Melatonin reduces cell motility and antioxidant defenses in ovarian cancer cell lines

Melatonin reduces cell motility and antioxidant defenses in ovarian cancer cell lines | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Melatonin reduces cell motility and antioxidant defenses in ovarian cancer cell lines Henrique Spaulonci Silveira, Roberta Carvalho Cesário, Vinicius Augusto Simão, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5924048/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 Ovarian cancer (OC), a highly recurrent and fatal tumor, poses diagnostic challenges due to generic symptoms and chemoresistance. Melatonin (Mel) is an indoleamine acting against tumor progression and exhibiting pro-oxidative actions in tumor cells. This study explores the impact of Mel on antioxidant defenses of OC cells (SKOV-3 and CAISMOV-24 lines), focusing on its receptor-dependent and -independent effects. Cell viability was assessed using the MTT method and the antioxidant system was analyzed by preparing supernatants for assessing glutathione (GS), reduced glutathione (GSH), oxidized glutathione (GSSG), catalase (CAT), glutathione S-transferase (GST), and superoxide dismutase (SOD). Mel stimulated its own intracellular levels, reducing cell viability in both cell lines. Notably, Mel independently of its membrane receptors, inhibited migration and invasion, thus showing its anti-tumoral potential. By investigating melatonin’s actions, we observed an impact on the antioxidant system primarily through the reduced activity of CAT and the GS axis. The modulation of these antioxidants by Mel demonstrates its multifaceted role in OC, emphasizing its therapeutic potential. We also demonstrated, for the first time, the theoretical ability of Mel to bind to CAT, which may be responsible for the reduction in enzyme activity. This study contributes with novel insights into Mel's receptor-independent actions, providing a foundation for further research in OC therapy. Ovarian cancer Melatonin Oxidative stress Antioxidant defense Catalase Cell invasion and migration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Ovarian cancer (OC) is one of the most common female tumor subtypes which has a high recurrence and mortality rate [ 1 ]. This mortality rate is associated with its late diagnosis due to multiple symptoms that are often generic and their aggressive recurrence even with already established treatments and surgical removal [ 2 , 3 ]. Modifications in cellular metabolism occur when tumors redefine how they obtain nutrients and navigate metabolic routes to fulfill the requirements for biological energy, duplication, and the control of reactive oxygen species (ROS) levels in the cancer cells examined [ 4 ]. These changes are recognized as key aspects of cancer metabolism, marked by features such as tissue invasion and metastasis, loss of growth inhibition, rapid proliferation, sustained cell viability, resistance against cell death, initiation of the angiogenesis pathway, evasion of the immune response, and modified cellular vitality [ 5 , 6 ]. Melatonin (Mel; N-acetyl-5 - methoxytryptamine) is a lipophilic molecule converted from serotonin by the enzymes arylalkylamine N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT) [ 7 ]. Mel has been characterized by actions that may either depend on or be independent of its membrane receptors (MT1 and MT2). While its main production occurs in the pineal gland at night, other reports have shown its synthesis in different tissues and cells with local actions [ 8 ]. Mel has numerous functions under normal metabolic conditions including its antioxidant actions by removing reactive oxygen and nitrogen species and stimulating DNA repair mechanisms [ 9 ]. In many cancer cell types, Mel has oncostatic functions associated with pro-oxidative function while concurrently reducing the migration and invasiveness of cancer cells and inducing apoptosis. [ 10 , 11 ]. Free radicals (FR) are a generic term that encompasses unstable and reactive atoms or molecules. In biological systems, FR can be separated into two classes: reactive oxygen species (ROS) and reactive nitrogen species (RNS) [ 12 ]. Superoxide anion (O2•−), hydroxyl radical (OH•), alkoxy radical (RO•), hydrogen peroxide (H 2 O 2 ), and hypochlorous acid are considered ROS and they are normally generated during cellular metabolism; nitrogen dioxide (NO 2 ), nitroxyl (HNO) and peroxynitrite (ONOO-) are examples of RNS produced from nitric oxide and by the NADPH oxidase (NOX) system [ 12 ]. In healthy cells, there is a delicate balance between the production and removal of FR, which ensures redox homeostasis and their optimal physiological effects [ 13 ]. FR affects gene expression, cell growth and differentiation, modulate metabolic reactions, and regulate transcription factor activation, in addition to acting as intra and intercellular signaling molecules [ 14 – 17 ]. In OC cell lines, there is initially a significant increase in ROS production, thus favoring tumorigenesis. This elevated ROS results from NADPH oxidase activity and mitochondrial metabolism, as this increase is diminished by mitochondrial complex I inhibitors [ 18 ]. Elevated ROS which occurs at the level of the electron transport chain (ETC) is associated with chemoresistance to platinum-derived therapies leading to a poor prognosis and accelerating OC growth [ 19 ]. In addition, the increase in ROS is associated with dysregulation of microRNAs (miRNAs) which are linked to tumor progression. In serous epithelial OC, there is overexpression of specific miRNAs, an action that does not occur in the non-serous OC [ 20 – 23 ]. To avoid the damage caused by ROS, cells have antioxidant systems which are either enzymatic or non-enzymatic in nature. Among the main anti-oxidative enzymes are superoxide dismutase (SOD), catalase (CAT,), glutathione peroxidase (GSH-Px), glutathione reductase (GSH-Rd), among others. It is important to consider that a specific antioxidant, to be efficient, does not necessarily need to be ubiquitous, but must act at the diffusion rate of the FR and be in the proper position in the immediate vicinity of where the radical is generated [ 24 ]. With the exception of H 2 O 2 , FR do not cross biological barriers and have a very short half-life. Mel is an antioxidant under normal circumstances but can serve a pro-oxidant under specific pathologies [ 25 ]. Although additional investigation is needed, Mel has been documented to promote an increase in ROS via different mechanisms [ 26 , 27 ]. In head and neck cancer cells, Mel induced ROS by reversing mitochondrial ETC which generated the anti-growth behavior [ 28 ]. Mel also induces apoptosis mediated by the stimulation of ROS synthesis in breast cancer as well as in mesangial cells, thus documenting that the anti-cancer actions of melatonin may be mediated by excessive FR generation effects [ 26 , 29 , 30 ]. To date, the role of Mel on oxidative processes in OC have not been examined. Herein, we investigated the impact of Mel, acting via its membrane receptors or not, on antioxidant defenses of OC cells as well as its protection against cell migration and invasiveness. Materials and Methods Cell culture and reagents SKOV-3 cell line (ATCC® HTB-77) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), while the CAISMOV-24 cell line was obtained from the Women’s Hospital Prof. Dr. José Aristodemo Pinotti Caism (UNICAMP, Campinas, SP, BRAZIL). SKOV-3 cells were cultivated using RPMI medium (Gibco, Paisley, UK), whereas CAISMOV-24 cells were nurtured in DMEN HAN’S F-12 (LGC, Cotia, BR). Both cell types received 10% fetal bovine serum (FBS) (Gibco) and penicillin at 100 IU/ml, as well as streptomycin at 100 µg/ml (Gibco). The cells were placed in a humidified environment at 37°C with 5% CO 2 . Cellular expansion was carried out in distinct 75 and 25 cm 2 culture flasks (Costar, Cambridge, MA, USA). Upon achieving 80% of confluence, the cell supernatant was carefully extracted. Afterwards, the cells underwent two washes with 10% phosphate-buffered saline (PBS; Oxoid Limited, Hampshire, UK). Subsequently, trypsin/EDTA (Gibco) was employed to interrupt adhesion to the flasks. Experimental design and treatments To examine the effects of Mel and its combination with luzindole (Luz) on OC cells, we first determined the safe concentrations according to the IC 50 method (MTT assay). Concentrations of 3.4 µM and 7 µM of Mel were chosen respectively to treat SKOV-3 and CAISMOV-24 cells for a period of 24h. The concentration of Luz (a Mel receptor antagonist) was defined as 1 µM for both cell lines. To understand the effect of Mel in combination with Luz on cancer cells, we divided each cancer cell line into three experimental groups: Control: cells exposed only to the culture medium containing 100 µL of DMSO solution as the vehicle; Mel: cells exposed to Mel at concentrations of 3.4 and 7 µM for SKOV-3 and CAISMOV-24, respectively, plus vehicle; and Mel + Luz: cells exposed to the combination of Mel and Luz plus vehicle. For the combination group, Luz was first administered, and after 30 min, the pre-established concentration of Mel was added. Mel and Luz were dissolved in DMSO (5%) maintaining the molarity values indicated by the manufacturers. All experimental assays were performed in biological and technical triplicates. Cell viability (MTT assay) Cell viability was analyzed using the MTT method based on the IC 50 to determine proper Mel concentrations. After the confluence rate, SKOV-3 and CAISMOV-24 cells were trypsinized, seeded in 6-well plates at a density of 5x10 5 cells per well, and cultured with appropriated medium supplemented with 10% FBS. After cell adherence, Mel and Luz were added to culture medium following the experiment design with specific concentrations (2.0, 3.4, 5.0, and 7.0 µM). Viability curves were estimated after 24h of treatment using the MTT solution (5mg/mL). To detect cytotoxicity, the reactions were replicated in 96-wells plate and read in a microplate reader (Epoch, Bio Tek Instruments, USA). Cell invasion and migration assays The evaluation of SKOV-3 and CAISMOV-24 cell invasiveness was performed using 24-well plates. A thin membrane of Geltrex® was added to each well, occluding the lower polyethylene terephthalate (PET) membrane. SKOV-3 and CAISMOV-24 (1x10 5 cells) were added to the top of the insert and received standard medium without FBS. The invasive potential was analyzed based on the ability of cells to cross the gel barrier and the PET membrane through the pores, being attracted chemotactically by inferior coverage of culture medium containing 5% FBS. The plates were placed in a CO 2 atmosphere at 37ºC for 24h. After the incubation period, cells were fixed in methanol for 10 min, and the remaining cells were removed by scraping. Migrated cells were stained with a 0.1% toluidine blue solution and photographed with a 5X objective in an inverted microscope (ZeissAxiovert®). For migration assay, a similar experimental procedure was used, except for Geltrex® which was not added to the transwell chamber. All experiments were performed in triplicate based on four fields and submitted to automatic cell count LUNA-II® (Logos Biosystems, South Korea) Measurement of melatonin concentration Mel levels were determined in both SKOV-3 and CAISMOV-24 cells using a human-specific commercial ELISA kit (EH3344, Fine Test), according to manufacturer’s instructions. The absorbance was read at 450 nm on a microplate reader (Epoch, Bio Tek Instruments, USA). Results were interpolated from standard curves generated by plotting the concentration of the standards against their absorbance. The concentrations are presented in pg/mL. Preparation of the supernatant for analyzing the antioxidant system After Mel treatment, the culture medium was discarded, and PBS was added to the cells. The cells were then lysed through three cycles of freezing (-80 ºC) and thawing (37 ºC) for 30 min each. After this process, protein levels were quantified and used to normalize the results of the total (GS), reduced (GSH), and oxidized glutathione (GSSG), catalase (CAT), glutathione S-transferase (GST), and superoxide dismutase (SOD). Activity of SOD and CAT After treatment with Mel and Luz, the cells (5 x 10 5 cells/mL) were washed with PBS (pH 7.4) and re-plated for all the analyzes. SOD enzyme activity assay was performed according to [ 31 ] with some modifications. The reaction medium was composed of methionine (13 mM), NBT (75 µM), and riboflavin (4 mM) in phosphate buffer (50 mM, pH 7.8). 