Apocynin, a natural acetophenone suppresses cell proliferation, migration and induces cell cycle arrest, apoptosis in ovarian carcinoma cell line A2780

Apocynin, a natural acetophenone suppresses cell proliferation, migration and induces cell cycle arrest, apoptosis in ovarian carcinoma cell line A2780 | 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 Apocynin, a natural acetophenone suppresses cell proliferation, migration and induces cell cycle arrest, apoptosis in ovarian carcinoma cell line A2780 Qian Zhu, Xuyuan Tang, Ke Wang, Wei Cheng, Dongmei Zhou, Yanhong Huang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4685264/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 In this study, the ameliorative effects of apocynin against ovarian cancer cell proliferation, migration, and induction of apoptosis were studied in vitro. A2780 human ovarian carcinoma cells and Vero normal epithelial cells were treated with apocynin and subjected to cytotoxicity assays. Lipid peroxidation and antioxidant status were quantified in apocynin-treated A2780 cells to assess the anticancer effect of apocynin. Staining techniques with DCFH-DA, Rhodamine-123, and AO/EtBr were done to analyze the ROS-induced apoptosis in A2780 cells. A wound scratch assay was performed to examine the effect of apocynin on cell migration. Flow cytometric analysis was done to analyze cell cycle arrest in apocynin-treated A2780 cells. To confirm the apoptosis in apocynin-treated cells, the apoptotic proteins were quantified using kits. Apocynin treatment significantly inhibited growth andpromoted oxidative stress and apoptosis in A2780 cells. The results of fluorescent staining assays clearly state that apocynin increases ROS levels and thereby induces lipid peroxidation, which leads to cell death. Apocynin treatment caused cell cycle arrest and promoted apoptosis in A2780 cells, which were confirmed by the flow cytometry results and an increase in caspases, bax, and a decrease in bcl2 levels, respectively. Apocynin treatment also inhibited cell migration, which was evidenced by our wound scratch assay. Overall, our findings confirm that apocynin significantly inhibits cell proliferation, cell migration, and induced apoptosis in ovarian cancer A2780 cells. Ovarian cancer A2780 cells Apocynin Cell migration Cell cycle arrest Apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Ovarian cancer is a serious type of cancer that was mostly diagnosed during the advanced stage. Among gynecological-related malignancies, ovarian cancer is a severe disease and the most frequently diagnosed type of cancer in females [ 1 – 4 ]. The Global Ovarian Cancer Charter reported that globally in 2020, about 3.1 lakh women were estimated to be diagnosed with ovarian cancer, and about 2 lakh mortalities were reported due to ovarian cancer [ 5 ]. Women in the post-menopause period have a higher incidence of ovarian cancer compared to other females[ 6 ]. The symptoms are imprecise, such as bloating, abdominal pain, feeling satiety, and urgency in urination [ 7 , 8 ]. Since the symptoms are vague, 70% of ovarian cancer patients were diagnosed at an advanced stage [ 9 ]. The tumors that occurs in the ovaries, fallopian tubes, and primary peritoneal cavity are collectively named ovarian cancer. 90% of ovarian cancer arises from the ovarian epithelial cells, which is high-grade serous ovarian cancer [ 10 ]. Other than age, family history, hormonal and reproductive disorders, race, and lifestyle also determine the occurrence of ovarian cancer [ 11 , 12 ]. Genetic mutations in BRCA1, BRCA2, and TP53 were exhibited in 10–15% of ovarian cancer patients [ 13 ]. Since the symptoms are common and there are no particular screening tests for ovarian cancer, treating it is a greater task. Surgery followed by chemotherapy used to be the first-line treatment given to patients diagnosed with ovarian cancer [ 14 , 15 ]. Due to intra-tumor heterogeneity, high-grade serous ovarian cancer shows resistance to chemotherapy drugs such as paclitaxel, carboplatin, olaprib,etc. [ 16 ]. If diagnosed at the early stage, the five-year survival rate of ovarian cancer is about 93%, whereas at the advanced stage, due to chemotherapy resistance the five-year survival rate is 29% [ 17 ]. Since ovarian cancer is mostly diagnosed at the advanced stage, an effective drug is needed today to treat it. Cancer chemotherapy is staged into three categories based on the patient’s risk and disease stage. Primary chemotherapy is a preventive measure given to the high-risk population with a genetic predisposition to cancer. Secondary chemotherapy treatment was given to inhibit the progression of cancer in patients at an early stage. Tertiary chemotherapy was given to the patients to prevent cancer relapse [ 18 , 19 ]. Phytochemicals play a substantial role in providing primary chemotherapy,and they are also reported to render secondary and tertiary chemotherapy [ 20 ]. Apocynin, 4-hydroxy-3-methoxy-acetophenone, is a polyphenolic compound present in extracts of Picrorhizakurroa andApocynumcannabinum used to treat cardiac, hepatic, respiratory, and rheumatoid illnesses in Ayurveda treatment [ 21 – 23 ]. Apocynin is a potent NADPH oxidase inhibitor that provides anti-inflammatory [ 24 ], neuroprotective [ 25 ], and anti-ageing effects [ 26 ]. Apocynin has been reported to suppress cancer proliferation in in vitro and animal models[ 27 – 28 ].The ameliorative effect of apocynin on most lethal cancers, like ovarian cancer,has not yet been elucidated.In this study, we analyzed the anticancer effect of apocynin on ovarian cancer cell proliferation and migration in an in vitro model. Materials and methods Apocynin, doxorubicin, and the reagents MTT and DMSO were procured from Sigma Aldrich, USA. Cell culture medium (DMEM, RPMI-1640), culture reagents, antibiotic-anitmycotic solutions, and Trypsin EDTA were purchased from ThermoFischer Scientific, USA. Staining kits DCFH-DA, Acridine Orange, Ethidium Bromide, and Rhodamine 123 were also procured from Sigma Aldrich, USA. Only analytical-grade chemicals and reagents were used for the experiments. Cell line culture Human ovarian carcinoma A2780 cells and non-malignant Vero cells were purchased from ATCC, USA. The cells were examined for contamination and cell growth under a light microscope before incubating in a CO2 incubator. The medium was replaced with 10% FBS-supplemented RPMI-1640 and incubated for 24h at 37ºC in a 5% CO2 incubator. The cells were trypsinized with Trypsin-EDTA and subcultured for further experiments. Cytotoxicity analysis of apocynin The cytotoxicity effect of apocynin was examined with the ovarian carcinoma A2780 human cell line and normal epithelial Vero cell line. A2780 cell lines were cultured in 10% FBS-supplemented RPMI-1640 medium, whereas Vero cells were cultured in DMEM medium. Both cells were treated with different concentrations of apocynin ranging from 0–15 µM. The drug-treated cells were incubated for 24h at 37ºC in a 5%CO2 incubator and then subjected to MTT analysis. The cells were then treated with 5mg/ml of freshly prepared MTT solution and incubated for 3h at 37ºC in a 5%CO2 incubator. The formazan crystals formed due to the cleavage of MTT by viable cells were measured by dissolving the crystals with DMSO. The final absorbance of the solution was measured at 540nm. The experiments were performed in triplicate, and the percentage of cell viability with different dosages of apocynin was calculated. The IC50 value was calculated, and the dose was used for the analysis of further experiments. Assessment of antioxidant property of apocynin TBARS assay Lipid peroxidation in cells induced by oxidative stress was quantified by measuring the final end product, TBARS. A2780 ovarian carcinoma cells were treated with 7.5µM of apocynin and 2µg of the standard drug doxorubicin for 24h and then subjected to TBARS quantification. TBARS were quantified using the MDA assay kit (ab233471), purchased from Abcam, USA. The final reaction mixture was incubated at room temperature for 45 minutes. The end red product formed due to the reaction of thiobarbituric acid and malondialdehyde was measured at 695nm. Superoxide dismutase assay The antioxidant SOD levels in apocynin-treated A2780 cells were measured with a superoxide dismutase kit procured from Sigma Aldrich, USA. WST-1, upon reduction with superoxide anion, forms formazan dye, whereas the rate of reduction with superoxide anion is linear to xanthine oxidase activity, which is inhibited by SOD. Hence, the SOD inhibition activity was measured by incubating the final mixture at 37ºC for 20min and then measured at 450nm. Glutathione assay Glutathione levels in apocynin- and doxorubicin-treated A2780 cells were quantified using a commercially available colorimetric kit procured from ThermoFischer Scientific, USA. The assay was performed according to the manufacturer’s guidelines. The final absorbance was measured at 405nm. DCFH-DA staining Apocynin treated A2780 cells were subjected to 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining to quantify the reactive oxygen species generated. A2780 cells were treated for 24h with 7.5µM apocynin, and as a positive control a set of cells were treated with 2µg of the standard anticancer drug doxorubicin. The control and drug-treated cells were stained with DCFDA/H2DCFDA (Cellular ROS Assay Kit, Abcam, USA). The cells were viewed under a fluorescent microscope, and the fluorescence intensity was quantified with ImageJ software. Rhodamine 123 staining Rhodamine 123 staining was performed on apocynin-treated A2780 cells to analyze the mitochondrial membrane potential of the cells. A2780 cells were treated with apocynin for 24h and then subjected to rhodamine 123 staining. The drug-treated cells were washed with PBS and stained with rhodamine 123 for 30 min in the dark. The cells were then viewed undera fluorescent microscope, and the intensity of fluorescence was quantified with ImageJ software. AO/EtBr staining A dual staining technique with acridine orange and ethidium bromide was used to analyze the percentage of live and dead cells after apocynin treatment. A2780 