Mechanism by which norcantharidin enhances radiotherapy sensitivity in colorectal cancer cells: Apoptosis induction via the mitochondrial dysfunction–reactive oxygen species–ERK/p38 MAPK/AKT pathway axis

Mechanism by which norcantharidin enhances radiotherapy sensitivity in colorectal cancer cells: Apoptosis induction via the mitochondrial dysfunction–reactive oxygen species–ERK/p38 MAPK/AKT pathway axis | 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 Mechanism by which norcantharidin enhances radiotherapy sensitivity in colorectal cancer cells: Apoptosis induction via the mitochondrial dysfunction–reactive oxygen species–ERK/p38 MAPK/AKT pathway axis qiong xu, hen zhang, dan dan gao, ying lu bi, ke yi wang, wei zhao, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8609994/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background Radiotherapy constitutes a vital treatment modality for rectal cancer; however, tumor cell radioresistance frequently leads to treatment failure. Although norcantharidin (NCTD) exhibits antitumor activity, its potential as a radiosensitizer for rectal cancer and the underlying molecular mechanisms remain unclear. This study investigated the effect NCTD has on colorectal cancer (CRC) cell radiosensitivity and its potential mechanisms of action. Methods The effects of NCTD combined with ionizing radiation (IR) on CRC cell viability were assessed using CCK-8 and colony formation assays. Apoptosis and intracellular reactive oxygen species (ROS) levels were detected via flow cytometry. The JC-1 probe was employed to evaluate mitochondrial membrane potential and function. Western blotting analysis assessed apoptosis-related proteins and phosphorylation levels of proteins within the ERK/p38MAPK/AKT signaling pathways. Concurrently, a nude mouse xenograft model was established for in vivo validation. Results NCTD significantly enhanced the growth-inhibitory and clonogenic suppression effects of IR on CRC cells whilst synergistically inducing apoptosis in vitro. Mechanistic investigations revealed that NCTD and IR treatment induced mitochondrial dysfunction, leading to a marked increase in intracellular ROS levels, which further activated ERK, p38MAPK, and AKT signaling pathways. Blocking these pathways using N -acetylcysteine significantly reversed the apoptosis-inducing effects and radiosensitizing activity of NCTD and IR. Conclusions NCTD enhances radiotherapy sensitivity in CRC cells by inducing mitochondrial dysfunction, promoting ROS production, and subsequently activating the ROS/ERK/p38MAPK/AKT signaling pathway axis, ultimately inducing apoptosis. This indicates that NCTD holds potential as a radiosensitizer for rectal cancer, offering novel strategies and theoretical foundations for overcoming clinical radiation resistance. Colorectal cancer Mitochondrial dysfunction Norcantharidin Radiotherapy sensitization Reactive oxygen species Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Colorectal cancer (CRC) remains a major global health challenge, ranking as the third most diagnosed cancer and second leading cause of cancer-related deaths [ 1 ]. Radiotherapy plays a crucial role in the multidisciplinary management of rectal cancer, effectively reducing tumor burden and improving patient survival [ 2 ]. However, radioresistance frequently leads to treatment failure and poor prognosis [ 3 ], which is attributable to tumor heterogeneity, the presence of tumor stem cells, and pro-survival signaling activation [ 4 ]. Current strategies to improve radiosensitivity, such as dose escalation and combination therapies, are often limited by increased toxicity and adverse effects [ 5 ], highlighting the urgent need for novel radiosensitizing agents. Norcantharidin (NCTD), a demethylated derivative of the traditional Chinese medicine, cantharidin, exhibits antitumor activity in various cancers [ 6 – 8 ]. Its pro-apoptotic effects are closely related to mitochondrial dysfunction induction and a marked rise in intracellular reactive oxygen species (ROS)[ 6 – 8 ]. Given that ROS generation is a primary mechanism of radiation-induced cell killing, these properties suggest that NCTD holds potential as a radiosensitizer. Nonetheless, the molecular mechanisms by which NCTD enhances radiosensitivity in CRC remain poorly defined. Ionizing radiation (IR) directly damages mitochondria, thereby worsening mitochondrial dysfunction and amplifying ROS production, which in turn enhances cytotoxic effects [ 9 , 10 ]. Notably, low ROS levels within cells following IR exposure may confer tolerance to such radiation in malignant tumors [ 11 , 12 ], making ROS augmentation a rational therapeutic strategy. Critically, ROS acts as a signaling molecule in cellular stress responses and secondary messenger to activate key downstream signaling pathways, including the MAPK family (such as ERK and p38) and AKT pathways. These pathways play a decisive role in determining cellular fate. Notably, the p38 MAPK pathway can be activated via oxidative stress and exerts a survival-dependent effect on cells depending on the therapeutic context [ 13 , 14 ]. Therefore, this study aimed to investigate whether NCTD enhances CRC radiosensitivity by synergizing with IR to aggravate mitochondrial dysfunction and ROS production to favor apoptosis. Our findings provide crucial mechanistic insights into the action of NCTD as a radiosensitizer and support its potential clinical application. Materials and Methods Cell culture Human colon cancer cell lines (HCT-116 and DLD-1) were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). Cells were cultured in RPMI-1640 medium (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco), 1% penicillin, and 0.1% streptomycin (Thermo Fisher Scientific) at 5% CO 2 and 37°C. Cell irradiation and treatment Cells were irradiated using 6 MeV high-energy X-rays generated with a linear accelerator (Varian Clinic 21ES; Palo Alto, CA, USA) at a dose rate of 3.0 Gy/min. Sealed sterile cell culture plates were placed in the center of the irradiation chamber for treatment. NCTD (Yuanye Biotechnology, Shanghai, China) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 100 mM stock solution and then stored at − 20°C. Working concentrations were prepared by diluting the stock solution in complete RPMI-1640 medium immediately before use. Cell viability assays Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Solarbio, Beijing, China). Cells in logarithmic growth were seeded into 96-well plates at a density of 5,000 cells/well. After 24 h, cells were treated with NCTD and/or IR. Following incubation for 24, 48, or 72 h, 10 µL CCK-8 solution was added to each well and incubated for 2–4 h at 37°C. Absorbance was measured at 450 nm using an enzyme-labeled instrument (Bio-Rad, Hercules, CA, USA). Colony formation assay Cells were seeded at densities of 200, 400, 800, 2,000, and 10,000 cells/well in a 6-well plate, depending on the radiation dose (0, 2, 4, 6, or 8 Gy, respectively). After 24 h, cells were treated with NCTD (5 or 50 µM) for 2 h prior to irradiation and then incubated for 10–14 days at 37°C with 5% CO 2 . Colonies were fixed with methanol, stained with 0.1% crystal violet, and counted using ImageJ (National Institutes of Health, Bethesda, MD, USA). Survival curves were fitted, and the sensitization enhancement ratio calculated using the multi-target single-hit model in GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). Wound healing assay Cells were seeded in 6-well plates and grown to 90–95% confluence. A scratch wound was created using a sterile 10 µL pipette tip. After washing with phosphate-buffered saine (PBS) to remove detached cells, fresh medium containing NCTD was added. Wound gaps were imaged at 0, 24, 48, and 72 h using an inverted microscope (Olympus, Tokyo, Japan). Migration distance was quantified by measuring gap width at multiple points using ImageJ. Cell apoptosis assays For the TUNEL assay (Yeasen, Shanghai, China), paraffin-embedded tissue sections or cell smears were deparaffinized and rehydrated. Sections were fixed with 4% paraformaldehyde for 30 min at room temperature (23–25°C) and permeabilized with 20 µg/mL proteinase K for 15 min at 37°C. After washing with PBS, samples were incubated with TUNEL reaction mixture for 60 min at 37°C in the dark, according to the manufacturer’s instructions. Apoptotic cells were visualized under a fluorescence microscope (Nikon, Tokyo, Japan). For quantitative analysis of apoptosis in cultured cells, an Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (Abbkine, Wuhan, China) was used. After treatment, cells were harvested, washed twice with cold PBS, and resuspended in 200 µL of 1× binding buffer. Samples were stained with 5 µL Annexin V-FITC and 2 µL PI for 15 min at room temperature in the dark. Apoptosis was immediately analyzed using a flow cytometer (BD Biosciences, San Jose, CA, USA), and the data processed with FlowJo v.10.8.1. Single-cell gel electrophoresis assay The alkaline comet assay was performed using a kit (ELK Biotechnology, Wuhan, China). Cells were suspended in PBS at 1 × 10 6 cells/mL, mixed with low-melting-point agarose, and spread onto slides. Slides were lysed in a lysis solution at 4℃ for 2 h, then incubated in alkaline electrophoresis buffer (200 mM NaOH, 1 mM EDTA) for 20 min at room temperature. Slides were left at room temperature for 60 min to maintain DNA under alkaline conditions, followed by electrophoresis at 25 V and 300 mA for 30 min. Subsequently, slides were washed with 0.4 mM Tris–HCl (pH 7.5) and stained with PI (10 µg/mL) for 10 min. Comet tails were visualized using a fluorescence microscope. DCFH-DA ROS assay Cells were seeded in 6-well plates and then treated when reaching 50–70% confluence. After treatment, cells were incubated with 10 µM DCFH-DA (diluted in serum-free medium) for 20 min at 37°C in the dark. Cells were washed thrice with serum-free medium, and fluorescence observed using a fluorescence microscope. RNA isolation and quantitative real-time PCR Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The cDNA was synthesized using the Hifair II 1st Strand cDNA Synthesis Kit (Yeasen). The quantitative real-time PCR assay was performed using qPCR SYBR Green Master Mix (Yeasen) on a QuantStudio 6 system (Applied Biosystems, Waltham, MA, USA). Gene expression was normalized to that of GAPDH. Primer sequences are listed in Supplementary Table S1. Western blotting Total protein was extracted using RIPA buffer (Beyotime, Shanghai, China) and quantified with the BCA Protein Assay Kit (Beyotime). Proteins (30 µg/lane) were separated via SDS-PAGE and transferred to PVDF membranes (0.22 µm; Millipore, Burlington, MA, USA). Membranes were blocked with 5% skim milk for 1 h at room temperature, then incubated with primary antibodies overnight at 4°C, followed by HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence reagent (Millipore). Antibody details are provided in Supplementary Table S2. Network pharmacology The SMILES number of NCTD was found on PubChem ( https://pubchem.ncbi.nlm.nih.gov ), and target prediction then performed ( http://swisstargetprediction.ch ). Predicted results were analyzed in Metascape ( https://metascape.org/gp/index.html ) and subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Protein-protein interaction (PPI) networks were obtained from the STRING database. Mitochondrial isolation and protein extraction Mitochondria were isolated using a Mitochondrial Isolation Kit (Beyotime). Briefly, cells were washed, centrifuged, and homogenized in ice-cold cytoplasmic extraction buffer. Following confirmation of cell disruption, the homogenate was centrifuged (600 × g , 10 min) to remove nuclei and cellular debris. The resulting supernatant, was carefully collected, whereas the pellet containing intact mitochondria was retained. The mitochondrial pellet was subsequently washed, resuspended in lysis buffer, and incubated on ice for complete lysis. Thereafter, the sample was centrifuged (12,000 × g , 15 min) to pellet insoluble material. The resulting supernatant, which contains mitochondrial proteins, was collected for further analysis. All subsequent procedures were performed as described in the Western blotting section. MitoTracker Red CMXRos To prepare a 1 mM stock solution, 94.1 µL DMSO was added to 50 µg lyophilized solids for dissolution and then diluted to 200 nM in complete medium. Cells were incubated with the working solution for 30 min at 37°C in the dark. After washing with PBS, cells were fixed with cold methanol at − 20°C for 15 min (if required for co-staining) and imaged using a confocal microscope (Leica, Wetzlar, Germany). Mitochondrial membrane potential assay Mitochondrial membrane potential (ΔΨm) was assessed using the JC-1 fluorescent probe (Yeasen). Cells were treated with NCTD (10, 50 µM) or IR (6 Gy) for 24 h. Following incubation with JC-1 working solution for 15 min and washing with PBS, cells were examined under a fluorescence microscope. In viable