10 µL of the cell extract was added, and the samples were exposed to fluorescent light (13W) for 5 min. Control samples (no enzymes) and the blank (containing all reagents but not exposed to light) were used. Readings were performed using a MultiSkan-Skyhigh microplate reader at 560 nm and at 25°C. For the evaluation of CAT activity, cells were lysed, and a reaction medium was prepared as described by Aebi et al. [ 32 ]. The reading was performed by a spectrophotometer at 240 nm for 1 min at 15-second intervals. Measurement of GSH, GT, GSSG, and GST. To quantify the levels of reduced glutathione (GSH) and total glutathione (GT), the protocol proposed by Rahman, Kode and Biswas [ 33 ] was carried out with modifications, using 5,5'-ditiobis 20-nitrobenzoic acid (DTNB) in the homogenate, evidenced by yellow formation. To determine total glutathione (TG) levels, DTNB, nicotinamide-adenine dinucleotide phosphate (NADPH), and glutathione reductase were used in the homogenate. GT and GSH levels were measured at 412 nm (Multiskan GO, Thermo Scientific), and results were expressed as µmol/mg protein. GSSG levels were calculated [ 34 ], considering the stoichiometry of the reactions. To evaluate glutathione S-transferase (GST) activity, 1-chloro-2,4-dinitrobenzene (CDNB) and potassium phosphate buffer were used. The absorbance of the samples was measured at 340 nm for 160 seconds, with 5 measurements at 40-second intervals [ 35 ]. In silico molecular blind docking The target protein used in this in silico study was human liver mitochondrial catalase (PDB ID: 8SGV), which was downloaded from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) database ( https://www.rcsb.org/ ; accessed on 10 January 2025) [ 36 ]. Catalase is a tetramer composed of four identical subunits (A,B,C, and D) with over 500 amino acid residues. Each chain is coordinate with iron-heme ring and binds NADPH as a cofactor. We conducted our analysis considering the A subunit isolated. The preparation of the protein for the molecular docking was conducted in the UCSF ChimeraX software (version 1.8) [ 37 ]. Water molecules, nonstandard residues and the chains B, C and D were deleted. The 3D structure of melatonin molecule (CID: 896) was downloaded from PubChem ( https://pubchem.ncbi.nlm.nih.gov/ ; accessed on 10 January 2025) [ 38 ] and was prepared in the Avogrado software (version 1.2.0) by adding hydrogens and optimizing the molecule geometry. The next steps were done in the Autodock Tools software (MGLTools-1.5.6). In that stage, any residual water molecule was deleted, polar hydrogens and Kollman charges were attached in the protein. The melatonin structure was forced to let all rotatable bonds rotatable. The grid box was set to 126, 126, and 126 Å along the X-, Y-, and Z-axis with a grid spacing of 0.925 Å in order to recognize the whole catalase subunit. The AutoDocking parameters adopted were: GA runs = 500; population size = 150; maximum number of energy evaluations = 25.000.000; GA crossover mode = two points. The Lamarckian Genetic algorithm was selected to search for the best conformations and the lowest binding energy conformer was chosen for further analysis. The Chimera X and the Biovia Discovery Studio Visualizer (version 24.1.0) were used to analyze and visualize the docking results. Statistical analysis Data were processed using an analysis of variance (One-way ANOVA) for one factor and presented as the mean ± standard deviation (SD). Significant results were complemented by Tukey’s test. Statistical significance was set at P < 0.05 for all analyses. Results were analyzed and generated using GraphPad Prism 9.0 scientific graphing software (GraphPad Software, San Diego, CA). Results Mel treatment enhances its intracellular concentration in OC cells. Based on MTT analysis, we identified the cytotoxic effect of Mel (IC 50 ) which was previously established at 3.4 µM for SKOV-3 cells and 7 µM for CAISMOV-24 cells. Luzindole, an antagonist of MT1/2 Mel receptors, was used to determine whether the Mel’s effect is dependent or independent of their membrane receptors. The intracellular concentrations of Mel were measured in the OC cells by the enzyme assay. Mel treatment elevated the levels of intracellular Mel compared to the control group, especially in SKOV-3 cells. After blocking MT1/2 receptors, Mel promoted an increase in its own intracellular levels in both SKOV-3 and CAISMOV-24 cells (1.96-fold increase vs. Control and 2.08-fold increase vs. Control, respectively). More importantly, Mel alone in the presence of its receptors, increased its intracellular levels in SKOV-3 cells (1.75-fold increase vs. Control), thus restoring Mel concentration in OC cells where they are normally depressed (Fig. 1 ). Mel reduces the migratory and invasive capacity of SKOV-3 and CAISMOV-24 cells regardless of the MT1/2 receptor activation. To investigate the effects of Mel and luzindole on the migratory and invasive potential of SKOV-3 and CAISMOV-24 cells, we used transwell inserts in 24-well plates. The invasive potential of SKOV-3 and CAISMOV-24 cells was reduced by Mel alone (2.45- and 3.58-fold decrease vs Control groups, respectively), and after the combination of Mel and luzindole (2.17-fold decrease vs Control group in SKOV-3 cells and 2.38-fold decrease vs Control group in CAISMOV-24 cells; Figs. 2 and 3 A, B). The results also showed that Mel alone significantly reduced the migration of both SKOV-3 and CAISMOV-24 cells (2.03- and 2.29-fold decrease vs Control group, respectively; Figs. 2 and 3 C, D). When Mel was combined with luzindole, a significant decrease in cell migration was also observed considering both OC cells (1.67-fold decrease vs. Control in SKOV-3 cells and 2.02-fold decrease vs. Control in CAISMOV-24). Mel differentially regulates the antioxidant enzymes in OC cells. SOD levels were only reduced by Mel in combination with luzindole in the CAISMOV-24 cells (p = 0.027). Regarding Catalase levels, a significant reduction was observed after Mel treatment in both OC cells (p < 0.05 for CAISMOV-24 and p = 0.012 for SKOV-3). The presence of luzindole restored catalase levels close to that of control groups, thus showing a receptor dependent response; it has been noted that the action of melatonin on antioxidant enzymes are receptor mediated. The changes in glutathione activities following treatments with Mel and luzindole varied differently between the two OC cell lines (Figs. 4 and 5 ). In SKOV-3 cells, Mel alone significantly decreased the levels of TG (p < 0.001) and GSSG (p < 0.001) compared with the control group. The combination of Mel and luzindole also showed a decrease in the levels of TG (p = 0.003) and GSSG (p < 0.001). Conversely, this combination increased the levels of GST compared with control group (p < 0.001) and Mel alone (p = 0.002). Notably, in CAISMOV-24 cells, Mel alone or combined with luzindole significantly reduced the activities of TG (p < 0.05), GST (p < 0.05), GSSG (p < 0.05), and GSH (p < 0.05) compared with the control group. These results demonstrate an opposite effect of Mel favoring pro-oxidative processes in the OC cells. Mel directly interacts with both the protein portion of catalase and its heme group. Through in vitro molecular docking assays, the ability of melatonin to interact with catalase was observed. Out of the 500 poses tested, the five with the lowest binding energies are presented in Table 1 . Subsequently, experiments were conducted to demonstrate the physical interaction between melatonin and catalase. Melatonin bound to an enzyme pocket in the β barrel domain, very close to the binding site of the heme prosthetic group (Fig. 6 A). Melatonin directly interacts with the polypeptide chain of catalase through hydrogen bonds involving residues Arg72, Tyr358, His362, and Arg365. Additionally, alkyl interactions with Val73, Val74, and Val146, as well as π-interactions with His75, are also observed (Fig. 6 B). As melatonin was found to be positioned near the porphyrin ring (Fig. 7 A), we further docked the interaction between these two molecules. The results show a close distance of approximately 7 Å between melatonin and the heme group, suggesting that the structures are likely oriented in a specific way that facilitates interaction (Fig. 7 B). Finally, it can be observed that some amino acids involved in anchoring the porphyrin ring to the protein may also interact with the indolamine (Fig. 8 ). These potential interactions are of significant value and warrant further investigation. Table 1 Docking results for interaction between the Melatonin and Catalase (subunit A). Pose BE K i vdW + Hbond + desolv Eletrostatic Torsional Unbound 453 -5.99 40.74 -6.99 -0.19 +1.19 -0.49 82 -5.92 45.87 -6.66 -0.45 +1.19 -0.71 214 -5.72 64.28 -6.93 + 0.02 +1.19 -0.38 146 -5.71 65.11 -6.93 + 0.03 +1.19 -0.33 475 -5.57 82.37 -6.77 + 0.01 +1.19 -0.37 BE: Binding energy; Ki: Estimated Inhibition Constant (micromole. L -1 ); vdW: van der Waals interactions; desolv: desolvation energy. Energy parameters are expressed as kcal.mol -1 . Discussion Herein we report that exogenously applied melatonin to the incubation medium increases the uptake of melatonin to improve its intracellular levels accompanied by attenuation of the invasive and migratory capacity of OC cells in a receptor-independent manner. Melatonin has been shown to be taken up by cells [ 39 ]. We also bring new information regarding the interference of Mel in the enzymatic antioxidant system of OC cells which may negatively impact their survival and growth (Fig. 9 ). Firstly, we observed an increased concentration of Mel in both OC cell lines, mainly in SKOV-3 cells following Mel treatment, in the presence of Luz or not, and in CAISMOV-24 cells only after the combination of Mel and Luz. We previously documented an increase in the intracellular concentrations of Mel in SKOV-3 cells after treatment with 3.2 mM of Mel [ 40 ]. A greater Mel level may contribute to the antiangiogenic process, pro-oxidative, and pro-apoptotic pathways and may promote modifications in the metabolic profile of tumor cells [ 41 ]. The elevation in intracellular levels of Mel appear to be receptor-independent since the combination of Mel with luzindole also promoted an increase in the availability of the indolamine. Treatment with Mel can disinhibit the enzymatic activity of the pyruvate dehydrogenase complex (PDC), either directly [ 42 ] or by first reducing hypoxia-inducible factor 1-alpha (HIF-1α), a factor highly expressed in hypoxic conditions of the tumor microenvironment, which stimulates the pyruvate dehydrogenase kinase (PDK) enzyme. After PDC disinhibition, Mel can be synthesized in mitochondria, and oxidative phosphorylation is reestablished [ 40 , 43 ]. The fact that Mel promotes a reduction in the invasive and migratory capacity of OC cells is well known [ 44 – 46 ]. Recently, our group showed that MT1 knockdown in SKOV-3 cells treated with 3.2- or 4-mM Mel presented a remarkable reduction in cell migration and invasion [ 46 ]; we obtained similar results using luzindole, a non-selective antagonist of both MT1 and MT2 receptors, in two different OC cell lines via transwell inserts. Despite the use of concentrations ranging from 3.4 and 7 µM of Mel to SKOV-3 and CAISMOV-24 cells, respectively, our results clearly demonstrated a reduction in the invasive and migratory capacity of OC cells, regardless of the activation of Mel receptors. The precise mechanisms by which Mel regulates OC cell migration and invasion are not yet fully understood. Previous studies have shown that Mel exerts anti-migratory effects via the MAPK and PI3K pathways in OC cells [ 44 , 46 ]. In other cancer types, such as colon and breast cancer, Mel attenuated cell invasion, migration, and survival by targeting PI3K/AKT and NF-κB signaling pathway [ 47 ] or by downregulating the p38 MAPK pathway [ 48 ]. In addition to its passive diffusion, Mel can alternatively enter tumor cells using other membrane transporters such as PEPT1/2 and GLUT1 [ 46 , 49 ], therefore it may act in an MT1/2 receptor-independent manner to exert the anti-tumoral effects on the migratory and invasive potential of OC cells. Defects in mitochondrial metabolism can increase the production of ROS and RNS, leading to OS. To combat the excessive and harmful production of these radical and non-radical species, cells are equipped with an antioxidant defense system that includes enzymes such as SOD, CAT, and glutathione derivatives [ 50 ]. An excessive and permanent generation of OS is related to genetic/epigenetic events that could facilitate the tumorigenic process; however, increasing the generation of ROS and RNS in tumor cells is paradoxically used as a strategy to induce their death [ 51 , 52 ]. A potential actor in this approach is the use of Mel, known for its antiproliferative effects and for interfering with angiogenesis and metastasis events; Mel acts by modulating the oxidant/antioxidant state of cells [ 52 – 56 ]. Moreover, the effects of Mel on tumor cells seem to be opposite of those occurring in normal healthy cells, that is, Mel acts as a pro-oxidant agent in tumor cells, thus reducing their antioxidant defenses [ 57 , 58 ]. Our results reinforce the apparent pro-oxidant properties of Mel in OC cells, especially in CAISMOV-24 cells. We are the first to demonstrate a decrease in SOD activity when Mel was combined with luzindole, i.e., a receptor-independent response, and a significant reduction in CAT levels after Mel treatment in both OC cells, suggesting that Mel acts directly as a modulator of these two antioxidant enzymes. Mel treatments have also been associated with lower enzymatic activities of SOD and CAT accompanied by an increased level of ROS observed in human colorectal cancer cells and in the hepatocellular carcinoma cells [ 59 , 60 ], resulting in apoptosis due to an excessive oxidative damage. In healthy tissues, Mel generally upregulates antioxidant enzymes, including the