cells were treated with 7.5µM of apocynin and 2µg of doxorubicin for 24h. Then the cells were stained with a cocktail mixture of AO and EtBr which was prepared in an equal ratio. The stained cells were incubated in the dark for 5min and then viewed under a fluorescent microscope. The fluorescence intensity was quantified with ImageJ software to assess the percentage of live and dead cells. Wound scratch assay A2780 cells were cultured on 6-wellplate and upon attaining 100% confluency, the cells were scratched horizontally with the tip of a sterile micropipette at the center of the wells to generate a wound on the cultured cells. The cells were then treated with 7.5µM of apocynin and 2µg of doxorubicin and incubated for 24h. The cells were then viewed under a microscope to assess the cell migration of control and drug-treated cells. Cell cycle analysis Annexin V/PI flow cytometry analysis was done to analyze the apocynin-induced apoptosis in ovarian carcinoma A2780 cells. The cells were treated with 7.5µM of apocynin and 2µg of doxorubicin and incubated for 24h. The cells were harvested and subjected to ethanol fixing. The ethanol-fixed cells were stained with a propidium iodide solution for 30min and then scanned with FACSCanto II and BD FACSDiva software. Assessment of apoptotic markers Ovarian carcinoma A2780 cells were treated with apocynin and doxorubicin for 24h and subjected to quantification of caspase 3, caspase 9, bax, and bcl2 using a commercially available ELISA kit from Abcam USA. The assay was done as per the guidelines provided in the kit, and the levels were calculated using the standard curve plot. Statistical Analysis All the results were presented as the mean value ± SEM of three independent observations done in triplicate. A one way analysis of variance followed by a Student’s t test was performed to analyze the statistical significance of the data. Statistical significance was considered to be with a difference p value of 0.05. Results Apocynin significantly induced cytotoxic effect in A2780 cells Figure 1 depicts the results of the MTT assay performed with A2780 human ovarian cancer cells and Vero normal epithelial cells treated with different concentrations of apocynin. Apocynin significantly induced cell death in A2780 cells, whereas only a minimal percentage of cell death was reported in normal Vero cells. The minimal concentration of 1µM apocynin treatment showed 23 ± 0.04% of cell death, whereas the highest concentration of 15µM apocynin treatment showed 85 ± 0.08% of cell death in A2780 cells. The IC50 value of apocynin for A2780 cells was obtained at a concentration of 7.5µM. In Vero cells, even the highest concentration (15µM) apocynin treatment showed only 10.2 ± 0.09% cell death. Apocynin decreased antioxidant levels in A2780 cells Antioxidants are prime players in cell death and proliferation; excess synthesis of antioxidants leads to uncontrolled proliferation of cells. Apocynin treatment significantly decreased the levels of enzymatic antioxidant SOD to 27 ± 0.09 units/mg protein, whereas it was 44 ± 0.2 units/mg protein in control cells. It also decreased the levels of the non-enzymatic antioxidant glutathione to 21 ± 0.05 units/mg protein, whereas it was 37 ± 0.09 units/mg protein in control cells. Apocynin treatment significantly increased the lipid peroxidation by 32 ± 0.08nmol/mg protein, whereas it was less than 1 nmol/mg protein in control cells (Fig. 2 ). Apocynin increased reactive oxygen species in A2780 cells Intracellular reactive oxygen species synthesized by apocynin in A2780 cells were quantified with fluorescent DHCF-DA stain, and the results are depicted in Fig. 3 . Apocynin significantly increased the levels of ROS in A2780 cells, which was evidenced by a 38 ± 2% fluorescent intensity compared to the control untreated cells. Standard drug doxorubicin treatment has shown a significantly increased fluorescent intensity of 64 ± 1.8%. Apocynin decreased mitochondrial membrane potential in A2780 cells Figure 4 illustrates the levels of mitochondrial membrane potential in control, apocynin-, and doxorubicin-treated A2780 cells. Apocynin treatment significantly decreased MMP levels in A2780 cells compared to the control cells. Compared to doxorubicin, the fluorescent intensity increased in apocynin-treated cells. Apocynin induced apoptosis in A2780 cells The induction of apoptosis in A2780 cells by apocynin was estimated with the dual staining technique, and the results are represented in Fig. 5 . Control cells showed increased green fluorescence, indicating the presence of live cells, whereas the apocynin-treated cells showed increased levels of yellowish-orange fluorescence, indicating early apoptotic cells. Compared to doxorubicin, apocynin shows decreased levels of reddish-orange fluorescence, which indicates late apoptotic cells. Apocynin treatment induced 34 ± 2% cell death in A2780 cells. Apocynin inhibited cell migration in A2780 cells Figure 6 represents the results of the wound scratch assay performed on control and apocynin-treated cells. Apocynin treatment effectively inhibited the migration of A2780 cells compared to the control. It was evident from our microscopic image that only a few cells were observed in the scratched site of apocynin-treated cells, whereas numerous cells can be seen in the scratched site of control cells. Apocynin inhibits cell proliferation in A2780 cells Figure 7 represents the flow cytometric analysis was done in control. Apocynin and doxorubicin treated cells to analyze the effect of drugs on cell proliferation. Apocynin treatment retained the cells in G0 andG1, compared to the control cells.A decrease in cells with S-phase and G2-phase was observed in the apocynin-treated cells. Doxorubicin significantly decreased the S-phase and G2 phase cells compared to the control and the apocynin-treated cells. Apocynin stimulates apoptotic protein synthesis in A2780 cells Figure 8 illustrates the levels of apoptotic proteins Caspase 3, Caspase 9, Bcl2, and Baxin apocynin-treated A2780 cells. Apocynin treatment significantly increased the levels of both caspases 3 and 9 in A2780 cells. It significantly decreased the levels of the antiapototic protein Bcl2 and increased the proapoptoticBax levels in A2780 cells. Discussion Ovarian cancer remains the most common cause of gynecological-related mortalities in post-menopausal women. 75% of ovarian cancers were diagnosed at the advanced stage. Debulking surgery and platinum-based chemotherapy are the first lines of treatment provided to ovarian cancer patients [ 29 , 30 ]. A combination of platinum, along with paclitaxel, olaprib, bevacizumab, and niraparib, are some of the drugs prescribed for treating ovarian cancer. In the initial stage, the patient responds to these drugs, whereas later, due to platinum resistance, 80% of the patients are prone to relapses of cancer, and their survival is questionable [ 31 ]. At present, targeted therapies are the new hope for treating ovarian cancer, but the recurrence rate of cancer is still in considerable numbers [ 32 ]. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is an enzyme with multiple subunits expressed in various tissues throughout the body. The prime function of NOX is to catalyze the generation of free radicals to facilitate adaptive immunity [ 33 – 35 ]. NOX were classified into two categories based on their activity: phagocyte-derived and non-phagocyte-derived NADPH oxidase. Overexpression of non-phagocytotic-derived NADPH oxidase, specifically the NOX-1 subtype, was observed in various cancer cells [ 36 – 38 ]. Ovarian cancer cells are also reported to overexpress NOX-1; hence, inhibiting NOX-1 may suppress cancer cell proliferation [ 39 ]. Apocynin, a NOX inhibitor, had been reported to suppress intestinal tumorigenesis in obese mice [ 40 ], prostate cancer [ 41 , 42 ], breast cancer [ 43 ], and bladder cancer [ 44 ]. In this study, we investigated the anticancer effect of apocycin against ovarian cancer cells and normal epithelial cells. Apocynin treatment significantly inhibited the cell proliferation of ovarian cancer cells (A2780), whereas a only minimal percentage of cell death was observed in normal epithelial vero cells. This confirms the targeted cell toxicity of apocynin. Oxidative stress created due to a reactive oxygen species imbalance imparts a dual response in cells. At the physiological level, ROS is the prime signaling pathway that regulates the growth, proliferation, metabolism, and apoptosis of cells [ 45 , 46 ], whereas excessive generation of ROS during pathogenic conditions hinders normal cellular functions [ 47 , 48 ]. Generation and inhibition of these reactive oxygen species play a vital role in treating various inflammatory diseases, including cancer [ 49 , 50 ]. Induction of ROS generation in ovarian cancer is proposed to render an anticancer effect by causing membrane lipid peroxidation, altering the genetic material, and inducing apoptosis in cancer cells [ 51 ]. Our study also correlated with the previous findings that apocynin treatment significantly increased lipid peroxidation in the A2780 cells via decreasing the enzymatic antioxidant superoxide and the non-enzymatic antioxidant glutathione levels, thereby causing apoptosis. Further, to confirm the apocynin-induced free radical generation-mediated apoptosis in A2780 cells, the apocynin-treated cells were stained with DCHF-DA stain, Rhodamine 123, and AO/EtBr stain. Our staining proves apocynin treatment significantly increased reactive oxygen species generation, thereby decreasing mitochondrial membrane potential and leading to cellular apoptosis. The mitochondrial disruption triggers the apoptotic proteins, both the pro-apoptotic proteins Bid, Bax, andcaspases, and the anti-apoptotic protein Bcl2. The anti-apoptotic protein Bcl2 prevents the mitochondrial release of apoptosis-inducing factors and also caspase activities. Hence, targeting Bcl2 may promote apoptosis in cancer cells [ 52 ]. In our study, apocynin treatment significantly decreased the levels of Bcl2 protein and also increased the levels of pro-apoptotic proteins Bax and caspases. The increased cell death