Antiproliferative Effects of Reynoutrin via Modulation of Oxidative Stress, Inflammation, and Apoptosis in Breast (MCF-7) and Prostate Cancer (PC-3) Cells Lines

Antiproliferative Effects of Reynoutrin via Modulation of Oxidative Stress, Inflammation, and Apoptosis in Breast (MCF-7) and Prostate Cancer (PC-3) Cells Lines | 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 Antiproliferative Effects of Reynoutrin via Modulation of Oxidative Stress, Inflammation, and Apoptosis in Breast (MCF-7) and Prostate Cancer (PC-3) Cells Lines Sedat GÖKMEN, İrfan ÇINAR This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8431324/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Reynoutrin, a bioactive flavonoid, is recognized for its biological effects. Despite toxic side effects, cisplatin is widely used to treat malignancies. This study aimed to assess the in vitro antiproliferative activity of reynoutrin in MCF-7 and PC-3 cell lines by evaluating its modulation of intracellular signaling pathways. Methods The cytotoxic effects of reynoutrin on MCF-7 and PC-3 cells were evaluated using an MTT assay after 24 h. Oxidative stress levels were measured using enzyme-linked immunosorbent assay (ELISA) with total antioxidant and oxidant status kits. The expression levels of tumor necrosis factor-α (TNF‑α), interleukin (IL)‑1β and IL‑6, caspase 3, caspase 9, B‑cell lymphoma‑2 (Bcl‑2), and Bcl‑2‑associated X protein (Bax) were detected by RT-PCR. Apoptosis was verified using Hoechst staining. Results Reynoutrin inhibited MCF-7 and PC-3 cell viability in a dose-dependent manner, with IC₅₀ values of 220 and 412 µg/mL, respectively. Reynoutrin increased the total oxidant status, decreased the total antioxidant enzyme activity, and enhanced oxidative stress (p < 0.05). RT-PCR showed that TNF-α, IL-1β, and IL-6 expression levels decreased in a dose-dependent manner following reynoutrin administration in MCF-7 and PC-3 cells (p < 0.05), indicating its anti-inflammatory activity. In cancer cells, reynoutrin increased caspase-3 and caspase-9 levels and decreased the Bcl-2/Bax ratio in a dose-dependent manner (p < 0.05). Conclusion These findings demonstrate that reynoutrin exerts antiproliferative activity by regulating inflammation, oxidative stress, and apoptotic pathways in cancer cells. Reynoutrin MCF-7 PC-3 Apoptosis Oxidative stress Anti-inflammatory Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Highlights Reynoutrin inhibited the viability of MCF-7 and PC-3 cells in a dose-dependent manner. Reynoutrin increased oxidative stress by elevating TOS and decreasing TAS levels. Reynoutrin suppressed pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 expression. Reynoutrin induced apoptosis by upregulating caspase-3, caspase-9, and Bax while downregulating Bcl-2. Reynoutrin shows promise as a potential therapeutic agent against breast and prostate cancers. Introduction Cancer is the second leading cause of death worldwide, represents a major global public health challenge, and is one of the leading contributor to mortality and morbidity. Reports indicate that 19,976,499 new cancer cases and 9,743,832 cancer-related deaths occurred worldwide in 2022. Breast (11.5%) and prostate (7.3%) cancers are among the top five types of cancers with the highest incidence rates worldwide [ 1 ]. Antineoplastic agents are fundamental in cancer therapy, often administered alongside radiotherapy, surgery, and immunotherapy [ 2 ]. However, their use is frequently limited by significant adverse effects and drug resistance in tumor cells. These drugs can cause mild to life-threatening effects, including gastrointestinal toxicity, hepatotoxicity, nephrotoxicity, hematopoietic system damage, cardiotoxicity, and neurotoxicity [ 3 – 7 ]. Cancer cells frequently acquire resistance to chemotherapeutic agents following treatment. These problems reduce patients' quality of life and drug efficacy, restrict clinical use, and can lead to treatment failure [ 8 , 9 ]. Therefore, more effective strategies for managing chemotherapy-related side effects are required. Alternative approaches have been investigated to reduce side effects, ensure effective doses, and reduce health care costs. Phytochemicals have been extensively studied for their synergistic effects on chemotherapy regimens and cancer prevention [ 8 – 10 ]. Currently, flavonoids are extensively used in both in vivo and in vitro studies to explore their pharmacological properties, particularly for cancer treatment. Flavonoids are used to treat a range of diseases including cancer, diabetes, cardiovascular diseases and ulcers. Flavonoids exert cytotoxic effects on cancer cells through multiple mechanisms, including anti-inflammatory activity, induction of apoptosis, and disruption of oxidative stress balance. Among the studies conducted on the anticancer activity of these compounds, hesperidin, quercetin, cyanidin, catechins, apigenin, and quercetin-3- O -glucoside are of particular interest [ 11 – 13 ]. Studies that have reached the final phase have demonstrated promising results, indicating that these compounds may serve as effective adjuvant agents in the treatment or reduction of the risk of various types of cancer [ 14 ]. Reynoutrin, also known as quercetin-3-D-xyloside, is a flavonoid naturally found in the fruits and leaves of various plants [ 15 ]. Although studies of reynoutrin are limited, existing research suggests that it exhibits a wide range of pharmacological activities. These include antitumor, antiviral, and antidiabetic effects [ 15 – 17 ]. Additionally, reynoutrin has been associated with wound healing properties and has potential benefits in improving ischemic heart failure [ 18 ]. Oxidative stress, an imbalance between reactive oxygen species (ROS) production and antioxidant defense systems, plays a significant role in carcinogenesis, tumor progression, and chemoresistance. Flavonoids have a dual role in ROS homeostasis; they act as antioxidants under physiological conditions but as pro-oxidants under pathological conditions, such as cancer, initiating apoptotic pathways. Pro-oxidant activity is a key mechanism underlying the anticancer potential of flavonoids [ 19 ]. Flavonoids, including daidzein, hesperidin, and naringenin, exert pro-oxidant effects in cancer cell lines such as MCF-7 and PC-3 by elevating intracellular ROS levels and enhancing oxidative stress. This increase in ROS disrupts cellular redox homeostasis, leading to the suppression of cancer cell proliferation and migration, and the induction of cell death [ 20 ]. Inflammation causes tumor development and regulates cellular differentiation, survival, proliferation, invasion, metastasis, and angiogenesis [ 21 ]. Inflammation facilitates tumor initiation and progression by promoting proliferative signaling, angiogenesis, and resistance to apoptosis. Proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) contribute to tumorigenesis. Flavonoids (such as apigenin, rutin, and pelargonidin) suppress inflammation by downregulating pro-inflammatory cytokines, such as IL-6, TNF-α, and IL-1β, thereby inhibiting tumor development. IL-6, IL-1β, and TNF-α regulate the functions of various cell types, including breast [ 22 ] and prostate [ 23 ] cancers, particularly tumor growth, invasion, and metastasis [ 24 ]. Apoptosis is a type of programmed cell death that maintains homeostasis and eliminates the damaged cells. The Bcl-2-associated X protein (Bax) and B-cell lymphoma 2 (Bcl-2) are critical regulators of apoptosis and cell survival, respectively. Bax promotes cell death, whereas Bcl-2 promotes cell survival. A decreased Bcl-2/Bax ratio indicates increased susceptibility to apoptosis [ 25 ]. The intrinsic apoptotic pathway is regulated by pro-apoptotic proteins such as Bax and anti-apoptotic proteins such as Bcl-2, which control mitochondrial membrane permeability and cytochrome c release. This activates caspase-9 and caspase-3, leading to DNA fragmentation and cell death [ 26 ]. Research has shown that caspase-3 and caspase-9 are essential mediators of flavonoid-induced apoptosis in cancer cells [ 27 ]. Caspase-9 initiates the apoptotic cascade, whereas caspase-3 cleaves cellular substrates during apoptosis [ 28 ]. Studies have shown that flavonoids induce apoptosis by upregulating pro-apoptotic markers and downregulating Bcl-2 [ 29 ]. Currently, studies on reynoutrin are limited, and its antiproliferative activities, particularly in breast and prostate cancers, have not been clarified. In this study, we investigated the antiproliferative potential of reynoutrin by evaluating its effects on oxidative stress parameters (total antioxidant and oxidant status), proinflammatory cytokines (TNF-α, IL-1β, and IL-6), and key apoptotic markers (caspase-3, caspase-9, and Bcl-2/Bax) in MCF-7 and PC-3 cells. These findings provide valuable insights into the molecular mechanisms underlying the anticancer effects of reynoutrin, and support its potential as a complementary cancer therapy. Materials and methods Chemicals and reagents Reynoutrin (Cas number: 549-32-6; purity > 97%) and cisplatin (Cas number: 15663-27-1; purity > 99.84%) were purchased from MedChemExpress (Manmouth Junctions, NJ, USA). Dulbecco’s modified Eagle’s medium (DMEM), cell culture medium, and reagents such as fetal bovine serum (FBS), penicillin/streptomycin, and trypsin–ethylenediaminetetraacetic acid (trypsin/EDTA) were obtained from Gibco (Invitrogen Inc., Grand Island, New York, USA). Cell culture procedures Human breast (MCF-7) and prostate (PC-3) cancer cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cell lines stored in the liquid nitrogen tank were briefly thawed in a 37°C water bath and subsequently transferred into T75 cm² flasks for culture. The cells were cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin (100 IU/mL) at 37°C in a humidified 5% CO₂ incubator. All incubations conducted on the cells throughout the study were performed in a CO₂ incubator (PHCbi cell culture incubator, Loughborough, UK) at 37°C in a medium containing 5% CO₂. Upon reaching approximately 80% confluence, cells were detached with 0.25% trypsin/EDTA and subsequently seeded into 6-well plates for RNA extraction and biochemical analyses and into 96-well plates for MTT assays. After the cells reached approximately 80% confluence, they were detached with 0.25% trypsin/EDTA and plated for RNA extraction and biochemical investigations in 6-well plates, and for the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyltetrazolium bromide] assay in 96-well plates. All cell culture procedures were performed in compliance with standards described in the literature. Cell viability assay The MTT (Thermo Fisher Scientific, Waltham, MA, USA) assay was performed to determine cell viability according to the manufacturer’s instructions. MCF-7 and PC-3 cells were cultured in 96-well plates (5 × 10³ cells/well) and were allowed to attach. The cells were then exposed to different concentrations (10–2000 µg/mL) of reynoutrin, dissolved in dimethyl sulfoxide (DMSO) as the solvent. The final DMSO concentration was maintained at 0.1% in all treatment and control groups, and incubated for 24 h to determine the IC₅₀ (half-maximal inhibitory concentration). These concentrations were investigated in previous studies. Following the completion of exposure, 20 µL of MTT solution (5 mg/mL in phosphate-buffered saline) was added to each well, and the plate was incubated at 37°C for 4 h. Dark crystals of formazan formed in intact cells were dissolved in 200 µL of DMSO. After shaking the plates for 15 min, the absorbance at 570 nm was measured using a microplate spectrophotometer (Epoch Microplate Spectrophotometer, BioTek, USA) in accordance with the manufacturer's protocol (Roche, Germany). The cell viability rate (%) was calculated using the following equation. $$\:\text{\%}\:\text{C}\text{e}\text{l}\text{l}\:\text{v}\text{i}\text{a}\text{b}\text{i}\text{l}\text{i}\text{t}\text{y}\:\text{r}\text{a}\text{t}\text{e}\:=\frac{{OD}_{test\:sample}\:-\:{OD}_{blank}}{{OD}_{control}\:-\:{OD}_{blank}}\times\:100$$ OD test sample : Optical density (absorbance) measured in the wells containing the test sample. OD blank : Absorbance of a blank well (medium without cells) subtracted from the background. OD control : Absorbance of the untreated control well The IC 50 concentrations were calculated using Graphpad Probit Analysis Program (Version 9.3.0). Experimental groups To investigate the antiproliferative effects of reynoutrin, six experimental groups were established for both the MCF-7 and PC-3 cell lines. The groups were designed as follows: Group I (Control; C): No chemical treatment was administered to MCF-7 or PC-3 cells. Group II (DMSO): MCF-7 and PC-3 cells treated with %0.1 DMSO for 24 h. Group III (Cis50): MCF-7 and PC-3 cells were exposed to 50 µM cisplatin for 24 h. Group IV (Rey Low Dose): Cells were treated for 24 h with a lower concentration of reynoutrin: 110 µg/mL for MCF-7 cells and 206 µg/mL for PC-3 cells. Group V (Rey Mid-level Dose): Cells were treated for 24 h with moderate concentrations of reynoutrin, 220 µg/mL for MCF-7 and 412 µg/mL for PC-3. Group VI (Rey High Dose): Cells were treated for 24 h with a higher concentration of reynoutrin, 330 µg/mL for MCF-7 and 618 µg/mL for PC-3. Determination of intracellular antioxidant–oxidant status and oxidative stress index The total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) are oxidative profiles that determine the intracellular oxidative. We measured TAS and TOS using Rel Assay Diagnostics Assay Kits (Rel Assay Diagnostics, Gaziantep, Turkey). The parameters were measured using a spectrophotometric method with an Epoch microplate reader following the manufacturer's instructions. The color change was measured at a wavelength of 660 nm, and the TAS results were expressed as mmol Trolox equivalents per mg of protein, whereas the TOS levels were determined based on the color change measured at 530 nm and expressed as µmol H₂O₂ equivalents per mg of protein. OSI was calculated using the following formula [ 31 ]: $$\:\text{O}\text{S}\text{I}\:(\text{A}\text{r}\text{b}\text{i}\text{t}\text{r}\text{a}\text{r}\text{y}\:\text{U}\text{n}\text{i}\text{t},\:\text{A}\text{U})=\frac{\text{T}\text{O}\text{S}\:({\mu\:}\text{m}\text{o}\text{l}\:{H}_{2}{O}_{2}\:\text{E}\text{q}\text{u}\text{i}\text{v}./\text{m}\text{g}\:\text{p}\text{r}\text{o}\text{t}\text{e}\text{i}\text{n})}{\text{T}\text{A}\text{S}\:(\text{m}\text{m}\text{o}\text{l}\:\text{T}\text{r}\text{o}\text{l}\text{o}\text{x}\:\text{E}\text{q}\text{u}\text{i}\text{v}./\text{m}\text{g}\:\text{p}\text{r}\text{o}\text{t}\text{e}\text{i}\text{n})}\times\:10$$ Each experiment was conducted in three independent replicates. Quantitative determination of mRNA expression by RT-PCR The mRNA expression levels of IL-1β, IL-6, TNF-α, caspase-3, caspase-9, Bax, and Bcl-2 were analyzed using Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The cells were collected from a 6-plate using a scraper and 350 µL of RLT solution was added separately to each group, followed by homogenization using a Tissue Lyser II device (Qiagen, Germany). RNA extraction was performed according to the manufacturer's instructions using an isolation kit (Qiagen RNA, Cat. No: 74,104, Qiagen, Hille, Germany). The RNA concentration was measured using a NanoDrop spectrophotometer (Epoch 2, BioTek, USA). The quality of RNA was determined using optical densities (OD260/280) per nanodrop, and RNAs with OD260/280 ratios between 1.7 and 2.2 (or ~ 1.8) were used in the study. For cDNA synthesis, the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, USA) was used for cDNA synthesis. The process was performed on a Veriti 96-Well Thermal Cycler (Applied Biosystems), and the synthesized cDNA was preserved at -20°C. The mRNA expression of IL-1β (Hs01555410_m1), IL-6 (Hs00174131_m1), TNF-α (Hs00174128_m1), Caspase-3 (Hs00234387_m1), Caspase-9 (Hs00154260_m1), Bax (Hs00180269_m1), and Bcl-2 (Hs04986394_s1) was measured using the TaqMan Master Mix kit. RT-PCR amplification and quantification were conducted using a StepOnePlus device (Applied Biosystems). Gene Expression levels were normalized to the β-actin reference gene Hs01060665_g1 TaqMan® Gene Expression, which was modified to the PCR program according to the kit’s instructions, and the process was initiated. All results are represented as fold-changes in expression compared with the groups using the 2 −ΔΔCt method. Each gene was analyzed using the approach described in our previous study [ 30 ]. Each experiment was conducted in triplicate. Hoechst 33342 staining As characteristic changes in nuclear morphology typically occur during apoptosis, these changes can serve as indicators of programmed cell death. Apoptotic cell death was determined by Hoechst 33342 staining. Hoechst 33342 fluorescence staining was used to evaluate apoptotic cells according to their nuclear morphology. Hoechst 33342, a blue fluorescent membrane-permeable dye, stains condensed chromatin more brightly in apoptotic cells than in normal cells. For this purpose, MCF-7 and PC-3 cells (2 × 10 5 cells/well) were seeded onto 6-well plates prior to treatment with reynoutrin or cisplatin and incubated (5% CO 2 , 37°C) for 24 h. The following day, cells were treated with different concentrations of either reynoutrin or cisplatin and incubated for 24 h. After washing three times with 1XPBS, the cells were stained with 1 mL Hoechst 33342 (10 µg/mL) solution for 15 min in the dark at 37°C. Apoptotic cells (alterations in nuclear morphology) were detected using Hoechst 33342 staining under an inverted fluorescence microscope (Leica, DMIL LED, Germany). Statistical analysis Statistical analyses were performed using the IBM SPSS Statistics software (version 25.0). Numerical data obtained from biochemical analyses and RT-PCR results were analyzed using one-way ANOVA post-hoc Duncan test. Results are presented as the mean ± standard deviation (SD). Additionally, IC₅₀ values were calculated using probit analysis. Statistical significance was set at p < 0.05. Results Reynoutrin reduces MCF-7 and PC-3 cells proliferation The viability of MCF-7 and PC-3 cells treated with different concentrations of reynoutrin (10-2000 µg/mL) was measured using the MTT assay, and the percentage of viable cells was monitored over 24 h. DMSO vehicle control had no effect on cell viability. These results revealed that reynoutrin reduced the viability of MCF-7 and PC-3 cells in a concentration-dependent manner. Probit analysis revealed that the concentrations required to achieve 50% inhibition (IC₅₀) were 220 µg/mL and 412 µg/mL for MCF-7 and PC-3 cells, respectively. Therefore, in the following experiments, reynoutrin was selected at concentrations corresponding to low, mid-level, and high values relative to its IC₅₀. Concentrations lower than 110 and 206 µg/mL did not cause a significant change in the proliferation of MCF-7 and PC-3 cells, respectively. To evaluate the effects of reynoutrin, MCF-7 cells were treated with concentrations of 110, 220, and 330 µg/mL, whereas PC-3 cells were treated with 206, 412, and 618 µg/mL reynoutrin. Reynoutrin induced oxidative stress in MCF-7 cells Figure 1 illustrates the effects of reynoutrin on TAS, TOS, and OSI in MCF-7 cells. In MCF-7 cells, a significant increase in TOS levels and a reduction in TAS levels were observed in the cisplatin-treated group compared with those in the control group (p < 0.05). Similarly, reynoutrin induced a concentration-dependent increase in TOS levels and a significant decrease in TAS levels relative to the control group (p < 0.05) (Fig. 1 a, 1 b). Moreover, reynoutrin markedly elevated OSI levels in MCF-7 cells (Fig. 1 c). Collectively, these findings suggested that reynoutrin exerted a pro-oxidant effect by enhancing oxidative stress in MCF-7 cells. Reynoutrin induced oxidative stress in PC-3 cells Figure 2 illustrates the effects of reynoutrin on TAS, TOS, and OSI in PC-3 cells. In PC-3 cells, a significant increase in TOS levels and a reduction in TAS levels were observed in the cisplatin-treated group compared with those in the control group (p < 0.05). Similarly, reynoutrin induced a concentration-dependent increase in TOS levels and a significant decrease in TAS levels relative to the control group (p 0.05). Moreover, reynoutrin markedly elevated OSI levels in PC-3 cells (Fig. 2 c). Collectively, these findings suggest that reynoutrin exerts a pro-oxidant effect by increasing oxidative stress in the PC-3 cells. Expression results of TNF-α, IL-1β, IL-6, caspase-3, caspase-9, and Bcl-2/Bax mRNA in MCF-7 cells Analysis of inflammatory markers showed that cisplatin (50 µM) treatment significantly increased the mRNA expression of TNF-α, IL-1β, and IL-6 in MCF-7 cells compared to that in the control group (p < 0.05). Conversely, reynoutrin administration (110, 220, and 330 µg/mL) significantly reduced TNF-α, IL-1β, and IL-6 mRNA levels in a concentration-dependent manner (p < 0.05; Fig. 3 ). These RT-PCR results demonstrated the anti-inflammatory potential of reynoutrin via the suppression of pro-inflammatory cytokine gene expression. Analysis of apoptotic parameters demonstrated that cisplatin significantly elevated caspase-3 and caspase-9 mRNA expression and significantly decreased the Bcl-2/Bax ratio in MCF-7 cells compared with that in the control group (p < 0.05). Similarly, medium and high concentrations of reynoutrin (220 and 330 µg/mL, respectively), but not low concentrations (110 µg/mL), induced a significant increase in caspase-3 and caspase-9 expression and a reduction in the Bcl-2/Bax ratio (p < 0.05; Fig. 4 ). Interestingly, the effect of high-dose reynoutrin on caspase-9 expression was similar to that of cisplatin alone. These results indicate that reynoutrin promoted apoptosis in MCF-7 cells in a concentration-dependent manner. The PCR results showed that reynoutrin exhibited anti-inflammatory activity and induced apoptotic cell death in MCF-7 cells. Expression levels of TNF-α, IL-1β, IL-6, caspase-3, caspase-9, and Bcl-2/Bax mRNA in PC-3 cells Analysis of inflammatory markers showed that cisplatin treatment significantly increased the mRNA expression of TNF-α, IL-1β, and IL-6 in PC-3 cells compared with that in the control group (p < 0.05). Conversely, administration of reynoutrin (206, 412, and 618 µg/mL) resulted in a significant concentration-dependent reduction in TNF-α, IL-1β, and IL-6 mRNA expression relative to the control group (p < 0.05; Fig. 5 ). These PCR results demonstrate the anti-inflammatory potential of reynoutrin via the suppression of pro-inflammatory cytokine gene expression. Analysis of apoptotic parameters demonstrated that cisplatin significantly elevated caspase-3 and caspase-9 mRNA expression and significantly decreased the Bcl-2/Bax ratio in PC-3 cells compared with that in the control group (p < 0.05). Similarly, reynoutrin induced a significant increase in caspase-3 and caspase-9 expression and a reduction—excluding the low concentration (206 µg/mL)— in the Bcl-2/Bax ratio (p < 0.05; Fig. 6 ). These results indicate that reynoutrin promoted apoptosis in PC-3 cells in a concentration-dependent manner. RT-PCR results showed that reynoutrin exhibited anti-inflammatory activity and induced apoptosis in PC-3 cells. Hoechst 33342 staining results Figure 7 demonstrates the effect of reynoutrin on Hoechst 33342 staining in MCF-7 and PC-3 cells. Reynoutrin induced remarkable morphological alterations in the nuclei of MCF-7 and PC-3 cells compared with those in the control group. Increasing concentrations of reynoutrin led to nuclear shrinkage, fragmentation in both PC-3 and MCF-7 cells, and chromatin condensation, which are the hallmark features of apoptosis. In contrast to the control group, in which the cells displayed intact and healthy blue nuclei, those treated with cisplatin or reynoutrin exhibited enhanced blue fluorescence, which is indicative of the early apoptotic stages. These findings suggest that reynoutrin triggers apoptosis-related morphological changes in MCF-7 and PC-3 cells. Discussion Cancer is a leading cause of mortality worldwide. Among various cancer types, breast cancer in women and prostate cancer in men rank as the second most fatal malignancy in their respective populations. Currently, chemotherapy, surgical intervention, and radiotherapy are the primary therapeutic approaches. However, these modalities often exhibit limited efficacy in cases of high incidence or low survival rates, and are frequently associated with severe adverse effects. Therefore, there is an urgent need to explore and develop novel anticancer agents derived from natural sources that are less toxic, more cost-effective, and have fewer side effects [ 32 ]. Naturally occurring compounds, particularly flavonoids, are regarded as valuable sources for the development of novel anticancer agents and have demonstrated significant potential in this field [ 33 ]. Flavonoids are naturally occurring phytochemicals with diverse pharmacological properties that have been the subject of exponentially increasing research on their pharmacological applications [ 34 ]. Flavonoids have demonstrated significant anticancer activities through the induction of oxidative stress, cell cycle arrest, attenuation of inflammatory responses, stimulation of apoptosis and autophagy, and suppression of cancer cell proliferation and invasion [ 19 , 34 ]. In this study, we investigated the antiproliferative, anti-inflammatory, and apoptotic activities of reynoutrin in the human prostate cancer cell line PC-3 and the human mammary cancer cell line MCF-7. In the present study, the IC₅₀ values of reynoutrin were 110 µg/mL for MCF-7 cells and 206 µg/mL for PC-3 cells. In contrast, Yüksel et al. reported a higher IC₅₀ value of 400 µg/mL in MCF-7 cells, suggesting that variations in experimental design, treatment duration, and cellular responsiveness may influence cytotoxic potency [ 15 ]. The relatively high IC₅₀ values observed in both studies may be attributed to the glycosylated structure of reynoutrin, which is known to limit membrane permeability and reduce intracellular accumulation compared with aglycone flavonoids. When the balance between prooxidant activity and antioxidant defense is disrupted, ROS levels increase, leading to the accumulation of free radicals. Excessive ROS production results in oxidative stress, which is a key factor in promoting the inflammatory processes associated with cancer development. Flavonoids play a multifaceted role in cancer prevention and treatment, because their antioxidant or pro-oxidant behavior is influenced by their concentration and cellular conditions. Under normal physiological conditions, flavonoids function as antioxidants, maintaining ROS homeostasis; however, in cancer cells, they can act as potent pro-oxidants, initiating apoptotic pathways and suppressing pro-inflammatory signaling. Although their antioxidant properties protect healthy cells from oxidative stress and potentially lower cancer risk, their pro-oxidant effects at higher concentrations can induce stress in cancer cells, potentially increasing the efficacy of anticancer therapies. This dual action highlights the context-dependent therapeutic potential of flavonoids, which enables them to safeguard normal tissues while rendering cancer cells susceptible to treatment [ 19 , 25 ]. Research conducted on various cancer cell lines, such as HepG2, HCT116, and OVCAR-3, has shown that different flavonoids can induce oxidative stress in cancer cells by increasing ROS levels. Studies have indicated that daidzein, an isoflavone derived from plants [ 35 ], along with flavanone hesperidin [ 36 ], both contribute to increased ROS production in MCF-7 cells. Similarly, naringin, a glycoside flavonoid, has been shown to have a comparable effect on PC-3 cells. Elevated ROS levels inhibit cell proliferation and migration, and promote apoptosis in cancer cells. In our study, reynoutrin increased OSI in a dose-dependent manner by increasing TOS and decreasing TAS levels in MCF-7 and PC-3 cells. Notably, the effect of high-dose reynoutrin (616 µg/mL) on oxidative stress in PC-3 cells was similar to that of cisplatin (50 µM). These findings suggest that reynoutrin exhibits a stronger pro-oxidant activity in PC-3 cells than in MCF-7 cells. When the inflammatory response is not properly regulated, it leads to an increase in the concentration of inflammatory mediators, which can contribute to the development of various chronic diseases including