glutathione system that plays a central role against OS [ 56 , 61 ]. Here, we observed a marked decrease in total glutathione (GT) levels in both OC cell lines following treatment with Mel alone. When Mel receptors were blocked by luzindole, only SKOV-3 cells showed a significant reduction in GT levels, while CAISMOV-24 cells showed only a trend. The reduced form of glutathione (GSH) is one of the most important machineries on the front line of the antioxidant defense system. GSH maintains a redox state in subcellular compartments such as mitochondria and cytosol, and its increased levels may be involved in the chemoresistance of cancer cells [ 62 ]. In this study, GSH content was only reduced with CAISMOV-24 cells treated with Mel alone. It is also reported that Mel can exert a depletion on GSH levels in HepG2 cells [ 52 , 63 ], and in human myeloid leukemia cell line (U937) [ 60 , 64 ]. Under OS conditions, GSH can be directly converted to oxidized glutathione (GSSG) in the presence of ROS [ 52 ]. We observed a notable reduction in GSSG levels in both OC cells. Particularly, for SKOV-3 cells, GSH levels were like that of the control group, while GSSG was significantly decreased by both treatments. Based on this GSH:GSSG ratio that favors GSH, we can hypothesize a more pronounced resistance of SKOV-3 cells to OS, which leads to the maintenance of GSH homeostasis, differing from what was observed with CAISMOV-24 cells. Cancer cells can also gain resistance through overexpression of enzymes that can increase detoxification capacity and avoid the cytotoxic action of antitumor drugs [ 65 ]. More specifically, overexpression of glutathione S-transferases (GST) and efflux pumps in tumor cells can reduce the reactivity of various anticancer drugs, such as cyclophosphamide [ 66 ], cisplatin [ 67 ], and others [ 65 ]. In this study, OC cell lines showed opposite results regarding GST. While in CAISMOV-24 cells both treatments decreased GST activities, in SKOV-3 cells the combination of Mel with luzindole increased its activity. As reviewed in detail by Sau et al. [ 65 ], the increase in GST levels occurs through transcriptional activation mediated by the nuclear factor erythroid 2 p45-related factor 2 (Nrf2). However, further studies are needed to precisely determine the role of Mel and its receptors in the molecular pathways that regulate the activity and expression of genes in the glutathione system. Overexpression of GST and high levels of GSH are linked to the development and expression of chemoresistance [ 68 ]. In this context, our treatment with Mel alone at 7 µM successfully promotes a decrease in GST and GSH levels in CAISMOV-24, a recently established human low-grade serous ovarian carcinoma cell line [ 69 ]. SKOV-3 cells, a non-serous ovarian cancer cell line, in turn, remained unchanged for these parameters after treatment of 3.4 µM of Mel alone, thus suggesting further evaluations with higher concentrations. This sounds plausible since it has been shown that high levels of Mel are required to induce ROS production in tumor cells [ 27 ]. In our previous study using the same OC cell lines and melatonin concentrations, we observed a reduction in Warburg-type metabolism and potentially glutaminolysis, which further attenuated oncogenic targets associated with OC progression and invasion [ 70 ]. As stated below, melatonin's capacity to modulate antioxidant enzymes activity is well documented. However, it remains unclear whether melatonin interacts directly with specific enzymes, and the precise nature of such interactions has not been fully explored. To bridge this gap, and given that catalase activity was reduced in the two OC cell lines, we employed in silico molecular docking to investigate the interaction between melatonin and catalase. Catalase’s catalytic mechanism occurs in two stages. Initially, the heme Fe 3+ reduces a hydrogen peroxide molecule to water, forming a covalent oxyferryl species, along with a porphyrin π-cation radical. This radical then oxidizes another hydrogen peroxide molecule into molecular oxygen, while the ferryl oxygen species is released as water. Several amino acid residues, including tyrosine, play crucial roles in facilitating these reactions at the active site [ 71 ]. The docking results revealed that melatonin binds most effectively on a structural pocket between β-sheet and wrapping domain, near the heme-binding cavity of catalase. Melatonin binding was stabilized with hydrogens and hydrophobic interactions, with a binding energy of -5.99 kcal.mol − 1 . The amino acid residues Arg72, Tyr, 358, His362, and Arg365 play major role in the binding of melatonin on catalase subunit. Melatonin forms four conventional hydrogen bonds with those amino acids, along with additional potential interactions. Notably, the tyrosine residue at position 358 interacts closely with the iron in the porphyrin ring, and melatonin also engages with this residue. Given the critical role of the heme group in catalase's catalytic activity, this interaction with Tyr358 may provide valuable insight into the mechanism by which melatonin reduces the enzyme's activity. Additionally, melatonin’s interaction with other amino acid residues could alter the microenvironment of the catalase active site, further contributing to the observed effect in enzyme activity. The binding of farnesiferol C, a sesquiterpene coumarin, to bovine liver catalase yielded similar findings, affecting both the enzyme’s affinity and catalytic activity [ 72 ]. The discovery of specific, non-toxic catalase inhibitors presents significant potential for use in cancer co-therapy. For instance, a Zn(II) complex tested against human colon cancer cells demonstrated cytotoxic effects by inhibiting catalase through mixed-type inhibition kinetics. Interestingly, the binding site of the complex also involved hydrogen bonding and exhibited a free energy of -7.13 kcal/mol [ 73 ], similar to the binding characteristics observed for melatonin. An intriguing and unexpected finding in our study was the close proximity of melatonin to the heme group of catalase. To explore this further, we evaluated the potential physical interaction between melatonin and the porphyrin ring. The binding free energy between melatonin and the heme group was calculated to be -4.68 kcal/mol, suggesting various interaction types. More importantly, several amino acid residues critical for maintaining the stability of the heme group – such as Val73, Val74, Arg72, His75, and Val146 – were found to interact with melatonin. These interactions may contribute to melatonin’s inhibition of catalase activity, a possibility that warrants further investigation. Conclusion Collectively, Mel treatment attenuates the migratory and invasive capacity of OC cells in a receptor independent manner while stimulating its intracellular concentration. Moreover, the antioxidant enzymatic defenses were dampened by Mel, especially in the CAISMOV-24 cells. The blockage of MT1/2 receptors by the antagonist luzindole followed by Mel administration showed a tendency to soften the results. Indeed, pre-incubation of SKOV-3 cells with 10 µM luzindole revealed an increase in cell viability [ 44 ]. Based on our results, we believe that the function of Mel in SKOV3 and CAISMOV-24 is partially mediated by MT1 and MT2 receptors. Also, our data provide valuable insights into the regulatory role of Mel in modulating antioxidant status in OC cells. Based on the molecular docking study, future investigations using molecular dynamics simulations and enzyme kinetics assays will be valuable and necessary to confirm the predicted interactions between catalase and melatonin as pivotal to enzyme modulation. Declarations Conflicts of Interest: The authors declare no competing interest. Funding: This research was funded by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil), grant number 88887.482443/2020-00, National Council for Scientific and Technological Development (CNPQ, Process numbers 304108/2020-0 and 306117/2023-1 to LGAC) and São Paulo Research Foundation (FAPESP grant #2021/12971-7 to LGAC). Author Contributions: Conceptualization, HS, FRS and LGAC; Data curation, HS; Formal analysis: RCC, FG, FRS, VAS, GSAF, MCS, DAPCZ and LGAC; Investigation, HS, FRS and DAPCZ; Methodology, HS, RCC, FG and DAPCZ; Visualization, RR; Writing – original draft, HS and LGAC; Writing – review & editing, RR. The authors read and approved the final version of this manuscript. Acknowledgments: We would like to extend our gratitude to the technicians, colleagues, and university staff whose unwavering support and valuable contributions were instrumental in the successful completion of this scientific article. References Siegel RL, Miller KD, Fuchs HE, Jemal A (2022) Cancer statistics, 2022. CA Cancer J Clin. 2022. 72, 7–33 Gabra H (2019) Epithelial ovarian cancer. Dewhurst’s Textbook of Obstetrics & Gynaecology, Seventh Edition. 2019. 625–635 Reid BM, Permuth JB, Sellers TA (2017) Epidemiology of ovarian cancer: A review. Cancer Biol Med. 2017. 14, 9–32 De Berardinis RJ, Chandel NS (2016) Fundamentals of cancer metabolism. Sci Adv. 2016. 2, 1–18 Hanahan D, Weinberg RA (2011) Hallmarks of cancer: The next generation. Cell. 2011. 144, 646–674 Pavlova NN, Thompson CB (2016) The emerging hallmarks of cancer metabolism. Cell Metab. 2016. 23, 27–47 Reiter RJ, Rosales-Corral SA, Tan DX, Acuna-Castroviejo D, Qin L, Yang SF, Xu K (2017) Melatonin, a full-service anti-cancer agent: Inhibition of initiation, progression, and metastasis. Int J Mol Sci. 2017. 18, 843 Zhao D, Yu Y, Shen Y, Liu Q, Zhao Z, Sharma R, Reiter RJ (2019) Melatonin synthesis and function: Evolutionary history in animals and plants. Front Endocrinol (Lausanne). 2019. 10, 1–16 Talib WH (2018) Melatonin and cancer hallmarks. Molecules. 2018. 23, 518 Zhang H-M, Zhang Y (2014) Melatonin: A well-documented antioxidant with conditional pro-oxidant actions. J Pineal Res. 2014. 57, 131–146 Cheng L, Li S, He K, Kang Y, Li T, Li C, Zhang Y, Zhang W, Huang Y (2023) Melatonin regulates cancer migration and stemness and enhances the anti-tumour effect of cisplatin. J Cell Mol Med. 2023. 27, 2215–2227 Byrne NJ, Rajasekaran NS, Abel ED, Bugger H (2021) Therapeutic potential of targeting oxidative stress in diabetic cardiomyopathy. Free Radic Biol Med. 2021. 169, 317–342 Galano A, Tan DX, Reiter RJ (2011) Melatonin as a natural ally against oxidative stress: A physicochemical examination. J Pineal Res. 2011. 51, 1–16 Rahal A, Kumar A, Singh V, Yadav B, Tiwari R, Chakraborty S, Dhama K (2014) Oxidative stress, prooxidants, and antioxidants: The interplay. Biomed Res Int. 2014 Kohen R, Nyska A (2002) Oxidation of biological systems: Oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification. Toxicol Pathol. 2002. 30, 620–650 D’Souza LC, Mishra S, Chakraborty A, Shekher A, Sharma A, Gupta SC (2020) Oxidative stress and cancer development: Are noncoding RNAs the missing links? Antioxid Redox Signal. 2020. 33, 1209–1229 Cheung EC, Vousden KH (2022) The role of ROS in tumour development and progression. Nat Rev Cancer. 2022. 22, 280–297 Xia C, Meng Q, Liu LZ, Rojanasakul Y, Wang XR, Jiang BH (2007) Reactive oxygen species regulate angiogenesis and tumor growth through vascular endothelial growth factor. Cancer Res. 2007. 67, 10823–10830 Jiang Y, Song L, Lin Y, Nowialis P, Gao Q, Li T, Li B, Mao X, Song Q, Xing C et al (2023) ROS-mediated SRMS activation confers platinum resistance in ovarian cancer. Oncogene. 2023. 42, 1672–1684 Stieg DC, Wang Y, Liu LZ, Jiang BH (2022) ROS and miRNA dysregulation in ovarian cancer development, angiogenesis and therapeutic resistance. Int J Mol Sci. 2022. 23 6702 Simone NL, Soule BP, Ly D, Saleh AD, Savage JE, DeGraff W, Cook J, Harris CC, Gius D, Mitchell JB (2009) Ionizing radiation-induced oxidative stress alters miRNA expression. PLoS One. 2009. 4, 6377 Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, Sweet-Cordero A, Ebert BL, Mak RH, Ferrando AA, Downing JR, Jacks T, Horvitz HR, Golub TR (2005) MicroRNA expression profiles classify human cancers. Nature. 2005. 435, 834–838 Rattanapan Y, Korkiatsakul V, Kongruang A, Siriboonpiputtana T, Rerkamnuaychoke B, Chareonsirisuthigul T (2020) MicroRNA expression profiling of epithelial ovarian cancer identifies new markers of tumor subtype. Microrna. 2020. 9, 289–294 Reiter RJ, Sharma RN, Chuffa LG, Silva DGH, Rosales-Corral S (2024) Intrinsically synthesized melatonin in mitochondria and factors controlling its production. Histol Histopathol. 2024. 7, 18776 Zhang H-M, Zhang Y (2014) Melatonin: A well-documented antioxidant with conditional pro-oxidant actions. J Pineal Res. 2014. 57, 131–146 Mortezaee K, Najafi M, Farhood B, Ahmadi A, Potes Y, Shabeeb D, Musa AE (2019) Modulation of apoptosis by melatonin for improving cancer treatment efficiency: An updated review. Life Sci. 2019. 228, 228–241 Florido J, Rodriguez-Santana C, Martinez-Ruiz L, López-Rodríguez A, Acuña-Castroviejo D, Rusanova I, Escames G (2022) Understanding the mechanism of action of melatonin, which induces ROS production in cancer cells. Antioxidants (Basel). 2022. 