observed in AO/EtBr-stained apocynin-treated A2780 cells may be due to the increase in the pro-apoptotic protein and the decrease in the anti-apoptotic protein Bcl2 levels. Anti-cancer treatment disrupts the homeostasis of the cells by generating reactive oxygen species, thereby damaging the nuclear content [ 53 , 54 ]. The oxidative stress created causes a damaged DNA response, which shifts the cells to cell cycle arrest and eventually leads to apoptosis [ 55 ]. Our flow cytometric analysis of apocynin-treated cells confirmed the cell cycle arrest in the A2780 cell. Apocynin treatment retained an increased number of A2780 cells in the G0/G1phase, and a decreased number of cells were observed in the S and G2/M phases compared to the control cells. Apart from inhibition of cell proliferation and induction of apoptosis, apocynin treatment also inhibited cell migration of A2780, as evidenced by our wound scratch assay. Conclusion Apocynin, a natural acetophenone, was examined for its anticancer effect against ovarian carcinoma A2780 cells. Apocynin significantly inhibited the cell proliferation of ovarian carcinoma A2780 cells, and a minimal cytotoxic effect was observed in normal epithelial verocells. Apocynin treatment significantly generated ROS and decreased antioxidant levels in A2780, which in turn induced cell cycle arrest and eventually apoptosis in A2780. It also potently inhibited the cell migration property of A2780. All together, our results prove apocynin is a potent anticancer agent that effectively induces apoptosis in in vitro conditions. Further studies may be conducted to formulate apocynin as a drug to treat ovarian cancer. Declarations Ethical approval and consent to participate: This article does not contain any studies with human participants or animals performed by any of the authors. Consent for publication: We consent to the publication of the article. Author Contribution: Qian Zhu, Dongmei Zhou : Conceptualization, Investigation, Formal analysis, Writing – original draft, Writing – review & editing. Ke Wang, Xuyuan Tang : Formal analysis, Validation, Writing – original draft, Writing – review & editing. Yanhong Huang : Validation, Writing – review & editing. Funding Xi'an International Medical Center Hospital, Youth project Funding number 2020QN010. Competing interest: The authors declare no competing of interests. Availability of Data and Materials: All data analyzed during this study are included in this published article. References Coburn SB, Bray F, Sherman ME, Trabert B. International patterns and trends in ovarian cancer incidence, overall and by histologic subtype. Int J Cancer. 2017 Jun 1;140(11):2451-2460 Momenimovahed Z, Tiznobaik A, Taheri S, Salehiniya H. Ovarian cancer in the world: epidemiology and risk factors. Int J Womens Health. 2019 Apr 30;11:287-299 Gaona-Luviano P, Medina-Gaona LA, Magaña-Pérez K. Epidemiology of ovarian cancer. Chin Clin Oncol. 2020 Aug;9(4):47 Nash Z, Menon U. Ovarian cancer screening: current status and future directions. Best Pract Res ClinObstetGynaecol. 2020;65:32- 45. Frances R & Anmol B. Global Trends in Incidence, Mortality, and Survival. The World Ovarian Cancer Coalition Atlas 2023. 2023: 1-65 Lengyel E. Ovarian cancer development and metastasis. Am J Pathol. 2010 Sep;177(3):1053-64 Goff BA, Mandel LS, Melancon CH, Muntz HG. Frequency of Symptoms of Ovarian Cancer in Women Presenting to Primary Care Clinics. JAMA. 2004; 291(22):2705–2712 Goff BA, Mandel LS, Drescher CW, Urban N, Gough S, Schurman KM, Patras J, Mahony BS, Andersen MR. Development of an ovarian cancer symptom index: possibilities for earlier detection. Cancer. 2007 Jan 15;109(2):221-7 Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018 Nov;68(6):394-424. doi: 10.3322/caac.21492. Epub 2018 Sep 12. Erratum in: CA Cancer J Clin. 2020 Jul;70(4):313 Kurman RJ, Carcangiu ML, Herrington CS, et al. WHO classification of tumours of the female reproductive organs. Lyon: WHO Press; 2014. Lu KH, Daniels M. Endometrial and ovarian cancer in women with Lynch syndrome: update in screening and prevention. Fam Cancer. 2013;12(2):273-277. Doi:10.1007/s10689-013-9664-5. 41 Momenimovahed Z, Tiznobaik A, Taheri S, Salehiniya H. Ovarian cancer in the world: epidemiology and risk factors. Int J Womens Health. 2019;11:287–299. Published 2019 Apr 30. doi:10.2147/IJWH.S197604 Bast RC Jr, Hennessy B, Mills GB. The biology of ovarian cancer: new opportunities for translation. Nat Rev Cancer. 2009 Jun;9(6):415-28 Franzese E, Diana A, Centonze S, Pignata S, De Vita F, Ciardiello F, Orditura M. PARP Inhibitors in First-Line Therapy of Ovarian Cancer: Are There Any Doubts? Front Oncol. 2020 Jun 12;10:782 Haddad FG, Karam E, Moujaess E, Kourie HR. Poly-(ADP-ribose) polymerase inhibitors: paradigm shift in the first-line treatment of newly diagnosed advanced ovarian cancer. Pharmacogenomics. 2020 Jul;21(10):721-727 Ingles Russo Garces AH. Drug-induced evolutionary dynamics in BRCA-mutant/nonmutant ovarian cancer models. Abstract 1697P. ESMO Congress 2022, 10 September, 2022. Lukanović D, Herzog M, Kobal B, Černe K. The contribution of copper efflux transporters ATP7A and ATP7B to chemoresistance and personalized medicine in ovarian cancer. Biomed Pharmacother. 2020 Sep;129:110401 Jinghua L, Linmei S, Ping H, Gothai S, Muniandy K, Kumar SS, Esa NM, Arulselvan P. Amelioration of oxidative stress through apoptosis-mediated pathway in colon cancer cells by hexane fraction of Moringa oleifera extract. Phcog Mag 2018;14:S311-8. De Flora S, Ferguson LR. Overview of mechanisms of cancer chemopreventive agents. Mutat Res. 2005 Dec 11;591(1-2):8-15 Ranjan A, Ramachandran S, Gupta N, Kaushik I, Wright S, Srivastava S, Das H, Srivastava S, Prasad S, Srivastava SK. Role of Phytochemicals in Cancer Prevention. Int J Mol Sci. 2019 Oct 9;20(20):4981 Ram VJ. Herbal preparations as a source of hepatoprotective agents. Drug News Perspect. 2001; 14:353–363 Stefanska J, Pawliczak R. Apocynin: molecular aptitudes. Mediators Inflamm. 2008;2008:106507 Savla SR, Laddha AP, Kulkarni YA. Pharmacology of apocynin: a natural acetophenone. Drug Metab Rev. 2021 Nov;53(4):542-562. Qin YY, Li M, Feng X, Wang J, Cao L, Shen XK, Chen J, Sun M, Sheng R, Han F, Qin ZH. Combined NADPH and the NOX inhibitor apocynin provides greater anti-inflammatory and neuroprotective effects in a mouse model of stroke. Free Radic Biol Med. 2017 Mar;104:333-345 Du ZD, Yu S, Qi Y, Qu TF, He L, Wei W, Liu K, Gong SS. NADPH oxidase inhibitor apocynin decreases mitochondrial dysfunction and apoptosis in the ventral cochlear nucleus of D-galactose-induced aging model in rats. Neurochem Int. 2019 Mar;124:31-40 Wang Q, Tompkins KD, Simonyi A, Korthuis RJ, Sun AY, Sun GY. Apocynin protects against global cerebral ischemia-reperfusion-induced oxidative stress and injury in the gerbil hippocampus. Brain Res. 2006 May 23;1090(1):182-9 Jantaree P, Lirdprapamongkol K, Kaewsri W, Thongsornkleeb C, Choowongkomon K, Atjanasuppat K, Ruchirawat S, Svasti J. Homodimers of Vanillin and Apocynin Decrease the Metastatic Potential of Human Cancer Cells by Inhibiting the FAK/PI3K/Akt Signaling Pathway. J Agric Food Chem. 2017 Mar 22;65(11):2299-2306 Paul S, Chakrabarty S, Ghosh S, Nag D, Das A, Dastidar DG, Dasgupta M, Dutta N, Kumari M, Pal M, Chakrabarti G. Targeting cellular microtubule by phytochemical apocynin exhibits autophagy-mediated apoptosis to inhibit lung carcinoma progression and tumorigenesis. Phytomedicine. 2020 Feb;67:153152 Sambasivan S. Epithelial ovarian cancer: Review article. Cancer Treat Res Commun. 2022;33:100629 Ortiz M, Wabel E, Mitchell K, Horibata S. Mechanisms of chemotherapy resistance in ovarian cancer. Cancer Drug Resist. 2022 Apr 3;5(2):304-316 Damia G, Broggini M. Platinum Resistance in Ovarian Cancer: Role of DNA Repair. Cancers (Basel). 2019 Jan 20;11(1):119 Jacobs IJ, Menon U, Ryan A, Gentry-Maharaj A, Burnell M, Kalsi JK, Amso NN, Apostolidou S, Benjamin E, Cruickshank D, Crump DN, Davies SK, Dawnay A, Dobbs S, Fletcher G, Ford J, Godfrey K, Gunu R, Habib M, Hallett R, Herod J, Jenkins H, Karpinskyj C, Leeson S, Lewis SJ, Liston WR, Lopes A, Mould T, Murdoch J, Oram D, Rabideau DJ, Reynolds K, Scott I, Seif MW, Sharma A, Singh N, Taylor J, Warburton F, Widschwendter M, Williamson K, Woolas R, Fallowfield L, McGuire AJ, Campbell S, Parmar M, Skates SJ. Ovarian cancer screening and mortality in the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS): a randomised controlled trial. Lancet. 2016 Mar 5;387(10022):945-956. doi: 10.1016/S0140-6736(15)01224-6. Epub 2015 Dec 17. Erratum in: Lancet. 2016 Mar 5;387(10022):944. Erratum in: Lancet. 2016 Mar 5;387(10022):944 Nauseef WM. Biological roles for the NOX family NADPH oxidases. J Biol Chem. 2008 Jun 20;283(25):16961-5 Kleniewska P, Piechota A, Skibska B, Gorąca A. The NADPH oxidase family and its inhibitors. Arch Immunol TherExp (Warsz). 2012 Aug;60(4):277-94 Katsuyama M, Matsuno K, Yabe-Nishimura C. Physiological roles of NOX/NADPH oxidase, the superoxide-generating enzyme. J ClinBiochemNutr. 2012 Jan;50(1):9-22 Fukuyama M, Rokutan K, Sano T, Miyake H, Shimada M, Tashiro S. Overexpression of a novel superoxide-producing enzyme, NADPH oxidase 1, in adenoma and well differentiated adenocarcinoma of the human colon. Cancer Lett. 2005 Apr 18;221(1):97-104 Lim SD, Sun C, Lambeth JD, Marshall F, Amin M, Chung L, Petros JA, Arnold RS. Increased Nox1 and hydrogen peroxide in prostate cancer. Prostate. 2005 Feb 1;62(2):200-7 Laurent E, McCoy JW 3rd, Macina RA, Liu W, Cheng G, Robine S, Papkoff J, Lambeth JD. Nox1 is over-expressed in human colon cancers and correlates with activating mutations in K-Ras. Int J Cancer. 