cells, mitochondria exhibit red fluorescence (Ex/Em: 550/600 nm), whereas apoptotic or necrotic cells display green fluorescence (Ex/Em: 485/535 nm). Cellular status was assessed by evaluating the red-to-green fluorescence intensity ratio. Immunohistochemical analysis For immunohistochemistry (IHC), deparaffinized and rehydrated sections underwent antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) at 95°C for 20 min. Endogenous peroxidase was blocked using 3% H 2 O 2 , and nonspecific binding reduced with 5% normal goat serum. Primary antibodies—anti-p-p38 (1:1000) and anti-cleaved caspase-3 (1:1800)—were applied overnight at 4°C, followed by incubation with HRP-conjugated secondary antibodies for 1 h at room temperature. Staining was visualized with DAB, counterstained, and three random fields per section then imaged and quantified using ImageJ (AOD = IOD/area). Immunofluorescence assay Cells on coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, then blocked with 5% bovine serum albumin. Incubation with anti-γ-H2AX (1:200) was carried out overnight at 4°C, then stained with Alexa Fluor 488-conjugated secondary antibody and DAPI. Images were obtained using a Leica Stellaris 8 confocal microscope. Animals All animal procedures were performed according to the Institutional Animal Care and Use Committee of the Institute of Radiation Medicine, China Academy of Chinese Medical Sciences (Approval No: IRM/2-IACUC-2401-011) and conducted in accordance with NIH guidelines. Female BALB/c-nu mice (4 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). HCT-116 cells (1 × 10 7 cells in 100 µL PBS) were subcutaneously injected into the left inguinal area. When tumors reached approximately 5 mm in diameter, mice were randomly divided into three groups (n = 4 per group): control, IR alone, and combination (NCTD + IR) groups. The IR group received 10 Gy X-ray irradiation, and the combination group received NCTD (5 mg/kg, intraperitoneal injection) daily for 14 days post-irradiation. Tumor volume was measured every three days and calculated as 0.5 × length × width 2 . At the end of the experiment, tumors and major organs were harvested for analysis. Statistical analyses All experiments were performed in triplicate. Data are presented as mean ± standard deviation. Statistical analysis was conducted using GraphPad Prism 9.0. One-way analysis of variance followed by Tukey’s post-hoc test was used for multiple comparisons. A p-value < 0.05 was considered statistically significant. Results NCTD inhibits proliferation and enhances radiosensitivity in CRC cells To evaluate the cytotoxic effects of NCTD on CRC cells, we performed CCK-8 and colony formation assays. NCTD significantly inhibited the viability of HCT-116 and DLD-1 cells in a time- and dose-dependent manner (Fig. 1 A, B). A wound healing assay further demonstrated that NCTD effectively suppressed cell migration, as evidenced by a significantly reduced scratch closure measured in treated groups compared to that in controls at 24, 48, and 72 h (p < 0.05; Fig. 1 C, D). Consistent with these findings, western blotting analysis revealed that NCTD upregulated E-cadherin and downregulated N-cadherin expression levels (Fig. 1 E, F), suggesting inhibition of the epithelial–mesenchymal transition. Based on these results, IC 20 values (5 µM for HCT-116 and 50 µM for DLD-1) were selected for subsequent experiments. Colony formation assays under X-ray irradiation (0–8 Gy) showed that NCTD pretreatment significantly enhanced radiosensitivity, particularly in HCT-116 cells (Fig. 1 G, H). These data indicate that NCTD suppresses CRC cell proliferation and migration while increasing radiosensitivity. NCTD enhances IR-induced DNA damage and apoptosis We assessed how NCTD affects radiation-induced DNA damage using the comet assay. The NCTD + IR combination resulted in significantly longer comet tails compared to those of IR alone (Fig. 2 A–C), indicating enhanced DNA breakage. Western blotting analysis corroborated these findings, showing increased DNA damage marker levels in the combination group (Fig. 2 D, E). We then evaluated apoptosis induction: combined treatment led to pronounced apoptotic morphology, increased TUNEL-positive cells (Fig. 2 F–G), and elevated Annexin V/PI staining (Fig. 2 H, I). Molecular analysis revealed that NCTD synergized with IR to upregulate pro-apoptotic proteins (cleaved caspase-3, Bim, Bax) and downregulate anti-apoptotic factors (Bcl-xL, Survivin) at both protein and mRNA levels (Fig. 2 J–L). These results demonstrate that NCTD potentiates IR-induced DNA damage and apoptosis in CRC cells. NCTD induces mitochondrial-dependent apoptosis We further investigated whether NCTD promoted apoptosis through the mitochondrial pathway. Cytochrome c (Cyt c) release is a critical step in this process; therefore, we examined its expression in total protein, cytoplasm, and mitochondria. Western blotting analysis of subcellular fractions revealed that, compared to IR alone, NCTD + IR treatment significantly increased total Cyt c expression (p < 0.05; Fig. 3 A, B) and promoted its translocation from mitochondria to the cytoplasm—elevated cytoplasmic Cyt c levels were observed alongside reduced mitochondrial Cyt c levels (p < 0.05; Fig. 3 D, E). These findings indicate that NCTD potentiates IR-induced Cyt c release, potentially representing a key mechanism in CRC cell apoptosis. NCTD induces mitochondrial dysfunction We found that apoptosis was induced via the mitochondrial pathway, and that mitochondrial changes were critical for both the effects and induction of apoptosis. Therefore, we examined whether NCTD affects mitochondrial integrity and function. Initially, we used confocal microscopy to observe mitochondrial morphology. Results showed that in the control group, mitochondria were mostly “rod-shaped,” and after NCTD + IR treatment, a significant increase in mitochondrial fragmentation and small round dots were observed (Fig. 4 A). ΔΨm loss is a key step in apoptosis, triggering the initiation of the apoptotic cascade and cell death. ΔΨm loss was assessed using JC-1 staining. Results demonstrated that following IR exposure, green-fluorescent cell signals were enhanced compared to those in the control group. After NCTD + IR administration, the intracellular green-fluorescent signal was further amplified, indicating a collapse of the ΔΨm (Fig. 4 B, C). As mitochondria are major sources of cellular ROS, we measured ROS levels and found that NCTD alone elevated them, and NCTD + IR further enhanced ROS production compared to that of IR alone (p < 0.05; Fig. 4 D). These data suggest that NCTD exacerbates IR-induced mitochondrial dysfunction, resulting in ΔΨm loss and increased ROS. NCTD-mediated IR regulates mitochondrial autophagy Multiple studies have shown that mitochondrial autophagy is associated with CRC cell radiosensitivity[ 15 – 17 ]. Therefore, we evaluated whether NCTD affects mitochondrial autophagy in CRC cells. The Parkin/PTEN-induced kinase 1 (PINK1) pathway is a typical mitochondrial autophagy pathway[ 18 ]. To confirm that autophagy is initiated through Parkin reorganization, we examined the subcellular distribution of Parkin. Immunofluorescence staining showed that IR promoted Parkin translocation to mitochondria, which was inhibited by NCTD co-treatment (Fig. 5 A, B). Western blotting analysis confirmed that NCTD downregulated PINK1 expression and Parkin phosphorylation induced by IR (Fig. 5 C, D). BNIP3, another mitophagy regulator, was also suppressed under combination treatment (Fig. 5 E, F). These results indicate that NCTD inhibits IR-induced mitophagy, possibly leading to the accumulation of dysfunctional mitochondria. ROS mediates NCTD-induced radiosensitization ROS constitutes a key mechanism for radiotherapy tolerance[ 19 , 20 ]. We hypothesized that ROS may mediate the radiosensitizing effect of NCTD in CRC cells. To test this, we employed the ROS scavenger, N -acetylcysteine (NAC; 5mM). Pretreatment with NAC significantly alleviated cell viability loss induced by NCTD + IR (Fig. 6 A). Flow cytometry further revealed that NAC attenuated NCTD-enhanced apoptosis (Fig. 6 B, C). Given the established role of ROS in provoking DNA damage, we then evaluated DNA damage levels via immunofluorescence. Results showed a pronounced increase in fluorescence intensity in the NCTD + IR group, which was substantially suppressed upon NAC addition (Fig. 6 D, E). Consistent with these observations, western blotting analysis indicated that the upregulation of pro-apoptotic proteins under combination treatment was reversed by NAC (Fig. 6 F, G). These findings support that NCTD enhances IR-induced DNA damage and apoptosis in CRC cells through ROS-dependent mechanisms. NCTD enhances radiosensitivity through ROS-mediated modulation of MAPK and AKT pathways To systematically investigate the mechanisms through which NCTD enhances radiosensitivity, we conducted an integrated network pharmacology analysis. Potential NCTD targets were predicted using the SwissTargetPrediction database, whereas CRC-related genes were obtained from the GeneCards database. Thirty overlapping target genes were identified (Fig. 7A), and a compound–target–disease network constructed for visualization (Fig. 7B). PPI network analysis further revealed core targets potentially involved in NCTD-mediated radiosensitization (Fig. 7C). GO enrichment analysis indicated significant enrichment in 30 biological processes, 10 cellular components, and 20 molecular functions among the overlapping targets (Fig. 7E). KEGG pathway analysis highlighted 15 significantly enriched pathways, including those related to DNA damage response and cell cycle regulation, as well as PI3K-AKT, MAPK, and EGFR signaling (Fig. 7D). Figure 7. Analysis of targets associated with NCTD and CRC. (A) Venn diagram of NCTD and CRC-associated targets. The diagram shows 74 NCTD-associated targets (left), 1,965 CRC-associated targets (right), and 26 common NCTD and CRC targets (center). (B) NCTD anti-CRC PPI network. Larger areas represent larger nodes; blue indicates stronger association, while lighter shades represent weaker association. Core targets are those within the inner circle. (C) Protein interaction network of NCTD and CRC was constructed using the STRING platform. (D) KEGG pathway enrichment analysis (DAVID). Pathways (Y-axis), FDR (X-axis), and P-values (color change). Bubble size indicates the number of genes enriched in each pathway. (E) GO enrichment analysis showing fold enrichment (y-axis), term (x-axis); green, orange, and purple represent the 15 core results for BP, CC, and MF, respectively. Given the established roles MAPK and AKT pathways play in regulating apoptosis [ 21 – 23 ], and considering that excessive ROS can trigger apoptosis through JNK and p38MAPK signaling cascades[ 24 ], we hypothesized that NCTD enhances radiosensitivity through these pathways. To test this, we examined the phosphorylation levels of key signaling molecules. Western blotting analysis showed that irradiation alone induced the phosphorylation of ERK, p38, and AKT, but not JNK; contrastingly, NCTD + IR treatment significantly suppressed their phosphorylation (Fig. 8 A–D). To assess whether ROS mediated these effects, we pretreated cells with NAC. NAC pretreatment markedly restored p38, ERK, and AKT phosphorylation levels (Fig. 8 E–H), indicating that NCTD inhibits these pathways in a ROS-dependent manner. These findings indicate that NCTD enhances radiosensitivity by inhibiting ROS-mediated ERK, p38 MAPK, and AKT signaling pathways. NCTD enhances radiotherapy efficacy in colorectal cancer xenografts To validate our in vitro findings, we established a CRC xenograft model. Both IR alone and NCTD + IR significantly reduced tumor volume and weight compared to those in controls, with the combination group showing superior antitumor effects (p < 0.01; Fig. 9 A–C). TUNEL staining demonstrated that NCTD + IR significantly increased apoptosis in tumor tissues compared to that of IR alone (Fig. 9 D). Consistent with our in vitro results, the combination treatment significantly suppressed p38, ERK1/2, and AKT phosphorylation in tumor tissues (p < 0.01; Fig. 9 E–H). IHC analysis further confirmed that NCTD enhanced cleaved caspase-3 expression while suppressing p-p38 expression following irradiation (Fig. 9 I). These in vivo findings demonstrate that NCTD overcomes radiation resistance in CRC through modulating MAPK and AKT signaling pathways. Discussion Herein, we demonstrate that NCTD acts as a potent radiosensitizer that significantly enhances IR-induced DNA damage, suppresses mitochondrial autophagy, and promotes apoptosis in CRC cells. We propose that these effects are primarily mediated through NCTD-induced ROS generation, which initiates a self-amplifying cycle of mitochondrial dysfunction, disrupts cellular redox homeostasis, and consequently inhibits pro-survival MAPK signaling while activating the intrinsic apoptotic pathway. Our findings establish ROS as a central