoxicity with apoptosis-related transcriptional changes and cell-cycle perturbations.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and reagents\u003c/h2\u003e \u003cp\u003eDimethyl sulfoxide (DMSO), curcumin (\u0026ge;\u0026thinsp;96% purity), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), fetal bovine serum (FBS), and trypan blue were purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640 medium, HEPES buffer solution, L-glutamine, gentamicin, and 0.25% trypsin-EDTA were obtained from Biowest (Nuaill\u0026eacute;, France). Acridine orange and ethidium bromide were purchased from Merck KGaA (Darmstadt, Germany).\u003c/p\u003e \u003cp\u003eTotal RNA was isolated using TransZol reagent (TransGen Biotech, China). First-strand cDNA was synthesized with the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). Quantitative real-time PCR (qPCR) was performed using Maxima SYBR Green qPCR Master Mix (Thermo Scientific). Propidium iodide (PI), RNase A, binding buffer, and the CycleTEST\u0026trade; PLUS DNA reagent kit were obtained from BD (Franklin Lakes, NJ, USA). All other reagents were of analytical grade.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell line and culture conditions\u003c/h3\u003e\n\u003cp\u003eThe human colorectal carcinoma cell line HCT-116 (epithelial morphology; large intestine/colon carcinoma) was used as an in vitro model of colorectal cancer. This cell line was originally derived from the colon of an adult male with colon cancer and harbours a mutation in codon 13 of the RAS proto-oncogene.\u003c/p\u003e \u003cp\u003eCells were maintained in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated FBS, 1% penicillin/streptomycin (100 U/mL penicillin and 100 \u0026micro;g/mL streptomycin), 2 mM L-glutamine, and 10 mM HEPES. Cultures were incubated at 37\u0026deg;C in a humidified atmosphere of 5% CO₂ and 95% air. The culture medium was replaced every 2\u0026ndash;3 days, and cells were sub-cultured at 70\u0026ndash;90% confluence using 0.25% trypsin-EDTA. Cell number and viability were routinely assessed by trypan blue exclusion.\u003c/p\u003e \u003cp\u003eAll in vitro experiments (MTT assay, AO/EtBr staining, qPCR, and cell-cycle analysis) were performed in at least three independent experiments unless otherwise stated.\u003c/p\u003e\n\u003ch3\u003ePreparation of curcumin stock solution and treatment\u003c/h3\u003e\n\u003cp\u003eCurcumin was dissolved in DMSO to prepare a 10 mg/mL stock solution, which was aliquoted and stored at -20\u0026deg;C in the dark until use. Immediately before each experiment, the stock solution was diluted in complete RPMI-1640 medium to obtain the desired working concentrations (0, 5, 10, 20, 40, 50, and 100 \u0026micro;g/mL) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The final DMSO concentration in all wells, including vehicle controls, did not exceed 0.5% (v/v).\u003c/p\u003e \u003cp\u003eFor treatments, HCT-116 cells were seeded and allowed to attach for 24 h, then exposed to curcumin at the indicated concentrations for 24, 48, or 72 h. Untreated cells and DMSO vehicle controls (0.5% DMSO) were included in each experiment [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eCell viability assay (MTT)\u003c/h3\u003e\n\u003cp\u003eCurcumin-induced cytotoxicity was quantified using the MTT assay, which measures the reduction of MTT to insoluble formazan by mitochondrial dehydrogenases in viable cells.\u003c/p\u003e \u003cp\u003eHCT-116 cells were suspended in complete medium and seeded into flat-bottom 96-well plates at 5 \u0026times; 10⁴ cells/well in 100 \u0026micro;L. Plates were incubated for 24 h at 37\u0026deg;C to allow cell attachment. The culture medium was then carefully aspirated and replaced with fresh medium containing curcumin at final concentrations of 0, 5, 10, 20, 40, 50, or 100 \u0026micro;g/mL, or vehicle control (0.5% DMSO). Each concentration was tested in triplicate wells in each independent experiment. Plates were incubated for 24, 48, or 72 h.\u003c/p\u003e \u003cp\u003eAt each time point, the medium was gently removed and replaced with 90 \u0026micro;L fresh RPMI-1640 and 10 \u0026micro;L MTT stock solution (5 mg/mL in PBS; final MTT concentration 0.5 mg/mL) per well. Plates were incubated for 4 h at 37\u0026deg;C to allow viable cells to form purple formazan crystals. The supernatant was then discarded, and the formazan crystals were solubilised in 50 \u0026micro;L DMSO per well, followed by gentle mixing on a plate shaker for 5 min.\u003c/p\u003e \u003cp\u003eThe optical density (OD) was measured at an appropriate wavelength range (530\u0026ndash;630 nm) using a microplate reader. Cell viability (%) was calculated as:\u003c/p\u003e \u003cp\u003eViability (%) = (OD\u003csub\u003et\u003c/sub\u003e/OD\u003csub\u003ec\u003c/sub\u003e)\u0026times;100.\u003c/p\u003e \u003cp\u003eWhere ODt is the mean optical density of wells treated with the tested sample, and ODc is the mean optical density of untreated cells. Dose-response curves were plotted, and half-maximal inhibitory concentration (IC₅₀) values at 24, 48, and 72 h were estimated by non-linear regression of the dose-response data using GraphPad Prism (GraphPad Software, San Diego, CA, USA), according to the general principles described by Mosmann [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eAnalysis of apoptosis and necrosis by acridine orange/ethidium bromide staining\u003c/h3\u003e\n\u003cp\u003eThe mode of cell death (viable, early apoptotic, late apoptotic, and necrotic) induced by curcumin was evaluated using acridine orange/ethidium bromide (AO/EtBr) dual staining.\u003c/p\u003e \u003cp\u003eHCT-116 cells were cultured on 8-well chamber slides (SPL, Seoul, South Korea) at a density of 1 \u0026times; 10⁴ cells/well and allowed to adhere for 24 h. Cells were then treated with curcumin at 35 \u0026micro;g/mL (approximately the 48-h IC₅₀ concentration) for 48 h; untreated cells (0 \u0026micro;g/mL) served as controls. At the end of treatment, the medium was aspirated, and cells were gently washed with PBS. A freshly prepared AO/EtBr staining solution (100 \u0026micro;g/mL acridine orange and 100 \u0026micro;g/mL ethidium bromide in PBS, mixed 1:1) was added to each well.\u003c/p\u003e \u003cp\u003eStained cells were immediately examined under a fluorescence microscope (Axio Imager Z2, Zeiss, Jena, Germany). Several random fields were analysed per condition. Cells with uniformly green nuclei were scored as viable; cells with green/yellow fragmented or condensed nuclei as early apoptotic; cells with bright orange/red condensed or fragmented nuclei as late apoptotic; and cells with uniformly red nuclei without chromatin condensation as necrotic. The percentage of cells in each category was calculated.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and quantitative real-time PCR\u003c/h2\u003e \u003cp\u003eAll solutions were prepared using sterile RNase-free water, and all tubes and pipette tips were handled with appropriate precautions to minimise RNase contamination.