11, 1621 Florido J, Martinez-Ruiz L, Rodriguez-Santana C, López-Rodríguez A, Hidalgo-Gutiérrez A, Cottet-Rousselle C, Lamarche F, Schlattner U, Guerra-Librero A, Aranda-Martínez P et al (2022) Melatonin drives apoptosis in head and neck cancer by increasing mitochondrial ROS generated via reverse electron transport. J Pineal Res. 2022. 73, 12824 Zhang H-M, Zhang Y, Zhang B-X (2011) The role of mitochondrial complex III in melatonin-induced ROS production in cultured mesangial cells. J Pineal Res. 2011. 50, 78–82 Gurer-Orhan H, Ince E, Konyar D, Saso L, Suzen S (2018) The role of oxidative stress modulators in breast cancer. Curr Med Chem. 2018. 25, 4084–4101 Marklund S, Marklund G (1974) Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem. 1974. 47, 469–474 Aebi H (1974) Catalase. In Bergmeyer, H.U., Ed., Methods of Enzymatic Analysis. 1974. Verlag Chemie/Academic Press Inc., Weinheim/NewYork, 673–684 Rahman I, Kode A, Biswas SK (2007) Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nature Protocols. 2007. 1, 3159–3165 Carrara IM, Melo GP, Bernardes SS, Neto FS, Ramalho LNZ, Marinello PC, Luiz RC, Cecchini R, Cecchini AL (2019) Looking beyond the skin: cutaneous and systemic oxidative stress in UVB-induced squamous cell carcinoma in hairless mice. J Photochem Photobiol B. 2019. 195, 17–26 Keen JH, Habig WH, Jakoby WB (1976) Mechanism for the several activities of the glutathione S-transferases. J Biol Chem. 1976. 251, 6183–6188 Berman HM, Battistuz T, Bhat TN, Bluhm WF, Bourne PE, Burkhardt K, Feng Z, Gilliland GL, Iype L, Jain S et al (2002) The Protein Data Bank. Acta Crystallogr D Biol Crystallogr. 2002. 58, 899–907 Meng EC, Goddard TD, Pettersen EF, Couch GS, Pearson ZJ, Morris JH, Ferrin TE (2023) UCSF ChimeraX: Tools for structure building and analysis. Protein Sci. 2023. 32, 4792 Kim S, Thiessen PA, Bolton EE, Chen J, Fu G, Gindulyte A, Han L, He J, He S, Shoemaker BA et al (2016) PubChem Substance and Compound databases. Nucleic Acids Res. 2016. 44, 1202–1213 Acuña-Castroviejo D, Noguiera-Navarro MT, Reiter RJ, Escames G (2018) Melatonin actions in the heart; more than a hormone. Melatonin Res. 2018. 1, 21–26 Cucielo MS, Cesário RC, Silveira HS, Gaiotte LB, Dos Santos SAA, de Campos Zuccari DAP, Seiva FRF, Reiter RJ, de Almeida Chuffa LG (2022) Melatonin reverses the Warburg-type metabolism and reduces mitochondrial membrane potential of ovarian cancer cells independent of MT1 receptor activation. Molecules. 2022. 27, 4350 Chuffa LGA, Reiter RJ, Lupi LA (2017) Melatonin as a promising agent to treat ovarian cancer: Molecular mechanisms. Carcinogenesis. 2017. 38, 945–952 Chen X, Hao B, Li D, Reiter RJ, Bai Y, Abay B, Chen G, Lin S, Zheng T, Ren Y et al (2021) Melatonin inhibits lung cancer development by reversing the Warburg effect via stimulating the SIRT3/PDH axis. J Pineal Res. 2021. 71, 12755 De Lima Mota A, Victorasso Jardim-Perassi B, Bergamin De Castro T, Colombo J, Sonehara NM, Gomes Nishiyama VK, Gonçalves Pierri VA, Aparecida D, De Campos Zuccari P (2019) Melatonin modifies tumor hypoxia and metabolism by inhibiting HIF-1α and energy metabolic pathway in the in vitro and in vivo models of breast cancer. Melatonin Res. 2019. 2, 83–98 Akbarzadeh M, Movassaghpour AA, Ghanbari H, Kheirandish M, Fathi Maroufi N, Rahbarghazi R, Nouri M, Samadi N (2017) The potential therapeutic effect of melatonin on human ovarian cancer by inhibition of invasion and migration of cancer stem cells. Sci Rep. 2017, 7, 1–11 Bu S, Wang Q, Sun J, Li X, Gu T, Lai D (2020) Melatonin suppresses chronic restraint stress-mediated metastasis of epithelial ovarian cancer via NE/AKT/β-catenin/SLUG axis. Cell Death Dis. 2020, 11, 1–17 Cucielo MS, Freire PP, Emílio-Silva MT, Romagnoli GG, Carvalho RF, Kaneno R, Hiruma-Lima CA, Delella FK, Reiter RJ, de Chuffa LG (2023) A. Melatonin enhances cell death and suppresses the metastatic capacity of ovarian cancer cells by attenuating the signaling of multiple kinases. Pathol Res Pract. 2023, 248,154637 Gao Y, Xiao X, Zhang C, Yu W, Guo W, Zhang Z, Li Z, Feng X, Hao J, Zhang K, Xiao B, Chen M, Huang W, Xiong S, Wu X, Deng W (2017) Melatonin synergizes the chemotherapeutic effect of 5-fluorouracil in colon cancer by suppressing PI3K/AKT and NF-κB/iNOS signaling pathways. J Pineal Res. 2017. 62, 12380 Mao L, Yuan L, Slakey LM, Jones FE, Burow ME, Hill SM (2010) Inhibition of breast cancer cell invasion by melatonin is mediated through regulation of the p38 mitogen-activated protein kinase signaling pathway. Breast Cancer Res. 2010. 12, 107 Reiter RJ, Sharma R, Rosales-Corral S, Manucha W, Chuffa LG, de Zuccari A (2021) C Melatonin and pathological cell interactions: mitochondrial glucose processing in cancer cells. Int J Mol Sci. 2021. 22, 12494 Taucher E, Mykoliuk I, Fediuk M, Smolle-Juettner FM (2022) Autophagy, oxidative stress, and cancer development. Cancers (Basel). 2022. 14, 1637 Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB (2010) Oxidative stress, inflammation, and cancer: How are they linked? Free Radic Biol Med. 2010. 49, 1603 Cruz EMS, Concato VM, de Morais JMB, Silva TF, Inoue FSR, de Souza Cremer M, Bidóia DL, Machado RRB, de Almeida Chuffa LG, Mantovani MS, Panis C, Pavanelli WR, Seiva FRF (2023) Melatonin modulates the Warburg effect and alters the morphology of hepatocellular carcinoma cell line resulting in reduced viability and migratory potential. Life Sci. 2023. 319, 121530 Prieto-Domínguez N, Ordónez R, Fernández A, Méndez-Blanco C, Baulies A, Garcia-Ruiz C, Fernández-Checa JC, Mauriz JL, González-Gallego J (2016) Melatonin-induced increase in sensitivity of human hepatocellular carcinoma cells to sorafenib is associated with reactive oxygen species production and mitophagy. J Pineal Res. 2016. 61, 396–407 Reiter RJ, Mayo JC, Tan DX, Sainz RM, Alatorre-Jimenez M, Qin L (2016) Melatonin as an antioxidant: under promises but over delivers. J Pineal Res. 2016. 61, 253–278 Zare H, Shafabakhsh R, Reiter RJ, Asemi Z (2019) Melatonin is a potential inhibitor of ovarian cancer: molecular aspects. J Ovarian Res. 2019. 12, 1–8 Reiter RJ, Sharma R, Rosales-Corral S, de Campos Zuccari DAP, de Almeida Chuffa LG (2022) Melatonin: A mitochondrial resident with a diverse skill set. Life Sci. 2022. 301, 120612 Mehrzadi S, Safa M, Kamrava SK, Darabi R, Hayat P, Motevalian M (2017) Protective mechanisms of melatonin against hydrogen-peroxide-induced toxicity in human bone-marrow-derived mesenchymal stem cells. Can J Physiol Pharmacol. 2017. 95, 773–786 Reiter RJ, Sharma R, Tan DX, Huang G, de Almeida Chuffa LG, Anderson G (2023) Melatonin modulates tumor metabolism and mitigates metastasis. Expert Rev Endocrinol Metab. 2023. 18, 321–336 Mihanfar A, Yousefi B, Darband SG, Sadighparvar S, Kaviani M, Majidinia M (2021) Melatonin increases 5-fluorouracil-mediated apoptosis of colorectal cancer cells through enhancing oxidative stress and downregulating survivin and XIAP. Bioimpacts. 2021. 11, 253–261 Ammar OA, El-Missiry MA, Othman AI, Amer ME (2022) Melatonin is a potential oncostatic agent to inhibit HepG2 cell proliferation through multiple pathways. Heliyon. 2022. 8, 08837 Kopustinskiene DM, Bernatoniene J (2021) Molecular mechanisms of melatonin-mediated cell protection and signaling in health and disease. Pharmaceutics. 2021. 13, 1–19 Balendiran GK, Dabur R, Fraser D (2004) The role of glutathione in cancer. Cell Biochem Funct. 2004. 22, 343–352 Osseni RA, Rat P, Bogdan A, Warnet JM, Touitou Y (2000) Evidence of prooxidant and antioxidant action of melatonin on human liver cell line HepG2. Life Sci. 2000. 68, 387–399 Albertini MC, Radogna F, Accorsi A, Uguccioni F, Paternoster L, Cerella C, De Nicola M, D’Alessio M, Bergamaschi A, Magrini A, Ghibelli L (2006) Intracellular pro-oxidant activity of melatonin deprives U937 cells of reduced glutathione without affecting glutathione peroxidase activity. Ann N Y Acad Sci. 2006. 1091, 10–16 Sau A, Pellizzari Tregno F, Valentino F, Federici G, Caccuri AM (2010) Glutathione transferases and development of new principles to overcome drug resistance. Arch Biochem Biophys. 2010. 500, 116–122 Audemard-Verger A, Silva NM, Verstuyft C, Costedoat-Chalumeau N, Hummel A, Le Guern V, Sacré K, Meyer O, Daugas E, Goujard C, Sultan A, Lobbedez T, Galicier L, Pourrat J, Le Hello C, Godin M, Morello R, Lambert M, Hachulla E, Vanhille P, Queffeulou G, Potier J, Dion JJ, Bataille P, Chauveau D, Moulis G, Farge-Bancel D, Duhaut P, Saint-Marcoux B, Deroux A, Manuzak J, Francès C, Aumaitre O, Bezanahary H, Becquemont L, Bienvenu B (2016) Glutathione S Transferases Polymorphisms Are Independent Prognostic Factors in Lupus Nephritis Treated with Cyclophosphamide. PLoS One. 2016, 11, 0151696 Li S, Li C, Jin S, Liu J, Xue X, Eltahan AS, Sun J, Tan J, Dong J, Liang XJ (2017) Overcoming Resistance to Cisplatin by Inhibition of Glutathione S-Transferases (GSTs) with Ethacraplatin Micelles in Vitro and in Vivo. Biomaterials 2017, 144, 119–129 Townsend DM, Tew KD (2003) The role of glutathione-S-transferase in anti-cancer drug resistance. Oncogene 22:7369–7375 Da Silva RF, Cardozo DM, Rodrigues GOL, Souza-Araújo CN, Migita NA, Andrade LAL, Derchain S, Yunes JA, Guimarães F (2017) CAISMOV24, a new human low-grade serous ovarian carcinoma cell line. BMC Cancer. 2017. 17, 756 Silveira HS, Cesário RC, Vígaro RA, Gaiotte LB, Cucielo MS, Guimarães F, Seiva FRF, Zuccari DAPC, Reiter RJ, Chuffa LGA (2024) Melatonin changes energy metabolism and reduces oncogenic signaling in ovarian cancer cells. Mol Cell Endocrinol. 2024. 592, 112296 Putnam CD, Arvai AS, Bourne Y, Tainer JA (2000) Active and inhibited human catalase structures: ligand and NADPH binding and catalytic mechanism. J Mol Biol. 2000. 296, 295–309 Yekta R, Dehghan G, Rashtbari S, Ghadari R, Moosavi-Movahedi AA (2018) The inhibitory effect of farnesiferol C against catalase; Kinetics, interaction mechanism and molecular docking simulation. Int J Biol Macromol. 2018. 113, 1258–1265 Shahraki S, Saeidifar M, Delarami HS, Kazemzadeh H (2020) Molecular docking and inhibitory effects of a novel cytotoxic agent with bovine liver catalase. J Mol Struct. 2020. 1205, 127590 Additional Declarations The authors declare no competing interests. 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-5924048","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":408791825,"identity":"fa3b852c-86f4-4ac1-b550-a32d22415353","order_by":0,"name":"Henrique Spaulonci Silveira","email":"","orcid":"","institution":"Department of Structural and Functional Biology, UNESP - São Paulo State University, Institute of Bioscences, Botucatu, 18618-689, São Paulo, Brazil","correspondingAuthor":false,"prefix":"","firstName":"Henrique","middleName":"Spaulonci","lastName":"Silveira","suffix":""},{"id":408791826,"identity":"d7ad115a-a5e0-48de-bccf-53ae365d7abe","order_by":1,"name":"Roberta Carvalho Cesário","email":"","orcid":"","institution":"Department of Structural and Functional Biology, UNESP - São Paulo State University, Institute of Bioscences, Botucatu, 18618-689, São Paulo, Brazil","correspondingAuthor":false,"prefix":"","firstName":"Roberta","middleName":"Carvalho","lastName":"Cesário","suffix":""},{"id":408791827,"identity":"61a85503-ca6c-461f-9f80-020d06a41021","order_by":2,"name":"Vinicius Augusto Simão","email":"","orcid":"","institution":"Department of Structural and Functional Biology, UNESP - São Paulo State University, Institute of Bioscences, Botucatu, 18618-689, São Paulo, Brazil","correspondingAuthor":false,"prefix":"","firstName":"Vinicius","middleName":"Augusto","lastName":"Simão","suffix":""},{"id":408791828,"identity":"67f08b02-9b33-429e-92d6-0dd092762564","order_by":3,"name":"Fernando Guimarães","email":"","orcid":"","institution":"Hospital da Mulher “Professor Doutor José Aristodemo Pinotti” – CAISM, UNICAMP; Campinas; São Paulo; Brasil","correspondingAuthor":false,"prefix":"","firstName":"Fernando","middleName":"","lastName":"Guimarães","suffix":""},{"id":408791829,"identity":"096ad5ed-16f3-46ad-b8f0-a324d7bdbbec","order_by":4,"name":"Fábio Rodrigues Ferreira Seiva","email":"","orcid":"","institution":"UNESP","correspondingAuthor":false,"prefix":"","firstName":"Fábio","middleName":"Rodrigues Ferreira","lastName":"Seiva","suffix":""},{"id":408791830,"identity":"c83ba822-e2a1-4281-8f5c-78b609b1c2c1","order_by":5,"name":"Debora Aparecida P. C. Zuccari","email":"","orcid":"","institution":"Faculdade de Medicina de São José do Rio Preto, São José do Rio Preto, SP, 15090–000, Brazil","correspondingAuthor":false,"prefix":"","firstName":"Debora","middleName":"Aparecida P. C.","lastName":"Zuccari","suffix":""},{"id":408791831,"identity":"1bfd3a2a-fb36-483e-9ab7-c9737bcde5a3","order_by":6,"name":"Glaura Scantamburlo Alves Fernandes","email":"","orcid":"","institution":"General Biology Department, Biological Sciences Center, State University of Londrina, Londrina (UEL), PR, Brazil","correspondingAuthor":false,"prefix":"","firstName":"Glaura","middleName":"Scantamburlo Alves","lastName":"Fernandes","suffix":""},{"id":408791832,"identity":"444c8b2b-ba6e-4611-9703-00bdb54cc1eb","order_by":7,"name":"Milena Cremer de Souza","email":"","orcid":"","institution":"North of Paraná State University (UENP), Biological Science Center, Bandeirantes, PR, Brazil","correspondingAuthor":false,"prefix":"","firstName":"Milena","middleName":"Cremer","lastName":"de Souza","suffix":""},{"id":408791833,"identity":"53220f86-2473-4e6f-8170-f00f8b0dd281","order_by":8,"name":"Russel