regulator of NCTD-mediated radiosensitization. Various stimuli, including inflammatory responses, IR, and chemotherapeutic agents, can induce substantial ROS production, which can lead to cellular damage, autophagy, and apoptosis[ 25 ]. We observed that NCTD pretreatment significantly increased intracellular ROS levels, consistent with previous reports on the pro-oxidant properties of NCTD in other cancer models[ 20 , 26 , 27 ]. This ROS surge directly contributes to DNA damage, synergizing with IR to induce genomic instability. Beyond DNA damage, our data suggest that NCTD-induced ROS initiates a positive feedback loop that perpetuates mitochondrial dysfunction. The initial ROS wave damages mitochondrial components, leading to membrane depolarization and permeability transition pore opening. These compromised mitochondria then contribute additional ROS through electron leakage, creating a self-sustaining cycle that overwhelms cellular antioxidant defenses. Our results demonstrate that NCTD + IR triggers the mitochondrial apoptosis pathway through Cyt c-mediated mechanisms. As shown in our subcellular fractionation experiments, the combination treatment significantly promoted Cyt c release from mitochondria into the cytoplasm. This translocation represents a pivotal commitment step in the intrinsic apoptotic pathway. Released Cyt c forms the apoptosome complex with Apaf-1 and caspase-9, causing caspase-9 activation that then cleaves and activates executioner caspases[ 28 ], ultimately leading to programmed cell death. Our observation of increased cleaved caspase-3 levels following combination treatment confirms the functional activation of this pathway, providing a direct mechanistic link between mitochondrial damage and apoptosis execution. Concurrently, we observed altered mitophagy regulation. Mitophagy promotes tumor cell survival, thereby antagonizing the effects of anticancer therapies and reducing the radiosensitivity of cancer cells[ 29 ]. Therefore, inhibiting mitophagy holds potential to improve overall CRC outcomes. However, in the context of severe mitochondrial damage induced by NCTD and IR, excessive mitophagy may deplete ATP reserves and release pro-apoptotic factors, shifting its role from pro-survival to pro-death[ 30 ]. Parkin is localized within mitochondria and implicated in the generation of mitochondrial ROS in CRC cells[ 31 ]. Our immunofluorescence data showed that IR promoted Parkin translocation to mitochondria, whereas NCTD co-treatment suppressed this redistribution, thereby inhibiting mitophagy. Furthermore, NCTD + IR suppressed the activation of ERK and p38 MAPK pathways. NCTD participates in the regulation of various diseases through multiple signaling targets[ 32 , 33 ]. Typically, IR activates ERK/P38 signaling as part of the cellular stress response, often associated with promoting survival and DNA repair[ 34 – 37 ]. Our results, however, indicate that the hyper-elevated ROS environment generated by combination therapy supersedes this conventional activation. We propose several non-mutually exclusive mechanisms for this critical observation. First, oxidative stress-induced signal exhaustion: sustained high levels of ROS can lead to the oxidative inactivation of key kinases within the MAPK pathway through the oxidation of critical cysteine residues in their catalytic domains. Second, phosphatase activation: ROS is a potent activator of certain dual-specificity phosphatases that dephosphorylate and inactivate both p-ERK and p-P38, effectively terminating the survival signal[ 38 , 39 ]. Finally, cellular fate decision-making: when the apoptotic stimulus is overwhelming, a cell may actively downregulate pro-survival pathways to commit to apoptosis. This concentration-dependent dual role of ROS is gaining recognition and aligns with our results[ 40 , 41 ]. In conclusion, our data reveal that NCTD potentiates the cytotoxic effects of IR by instigating a catastrophic ROS-mitochondria feedback loop. This oxidative crisis suppresses pro-survival ERK/P38 signaling and activates mitophagy. Targeting this axis with NCTD may present a novel therapeutic strategy to overcome radioresistance. Abbreviations ΔΨm mitochondrial membrane potential CCK-8 Cell Counting Kit-8 CRC colorectal cancer Cyt c cytochrome c DMSO dimethyl sulfoxide GO Gene Ontology IHC immunohistochemistry IR ionizing radiation KEGG Kyoto Encyclopedia of Genes and Genomes NAC N -acetylcysteine NCTD norcantharidin PBS phosphate-buffered saline PI propidium iodide PINK1 PTEN-induced kinase 1 PPI protein-protein interaction ROS reactive oxygen species. Declarations Ethics approval The experimental protocol (IRM/2-IACUC-2401-011) for the tumor xenograft model in nude mice was reviewed and approved by the Experimental Animal Ethics Committee of the Institute of Radiation Medicine, Chinese Academy of Medical Sciences. Consent for publication Not applicable Competing interests The authors declare that they have no competing interests. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Funding This study is supported by Beijing Municipal Traditional Chinese Medicine Science and Technology Development Fund Project (BJZY-2025-08-TJ); National Natural Science Foundation of China (81573089,81972847); Tianjin key Medical Discipline (Specialty) Construction Project (TJYXZDXK-053B) and Tianjin Science and Technology Planning Project (21JCQNJC01900). Author Contribution Qiong Xu: Writing – original draft, Software, Methodology, Investigation, Formal analysis, Data curation. Heng Zhang:Validation, Software, Resources, Investigation. Dan-dan Gao:Software, Methodology.Yinglu Bi: Software. Keyi Wang: Data curation. Wei Zhao: Methodology, Data curation. Yuling Wang: Writing – review & editing, Project administration, Funding acquisition. Acknowledgement Thanks to “Editage (https://www.editage.cn/) ” for the effort in polishing the English content of this manuscript. Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request. References Rawla P, Sunkara T, Barsouk A. Epidemiology of colorectal cancer: incidence, mortality, survival, and risk factors. GASTROENTEROL REV. 2019;14(2):89–103. Feeney G, Sehgal R, Sheehan M, Hogan A, Regan M, Joyce M, Kerin M. Neoadjuvant radiotherapy for rectal cancer management. WORLD J GASTROENTERO. 2019;25(33):4850–69. Li J, Zhang HL, Yin HK, Zhang HK, Wang Y, Xu SN, Ma F, Gao JB, Li HL, Qu JR. Comparison of MRI and CT-Based Radiomics and Their Combination for Early Identification of Pathological Response to Neoadjuvant Chemotherapy in Locally Advanced Gastric Cancer. J MAGN RESON IMAGING. 2023;58(3):907–23. Haynes J, Manogaran P. Mechanisms and Strategies to Overcome Drug Resistance in Colorectal Cancer. INT J MOL SCI 2025, 26(5). Chevalier F. Counteracting Radio-Resistance Using the Optimization of Radiotherapy. INT J MOL SCI 2020, 21(5). Lin CL, Chen CM, Lin CL, Cheng CW, Lee CH, Hsieh YH. Norcantharidin induces mitochondrial-dependent apoptosis through Mcl-1 inhibition in human prostate cancer cells. BBA-MOL CELL RES. 2017;1864(10):1867–76. Zheng LC, Yang MD, Kuo CL, Lin CH, Fan MJ, Chou YC, Lu HF, Huang WW, Peng SF, Chung JG. Norcantharidin-induced Apoptosis of AGS Human Gastric Cancer Cells Through Reactive Oxygen Species Production, and Caspase- and Mitochondria-dependent Signaling Pathways. ANTICANCER RES. 2016;36(11):6031–42. Chang C, Zhu YQ, Mei JJ, Liu SQ, Luo J. Involvement of mitochondrial pathway in NCTD-induced cytotoxicity in human hepG2 cells. J EXP CLIN CANC RES. 2010;29(1):145. Reichstein DA, Brock AL. Radiation therapy for uveal melanoma: a review of treatment methods available in 2021. CURR OPIN OPHTHALMOL. 2021;32(3):183–90. Sridharan DM, Asaithamby A, Bailey SM, Costes SV, Doetsch PW, Dynan WS, Kronenberg A, Rithidech KN, Saha J, Snijders AM, et al. Understanding cancer development processes after HZE-particle exposure: roles of ROS, DNA damage repair and inflammation. RADIAT RES. 2015;183(1):1–26. Chen Y, Li Y, Huang L, Du Y, Gan F, Li Y, Yao Y. Antioxidative Stress: Inhibiting Reactive Oxygen Species Production as a Cause of Radioresistance and Chemoresistance. OXID MED CELL LONGEV 2021, 2021:6620306. Nguyen L, Dobiasch S, Schneider G, Schmid RM, Azimzadeh O, Kanev K, Buschmann D, Pfaffl MW, Bartzsch S, Schmid TE, et al. Impact of DNA repair and reactive oxygen species levels on radioresistance in pancreatic cancer. RADIOTHER ONCOL. 2021;159:265–76. Pranteda A, Piastra V, Stramucci L, Fratantonio D, Bossi G. The p38 MAPK Signaling Activation in Colorectal Cancer upon Therapeutic Treatments. INT J MOL SCI 2020, 21(8). Lim S, Lee Y, Lee E. p38MAPK inhibitor SB203580 sensitizes human SNU-C4 colon cancer cells to exisulind-induced apoptosis. ONCOL REP. 2006;16(5):1131–5. Ren Y, Yang P, Li C, Wang WA, Zhang T, Li J, Li H, Dong C, Meng W, Zhou H. Ionizing radiation triggers mitophagy to enhance DNA damage in cancer cells. CELL DEATH DISCOV. 2023;9(1):267. Chen X, Zhuo S, Xu W, Chen X, Huang D, Sun X, Cheng Y. Isocitrate dehydrogenase 2 contributes to radiation resistance of oesophageal squamous cell carcinoma via regulating mitochondrial function and ROS/pAKT signalling. BRIT J CANCER. 2020;123(1):126–36. Song C, Pan S, Zhang J, Li N, Geng Q. Mitophagy: A novel perspective for insighting into cancer and cancer treatment. CELL PROLIFERAT. 2022;55(12):e13327. Vives-Bauza C, Zhou C, Huang Y, Cui M, de Vries RLA, Kim J, May J, Tocilescu MA, Liu W, Ko HS, et al. PINK1-dependent recruitment of Parkin to mitochondria in mitophagy. P NATL ACAD SCI USA. 2010;107(1):378–83. Chen Y, Li Y, Huang L, Du Y, Gan F, Li Y, Yao Y. Antioxidative Stress: Inhibiting Reactive Oxygen Species Production as a Cause of Radioresistance and Chemoresistance. OXID MED CELL LONGEV 2021, 2021:6620306. Nguyen L, Dobiasch S, Schneider G, Schmid RM, Azimzadeh O, Kanev K, Buschmann D, Pfaffl MW, Bartzsch S, Schmid TE, et al. Impact of DNA repair and reactive oxygen species levels on radioresistance in pancreatic cancer. RADIOTHER ONCOL. 2021;159:265–76. Kwak AW, Kim WK, Lee SO, Yoon G, Cho SS, Kim KT, Lee MH, Choi YH, Lee JY, Park JW et al. Licochalcone B Induces ROS-Dependent Apoptosis in Oxaliplatin-Resistant Colorectal Cancer Cells via p38/JNK MAPK Signaling. ANTIOXIDANTS-BASEL 2023, 12(3). Kwak AW, Lee JY, Lee SO, Seo JH, Park JW, Choi YH, Cho SS, Yoon G, Lee MH, Shim JH. Echinatin induces reactive oxygen species-mediated apoptosis via JNK/p38 MAPK signaling pathway in colorectal cancer cells. PHYTOTHER RES. 2023;37(2):563–77. Yu Y, Chen D, Wu T, Lin H, Ni L, Sui H, Xiao S, Wang C, Jiang S, Pan H, et al. Dihydroartemisinin enhances the anti-tumor activity of oxaliplatin in colorectal cancer cells by altering PRDX2-reactive oxygen species-mediated multiple signaling pathways. Phytomedicine. 2022;98:153932. Wei F, Nian Q, Zhao M, Wen Y, Yang Y, Wang J, He Z, Chen X, Yin X, Wang J, et al. Natural products and mitochondrial allies in colorectal cancer therapy. BIOMED PHARMACOTHER. 2023;167:115473. Cheung EC, Vousden KH. The role of ROS in tumour development and progression. NAT REV CANCER. 2022;22(5):280–97. Lin CL, Chen CM, Lin CL, Cheng CW, Lee CH, Hsieh YH. Norcantharidin induces mitochondrial-dependent apoptosis through Mcl-1 inhibition in human prostate cancer cells. BBA-MOL CELL RES. 2017;1864(10):1867–76. Zheng LC, Yang MD, Kuo CL, Lin CH, Fan MJ, Chou YC, Lu HF, Huang WW, Peng SF, Chung JG. Norcantharidin-induced Apoptosis of AGS Human Gastric Cancer Cells Through Reactive Oxygen Species Production, and Caspase- and Mitochondria-dependent Signaling Pathways. ANTICANCER RES. 2016;36(11):6031–42. Xie H, Song L, Katz S, Zhu J, Liu Y, Tang J, Cai L, Hildebrandt P, Han XX. Electron transfer between cytochrome c and microsomal monooxygenase generates reactive oxygen species that accelerates apoptosis. REDOX BIOL. 2022;53:102340. Zhang Y, Pang C, Zhang C, Wang Y, Wang P, Chen Y, Wang J, Hu Y, Liu C, Liang H, et al. HILPDA-mediated lipidomic remodelling promotes radiotherapy resistance in nasopharyngeal carcinoma by accelerating mitophagy. CELL MOL LIFE SCI. 2023;80(9):242. Xia J, Jin J, Dai S, Fan H, Chen K, Li J, Luo F, Peng X. Mitophagy: A key regulator of radiotherapy resistance in the tumor immune microenvironment. MOL ASPECTS MED. 2025;105:101385. Lin Q, Li S, Jiang N, Shao X, Zhang M, Jin H, Zhang Z, Shen J, Zhou Y, Zhou W, et al. PINK1-parkin pathway of mitophagy protects against contrast-induced acute kidney injury via decreasing mitochondrial ROS and NLRP3 inflammasome activation. REDOX BIOL. 2019;26:101254. Wang Y, Dong S, Hu K, Xu L, Feng Q, Li B, Wang G, Chen G, Zhang B, Jia X, et al. The novel norcantharidin derivative DCZ5417 suppresses multiple myeloma progression by targeting the TRIP13-MAPK-YWHAE signaling pathway. J TRANSL MED. 2023;21(1):858. Yousef EH, El-Magd NFA, El Gayar AM. Norcantharidin potentiates sorafenib antitumor activity in hepatocellular carcinoma rat model through inhibiting IL-6/STAT3 pathway. TRANSL RES. 2023;260:69–82. Liu Y, Lin CH, Chen K, Lai D, Hsu F. Inactivation of EGFR/ERK/NF-kappaB signalling associates with radiosensitizing effect of 18beta-glycyrrhetinic acid on progression of hepatocellular carcinoma. J CELL MOL MED. 2023;27(11):1539–49. Paramanantham A, Jung EJ, Go S, Jeong BK, Jung J, Hong SC, Kim GS, Lee WS. Activated ERK Signaling Is One of the Major Hub Signals Related to the Acquisition of Radiotherapy-Resistant MDA-MB-231 Breast Cancer Cells. INT J MOL SCI 2021, 22(9). Irani K, Xia Y, Zweier JL, Sollott SJ, Der CJ, Fearon ER, Sundaresan M, Finkel T, Goldschmidt-Clermont PJ. Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts. Science. 1997;275(5306):1649–52. Reddy KB, Glaros S. Inhibition of the MAP kinase activity suppresses estrogen-induced breast tumor growth both in vitro and in vivo. INT J ONCOL. 2007;30(4):971–5. Li Z, Fei T, Zhang J, Zhu G, Wang L, Lu D, Chi X, Teng Y, Hou N, Yang X, et al. BMP4 Signaling Acts via dual-specificity phosphatase 9 to control ERK activity in mouse embryonic stem cells. Cell Stem Cell. 2012;10(2):171–82. Kucera J, Netusilova J, Sladecek S, Lanova M, Vasicek O, Stefkova K, Navratilova J, Kubala L, Pachernik J. Hypoxia Downregulates MAPK/ERK but Not STAT3 Signaling in ROS-Dependent and HIF-1-Independent Manners in Mouse Embryonic Stem Cells. OXID MED CELL LONGEV. 2017;2017:4386947. Moloney JN, Cotter TG. ROS signalling in the biology of cancer. SEMIN CELL DEV BIOL. 2018;80:50–64. Idelchik MDPS, Begley U, Begley TJ, Melendez JA. Mitochondrial ROS control of cancer. SEMIN CANCER BIOL. 2017;47:57–66. Additional Declarations No competing interests reported. Supplementary Files TableS1S2.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 04 May, 2026 Editor assigned by journal 30 Mar, 2026 Submission checks completed at journal 30 Mar, 2026 First submitted to journal 15 Jan, 2026 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. 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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-8609994","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":634147422,"identity":"f9777454-1fd7-4166-ad37-64c1af3f8cfb","order_by":0,"name":"qiong xu","email":"","orcid":"","institution":"Tianjin Academy of Traditional Chinese Medicine Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"qiong","middleName":"","lastName":"xu","suffix":""},{"id":634147423,"identity":"8110d551-bb97-485e-a312-dfd6e19eabcd","order_by":1,"name":"hen zhang","email":"","orcid":"","institution":"Nankai University","correspondingAuthor":false,"prefix":"","firstName":"hen","middleName":"","lastName":"zhang","suffix":""},{"id":634147424,"identity":"a7741414-62e9-431b-be9d-b1d10b9effa2","order_by":2,"name":"dan dan gao","email":"","orcid":"","institution":"Jinan Maternity and Child Care Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"dan","middleName":"dan","lastName":"gao","suffix":""},{"id":634147425,"identity":"0abc88ec-6c19-4794-808b-0e214d685869","order_by":3,"name":"ying lu bi","email":"","orcid":"","institution":"Tianjin Academy of Traditional Chinese Medicine Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"ying","middleName":"lu","lastName":"bi","suffix":""},{"id":634147426,"identity":"bba250af-455a-4333-9b01-71d85df72e8c","order_by":4,"name":"ke yi wang","email":"","orcid":"","institution":"Tianjin Academy of Traditional Chinese Medicine Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"ke","middleName":"yi","lastName":"wang","suffix":""},{"id":634147427,"identity":"30b31454-11e8-42b8-a873-b7ae711e2edc","order_by":5,"name":"wei zhao","email":"","orcid":"","institution":"Tianjin Academy of Traditional Chinese Medicine Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"wei","middleName":"","lastName":"zhao","suffix":""},{"id":634147428,"identity":"a887b8f2-dd72-45a5-a38d-299193403e70","order_by":6,"name":"yulin wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYBCDBAZmxsYHHwxs7EjRwnzYcEZBWjIJWhjY0qR5PhxibCCklJ//jJnEzx21eQbHeQykbQwOMDOwHz66AZ8WyYZjaZK9Z44XGxzmMTDOMbjDx8CTlnYDnxaDg83HbvC2HUvcANSSnGPwjJlBgscMrxb7w4xtN/9CtRy2MDjM2EBIiwEb87HbvG01QC1sic0MxGiROMOW/lu27UDizMPMhxl7DNKS2Qj5hb//jLHh27a6xL7zB9t//PhjY8fPfvgYXi1QcBjBZCNCOQjUEaluFIyCUTAKRiQAAHsPTt/W+NP+AAAAAElFTkSuQmCC","orcid":"","institution":"Tianjin Academy of Traditional Chinese Medicine Affiliated Hospital","correspondingAuthor":true,"prefix":"","firstName":"yulin","middleName":"","lastName":"wang","suffix":""}],"badges":[],"createdAt":"2026-01-15 11:38:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8609994/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8609994/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109097573,"identity":"8b9ac937-ba0b-45de-aa52-c94468c71859","added_by":"auto","created_at":"2026-05-12 14:09:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1345833,"visible":true,"origin":"","legend":"\u003cp\u003eNCTD enhances the radiosensitivity of colorectal cancer (CRC) cells in vitro. (A–B) CCK-8 assay was performed to evaluate the viability of CRC cells (HCT-116, DLD-1). (C) Wound-healing assay. (D) Quantitative analysis of CRC cell migration rate. (E) Expression of epithelial-mesenchymal transition (EMT)-related proteins. (F) Quantitative analysis of EMT-related proteins (n = 3). (G–H) Colony formation assay showing enhanced radiosensitivity of CRC cells in vitro, as evaluated using the multi-target single-hit model, after NCTD (5 and 50 µM) treatment.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8609994/v1/76ba0dc5104bf30f5a7cdfcc.png"},{"id":109097386,"identity":"ab0424f0-3b75-477c-8377-58b405016e8e","added_by":"auto","created_at":"2026-05-12 14:08:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":946613,"visible":true,"origin":"","legend":"\u003cp\u003eNCTD-induced DNA damage and apoptosis following IR exposure. (A–B) Detection of DNA damage using the comet assay. (C) Casplab software was used to process and analyze the comet images, and the tail lengths of the four treatment groups were analyzed after different interventions. (D) Expression of DNA damage-related proteins. (E) Quantitative analysis of DNA damage-related proteins. (F–G) Following Annexin V-FITC/PI staining, the percentage of apoptotic cells was determined by flow cytometry. (H) Expression levels of cleaved caspase-3, Bcl-xL, Bim, Survivin, and Bax. GAPDH was used as the control. (I) Quantitative analysis of apoptosis-related proteins (n = 3). **p \u0026lt; 0.01, ***p \u0026lt; 0.001 in comparison with the control. (J) mRNA analysis of apoptosis-related proteins in CRC cells.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8609994/v1/144ba13f3e2fdfe0450eb16d.png"},{"id":109097568,"identity":"3ede6de5-722b-4e1c-9317-f6b8a760fd32","added_by":"auto","created_at":"2026-05-12 14:08:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":498813,"visible":true,"origin":"","legend":"\u003cp\u003eNCTD promotes the translocation of mitochondrial Cyt c to the cytoplasm following IR. (A) Total Cyt c protein levels. (B) Quantitative analysis of Cyt c protein. (C) Cyt c mRNA levels. (D) Cyt c expression levels in the cytoplasm and mitochondria of two cell lines. (E) Quantitative analysis of Cyt c protein expression changes in cytoplasm and mitochondria of HCT-116 and DLD-1 cell lines. ns \u0026gt; 0.05，*** p \u0026lt; 0.001，**** p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8609994/v1/f6225d8db059115cff278ab2.png"},{"id":109097526,"identity":"569de755-64e8-4469-8cfc-01ca6a75e2ce","added_by":"auto","created_at":"2026-05-12 14:08:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1221892,"visible":true,"origin":"","legend":"\u003cp\u003eNCTD promotes mitochondrial dysfunction following IR. (A) Mitochondria morphology in CRC cells treated with IR, NCTD, or both, as observed by confocal microscopy. Scale bar = 10 µm. (B) JC-1 staining for the detection of mitochondrial membrane potential. Scale bar = 100 μm. (C) Quantification of mitochondrial membrane potential. (D) Detection of intracellular ROS levels by flow cytometry after treatment. *p \u0026lt; 0.05, **p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8609994/v1/3893ee145fa1b1d1cbc4d80e.png"},{"id":109097572,"identity":"fd10fc78-5a2c-4dd5-9817-c79f770ca180","added_by":"auto","created_at":"2026-05-12 14:09:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1002751,"visible":true,"origin":"","legend":"\u003cp\u003eNCTD promotes mitochondrial autophagy following IR. (A–B) Representative confocal images showing Parkin immunofluorescence and mitochondria stained with MitoTracker®. Nuclei were counterstained with DAPI. Scale bar = 10 µm. (C) Expression levels of Parkin, PINK1, and their phosphorylated forms. (D) Quantitative analysis of mitochondrial autophagy-related proteins. (E) Expression levels of the mitochondrial autophagy protein BNIP3. (F) Quantitative analysis of BNIP3 protein expression. ns \u0026gt; 0.05, *p \u0026lt; 0.05, **p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8609994/v1/485dbeb711f70ff729b61881.png"},{"id":109097387,"identity":"c0196210-86e7-415a-a774-abaf4118ac62","added_by":"auto","created_at":"2026-05-12 14:08:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":670802,"visible":true,"origin":"","legend":"\u003cp\u003eNCTD-IR–mediated ROS generation promotes DNA damage and apoptosis in CRC cells. (A) Cells were pretreated with NAC (5 mM, \u0026nbsp;2 h) followed by NCTD and IR treatment. Cell viability was assessed using the CCK-8 assay. (B) Flow cytometry was performed to detect the percentage of apoptotic cells. (C) Quantitative bar chart showing apoptosis rate following ROS inhibitor treatment. (D) Effect of the ROS inhibitor NAC on DNA damage, as detected by immunofluorescence staining. (E) Quantification of γ-H2AX expression following ROS inhibitor treatment. (F) Expression levels of cleaved caspase-3, Survivin, and Bax. (G) Quantitative analysis of apoptosis-related proteins after NAC treatment. **p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8609994/v1/55e62e17d72e6b49a06aa95c.png"},{"id":109097384,"identity":"fb1d2bf7-6b16-4dee-964e-9e0d56fa3d19","added_by":"auto","created_at":"2026-05-12 14:08:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":635345,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of targets associated with NCTD and CRC. (A) Venn diagram of NCTD and CRC-associated targets. The diagram shows 74 NCTD-associated targets (left), 1,965 CRC-associated targets (right), and 26 common NCTD and CRC targets (center). (B) NCTD anti-CRC PPI network. Larger areas represent larger nodes; blue indicates stronger association, while lighter shades represent weaker association. Core targets are those within the inner circle. (C) Protein interaction network of NCTD and CRC was constructed using the STRING platform. (D) KEGG pathway enrichment analysis (DAVID). Pathways (Y-axis), FDR (X-axis), and P-values (color change). Bubble size indicates the number of genes enriched in each pathway. (E) GO enrichment analysis showing fold enrichment (y-axis), term (x-axis); green, orange, and purple represent the 15 core results for BP, CC, and MF, respectively.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8609994/v1/7bfe280eb4883c543003a8d7.png"},{"id":109204830,"identity":"c456afbb-6640-4d63-9d1e-ce56c0fa726c","added_by":"auto","created_at":"2026-05-13 15:02:31","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":836497,"visible":true,"origin":"","legend":"\u003cp\u003eNCTD promotes ROS production following IR and inhibits the p38, ERK, and AKT signaling pathways to induce apoptosis. (A–B) p38 and ERK protein phosphorylation and total protein levels in CRC cells treated with NCTD and IR. (C–D) AKT phosphorylation and total protein levels in CRC cells treated with NCTD and IR. (E–F) p38 and ERK phosphorylation and total protein levels. CRC cells were pretreated with NAC (5 mM, 2 h) followed by treatment with NCTD and IR. (G–H) AKT phosphorylation and total protein levels. Rectal carcinoma cells were pretreated with NAC (5 mM, 2 h) followed by treatment with NCTD and IR.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8609994/v1/c5217d684ffa6d696ff14a11.png"},{"id":109097525,"identity":"8973b381-8a11-45d3-bc88-81d1320f369c","added_by":"auto","created_at":"2026-05-12 14:08:53","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":927253,"visible":true,"origin":"","legend":"\u003cp\u003eNCTD enhances the radiosensitivity of CRC cells in vivo. (A) Tumors from nude mouse xenografts. (B) Tumor volume. (C) Tumor weight. (D) Detection of apoptosis in tumor tissue using the TUNEL assay. (E–G) Expression levels of p38, ERK, and AKT phosphorylation and total proteins in tumor tissue. (H) Quantification of p38, ERK, and AKT protein expression. (I) Immunohistochemical detection of cleaved caspase-3 and p-p38 expression in tumor tissue.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8609994/v1/0c99012e419cf181c2ab86a3.png"},{"id":109097219,"identity":"1d9f4640-0ecc-4f5a-92d2-9e0f24a0573f","added_by":"auto","created_at":"2026-05-12 14:07:32","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":56885,"visible":true,"origin":"","legend":"\u003cp\u003eMechanism of action of NCTD in increasing the sensitivity to radiotherapy in CRC.