\u003c/p\u003e \u003cp\u003eHCT-116 cells were treated with curcumin at 35 \u0026micro;g/mL or vehicle control for 48 h, then harvested for RNA extraction. Total RNA was extracted using TransZol reagent according to the manufacturer\u0026rsquo;s protocol. Briefly, 1 mL cold TransZol was added to each sample, mixed thoroughly, and incubated for 5 min at room temperature. Chloroform (0.2 mL per 1 mL TransZol) was added, samples were vigorously shaken for 30 s, incubated for 3 min, and centrifuged at 10,000 \u0026times; g for 15 min at 4\u0026deg;C. The aqueous phase was transferred to a new tube, mixed with 0.5 mL isopropanol per 1 mL TransZol, incubated for 10 min, and centrifuged at 10,000 \u0026times; g for 10 min at 4\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe RNA pellet was washed with 1 mL 75% ethanol, centrifuged at 7,500 \u0026times; g for 5 min at 4\u0026deg;C, air-dried for approximately 15 min, and dissolved in 50 \u0026micro;L RNase-free water. RNA concentration and purity (A₂₆₀/A₂₈₀) were determined using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). RNA samples were stored at -80\u0026deg;C until use.\u003c/p\u003e \u003cp\u003eFirst-strand cDNA was synthesised from 1 \u0026micro;g total RNA using the RevertAid First Strand cDNA Synthesis Kit according to the manufacturer\u0026rsquo;s instructions. Gene expression of TP53 (p53), BAX, and BCL-2 was quantified by SYBR Green-based qPCR using Maxima SYBR Green qPCR Master Mix in a real-time PCR system (DTlite, DNA-Technology). GAPDH was used as the endogenous control.\u003c/p\u003e \u003cp\u003eEach 25 \u0026micro;L qPCR reaction contained 12.5 \u0026micro;L 2\u0026times; SYBR Green master mix, 1 \u0026micro;L of each forward and reverse primer, cDNA template (\u0026le;\u0026thinsp;100 ng), and nuclease-free water. All reactions were run in technical triplicate for each biological replicate. Relative mRNA expression was calculated using the 2⁻ΔΔCq method, normalising target genes to GAPDH and comparing curcumin-treated samples to untreated controls.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell cycle analysis by flow cytometry\u003c/h3\u003e\n\u003cp\u003eCell-cycle distribution and the hypodiploid (sub-G1) population were analysed by propidium iodide staining and flow cytometry. HCT-116 cells were cultured in flasks until approximately 70\u0026ndash;80% confluence, then treated with curcumin at the 48-h IC₅₀ concentration (\u0026asymp;\u0026thinsp;35 \u0026micro;g/mL) for 48 h or left untreated (vehicle only) as controls. At the end of the incubation period, both floating and adherent cells were collected; adherent cells were detached by trypsinisation and pooled with floating cells, pelleted by centrifugation, and washed twice with cold PBS.\u003c/p\u003e \u003cp\u003eCell pellets were gently resuspended in PBS, and ice-cold 70% ethanol was added dropwise while vortexing to achieve fixation. Samples were fixed at -20\u0026deg;C for at least 1 h. Fixed cells were then centrifuged to remove ethanol, washed with PBS, and resuspended in staining solution containing PI and RNase A using the CycleTEST\u0026trade; PLUS DNA reagent kit (BD Biosciences) according to the manufacturer\u0026rsquo;s instructions. Samples were incubated in the dark at room temperature for 30\u0026ndash;60 min before acquisition.\u003c/p\u003e \u003cp\u003ePI fluorescence histograms were generated, and the proportions of cells with hypodiploid DNA content (sub-G1 population, used as an index of late apoptosis) and with diploid/tetraploid DNA content corresponding to G0/G1, S, and G2/M phases were determined using the instrument\u0026rsquo;s analysis software [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Treated and control samples were compared to evaluate curcumin-induced alterations in cell-cycle distribution and in the sub-G1 fraction.\u003c/p\u003e\n\u003ch3\u003eStatistical data analysis\u003c/h3\u003e\n\u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) from at least three independent experiments. Dose-response curves for cell viability were fitted using non-linear regression with a five-parameter logistic model in GraphPad Prism version 10.3.1 (GraphPad Software, San Diego, CA, USA), and IC₅₀ values were obtained from the fitted curves. For other endpoints, comparisons between control and curcumin-treated groups were performed using two-tailed unpaired Student\u0026rsquo;s t-tests when data were approximately normally distributed and the Wilcoxon rank-sum test when distributional assumptions were not met. A P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCurcumin reduces viability of HCT-116 cells in a time- and concentration-dependent manner\u003c/h2\u003e \u003cp\u003eThe effect of curcumin on the viability of HCT-116 colorectal carcinoma cells was evaluated by MTT assay after 24, 48, and 72 h of exposure to 0-100 \u0026micro;g/mL curcumin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAt 24 h, cell viability remained above 80% at 5\u0026ndash;20 \u0026micro;g/mL (81.03\u0026thinsp;\u0026plusmn;\u0026thinsp;2.23%, 99.73\u0026thinsp;\u0026plusmn;\u0026thinsp;11.14%, and 83.75\u0026thinsp;\u0026plusmn;\u0026thinsp;12.31%, respectively) but decreased to 64.06\u0026thinsp;\u0026plusmn;\u0026thinsp;14.76% at 40 \u0026micro;g/mL, 48.87\u0026thinsp;\u0026plusmn;\u0026thinsp;2.68% at 50 \u0026micro;g/mL, and 39.44\u0026thinsp;\u0026plusmn;\u0026thinsp;5.88% at 100 \u0026micro;g/mL (P\u0026thinsp;=\u0026thinsp;0.0058 for overall effect vs control). The estimated IC₅₀ at 24 h was 48.87 \u0026micro;g/mL.\u003c/p\u003e \u003cp\u003eAt 48 h, curcumin produced a more marked reduction in viability (P\u0026thinsp;=\u0026thinsp;0.001). Viability declined from 100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;7.56% in untreated cells to 97.00\u0026thinsp;\u0026plusmn;\u0026thinsp;14.19% at 5 \u0026micro;g/mL, 86.61\u0026thinsp;\u0026plusmn;\u0026thinsp;18.33% at 10 \u0026micro;g/mL, 85.95\u0026thinsp;\u0026plusmn;\u0026thinsp;9.77% at 20 \u0026micro;g/mL, and sharply to 18.15\u0026thinsp;\u0026plusmn;\u0026thinsp;11.13%, 17.38\u0026thinsp;\u0026plusmn;\u0026thinsp;6.70%, and 14.89\u0026thinsp;\u0026plusmn;\u0026thinsp;4.43% at 40, 50, and 100 \u0026micro;g/mL, respectively. The corresponding 48-h IC₅₀ was 31.0 \u0026micro;g/mL.\u003c/p\u003e \u003cp\u003eAt 72 h, curcumin markedly suppressed cell viability (P\u0026thinsp;=\u0026thinsp;0.0015). Viability remained relatively high at 5\u0026ndash;10 \u0026micro;g/mL (81.24\u0026thinsp;\u0026plusmn;\u0026thinsp;17.25% and 78.13\u0026thinsp;\u0026plusmn;\u0026thinsp;19.00%), but decreased to 38.33\u0026thinsp;\u0026plusmn;\u0026thinsp;14.36% at 20 \u0026micro;g/mL and to 6.31\u0026thinsp;\u0026plusmn;\u0026thinsp;2.92%, 5.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85%, and 5.41\u0026thinsp;\u0026plusmn;\u0026thinsp;2.23% at 40, 50, and 100 \u0026micro;g/mL, respectively. The 72-h IC₅₀ was 17.5 \u0026micro;g/mL.