cancer. Inflammation is a hallmark of cancer and plays a critical role in the progression of many types of cancers. Elevated levels of cytokines, such as TNF-α, IL-1β, and IL-6, have been associated with cancer; therefore, cytokine inhibition may represent an important therapeutic target for cancer [ 20 , 22 ]. Experimental studies have linked TNF-α, IL- 1β, and IL-6 levels with cancer progression. Several studies have demonstrated that various flavonoids, including pelargonidin [ 37 ], apigenin [ 38 ], quercetin, rutin [ 39 ], and luteolin [ 40 ], suppress the expression of cytokines, such as TNF-α, IL-1β, IL-6, and inhibit tumor growth. The flavanone hesperetin has been reported to inhibit the secretion of TNF-α, IL-6, and IL-1β in RAW 264.7 cells [ 41 ]. Flavon apigenin downregulated TNF-α-associated inflammatory signaling in the A375 human melanoma cell line [ 38 ]. Flavonoids, such as gossypin [ 42 , 43 ], chalcone derivatives [ 44 ], and curcumin [ 45 ], exhibit anti-inflammatory activity in PC-3 cells by suppressing TNF-α, IL-1β, and IL-6. Our findings support those of previous studies on the expression of proinflammatory cytokines. In the present study, reynoutrin demonstrated antiproliferative activity in both MCF-7 and PC-3 cells by reducing the levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in breast and prostate cancer cells. Apoptosis, a form of programmed cell death, plays a vital role in the elimination of irreversibly damaged cells from the body. Consequently, it is critical in the context of cancer, and the regulation of pro- and anti-apoptotic proteins has been extensively studied. Among the principal regulators of apoptosis, caspase-3, caspase-9, and Bax act as pro-apoptotic factors, while Bcl-2 functions as an anti-apoptotic factor. Flavonoids, as modulators of the apoptotic pathway, have been shown to promote proapoptotic mechanisms both in vitro and in vivo . These compounds induce apoptosis in cancer cells by downregulating antiapoptotic proteins and upregulating pro-apoptotic proteins [ 14 ]. Numerous studies have demonstrated that genistein in colon cancer cells (HT29) [ 46 ], luteolin in breast cancer cells (MDA-MB-231) [ 47 ], naringenin in hepatocellular carcinoma (HepG2) [ 48 ], and vitexin [ 49 ] in kidney cancer cells upregulate caspase-3., a key pro-apoptotic protein. Similarly, increased Bax levels were reported in cells treated with quercetin, taxifolin, luteolin [ 47 ], daidzein [ 50 ], vitexin [ 49 ], apigenin [ 51 ], myricetin [ 52 ], naringenin [ 48 ], and genistein [ 53 ]. In addition, studies have reported that treatment with epigallocatechin [ 54 ], myricetin [ 52 ], apigenin [ 51 ], genistein [ 53 ], and daidzein [ 55 ] results in significant downregulation of Bcl-2 in prostate cancer, hepatocellular carcinoma, colon cancer, human leukemia, and renal carcinoma cells compared to controls [ 14 ]. In our study, RT-PCR analysis revealed significant upregulation of Bax, caspase-3, and caspase-9 gene expression in both MCF-7 and PC-3 cells, which is consistent with the findings of previous studies. Moreover, medium and high concentrations of reynoutrin reduced the Bcl-2/Bax ratio in both cell lines, which is consistent with earlier reports. These results suggest that reynoutrin inhibits anti-apoptotic mechanisms, thereby promoting pro-apoptotic signaling during apoptosis. Apoptosis was assessed using Hoechst staining. Reynoutrin significantly increased nuclear fragmentation in MCF-7 and PC-3 cells in a dose-dependent manner. These findings indicate that reynoutrin induced apoptosis, particularly at higher concentrations. Conclusion In summary, our results demonstrated that reynoutrin induces cytotoxic effects by inducing apoptosis and oxidative stress in breast and prostate cancer cells in vitro and demonstrated its anti-inflammatory effects. However, further research, especially in vivo experiments, are needed to confirm the detailed mechanisms involved. Our report strongly supports the use of reynoutrin as a potential medicinal agent in the development of novel antiproliferative agents. Declarations Ethical approval was not required because it was a cell culture study. Consent for publication All authors agreed to the publication of this paper. Competing interests The authors declare no competing interests Funding This research was supported by the Kastamonu University Scientific Research Projects Coordination Unit (KU BAP) (KÜBAP-01/2023-25). Author Contribution Sedat Gokmen and Irfan Cinar Investigation, Conceptualization, Methodology, Writing- Original Draft, Data Curation, Software, and Validation. All authors have read and approved the final version of the manuscript. Acknowledgement This research was supported by the Kastamonu University Scientific Research Projects Coordination Unit (KU BAP) (KÜBAP-01/2023-25). Data Availability The data supporting the findings of this study are available from the corresponding author upon reasonable request. References International Agency for Research on Cancer. (2022) Estimated age-standardized incidence and mortality rates (World) in 2020, worldwide, both sexes, all ages. IARC. https://gco.iarc.fr/ . Accessed 2 April 2023. Valentovic MA. Evaluation of Resveratrol in Cancer Patients and Experimental Models. In: Tew KD, Fisher PB, editors. Advances in Cancer Research. Volume 137. London: Academic; 2018. pp. 171–88. Amjad E, Asnaashari S, Sokouti B, Dastmalchi S. (2020) Systems biology comprehensive analysis on breast cancer for identification of key gene modules and genes associated with TNM-based clinical stages. Sci Rep. 10(1):10816. https://doi.org/10.1038/s41598-020-67643-w . PMID: 32616754. Bukowski K, Kciuk M, Kontek R. Mechanisms of Multidrug Resistance in Cancer Chemotherapy. Int J Mol Sci. 2020;21(9):3233. https://doi.org/10.3390/ijms21093233 . Moradi Z, Mohammadian M, Saberi H, Ebrahimifar M, Mohammadi Z, Ebrahimpour M, Behrouzkia Z. Anti-cancer effects of chemotherapeutic agent; 17-AAG, in combined with gold nanoparticles and irradiation in human colorectal cancer cells. Daru. 2019;27(1):111–9. https://doi.org/10.1007/s40199-019-00251-w . Nikolaou M, Pavlopoulou A, Georgakilas AG, Kyrodimos E. The challenge of drug resistance in cancer treatment: a current overview. Clin Exp Metastasis. 2018;35(4):309–18. https://doi.org/10.1007/s10585-018-9903-0 . Schirrmacher V. From chemotherapy to biological therapy: A review of novel concepts to reduce the side effects of systemic cancer treatment (Review). Int J Oncol. 2019;54(2):407–19. https://doi.org/10.3892/ijo.2018.4661 . Chen T, Xiao Z, Liu X, Wang T, Wang Y, Ye F, Su J, Yao X, Xiong L, Yang DH. Natural products for combating multidrug resistance in cancer. Pharmacol Res. 2024;202:107099. https://doi.org/10.1016/j.phrs.2024.107099 . Zhang QY, Wang FX, Jia KK, Kong LD. Natural Product Interventions for Chemotherapy and Radiotherapy-Induced Side Effects. Front Pharmacol. 2018;9:1253. https://doi.org/10.3389/fphar.2018.01253 . Poormolaie N, Mohammadi M, Mir A, Asadi M, Kararoudi AN, Vahedian V, Rashidi M, Maroufi NF. Xanthomicrol: Effective therapy for cancer treatment. Toxicol Rep. 2023;10:436–40. https://doi.org/10.1016/j.toxrep.2023.02.008 . Clere N, Faure S, Martinez MC, Andriantsitohaina R. Anticancer properties of flavonoids: roles in various stages of carcinogenesis. Cardiovasc Hematol Agents Med Chem. 2011;9(2):62–77. https://doi.org/10.2174/187152511796196498 . González-Gallego J, Sánchez-Campos S, Tuñón MJ. Anti-inflammatory properties of dietary flavonoids. Nutr Hosp. 2007;22(3):287–93. Li H, Zeng Y, Zi J, Hu Y, Ma G, Wang X, Shan S, Cheng G, Xiong J. Dietary Flavonoids Consumption and Health: An Umbrella Review. Mol Nutr Food Res. 2024;68(12):e2300727. https://doi.org/10.1002/mnfr.202300727 . Ponte LGS, Pavan ICB, Mancini MCS, da Silva LGS, Morelli AP, Severino MB, Bezerra RMN, Simabuco FM. The Hallmarks of Flavonoids in Cancer. Molecules. 2021;26(7):2029. https://doi.org/10.3390/molecules26072029 . Yüksel TN, Bozgeyik E, Yayla M. The Effect of Quercetin and Quercetin-3-d-xyloside on Breast Cancer Proliferation and Migration. JBACHS. 2022;6(2):569–78. 10.30621/jbachs.1056769 . Ho R, Violette A, Cressend D, Raharivelomanana P, Carrupt PA, Hostettmann K. Antioxidant potential and radical-scavenging effects of flavonoids from the leaves of Psidium cattleianum grown in French Polynesia. Nat Prod Res. 2012;26(3):274–7. https://doi.org/10.1080/14786419.2011.585610 . Eldin Elhawary SS, Elmotyam AKE, Alsayed DK, Zahran EM, Fouad MA, Sleem AA, Elimam H, Rashed MH, Hayallah AM, Mohammed AF, Abdelmohsen UR. (2020) Cytotoxic and anti-diabetic potential, metabolic profiling and insilico studies of Syzygium cumini (L.) Skeels belonging to family Myrtaceae. Nat Prod Res. 2022 36(4):1026–1030. https://doi.org/10.1080/14786419.2020.1843032 Yang W, Tu H, Tang K, Huang H, Ou S, Wu J. Reynoutrin Improves Ischemic Heart Failure in Rats Via Targeting S100A1. Front Pharmacol. 2021;12:703962. https://doi.org/10.3389/fphar.2021.703962 . Kopustinskiene DM, Jakstas V, Savickas A, Bernatoniene J. Flavonoids as Anticancer Agents. Nutrients. 2020;12(2):457. https://doi.org/10.3390/nu12020457 . Lim W, Park S, Bazer FW, Song G. Naringenin-Induced Apoptotic Cell Death in Prostate Cancer Cells Is Mediated via the PI3K/AKT and MAPK Signaling Pathways. J Cell Biochem. 2017;118(5):1118–31. https://doi.org/10.1002/jcb.25729 . Gupta SC, Kunnumakkara AB, Aggarwal S, Aggarwal BB. Inflammation, a Double-Edge Sword for Cancer and Other Age-Related Diseases. Front Immunol. 2018;9:2160. https://doi.org/10.3389/fimmu.2018.02160 . Chukiatsiri S, Siriwong S, Thumanu K. Pupae protein extracts exert anticancer effects by downregulating the expression of IL-6, IL-1β and TNF-α through biomolecular changes in human breast cancer cells. Biomed Pharmacother. 2020;128:110278. https://doi.org/10.1016/j.biopha.2020.110278 . Golovko O, Nazarova N, Tuohimaa P. Vitamin D-induced up-regulation of tumour necrosis factor alpha (TNF-alpha) in prostate cancer cells. Life Sci. 2005;77(5):562–77. https://doi.org/10.1016/j.lfs.2004.10.072 . Adekoya TO, Richardson RM. Cytokines and Chemokines as Mediators of Prostate Cancer Metastasis. Int J Mol Sci. 2020;21(12):4449. https://doi.org/10.3390/ijms21124449 . Aslan A, Seçme M. Effects of Pelargonium Sidoides Extract on Apoptosis and Oxidative Stress in Human Neuroblastoma Cells. Med (Kaunas). 2024;60(12):2110. https://doi.org/10.3390/medicina60122110 . O'Brien MA, Kirby R. Apoptosis: A review of pro-apoptotic and anti‐apoptotic pathways and dysregulation in disease. J Vet Emerg Crit Care. 2008;18(6):572–85. https://doi.org/10.1111/j.1476-4431.2008.00363.x . Jakimiuk K, Szoka Ł, Surażyński A, Tomczyk M. Using Flavonoid Substitution Status to Predict Anticancer Effects in Human Melanoma Cancers: An In Vitro Study. Cancers (Basel). 2024;16(3):487. https://doi.org/10.3390/cancers16030487 . Sahoo G, Samal D, Khandayataray P, Murthy MK. A Review on Caspases: Key Regulators of Biological Activities and Apoptosis. Mol Neurobiol. 2023;60(10):5805–37. https://doi.org/10.1007/s12035-023-03433-5 . Chimento A, De Luca A, D'Amico M, De Amicis F, Pezzi V. The Involvement of Natural Polyphenols in Molecular Mechanisms Inducing Apoptosis in Tumor Cells: A Promising Adjuvant in Cancer Therapy. Int J Mol Sci. 2023;24(2):1680. https://doi.org/10.3390/ijms24021680 . Çınar İ, Gıdık B, Dirican E. Determination of anti-cancer effects of Nigella sativa seed oil on MCF7 breast and AGS gastric cancer cells. Mol Biol Rep. 2024;51(1):491. https://doi.org/10.1007/s11033-024-09453-1 . Tülüce Y, Lak PTA, Koyuncu İ, Kılıç A, Durgun M, Özkol H. The apoptotic, cytotoxic and genotoxic effect of novel binuclear boron-fluoride complex on endometrial cancer. Biometals. 2017;30(6):933–44. https://doi.org/10.1007/s10534-017-0060-8 . Marathe K, Naik J, Maheshwari V. Synthesis, characterisation and in vitro anticancer activity of conjugated protease inhibitor-silver nanoparticles (AgNPs-PI) against human breast MCF-7 and prostate PC-3 cancer cell lines. Bioprocess Biosyst Eng. 2024;47(6):931–42. https://doi.org/10.1007/s00449-024-03023-2 . Wang X, Gong M, Zhu Z, Zhang B, Han L, Li W, Wu Z, Ma Q, Wang Z, Qian W. Rutin protects the pancreas from inflammatory injury and oncogene-driven tumorigenesis by inhibiting acinar to ductal metaplasia. Eur J Pharmacol. 2025;998:177536. https://doi.org/10.1016/j.ejphar.2025.177536 . de Luna FCF, Ferreira WAS, Casseb SMM, de Oliveira EHC. Anticancer Potential of Flavonoids: An Overview with an Emphasis on Tangeretin. Pharmaceuticals (Basel). 2023;16(9):1229. https://doi.org/10.3390/ph16091229 . Jin S, Zhang QY, Kang XM, Wang JX, Zhao WH. Daidzein induces MCF-7 breast cancer cell apoptosis via the mitochondrial pathway. Ann Oncol. 2010;21(2):263–8. https://doi.org/10.1093/annonc/mdp499 . Palit S, Kar S, Sharma G, Das PK. Hesperetin Induces Apoptosis in Breast Carcinoma by Triggering Accumulation of ROS and Activation of ASK1/JNK Pathway. J Cell Physiol. 2015;230(8):1729–39. https://doi.org/10.1002/jcp.24818 . Lee WJ, Chen WK, Wang CJ, Lin WL, Tseng TH. Apigenin inhibits HGF-promoted invasive growth and metastasis involving blocking PI3K/Akt pathway and beta 4 integrin function in MDA-MB-231 breast cancer cells. Toxicol Appl Pharmacol. 2008;226(2):178–91. https://doi.org/10.1016/j.taap.2007.09.013 . Ghițu A, Schwiebs A, Radeke HH, Avram S, Zupko I, Bor A, Pavel IZ, Dehelean CA, Oprean C, Bojin F, Farcas C, Soica C, Duicu O, Danciu C. A Comprehensive Assessment of Apigenin as an Antiproliferative, Proapoptotic, Antiangiogenic and Immunomodulatory Phytocompound. Nutrients. 2019;11(4):858. https://doi.org/10.3390/nu11040858 . do Nascimento RP, Dos Santos BL, Amparo JAO, Soares JRP, da Silva KC, Santana MR, Almeida ÁMAN, da Silva VDA, Costa MFD, Ulrich H, Moura-Neto V, Lopes GPF, Costa SL. Neuroimmunomodulatory Properties of Flavonoids and Derivates: A Potential Action as Adjuvants for the Treatment of Glioblastoma. Pharmaceutics. 2022;14(1):116. https://doi.org/10.3390/pharmaceutics14010116 . Yao Q, Luo Y, Sun L, Wang H, Li W. Luteolin suppressed growth of colon tumor via inflammation, oxidative stress, and NLRP3/IL-1β signal axis. Pharmacognosy Magazine. 2022;18(78):494–501. 