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-8609994/v1/bbc7745b8c5820244ad57744.png"},{"id":109207225,"identity":"08e63f87-5a11-4114-9baf-de48573ad692","added_by":"auto","created_at":"2026-05-13 15:18:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8200383,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8609994/v1/20aed76d-1302-4a2a-b673-ceac5d8a8192.pdf"},{"id":109097390,"identity":"e25a6653-3467-40ef-b1a4-801649a0d1e0","added_by":"auto","created_at":"2026-05-12 14:08:28","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17737,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1S2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8609994/v1/5b3f188a28065162d928a631.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mechanism by which norcantharidin enhances radiotherapy sensitivity in colorectal cancer cells: Apoptosis induction via the mitochondrial dysfunction–reactive oxygen species–ERK/p38 MAPK/AKT pathway axis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer (CRC) remains a major global health challenge, ranking as the third most diagnosed cancer and second leading cause of cancer-related deaths [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Radiotherapy plays a crucial role in the multidisciplinary management of rectal cancer, effectively reducing tumor burden and improving patient survival [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, radioresistance frequently leads to treatment failure and poor prognosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], which is attributable to tumor heterogeneity, the presence of tumor stem cells, and pro-survival signaling activation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Current strategies to improve radiosensitivity, such as dose escalation and combination therapies, are often limited by increased toxicity and adverse effects [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], highlighting the urgent need for novel radiosensitizing agents.\u003c/p\u003e \u003cp\u003eNorcantharidin (NCTD), a demethylated derivative of the traditional Chinese medicine, cantharidin, exhibits antitumor activity in various cancers [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Its pro-apoptotic effects are closely related to mitochondrial dysfunction induction and a marked rise in intracellular reactive oxygen species (ROS)[\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Given that ROS generation is a primary mechanism of radiation-induced cell killing, these properties suggest that NCTD holds potential as a radiosensitizer. Nonetheless, the molecular mechanisms by which NCTD enhances radiosensitivity in CRC remain poorly defined.\u003c/p\u003e \u003cp\u003eIonizing radiation (IR) directly damages mitochondria, thereby worsening mitochondrial dysfunction and amplifying ROS production, which in turn enhances cytotoxic effects [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Notably, low ROS levels within cells following IR exposure may confer tolerance to such radiation in malignant tumors [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], making ROS augmentation a rational therapeutic strategy. Critically, ROS acts as a signaling molecule in cellular stress responses and secondary messenger to activate key downstream signaling pathways, including the MAPK family (such as ERK and p38) and AKT pathways. These pathways play a decisive role in determining cellular fate. Notably, the p38 MAPK pathway can be activated via oxidative stress and exerts a survival-dependent effect on cells depending on the therapeutic context [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, this study aimed to investigate whether NCTD enhances CRC radiosensitivity by synergizing with IR to aggravate mitochondrial dysfunction and ROS production to favor apoptosis. Our findings provide crucial mechanistic insights into the action of NCTD as a radiosensitizer and support its potential clinical application.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eCell culture\u003c/h2\u003e\n \u003cp\u003eHuman colon cancer cell lines (HCT-116 and DLD-1) were purchased from Procell Life Science \u0026amp; Technology Co., Ltd. (Wuhan, China). Cells were cultured in RPMI-1640 medium (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco), 1% penicillin, and 0.1% streptomycin (Thermo Fisher Scientific) at 5% CO\u003csub\u003e2\u003c/sub\u003e and 37\u0026deg;C.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eCell irradiation and treatment\u003c/h3\u003e\n\u003cp\u003eCells were irradiated using 6 MeV high-energy X-rays generated with a linear accelerator (Varian Clinic 21ES; Palo Alto, CA, USA) at a dose rate of 3.0 Gy/min. Sealed sterile cell culture plates were placed in the center of the irradiation chamber for treatment. NCTD (Yuanye Biotechnology, Shanghai, China) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 100 mM stock solution and then stored at \u0026minus;\u0026thinsp;20\u0026deg;C. Working concentrations were prepared by diluting the stock solution in complete RPMI-1640 medium immediately before use.\u003c/p\u003e\n\u003ch3\u003eCell viability assays\u003c/h3\u003e\n\u003cp\u003eCell viability was assessed using the Cell Counting Kit-8 (CCK-8; Solarbio, Beijing, China). Cells in logarithmic growth were seeded into 96-well plates at a density of 5,000 cells/well. After 24 h, cells were treated with NCTD and/or IR. Following incubation for 24, 48, or 72 h, 10 \u0026micro;L CCK-8 solution was added to each well and incubated for 2\u0026ndash;4 h at 37\u0026deg;C. Absorbance was measured at 450 nm using an enzyme-labeled instrument (Bio-Rad, Hercules, CA, USA).\u003c/p\u003e\n\u003ch3\u003eColony formation assay\u003c/h3\u003e\n\u003cp\u003eCells were seeded at densities of 200, 400, 800, 2,000, and 10,000 cells/well in a 6-well plate, depending on the radiation dose (0, 2, 4, 6, or 8 Gy, respectively). After 24 h, cells were treated with NCTD (5 or 50 \u0026micro;M) for 2 h prior to irradiation and then incubated for 10\u0026ndash;14 days at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Colonies were fixed with methanol, stained with 0.1% crystal violet, and counted using ImageJ (National Institutes of Health, Bethesda, MD, USA). Survival curves were fitted, and the sensitization enhancement ratio calculated using the multi-target single-hit model in GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA).\u003c/p\u003e\n\u003ch3\u003eWound healing assay\u003c/h3\u003e\n\u003cp\u003eCells were seeded in 6-well plates and grown to 90\u0026ndash;95% confluence. A scratch wound was created using a sterile 10 \u0026micro;L pipette tip. After washing with phosphate-buffered saine (PBS) to remove detached cells, fresh medium containing NCTD was added. Wound gaps were imaged at 0, 24, 48, and 72 h using an inverted microscope (Olympus, Tokyo, Japan). Migration distance was quantified by measuring gap width at multiple points using ImageJ.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eCell apoptosis assays\u003c/h2\u003e\n \u003cp\u003eFor the TUNEL assay (Yeasen, Shanghai, China), paraffin-embedded tissue sections or cell smears were deparaffinized and rehydrated. Sections were fixed with 4% paraformaldehyde for 30 min at room temperature (23\u0026ndash;25\u0026deg;C) and permeabilized with 20 \u0026micro;g/mL proteinase K for 15 min at 37\u0026deg;C. After washing with PBS, samples were incubated with TUNEL reaction mixture for 60 min at 37\u0026deg;C in the dark, according to the manufacturer\u0026rsquo;s instructions. Apoptotic cells were visualized under a fluorescence microscope (Nikon, Tokyo, Japan).\u003c/p\u003e\n \u003cp\u003eFor quantitative analysis of apoptosis in cultured cells, an Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (Abbkine, Wuhan, China) was used. After treatment, cells were harvested, washed twice with cold PBS, and resuspended in 200 \u0026micro;L of 1\u0026times; binding buffer. Samples were stained with 5 \u0026micro;L Annexin V-FITC and 2 \u0026micro;L PI for 15 min at room temperature in the dark. Apoptosis was immediately analyzed using a flow cytometer (BD Biosciences, San Jose, CA, USA), and the data processed with FlowJo v.10.8.1.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eSingle-cell gel electrophoresis assay\u003c/h3\u003e\n\u003cp\u003eThe alkaline comet assay was performed using a kit (ELK Biotechnology, Wuhan, China). Cells were suspended in PBS at 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mL, mixed with low-melting-point agarose, and spread onto slides. Slides were lysed in a lysis solution at 4℃ for 2 h, then incubated in alkaline electrophoresis buffer (200 mM NaOH, 1 mM EDTA) for 20 min at room temperature. Slides were left at room temperature for 60 min to maintain DNA under alkaline conditions, followed by electrophoresis at 25 V and 300 mA for 30 min. Subsequently, slides were washed with 0.4 mM Tris\u0026ndash;HCl (pH 7.5) and stained with PI (10 \u0026micro;g/mL) for 10 min. Comet tails were visualized using a fluorescence microscope.\u003c/p\u003e\n\u003ch3\u003eDCFH-DA ROS assay\u003c/h3\u003e\n\u003cp\u003eCells were seeded in 6-well plates and then treated when reaching 50\u0026ndash;70% confluence. After treatment, cells were incubated with 10 \u0026micro;M DCFH-DA (diluted in serum-free medium) for 20 min at 37\u0026deg;C in the dark. Cells were washed thrice with serum-free medium, and fluorescence observed using a fluorescence microscope.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eRNA isolation and quantitative real-time PCR\u003c/h2\u003e\n \u003cp\u003eTotal RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA).\u003c/p\u003e\n \u003cp\u003eThe cDNA was synthesized using the Hifair II 1st Strand cDNA Synthesis Kit (Yeasen). The quantitative real-time PCR assay was performed using qPCR SYBR Green Master Mix (Yeasen) on a QuantStudio 6 system (Applied Biosystems, Waltham, MA, USA). Gene expression was normalized to that of GAPDH. Primer sequences are listed in Supplementary Table S1.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eWestern blotting\u003c/h2\u003e\n \u003cp\u003eTotal protein was extracted using RIPA buffer (Beyotime, Shanghai, China) and quantified with the BCA Protein Assay Kit (Beyotime). Proteins (30 \u0026micro;g/lane) were separated via SDS-PAGE and transferred to PVDF membranes (0.22 \u0026micro;m; Millipore, Burlington, MA, USA). Membranes were blocked with 5% skim milk for 1 h at room temperature, then incubated with primary antibodies overnight at 4\u0026deg;C, followed by HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence reagent (Millipore). Antibody details are provided in Supplementary Table S2.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eNetwork pharmacology\u003c/h2\u003e\n \u003cp\u003eThe SMILES number of NCTD was found on PubChem (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov\u003c/span\u003e\u003c/span\u003e), and target prediction then performed (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://swisstargetprediction.ch\u003c/span\u003e\u003c/span\u003e). Predicted results were analyzed in Metascape (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://metascape.org/gp/index.html\u003c/span\u003e\u003c/span\u003e) and subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Protein-protein interaction (PPI) networks were obtained from the STRING database.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eMitochondrial isolation and protein extraction\u003c/h2\u003e\n \u003cp\u003eMitochondria were isolated using a Mitochondrial Isolation Kit (Beyotime). Briefly, cells were washed, centrifuged, and homogenized in ice-cold cytoplasmic extraction buffer. Following confirmation of cell disruption, the homogenate was centrifuged (600 \u0026times; \u003cem\u003eg\u003c/em\u003e, 10 min) to remove nuclei and cellular debris. The resulting supernatant, was carefully collected, whereas the pellet containing intact mitochondria was retained. The mitochondrial pellet was subsequently washed, resuspended in lysis buffer, and incubated on ice for complete lysis. Thereafter, the sample was centrifuged (12,000 \u0026times; \u003cem\u003eg\u003c/em\u003e, 15 min) to pellet insoluble material. The resulting supernatant, which contains mitochondrial proteins, was collected for further analysis. All subsequent procedures were performed as described in the \u003cem\u003eWestern blotting\u003c/em\u003e section.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eMitoTracker Red CMXRos\u003c/h2\u003e\n \u003cp\u003eTo prepare a 1 mM stock solution, 94.1 \u0026micro;L DMSO was added to 50 \u0026micro;g lyophilized solids for dissolution and then diluted to 200 nM in complete medium. Cells were incubated with the working solution for 30 min at 37\u0026deg;C in the dark. After washing with PBS, cells were fixed with cold methanol at \u0026minus;\u0026thinsp;20\u0026deg;C for 15 min (if required for co-staining) and imaged using a confocal microscope (Leica, Wetzlar, Germany).