\u003c/p\u003e \u003cp\u003eCurcumin reduced HCT-116 cell viability in a time- and concentration-dependent manner, with IC₅₀ values of 48.87 \u0026micro;g/mL at 24 h, 31.0 \u0026micro;g/mL at 48 h, and 17.5 \u0026micro;g/mL at 72 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCurcumin at 35 \u0026micro;g/mL induces apoptosis and necrosis in HCT-116 cells\u003c/h2\u003e \u003cp\u003eTo characterize the mode of cell death at a cytotoxic concentration, HCT-116 cells were exposed to 35 \u0026micro;g/mL curcumin (approximately the 48-h IC₅₀) for 48 h and stained with acridine orange/ethidium bromide. Representative fluorescence micrographs are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, and quantitative data are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eIn untreated controls, the majority of cells were viable (95%), with a small fraction (5%) exhibiting features of early apoptosis; late apoptotic and necrotic cells were not observed (0% each). After treatment with 35 \u0026micro;g/mL curcumin, the proportion of viable cells decreased to 40%, while 10% of cells were in early apoptosis, 32% in late apoptosis, and 18% were necrotic (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCurcumin-treated cells exhibited morphological hallmarks of apoptosis, including cell shrinkage, membrane blebbing, and chromatin condensation/fragmentation, together with a subset of cells showing necrotic morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCurcumin at 35 \u0026micro;g/mL modulates p53, BAX, and BCL-2 expression\u003c/h2\u003e \u003cp\u003eThe impact of curcumin on apoptosis-related genes was assessed by SYBR Green qPCR after 48 h exposure of HCT-116 cells to 35 \u0026micro;g/mL curcumin. Expression levels were normalized to GAPDH and calculated using the 2⁻ΔΔCq method, with untreated cells as calibrator (RQ\u0026thinsp;=\u0026thinsp;1.0). Detailed Ct, ΔCt, ΔΔCt, and RQ values are provided in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003eCurcumin significantly increased BAX mRNA expression compared with control cells, with a mean fold change (RQ) of 1.70 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The tumor suppressor TP53 (p53) was also significantly upregulated, with a mean fold change of 2.74 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eIn contrast, curcumin treatment reduced the expression of the anti-apoptotic gene BCL-2, with a mean relative expression of 0.37 compared with control (\u0026asymp;\u0026thinsp;63% decrease; P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eAt 35 \u0026micro;g/mL, curcumin increased p53 and BAX mRNA levels and decreased BCL-2 expression in HCT-116 cells (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Supplementary Table\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCurcumin at 35 \u0026micro;g/mL induces S-phase accumulation in HCT-116 cells\u003c/h2\u003e \u003cp\u003eTo examine the effect of curcumin on cell-cycle distribution, HCT-116 cells were treated with 35 \u0026micro;g/mL curcumin for 48 h, stained with propidium iodide, and analyzed by flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn untreated control cells, 71.58% were in G1 phase, 7.50% in S phase, 0.53% in G2/M, and 19.33% in the sub-G1 (hypodiploid) population, which was used as an index of late apoptosis (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). After treatment with curcumin, the fraction of cells in G1 decreased to 60.26%, whereas the proportion in S phase increased to 18.69%. The G2/M fraction remained low (0.64%), and the sub-G1 population showed a slight increase to 20.33% (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAt 35 \u0026micro;g/mL, 48-h curcumin exposure was associated with a redistribution of HCT-116 cells from G1 into S phase, i.e., S-phase accumulation, with minimal change in the G2/M fraction (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTime- and concentration-dependent effects of curcumin on viability of HCT-116 colorectal carcinoma cells (MTT assay)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCurcumin (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24 h viability % (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48 h viability % (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e72 h viability % (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0 (control)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;6.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;7.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;6.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e81.03\u0026thinsp;\u0026plusmn;\u0026thinsp;2.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e97.00\u0026thinsp;\u0026plusmn;\u0026thinsp;14.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e81.24\u0026thinsp;\u0026plusmn;\u0026thinsp;17.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e99.73\u0026thinsp;\u0026plusmn;\u0026thinsp;11.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e86.61\u0026thinsp;\u0026plusmn;\u0026thinsp;18.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e78.13\u0026thinsp;\u0026plusmn;\u0026thinsp;19.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e20\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e83.75\u0026thinsp;\u0026plusmn;\u0026thinsp;12.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e85.95\u0026thinsp;\u0026plusmn;\u0026thinsp;9.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e38.33\u0026thinsp;\u0026plusmn;\u0026thinsp;14.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e40\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e64.06\u0026thinsp;\u0026plusmn;\u0026thinsp;14.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e18.15\u0026thinsp;\u0026plusmn;\u0026thinsp;11.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e6.31\u0026thinsp;\u0026plusmn;\u0026thinsp;2.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e50\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e48.87\u0026thinsp;\u0026plusmn;\u0026thinsp;2.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e17.38\u0026thinsp;\u0026plusmn;\u0026thinsp;6.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e5.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e100\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e39.44\u0026thinsp;\u0026plusmn;\u0026thinsp;5.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e14.89\u0026thinsp;\u0026plusmn;\u0026thinsp;4.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e5.41\u0026thinsp;\u0026plusmn;\u0026thinsp;2.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFootnote.\u003c/b\u003e Values are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) of percentage viability relative to untreated control (100%) from three independent experiments (each performed in triplicate). Curcumin significantly reduced HCT-116 cell viability in a concentration-dependent manner at all time points (overall effect vs control: P\u0026thinsp;=\u0026thinsp;0.0058 at 24 h, P\u0026thinsp;=\u0026thinsp;0.001 at 48 h, and P\u0026thinsp;=\u0026thinsp;0.0015 at 72 h). The corresponding IC₅₀ values, estimated by non-linear regression of the dose\u0026ndash;response curves, were 48.87 \u0026micro;g/mL (24 h), 31.0 \u0026micro;g/mL (48 h), and 17.5 \u0026micro;g/mL (72 h).