J. Reiter","email":"","orcid":"","institution":"Department of Cellular and Structural Biology, UTHealth, San Antonio, TX 78229, USA","correspondingAuthor":false,"prefix":"","firstName":"Russel","middleName":"J.","lastName":"Reiter","suffix":""},{"id":408791834,"identity":"9f5b2081-2d60-4288-8616-514d9a2e214b","order_by":9,"name":"Luiz Gustavo de Almeida Chuffa","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-0199-3396","institution":"Department of Structural and Functional Biology, UNESP - São Paulo State University, Institute of Bioscences, Botucatu, 18618-689, São Paulo, Brazil","correspondingAuthor":true,"prefix":"","firstName":"Luiz","middleName":"Gustavo de Almeida","lastName":"Chuffa","suffix":""}],"badges":[],"createdAt":"2025-01-29 12:02:50","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5924048/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5924048/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75135184,"identity":"194ce6f8-e3bc-494f-9491-b54daa916a77","added_by":"auto","created_at":"2025-01-31 02:27:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":130566,"visible":true,"origin":"","legend":"\u003cp\u003eIntracellular Mel concentration after treatment with Mel and Luz in the SKOV-3 and CAISMOV-24 cell lines. Data were expressed as mean ± SEM of triplets. * P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5924048/v1/38953a87cb2314f0f3fff50e.png"},{"id":75135188,"identity":"7a6bf05c-31ed-4c4e-a028-3e3b5b686392","added_by":"auto","created_at":"2025-01-31 02:27:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5238431,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Mel and Luz on the invasion and migration of SKOV-3 cells. A) Effect of Mel and Luz on the invasive capacity of SKOV-3 cells. B) Images of invaded cells after treatments. C) Effect of Mel and Luz on the migratory potential of SKOV-3 cells. D) Images of migrated cells after treatments. Mel: melatonin; Luz: luzindole. Data were expressed as mean ± SEM of the triplets. *P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5924048/v1/f6157ab3ef6929a2d4cf778b.png"},{"id":75135186,"identity":"fa35e545-a4bc-4b3f-83ba-f206f1748e07","added_by":"auto","created_at":"2025-01-31 02:27:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4576832,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Mel and Luzon the invasion and migration of CAISMOV-24 cells. A) Effect of Mel and Luz on the invasive capacity of CAISMOV-24 cells. B) Images of invaded cells after treatments. C) Effect of Mel and Luz on the migratory potential of CAISMOV-24 cells. D) Images of migrated cells after treatments. Mel: melatonin; Luz: luzindole. Data were expressed as mean ± SEM of the triplets. *P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5924048/v1/6b8c07939ebe09e56bbd178f.png"},{"id":75135196,"identity":"ee2c251d-c05f-47df-97a3-f0d9b827ea9b","added_by":"auto","created_at":"2025-01-31 02:27:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":321203,"visible":true,"origin":"","legend":"\u003cp\u003eEnzymatic activities of SOD, Catalase, GT, GST, GSSG and GSH in SKOV-3 cells in response to Mel and luzindole after 24h of treatment exposure. Data are expressed as the mean ± SD. *P\u0026lt;0.05. All samples were assayed in triplicate and in the same run. One-way ANOVA complemented by Tukey’s test. SOD: superoxide dismutase, TG: Total Glutathione, GST: Glutathione-S-transferase, GSSG: Oxidized glutathione, GSH: Reduced glutathione.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5924048/v1/67993abc84d5485da09eb57f.png"},{"id":75135191,"identity":"35f809c6-86e8-419e-b62f-c3d8462d1244","added_by":"auto","created_at":"2025-01-31 02:27:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":332709,"visible":true,"origin":"","legend":"\u003cp\u003eEnzymatic activities of SOD, Catalase, GT, GST, GSSG and GSH in CAISMOV-24 cells in response to Mel and luzindole after 24h of treatment exposure. Data are expressed as the mean ± SD. *P\u0026lt;0.05. All samples were assayed in triplicate and in the same run. One-way ANOVA complemented by Tukey’s test. SOD: superoxide dismutase, TG: Total Glutathione, GST: Glutathione-S-transferase, GSSG: Oxidized glutathione, GSH: Reduced glutathione.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5924048/v1/fa20782aba159dc29cb2e77c.png"},{"id":75135261,"identity":"165b4114-0b4a-42fc-8565-cf96e441f76c","added_by":"auto","created_at":"2025-01-31 02:35:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2456673,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking results showing the interaction between melatonin and catalase. A) Structure of a catalase subunit evidencing its domains and melatonin localization. B) Specific amino acids residues involved in melatonin binding.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5924048/v1/95ae2cd716b7e52abe210c73.png"},{"id":75135195,"identity":"e69b373e-3f38-4b72-b64a-8b18d8e2bcbe","added_by":"auto","created_at":"2025-01-31 02:27:49","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2754607,"visible":true,"origin":"","legend":"\u003cp\u003eDocking view illustrating the close association between melatonin (green) and the heme group (red). A) Melatonin binds in the same pocket as the heme group. B) Theoretical interaction between melatonin and the heme group.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-5924048/v1/c178ddcb663e634919a55dbe.png"},{"id":75135210,"identity":"7bebed47-c494-4ccc-a73e-20cea54abc9c","added_by":"auto","created_at":"2025-01-31 02:27:50","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1026649,"visible":true,"origin":"","legend":"\u003cp\u003eCommon amino acid residues (indicated by arrows) that form heme-binding site and may also contribute to melatonin binding.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-5924048/v1/7b57edee4172966a140334f2.png"},{"id":75135199,"identity":"b7a86074-fcfe-43b7-8e74-0fc06c2106d9","added_by":"auto","created_at":"2025-01-31 02:27:49","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":102387,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the effects of Mel against agents related to oxidative stress in SKOV-3 and CAISMOV-24 OC. SOD: superoxide dismutase, GT: Total Glutathione, GST: Glutathione-S-transferase, GSSG: Oxidized glutathione, GSH: Reduced glutathione.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-5924048/v1/79f16ed902b5c26ee9c02458.png"},{"id":75135833,"identity":"42a6fc8a-a6da-498e-a4d7-17293a5a1470","added_by":"auto","created_at":"2025-01-31 02:43:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17227817,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5924048/v1/3638429b-6771-4ae9-992e-c445af3b0db2.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eMelatonin reduces cell motility and antioxidant defenses in ovarian cancer cell lines\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOvarian cancer (OC) is one of the most common female tumor subtypes which has a high recurrence and mortality rate [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This mortality rate is associated with its late diagnosis due to multiple symptoms that are often generic and their aggressive recurrence even with already established treatments and surgical removal [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Modifications in cellular metabolism occur when tumors redefine how they obtain nutrients and navigate metabolic routes to fulfill the requirements for biological energy, duplication, and the control of reactive oxygen species (ROS) levels in the cancer cells examined [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These changes are recognized as key aspects of cancer metabolism, marked by features such as tissue invasion and metastasis, loss of growth inhibition, rapid proliferation, sustained cell viability, resistance against cell death, initiation of the angiogenesis pathway, evasion of the immune response, and modified cellular vitality [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMelatonin (Mel; N-acetyl-5\u003cem\u003e-\u003c/em\u003emethoxytryptamine) is a lipophilic molecule converted from serotonin by the enzymes arylalkylamine N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Mel has been characterized by actions that may either depend on or be independent of its membrane receptors (MT1 and MT2). While its main production occurs in the pineal gland at night, other reports have shown its synthesis in different tissues and cells with local actions [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Mel has numerous functions under normal metabolic conditions including its antioxidant actions by removing reactive oxygen and nitrogen species and stimulating DNA repair mechanisms [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In many cancer cell types, Mel has oncostatic functions associated with pro-oxidative function while concurrently reducing the migration and invasiveness of cancer cells and inducing apoptosis. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFree radicals (FR) are a generic term that encompasses unstable and reactive atoms or molecules. In biological systems, FR can be separated into two classes: reactive oxygen species (ROS) and reactive nitrogen species (RNS) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Superoxide anion (O2\u0026bull;\u0026minus;), hydroxyl radical (OH\u0026bull;), alkoxy radical (RO\u0026bull;), hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), and hypochlorous acid are considered ROS and they are normally generated during cellular metabolism; nitrogen dioxide (NO\u003csub\u003e2\u003c/sub\u003e), nitroxyl (HNO) and peroxynitrite (ONOO-) are examples of RNS produced from nitric oxide and by the NADPH oxidase (NOX) system [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In healthy cells, there is a delicate balance between the production and removal of FR, which ensures redox homeostasis and their optimal physiological effects [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFR affects gene expression, cell growth and differentiation, modulate metabolic reactions, and regulate transcription factor activation, in addition to acting as intra and intercellular signaling molecules [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In OC cell lines, there is initially a significant increase in ROS production, thus favoring tumorigenesis. This elevated ROS results from NADPH oxidase activity and mitochondrial metabolism, as this increase is diminished by mitochondrial complex I inhibitors [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Elevated ROS which occurs at the level of the electron transport chain (ETC) is associated with chemoresistance to platinum-derived therapies leading to a poor prognosis and accelerating OC growth [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In addition, the increase in ROS is associated with dysregulation of microRNAs (miRNAs) which are linked to tumor progression. In serous epithelial OC, there is overexpression of specific miRNAs, an action that does not occur in the non-serous OC [\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo avoid the damage caused by ROS, cells have antioxidant systems which are either enzymatic or non-enzymatic in nature. Among the main anti-oxidative enzymes are superoxide dismutase (SOD), catalase (CAT,), glutathione peroxidase (GSH-Px), glutathione reductase (GSH-Rd), among others. It is important to consider that a specific antioxidant, to be efficient, does not necessarily need to be ubiquitous, but must act at the diffusion rate of the FR and be in the proper position in the immediate vicinity of where the radical is generated [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. With the exception of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, FR do not cross biological barriers and have a very short half-life. Mel is an antioxidant under normal circumstances but can serve a pro-oxidant under specific pathologies [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Although additional investigation is needed, Mel has been documented to promote an increase in ROS via different mechanisms [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In head and neck cancer cells, Mel induced ROS by reversing mitochondrial ETC which generated the anti-growth behavior [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Mel also induces apoptosis mediated by the stimulation of ROS synthesis in breast cancer as well as in mesangial cells, thus documenting that the anti-cancer actions of melatonin may be mediated by excessive FR generation effects [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. To date, the role of Mel on oxidative processes in OC have not been examined. Herein, we investigated the impact of Mel, acting via its membrane receptors or not, on antioxidant defenses of OC cells as well as its protection against cell migration and invasiveness.