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eMitochondrial membrane potential assay\u003c/h2\u003e\n \u003cp\u003eMitochondrial membrane potential (\u0026Delta;\u0026Psi;m) was assessed using the JC-1 fluorescent probe (Yeasen). Cells were treated with NCTD (10, 50 \u0026micro;M) or IR (6 Gy) for 24 h. Following incubation with JC-1 working solution for 15 min and washing with PBS, cells were examined under a fluorescence microscope. In viable cells, mitochondria exhibit red fluorescence (Ex/Em: 550/600 nm), whereas apoptotic or necrotic cells display green fluorescence (Ex/Em: 485/535 nm). Cellular status was assessed by evaluating the red-to-green fluorescence intensity ratio.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eImmunohistochemical analysis\u003c/h2\u003e\n \u003cp\u003eFor immunohistochemistry (IHC), deparaffinized and rehydrated sections underwent antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) at 95\u0026deg;C for 20 min. Endogenous peroxidase was blocked using 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and nonspecific binding reduced with 5% normal goat serum. Primary antibodies\u0026mdash;anti-p-p38 (1:1000) and anti-cleaved caspase-3 (1:1800)\u0026mdash;were applied overnight at 4\u0026deg;C, followed by incubation with HRP-conjugated secondary antibodies for 1 h at room temperature. Staining was visualized with DAB, counterstained, and three random fields per section then imaged and quantified using ImageJ (AOD\u0026thinsp;=\u0026thinsp;IOD/area).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eImmunofluorescence assay\u003c/h2\u003e\n \u003cp\u003eCells on coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, then blocked with 5% bovine serum albumin. Incubation with anti-\u0026gamma;-H2AX (1:200) was carried out overnight at 4\u0026deg;C, then stained with Alexa Fluor 488-conjugated secondary antibody and DAPI. Images were obtained using a Leica Stellaris 8 confocal microscope.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eAnimals\u003c/h2\u003e\n \u003cp\u003eAll animal procedures were performed according to the Institutional Animal Care and Use Committee of the Institute of Radiation Medicine, China Academy of Chinese Medical Sciences (Approval No: IRM/2-IACUC-2401-011) and conducted in accordance with NIH guidelines. Female BALB/c-nu mice (4 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). HCT-116 cells (1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells in 100 \u0026micro;L PBS) were subcutaneously injected into the left inguinal area. When tumors reached approximately 5 mm in diameter, mice were randomly divided into three groups (n\u0026thinsp;=\u0026thinsp;4 per group): control, IR alone, and combination (NCTD\u0026thinsp;+\u0026thinsp;IR) groups. The IR group received 10 Gy X-ray irradiation, and the combination group received NCTD (5 mg/kg, intraperitoneal injection) daily for 14 days post-irradiation. Tumor volume was measured every three days and calculated as 0.5 \u0026times; length \u0026times; width\u003csup\u003e2\u003c/sup\u003e. At the end of the experiment, tumors and major organs were harvested for analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analyses\u003c/h2\u003e\n \u003cp\u003eAll experiments were performed in triplicate. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical analysis was conducted using GraphPad Prism 9.0. One-way analysis of variance followed by Tukey\u0026rsquo;s post-hoc test was used for multiple comparisons. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eNCTD inhibits proliferation and enhances radiosensitivity in CRC cells\u003c/h2\u003e \u003cp\u003eTo evaluate the cytotoxic effects of NCTD on CRC cells, we performed CCK-8 and colony formation assays. NCTD significantly inhibited the viability of HCT-116 and DLD-1 cells in a time- and dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). A wound healing assay further demonstrated that NCTD effectively suppressed cell migration, as evidenced by a significantly reduced scratch closure measured in treated groups compared to that in controls at 24, 48, and 72 h (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). Consistent with these findings, western blotting analysis revealed that NCTD upregulated E-cadherin and downregulated N-cadherin expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, F), suggesting inhibition of the epithelial\u0026ndash;mesenchymal transition. Based on these results, IC\u003csub\u003e20\u003c/sub\u003e values (5 \u0026micro;M for HCT-116 and 50 \u0026micro;M for DLD-1) were selected for subsequent experiments. Colony formation assays under X-ray irradiation (0\u0026ndash;8 Gy) showed that NCTD pretreatment significantly enhanced radiosensitivity, particularly in HCT-116 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, H). These data indicate that NCTD suppresses CRC cell proliferation and migration while increasing radiosensitivity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eNCTD enhances IR-induced DNA damage and apoptosis\u003c/h2\u003e \u003cp\u003eWe assessed how NCTD affects radiation-induced DNA damage using the comet assay. The NCTD\u0026thinsp;+\u0026thinsp;IR combination resulted in significantly longer comet tails compared to those of IR alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;C), indicating enhanced DNA breakage. Western blotting analysis corroborated these findings, showing increased DNA damage marker levels in the combination group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, E). We then evaluated apoptosis induction: combined treatment led to pronounced apoptotic morphology, increased TUNEL-positive cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF\u0026ndash;G), and elevated Annexin V/PI staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, I). Molecular analysis revealed that NCTD synergized with IR to upregulate pro-apoptotic proteins (cleaved caspase-3, Bim, Bax) and downregulate anti-apoptotic factors (Bcl-xL, Survivin) at both protein and mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ\u0026ndash;L). These results demonstrate that NCTD potentiates IR-induced DNA damage and apoptosis in CRC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eNCTD induces mitochondrial-dependent apoptosis\u003c/h2\u003e \u003cp\u003eWe further investigated whether NCTD promoted apoptosis through the mitochondrial pathway. Cytochrome \u003cem\u003ec\u003c/em\u003e (Cyt c) release is a critical step in this process; therefore, we examined its expression in total protein, cytoplasm, and mitochondria. Western blotting analysis of subcellular fractions revealed that, compared to IR alone, NCTD\u0026thinsp;+\u0026thinsp;IR treatment significantly increased total Cyt c expression (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B) and promoted its translocation from mitochondria to the cytoplasm\u0026mdash;elevated cytoplasmic Cyt c levels were observed alongside reduced mitochondrial Cyt c levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E). These findings indicate that NCTD potentiates IR-induced Cyt c release, potentially representing a key mechanism in CRC cell apoptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eNCTD induces mitochondrial dysfunction\u003c/h2\u003e \u003cp\u003eWe found that apoptosis was induced via the mitochondrial pathway, and that mitochondrial changes were critical for both the effects and induction of apoptosis. Therefore, we examined whether NCTD affects mitochondrial integrity and function. Initially, we used confocal microscopy to observe mitochondrial morphology. Results showed that in the control group, mitochondria were mostly \u0026ldquo;rod-shaped,\u0026rdquo; and after NCTD\u0026thinsp;+\u0026thinsp;IR treatment, a significant increase in mitochondrial fragmentation and small round dots were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). ΔΨm loss is a key step in apoptosis, triggering the initiation of the apoptotic cascade and cell death. ΔΨm loss was assessed using JC-1 staining. Results demonstrated that following IR exposure, green-fluorescent cell signals were enhanced compared to those in the control group. After NCTD\u0026thinsp;+\u0026thinsp;IR administration, the intracellular green-fluorescent signal was further amplified, indicating a collapse of the ΔΨm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). As mitochondria are major sources of cellular ROS, we measured ROS levels and found that NCTD alone elevated them, and NCTD\u0026thinsp;+\u0026thinsp;IR further enhanced ROS production compared to that of IR alone (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These data suggest that NCTD exacerbates IR-induced mitochondrial dysfunction, resulting in ΔΨm loss and increased ROS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eNCTD-mediated IR regulates mitochondrial autophagy\u003c/h2\u003e \u003cp\u003eMultiple studies have shown that mitochondrial autophagy is associated with CRC cell radiosensitivity[\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, we evaluated whether NCTD affects mitochondrial autophagy in CRC cells. The Parkin/PTEN-induced kinase 1 (PINK1) pathway is a typical mitochondrial autophagy pathway[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. To confirm that autophagy is initiated through Parkin reorganization, we examined the subcellular distribution of Parkin. Immunofluorescence staining showed that IR promoted Parkin translocation to mitochondria, which was inhibited by NCTD co-treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Western blotting analysis confirmed that NCTD downregulated PINK1 expression and Parkin phosphorylation induced by IR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). BNIP3, another mitophagy regulator, was also suppressed under combination treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F). These results indicate that NCTD inhibits IR-induced mitophagy, possibly leading to the accumulation of dysfunctional mitochondria.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eROS mediates NCTD-induced radiosensitization\u003c/h2\u003e \u003cp\u003eROS constitutes a key mechanism for radiotherapy tolerance[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. We hypothesized that ROS may mediate the radiosensitizing effect of NCTD in CRC cells. To test this, we employed the ROS scavenger, \u003cem\u003eN\u003c/em\u003e-acetylcysteine (NAC; 5mM). Pretreatment with NAC significantly alleviated cell viability loss induced by NCTD\u0026thinsp;+\u0026thinsp;IR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Flow cytometry further revealed that NAC attenuated NCTD-enhanced apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C). Given the established role of ROS in provoking DNA damage, we then evaluated DNA damage levels via immunofluorescence. Results showed a pronounced increase in fluorescence intensity in the NCTD\u0026thinsp;+\u0026thinsp;IR group, which was substantially suppressed upon NAC addition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, E). Consistent with these observations, western blotting analysis indicated that the upregulation of pro-apoptotic proteins under combination treatment was reversed by NAC (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, G). These findings support that NCTD enhances IR-induced DNA damage and apoptosis in CRC cells through ROS-dependent mechanisms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eNCTD enhances radiosensitivity through ROS-mediated modulation of MAPK and AKT pathways\u003c/h2\u003e \u003cp\u003eTo systematically investigate the mechanisms through which NCTD enhances radiosensitivity, we conducted an integrated network pharmacology analysis. Potential NCTD targets were predicted using the SwissTargetPrediction database, whereas CRC-related genes were obtained from the GeneCards database. Thirty overlapping target genes were identified (Fig.\u0026nbsp;7A), and a compound\u0026ndash;target\u0026ndash;disease network constructed for visualization (Fig.\u0026nbsp;7B). PPI network analysis further revealed core targets potentially involved in NCTD-mediated radiosensitization (Fig.\u0026nbsp;7C). GO enrichment analysis indicated significant enrichment in 30 biological processes, 10 cellular components, and 20 molecular functions among the overlapping targets (Fig.\u0026nbsp;7E). KEGG pathway analysis highlighted 15 significantly enriched pathways, including those related to DNA damage response and cell cycle regulation, as well as PI3K-AKT, MAPK, and EGFR signaling (Fig.\u0026nbsp;7D).