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDistribution of viable, apoptotic, and necrotic HCT-116 cells after 48 h of curcumin treatment was assessed by acridine orange/ethidium bromide staining\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCondition\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCurcumin (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eViable cells (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEarly apoptosis (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLate apoptosis (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNecrosis (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCurcumin-treated\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cb\u003eFootnote.\u003c/b\u003e Percentages represent the proportion of cells in each death category after 48 h exposure of HCT-116 cells to curcumin at a concentration close to the 48-h IC₅₀ (\u0026asymp;\u0026thinsp;35 \u0026micro;g/mL), compared with untreated controls, as determined by AO/EtBr staining and morphological assessment under a fluorescence microscope.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRelative expression of apoptosis-related genes in HCT-116 cells after 48 h of curcumin treatment (qPCR)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCurcumin (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFold change vs untreated control (RQ)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRegulation vs control\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBAX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUpregulated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTP53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUpregulated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCL-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDownregulated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003cb\u003eFootnote.\u003c/b\u003e Gene expression was quantified by SYBR Green qPCR after 48 h exposure of HCT-116 cells to curcumin at a concentration close to the 48-h IC₅₀ (35 \u0026micro;g/mL or \u0026asymp;\u0026thinsp;35 \u0026micro;g/mL, depending on the notation you choose to standardize). Expression levels were normalized to GAPDH and calculated using the 2⁻ΔΔCq method, with untreated cells as the calibrator (RQ\u0026thinsp;=\u0026thinsp;1.0). The RQ values shown correspond to the mean relative quantities derived from the underlying calculations (BAX control RQ\u0026thinsp;\u0026asymp;\u0026thinsp;1.01, treated 1.70; TP53 control RQ\u0026thinsp;\u0026asymp;\u0026thinsp;1.00, treated 2.74; BCL-2 control RQ\u0026thinsp;\u0026asymp;\u0026thinsp;1.00, treated 0.37), which are presented in detail in Supplementary Table\u0026nbsp;1. All genes showed significant differences compared with the control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCell-cycle distribution of HCT-116 cells after 48 h of curcumin treatment at the 48-h IC₅₀\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCell-cycle phase\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl (0 \u0026micro;g/mL), n (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCurcumin (\u0026asymp;\u0026thinsp;35 \u0026micro;g/mL), n (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSub-G1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e71.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18.69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG2/M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003cb\u003eFootnote.\u003c/b\u003e HCT-116 cells were treated for 48 h with curcumin at a concentration approximating the 48-h IC₅₀ (\u0026asymp;\u0026thinsp;35 \u0026micro;g/mL) or left untreated. DNA content was assessed by propidium iodide staining and flow cytometry. Sub-G1 represents the hypodiploid/apoptotic fraction. Cells in G0 and G1 phases cannot be distinguished by DNA content alone and are therefore reported together as G0/G1\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis in vitro study examined the effects of free curcumin on cell viability, cell death morphology, apoptosis-related gene expression, and cell cycle distribution in HCT-116 colorectal carcinoma cells. Curcumin reduced viability in a clear time- and concentration-dependent manner, with IC₅₀ values decreasing from 48.87 \u0026micro;g/mL at 24 h to 31.0 \u0026micro;g/mL at 48 h and 17.5 \u0026micro;g/mL at 72 h. At a cytotoxic concentration close to the 48-h IC₅₀ (35 \u0026micro;g/mL), curcumin induced predominantly apoptotic cell death with a smaller necrotic component, increased TP53 and BAX mRNA levels, decreased BCL2 mRNA, and produced S-phase accumulation with minimal change in G₂/M. Collectively, these findings support a model in which curcumin imposes replication-associated stress and engages a p53-centred programme that shifts the Bax/Bcl-2 balance toward mitochondrial apoptosis in this p53-competent CRC line.\u003c/p\u003e \u003cp\u003eThe progressive leftward shift in IC₅₀ values over 72 h indicates that both exposure time and concentration are critical determinants of curcumin cytotoxicity in this system. At 5\u0026ndash;10 \u0026micro;g/mL, viability remained relatively preserved, particularly at earlier time points, whereas at \u0026ge;\u0026thinsp;40 \u0026micro;g/mL, viability declined sharply by 48\u0026ndash;72 h. This profile is more consistent with a compound that perturbs multiple cellular networks over time than with an immediate membrane toxin. In colorectal cancer models, curcumin and related formulations typically suppress viability and clonogenic growth over tens of micromolar concentrations and multi-day exposures, and these effects are linked to modulation of NF-κB, STAT3, PI3K/Akt, AMPK-COX-2, and redox homeostasis rather than a single dominant target [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Consistent with this, in HCT-116 cells, nanocurcumin and other advanced formulations often achieve comparable or stronger effects at lower nominal doses due to improved solubility and uptake [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Under the relatively stringent conditions used here (free curcumin in standard medium), the marked loss of viability by 48\u0026ndash;72 h supports a genuine antiproliferative and cytotoxic effect.\u003c/p\u003e \u003cp\u003eThe dose-response pattern (modest effects at 5\u0026ndash;10 \u0026micro;g/mL with a steep decline above ~\u0026thinsp;40 \u0026micro;g/mL at later time points) suggests threshold behaviour. Below a certain intracellular burden, cells appear able to accommodate curcumin-induced stress; above that threshold, damage accumulates and commitment to death increases. Mechanistically, curcumin is known to disrupt cellular redox balance and increase reactive oxygen species (ROS) in colon cancer cells, with superoxide and other ROS contributing to mitochondrial dysfunction and apoptosis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Curcumin can also interfere with survival signalling (including NF-κB, STAT3, and PI3K/Akt) and sensitize CRC cells to agents such as 5-fluorouracil and oxaliplatin [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The temporal IC₅₀ shift observed here is compatible with progressive amplification of stress signalling and cumulative disruption of adaptive responses.