10.4103/pm.pm_284_21 . Ren H, Hao J, Liu T, Zhang D, Lv H, Song E, Zhu C. Hesperetin Suppresses Inflammatory Responses in Lipopolysaccharide-Induced RAW 264.7 Cells via the Inhibition of NF-κB and Activation of Nrf2/HO-1 Pathways. Inflammation. 2016;39(3):964–73. https://doi.org/10.1007/s10753-016-0311-9 . Cinar I. Apoptosis-Inducing Activity and Antiproliferative Effect of Gossypin on PC-3 Prostate Cancer Cells. Anticancer Agents Med Chem. 2021;21(4):445–50. https://doi.org/10.2174/1871520620666200721103422 . Song B, Shen X, Tong C, Zhang S, Chen Q, Li Y, Li S. Gossypin: A flavonoid with diverse pharmacological effects. Chem Biol Drug Des. 2023;101(1):131–7. https://doi.org/10.1111/cbdd.14152 . Horta B, Freitas-Silva J, Silva J, Dias F, Teixeira AL, Medeiros R, Cidade H, Pinto M, Cerqueira F. Antitumor Effect of Chalcone Derivatives against Human Prostate (LNCaP and PC-3), Cervix HPV-Positive (HeLa) and Lymphocyte (Jurkat) Cell Lines and Their Effect on Macrophage Functions. Molecules. 2023;28(5):2159. https://doi.org/10.3390/molecules28052159 . Pellegrino M, Bevacqua E, Frattaruolo L, Cappello AR, Aquaro S, Tucci P. Enhancing the Anticancer and Anti-Inflammatory Properties of Curcumin in Combination with Quercetin, for the Prevention and Treatment of Prostate Cancer. Biomedicines. 2023;11(7):2023. https://doi.org/10.3390/biomedicines11072023 . Shafiee G, Saidijam M, Tavilani H, Ghasemkhani N, Khodadadi I. Genistein Induces Apoptosis and Inhibits Proliferation of HT29 Colon Cancer Cells. Int J Mol Cell Med. 2016;5(3):178–91. Huang L, Jin K, Lan H. Luteolin inhibits cell cycle progression and induces apoptosis of breast cancer cells through downregulation of human telomerase reverse transcriptase. Oncol Lett. 2019;17(4):3842–50. https://doi.org/10.3892/ol.2019.10052 . Arul D, Subramanian P. Naringenin (citrus flavonone) induces growth inhibition, cell cycle arrest and apoptosis in human hepatocellular carcinoma cells. Pathol Oncol Res. 2013;19(4):763–70. https://doi.org/10.1007/s12253-013-9641-1 . Li Y, Sun Q, Li H, Yang B, Wang M. Vitexin suppresses renal cell carcinoma by regulating mTOR pathways. Transl Androl Urol. 2020;9(4):1700–11. https://doi.org/10.21037/tau-20-1094 . Zheng W, Liu T, Sun R, Yang L, An R, Xue Y. Daidzein induces choriocarcinoma cell apoptosis in a dose-dependent manner via the mitochondrial apoptotic pathway. Mol Med Rep. 2018;17(4):6093–9. https://doi.org/10.3892/mmr.2018.8604 . Shukla S, Fu P, Gupta S. Apigenin induces apoptosis by targeting inhibitor of apoptosis proteins and Ku70-Bax interaction in prostate cancer. Apoptosis. 2014;19(5):883–94. https://doi.org/10.1007/s10495-014-0971-6 . Kim ME, Ha TK, Yoon JH, Lee JS. Myricetin induces cell death of human colon cancer cells via BAX/BCL2-dependent pathway. Anticancer Res. 2014;34(2):701–6. Hsiao YC, Peng SF, Lai KC, Liao CL, Huang YP, Lin CC, Lin ML, Liu KC, Tsai CC, Ma YS, Chung JG. Genistein induces apoptosis in vitro and has antitumor activity against human leukemia HL-60 cancer cell xenograft growth in vivo. Environ Toxicol. 2019;34(4):443–56. https://doi.org/10.1002/tox.22698 . Wei R, Zhu G, Jia N, Yang W. Epigallocatechin-3-gallate Sensitizes Human 786-O Renal Cell Carcinoma Cells to TRAIL-Induced Apoptosis. Cell Biochem Biophys. 2015;72(1):157–64. https://doi.org/10.1007/s12013-014-0428-0 . Han BJ, Li W, Jiang GB, Lai SH, Zhang C, Zeng CC, Liu YJ. Effects of daidzein in regards to cytotoxicity in vitro, apoptosis, reactive oxygen species level, cell cycle arrest and the expression of caspase and Bcl-2 family proteins. Oncol Rep. 2015;34(3):1115–20. https://doi.org/10.3892/or.2015.4133 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8431324","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":602631876,"identity":"aae09f8a-a033-42a9-b9e3-b7f6de9b2dda","order_by":0,"name":"Sedat GÖKMEN","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/UlEQVRIiWNgGAWjYHACZhAhJ4EsJIFdJaoWY6AqxgYkLQYEtSTOIFqLfP/iw0Y3Ku6kz2w//vzBzxybPIMDzAdv8zD8ycelxeDGs+TknDPPcmfz5Bg29m5LKzY4wJZszcNgYNmAS4vEGePDuW2Hc+cx5DA28G47nLjhAI+ZNFALTpfJzwBp+Xc4XY7/+cPGv2At/N/wamE432OcnNtwOEFaIsGwGWoLG14tBjfYko1zjh02nDnjjeFs2W1piTMPsxlbzjEwxu2w/sOHpXNqDstLnE9/8PHtNpvEvuPND2+8qZDD7TCJBHQRcDzhi0n+A3gkR8EoGAWjYBSAAADOaVlZXX4KzwAAAABJRU5ErkJggg==","orcid":"","institution":"Kastamonu University","correspondingAuthor":true,"prefix":"","firstName":"Sedat","middleName":"","lastName":"GÖKMEN","suffix":""},{"id":602631877,"identity":"22285e91-57cd-4cff-8a52-737ca757bdea","order_by":1,"name":"İrfan ÇINAR","email":"","orcid":"","institution":"Kastamonu University","correspondingAuthor":false,"prefix":"","firstName":"İrfan","middleName":"","lastName":"ÇINAR","suffix":""}],"badges":[],"createdAt":"2025-12-23 07:38:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8431324/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8431324/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104405507,"identity":"d7e7664c-da4e-4f5f-95cb-5140dc77fa06","added_by":"auto","created_at":"2026-03-11 12:23:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":12734,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e.\u003c/strong\u003e (a) Total oxidant status (TOS), (b) total antioxidant status (TAS), and (c) oxidative stress index (OSI) levels among different treatment groups following reynoutrin or cisplatin administration in MCF-7 cells. \u003csup\u003ea–d\u003c/sup\u003e Values not sharing a common superscript differ significantly between groups at p\u0026lt;0.05 according to Duncan’s Multiple Range Test (DMRT). C: control, Cis: cisplatin, REY: reynoutrin, DMSO: dimethyl sulfoxide.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8431324/v1/ba9ed6e3bd47b2cc0bb59254.png"},{"id":104350185,"identity":"f8e6b4d6-1727-40e2-ba56-d0b636c07640","added_by":"auto","created_at":"2026-03-10 19:13:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":12102,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Total oxidant status (TOS), (b) total antioxidant status (TAS), and (c) oxidative stress index (OSI) levels among different treatment groups following reynoutrin or cisplatin administration in PC-3 cells. \u003csup\u003ea–d\u003c/sup\u003e Values not sharing a common superscript differ significantly between groups at p\u0026lt;0.05 according to Duncan’s Multiple Range Test (DMRT). C: control, Cis: cisplatin, REY: reynoutrin, DMSO: dimethyl sulfoxide.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8431324/v1/04c93858dd896e8f4d708ee9.png"},{"id":104406176,"identity":"ac51970f-c9f3-45b7-90cc-941835656d52","added_by":"auto","created_at":"2026-03-11 12:24:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":8317,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of reynoutrin on inflammation parameters in MCF-7 cells. \u003csup\u003ea–e\u003c/sup\u003e Values not sharing a common superscript differ significantly between groups at p\u0026lt;0.05 according to Duncan’s Multiple Range Test (DMRT). C: control, Cis: cisplatin, REY: reynoutrin, DMSO: dimethyl sulfoxide.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8431324/v1/1b6ba09f16f57a934a4ffbc3.png"},{"id":104350191,"identity":"5306d87f-e805-415b-9c53-f572b740603d","added_by":"auto","created_at":"2026-03-10 19:13:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":9099,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of reynoutrin on apoptosis parameters in MCF-7 cells. \u003csup\u003ea–e\u003c/sup\u003e Values not sharing a common superscript differ significantly between groups at p\u0026lt;0.05 according to Duncan’s Multiple Range Test (DMRT). C: control, Cis: cisplatin, REY: reynoutrin, DMSO: dimethyl sulfoxide.\u003c/p\u003e\n\u003cp\u003eThe PCR results showed that reynoutrin exhibited anti-inflammatory activity and induced apoptotic cell death in MCF-7 cells.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8431324/v1/164d051d291a18de3da56053.png"},{"id":104350189,"identity":"4da2808f-dbc2-4dd8-b216-5b0d969a482b","added_by":"auto","created_at":"2026-03-10 19:13:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":45864,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of reynoutrin on inflammation parameters in PC-3 cells. \u003csup\u003ea–e\u003c/sup\u003e Values not sharing a common superscript differ significantly between groups at p\u0026lt;0.05 according to Duncan’s Multiple Range Test (DMRT). C: control, Cis: cisplatin, REY: reynoutrin, DMSO: dimethyl sulfoxide.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8431324/v1/4ae97a301de3b57ee2a1a0bb.png"},{"id":104350187,"identity":"0cf5d060-7c4d-4ea9-8dcf-cae60f70f0e4","added_by":"auto","created_at":"2026-03-10 19:13:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":71984,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of reynoutrin on apoptosis in PC-3 cells. \u003csup\u003ea–e\u003c/sup\u003e Values not sharing a common superscript differ significantly between groups at p\u0026lt;0.05 according to Duncan’s Multiple Range Test (DMRT). C: control, Cis: cisplatin, REY: reynoutrin, DMSO: dimethyl sulfoxide.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8431324/v1/fce5545e0b88bac6a1740e66.png"},{"id":104350190,"identity":"c55b852a-1bd7-4602-bc88-230c892210e0","added_by":"auto","created_at":"2026-03-10 19:13:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":482756,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescent microscope findings after Hoechst 33342 staining in MCF-7 and PC-3 cells treated with reynoutrin (40× magnification). Nuclear morphological changes in reynoutrin-treated MCF-7 and PC-3 cells. MCF-7 breast cancer and PC-3 prostate cancer cells were treated with different concentrations of the compound for 24 h, stained with HO33342, and visualized under a fluorescence microscope. Untreated (control) cells showed healthy blue nuclei, whereas treated cells exhibited apoptotic features including nuclear chromatin condensation, shrinkage, and fragmentation. Early apoptotic events were identified by the bright blue fluorescence of the condensed chromatin. Cis: cisplatin, REY: reynoutrin, DMSO: dimethyl sulfoxide.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8431324/v1/ca0bd75b4f3b5e24ee018381.png"},{"id":105405373,"identity":"b78d7d53-4fea-4287-8b0a-25ce9e174c7a","added_by":"auto","created_at":"2026-03-25 16:11:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1520061,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8431324/v1/a4883518-013b-41d1-a6a3-b6388388cf91.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antiproliferative Effects of Reynoutrin via Modulation of Oxidative Stress, Inflammation, and Apoptosis in Breast (MCF-7) and Prostate Cancer (PC-3) Cells Lines","fulltext":[{"header":"Highlights","content":"\u003cp\u003eReynoutrin inhibited the viability of MCF-7 and PC-3 cells in a dose-dependent manner.\u003c/p\u003e\u003cp\u003eReynoutrin increased oxidative stress by elevating TOS and decreasing TAS levels.\u003c/p\u003e\u003cp\u003eReynoutrin suppressed pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 expression.\u003c/p\u003e\u003cp\u003eReynoutrin induced apoptosis by upregulating caspase-3, caspase-9, and Bax while downregulating Bcl-2.\u003c/p\u003e\u003cp\u003eReynoutrin shows promise as a potential therapeutic agent against breast and prostate cancers.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eCancer is the second leading cause of death worldwide, represents a major global public health challenge, and is one of the leading contributor to mortality and morbidity. Reports indicate that 19,976,499 new cancer cases and 9,743,832 cancer-related deaths occurred worldwide in 2022. Breast (11.5%) and prostate (7.3%) cancers are among the top five types of cancers with the highest incidence rates worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAntineoplastic agents are fundamental in cancer therapy, often administered alongside radiotherapy, surgery, and immunotherapy [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, their use is frequently limited by significant adverse effects and drug resistance in tumor cells. These drugs can cause mild to life-threatening effects, including gastrointestinal toxicity, hepatotoxicity, nephrotoxicity, hematopoietic system damage, cardiotoxicity, and neurotoxicity [\u003cspan additionalcitationids=\"CR4 CR5 CR6\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Cancer cells frequently acquire resistance to chemotherapeutic agents following treatment. These problems reduce patients' quality of life and drug efficacy, restrict clinical use, and can lead to treatment failure [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, more effective strategies for managing chemotherapy-related side effects are required. Alternative approaches have been investigated to reduce side effects, ensure effective doses, and reduce health care costs. Phytochemicals have been extensively studied for their synergistic effects on chemotherapy regimens and cancer prevention [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Currently, flavonoids are extensively used in both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e studies to explore their pharmacological properties, particularly for cancer treatment. Flavonoids are used to treat a range of diseases including cancer, diabetes, cardiovascular diseases and ulcers. Flavonoids exert cytotoxic effects on cancer cells through multiple mechanisms, including anti-inflammatory activity, induction of apoptosis, and disruption of oxidative stress balance. Among the studies conducted on the anticancer activity of these compounds, hesperidin, quercetin, cyanidin, catechins, apigenin, and quercetin-3-\u003cem\u003eO\u003c/em\u003e-glucoside are of particular interest [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Studies that have reached the final phase have demonstrated promising results, indicating that these compounds may serve as effective adjuvant agents in the treatment or reduction of the risk of various types of cancer [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eReynoutrin, also known as quercetin-3-D-xyloside, is a flavonoid naturally found in the fruits and leaves of various plants [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Although studies of reynoutrin are limited, existing research suggests that it exhibits a wide range of pharmacological activities. These include antitumor, antiviral, and antidiabetic effects [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Additionally, reynoutrin has been associated with wound healing properties and has potential benefits in improving ischemic heart failure [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOxidative stress, an imbalance between reactive oxygen species (ROS) production and antioxidant defense systems, plays a significant role in carcinogenesis, tumor progression, and chemoresistance. Flavonoids have a dual role in ROS homeostasis; they act as antioxidants under physiological conditions but as pro-oxidants under pathological conditions, such as cancer, initiating apoptotic pathways. Pro-oxidant activity is a key mechanism underlying the anticancer potential of flavonoids [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Flavonoids, including daidzein, hesperidin, and naringenin, exert pro-oxidant effects in cancer cell lines such as MCF-7 and PC-3 by elevating intracellular ROS levels and enhancing oxidative stress. This increase in ROS disrupts cellular redox homeostasis, leading to the suppression of cancer cell proliferation and migration, and the induction of cell death [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInflammation causes tumor development and regulates cellular differentiation, survival, proliferation, invasion, metastasis, and angiogenesis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Inflammation facilitates tumor initiation and progression by promoting proliferative signaling, angiogenesis, and resistance to apoptosis. Proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) contribute to tumorigenesis. Flavonoids (such as apigenin, rutin, and pelargonidin) suppress inflammation by downregulating pro-inflammatory cytokines, such as IL-6, TNF-α, and IL-1β, thereby inhibiting tumor development. IL-6, IL-1β, and TNF-α regulate the functions of various cell types, including breast [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and prostate [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] cancers, particularly tumor growth, invasion, and metastasis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eApoptosis is a type of programmed cell death that maintains homeostasis and eliminates the damaged cells. The Bcl-2-associated X protein (Bax) and B-cell lymphoma 2 (Bcl-2) are critical regulators of apoptosis and cell survival, respectively. Bax promotes cell death, whereas Bcl-2 promotes cell survival. A decreased Bcl-2/Bax ratio indicates increased susceptibility to apoptosis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The intrinsic apoptotic pathway is regulated by pro-apoptotic proteins such as Bax and anti-apoptotic proteins such as Bcl-2, which control mitochondrial membrane permeability and cytochrome \u003cem\u003ec\u003c/em\u003e release. This activates caspase-9 and caspase-3, leading to DNA fragmentation and cell death [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Research has shown that caspase-3 and caspase-9 are essential mediators of flavonoid-induced apoptosis in cancer cells [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Caspase-9 initiates the apoptotic cascade, whereas caspase-3 cleaves cellular substrates during apoptosis [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Studies have shown that flavonoids induce apoptosis by upregulating pro-apoptotic markers and downregulating Bcl-2 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently, studies on reynoutrin are limited, and its antiproliferative activities, particularly in breast and prostate cancers, have not been clarified. In this study, we investigated the antiproliferative potential of reynoutrin by evaluating its effects on oxidative stress parameters (total antioxidant and oxidant status), proinflammatory cytokines (TNF-α, IL-1β, and IL-6), and key apoptotic markers (caspase-3, caspase-9, and Bcl-2/Bax) in MCF-7 and PC-3 cells. These findings provide valuable insights into the molecular mechanisms underlying the anticancer effects of reynoutrin, and support its potential as a complementary cancer therapy.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and reagents\u003c/h2\u003e \u003cp\u003eReynoutrin (Cas number: 549-32-6; purity\u0026thinsp;\u0026gt;\u0026thinsp;97%) and cisplatin (Cas number: 15663-27-1; purity\u0026thinsp;\u0026gt;\u0026thinsp;99.84%) were purchased from MedChemExpress (Manmouth Junctions, NJ, USA). Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM), cell culture medium, and reagents such as fetal bovine serum (FBS), penicillin/streptomycin, and trypsin\u0026ndash;ethylenediaminetetraacetic acid (trypsin/EDTA) were obtained from Gibco (Invitrogen Inc., Grand Island, New York, USA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell culture procedures\u003c/h3\u003e\n\u003cp\u003eHuman breast (MCF-7) and prostate (PC-3) cancer cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cell lines stored in the liquid nitrogen tank were briefly thawed in a 37\u0026deg;C water bath and subsequently transferred into T75 cm\u0026sup2; flasks for culture. The cells were cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin (100 IU/mL) at 37\u0026deg;C in a humidified 5% CO₂ incubator. All incubations conducted on the cells throughout the study were performed in a CO₂ incubator (PHCbi cell culture incubator, Loughborough, UK) at 37\u0026deg;C in a medium containing 5% CO₂. Upon reaching approximately 80% confluence, cells were detached with 0.25% trypsin/EDTA and subsequently seeded into 6-well plates for RNA extraction and biochemical analyses and into 96-well plates for MTT assays. After the cells reached approximately 80% confluence, they were detached with 0.25% trypsin/EDTA and plated for RNA extraction and biochemical investigations in 6-well plates, and for the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyltetrazolium bromide] assay in 96-well plates. All cell culture procedures were performed in compliance with standards described in the literature.\u003c/p\u003e\n\u003ch3\u003eCell viability assay\u003c/h3\u003e\n\u003cp\u003eThe MTT (Thermo Fisher Scientific, Waltham, MA, USA) assay was performed to determine cell viability according to the manufacturer\u0026rsquo;s instructions. MCF-7 and PC-3 cells were cultured in 96-well plates (5 \u0026times; 10\u0026sup3; cells/well) and were allowed to attach. The cells were then exposed to different concentrations (10\u0026ndash;2000 \u0026micro;g/mL) of reynoutrin, dissolved in dimethyl sulfoxide (DMSO) as the solvent. The final DMSO concentration was maintained at 0.1% in all treatment and control groups, and incubated for 24 h to determine the IC₅₀ (half-maximal inhibitory concentration). These concentrations were investigated in previous studies. Following the completion of exposure, 20 \u0026micro;L of MTT solution (5 mg/mL in phosphate-buffered saline) was added to each well, and the plate was incubated at 37\u0026deg;C for 4 h. Dark crystals of formazan formed in intact cells were dissolved in 200 \u0026micro;L of DMSO. After shaking the plates for 15 min, the absorbance at 570 nm was measured using a microplate spectrophotometer (Epoch Microplate Spectrophotometer, BioTek, USA) in accordance with the manufacturer's protocol (Roche, Germany). The cell viability rate (%) was calculated using the following equation.\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{\\%}\\:\\text{C}\\text{e}\\text{l}\\text{l}\\:\\text{v}\\text{i}\\text{a}\\text{b}\\text{i}\\text{l}\\text{i}\\text{t}\\text{y}\\:\\text{r}\\text{a}\\text{t}\\text{e}\\:=\\frac{{OD}_{test\\:sample}\\:-\\:{OD}_{blank}}{{OD}_{control}\\:-\\:{OD}_{blank}}\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eOD\u003csub\u003etest sample\u003c/sub\u003e: Optical density (absorbance) measured in the wells containing the test sample.\u003c/p\u003e \u003cp\u003eOD\u003csub\u003eblank\u003c/sub\u003e: Absorbance of a blank well (medium without cells) subtracted from the background.\u003c/p\u003e \u003cp\u003eOD\u003csub\u003econtrol\u003c/sub\u003e: Absorbance of the untreated control well\u003c/p\u003e \u003cp\u003eThe IC\u003csub\u003e50\u003c/sub\u003e concentrations were calculated using Graphpad Probit Analysis Program (Version 9.3.0).\u003c/p\u003e\n\u003ch3\u003eExperimental groups\u003c/h3\u003e\n\u003cp\u003eTo investigate the antiproliferative effects of reynoutrin, six experimental groups were established for both the MCF-7 and PC-3 cell lines. The groups were designed as follows:\u003c/p\u003e \u003cp\u003eGroup I (Control; C): No chemical treatment was administered to MCF-7 or PC-3 cells.\u003c/p\u003e \u003cp\u003eGroup II (DMSO): MCF-7 and PC-3 cells treated with %0.1 DMSO for 24 h.\u003c/p\u003e \u003cp\u003eGroup III (Cis50): MCF-7 and PC-3 cells were exposed to 50 \u0026micro;M cisplatin for 24 h.\u003c/p\u003e \u003cp\u003eGroup IV (Rey Low Dose): Cells were treated for 24 h with a lower concentration of reynoutrin: 110 \u0026micro;g/mL for MCF-7 cells and 206 \u0026micro;g/mL for PC-3 cells.\u003c/p\u003e \u003cp\u003eGroup V (Rey Mid-level Dose): Cells were treated for 24 h with moderate concentrations of reynoutrin, 220 \u0026micro;g/mL for MCF-7 and 412 \u0026micro;g/mL for PC-3.\u003c/p\u003e \u003cp\u003eGroup VI (Rey High Dose): Cells were treated for 24 h with a higher concentration of reynoutrin, 330 \u0026micro;g/mL for MCF-7 and 618 \u0026micro;g/mL for PC-3.\u003c/p\u003e\n\u003ch3\u003eDetermination of intracellular antioxidant–oxidant status and oxidative stress index\u003c/h3\u003e\n\u003cp\u003eThe total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) are oxidative profiles that determine the intracellular oxidative. We measured TAS and TOS using Rel Assay Diagnostics Assay Kits (Rel Assay Diagnostics, Gaziantep, Turkey). The parameters were measured using a spectrophotometric method with an Epoch microplate reader following the manufacturer's instructions. The color change was measured at a wavelength of 660 nm, and the TAS results were expressed as mmol Trolox equivalents per mg of protein, whereas the TOS levels were determined based on the color change measured at 530 nm and expressed as \u0026micro;mol H₂O₂ equivalents per mg of protein. OSI was calculated using the following formula [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\text{O}\\text{S}\\text{I}\\:(\\text{A}\\text{r}\\text{b}\\text{i}\\text{t}\\text{r}\\text{a}\\text{r}\\text{y}\\:\\text{U}\\text{n}\\text{i}\\text{t},\\:\\text{A}\\text{U})=\\frac{\\text{T}\\text{O}\\text{S}\\:({\\mu\\:}\\text{m}\\text{o}\\text{l}\\:{H}_{2}{O}_{2}\\:\\text{E}\\text{q}\\text{u}\\text{i}\\text{v}./\\text{m}\\text{g}\\:\\text{p}\\text{r}\\text{o}\\text{t}\\text{e}\\text{i}\\text{n})}{\\text{T}\\text{A}\\text{S}\\:(\\text{m}\\text{m}\\text{o}\\text{l}\\:\\text{T}\\text{r}\\text{o}\\text{l}\\text{o}\\text{x}\\:\\text{E}\\text{q}\\text{u}\\text{i}\\text{v}./\\text{m}\\text{g}\\:\\text{p}\\text{r}\\text{o}\\text{t}\\text{e}\\text{i}\\text{n})}\\times\\:10$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eEach experiment was conducted in three independent replicates.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative determination of mRNA expression by RT-PCR\u003c/h2\u003e \u003cp\u003eThe mRNA expression levels of IL-1β, IL-6, TNF-α, caspase-3, caspase-9, Bax, and Bcl-2 were analyzed using Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The cells were collected from a 6-plate using a scraper and 350 \u0026micro;L of RLT solution was added separately to each group, followed by homogenization using a Tissue Lyser II device (Qiagen, Germany). RNA extraction was performed according to the manufacturer's instructions using an isolation kit (Qiagen RNA, Cat. No: 74,104, Qiagen, Hille, Germany). The RNA concentration was measured using a NanoDrop spectrophotometer (Epoch 2, BioTek, USA). The quality of RNA was determined using optical densities (OD260/280) per nanodrop, and RNAs with OD260/280 ratios between 1.7 and 2.2 (or ~\u0026thinsp;1.8) were used in the study. For cDNA synthesis, the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, USA) was used for cDNA synthesis. The process was performed on a Veriti 96-Well Thermal Cycler (Applied Biosystems), and the synthesized cDNA was preserved at -20\u0026deg;C. The mRNA expression of IL-1β (Hs01555410_m1), IL-6 (Hs00174131_m1), TNF-α (Hs00174128_m1), Caspase-3 (Hs00234387_m1), Caspase-9 (Hs00154260_m1), Bax (Hs00180269_m1), and Bcl-2 (Hs04986394_s1) was measured using the TaqMan Master Mix kit. RT-PCR amplification and quantification were conducted using a StepOnePlus device (Applied Biosystems). Gene Expression levels were normalized to the β-actin reference gene Hs01060665_g1 TaqMan\u0026reg; Gene Expression, which was modified to the PCR program according to the kit\u0026rsquo;s instructions, and the process was initiated. All results are represented as fold-changes in expression compared with the groups using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method. Each gene was analyzed using the approach described in our previous study [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Each experiment was conducted in triplicate.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHoechst 33342 staining\u003c/h3\u003e\n\u003cp\u003eAs characteristic changes in nuclear morphology typically occur during apoptosis, these changes can serve as indicators of programmed cell death. Apoptotic cell death was determined by Hoechst 33342 staining. Hoechst 33342 fluorescence staining was used to evaluate apoptotic cells according to their nuclear morphology. Hoechst 33342, a blue fluorescent membrane-permeable dye, stains condensed chromatin more brightly in apoptotic cells than in normal cells. For this purpose, MCF-7 and PC-3 cells (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well) were seeded onto 6-well plates prior to treatment with reynoutrin or cisplatin and incubated (5% CO\u003csub\u003e2\u003c/sub\u003e, 37\u0026deg;C) for 24 h. The following day, cells were treated with different concentrations of either reynoutrin or cisplatin and incubated for 24 h. After washing three times with 1XPBS, the cells were stained with 1 mL Hoechst 33342 (10 \u0026micro;g/mL) solution for 15 min in the dark at 37\u0026deg;C. Apoptotic cells (alterations in nuclear morphology) were detected using Hoechst 33342 staining under an inverted fluorescence microscope (Leica, DMIL LED, Germany).