\u003c/p\u003e \u003cp\u003eFigure 7. Analysis of targets associated with NCTD and CRC. (A) Venn diagram of NCTD and CRC-associated targets. The diagram shows 74 NCTD-associated targets (left), 1,965 CRC-associated targets (right), and 26 common NCTD and CRC targets (center). (B) NCTD anti-CRC PPI network. Larger areas represent larger nodes; blue indicates stronger association, while lighter shades represent weaker association. Core targets are those within the inner circle. (C) Protein interaction network of NCTD and CRC was constructed using the STRING platform. (D) KEGG pathway enrichment analysis (DAVID). Pathways (Y-axis), FDR (X-axis), and P-values (color change). Bubble size indicates the number of genes enriched in each pathway. (E) GO enrichment analysis showing fold enrichment (y-axis), term (x-axis); green, orange, and purple represent the 15 core results for BP, CC, and MF, respectively.\u003c/p\u003e \u003cp\u003eGiven the established roles MAPK and AKT pathways play in regulating apoptosis [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and considering that excessive ROS can trigger apoptosis through JNK and p38MAPK signaling cascades[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], we hypothesized that NCTD enhances radiosensitivity through these pathways. To test this, we examined the phosphorylation levels of key signaling molecules. Western blotting analysis showed that irradiation alone induced the phosphorylation of ERK, p38, and AKT, but not JNK; contrastingly, NCTD\u0026thinsp;+\u0026thinsp;IR treatment significantly suppressed their phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eA\u0026ndash;D). To assess whether ROS mediated these effects, we pretreated cells with NAC. NAC pretreatment markedly restored p38, ERK, and AKT phosphorylation levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eE\u0026ndash;H), indicating that NCTD inhibits these pathways in a ROS-dependent manner. These findings indicate that NCTD enhances radiosensitivity by inhibiting ROS-mediated ERK, p38 MAPK, and AKT signaling pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eNCTD enhances radiotherapy efficacy in colorectal cancer xenografts\u003c/h2\u003e \u003cp\u003eTo validate our in vitro findings, we established a CRC xenograft model. Both IR alone and NCTD\u0026thinsp;+\u0026thinsp;IR significantly reduced tumor volume and weight compared to those in controls, with the combination group showing superior antitumor effects (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eA\u0026ndash;C). TUNEL staining demonstrated that NCTD\u0026thinsp;+\u0026thinsp;IR significantly increased apoptosis in tumor tissues compared to that of IR alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eD). Consistent with our in vitro results, the combination treatment significantly suppressed p38, ERK1/2, and AKT phosphorylation in tumor tissues (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eE\u0026ndash;H). IHC analysis further confirmed that NCTD enhanced cleaved caspase-3 expression while suppressing p-p38 expression following irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003eI). These in vivo findings demonstrate that NCTD overcomes radiation resistance in CRC through modulating MAPK and AKT signaling pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHerein, we demonstrate that NCTD acts as a potent radiosensitizer that significantly enhances IR-induced DNA damage, suppresses mitochondrial autophagy, and promotes apoptosis in CRC cells. We propose that these effects are primarily mediated through NCTD-induced ROS generation, which initiates a self-amplifying cycle of mitochondrial dysfunction, disrupts cellular redox homeostasis, and consequently inhibits pro-survival MAPK signaling while activating the intrinsic apoptotic pathway.\u003c/p\u003e \u003cp\u003eOur findings establish ROS as a central regulator of NCTD-mediated radiosensitization. Various stimuli, including inflammatory responses, IR, and chemotherapeutic agents, can induce substantial ROS production, which can lead to cellular damage, autophagy, and apoptosis[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. We observed that NCTD pretreatment significantly increased intracellular ROS levels, consistent with previous reports on the pro-oxidant properties of NCTD in other cancer models[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This ROS surge directly contributes to DNA damage, synergizing with IR to induce genomic instability. Beyond DNA damage, our data suggest that NCTD-induced ROS initiates a positive feedback loop that perpetuates mitochondrial dysfunction. The initial ROS wave damages mitochondrial components, leading to membrane depolarization and permeability transition pore opening. These compromised mitochondria then contribute additional ROS through electron leakage, creating a self-sustaining cycle that overwhelms cellular antioxidant defenses.\u003c/p\u003e \u003cp\u003eOur results demonstrate that NCTD\u0026thinsp;+\u0026thinsp;IR triggers the mitochondrial apoptosis pathway through Cyt c-mediated mechanisms. As shown in our subcellular fractionation experiments, the combination treatment significantly promoted Cyt c release from mitochondria into the cytoplasm. This translocation represents a pivotal commitment step in the intrinsic apoptotic pathway. Released Cyt c forms the apoptosome complex with Apaf-1 and caspase-9, causing caspase-9 activation that then cleaves and activates executioner caspases[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], ultimately leading to programmed cell death. Our observation of increased cleaved caspase-3 levels following combination treatment confirms the functional activation of this pathway, providing a direct mechanistic link between mitochondrial damage and apoptosis execution.\u003c/p\u003e \u003cp\u003eConcurrently, we observed altered mitophagy regulation. Mitophagy promotes tumor cell survival, thereby antagonizing the effects of anticancer therapies and reducing the radiosensitivity of cancer cells[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, inhibiting mitophagy holds potential to improve overall CRC outcomes. However, in the context of severe mitochondrial damage induced by NCTD and IR, excessive mitophagy may deplete ATP reserves and release pro-apoptotic factors, shifting its role from pro-survival to pro-death[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Parkin is localized within mitochondria and implicated in the generation of mitochondrial ROS in CRC cells[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Our immunofluorescence data showed that IR promoted Parkin translocation to mitochondria, whereas NCTD co-treatment suppressed this redistribution, thereby inhibiting mitophagy.\u003c/p\u003e \u003cp\u003eFurthermore, NCTD\u0026thinsp;+\u0026thinsp;IR suppressed the activation of ERK and p38 MAPK pathways. NCTD participates in the regulation of various diseases through multiple signaling targets[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Typically, IR activates ERK/P38 signaling as part of the cellular stress response, often associated with promoting survival and DNA repair[\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Our results, however, indicate that the hyper-elevated ROS environment generated by combination therapy supersedes this conventional activation. We propose several non-mutually exclusive mechanisms for this critical observation. First, oxidative stress-induced signal exhaustion: sustained high levels of ROS can lead to the oxidative inactivation of key kinases within the MAPK pathway through the oxidation of critical cysteine residues in their catalytic domains. Second, phosphatase activation: ROS is a potent activator of certain dual-specificity phosphatases that dephosphorylate and inactivate both p-ERK and p-P38, effectively terminating the survival signal[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Finally, cellular fate decision-making: when the apoptotic stimulus is overwhelming, a cell may actively downregulate pro-survival pathways to commit to apoptosis. This concentration-dependent dual role of ROS is gaining recognition and aligns with our results[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn conclusion, our data reveal that NCTD potentiates the cytotoxic effects of IR by instigating a catastrophic ROS-mitochondria feedback loop. This oxidative crisis suppresses pro-survival ERK/P38 signaling and activates mitophagy. Targeting this axis with NCTD may present a novel therapeutic strategy to overcome radioresistance.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eΔΨm\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emitochondrial membrane potential\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCCK-8\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCell Counting Kit-8\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\"\u003eCyt c\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecytochrome \u003cem\u003ec\u003c/em\u003e\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\"\u003eGO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene Ontology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eimmunohistochemistry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eionizing radiation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKEGG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKyoto Encyclopedia of Genes and Genomes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNAC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eN\u003c/em\u003e-acetylcysteine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNCTD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enorcantharidin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphate-buffered saline\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\"\u003ePINK1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePTEN-induced kinase 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePPI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eprotein-protein interaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereactive oxygen species.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval\u003c/strong\u003e \u003cp\u003eThe experimental protocol (IRM/2-IACUC-2401-011) for the tumor xenograft model in nude mice was reviewed and approved by the Experimental Animal Ethics Committee of the Institute of Radiation Medicine, Chinese Academy of Medical Sciences.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study is supported by Beijing Municipal Traditional Chinese Medicine Science and Technology Development Fund Project (BJZY-2025-08-TJ); National Natural Science Foundation of China (81573089,81972847); Tianjin key Medical Discipline (Specialty) Construction Project (TJYXZDXK-053B) and Tianjin Science and Technology Planning Project (21JCQNJC01900).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eQiong Xu: Writing \u0026ndash; original draft, Software, Methodology, Investigation, Formal analysis, Data curation. Heng Zhang:Validation, Software, Resources, Investigation. Dan-dan Gao:Software, Methodology.Yinglu Bi: Software. Keyi Wang: Data curation. Wei Zhao: Methodology, Data curation. Yuling Wang: Writing \u0026ndash; review \u0026amp; editing, Project administration, Funding acquisition.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThanks to \u0026ldquo;Editage (https://www.editage.cn/) \u0026rdquo; for the effort in polishing the English content of this manuscript.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eThe datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRawla P, Sunkara T, Barsouk A. Epidemiology of colorectal cancer: incidence, mortality, survival, and risk factors. GASTROENTEROL REV. 2019;14(2):89\u0026ndash;103.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeeney G, Sehgal R, Sheehan M, Hogan A, Regan M, Joyce M, Kerin M. Neoadjuvant radiotherapy for rectal cancer management. WORLD J GASTROENTERO. 2019;25(33):4850\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, Zhang HL, Yin HK, Zhang HK, Wang Y, Xu SN, Ma F, Gao JB, Li HL, Qu JR. Comparison of MRI and CT-Based Radiomics and Their Combination for Early Identification of Pathological Response to Neoadjuvant Chemotherapy in Locally Advanced Gastric Cancer. J MAGN RESON IMAGING. 2023;58(3):907\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaynes J, Manogaran P. Mechanisms and Strategies to Overcome Drug Resistance in Colorectal Cancer. INT J MOL SCI 2025, 26(5).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChevalier F. Counteracting Radio-Resistance Using the Optimization of Radiotherapy. INT J MOL SCI 2020, 21(5).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin CL, Chen CM, Lin CL, Cheng CW, Lee CH, Hsieh YH. Norcantharidin induces mitochondrial-dependent apoptosis through Mcl-1 inhibition in human prostate cancer cells. BBA-MOL CELL RES. 2017;1864(10):1867\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng LC, Yang MD, Kuo CL, Lin CH, Fan MJ, Chou YC, Lu HF, Huang WW, Peng SF, Chung JG. Norcantharidin-induced Apoptosis of AGS Human Gastric Cancer Cells Through Reactive Oxygen Species Production, and Caspase- and Mitochondria-dependent Signaling Pathways. ANTICANCER RES. 2016;36(11):6031\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChang C, Zhu YQ, Mei JJ, Liu SQ, Luo J. Involvement of mitochondrial pathway in NCTD-induced cytotoxicity in human hepG2 cells. J EXP CLIN CANC RES. 2010;29(1):145.