2008 Jul 1;123(1):100-7 Desouki MM, Kulawiec M, Bansal S, Das GM, Singh KK. Cross talk between mitochondria and superoxide generating NADPH oxidase in breast and ovarian tumors. Cancer Biol Ther. 2005 Dec;4(12):1367-73 Komiya M, Fujii G, Miyamoto S, Takahashi M, Ishigamori R, Onuma W, Ishino K, Totsuka Y, Fujimoto K, Mutoh M. Suppressive effects of the NADPH oxidase inhibitor apocynin on intestinal tumorigenesis in obese KK-A(y) and Apc mutant Min mice. Cancer Sci. 2015 Nov;106(11):1499-505. Arulselvan P, Wen CC, Lan CW, Chen YH, Wei WC, Yang NS. Dietary Administration of Scallion Extract Effectively Inhibits Colorectal Tumor Growth: Cellular and Molecular Mechanisms in Mice. PLoS ONE. 2012; 7(9): e44658. Suzuki S, Shiraga K, Sato S, Punfa W, Naiki-Ito A, Yamashita Y, Shirai T, Takahashi S. Apocynin, an NADPH oxidase inhibitor, suppresses rat prostate carcinogenesis. Cancer Sci. 2013 Dec;104(12):1711-7 Klees RF, De Marco PC, Salasznyk RM, Ahuja D, Hogg M, Antoniotti S, Kamath L, Dordick JS, Plopper GE. Apocynin derivatives interrupt intracellular signaling resulting in decreased migration in breast cancer cells. J Biomed Biotechnol. 2006;2006(2):87246 Zhao W, Lao Y, Liu Y, Niu J, Xiao Z, Arulselvan P, Shen J. Escin induces apoptosis in ovarian cancer cell line by triggering S-phase cell cycle arrest and p38 MAPK/ERK pathway inhibition. J. King Saud Uni – Sci. 34(1), 2022, 101644. Chrissobolis S, Faraci FM. The role of oxidative stress and NADPH oxidase in cerebrovascular disease. Trends Mol Med. 2008;14:495–502 Kim J, Kim J, Bae JS. ROS homeostasis and metabolism: a critical liaison for cancer therapy. Exp Mol Med. 2016 Nov 4;48(11):e269 Gius D, Spitz DR. Redox signaling in cancer biology. Antioxid Redox Signal. 2006;8:1249–1252 Galaris D, Mantzaris M, Amorgianiotis C. Oxidative stress and aging: the potential role of iron. Hormones (Athens) 2008;7:114–122 Rojas A, Silva R, Figueroa H, Morales MA. Oxidative stress in tumor microenvironment--Its role in angiogenesis. ZhongguoFei Ai Za Zhi. 2008;11:297–305 Giannoni E, Parri M, Chiarugi P. EMT and oxidative stress: a bidirectional interplay affecting tumor malignancy. Antioxid Redox Signal. 2012;16:1248–1263 Kumar D, Mutreja I, Chitcholtan K, Sykes P. Cytotoxicity and cellular uptake of different sized gold nanoparticles in ovarian cancer cells. Nanotechnology . 2017;28(47):475101 Edlich F. BCL-2 proteins and apoptosis: recent insights and unknowns. BiochemBiophys Res Commun . 2018;500(1):26–34 Lord CJ, Ashworth A. The DNA damage response and cancer therapy. Nature. 2012 Jan 18;481(7381):287-94 Perillo B, Di Donato M, Pezone A, Di Zazzo E, Giovannelli P, Galasso G, Castoria G, Migliaccio A. ROS in cancer therapy: the bright side of the moon. Exp Mol Med. 2020 Feb;52(2):192-203 Kuczler MD, Olseen AM, Pienta KJ, Amend SR. ROS-induced cell cycle arrest as a mechanism of resistance in polyaneuploid cancer cells (PACCs). ProgBiophysMol Biol. 2021 Oct;165:3-7 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-4685264","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":333610651,"identity":"1887954e-691e-405b-af71-73021acd11e0","order_by":0,"name":"Qian Zhu","email":"","orcid":"","institution":"Xi'an International Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Zhu","suffix":""},{"id":333610652,"identity":"3ea968ca-c242-45c0-a9e2-a03c842ce4c7","order_by":1,"name":"Xuyuan Tang","email":"","orcid":"","institution":"Xi'an International Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Xuyuan","middleName":"","lastName":"Tang","suffix":""},{"id":333610653,"identity":"2c8e227a-f1f5-453d-b9ec-9c3f3242f5e3","order_by":2,"name":"Ke Wang","email":"","orcid":"","institution":"Xi'an International Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Wang","suffix":""},{"id":333610654,"identity":"b94d6e7c-615c-4ef0-b32e-97d8039091eb","order_by":3,"name":"Wei Cheng","email":"","orcid":"","institution":"Xi'an International Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Cheng","suffix":""},{"id":333610655,"identity":"8546d5f8-9714-4425-a1c0-a76f97d04d09","order_by":4,"name":"Dongmei Zhou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYDCCA2BSgsGAgYHxwYcKIJuZuYFoLcyGM86AtDASpYUBpIVNmLcNxCSghe947+FXNyos7M3Ze8wYeOfVRvO3A7X8qNiGU4vkmXNp1jlnJJgte86YPZDcdjx3xmHGBsaeM7dxajG4kWNmnNsmwQZkmBsYbjuW2wDUwszYRkjLPwkeEEMicc6x3PlEaDF+nNsgIQHWcrChJncDIS2SZ86YMecckzAwOHOs2LDh2IHcjUAtB/H5he94j/HnnJo6e4PjzRsf/6mpy513/vDBBz8qcGsBAjYJJM5hMHkAn3ogYP6AxKkjoHgUjIJRMApGIgAAN15fnxquS/QAAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0005-8681-5589","institution":"Xi'an International Medical Center","correspondingAuthor":true,"prefix":"","firstName":"Dongmei","middleName":"","lastName":"Zhou","suffix":""},{"id":333610656,"identity":"cfdcbe2a-7b36-4fe8-a4d4-21813af6576d","order_by":5,"name":"Yanhong Huang","email":"","orcid":"","institution":"Xi'an International Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Yanhong","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2024-07-04 09:07:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4685264/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4685264/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63262066,"identity":"0e18c28f-81c7-4754-b408-06eaa16de06e","added_by":"auto","created_at":"2024-08-26 09:21:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":466913,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eApocynin significantly induced cytotoxic effect in A2780 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Percentage of viable A2780 human ovarian cancer cells treated with different concentrations of apocynin for 24h. B) Percentage of viable Vero normal epithelial cells treated with different concentrations of apocynin for 24h. Data analysis was done with SPSS 11.0 software. One Way ANOVA and Student’s t test were done to determine statistical significance. Statistical significance *p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4685264/v1/d6b3038c94d8c171222e9b64.png"},{"id":63262992,"identity":"7e09416b-e360-434d-a870-37e7a9cb4a77","added_by":"auto","created_at":"2024-08-26 09:29:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":235526,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eApocynin decreased antioxidant levels in A2780 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eA) TBAR levels B) Enzymatic antioxidant SOD and C) Non-enzymatic antioxidant glutathione levels in A2780 human ovarian cancer cells treated with apocynin and doxorubicin 24h. Data analysis was done with SPSS 11.0 software. One Way ANOVA and Student’s t test were done to determine statistical significance. Statistical significance *,**p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4685264/v1/de5025c42e1e1a1439a4f780.png"},{"id":63262993,"identity":"5d301238-1b1b-4bcf-807d-51b47f1241d0","added_by":"auto","created_at":"2024-08-26 09:29:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":620298,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eApocynin increased reactive oxygen species in A2780 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative microphotograph of DCHF-DA stained A2780 human ovarian cancer cells treated with apocynin and doxorubicin 24h. Data analysis was done with SPSS 11.0 software. One Way ANOVA and Student’s t test were done to determine statistical significance. Statistical significance *,**p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4685264/v1/df77ea2f261c01852d2fc55d.png"},{"id":63262994,"identity":"51a8028c-ba86-4710-b437-f0743b6cb44d","added_by":"auto","created_at":"2024-08-26 09:29:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":796352,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eApocynin decreased mitochondrial membrane potential in A2780 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative microphotograph of Rhodamine 123 stained A2780 human ovarian cancer cells treated with apocynin and doxorubicin 24h. Data analysis was done with SPSS 11.0 software. One Way ANOVA and Student’s t test were done to determine statistical significance. Statistical significance *,**p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4685264/v1/275ca35f9c6b02f324c59bb4.png"},{"id":63262068,"identity":"483d6823-4c95-46c6-ab7d-41d7a1019d8c","added_by":"auto","created_at":"2024-08-26 09:21:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1275903,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eApocynin induced apoptosis in A2780 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative microphotograph of AO/EtBr stained A2780 human ovarian cancer cells treated with apocynin and doxorubicin 24h. Data analysis was done with SPSS 11.0 software. One Way ANOVA and Student’s t test were done to determine statistical significance. Statistical significance *,**p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4685264/v1/b1bfff1525cd057d55cca665.png"},{"id":63262071,"identity":"c233e434-429d-44ac-80f8-dfc5e57497c4","added_by":"auto","created_at":"2024-08-26 09:21:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1610571,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eApocynin inhibited cell migration in A2780 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative microphotograph of wound scratched A2780 human ovarian cancer cells treated with apocynin and doxorubicin 24h.