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eCell culture and reagents\u003c/h2\u003e\n\u003cp\u003eSKOV-3 cell line (ATCC\u0026reg; HTB-77) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), while the CAISMOV-24 cell line was obtained from the Women\u0026rsquo;s Hospital Prof. Dr. Jos\u0026eacute; Aristodemo Pinotti Caism (UNICAMP, Campinas, SP, BRAZIL). SKOV-3 cells were cultivated using RPMI medium (Gibco, Paisley, UK), whereas CAISMOV-24 cells were nurtured in DMEN HAN\u0026rsquo;S F-12 (LGC, Cotia, BR). Both cell types received 10% fetal bovine serum (FBS) (Gibco) and penicillin at 100 IU/ml, as well as streptomycin at 100 \u0026micro;g/ml (Gibco). The cells were placed in a humidified environment at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Cellular expansion was carried out in distinct 75 and 25 cm\u003csup\u003e2\u003c/sup\u003e culture flasks (Costar, Cambridge, MA, USA). Upon achieving 80% of confluence, the cell supernatant was carefully extracted. Afterwards, the cells underwent two washes with 10% phosphate-buffered saline (PBS; Oxoid Limited, Hampshire, UK). Subsequently, trypsin/EDTA (Gibco) was employed to interrupt adhesion to the flasks.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eExperimental design and treatments\u003c/h3\u003e\n\u003cp\u003eTo examine the effects of Mel and its combination with luzindole (Luz) on OC cells, we first determined the safe concentrations according to the IC\u003csub\u003e50\u003c/sub\u003e method (MTT assay). Concentrations of 3.4 \u0026micro;M and 7 \u0026micro;M of Mel were chosen respectively to treat SKOV-3 and CAISMOV-24 cells for a period of 24h. The concentration of Luz (a Mel receptor antagonist) was defined as 1 \u0026micro;M for both cell lines. To understand the effect of Mel in combination with Luz on cancer cells, we divided each cancer cell line into three experimental groups: Control: cells exposed only to the culture medium containing 100 \u0026micro;L of DMSO solution as the vehicle; Mel: cells exposed to Mel at concentrations of 3.4 and 7 \u0026micro;M for SKOV-3 and CAISMOV-24, respectively, plus vehicle; and Mel\u0026thinsp;+\u0026thinsp;Luz: cells exposed to the combination of Mel and Luz plus vehicle. For the combination group, Luz was first administered, and after 30 min, the pre-established concentration of Mel was added. Mel and Luz were dissolved in DMSO (5%) maintaining the molarity values indicated by the manufacturers. All experimental assays were performed in biological and technical triplicates.\u003c/p\u003e\n\u003ch3\u003eCell viability (MTT assay)\u003c/h3\u003e\n\u003cp\u003eCell viability was analyzed using the MTT method based on the IC\u003csub\u003e50\u003c/sub\u003e to determine proper Mel concentrations. After the confluence rate, SKOV-3 and CAISMOV-24 cells were trypsinized, seeded in 6-well plates at a density of 5x10\u003csup\u003e5\u003c/sup\u003e cells per well, and cultured with appropriated medium supplemented with 10% FBS. After cell adherence, Mel and Luz were added to culture medium following the experiment design with specific concentrations (2.0, 3.4, 5.0, and 7.0 \u0026micro;M). Viability curves were estimated after 24h of treatment using the MTT solution (5mg/mL). To detect cytotoxicity, the reactions were replicated in 96-wells plate and read in a microplate reader (Epoch, Bio Tek Instruments, USA).\u003c/p\u003e\n\u003ch3\u003eCell invasion and migration assays\u003c/h3\u003e\n\u003cp\u003eThe evaluation of SKOV-3 and CAISMOV-24 cell invasiveness was performed using 24-well plates. A thin membrane of Geltrex\u0026reg; was added to each well, occluding the lower polyethylene terephthalate (PET) membrane. SKOV-3 and CAISMOV-24 (1x10\u003csup\u003e5\u003c/sup\u003e cells) were added to the top of the insert and received standard medium without FBS. The invasive potential was analyzed based on the ability of cells to cross the gel barrier and the PET membrane through the pores, being attracted chemotactically by inferior coverage of culture medium containing 5% FBS. The plates were placed in a CO\u003csub\u003e2\u003c/sub\u003e atmosphere at 37\u0026ordm;C for 24h. After the incubation period, cells were fixed in methanol for 10 min, and the remaining cells were removed by scraping. Migrated cells were stained with a 0.1% toluidine blue solution and photographed with a 5X objective in an inverted microscope (ZeissAxiovert\u0026reg;). For migration assay, a similar experimental procedure was used, except for Geltrex\u0026reg; which was not added to the transwell chamber. All experiments were performed in triplicate based on four fields and submitted to automatic cell count LUNA-II\u0026reg; (Logos Biosystems, South Korea)\u003c/p\u003e\n\u003ch3\u003eMeasurement of melatonin concentration\u003c/h3\u003e\n\u003cp\u003eMel levels were determined in both SKOV-3 and CAISMOV-24 cells using a human-specific commercial ELISA kit (EH3344, Fine Test), according to manufacturer\u0026rsquo;s instructions. The absorbance was read at 450 nm on a microplate reader (Epoch, Bio Tek Instruments, USA). Results were interpolated from standard curves generated by plotting the concentration of the standards against their absorbance. The concentrations are presented in pg/mL.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003ePreparation of the supernatant for analyzing the antioxidant system\u003c/h2\u003e\n\u003cp\u003eAfter Mel treatment, the culture medium was discarded, and PBS was added to the cells. The cells were then lysed through three cycles of freezing (-80 \u0026ordm;C) and thawing (37 \u0026ordm;C) for 30 min each. After this process, protein levels were quantified and used to normalize the results of the total (GS), reduced (GSH), and oxidized glutathione (GSSG), catalase (CAT), glutathione S-transferase (GST), and superoxide dismutase (SOD).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eActivity of SOD and CAT\u003c/h3\u003e\n\u003cp\u003eAfter treatment with Mel and Luz, the cells (5 x 10\u003csup\u003e5\u003c/sup\u003e cells/mL) were washed with PBS (pH 7.4) and re-plated for all the analyzes. SOD enzyme activity assay was performed according to [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e] with some modifications. The reaction medium was composed of methionine (13 mM), NBT (75 \u0026micro;M), and riboflavin (4 mM) in phosphate buffer (50 mM, pH 7.8). 10 \u0026micro;L of the cell extract was added, and the samples were exposed to fluorescent light (13W) for 5 min. Control samples (no enzymes) and the blank (containing all reagents but not exposed to light) were used. Readings were performed using a MultiSkan-Skyhigh microplate reader at 560 nm and at 25\u0026deg;C. For the evaluation of CAT activity, cells were lysed, and a reaction medium was prepared as described by Aebi et al. [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]. The reading was performed by a spectrophotometer at 240 nm for 1 min at 15-second intervals.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMeasurement of GSH, GT, GSSG, and GST.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo quantify the levels of reduced glutathione (GSH) and total glutathione (GT),\u003c/p\u003e\n\u003cp\u003ethe protocol proposed by Rahman, Kode and Biswas [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e] was carried out with modifications, using 5,5'-ditiobis 20-nitrobenzoic acid (DTNB) in the homogenate, evidenced by yellow formation. To determine total glutathione (TG) levels, DTNB, nicotinamide-adenine dinucleotide phosphate (NADPH), and glutathione reductase were used in the homogenate. GT and GSH levels were measured at 412 nm (Multiskan GO, Thermo Scientific), and results were expressed as \u0026micro;mol/mg protein. GSSG levels were calculated [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e], considering the stoichiometry of the reactions. To evaluate glutathione S-transferase (GST) activity, 1-chloro-2,4-dinitrobenzene (CDNB) and potassium phosphate buffer were used. The absorbance of the samples was measured at 340 nm for 160 seconds, with 5 measurements at 40-second intervals [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eIn silico molecular blind docking\u003c/h3\u003e\n\u003cp\u003eThe target protein used in this \u003cem\u003ein silico\u003c/em\u003e study was human liver mitochondrial catalase (PDB ID: 8SGV), which was downloaded from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/\u003c/span\u003e\u003c/span\u003e; accessed on 10 January 2025) [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]. Catalase is a tetramer composed of four identical subunits (A,B,C, and D) with over 500 amino acid residues. Each chain is coordinate with iron-heme ring and binds NADPH as a cofactor. We conducted our analysis considering the A subunit isolated. The preparation of the protein for the molecular docking was conducted in the UCSF ChimeraX software (version 1.8) [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. Water molecules, nonstandard residues and the chains B, C and D were deleted. The 3D structure of melatonin molecule (CID: 896) was downloaded from PubChem (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003c/span\u003e; accessed on 10 January 2025) [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e] and was prepared in the Avogrado software (version 1.2.0) by adding hydrogens and optimizing the molecule geometry. The next steps were done in the Autodock Tools software (MGLTools-1.5.6). In that stage, any residual water molecule was deleted, polar hydrogens and Kollman charges were attached in the protein. The melatonin structure was forced to let all rotatable bonds rotatable. The grid box was set to 126, 126, and 126 \u0026Aring; along the X-, Y-, and Z-axis with a grid spacing of 0.925 \u0026Aring; in order to recognize the whole catalase subunit. The AutoDocking parameters adopted were: GA runs\u0026thinsp;=\u0026thinsp;500; population size\u0026thinsp;=\u0026thinsp;150; maximum number of energy evaluations\u0026thinsp;=\u0026thinsp;25.000.000; GA crossover mode\u0026thinsp;=\u0026thinsp;two points. The Lamarckian Genetic algorithm was selected to search for the best conformations and the lowest binding energy conformer was chosen for further analysis. The Chimera X and the Biovia Discovery Studio Visualizer (version 24.1.0) were used to analyze and visualize the docking results.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eData were processed using an analysis of variance (One-way ANOVA) for one factor and presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Significant results were complemented by Tukey\u0026rsquo;s test. Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for all analyses. Results were analyzed and generated using GraphPad Prism 9.0 scientific graphing software (GraphPad Software, San Diego, CA).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e\u003cem\u003eMel treatment enhances its intracellular concentration in OC cells.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eBased on MTT analysis, we identified the cytotoxic effect of Mel (IC\u003csub\u003e50\u003c/sub\u003e) which was previously established at 3.4 \u0026micro;M for SKOV-3 cells and 7 \u0026micro;M for CAISMOV-24 cells. Luzindole, an antagonist of MT1/2 Mel receptors, was used to determine whether the Mel\u0026rsquo;s effect is dependent or independent of their membrane receptors. The intracellular concentrations of Mel were measured in the OC cells by the enzyme assay. Mel treatment elevated the levels of intracellular Mel compared to the control group, especially in SKOV-3 cells. After blocking MT1/2 receptors, Mel promoted an increase in its own intracellular levels in both SKOV-3 and CAISMOV-24 cells (1.96-fold increase vs. Control and 2.08-fold increase vs. Control, respectively). More importantly, Mel alone in the presence of its receptors, increased its intracellular levels in SKOV-3 cells (1.75-fold increase vs. Control), thus restoring Mel concentration in OC cells where they are normally depressed (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cem\u003eMel reduces the migratory and invasive capacity of SKOV-3 and CAISMOV-24 cells regardless of the MT1/2 receptor activation.\u003c/em\u003e\u003c/p\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eTo investigate the effects of Mel and luzindole on the migratory and invasive potential of SKOV-3 and CAISMOV-24 cells, we used transwell inserts in 24-well plates. The invasive potential of SKOV-3 and CAISMOV-24 cells was reduced by Mel alone (2.45- and 3.58-fold decrease vs Control groups, respectively), and after the combination of Mel and luzindole (2.17-fold decrease vs Control group in SKOV-3 cells and 2.38-fold decrease vs Control group in CAISMOV-24 cells; Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). The results also showed that Mel alone significantly reduced the migration of both SKOV-3 and CAISMOV-24 cells (2.03- and 2.29-fold decrease vs Control group, respectively; Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). When Mel was combined with luzindole, a significant decrease in cell migration was also observed considering both OC cells (1.67-fold decrease vs. Control in SKOV-3 cells and 2.02-fold decrease vs. Control in CAISMOV-24).\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cem\u003eMel differentially regulates the antioxidant enzymes in OC cells.