\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using the IBM SPSS Statistics software (version 25.0). Numerical data obtained from biochemical analyses and RT-PCR results were analyzed using one-way ANOVA post-hoc Duncan test. Results are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Additionally, IC₅₀ values were calculated using probit analysis. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eReynoutrin reduces MCF-7 and PC-3 cells proliferation\u003c/h2\u003e \u003cp\u003eThe viability of MCF-7 and PC-3 cells treated with different concentrations of reynoutrin (10-2000 \u0026micro;g/mL) was measured using the MTT assay, and the percentage of viable cells was monitored over 24 h. DMSO vehicle control had no effect on cell viability. These results revealed that reynoutrin reduced the viability of MCF-7 and PC-3 cells in a concentration-dependent manner. Probit analysis revealed that the concentrations required to achieve 50% inhibition (IC₅₀) were 220 \u0026micro;g/mL and 412 \u0026micro;g/mL for MCF-7 and PC-3 cells, respectively. Therefore, in the following experiments, reynoutrin was selected at concentrations corresponding to low, mid-level, and high values relative to its IC₅₀. Concentrations lower than 110 and 206 \u0026micro;g/mL did not cause a significant change in the proliferation of MCF-7 and PC-3 cells, respectively. To evaluate the effects of reynoutrin, MCF-7 cells were treated with concentrations of 110, 220, and 330 \u0026micro;g/mL, whereas PC-3 cells were treated with 206, 412, and 618 \u0026micro;g/mL reynoutrin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eReynoutrin induced oxidative stress in MCF-7 cells\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e illustrates the effects of reynoutrin on TAS, TOS, and OSI in MCF-7 cells. In MCF-7 cells, a significant increase in TOS levels and a reduction in TAS levels were observed in the cisplatin-treated group compared with those in the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similarly, reynoutrin induced a concentration-dependent increase in TOS levels and a significant decrease in TAS levels relative to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Moreover, reynoutrin markedly elevated OSI levels in MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Collectively, these findings suggested that reynoutrin exerted a pro-oxidant effect by enhancing oxidative stress in MCF-7 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eReynoutrin induced oxidative stress in PC-3 cells\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e illustrates the effects of reynoutrin on TAS, TOS, and OSI in PC-3 cells. In PC-3 cells, a significant increase in TOS levels and a reduction in TAS levels were observed in the cisplatin-treated group compared with those in the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similarly, reynoutrin induced a concentration-dependent increase in TOS levels and a significant decrease in TAS levels relative to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Notably, a high concentration of reynoutrin (618 \u0026micro;g/mL) exhibited a similar effect on TAS and TOS levels to cisplatin (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Moreover, reynoutrin markedly elevated OSI levels in PC-3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Collectively, these findings suggest that reynoutrin exerts a pro-oxidant effect by increasing oxidative stress in the PC-3 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eExpression results of TNF-α, IL-1β, IL-6, caspase-3, caspase-9, and Bcl-2/Bax mRNA in MCF-7 cells\u003c/h2\u003e \u003cp\u003eAnalysis of inflammatory markers showed that cisplatin (50 \u0026micro;M) treatment significantly increased the mRNA expression of TNF-α, IL-1β, and IL-6 in MCF-7 cells compared to that in the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Conversely, reynoutrin administration (110, 220, and 330 \u0026micro;g/mL) significantly reduced TNF-α, IL-1β, and IL-6 mRNA levels in a concentration-dependent manner (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These RT-PCR results demonstrated the anti-inflammatory potential of reynoutrin via the suppression of pro-inflammatory cytokine gene expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalysis of apoptotic parameters demonstrated that cisplatin significantly elevated caspase-3 and caspase-9 mRNA expression and significantly decreased the Bcl-2/Bax ratio in MCF-7 cells compared with that in the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similarly, medium and high concentrations of reynoutrin (220 and 330 \u0026micro;g/mL, respectively), but not low concentrations (110 \u0026micro;g/mL), induced a significant increase in caspase-3 and caspase-9 expression and a reduction in the Bcl-2/Bax ratio (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Interestingly, the effect of high-dose reynoutrin on caspase-9 expression was similar to that of cisplatin alone. These results indicate that reynoutrin promoted apoptosis in MCF-7 cells in a concentration-dependent manner.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe PCR results showed that reynoutrin exhibited anti-inflammatory activity and induced apoptotic cell death in MCF-7 cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eExpression levels of TNF-α, IL-1β, IL-6, caspase-3, caspase-9, and Bcl-2/Bax mRNA in PC-3 cells\u003c/h2\u003e \u003cp\u003eAnalysis of inflammatory markers showed that cisplatin treatment significantly increased the mRNA expression of TNF-α, IL-1β, and IL-6 in PC-3 cells compared with that in the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Conversely, administration of reynoutrin (206, 412, and 618 \u0026micro;g/mL) resulted in a significant concentration-dependent reduction in TNF-α, IL-1β, and IL-6 mRNA expression relative to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These PCR results demonstrate the anti-inflammatory potential of reynoutrin via the suppression of pro-inflammatory cytokine gene expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalysis of apoptotic parameters demonstrated that cisplatin significantly elevated caspase-3 and caspase-9 mRNA expression and significantly decreased the Bcl-2/Bax ratio in PC-3 cells compared with that in the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similarly, reynoutrin induced a significant increase in caspase-3 and caspase-9 expression and a reduction\u0026mdash;excluding the low concentration (206 \u0026micro;g/mL)\u0026mdash; in the Bcl-2/Bax ratio (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These results indicate that reynoutrin promoted apoptosis in PC-3 cells in a concentration-dependent manner.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRT-PCR results showed that reynoutrin exhibited anti-inflammatory activity and induced apoptosis in PC-3 cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eHoechst 33342 staining results\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e demonstrates the effect of reynoutrin on Hoechst 33342 staining in MCF-7 and PC-3 cells. Reynoutrin induced remarkable morphological alterations in the nuclei of MCF-7 and PC-3 cells compared with those in the control group. Increasing concentrations of reynoutrin led to nuclear shrinkage, fragmentation in both PC-3 and MCF-7 cells, and chromatin condensation, which are the hallmark features of apoptosis. In contrast to the control group, in which the cells displayed intact and healthy blue nuclei, those treated with cisplatin or reynoutrin exhibited enhanced blue fluorescence, which is indicative of the early apoptotic stages. These findings suggest that reynoutrin triggers apoptosis-related morphological changes in MCF-7 and PC-3 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCancer is a leading cause of mortality worldwide. Among various cancer types, breast cancer in women and prostate cancer in men rank as the second most fatal malignancy in their respective populations. Currently, chemotherapy, surgical intervention, and radiotherapy are the primary therapeutic approaches. However, these modalities often exhibit limited efficacy in cases of high incidence or low survival rates, and are frequently associated with severe adverse effects. Therefore, there is an urgent need to explore and develop novel anticancer agents derived from natural sources that are less toxic, more cost-effective, and have fewer side effects [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Naturally occurring compounds, particularly flavonoids, are regarded as valuable sources for the development of novel anticancer agents and have demonstrated significant potential in this field [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Flavonoids are naturally occurring phytochemicals with diverse pharmacological properties that have been the subject of exponentially increasing research on their pharmacological applications [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Flavonoids have demonstrated significant anticancer activities through the induction of oxidative stress, cell cycle arrest, attenuation of inflammatory responses, stimulation of apoptosis and autophagy, and suppression of cancer cell proliferation and invasion [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In this study, we investigated the antiproliferative, anti-inflammatory, and apoptotic activities of reynoutrin in the human prostate cancer cell line PC-3 and the human mammary cancer cell line MCF-7.\u003c/p\u003e \u003cp\u003eIn the present study, the IC₅₀ values of reynoutrin were 110 \u0026micro;g/mL for MCF-7 cells and 206 \u0026micro;g/mL for PC-3 cells. In contrast, Y\u0026uuml;ksel et al. reported a higher IC₅₀ value of 400 \u0026micro;g/mL in MCF-7 cells, suggesting that variations in experimental design, treatment duration, and cellular responsiveness may influence cytotoxic potency [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The relatively high IC₅₀ values observed in both studies may be attributed to the glycosylated structure of reynoutrin, which is known to limit membrane permeability and reduce intracellular accumulation compared with aglycone flavonoids.\u003c/p\u003e \u003cp\u003eWhen the balance between prooxidant activity and antioxidant defense is disrupted, ROS levels increase, leading to the accumulation of free radicals. Excessive ROS production results in oxidative stress, which is a key factor in promoting the inflammatory processes associated with cancer development. Flavonoids play a multifaceted role in cancer prevention and treatment, because their antioxidant or pro-oxidant behavior is influenced by their concentration and cellular conditions. Under normal physiological conditions, flavonoids function as antioxidants, maintaining ROS homeostasis; however, in cancer cells, they can act as potent pro-oxidants, initiating apoptotic pathways and suppressing pro-inflammatory signaling. Although their antioxidant properties protect healthy cells from oxidative stress and potentially lower cancer risk, their pro-oxidant effects at higher concentrations can induce stress in cancer cells, potentially increasing the efficacy of anticancer therapies. This dual action highlights the context-dependent therapeutic potential of flavonoids, which enables them to safeguard normal tissues while rendering cancer cells susceptible to treatment [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Research conducted on various cancer cell lines, such as HepG2, HCT116, and OVCAR-3, has shown that different flavonoids can induce oxidative stress in cancer cells by increasing ROS levels. Studies have indicated that daidzein, an isoflavone derived from plants [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], along with flavanone hesperidin [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], both contribute to increased ROS production in MCF-7 cells. Similarly, naringin, a glycoside flavonoid, has been shown to have a comparable effect on PC-3 cells. Elevated ROS levels inhibit cell proliferation and migration, and promote apoptosis in cancer cells. In our study, reynoutrin increased OSI in a dose-dependent manner by increasing TOS and decreasing TAS levels in MCF-7 and PC-3 cells. Notably, the effect of high-dose reynoutrin (616 \u0026micro;g/mL) on oxidative stress in PC-3 cells was similar to that of cisplatin (50 \u0026micro;M). These findings suggest that reynoutrin exhibits a stronger pro-oxidant activity in PC-3 cells than in MCF-7 cells.\u003c/p\u003e \u003cp\u003eWhen the inflammatory response is not properly regulated, it leads to an increase in the concentration of inflammatory mediators, which can contribute to the development of various chronic diseases including cancer. Inflammation is a hallmark of cancer and plays a critical role in the progression of many types of cancers. Elevated levels of cytokines, such as TNF-α, IL-1β, and IL-6, have been associated with cancer; therefore, cytokine inhibition may represent an important therapeutic target for cancer [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Experimental studies have linked TNF-α, IL- 1β, and IL-6 levels with cancer progression. Several studies have demonstrated that various flavonoids, including pelargonidin [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], apigenin [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], quercetin, rutin [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and luteolin [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], suppress the expression of cytokines, such as TNF-α, IL-1β, IL-6, and inhibit tumor growth. The flavanone hesperetin has been reported to inhibit the secretion of TNF-α, IL-6, and IL-1β in RAW 264.7 cells [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Flavon apigenin downregulated TNF-α-associated inflammatory signaling in the A375 human melanoma cell line [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Flavonoids, such as gossypin [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], chalcone derivatives [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], and curcumin [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], exhibit anti-inflammatory activity in PC-3 cells by suppressing TNF-α, IL-1β, and IL-6. Our findings support those of previous studies on the expression of proinflammatory cytokines. In the present study, reynoutrin demonstrated antiproliferative activity in both MCF-7 and PC-3 cells by reducing the levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in breast and prostate cancer cells.