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4685264/v1/2d844ce0b77699ea425f2713.png"},{"id":63262069,"identity":"bd5d6c0f-cbca-4324-9c7f-6d7c4338c0f4","added_by":"auto","created_at":"2024-08-26 09:21:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":176315,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eApocynin inhibits cell proliferation in A2780 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eResults of Flow cytometric analysis of A2780 human ovarian cancer cells treated with apocynin and doxorubicin 24h. Data analysis was done with SPSS 11.0 software. One Way ANOVA and Student’s t test were done to determine statistical significance. Statistical significance *,**p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4685264/v1/14cd534f8bc46df6808a48ee.png"},{"id":63262072,"identity":"ee974e31-21d4-485e-853e-cb418b6e7c8f","added_by":"auto","created_at":"2024-08-26 09:21:50","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":345570,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eApocynin stimulates apoptotic protein synthesis in A2780 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eA) Caspase 3 B) Caspase 9 C) Bcl2 D) Bax levels in A2780 human ovarian cancer cells treated with apocynin and doxorubicin 24h. Apoptotic protein levels were quantified with ELISA technique. Data analysis was done with SPSS 11.0 software. One Way ANOVA and Student’s t test were done to determine statistical significance. Statistical significance *,**p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4685264/v1/b92240924f946f05c4739af2.png"},{"id":65212120,"identity":"42b81221-ebeb-4a1f-8a24-32aac85cecfd","added_by":"auto","created_at":"2024-09-24 20:40:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8084347,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4685264/v1/5c83abd8-8c4d-4211-839c-222aef180993.pdf"}],"financialInterests":"","formattedTitle":"Apocynin, a natural acetophenone suppresses cell proliferation, migration and induces cell cycle arrest, apoptosis in ovarian carcinoma cell line A2780","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOvarian cancer is a serious type of cancer that was mostly diagnosed during the advanced stage. Among gynecological-related malignancies, ovarian cancer is a severe disease and the most frequently diagnosed type of cancer in females [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The Global Ovarian Cancer Charter reported that globally in 2020, about 3.1 lakh women were estimated to be diagnosed with ovarian cancer, and about 2 lakh mortalities were reported due to ovarian cancer [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Women in the post-menopause period have a higher incidence of ovarian cancer compared to other females[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The symptoms are imprecise, such as bloating, abdominal pain, feeling satiety, and urgency in urination [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Since the symptoms are vague, 70% of ovarian cancer patients were diagnosed at an advanced stage [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The tumors that occurs in the ovaries, fallopian tubes, and primary peritoneal cavity are collectively named ovarian cancer. 90% of ovarian cancer arises from the ovarian epithelial cells, which is high-grade serous ovarian cancer [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOther than age, family history, hormonal and reproductive disorders, race, and lifestyle also determine the occurrence of ovarian cancer [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Genetic mutations in BRCA1, BRCA2, and TP53 were exhibited in 10\u0026ndash;15% of ovarian cancer patients [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Since the symptoms are common and there are no particular screening tests for ovarian cancer, treating it is a greater task. Surgery followed by chemotherapy used to be the first-line treatment given to patients diagnosed with ovarian cancer [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Due to intra-tumor heterogeneity, high-grade serous ovarian cancer shows resistance to chemotherapy drugs such as paclitaxel, carboplatin, olaprib,etc. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. If diagnosed at the early stage, the five-year survival rate of ovarian cancer is about 93%, whereas at the advanced stage, due to chemotherapy resistance the five-year survival rate is 29% [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Since ovarian cancer is mostly diagnosed at the advanced stage, an effective drug is needed today to treat it.\u003c/p\u003e \u003cp\u003eCancer chemotherapy is staged into three categories based on the patient\u0026rsquo;s risk and disease stage. Primary chemotherapy is a preventive measure given to the high-risk population with a genetic predisposition to cancer. Secondary chemotherapy treatment was given to inhibit the progression of cancer in patients at an early stage. Tertiary chemotherapy was given to the patients to prevent cancer relapse [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Phytochemicals play a substantial role in providing primary chemotherapy,and they are also reported to render secondary and tertiary chemotherapy [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Apocynin, 4-hydroxy-3-methoxy-acetophenone, is a polyphenolic compound present in extracts of Picrorhizakurroa andApocynumcannabinum used to treat cardiac, hepatic, respiratory, and rheumatoid illnesses in Ayurveda treatment [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Apocynin is a potent NADPH oxidase inhibitor that provides anti-inflammatory [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], neuroprotective [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and anti-ageing effects [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Apocynin has been reported to suppress cancer proliferation in in vitro and animal models[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].The ameliorative effect of apocynin on most lethal cancers, like ovarian cancer,has not yet been elucidated.In this study, we analyzed the anticancer effect of apocynin on ovarian cancer cell proliferation and migration in an in vitro model.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eApocynin, doxorubicin, and the reagents MTT and DMSO were procured from Sigma Aldrich, USA. Cell culture medium (DMEM, RPMI-1640), culture reagents, antibiotic-anitmycotic solutions, and Trypsin EDTA were purchased from ThermoFischer Scientific, USA. Staining kits DCFH-DA, Acridine Orange, Ethidium Bromide, and Rhodamine 123 were also procured from Sigma Aldrich, USA. Only analytical-grade chemicals and reagents were used for the experiments.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell line culture\u003c/h2\u003e \u003cp\u003eHuman ovarian carcinoma A2780 cells and non-malignant Vero cells were purchased from ATCC, USA. The cells were examined for contamination and cell growth under a light microscope before incubating in a CO2 incubator. The medium was replaced with 10% FBS-supplemented RPMI-1640 and incubated for 24h at 37\u0026ordm;C in a 5% CO2 incubator. The cells were trypsinized with Trypsin-EDTA and subcultured for further experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCytotoxicity analysis of apocynin\u003c/h2\u003e \u003cp\u003eThe cytotoxicity effect of apocynin was examined with the ovarian carcinoma A2780 human cell line and normal epithelial Vero cell line. A2780 cell lines were cultured in 10% FBS-supplemented RPMI-1640 medium, whereas Vero cells were cultured in DMEM medium. Both cells were treated with different concentrations of apocynin ranging from 0\u0026ndash;15 \u0026micro;M. The drug-treated cells were incubated for 24h at 37\u0026ordm;C in a 5%CO2 incubator and then subjected to MTT analysis. The cells were then treated with 5mg/ml of freshly prepared MTT solution and incubated for 3h at 37\u0026ordm;C in a 5%CO2 incubator. The formazan crystals formed due to the cleavage of MTT by viable cells were measured by dissolving the crystals with DMSO. The final absorbance of the solution was measured at 540nm. The experiments were performed in triplicate, and the percentage of cell viability with different dosages of apocynin was calculated. The IC50 value was calculated, and the dose was used for the analysis of further experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of antioxidant property of apocynin\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eTBARS assay\u003c/h2\u003e \u003cp\u003eLipid peroxidation in cells induced by oxidative stress was quantified by measuring the final end product, TBARS. A2780 ovarian carcinoma cells were treated with 7.5\u0026micro;M of apocynin and 2\u0026micro;g of the standard drug doxorubicin for 24h and then subjected to TBARS quantification. TBARS were quantified using the MDA assay kit (ab233471), purchased from Abcam, USA. The final reaction mixture was incubated at room temperature for 45 minutes. The end red product formed due to the reaction of thiobarbituric acid and malondialdehyde was measured at 695nm.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSuperoxide dismutase assay\u003c/h2\u003e \u003cp\u003eThe antioxidant SOD levels in apocynin-treated A2780 cells were measured with a superoxide dismutase kit procured from Sigma Aldrich, USA. WST-1, upon reduction with superoxide anion, forms formazan dye, whereas the rate of reduction with superoxide anion is linear to xanthine oxidase activity, which is inhibited by SOD. Hence, the SOD inhibition activity was measured by incubating the final mixture at 37\u0026ordm;C for 20min and then measured at 450nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGlutathione assay\u003c/h2\u003e \u003cp\u003eGlutathione levels in apocynin- and doxorubicin-treated A2780 cells were quantified using a commercially available colorimetric kit procured from ThermoFischer Scientific, USA. The assay was performed according to the manufacturer\u0026rsquo;s guidelines. The final absorbance was measured at 405nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDCFH-DA staining\u003c/h2\u003e \u003cp\u003eApocynin treated A2780 cells were subjected to 2\u0026prime;,7\u0026prime;-dichlorodihydrofluorescein diacetate (DCFH-DA) staining to quantify the reactive oxygen species generated. A2780 cells were treated for 24h with 7.5\u0026micro;M apocynin, and as a positive control a set of cells were treated with 2\u0026micro;g of the standard anticancer drug doxorubicin. The control and drug-treated cells were stained with DCFDA/H2DCFDA (Cellular ROS Assay Kit, Abcam, USA). The cells were viewed under a fluorescent microscope, and the fluorescence intensity was quantified with ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eRhodamine 123 staining\u003c/h2\u003e \u003cp\u003eRhodamine 123 staining was performed on apocynin-treated A2780 cells to analyze the mitochondrial membrane potential of the cells. A2780 cells were treated with apocynin for 24h and then subjected to rhodamine 123 staining. The drug-treated cells were washed with PBS and stained with rhodamine 123 for 30 min in the dark. The cells were then viewed undera fluorescent microscope, and the intensity of fluorescence was quantified with ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAO/EtBr staining\u003c/h2\u003e \u003cp\u003eA dual staining technique with acridine orange and ethidium bromide was used to analyze the percentage of live and dead cells after apocynin treatment. A2780 cells were treated