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSOD levels were only reduced by Mel in combination with luzindole in the CAISMOV-24 cells (p\u0026thinsp;=\u0026thinsp;0.027). Regarding Catalase levels, a significant reduction was observed after Mel treatment in both OC cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for CAISMOV-24 and p\u0026thinsp;=\u0026thinsp;0.012 for SKOV-3). The presence of luzindole restored catalase levels close to that of control groups, thus showing a receptor dependent response; it has been noted that the action of melatonin on antioxidant enzymes are receptor mediated. The changes in glutathione activities following treatments with Mel and luzindole varied differently between the two OC cell lines (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). In SKOV-3 cells, Mel alone significantly decreased the levels of TG (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and GSSG (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared with the control group. The combination of Mel and luzindole also showed a decrease in the levels of TG (p\u0026thinsp;=\u0026thinsp;0.003) and GSSG (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Conversely, this combination increased the levels of GST compared with control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and Mel alone (p\u0026thinsp;=\u0026thinsp;0.002). Notably, in CAISMOV-24 cells, Mel alone or combined with luzindole significantly reduced the activities of TG (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), GST (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), GSSG (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and GSH (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared with the control group. These results demonstrate an opposite effect of Mel favoring pro-oxidative processes in the OC cells.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMel directly interacts with both the protein portion of catalase and its heme group.\u003c/em\u003e\u003c/p\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThrough in vitro molecular docking assays, the ability of melatonin to interact with catalase was observed. Out of the 500 poses tested, the five with the lowest binding energies are presented in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Subsequently, experiments were conducted to demonstrate the physical interaction between melatonin and catalase. Melatonin bound to an enzyme pocket in the \u0026beta; barrel domain, very close to the binding site of the heme prosthetic group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). Melatonin directly interacts with the polypeptide chain of catalase through hydrogen bonds involving residues Arg72, Tyr358, His362, and Arg365. Additionally, alkyl interactions with Val73, Val74, and Val146, as well as \u0026pi;-interactions with His75, are also observed (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). As melatonin was found to be positioned near the porphyrin ring (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA), we further docked the interaction between these two molecules. The results show a close distance of approximately 7 \u0026Aring; between melatonin and the heme group, suggesting that the structures are likely oriented in a specific way that facilitates interaction (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB). Finally, it can be observed that some amino acids involved in anchoring the porphyrin ring to the protein may also interact with the indolamine (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). These potential interactions are of significant value and warrant further investigation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDocking results for interaction between the Melatonin and Catalase (subunit A).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eK\u003csub\u003ei\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003evdW\u0026thinsp;+\u0026thinsp;Hbond\u0026thinsp;+\u0026thinsp;desolv\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eEletrostatic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eTorsional\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eUnbound\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e453\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-5.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e40.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-6.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e-0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e+1.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e-0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-5.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e45.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-6.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e-0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e+1.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e-0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e214\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-5.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e64.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-6.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e+\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e+1.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e-0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e146\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-5.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e65.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-6.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e+\u0026thinsp;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e+1.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e-0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e475\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-5.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e82.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-6.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e+\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e+1.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e-0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eBE: Binding energy; Ki: Estimated Inhibition Constant (micromole. L\u003csup\u003e-1\u003c/sup\u003e); vdW: van der Waals interactions; desolv: desolvation energy. Energy parameters are expressed as kcal.mol\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eHerein we report that exogenously applied melatonin to the incubation medium increases the uptake of melatonin to improve its intracellular levels accompanied by attenuation of the invasive and migratory capacity of OC cells in a receptor-independent manner. Melatonin has been shown to be taken up by cells [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. We also bring new information regarding the interference of Mel in the enzymatic antioxidant system of OC cells which may negatively impact their survival and growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Firstly, we observed an increased concentration of Mel in both OC cell lines, mainly in SKOV-3 cells following Mel treatment, in the presence of Luz or not, and in CAISMOV-24 cells only after the combination of Mel and Luz. We previously documented an increase in the intracellular concentrations of Mel in SKOV-3 cells after treatment with 3.2 mM of Mel [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. A greater Mel level may contribute to the antiangiogenic process, pro-oxidative, and pro-apoptotic pathways and may promote modifications in the metabolic profile of tumor cells [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The elevation in intracellular levels of Mel appear to be receptor-independent since the combination of Mel with luzindole also promoted an increase in the availability of the indolamine. Treatment with Mel can disinhibit the enzymatic activity of the pyruvate dehydrogenase complex (PDC), either directly [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] or by first reducing hypoxia-inducible factor 1-alpha (HIF-1α), a factor highly expressed in hypoxic conditions of the tumor microenvironment, which stimulates the pyruvate dehydrogenase kinase (PDK) enzyme. After PDC disinhibition, Mel can be synthesized in mitochondria, and oxidative phosphorylation is reestablished [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe fact that Mel promotes a reduction in the invasive and migratory capacity of OC cells is well known [\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Recently, our group showed that MT1 knockdown in SKOV-3 cells treated with 3.2- or 4-mM Mel presented a remarkable reduction in cell migration and invasion [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]; we obtained similar results using luzindole, a non-selective antagonist of both MT1 and MT2 receptors, in two different OC cell lines via transwell inserts. Despite the use of concentrations ranging from 3.4 and 7 \u0026micro;M of Mel to SKOV-3 and CAISMOV-24 cells, respectively, our results clearly demonstrated a reduction in the invasive and migratory capacity of OC cells, regardless of the activation of Mel receptors. The precise mechanisms by which Mel regulates OC cell migration and invasion are not yet fully understood. Previous studies have shown that Mel exerts anti-migratory effects via the MAPK and PI3K pathways in OC cells [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In other cancer types, such as colon and breast cancer, Mel attenuated cell invasion, migration, and survival by targeting PI3K/AKT and NF-κB signaling pathway [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] or by downregulating the p38 MAPK pathway [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In addition to its passive diffusion, Mel can alternatively enter tumor cells using other membrane transporters such as PEPT1/2 and GLUT1 [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], therefore it may act in an MT1/2 receptor-independent manner to exert the anti-tumoral effects on the migratory and invasive potential of OC cells.\u003c/p\u003e \u003cp\u003eDefects in mitochondrial metabolism can increase the production of ROS and RNS, leading to OS. To combat the excessive and harmful production of these radical and non-radical species, cells are equipped with an antioxidant defense system that includes enzymes such as SOD, CAT, and glutathione derivatives [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. An excessive and permanent generation of OS is related to genetic/epigenetic events that could facilitate the tumorigenic process; however, increasing the generation of ROS and RNS in tumor cells is paradoxically used as a strategy to induce their death [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. A potential actor in this approach is the use of Mel, known for its antiproliferative effects and for interfering with angiogenesis and metastasis events; Mel acts by modulating the oxidant/antioxidant state of cells [\u003cspan additionalcitationids=\"CR53 CR54 CR55\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Moreover, the effects of Mel on tumor cells seem to be opposite of those occurring in normal healthy cells, that is, Mel acts as a pro-oxidant agent in tumor cells, thus reducing their antioxidant defenses [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur results reinforce the apparent pro-oxidant properties of Mel in OC cells, especially in CAISMOV-24 cells. We are the first to demonstrate a decrease in SOD activity when Mel was combined with luzindole, i.e., a receptor-independent response, and a significant reduction in CAT levels after Mel treatment in both OC cells, suggesting that Mel acts directly as a modulator of these two antioxidant enzymes. Mel treatments have also been associated with lower enzymatic activities of SOD and CAT accompanied by an increased level of ROS observed in human colorectal cancer cells and in the hepatocellular carcinoma cells [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], resulting in apoptosis due to an excessive oxidative damage.\u003c/p\u003e \u003cp\u003eIn healthy tissues, Mel generally upregulates antioxidant enzymes, including the glutathione system that plays a central role against OS [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Here, we observed a marked decrease in total glutathione (GT) levels in both OC cell lines following treatment with Mel alone. When Mel receptors were blocked by luzindole, only SKOV-3 cells showed a significant reduction in GT levels, while CAISMOV-24 cells showed only a trend. The reduced form of glutathione (GSH) is one of the most important machineries on the front line of the antioxidant defense system. GSH maintains a redox state in subcellular compartments such as mitochondria and cytosol, and its increased levels may be involved in the chemoresistance of cancer cells [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. In this study, GSH content was only reduced with CAISMOV-24 cells treated with Mel alone. It is also reported that Mel can exert a depletion on GSH levels in HepG2 cells [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], and in human myeloid leukemia cell line (U937) [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Under OS conditions, GSH can be directly converted to oxidized glutathione (GSSG) in the presence of ROS [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. We observed a notable reduction in GSSG levels in both OC cells. Particularly, for SKOV-3 cells, GSH levels were like that of the control group, while GSSG was significantly decreased by both treatments. Based on this GSH:GSSG ratio that favors GSH, we can hypothesize a more pronounced resistance of SKOV-3 cells to OS, which leads to the maintenance of GSH homeostasis, differing from what was observed with CAISMOV-24 cells.