\u003c/p\u003e \u003cp\u003eApoptosis, a form of programmed cell death, plays a vital role in the elimination of irreversibly damaged cells from the body. Consequently, it is critical in the context of cancer, and the regulation of pro- and anti-apoptotic proteins has been extensively studied. Among the principal regulators of apoptosis, caspase-3, caspase-9, and Bax act as pro-apoptotic factors, while Bcl-2 functions as an anti-apoptotic factor. Flavonoids, as modulators of the apoptotic pathway, have been shown to promote proapoptotic mechanisms both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. These compounds induce apoptosis in cancer cells by downregulating antiapoptotic proteins and upregulating pro-apoptotic proteins [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Numerous studies have demonstrated that genistein in colon cancer cells (HT29) [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], luteolin in breast cancer cells (MDA-MB-231) [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], naringenin in hepatocellular carcinoma (HepG2) [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], and vitexin [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] in kidney cancer cells upregulate caspase-3., a key pro-apoptotic protein. Similarly, increased Bax levels were reported in cells treated with quercetin, taxifolin, luteolin [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], daidzein [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], vitexin [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], apigenin [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], myricetin [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], naringenin [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], and genistein [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In addition, studies have reported that treatment with epigallocatechin [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], myricetin [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], apigenin [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], genistein [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], and daidzein [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] results in significant downregulation of Bcl-2 in prostate cancer, hepatocellular carcinoma, colon cancer, human leukemia, and renal carcinoma cells compared to controls [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In our study, RT-PCR analysis revealed significant upregulation of Bax, caspase-3, and caspase-9 gene expression in both MCF-7 and PC-3 cells, which is consistent with the findings of previous studies. Moreover, medium and high concentrations of reynoutrin reduced the Bcl-2/Bax ratio in both cell lines, which is consistent with earlier reports. These results suggest that reynoutrin inhibits anti-apoptotic mechanisms, thereby promoting pro-apoptotic signaling during apoptosis. Apoptosis was assessed using Hoechst staining. Reynoutrin significantly increased nuclear fragmentation in MCF-7 and PC-3 cells in a dose-dependent manner. These findings indicate that reynoutrin induced apoptosis, particularly at higher concentrations.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, our results demonstrated that reynoutrin induces cytotoxic effects by inducing apoptosis and oxidative stress in breast and prostate cancer cells \u003cem\u003ein vitro\u003c/em\u003e and demonstrated its anti-inflammatory effects. However, further research, especially \u003cem\u003ein vivo\u003c/em\u003e experiments, are needed to confirm the detailed mechanisms involved. Our report strongly supports the use of reynoutrin as a potential medicinal agent in the development of novel antiproliferative agents.\u003c/p\u003e "},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthical approval\u003c/strong\u003e \u003cp\u003ewas not required because it was a cell culture study.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eAll authors agreed to the publication of this paper.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was supported by the Kastamonu University Scientific Research Projects Coordination Unit (KU BAP) (K\u0026Uuml;BAP-01/2023-25).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSedat Gokmen and Irfan Cinar Investigation, Conceptualization, Methodology, Writing- Original Draft, Data Curation, Software, and Validation. All authors have read and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis research was supported by the Kastamonu University Scientific Research Projects Coordination Unit (KU BAP) (K\u0026Uuml;BAP-01/2023-25).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eInternational Agency for Research on Cancer. (2022) Estimated age-standardized incidence and mortality rates (World) in 2020, worldwide, both sexes, all ages. IARC. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gco.iarc.fr/\u003c/span\u003e\u003cspan address=\"https://gco.iarc.fr/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Accessed 2 April 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValentovic MA. Evaluation of Resveratrol in Cancer Patients and Experimental Models. In: Tew KD, Fisher PB, editors. Advances in Cancer Research. Volume 137. London: Academic; 2018. pp. 171\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmjad E, Asnaashari S, Sokouti B, Dastmalchi S. (2020) Systems biology comprehensive analysis on breast cancer for identification of key gene modules and genes associated with TNM-based clinical stages. Sci Rep. 10(1):10816. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-020-67643-w\u003c/span\u003e\u003cspan address=\"10.1038/s41598-020-67643-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PMID: 32616754.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBukowski K, Kciuk M, Kontek R. Mechanisms of Multidrug Resistance in Cancer Chemotherapy. Int J Mol Sci. 2020;21(9):3233. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms21093233\u003c/span\u003e\u003cspan address=\"10.3390/ijms21093233\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoradi Z, Mohammadian M, Saberi H, Ebrahimifar M, Mohammadi Z, Ebrahimpour M, Behrouzkia Z. Anti-cancer effects of chemotherapeutic agent; 17-AAG, in combined with gold nanoparticles and irradiation in human colorectal cancer cells. Daru. 2019;27(1):111\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s40199-019-00251-w\u003c/span\u003e\u003cspan address=\"10.1007/s40199-019-00251-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNikolaou M, Pavlopoulou A, Georgakilas AG, Kyrodimos E. The challenge of drug resistance in cancer treatment: a current overview. Clin Exp Metastasis. 2018;35(4):309\u0026ndash;18. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10585-018-9903-0\u003c/span\u003e\u003cspan address=\"10.1007/s10585-018-9903-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchirrmacher V. From chemotherapy to biological therapy: A review of novel concepts to reduce the side effects of systemic cancer treatment (Review). Int J Oncol. 2019;54(2):407\u0026ndash;19. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3892/ijo.2018.4661\u003c/span\u003e\u003cspan address=\"10.3892/ijo.2018.4661\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen T, Xiao Z, Liu X, Wang T, Wang Y, Ye F, Su J, Yao X, Xiong L, Yang DH. Natural products for combating multidrug resistance in cancer. Pharmacol Res. 2024;202:107099. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.phrs.2024.107099\u003c/span\u003e\u003cspan address=\"10.1016/j.phrs.2024.107099\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang QY, Wang FX, Jia KK, Kong LD. Natural Product Interventions for Chemotherapy and Radiotherapy-Induced Side Effects. Front Pharmacol. 2018;9:1253. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fphar.2018.01253\u003c/span\u003e\u003cspan address=\"10.3389/fphar.2018.01253\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoormolaie N, Mohammadi M, Mir A, Asadi M, Kararoudi AN, Vahedian V, Rashidi M, Maroufi NF. Xanthomicrol: Effective therapy for cancer treatment. Toxicol Rep. 2023;10:436\u0026ndash;40. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.toxrep.2023.02.008\u003c/span\u003e\u003cspan address=\"10.1016/j.toxrep.2023.02.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClere N, Faure S, Martinez MC, Andriantsitohaina R. Anticancer properties of flavonoids: roles in various stages of carcinogenesis. Cardiovasc Hematol Agents Med Chem. 2011;9(2):62\u0026ndash;77. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2174/187152511796196498\u003c/span\u003e\u003cspan address=\"10.2174/187152511796196498\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGonz\u0026aacute;lez-Gallego J, S\u0026aacute;nchez-Campos S, Tu\u0026ntilde;\u0026oacute;n MJ. Anti-inflammatory properties of dietary flavonoids. Nutr Hosp. 2007;22(3):287\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi H, Zeng Y, Zi J, Hu Y, Ma G, Wang X, Shan S, Cheng G, Xiong J. Dietary Flavonoids Consumption and Health: An Umbrella Review. Mol Nutr Food Res. 2024;68(12):e2300727. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/mnfr.202300727\u003c/span\u003e\u003cspan address=\"10.1002/mnfr.202300727\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePonte LGS, Pavan ICB, Mancini MCS, da Silva LGS, Morelli AP, Severino MB, Bezerra RMN, Simabuco FM. The Hallmarks of Flavonoids in Cancer. Molecules. 2021;26(7):2029. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules26072029\u003c/span\u003e\u003cspan address=\"10.3390/molecules26072029\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eY\u0026uuml;ksel TN, Bozgeyik E, Yayla M. The Effect of Quercetin and Quercetin-3-d-xyloside on Breast Cancer Proliferation and Migration. JBACHS. 2022;6(2):569\u0026ndash;78. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.30621/jbachs.1056769\u003c/span\u003e\u003cspan address=\"10.30621/jbachs.1056769\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHo R, Violette A, Cressend D, Raharivelomanana P, Carrupt PA, Hostettmann K. Antioxidant potential and radical-scavenging effects of flavonoids from the leaves of Psidium cattleianum grown in French Polynesia. Nat Prod Res. 2012;26(3):274\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/14786419.2011.585610\u003c/span\u003e\u003cspan address=\"10.1080/14786419.2011.585610\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEldin Elhawary SS, Elmotyam AKE, Alsayed DK, Zahran EM, Fouad MA, Sleem AA, Elimam H, Rashed MH, Hayallah AM, Mohammed AF, Abdelmohsen UR. (2020) Cytotoxic and anti-diabetic potential, metabolic profiling and insilico studies of Syzygium cumini (L.) Skeels belonging to family Myrtaceae. Nat Prod Res. 2022 36(4):1026\u0026ndash;1030. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/14786419.2020.1843032\u003c/span\u003e\u003cspan address=\"10.1080/14786419.2020.1843032\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang W, Tu H, Tang K, Huang H, Ou S, Wu J. Reynoutrin Improves Ischemic Heart Failure in Rats Via Targeting S100A1. Front Pharmacol. 2021;12:703962. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fphar.2021.703962\u003c/span\u003e\u003cspan address=\"10.3389/fphar.2021.703962\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKopustinskiene DM, Jakstas V, Savickas A, Bernatoniene J. Flavonoids as Anticancer Agents. Nutrients. 2020;12(2):457. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/nu12020457\u003c/span\u003e\u003cspan address=\"10.3390/nu12020457\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLim W, Park S, Bazer FW, Song G. Naringenin-Induced Apoptotic Cell Death in Prostate Cancer Cells Is Mediated via the PI3K/AKT and MAPK Signaling Pathways. J Cell Biochem. 2017;118(5):1118\u0026ndash;31. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jcb.25729\u003c/span\u003e\u003cspan address=\"10.1002/jcb.25729\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGupta SC, Kunnumakkara AB, Aggarwal S, Aggarwal BB. Inflammation, a Double-Edge Sword for Cancer and Other Age-Related Diseases. Front Immunol. 2018;9:2160. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fimmu.2018.02160\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2018.02160\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChukiatsiri S, Siriwong S, Thumanu K. Pupae protein extracts exert anticancer effects by downregulating the expression of IL-6, IL-1β and TNF-α through biomolecular changes in human breast cancer cells. Biomed Pharmacother. 2020;128:110278. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biopha.2020.110278\u003c/span\u003e\u003cspan address=\"10.1016/j.biopha.2020.110278\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGolovko O, Nazarova N, Tuohimaa P. Vitamin D-induced up-regulation of tumour necrosis factor alpha (TNF-alpha) in prostate cancer cells. Life Sci. 2005;77(5):562\u0026ndash;77. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.lfs.2004.10.072\u003c/span\u003e\u003cspan address=\"10.1016/j.lfs.2004.10.072\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAdekoya TO, Richardson RM. Cytokines and Chemokines as Mediators of Prostate Cancer Metastasis. Int J Mol Sci. 2020;21(12):4449. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms21124449\u003c/span\u003e\u003cspan address=\"10.3390/ijms21124449\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAslan A, Se\u0026ccedil;me M. Effects of Pelargonium Sidoides Extract on Apoptosis and Oxidative Stress in Human Neuroblastoma Cells. Med (Kaunas). 2024;60(12):2110. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/medicina60122110\u003c/span\u003e\u003cspan address=\"10.3390/medicina60122110\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO'Brien MA, Kirby R. Apoptosis: A review of pro-apoptotic and anti‐apoptotic pathways and dysregulation in disease. J Vet Emerg Crit Care. 2008;18(6):572\u0026ndash;85. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1476-4431.2008.00363.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1476-4431.2008.00363.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJakimiuk K, Szoka Ł, Surażyński A, Tomczyk M. Using Flavonoid Substitution Status to Predict Anticancer Effects in Human Melanoma Cancers: An In Vitro Study. Cancers (Basel). 2024;16(3):487. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/cancers16030487\u003c/span\u003e\u003cspan address=\"10.3390/cancers16030487\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSahoo G, Samal D, Khandayataray P, Murthy MK. A Review on Caspases: Key Regulators of Biological Activities and Apoptosis. Mol Neurobiol. 2023;60(10):5805\u0026ndash;37. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12035-023-03433-5\u003c/span\u003e\u003cspan address=\"10.1007/s12035-023-03433-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChimento A, De Luca A, D'Amico M, De Amicis F, Pezzi V. The Involvement of Natural Polyphenols in Molecular Mechanisms Inducing Apoptosis in Tumor Cells: A Promising Adjuvant in Cancer Therapy. Int J Mol Sci. 2023;24(2):1680. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms24021680\u003c/span\u003e\u003cspan address=\"10.3390/ijms24021680\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u0026Ccedil;ınar İ, Gıdık B, Dirican E. Determination of anti-cancer effects of Nigella sativa seed oil on MCF7 breast and AGS gastric cancer cells. Mol Biol Rep. 2024;51(1):491. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11033-024-09453-1\u003c/span\u003e\u003cspan address=\"10.1007/s11033-024-09453-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eT\u0026uuml;l\u0026uuml;ce Y, Lak PTA, Koyuncu İ, Kılı\u0026ccedil; A, Durgun M, \u0026Ouml;zkol H. The apoptotic, cytotoxic and genotoxic effect of novel binuclear boron-fluoride complex on endometrial cancer. Biometals. 2017;30(6):933\u0026ndash;44. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10534-017-0060-8\u003c/span\u003e\u003cspan address=\"10.1007/s10534-017-0060-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarathe K, Naik J, Maheshwari V. Synthesis, characterisation and in vitro anticancer activity of conjugated protease inhibitor-silver nanoparticles (AgNPs-PI) against human breast MCF-7 and prostate PC-3 cancer cell lines. Bioprocess Biosyst Eng. 2024;47(6):931\u0026ndash;42. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00449-024-03023-2\u003c/span\u003e\u003cspan address=\"10.1007/s00449-024-03023-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Gong M, Zhu Z, Zhang B, Han L, Li W, Wu Z, Ma Q, Wang Z, Qian W. Rutin protects the pancreas from inflammatory injury and oncogene-driven tumorigenesis by inhibiting acinar to ductal metaplasia. Eur J Pharmacol. 2025;998:177536. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ejphar.2025.177536\u003c/span\u003e\u003cspan address=\"10.1016/j.ejphar.2025.177536\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Luna FCF, Ferreira WAS, Casseb SMM, de Oliveira EHC. Anticancer Potential of Flavonoids: An Overview with an Emphasis on Tangeretin. Pharmaceuticals (Basel). 2023;16(9):1229. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ph16091229\u003c/span\u003e\u003cspan address=\"10.3390/ph16091229\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin S, Zhang QY, Kang XM, Wang JX, Zhao WH. Daidzein induces MCF-7 breast cancer cell apoptosis via the mitochondrial pathway. Ann Oncol. 2010;21(2):263\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/annonc/mdp499\u003c/span\u003e\u003cspan address=\"10.1093/annonc/mdp499\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePalit S, Kar S, Sharma G, Das PK. Hesperetin Induces Apoptosis in Breast Carcinoma by Triggering Accumulation of ROS and Activation of ASK1/JNK Pathway. J Cell Physiol. 2015;230(8):1729\u0026ndash;39. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jcp.24818\u003c/span\u003e\u003cspan address=\"10.1002/jcp.24818\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee WJ, Chen WK, Wang CJ, Lin WL, Tseng TH. Apigenin inhibits HGF-promoted invasive growth and metastasis involving blocking PI3K/Akt pathway and beta 4 integrin function in MDA-MB-231 breast cancer cells. Toxicol Appl Pharmacol. 2008;226(2):178\u0026ndash;91. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.taap.2007.09.013\u003c/span\u003e\u003cspan address=\"10.1016/j.taap.2007.09.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhițu A, Schwiebs A, Radeke HH, Avram S, Zupko I, Bor A, Pavel IZ, Dehelean CA, Oprean C, Bojin F, Farcas C, Soica C, Duicu O, Danciu C. A Comprehensive Assessment of Apigenin as an Antiproliferative, Proapoptotic, Antiangiogenic and Immunomodulatory Phytocompound. Nutrients. 2019;11(4):858. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/nu11040858\u003c/span\u003e\u003cspan address=\"10.3390/nu11040858\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003edo Nascimento RP, Dos Santos BL, Amparo JAO, Soares JRP, da Silva KC, Santana MR, Almeida \u0026Aacute;MAN, da Silva VDA, Costa MFD, Ulrich H, Moura-Neto V, Lopes GPF, Costa SL. Neuroimmunomodulatory Properties of Flavonoids and Derivates: A Potential Action as Adjuvants for the Treatment of Glioblastoma. Pharmaceutics. 2022;14(1):116. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/pharmaceutics14010116\u003c/span\u003e\u003cspan address=\"10.3390/pharmaceutics14010116\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYao Q, Luo Y, Sun L, Wang H, Li W. Luteolin suppressed growth of colon tumor via inflammation, oxidative stress, and NLRP3/IL-1β signal axis. Pharmacognosy Magazine. 2022;18(78):494\u0026ndash;501. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4103/pm.pm_284_21\u003c/span\u003e\u003cspan address=\"10.4103/pm.pm_284_21\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRen H, Hao J, Liu T, Zhang D, Lv H, Song E, Zhu C. Hesperetin Suppresses Inflammatory Responses in Lipopolysaccharide-Induced RAW 264.7 Cells via the Inhibition of NF-κB and Activation of Nrf2/HO-1 Pathways. Inflammation. 2016;39(3):964\u0026ndash;73. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10753-016-0311-9\u003c/span\u003e\u003cspan address=\"10.1007/s10753-016-0311-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCinar I. Apoptosis-Inducing Activity and Antiproliferative Effect of Gossypin on PC-3 Prostate Cancer Cells. Anticancer Agents Med Chem. 2021;21(4):445\u0026ndash;50. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2174/1871520620666200721103422\u003c/span\u003e\u003cspan address=\"10.2174/1871520620666200721103422\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong B, Shen X, Tong C, Zhang S, Chen Q, Li Y, Li S. Gossypin: A flavonoid with diverse pharmacological effects. Chem Biol Drug Des. 2023;101(1):131\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/cbdd.14152\u003c/span\u003e\u003cspan address=\"10.1111/cbdd.14152\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHorta B, Freitas-Silva J, Silva J, Dias F, Teixeira AL, Medeiros R, Cidade H, Pinto M, Cerqueira F. Antitumor Effect of Chalcone Derivatives against Human Prostate (LNCaP and PC-3), Cervix HPV-Positive (HeLa) and Lymphocyte (Jurkat) Cell Lines and Their Effect on Macrophage Functions. Molecules. 2023;28(5):2159. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules28052159\u003c/span\u003e\u003cspan address=\"10.3390/molecules28052159\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePellegrino M, Bevacqua E, Frattaruolo L, Cappello AR, Aquaro S, Tucci P. Enhancing the Anticancer and Anti-Inflammatory Properties of Curcumin in Combination with Quercetin, for the Prevention and Treatment of Prostate Cancer. Biomedicines. 2023;11(7):2023. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/biomedicines11072023\u003c/span\u003e\u003cspan address=\"10.3390/biomedicines11072023\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShafiee G, Saidijam M, Tavilani H, Ghasemkhani N, Khodadadi I. Genistein Induces Apoptosis and Inhibits Proliferation of HT29 Colon Cancer Cells. Int J Mol Cell Med. 2016;5(3):178\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang L, Jin K, Lan H. Luteolin inhibits cell cycle progression and induces apoptosis of breast cancer cells through downregulation of human telomerase reverse transcriptase. Oncol Lett. 2019;17(4):3842\u0026ndash;50. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3892/ol.2019.10052\u003c/span\u003e\u003cspan address=\"10.3892/ol.2019.10052\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArul D, Subramanian P. Naringenin (citrus flavonone) induces growth inhibition, cell cycle arrest and apoptosis in human hepatocellular carcinoma cells. Pathol Oncol Res. 2013;19(4):763\u0026ndash;70. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12253-013-9641-1\u003c/span\u003e\u003cspan address=\"10.1007/s12253-013-9641-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Sun Q, Li H, Yang B, Wang M. Vitexin suppresses renal cell carcinoma by regulating mTOR pathways. Transl Androl Urol. 2020;9(4):1700\u0026ndash;11. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.21037/tau-20-1094\u003c/span\u003e\u003cspan address=\"10.21037/tau-20-1094\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng W, Liu T, Sun R, Yang L, An R, Xue Y. Daidzein induces choriocarcinoma cell apoptosis in a dose-dependent manner via the mitochondrial apoptotic pathway. Mol Med Rep. 2018;17(4):6093\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3892/mmr.2018.8604\u003c/span\u003e\u003cspan address=\"10.3892/mmr.2018.8604\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShukla S, Fu P, Gupta S. Apigenin induces apoptosis by targeting inhibitor of apoptosis proteins and Ku70-Bax interaction in prostate cancer. Apoptosis. 2014;19(5):883\u0026ndash;94. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10495-014-0971-6\u003c/span\u003e\u003cspan address=\"10.1007/s10495-014-0971-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim ME, Ha TK, Yoon JH, Lee JS. Myricetin induces cell death of human colon cancer cells via BAX/BCL2-dependent pathway. Anticancer Res. 2014;34(2):701\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHsiao YC, Peng SF, Lai KC, Liao CL, Huang YP, Lin CC, Lin ML, Liu KC, Tsai CC, Ma YS, Chung JG. Genistein induces apoptosis in vitro and has antitumor activity against human leukemia HL-60 cancer cell xenograft growth in vivo. Environ Toxicol. 2019;34(4):443\u0026ndash;56. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/tox.22698\u003c/span\u003e\u003cspan address=\"10.1002/tox.22698\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei R, Zhu G, Jia N, Yang W. Epigallocatechin-3-gallate Sensitizes Human 786-O Renal Cell Carcinoma Cells to TRAIL-Induced Apoptosis. Cell Biochem Biophys. 2015;72(1):157\u0026ndash;64. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12013-014-0428-0\u003c/span\u003e\u003cspan address=\"10.1007/s12013-014-0428-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan BJ, Li W, Jiang GB, Lai SH, Zhang C, Zeng CC, Liu YJ. Effects of daidzein in regards to cytotoxicity in vitro, apoptosis, reactive oxygen species level, cell cycle arrest and the expression of caspase and Bcl-2 family proteins. Oncol Rep. 2015;34(3):1115\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3892/or.2015.4133\u003c/span\u003e\u003cspan address=\"10.3892/or.2015.4133\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Reynoutrin, MCF-7, PC-3, Apoptosis, Oxidative stress, Anti-inflammatory","lastPublishedDoi":"10.21203/rs.3.rs-8431324/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8431324/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eReynoutrin, a bioactive flavonoid, is recognized for its biological effects. Despite toxic side effects, cisplatin is widely used to treat malignancies. This study aimed to assess the \u003cem\u003ein vitro\u003c/em\u003e antiproliferative activity of reynoutrin in MCF-7 and PC-3 cell lines by evaluating its modulation of intracellular signaling pathways.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe cytotoxic effects of reynoutrin on MCF-7 and PC-3 cells were evaluated using an MTT assay after 24 h. Oxidative stress levels were measured using enzyme-linked immunosorbent assay (ELISA) with total antioxidant and oxidant status kits. The expression levels of tumor necrosis factor-α (TNF‑α), interleukin (IL)‑1β and IL‑6, caspase 3, caspase 9, B‑cell lymphoma‑2 (Bcl‑2), and Bcl‑2‑associated X protein (Bax) were detected by RT-PCR. Apoptosis was verified using Hoechst staining.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eReynoutrin inhibited MCF-7 and PC-3 cell viability in a dose-dependent manner, with IC₅₀ values of 220 and 412 \u0026micro;g/mL, respectively. Reynoutrin increased the total oxidant status, decreased the total antioxidant enzyme activity, and enhanced oxidative stress (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). RT-PCR showed that TNF-α, IL-1β, and IL-6 expression levels decreased in a dose-dependent manner following reynoutrin administration in MCF-7 and PC-3 cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating its anti-inflammatory activity. In cancer cells, reynoutrin increased caspase-3 and caspase-9 levels and decreased the Bcl-2/Bax ratio in a dose-dependent manner (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThese findings demonstrate that reynoutrin exerts antiproliferative activity by regulating inflammation, oxidative stress, and apoptotic pathways in cancer cells.\u003c/p\u003e","manuscriptTitle":"Antiproliferative Effects of Reynoutrin via Modulation of Oxidative Stress, Inflammation, and Apoptosis in Breast (MCF-7) and Prostate Cancer (PC-3) Cells Lines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-10 19:13:52","doi":"10.21203/rs.3.rs-8431324/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8bd125d3-3417-40f6-aac7-defa79449002","owner":[],"postedDate":"March 10th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-04T08:43:21+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-10 19:13:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8431324","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8431324","identity":"rs-8431324","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}