with 7.5\u0026micro;M of apocynin and 2\u0026micro;g of doxorubicin for 24h. Then the cells were stained with a cocktail mixture of AO and EtBr which was prepared in an equal ratio. The stained cells were incubated in the dark for 5min and then viewed under a fluorescent microscope. The fluorescence intensity was quantified with ImageJ software to assess the percentage of live and dead cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWound scratch assay\u003c/h2\u003e \u003cp\u003eA2780 cells were cultured on 6-wellplate and upon attaining 100% confluency, the cells were scratched horizontally with the tip of a sterile micropipette at the center of the wells to generate a wound on the cultured cells. The cells were then treated with 7.5\u0026micro;M of apocynin and 2\u0026micro;g of doxorubicin and incubated for 24h. The cells were then viewed under a microscope to assess the cell migration of control and drug-treated cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCell cycle analysis\u003c/h2\u003e \u003cp\u003eAnnexin V/PI flow cytometry analysis was done to analyze the apocynin-induced apoptosis in ovarian carcinoma A2780 cells. The cells were treated with 7.5\u0026micro;M of apocynin and 2\u0026micro;g of doxorubicin and incubated for 24h. The cells were harvested and subjected to ethanol fixing. The ethanol-fixed cells were stained with a propidium iodide solution for 30min and then scanned with FACSCanto II and BD FACSDiva software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of apoptotic markers\u003c/h2\u003e \u003cp\u003eOvarian carcinoma A2780 cells were treated with apocynin and doxorubicin for 24h and subjected to quantification of caspase 3, caspase 9, bax, and bcl2 using a commercially available ELISA kit from Abcam USA. The assay was done as per the guidelines provided in the kit, and the levels were calculated using the standard curve plot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll the results were presented as the mean value\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM of three independent observations done in triplicate. A one way analysis of variance followed by a Student\u0026rsquo;s t test was performed to analyze the statistical significance of the data. Statistical significance was considered to be with a difference p value of 0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eApocynin significantly induced cytotoxic effect in A2780 cells\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e depicts the results of the MTT assay performed with A2780 human ovarian cancer cells and Vero normal epithelial cells treated with different concentrations of apocynin. Apocynin significantly induced cell death in A2780 cells, whereas only a minimal percentage of cell death was reported in normal Vero cells. The minimal concentration of 1\u0026micro;M apocynin treatment showed 23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04% of cell death, whereas the highest concentration of 15\u0026micro;M apocynin treatment showed 85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08% of cell death in A2780 cells. The IC50 value of apocynin for A2780 cells was obtained at a concentration of 7.5\u0026micro;M. In Vero cells, even the highest concentration (15\u0026micro;M) apocynin treatment showed only 10.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09% cell death.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eApocynin decreased antioxidant levels in A2780 cells\u003c/h2\u003e \u003cp\u003eAntioxidants are prime players in cell death and proliferation; excess synthesis of antioxidants leads to uncontrolled proliferation of cells. Apocynin treatment significantly decreased the levels of enzymatic antioxidant SOD to 27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 units/mg protein, whereas it was 44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 units/mg protein in control cells. It also decreased the levels of the non-enzymatic antioxidant glutathione to 21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 units/mg protein, whereas it was 37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 units/mg protein in control cells. Apocynin treatment significantly increased the lipid peroxidation by 32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08nmol/mg protein, whereas it was less than 1 nmol/mg protein in control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eApocynin increased reactive oxygen species in A2780 cells\u003c/h2\u003e \u003cp\u003eIntracellular reactive oxygen species synthesized by apocynin in A2780 cells were quantified with fluorescent DHCF-DA stain, and the results are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Apocynin significantly increased the levels of ROS in A2780 cells, which was evidenced by a 38\u0026thinsp;\u0026plusmn;\u0026thinsp;2% fluorescent intensity compared to the control untreated cells. Standard drug doxorubicin treatment has shown a significantly increased fluorescent intensity of 64\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eApocynin decreased mitochondrial membrane potential in A2780 cells\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e illustrates the levels of mitochondrial membrane potential in control, apocynin-, and doxorubicin-treated A2780 cells. Apocynin treatment significantly decreased MMP levels in A2780 cells compared to the control cells. Compared to doxorubicin, the fluorescent intensity increased in apocynin-treated cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eApocynin induced apoptosis in A2780 cells\u003c/h2\u003e \u003cp\u003eThe induction of apoptosis in A2780 cells by apocynin was estimated with the dual staining technique, and the results are represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Control cells showed increased green fluorescence, indicating the presence of live cells, whereas the apocynin-treated cells showed increased levels of yellowish-orange fluorescence, indicating early apoptotic cells. Compared to doxorubicin, apocynin shows decreased levels of reddish-orange fluorescence, which indicates late apoptotic cells. Apocynin treatment induced 34\u0026thinsp;\u0026plusmn;\u0026thinsp;2% cell death in A2780 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eApocynin inhibited cell migration in A2780 cells\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e represents the results of the wound scratch assay performed on control and apocynin-treated cells. Apocynin treatment effectively inhibited the migration of A2780 cells compared to the control. It was evident from our microscopic image that only a few cells were observed in the scratched site of apocynin-treated cells, whereas numerous cells can be seen in the scratched site of control cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eApocynin inhibits cell proliferation in A2780 cells\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e represents the flow cytometric analysis was done in control. Apocynin and doxorubicin treated cells to analyze the effect of drugs on cell proliferation. Apocynin treatment retained the cells in G0 andG1, compared to the control cells.A decrease in cells with S-phase and G2-phase was observed in the apocynin-treated cells. Doxorubicin significantly decreased the S-phase and G2 phase cells compared to the control and the apocynin-treated cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eApocynin stimulates apoptotic protein synthesis in A2780 cells\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e illustrates the levels of apoptotic proteins Caspase 3, Caspase 9, Bcl2, and Baxin apocynin-treated A2780 cells. Apocynin treatment significantly increased the levels of both caspases 3 and 9 in A2780 cells. It significantly decreased the levels of the antiapototic protein Bcl2 and increased the proapoptoticBax levels in A2780 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOvarian cancer remains the most common cause of gynecological-related mortalities in post-menopausal women. 75% of ovarian cancers were diagnosed at the advanced stage. Debulking surgery and platinum-based chemotherapy are the first lines of treatment provided to ovarian cancer patients [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. A combination of platinum, along with paclitaxel, olaprib, bevacizumab, and niraparib, are some of the drugs prescribed for treating ovarian cancer. In the initial stage, the patient responds to these drugs, whereas later, due to platinum resistance, 80% of the patients are prone to relapses of cancer, and their survival is questionable [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. At present, targeted therapies are the new hope for treating ovarian cancer, but the recurrence rate of cancer is still in considerable numbers [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNicotinamide adenine dinucleotide phosphate oxidase (NOX) is an enzyme with multiple subunits expressed in various tissues throughout the body. The prime function of NOX is to catalyze the generation of free radicals to facilitate adaptive immunity [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. NOX were classified into two categories based on their activity: phagocyte-derived and non-phagocyte-derived NADPH oxidase. Overexpression of non-phagocytotic-derived NADPH oxidase, specifically the NOX-1 subtype, was observed in various cancer cells [\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Ovarian cancer cells are also reported to overexpress NOX-1; hence, inhibiting NOX-1 may suppress cancer cell proliferation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Apocynin, a NOX inhibitor, had been reported to suppress intestinal tumorigenesis in obese mice [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], prostate cancer [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], breast cancer [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], and bladder cancer [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In this study, we investigated the anticancer effect of apocycin against ovarian cancer cells and normal epithelial cells. Apocynin treatment significantly inhibited the cell proliferation of ovarian cancer cells (A2780), whereas a only minimal percentage of cell death was observed in normal epithelial vero cells. This confirms the targeted cell toxicity of apocynin.