\u003c/p\u003e \u003cp\u003eAt 35 \u0026micro;g/mL, AO/EtBr staining demonstrated that apoptosis predominated: the viable fraction decreased from 95% to 40%, late apoptotic cells accounted for 32%, early apoptotic cells for 10%, and necrotic cells for 18%. The morphological hallmarks (cell shrinkage, membrane blebbing, chromatin condensation, and nuclear fragmentation) support activation of regulated apoptosis rather than purely accidental necrosis. Similar patterns have been reported in HT-29 and other CRC models in which curcumin increases annexin V-positive fractions, induces DNA fragmentation, and activates caspase-3 and caspase-9, often accompanied by changes in p53, Bax, and Bcl-2 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The necrotic fraction observed here is plausible at a clearly cytotoxic dose: some cells may undergo primary necrosis when injury is too severe, while others enter apoptosis but progress to secondary necrosis in 2D culture in the absence of phagocytic clearance. Although we did not directly assess caspase activity or mitochondrial membrane potential, the combined morphology and transcriptional profile (below) support apoptosis as the dominant mode of death under these conditions.\u003c/p\u003e \u003cp\u003eThe transcriptional response to curcumin (TP53 up 2.74-fold; BAX up 1.70-fold; BCL2 reduced to 0.37-fold of control) is consistent with engagement of the p53\u0026ndash;Bax/Bcl-2 axis. Foundational work established that anti-apoptotic Bcl-2 can heterodimerise with pro-apoptotic Bax, and that the relative abundance of these proteins functions as a rheostat influencing susceptibility to mitochondrial apoptosis after stress [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. p53 integrates DNA damage and broader stress signals and can transcriptionally up-regulate BAX and other pro-apoptotic mediators while down-regulating BCL2 or functionally antagonising Bcl-2, thereby lowering the threshold for intrinsic apoptosis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Importantly, p53 outcomes are controlled by a complex network of post-translational modifications, protein-protein interactions, and changes in subcellular localisation rather than a single linear pathway [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWithin this framework, the coordinated increase in TP53 and BAX alongside suppression of BCL2 matches the direction of change expected to favour mitochondrial apoptosis. Prior CRC studies have reported comparable shifts in the pathway at the protein level. In HT-29 cells, for example, curcumin increased phospho-p53 (Ser15), up-regulated Bax, down-regulated Bcl-2, and triggered caspase activation, leading to apoptotic morphology [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In HCT-116 and related CRC systems, curcumin and curcumin-based formulations have also been reported to increase p53 and Bax while decreasing Bcl-2, with associated shifts in Bax/Bcl-2 balance and enhanced chemosensitivity [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Our data extend these observations by showing a coordinated transcriptional engagement of this axis under a defined exposure condition in wild-type p53 HCT-116 cells, consistent with the observed apoptotic phenotype.\u003c/p\u003e \u003cp\u003eCurcumin altered the cell-cycle distribution in a pattern more compatible with S-phase accumulation than with a classic G₂/M block. After 48 h at 35 \u0026micro;g/mL, G1 decreased from 71.58% to 60.26% while S phase increased from 7.50% to 18.69%; G₂/M remained low (~\u0026thinsp;0.6%), and the sub-G1 fraction rose only slightly (19.33% to 20.33%). This redistribution suggests that curcumin-treated cells enter S phase but fail to complete DNA replication efficiently, consistent with replication stress and/or DNA damage signalling. Across CRC models, curcumin and its analogues have been reported to induce arrest at G₀/G₁, S, and/or G₂/M depending on the cell line, dose, exposure time, and formulation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In HCT-116, curcumin has been linked to S-phase accumulation associated with DNA damage and impaired repair, whereas other settings report G₀/G₁ or G₂/M effects mediated via p21-Rb signalling and CDK modulation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Curcumin-based nano-prodrugs and phytosomal or dendrosomal formulations can induce multi-phase arrest and suppress CDKs and PLK1 with improved potency relative to free curcumin [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this context, our data indicate that free curcumin at 35 \u0026micro;g/mL over 48 h primarily delays S-phase progression in HCT-116 cells. The modest rise in sub-G1 by PI staining, despite substantial apoptosis on microscopy, is compatible with a dynamic state in which many cells have initiated apoptosis but have not yet progressed to extensive DNA fragmentation sufficient to be scored as hypodiploid. Mechanistically, the combination of S-phase accumulation and increased TP53 expression is consistent with replication stress engaging p53-dependent checkpoints, followed by Bax/Bcl-2 reprogramming and commitment to intrinsic apoptosis.\u003c/p\u003e \u003cp\u003eAlthough we did not directly assess cancer stem cell (CSC) markers, multidrug resistance (MDR) transporters, or drug combinations, the pattern observed here is relevant to these areas. CSC-enriched CRC models (often CD133- and ALDH-positive) exhibit increased resistance to 5-FU and oxaliplatin and are implicated in relapse after standard therapy [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Curcumin analogues, such as GO-Y030, inhibit STAT3, reduce ALDH⁺/CD133⁺ colon CSC populations, suppress tumor sphere formation, and inhibit tumor growth in vivo [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Curcumin has also been reported to modulate MDR mechanisms: in vincristine-resistant colon cancer cells, curcumin decreased P-glycoprotein and survivin, increased intracellular rhodamine-123 accumulation, and resensitized cells to multiple cytotoxics [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In HCT-116, curcumin enhanced 5-FU activity against viability, invasion, and migration [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and in inflammation-driven CRC models, phytosomal curcumin reduced tumor burden and modulated inflammatory and proliferative markers [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Taken together, our results in parental HCT-116 cells provide a mechanistic baseline for these observations: an S-phase-linked stress response, with activation of p53-Bax/Bcl-2 signalling, would be expected to lower the apoptotic threshold in proliferating cells, potentially increasing vulnerability to antimetabolites and DNA-damaging agents in combination settings.