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReichstein DA, Brock AL. Radiation therapy for uveal melanoma: a review of treatment methods available in 2021. CURR OPIN OPHTHALMOL. 2021;32(3):183\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSridharan DM, Asaithamby A, Bailey SM, Costes SV, Doetsch PW, Dynan WS, Kronenberg A, Rithidech KN, Saha J, Snijders AM, et al. Understanding cancer development processes after HZE-particle exposure: roles of ROS, DNA damage repair and inflammation. RADIAT RES. 2015;183(1):1\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Y, Li Y, Huang L, Du Y, Gan F, Li Y, Yao Y. Antioxidative Stress: Inhibiting Reactive Oxygen Species Production as a Cause of Radioresistance and Chemoresistance. \u003cem\u003eOXID MED CELL LONGEV\u003c/em\u003e 2021, 2021:6620306.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen L, Dobiasch S, Schneider G, Schmid RM, Azimzadeh O, Kanev K, Buschmann D, Pfaffl MW, Bartzsch S, Schmid TE, et al. Impact of DNA repair and reactive oxygen species levels on radioresistance in pancreatic cancer. RADIOTHER ONCOL. 2021;159:265\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePranteda A, Piastra V, Stramucci L, Fratantonio D, Bossi G. The p38 MAPK Signaling Activation in Colorectal Cancer upon Therapeutic Treatments. INT J MOL SCI 2020, 21(8).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLim S, Lee Y, Lee E. p38MAPK inhibitor SB203580 sensitizes human SNU-C4 colon cancer cells to exisulind-induced apoptosis. ONCOL REP. 2006;16(5):1131\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRen Y, Yang P, Li C, Wang WA, Zhang T, Li J, Li H, Dong C, Meng W, Zhou H. Ionizing radiation triggers mitophagy to enhance DNA damage in cancer cells. CELL DEATH DISCOV. 2023;9(1):267.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen X, Zhuo S, Xu W, Chen X, Huang D, Sun X, Cheng Y. Isocitrate dehydrogenase 2 contributes to radiation resistance of oesophageal squamous cell carcinoma via regulating mitochondrial function and ROS/pAKT signalling. BRIT J CANCER. 2020;123(1):126\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong C, Pan S, Zhang J, Li N, Geng Q. Mitophagy: A novel perspective for insighting into cancer and cancer treatment. CELL PROLIFERAT. 2022;55(12):e13327.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVives-Bauza C, Zhou C, Huang Y, Cui M, de Vries RLA, Kim J, May J, Tocilescu MA, Liu W, Ko HS, et al. PINK1-dependent recruitment of Parkin to mitochondria in mitophagy. P NATL ACAD SCI USA. 2010;107(1):378\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Y, Li Y, Huang L, Du Y, Gan F, Li Y, Yao Y. Antioxidative Stress: Inhibiting Reactive Oxygen Species Production as a Cause of Radioresistance and Chemoresistance. \u003cem\u003eOXID MED CELL LONGEV\u003c/em\u003e 2021, 2021:6620306.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen L, Dobiasch S, Schneider G, Schmid RM, Azimzadeh O, Kanev K, Buschmann D, Pfaffl MW, Bartzsch S, Schmid TE, et al. Impact of DNA repair and reactive oxygen species levels on radioresistance in pancreatic cancer. RADIOTHER ONCOL. 2021;159:265\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKwak AW, Kim WK, Lee SO, Yoon G, Cho SS, Kim KT, Lee MH, Choi YH, Lee JY, Park JW et al. Licochalcone B Induces ROS-Dependent Apoptosis in Oxaliplatin-Resistant Colorectal Cancer Cells via p38/JNK MAPK Signaling. ANTIOXIDANTS-BASEL 2023, 12(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKwak AW, Lee JY, Lee SO, Seo JH, Park JW, Choi YH, Cho SS, Yoon G, Lee MH, Shim JH. Echinatin induces reactive oxygen species-mediated apoptosis via JNK/p38 MAPK signaling pathway in colorectal cancer cells. PHYTOTHER RES. 2023;37(2):563\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu Y, Chen D, Wu T, Lin H, Ni L, Sui H, Xiao S, Wang C, Jiang S, Pan H, et al. Dihydroartemisinin enhances the anti-tumor activity of oxaliplatin in colorectal cancer cells by altering PRDX2-reactive oxygen species-mediated multiple signaling pathways. Phytomedicine. 2022;98:153932.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei F, Nian Q, Zhao M, Wen Y, Yang Y, Wang J, He Z, Chen X, Yin X, Wang J, et al. Natural products and mitochondrial allies in colorectal cancer therapy. BIOMED PHARMACOTHER. 2023;167:115473.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheung EC, Vousden KH. The role of ROS in tumour development and progression. NAT REV CANCER. 2022;22(5):280\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin CL, Chen CM, Lin CL, Cheng CW, Lee CH, Hsieh YH. Norcantharidin induces mitochondrial-dependent apoptosis through Mcl-1 inhibition in human prostate cancer cells. BBA-MOL CELL RES. 2017;1864(10):1867\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng LC, Yang MD, Kuo CL, Lin CH, Fan MJ, Chou YC, Lu HF, Huang WW, Peng SF, Chung JG. Norcantharidin-induced Apoptosis of AGS Human Gastric Cancer Cells Through Reactive Oxygen Species Production, and Caspase- and Mitochondria-dependent Signaling Pathways. ANTICANCER RES. 2016;36(11):6031\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie H, Song L, Katz S, Zhu J, Liu Y, Tang J, Cai L, Hildebrandt P, Han XX. Electron transfer between cytochrome c and microsomal monooxygenase generates reactive oxygen species that accelerates apoptosis. REDOX BIOL. 2022;53:102340.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Pang C, Zhang C, Wang Y, Wang P, Chen Y, Wang J, Hu Y, Liu C, Liang H, et al. HILPDA-mediated lipidomic remodelling promotes radiotherapy resistance in nasopharyngeal carcinoma by accelerating mitophagy. CELL MOL LIFE SCI. 2023;80(9):242.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXia J, Jin J, Dai S, Fan H, Chen K, Li J, Luo F, Peng X. Mitophagy: A key regulator of radiotherapy resistance in the tumor immune microenvironment. MOL ASPECTS MED. 2025;105:101385.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin Q, Li S, Jiang N, Shao X, Zhang M, Jin H, Zhang Z, Shen J, Zhou Y, Zhou W, et al. PINK1-parkin pathway of mitophagy protects against contrast-induced acute kidney injury via decreasing mitochondrial ROS and NLRP3 inflammasome activation. REDOX BIOL. 2019;26:101254.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Dong S, Hu K, Xu L, Feng Q, Li B, Wang G, Chen G, Zhang B, Jia X, et al. The novel norcantharidin derivative DCZ5417 suppresses multiple myeloma progression by targeting the TRIP13-MAPK-YWHAE signaling pathway. J TRANSL MED. 2023;21(1):858.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYousef EH, El-Magd NFA, El Gayar AM. Norcantharidin potentiates sorafenib antitumor activity in hepatocellular carcinoma rat model through inhibiting IL-6/STAT3 pathway. TRANSL RES. 2023;260:69\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, Lin CH, Chen K, Lai D, Hsu F. Inactivation of EGFR/ERK/NF-kappaB signalling associates with radiosensitizing effect of 18beta-glycyrrhetinic acid on progression of hepatocellular carcinoma. J CELL MOL MED. 2023;27(11):1539\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParamanantham A, Jung EJ, Go S, Jeong BK, Jung J, Hong SC, Kim GS, Lee WS. Activated ERK Signaling Is One of the Major Hub Signals Related to the Acquisition of Radiotherapy-Resistant MDA-MB-231 Breast Cancer Cells. INT J MOL SCI 2021, 22(9).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIrani K, Xia Y, Zweier JL, Sollott SJ, Der CJ, Fearon ER, Sundaresan M, Finkel T, Goldschmidt-Clermont PJ. Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts. Science. 1997;275(5306):1649\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReddy KB, Glaros S. Inhibition of the MAP kinase activity suppresses estrogen-induced breast tumor growth both in vitro and in vivo. INT J ONCOL. 2007;30(4):971\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Z, Fei T, Zhang J, Zhu G, Wang L, Lu D, Chi X, Teng Y, Hou N, Yang X, et al. BMP4 Signaling Acts via dual-specificity phosphatase 9 to control ERK activity in mouse embryonic stem cells. Cell Stem Cell. 2012;10(2):171\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKucera J, Netusilova J, Sladecek S, Lanova M, Vasicek O, Stefkova K, Navratilova J, Kubala L, Pachernik J. Hypoxia Downregulates MAPK/ERK but Not STAT3 Signaling in ROS-Dependent and HIF-1-Independent Manners in Mouse Embryonic Stem Cells. OXID MED CELL LONGEV. 2017;2017:4386947.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoloney JN, Cotter TG. ROS signalling in the biology of cancer. SEMIN CELL DEV BIOL. 2018;80:50\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIdelchik MDPS, Begley U, Begley TJ, Melendez JA. Mitochondrial ROS control of cancer. SEMIN CANCER BIOL. 2017;47:57\u0026ndash;66.\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":"cancer-cell-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccin","sideBox":"Learn more about [Cancer Cell International](http://cancerci.biomedcentral.com/)","snPcode":"12935","submissionUrl":"https://submission.nature.com/new-submission/12935/3","title":"Cancer Cell International","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Colorectal cancer, Mitochondrial dysfunction, Norcantharidin, Radiotherapy sensitization, Reactive oxygen species","lastPublishedDoi":"10.21203/rs.3.rs-8609994/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8609994/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eRadiotherapy constitutes a vital treatment modality for rectal cancer; however, tumor cell radioresistance frequently leads to treatment failure. Although norcantharidin (NCTD) exhibits antitumor activity, its potential as a radiosensitizer for rectal cancer and the underlying molecular mechanisms remain unclear. This study investigated the effect NCTD has on colorectal cancer (CRC) cell radiosensitivity and its potential mechanisms of action.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe effects of NCTD combined with ionizing radiation (IR) on CRC cell viability were assessed using CCK-8 and colony formation assays. Apoptosis and intracellular reactive oxygen species (ROS) levels were detected via flow cytometry. The JC-1 probe was employed to evaluate mitochondrial membrane potential and function. Western blotting analysis assessed apoptosis-related proteins and phosphorylation levels of proteins within the ERK/p38MAPK/AKT signaling pathways. Concurrently, a nude mouse xenograft model was established for in vivo validation.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eNCTD significantly enhanced the growth-inhibitory and clonogenic suppression effects of IR on CRC cells whilst synergistically inducing apoptosis in vitro. Mechanistic investigations revealed that NCTD and IR treatment induced mitochondrial dysfunction, leading to a marked increase in intracellular ROS levels, which further activated ERK, p38MAPK, and AKT signaling pathways. Blocking these pathways using \u003cem\u003eN\u003c/em\u003e-acetylcysteine significantly reversed the apoptosis-inducing effects and radiosensitizing activity of NCTD and IR.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eNCTD enhances radiotherapy sensitivity in CRC cells by inducing mitochondrial dysfunction, promoting ROS production, and subsequently activating the ROS/ERK/p38MAPK/AKT signaling pathway axis, ultimately inducing apoptosis. This indicates that NCTD holds potential as a radiosensitizer for rectal cancer, offering novel strategies and theoretical foundations for overcoming clinical radiation resistance.\u003c/p\u003e","manuscriptTitle":"Mechanism by which norcantharidin enhances radiotherapy sensitivity in colorectal cancer cells: Apoptosis induction via the mitochondrial dysfunction–reactive oxygen species–ERK/p38 MAPK/AKT pathway axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-12 14:00:28","doi":"10.21203/rs.3.rs-8609994/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-05-04T12:25:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-30T10:36:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-30T10:36:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Cell International","date":"2026-01-15T11:24:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-cell-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccin","sideBox":"Learn more about [Cancer Cell International](http://cancerci.biomedcentral.com/)","snPcode":"12935","submissionUrl":"https://submission.nature.com/new-submission/12935/3","title":"Cancer Cell International","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d7ca1d7c-4f42-4681-ab27-5e5bfff980a9","owner":[],"postedDate":"May 12th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewersInvited","content":"8","date":"2026-05-04T12:25:28+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-12T14:00:28+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-12 14:00:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8609994","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8609994","identity":"rs-8609994","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}