\u003c/p\u003e \u003cp\u003eOxidative stress created due to a reactive oxygen species imbalance imparts a dual response in cells. At the physiological level, ROS is the prime signaling pathway that regulates the growth, proliferation, metabolism, and apoptosis of cells [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], whereas excessive generation of ROS during pathogenic conditions hinders normal cellular functions [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Generation and inhibition of these reactive oxygen species play a vital role in treating various inflammatory diseases, including cancer [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Induction of ROS generation in ovarian cancer is proposed to render an anticancer effect by causing membrane lipid peroxidation, altering the genetic material, and inducing apoptosis in cancer cells [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Our study also correlated with the previous findings that apocynin treatment significantly increased lipid peroxidation in the A2780 cells via decreasing the enzymatic antioxidant superoxide and the non-enzymatic antioxidant glutathione levels, thereby causing apoptosis.\u003c/p\u003e \u003cp\u003eFurther, to confirm the apocynin-induced free radical generation-mediated apoptosis in A2780 cells, the apocynin-treated cells were stained with DCHF-DA stain, Rhodamine 123, and AO/EtBr stain. Our staining proves apocynin treatment significantly increased reactive oxygen species generation, thereby decreasing mitochondrial membrane potential and leading to cellular apoptosis. The mitochondrial disruption triggers the apoptotic proteins, both the pro-apoptotic proteins Bid, Bax, andcaspases, and the anti-apoptotic protein Bcl2. The anti-apoptotic protein Bcl2 prevents the mitochondrial release of apoptosis-inducing factors and also caspase activities. Hence, targeting Bcl2 may promote apoptosis in cancer cells [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. In our study, apocynin treatment significantly decreased the levels of Bcl2 protein and also increased the levels of pro-apoptotic proteins Bax and caspases. The increased cell death observed in AO/EtBr-stained apocynin-treated A2780 cells may be due to the increase in the pro-apoptotic protein and the decrease in the anti-apoptotic protein Bcl2 levels.\u003c/p\u003e \u003cp\u003eAnti-cancer treatment disrupts the homeostasis of the cells by generating reactive oxygen species, thereby damaging the nuclear content [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The oxidative stress created causes a damaged DNA response, which shifts the cells to cell cycle arrest and eventually leads to apoptosis [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Our flow cytometric analysis of apocynin-treated cells confirmed the cell cycle arrest in the A2780 cell. Apocynin treatment retained an increased number of A2780 cells in the G0/G1phase, and a decreased number of cells were observed in the S and G2/M phases compared to the control cells. Apart from inhibition of cell proliferation and induction of apoptosis, apocynin treatment also inhibited cell migration of A2780, as evidenced by our wound scratch assay.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eApocynin, a natural acetophenone, was examined for its anticancer effect against ovarian carcinoma A2780 cells. Apocynin significantly inhibited the cell proliferation of ovarian carcinoma A2780 cells, and a minimal cytotoxic effect was observed in normal epithelial verocells. Apocynin treatment significantly generated ROS and decreased antioxidant levels in A2780, which in turn induced cell cycle arrest and eventually apoptosis in A2780. It also potently inhibited the cell migration property of A2780. All together, our results prove apocynin is a potent anticancer agent that effectively induces apoptosis in in vitro conditions. Further studies may be conducted to formulate apocynin as a drug to treat ovarian cancer.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe consent to the publication of the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution: \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eQian Zhu, Dongmei Zhou\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e: \u003c/strong\u003eConceptualization, Investigation, Formal analysis, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003e\u003cem\u003eKe Wang, Xuyuan Tang\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e: \u003c/strong\u003eFormal analysis, Validation, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003e\u003cem\u003eYanhong Huang\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e: \u003c/strong\u003eValidation, Writing \u0026ndash; review \u0026amp; editing. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXi\u0026apos;an International Medical Center Hospital, Youth project Funding number 2020QN010.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing of interests. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data analyzed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eCoburn SB, Bray F, Sherman ME, Trabert B. International patterns and trends in ovarian cancer incidence, overall and by histologic subtype. Int J Cancer. 2017 Jun 1;140(11):2451-2460\u003c/li\u003e\n \u003cli\u003eMomenimovahed Z, Tiznobaik A, Taheri S, Salehiniya H. Ovarian cancer in the world: epidemiology and risk factors. Int J Womens Health. 2019 Apr 30;11:287-299\u003c/li\u003e\n \u003cli\u003eGaona-Luviano P, Medina-Gaona LA, Maga\u0026ntilde;a-P\u0026eacute;rez K. Epidemiology of ovarian cancer. Chin Clin Oncol. 2020 Aug;9(4):47\u003c/li\u003e\n \u003cli\u003eNash Z, Menon U. Ovarian cancer screening: current status and future directions. Best Pract Res ClinObstetGynaecol. 2020;65:32- 45.\u003c/li\u003e\n \u003cli\u003eFrances R \u0026amp; Anmol B. Global Trends in Incidence, Mortality, and Survival. The World Ovarian Cancer Coalition Atlas 2023. 2023: 1-65\u003c/li\u003e\n \u003cli\u003eLengyel E. Ovarian cancer development and metastasis. Am J Pathol. 2010 Sep;177(3):1053-64\u003c/li\u003e\n \u003cli\u003eGoff BA, Mandel LS, Melancon CH, Muntz HG. Frequency of Symptoms of Ovarian Cancer in Women Presenting to Primary Care Clinics.\u0026nbsp;JAMA.\u0026nbsp;2004; 291(22):2705\u0026ndash;2712\u003c/li\u003e\n \u003cli\u003eGoff BA, Mandel LS, Drescher CW, Urban N, Gough S, Schurman KM, Patras J, Mahony BS, Andersen MR. Development of an ovarian cancer symptom index: possibilities for earlier detection. Cancer. 2007 Jan 15;109(2):221-7\u003c/li\u003e\n \u003cli\u003eBray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018 Nov;68(6):394-424. doi: 10.3322/caac.21492. Epub 2018 Sep 12. Erratum in: CA Cancer J Clin. 2020 Jul;70(4):313\u003c/li\u003e\n \u003cli\u003eKurman RJ, Carcangiu ML, Herrington CS, et al. \u003cem\u003eWHO classification of tumours of the female reproductive organs.\u003c/em\u003e Lyon: WHO Press; 2014.\u003c/li\u003e\n \u003cli\u003eLu KH, Daniels M. Endometrial and ovarian cancer in women with Lynch syndrome: update in screening and prevention. Fam Cancer. 2013;12(2):273-277. Doi:10.1007/s10689-013-9664-5. 41\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMomenimovahed Z, Tiznobaik A, Taheri S, Salehiniya H. Ovarian cancer in the world: epidemiology and risk factors.\u0026nbsp;Int J Womens Health. 2019;11:287\u0026ndash;299. Published 2019 Apr 30. doi:10.2147/IJWH.S197604\u003c/li\u003e\n \u003cli\u003eBast RC Jr, Hennessy B, Mills GB. The biology of ovarian cancer: new opportunities for translation. Nat Rev Cancer. 2009 Jun;9(6):415-28\u003c/li\u003e\n \u003cli\u003eFranzese E, Diana A, Centonze S, Pignata S, De Vita F, Ciardiello F, Orditura M. PARP Inhibitors in First-Line Therapy of Ovarian Cancer: Are There Any Doubts? Front Oncol. 2020 Jun 12;10:782\u003c/li\u003e\n \u003cli\u003eHaddad FG, Karam E, Moujaess E, Kourie HR. Poly-(ADP-ribose) polymerase\u0026nbsp;inhibitors: paradigm shift in the first-line treatment of newly diagnosed advanced ovarian cancer. Pharmacogenomics. 2020 Jul;21(10):721-727\u003c/li\u003e\n \u003cli\u003eIngles Russo Garces AH. Drug-induced evolutionary dynamics in BRCA-mutant/nonmutant ovarian cancer models. Abstract 1697P. ESMO Congress 2022, 10 September, 2022.\u003c/li\u003e\n \u003cli\u003eLukanović D, Herzog M, Kobal B, Černe K. The contribution of copper efflux transporters ATP7A and ATP7B to chemoresistance and personalized medicine in ovarian cancer. Biomed Pharmacother. 2020 Sep;129:110401\u003c/li\u003e\n \u003cli\u003eJinghua L, Linmei S, Ping H, Gothai S, Muniandy K, Kumar SS, Esa NM, Arulselvan P. Amelioration of oxidative stress through apoptosis-mediated pathway in colon cancer cells by hexane fraction of Moringa oleifera extract. Phcog Mag 2018;14:S311-8.\u003c/li\u003e\n \u003cli\u003eDe Flora S, Ferguson LR. Overview of mechanisms of cancer chemopreventive agents. Mutat Res. 2005 Dec 11;591(1-2):8-15\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eRanjan A, Ramachandran S, Gupta N, Kaushik I, Wright S, Srivastava S, Das H, Srivastava S, Prasad S, Srivastava SK. Role of Phytochemicals in Cancer Prevention. Int J Mol Sci. 2019 Oct 9;20(20):4981\u003c/li\u003e\n \u003cli\u003eRam VJ. Herbal preparations as a source of hepatoprotective agents. Drug News Perspect. 2001; 14:353\u0026ndash;363\u003c/li\u003e\n \u003cli\u003eStefanska J, Pawliczak R. Apocynin: molecular aptitudes. Mediators Inflamm. 2008;2008:106507\u003c/li\u003e\n \u003cli\u003eSavla SR, Laddha AP, Kulkarni YA. Pharmacology of apocynin: a natural acetophenone. Drug Metab Rev. 2021 Nov;53(4):542-562.