\u003c/p\u003e \u003cp\u003eSeveral limitations should be considered. First, all experiments were performed in a single cell line (HCT-116; TP53 wild-type, KRAS mutant), and responses may differ in TP53-mutant CRC lines or in non-malignant colon epithelial cells [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Second, mechanistic readouts (AO/EtBr, qPCR, and cell-cycle analysis) were conducted at a single curcumin concentration (35 \u0026micro;g/mL) and a single time point (48 h), which does not define dose-response relationships or early versus late dynamics. Third, TP53, BAX, and BCL2 were measured only at the mRNA level; because both p53 and Bcl-2 family proteins are strongly regulated post-translationally, protein-level and functional confirmation (p53 stabilisation/phosphorylation, Bax/Bcl-2 protein ratio, mitochondrial membrane potential, caspase activation) is needed to strengthen causal inference [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], Fourth, AO/EtBr staining is informative but cannot distinguish primary from secondary necrosis and is less quantitative than annexin V/PI flow cytometry. Finally, the work was conducted in 2D monolayers; organoids, CSC-enriched systems, and in vivo models may yield different sensitivities and microenvironment-dependent effects [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFuture work should extend these findings in several directions. Time-course and multi-dose experiments would better map the temporal sequence among S-phase perturbation, p53 activation, Bax/Bcl-2 remodelling, and apoptosis execution, alongside upstream readouts such as ROS and DNA damage (e.g., γH2AX) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Studies in additional CRC lines with differing TP53 and KRAS status, together with normal colon epithelial models, would clarify genotype-dependent and selective effects [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Combination studies with 5-FU, oxaliplatin, or irinotecan (particularly in CSC-enriched and drug-resistant settings) could test whether curcumin-associated S-phase stress and apoptotic priming translate into additive or synergistic effects [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Finally, direct comparisons of free curcumin with nano-curcumin, dendrosomal, or phytosomal formulations under the same endpoints would clarify how improved delivery influences the S-phase-linked and p53\u0026ndash;Bax/Bcl-2-centred mechanisms identified here [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study shows that in HCT-116 colorectal carcinoma cells, free curcumin reduces viability in a clear time- and concentration-dependent manner and, at cytotoxic doses, induces predominantly apoptotic cell death with a smaller necrotic component. At 35 \u0026micro;g/mL, curcumin is associated with S-phase accumulation and coordinated modulation of the p53-Bax/Bcl-2 axis, characterised by up-regulation of TP53 and BAX and down-regulation of BCL2, consistent with activation of intrinsic, mitochondria-mediated apoptosis.\u003c/p\u003e \u003cp\u003eThese findings support the view that curcumin is not simply a non-specific cytotoxin but a multi-target agent that couples cell-cycle perturbation with intrinsic apoptotic signalling in CRC cells. In a KRAS-mutant, p53-wild-type background, this mechanistic profile provides a rational basis for further evaluation of curcumin and more bioavailable derivatives as adjuncts to standard colorectal cancer therapies, particularly in strategies aimed at lowering the apoptotic threshold and overcoming treatment resistance. Future work in additional genetic backgrounds, CSC-enriched and drug-resistant models, in vivo systems, and in combination with conventional chemotherapeutics and optimised formulations will be essential to define the translational potential of these observations.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAO/EtBr\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eacridine orange/ethidium bromide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCRC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecolorectal cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCt\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecycle threshold\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMSO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edimethyl sulfoxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efetal bovine serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGAPDH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglyceraldehyde 3-phosphate dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIC50\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehalf-maximal inhibitory concentration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMTT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epropidium iodide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eqPCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003equantitative polymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRT-qPCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereverse transcription quantitative polymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003estandard deviation.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable (in vitro study using an established human cell line).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo specific funding was received for this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAhmed Mahamed Abualshroud:\u003c/strong\u003e Conceptualization; Methodology; Investigation (cell culture experiments, MTT assay, AO/EtBr staining, flow cytometry, RT-qPCR); Data curation; Formal analysis; Writing original draft.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFaris Abdon:\u003c/strong\u003e Conceptualization; Methodology; Writing original draft; Writing review \u0026amp; editing; Critical revision for intellectual content; Corresponding author responsibilities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbdalla Ahmed Eldaw Elamin:\u003c/strong\u003e Methodology; Investigation; Data curation; Writing review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbdalla Ramadan ElGenaidi:\u003c/strong\u003e Investigation; Validation; Data curation; Writing review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAyman Balla Mustafa:\u003c/strong\u003e Methodology; Formal analysis; Visualization; Writing review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eManal M Sami:\u003c/strong\u003e Supervision; Methodology; Resources; Writing review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAll authors\u003c/strong\u003e contributed substantially to the work, reviewed and approved the final manuscript, and agree to be accountable for all aspects of the