\u003c/li\u003e\n \u003cli\u003eQin YY, Li M, Feng X, Wang J, Cao L, Shen XK, Chen J, Sun M, Sheng R, Han F, Qin ZH. Combined NADPH and the NOX inhibitor apocynin provides greater anti-inflammatory and neuroprotective effects in a mouse model of stroke. Free Radic Biol Med. 2017 Mar;104:333-345\u003c/li\u003e\n \u003cli\u003eDu ZD, Yu S, Qi Y, Qu TF, He L, Wei W, Liu K, Gong SS. NADPH oxidase inhibitor apocynin decreases mitochondrial dysfunction and apoptosis in the ventral cochlear nucleus of D-galactose-induced aging model in rats. Neurochem Int. 2019 Mar;124:31-40\u003c/li\u003e\n \u003cli\u003eWang Q, Tompkins KD, Simonyi A, Korthuis RJ, Sun AY, Sun GY. Apocynin protects against global cerebral ischemia-reperfusion-induced oxidative stress and injury in the gerbil hippocampus. Brain Res. 2006 May 23;1090(1):182-9\u003c/li\u003e\n \u003cli\u003eJantaree P, Lirdprapamongkol K, Kaewsri W, Thongsornkleeb C, Choowongkomon K, Atjanasuppat K, Ruchirawat S, Svasti J. Homodimers of Vanillin and Apocynin Decrease the Metastatic Potential of Human Cancer Cells by Inhibiting the FAK/PI3K/Akt Signaling Pathway. J Agric Food Chem. 2017 Mar 22;65(11):2299-2306\u003c/li\u003e\n \u003cli\u003ePaul S, Chakrabarty S, Ghosh S, Nag D, Das A, Dastidar DG, Dasgupta M, Dutta N, Kumari M, Pal M, Chakrabarti G. Targeting cellular microtubule by phytochemical apocynin exhibits autophagy-mediated apoptosis to inhibit lung carcinoma progression and tumorigenesis. Phytomedicine. 2020 Feb;67:153152\u003c/li\u003e\n \u003cli\u003eSambasivan S. Epithelial ovarian cancer: Review article. Cancer Treat Res Commun. 2022;33:100629\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eOrtiz M, Wabel E, Mitchell K, Horibata S. Mechanisms of chemotherapy resistance in ovarian cancer. Cancer Drug Resist. 2022 Apr 3;5(2):304-316\u003c/li\u003e\n \u003cli\u003eDamia G, Broggini M. Platinum Resistance in Ovarian Cancer: Role of DNA Repair. Cancers (Basel). 2019 Jan 20;11(1):119\u003c/li\u003e\n \u003cli\u003eJacobs IJ, Menon U, Ryan A, Gentry-Maharaj A, Burnell M, Kalsi JK, Amso NN, Apostolidou S, Benjamin E, Cruickshank D, Crump DN, Davies SK, Dawnay A, Dobbs S, Fletcher G, Ford J, Godfrey K, Gunu R, Habib M, Hallett R, Herod J, Jenkins H, Karpinskyj C, Leeson S, Lewis SJ, Liston WR, Lopes A, Mould T, Murdoch J, Oram D, Rabideau DJ, Reynolds K, Scott I, Seif MW, Sharma A, Singh N, Taylor J, Warburton F, Widschwendter M, Williamson K, Woolas R, Fallowfield L, McGuire AJ, Campbell S, Parmar M, Skates SJ. Ovarian cancer screening and mortality in the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS): a randomised controlled trial. Lancet. 2016 Mar 5;387(10022):945-956. doi: 10.1016/S0140-6736(15)01224-6. Epub 2015 Dec 17. Erratum in: Lancet. 2016 Mar 5;387(10022):944. Erratum in: Lancet. 2016 Mar 5;387(10022):944\u003c/li\u003e\n \u003cli\u003eNauseef WM. Biological roles for the NOX family NADPH oxidases. J Biol Chem. 2008 Jun 20;283(25):16961-5\u003c/li\u003e\n \u003cli\u003eKleniewska P, Piechota A, Skibska B, Gorąca A. The NADPH oxidase family and its inhibitors. Arch Immunol TherExp (Warsz). 2012 Aug;60(4):277-94\u003c/li\u003e\n \u003cli\u003eKatsuyama M, Matsuno K, Yabe-Nishimura C. Physiological roles of NOX/NADPH oxidase, the superoxide-generating enzyme. J ClinBiochemNutr. 2012 Jan;50(1):9-22\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eFukuyama M, Rokutan K, Sano T, Miyake H, Shimada M, Tashiro S. Overexpression of a novel superoxide-producing enzyme, NADPH oxidase 1, in adenoma and well differentiated adenocarcinoma of the human colon. Cancer Lett. 2005 Apr 18;221(1):97-104\u003c/li\u003e\n \u003cli\u003eLim SD, Sun C, Lambeth JD, Marshall F, Amin M, Chung L, Petros JA, Arnold RS. Increased Nox1 and hydrogen peroxide in prostate cancer. Prostate. 2005 Feb 1;62(2):200-7\u003c/li\u003e\n \u003cli\u003eLaurent E, McCoy JW 3rd, Macina RA, Liu W, Cheng G, Robine S, Papkoff J, Lambeth JD. Nox1 is over-expressed in human colon cancers and correlates with activating mutations in K-Ras. Int J Cancer. 2008 Jul 1;123(1):100-7\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eDesouki MM, Kulawiec M, Bansal S, Das GM, Singh KK. Cross talk between mitochondria and superoxide generating NADPH oxidase in breast and ovarian tumors. Cancer Biol Ther. 2005 Dec;4(12):1367-73\u003c/li\u003e\n \u003cli\u003eKomiya M, Fujii G, Miyamoto S, Takahashi M, Ishigamori R, Onuma W, Ishino K, Totsuka Y, Fujimoto K, Mutoh M. Suppressive effects of the NADPH oxidase inhibitor apocynin on intestinal tumorigenesis in obese KK-A(y) and Apc mutant Min mice. Cancer Sci. 2015 Nov;106(11):1499-505.\u003c/li\u003e\n \u003cli\u003eArulselvan P, Wen CC, Lan CW, Chen YH, Wei WC, Yang NS. Dietary Administration of Scallion Extract Effectively Inhibits Colorectal Tumor Growth: Cellular and Molecular Mechanisms in Mice. PLoS ONE. 2012; 7(9): e44658.\u003c/li\u003e\n \u003cli\u003eSuzuki S, Shiraga K, Sato S, Punfa W, Naiki-Ito A, Yamashita Y, Shirai T, Takahashi S. Apocynin, an NADPH oxidase inhibitor, suppresses rat prostate carcinogenesis. Cancer Sci. 2013 Dec;104(12):1711-7\u003c/li\u003e\n \u003cli\u003eKlees RF, De Marco PC, Salasznyk RM, Ahuja D, Hogg M, Antoniotti S, Kamath L, Dordick JS, Plopper GE. Apocynin derivatives interrupt intracellular signaling resulting in decreased migration in breast cancer cells. J Biomed Biotechnol. 2006;2006(2):87246\u003c/li\u003e\n \u003cli\u003eZhao W, Lao Y, Liu Y, Niu J, Xiao Z, Arulselvan P, Shen J. Escin induces apoptosis in ovarian cancer cell line by triggering S-phase cell cycle arrest and p38 MAPK/ERK pathway inhibition. J. King Saud Uni \u0026ndash; Sci. 34(1), 2022, 101644.\u003c/li\u003e\n \u003cli\u003eChrissobolis S, Faraci FM. The role of oxidative stress and NADPH oxidase in cerebrovascular disease. \u003cem\u003eTrends Mol Med.\u0026nbsp;\u003c/em\u003e2008;14:495\u0026ndash;502\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eKim J, Kim J, Bae JS. ROS homeostasis and metabolism: a critical liaison for cancer therapy. Exp Mol Med. 2016 Nov 4;48(11):e269\u003c/li\u003e\n \u003cli\u003eGius D, Spitz DR. Redox signaling in cancer biology.\u0026nbsp;\u003cem\u003eAntioxid Redox Signal.\u0026nbsp;\u003c/em\u003e2006;8:1249\u0026ndash;1252\u003c/li\u003e\n \u003cli\u003eGalaris D, Mantzaris M, Amorgianiotis C. Oxidative stress and aging: the potential role of iron. \u003cem\u003eHormones (Athens)\u0026nbsp;\u003c/em\u003e2008;7:114\u0026ndash;122\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eRojas A, Silva R, Figueroa H, Morales MA. Oxidative stress in tumor microenvironment--Its role in angiogenesis. \u003cem\u003eZhongguoFei Ai Za Zhi.\u0026nbsp;\u003c/em\u003e2008;11:297\u0026ndash;305\u003c/li\u003e\n \u003cli\u003eGiannoni E, Parri M, Chiarugi P. EMT and oxidative stress: a bidirectional interplay affecting tumor malignancy. \u003cem\u003eAntioxid Redox Signal.\u0026nbsp;\u003c/em\u003e2012;16:1248\u0026ndash;1263\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eKumar D, Mutreja I, Chitcholtan K, Sykes P. Cytotoxicity and cellular uptake of different sized gold nanoparticles in ovarian cancer cells. \u003cem\u003eNanotechnology\u003c/em\u003e. 2017;28(47):475101\u003c/li\u003e\n \u003cli\u003eEdlich F. BCL-2 proteins and apoptosis: recent insights and unknowns. \u003cem\u003eBiochemBiophys Res Commun\u003c/em\u003e. 2018;500(1):26\u0026ndash;34\u003c/li\u003e\n \u003cli\u003eLord CJ, Ashworth A. The DNA damage response and cancer therapy. Nature. 2012 Jan 18;481(7381):287-94\u003c/li\u003e\n \u003cli\u003ePerillo B, Di Donato M, Pezone A, Di Zazzo E, Giovannelli P, Galasso G, Castoria G, Migliaccio A. ROS in cancer therapy: the bright side of the moon. Exp Mol Med. 2020 Feb;52(2):192-203\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eKuczler MD, Olseen AM, Pienta KJ, Amend SR. ROS-induced cell cycle arrest as a mechanism of resistance in polyaneuploid cancer cells (PACCs). ProgBiophysMol Biol. 2021 Oct;165:3-7\u003c/li\u003e\n\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":"Ovarian cancer, A2780 cells, Apocynin, Cell migration, Cell cycle arrest, Apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-4685264/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4685264/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, the ameliorative effects of apocynin against ovarian cancer cell proliferation, migration, and induction of apoptosis were studied in vitro. A2780 human ovarian carcinoma cells and Vero normal epithelial cells were treated with apocynin and subjected to cytotoxicity assays. Lipid peroxidation and antioxidant status were quantified in apocynin-treated A2780 cells to assess the anticancer effect of apocynin. Staining techniques with DCFH-DA, Rhodamine-123, and AO/EtBr were done to analyze the ROS-induced apoptosis in A2780 cells. A wound scratch assay was performed to examine the effect of apocynin on cell migration. Flow cytometric analysis was done to analyze cell cycle arrest in apocynin-treated A2780 cells. To confirm the apoptosis in apocynin-treated cells, the apoptotic proteins were quantified using kits. Apocynin treatment significantly inhibited growth andpromoted oxidative stress and apoptosis in A2780 cells. The results of fluorescent staining assays clearly state that apocynin increases ROS levels and thereby induces lipid peroxidation, which leads to cell death. Apocynin treatment caused cell cycle arrest and promoted apoptosis in A2780 cells, which were confirmed by the flow cytometry results and an increase in caspases, bax, and a decrease in bcl2 levels, respectively. Apocynin treatment also inhibited cell migration, which was evidenced by our wound scratch assay. Overall, our findings confirm that apocynin significantly inhibits cell proliferation, cell migration, and induced apoptosis in ovarian cancer A2780 cells.\u003c/p\u003e","manuscriptTitle":"Apocynin, a natural acetophenone suppresses cell proliferation, migration and induces cell cycle arrest, apoptosis in ovarian carcinoma cell line A2780","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-26 09:21:45","doi":"10.21203/rs.3.rs-4685264/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":"786728a3-720d-4901-aa0c-90ed584b8a54","owner":[],"postedDate":"August 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-24T20:32:29+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-26 09:21:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4685264","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4685264","identity":"rs-4685264","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}