work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Kratzer TB, Giaquinto AN, Sung H, Jemal A. Cancer statistics, 2025. CA Cancer J Clin. 2025;75(1):10\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAggarwal BB, Sundaram C, Malani N, Ichikawa H. Curcumin: the Indian solid gold. Adv Exp Med Biol. 2007;595:1\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJokhadze N, Das A, Dizon DS. Global cancer statistics: A healthy population relies on population health. CA Cancer J Clin. 2024;74(3):224\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiegel RL, Wagle NS, Cercek A, Smith RA, Jemal A. Colorectal cancer statistics, 2023. CA Cancer J Clin. 2023;73(3):233\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIslami F, Goding Sauer A, Miller KD, Siegel RL, Fedewa SA, Jacobs EJ, et al. Proportion and number of cancer cases and deaths attributable to potentially modifiable risk factors in the United States. CA Cancer J Clin. 2018;68(1):31\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDienstmann R, Salazar R, Tabernero J. Personalizing colon cancer adjuvant therapy: selecting optimal treatments for individual patients. J Clin Oncol. 2015;33(16):1787\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. CA Cancer J Clin. 2024;74(1):12\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Porras VR, Layos L, Mart\u0026iacute;nez-Balibrea E, editors. Curcumin: A therapeutic strategy for colorectal cancer? Seminars in cancer biology. Elsevier; 2021.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBracci L, Fabbri A, Del Corno M, Conti L. Dietary polyphenols: promising adjuvants for colorectal cancer therapies. Cancers. 2021;13(18):4499.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKunnumakkara AB, Bordoloi D, Padmavathi G, Monisha J, Roy NK, Prasad S, et al. Curcumin, the golden nutraceutical: multitargeting for multiple chronic diseases. Br J Pharmacol. 2017;174(11):1325\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNautiyal J, Kanwar SS, Yu Y, Majumdar AP. Combination of dasatinib and curcumin eliminates chemo-resistant colon cancer cells. J Mol Signal. 2011;6(1):1\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLane DP. p53, guardian of the genome. Nature. 1992;358(6381).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHewlings SJ, Kalman DS, Curcumin. A review of its effects on human health. Foods. 2017;6(10):92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeng W, Goel A, editors. Curcumin and colorectal cancer: An update and current perspective on this natural medicine. Seminars in cancer biology. Elsevier; 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharma RA, Gescher AJ, Steward WP. Curcumin: the story so far. Eur J Cancer. 2005;41(13):1955\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSelvam C, Prabu SL, Jordan BC, Purushothaman Y, Umamaheswari A, Zare MSH, et al. Molecular mechanisms of curcumin and its analogs in colon cancer prevention and treatment. Life Sci. 2019;239:117032.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShanmugam MK, Rane G, Kanchi MM, Arfuso F, Chinnathambi A, Zayed M, et al. The multifaceted role of curcumin in cancer prevention and treatment. Molecules. 2015;20(2):2728\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVillegas I, S\u0026aacute;nchez-Fidalgo S, de la Alarcon C. New mechanisms and therapeutic potential of curcumin for colorectal cancer. Mol Nutr Food Res. 2008;52(9):1040\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVillegas I, S\u0026aacute;nchez-Fidalgo S, de la Lastra CA. Chemopreventive effect of dietary curcumin on inflammation‐induced colorectal carcinogenesis in mice. Mol Nutr Food Res. 2011;55(2):259\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi P, Pu S, Lin C, He L, Zhao H, Yang C, et al. Curcumin selectively induces colon cancer cell apoptosis and S cell cycle arrest by regulates Rb/E2F/p53 pathway. J Mol Struct. 2022;1263:133180.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu H, Wu H, Zhao Q, Zhao R, Liu J, Yang Z, et al. Curcumin suppresses colorectal cancer by inhibiting TRIM2 and mTOR signaling. Translational Oncol. 2025;61:102517.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiang L, He B, Liu Q, Hu D, Liao W, Li R, et al. Antitumor effects of curcumin on the proliferation, migration and apoptosis of human colorectal carcinoma HCT\u0026ndash;116 cells. Oncol Rep. 2020;44(5):1997\u0026ndash;2008.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi S, Zhu X-R, Qin B-C, Chen M-B. Curcumin in colorectal cancer: mechanistic insights, pharmacological limitations, and translational perspectives. Front Pharmacol. 2025;16:1667731.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu W-D, Qin Y, Yang C, Li L. Effect of curcumin on human colon cancer multidrug resistance in vitro and in vivo. Clinics. 2013;68:694\u0026ndash;701.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOltval ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programed cell death. Cell. 1993;74(4):609\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoule RJ, Strasser A. The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol. 2008;9(1):47\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVousden KH, Lane DP. p53 in health and disease. Nat Rev Mol Cell Biol. 2007;8(4):275\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLakin ND, Jackson SP. Regulation of p53 in response to DNA damage. Oncogene. 1999;18(53):7644\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrives C, Hall PA. The p53 pathway. J Pathol. 1999;187(1):112\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong G, Mao Y, Cai Q, Yao L, Ouyang G, Bao S. Curcumin induces human HT-29 colon adenocarcinoma cell apoptosis by activating p53 and regulating apoptosis-related protein expression. Braz J Med Biol Res. 2005;38:1791\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWatson JL, Hill R, Yaffe PB, Greenshields A, Walsh M, Lee PW, et al. Curcumin causes superoxide anion production and p53-independent apoptosis in human colon cancer cells. Cancer Lett. 2010;297(1):1\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu D, Feng X, Wan Y, Yang L, Gao Q, Yang Z, et al. Curcumin nano-prodrug induces multi-phase cell cycle arrest in colorectal cancer through suppression of CDKs and specific down-regulation of PLK1. Smart Mater Med. 2023;4:648\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePricci M, Girardi B, Giorgio F, Losurdo G, Ierardi E, Di Leo A. Curcumin and colorectal cancer: from basic to clinical evidences. Int J Mol Sci. 2020;21(7):2364.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen J. The cell-cycle arrest and apoptotic functions of p53 in tumor initiation and progression. Cold Spring Harbor Perspect Med. 2016;6(3):a026104.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShehata M, Rafaat Y, Gad NA, Gaafar HF. Selective Cytotoxic and Chemoprotective effect of nanocurcumin against human HCT-116 cell line. Biochem Lett. 2018;14(1):85\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGabr SA, Elsaed WM, Eladl MA, El-Sherbiny M, Ebrahim HA, Asseri SM, et al. Curcumin modulates oxidative stress, fibrosis, and apoptosis in drug-resistant cancer cell lines. Life. 2022;12(9):1427.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSritharan S, Sivalingam N. Curcumin induced apoptosis is mediated through oxidative stress in mutated p53 and wild type p53 colon adenocarcinoma cell lines. J Biochem Mol Toxicol. 2021;35(1):e22616.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKinzler KW, Vogelstein B. Lessons from hereditary colorectal cancer. Cell. 1996;87(2):159\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCiuca MD, Racovita RC, Curcumin. Overview of extraction methods, health benefits, and encapsulation and delivery using microemulsions and nanoemulsions. Int J Mol Sci. 2023;24(10):8874.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65(1\u0026ndash;2):55\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeif RC. Practical Flow Cytometry, 3rd Edition, by, Howard M, Shapiro MD, Wiley-Liss. Inc., New York, 1995, 542 pages, \u003cspan\u003e$\u003c/span\u003e79.95. Cytometry. 1995;19(4):376-.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShakibaei M, Mobasheri A, Lueders C, Busch F, Shayan P, Goel A. Curcumin enhances the effect of chemotherapy against colorectal cancer cells by inhibition of NF-κB and Src protein kinase signaling pathways. PLoS ONE. 2013;8(2):e57218.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDean RT, Fu S, Stocker R, Davies MJ. Biochemistry and pathology of radical-mediated protein oxidation. Biochem J. 1997;324(1):1\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin L, Liu Y, Li H, Li P, Fuchs J, Shibata H, et al. Targeting colon cancer stem cells using a new curcumin analogue, GO-Y030. Br J Cancer. 2011;105(2):212\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarjaneh RM, Rahmani F, Hassanian SM, Rezaei N, Hashemzehi M, Bahrami A, et al. RETRACTED: Phytosomal curcumin inhibits tumor growth in colitis-associated colorectal cancer. J Cell Physiol. 2018;233(10):6785\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKvansakul M, Hinds MG. The Bcl-2 family: structures, interactions and targets for drug discovery. Apoptosis. 2015;20(2):136\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu J-J, Cai Y-J, Ding J. Curcumin induces DNA damage and caffeine-insensitive cell cycle arrest in colorectal carcinoma HCT116 cells. Mol Cell Biochem. 2011;354(1):247\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-pharmacology-and-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"phat","sideBox":"Learn more about [BMC Pharmacology and Toxicology](http://bmcpharmacoltoxicol.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/phat/Default.aspx","title":"BMC Pharmacology and Toxicology","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"curcumin, colorectal cancer, HCT-116, apoptosis, cell cycle, p53, Bax, Bcl-2","lastPublishedDoi":"10.21203/rs.3.rs-8432900/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8432900/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCurcumin has been widely investigated as a bioactive phytochemical with anticancer potential, but its coupled effects on apoptosis-related transcriptional programs and cell-cycle distribution in colorectal cancer models remain incompletely characterized.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHCT-116 colorectal carcinoma cells were exposed to curcumin (0-100 \u0026micro;g/mL) for 24\u0026ndash;72 h, and viability was quantified by MTT to derive IC₅₀ values. Cell death morphology was assessed by acridine orange/ethidium bromide staining at a cytotoxic exposure. TP53, BAX, and BCL2 mRNA expression was quantified by SYBR Green RT-qPCR, and DNA-content profiling was performed by propidium iodide staining and flow cytometry.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCurcumin reduced HCT-116 viability with increasing exposure, with IC₅₀ values of 48.87 \u0026micro;g/mL (24 h), 31.0 \u0026micro;g/mL (48 h), and 17.5 \u0026micro;g/mL (72 h). At 35 \u0026micro;g/mL for 48 h, AO/EtBr staining showed 40% viable cells, 10% early apoptosis, 32% late apoptosis, and 18% necrosis. Curcumin increased TP53 (2.74-fold) and BAX (1.70-fold) and decreased BCL2 (0.37-fold) versus control (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Flow cytometry demonstrated a shift from G0/G1 (71.58% to 60.26%) toward S phase (7.50% to 18.69%), with minimal change in G2/M.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn HCT-116 cells, free curcumin is associated with S-phase accumulation and a pro-apoptotic transcriptional shift characterized by increased TP53/BAX and reduced BCL2, accompanied by predominantly apoptotic cell death. These findings support further evaluation of curcumin-based strategies (particularly optimized formulations and rational combinations) in colorectal cancer models.\u003c/p\u003e","manuscriptTitle":"Curcumin Triggers S-Phase Accumulation and p53-Bax/Bcl-2-Mediated Intrinsic Apoptosis in HCT-116 Colorectal Cancer Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-02 10:27:23","doi":"10.21203/rs.3.rs-8432900/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-02-17T14:34:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-09T14:10:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-07T14:24:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"327071189791435891017993155249733477879","date":"2026-02-07T01:58:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-02T13:53:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"96910022303387075508900053431757218892","date":"2026-02-02T13:01:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"122668642452278953357486773597700446326","date":"2026-01-31T21:45:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"326939470085251865993698328040460064887","date":"2026-01-30T02:09:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-29T19:48:03+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-09T15:35:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-25T08:57:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-25T08:55:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Pharmacology and Toxicology","date":"2025-12-23T11:09:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-pharmacology-and-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"phat","sideBox":"Learn more about [BMC Pharmacology and Toxicology](http://bmcpharmacoltoxicol.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/phat/Default.aspx","title":"BMC Pharmacology and Toxicology","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d3384e47-419d-4646-8e2b-34241686d709","owner":[],"postedDate":"February 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T10:27:23+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-02 10:27:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8432900","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8432900","identity":"rs-8432900","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}