\u003c/p\u003e \u003cp\u003eCancer cells can also gain resistance through overexpression of enzymes that can increase detoxification capacity and avoid the cytotoxic action of antitumor drugs [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. More specifically, overexpression of glutathione S-transferases (GST) and efflux pumps in tumor cells can reduce the reactivity of various anticancer drugs, such as cyclophosphamide [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], cisplatin [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], and others [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. In this study, OC cell lines showed opposite results regarding GST. While in CAISMOV-24 cells both treatments decreased GST activities, in SKOV-3 cells the combination of Mel with luzindole increased its activity. As reviewed in detail by Sau et al. [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e], the increase in GST levels occurs through transcriptional activation mediated by the nuclear factor erythroid 2 p45-related factor 2 (Nrf2). However, further studies are needed to precisely determine the role of Mel and its receptors in the molecular pathways that regulate the activity and expression of genes in the glutathione system. Overexpression of GST and high levels of GSH are linked to the development and expression of chemoresistance [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. In this context, our treatment with Mel alone at 7 \u0026micro;M successfully promotes a decrease in GST and GSH levels in CAISMOV-24, a recently established human low-grade serous ovarian carcinoma cell line [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. SKOV-3 cells, a non-serous ovarian cancer cell line, in turn, remained unchanged for these parameters after treatment of 3.4 \u0026micro;M of Mel alone, thus suggesting further evaluations with higher concentrations. This sounds plausible since it has been shown that high levels of Mel are required to induce ROS production in tumor cells [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In our previous study using the same OC cell lines and melatonin concentrations, we observed a reduction in Warburg-type metabolism and potentially glutaminolysis, which further attenuated oncogenic targets associated with OC progression and invasion [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eAs stated below, melatonin's capacity to modulate antioxidant enzymes activity is well documented. However, it remains unclear whether melatonin interacts directly with specific enzymes, and the precise nature of such interactions has not been fully explored. To bridge this gap, and given that catalase activity was reduced in the two OC cell lines, we employed in silico molecular docking to investigate the interaction between melatonin and catalase. Catalase\u0026rsquo;s catalytic mechanism occurs in two stages. Initially, the heme Fe\u003csup\u003e3+\u003c/sup\u003e reduces a hydrogen peroxide molecule to water, forming a covalent oxyferryl species, along with a porphyrin π-cation radical. This radical then oxidizes another hydrogen peroxide molecule into molecular oxygen, while the ferryl oxygen species is released as water. Several amino acid residues, including tyrosine, play crucial roles in facilitating these reactions at the active site [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe docking results revealed that melatonin binds most effectively on a structural pocket between β-sheet and wrapping domain, near the heme-binding cavity of catalase. Melatonin binding was stabilized with hydrogens and hydrophobic interactions, with a binding energy of -5.99 kcal.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The amino acid residues Arg72, Tyr, 358, His362, and Arg365 play major role in the binding of melatonin on catalase subunit. Melatonin forms four conventional hydrogen bonds with those amino acids, along with additional potential interactions. Notably, the tyrosine residue at position 358 interacts closely with the iron in the porphyrin ring, and melatonin also engages with this residue. Given the critical role of the heme group in catalase's catalytic activity, this interaction with Tyr358 may provide valuable insight into the mechanism by which melatonin reduces the enzyme's activity. Additionally, melatonin\u0026rsquo;s interaction with other amino acid residues could alter the microenvironment of the catalase active site, further contributing to the observed effect in enzyme activity. The binding of farnesiferol C, a sesquiterpene coumarin, to bovine liver catalase yielded similar findings, affecting both the enzyme\u0026rsquo;s affinity and catalytic activity [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. The discovery of specific, non-toxic catalase inhibitors presents significant potential for use in cancer co-therapy. For instance, a Zn(II) complex tested against human colon cancer cells demonstrated cytotoxic effects by inhibiting catalase through mixed-type inhibition kinetics. Interestingly, the binding site of the complex also involved hydrogen bonding and exhibited a free energy of -7.13 kcal/mol [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e], similar to the binding characteristics observed for melatonin.\u003c/p\u003e \u003cp\u003eAn intriguing and unexpected finding in our study was the close proximity of melatonin to the heme group of catalase. To explore this further, we evaluated the potential physical interaction between melatonin and the porphyrin ring. The binding free energy between melatonin and the heme group was calculated to be -4.68 kcal/mol, suggesting various interaction types. More importantly, several amino acid residues critical for maintaining the stability of the heme group \u0026ndash; such as Val73, Val74, Arg72, His75, and Val146 \u0026ndash; were found to interact with melatonin. These interactions may contribute to melatonin\u0026rsquo;s inhibition of catalase activity, a possibility that warrants further investigation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eCollectively, Mel treatment attenuates the migratory and invasive capacity of OC cells in a receptor independent manner while stimulating its intracellular concentration. Moreover, the antioxidant enzymatic defenses were dampened by Mel, especially in the CAISMOV-24 cells. The blockage of MT1/2 receptors by the antagonist luzindole followed by Mel administration showed a tendency to soften the results. Indeed, pre-incubation of SKOV-3 cells with 10 \u0026micro;M luzindole revealed an increase in cell viability [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Based on our results, we believe that the function of Mel in SKOV3 and CAISMOV-24 is partially mediated by MT1 and MT2 receptors. Also, our data provide valuable insights into the regulatory role of Mel in modulating antioxidant status in OC cells. Based on the molecular docking study, future investigations using molecular dynamics simulations and enzyme kinetics assays will be valuable and necessary to confirm the predicted interactions between catalase and melatonin as pivotal to enzyme modulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of Interest:\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis research was funded by CAPES (Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior \u0026ndash; Brasil), grant number 88887.482443/2020-00, National Council for Scientific and Technological Development (CNPQ, Process numbers 304108/2020-0 and 306117/2023-1 to LGAC) and S\u0026atilde;o Paulo Research Foundation (FAPESP grant #2021/12971-7 to LGAC).\u003c/p\u003e\u003ch2\u003eAuthor Contributions:\u003c/h2\u003e \u003cp\u003eConceptualization, HS, FRS and LGAC; Data curation, HS; Formal analysis: RCC, FG, FRS, VAS, GSAF, MCS, DAPCZ and LGAC; Investigation, HS, FRS and DAPCZ; Methodology, HS, RCC, FG and DAPCZ; Visualization, RR; Writing \u0026ndash; original draft, HS and LGAC; Writing \u0026ndash; review \u0026amp; editing, RR. The authors read and approved the final version of this manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments:\u003c/h2\u003e \u003cp\u003eWe would like to extend our gratitude to the technicians, colleagues, and university staff whose unwavering support and valuable contributions were instrumental in the successful completion of this scientific article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Miller KD, Fuchs HE, Jemal A (2022) Cancer statistics, 2022. CA Cancer J Clin. 2022. 72, 7\u0026ndash;33\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGabra H (2019) Epithelial ovarian cancer. Dewhurst\u0026rsquo;s Textbook of Obstetrics \u0026amp; Gynaecology, Seventh Edition. 2019. 625\u0026ndash;635\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReid BM, Permuth JB, Sellers TA (2017) Epidemiology of ovarian cancer: A review. Cancer Biol Med. 2017. 14, 9\u0026ndash;32\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Berardinis RJ, Chandel NS (2016) Fundamentals of cancer metabolism. Sci Adv. 2016. 2, 1\u0026ndash;18\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHanahan D, Weinberg RA (2011) Hallmarks of cancer: The next generation. Cell. 2011. 144, 646\u0026ndash;674\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePavlova NN, Thompson CB (2016) The emerging hallmarks of cancer metabolism. Cell Metab. 2016. 23, 27\u0026ndash;47\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReiter RJ, Rosales-Corral SA, Tan DX, Acuna-Castroviejo D, Qin L, Yang SF, Xu K (2017) Melatonin, a full-service anti-cancer agent: Inhibition of initiation, progression, and metastasis. Int J Mol Sci. 2017. 18, 843\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao D, Yu Y, Shen Y, Liu Q, Zhao Z, Sharma R, Reiter RJ (2019) Melatonin synthesis and function: Evolutionary history in animals and plants. Front Endocrinol (Lausanne). 2019. 10, 1\u0026ndash;16\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTalib WH (2018) Melatonin and cancer hallmarks. Molecules. 2018. 23, 518\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang H-M, Zhang Y (2014) Melatonin: A well-documented antioxidant with conditional pro-oxidant actions. J Pineal Res. 2014. 57, 131\u0026ndash;146\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng L, Li S, He K, Kang Y, Li T, Li C, Zhang Y, Zhang W, Huang Y (2023) Melatonin regulates cancer migration and stemness and enhances the anti-tumour effect of cisplatin. J Cell Mol Med. 2023. 27, 2215\u0026ndash;2227\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eByrne NJ, Rajasekaran NS, Abel ED, Bugger H (2021) Therapeutic potential of targeting oxidative stress in diabetic cardiomyopathy. Free Radic Biol Med. 2021. 169, 317\u0026ndash;342\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGalano A, Tan DX, Reiter RJ (2011) Melatonin as a natural ally against oxidative stress: A physicochemical examination. J Pineal Res. 2011. 51, 1\u0026ndash;16\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahal A, Kumar A, Singh V, Yadav B, Tiwari R, Chakraborty S, Dhama K (2014) Oxidative stress, prooxidants, and antioxidants: The interplay. Biomed Res Int. 2014\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKohen R, Nyska A (2002) Oxidation of biological systems: Oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification. Toxicol Pathol. 2002. 30, 620\u0026ndash;650\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD\u0026rsquo;Souza LC, Mishra S, Chakraborty A, Shekher A, Sharma A, Gupta SC (2020) Oxidative stress and cancer development: Are noncoding RNAs the missing links? Antioxid Redox Signal. 2020. 33, 1209\u0026ndash;1229\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheung EC, Vousden KH (2022) The role of ROS in tumour development and progression. Nat Rev Cancer. 2022. 22, 280\u0026ndash;297\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXia C, Meng Q, Liu LZ, Rojanasakul Y, Wang XR, Jiang BH (2007) Reactive oxygen species regulate angiogenesis and tumor growth through vascular endothelial growth factor. Cancer Res. 2007. 67, 10823\u0026ndash;10830\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang Y, Song L, Lin Y, Nowialis P, Gao Q, Li T, Li B, Mao X, Song Q, Xing C et al (2023) ROS-mediated SRMS activation confers platinum resistance in ovarian cancer. Oncogene. 2023. 42, 1672\u0026ndash;1684\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStieg DC, Wang Y, Liu LZ, Jiang BH (2022) ROS and miRNA dysregulation in ovarian cancer development, angiogenesis and therapeutic resistance. Int J Mol Sci. 2022. 23 6702\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimone NL, Soule BP, Ly D, Saleh AD, Savage JE, DeGraff W, Cook J, Harris CC, Gius D, Mitchell JB (2009) Ionizing radiation-induced oxidative stress alters miRNA expression. PLoS One. 2009. 4, 6377\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, Sweet-Cordero A, Ebert BL, Mak RH, Ferrando AA, Downing JR, Jacks T, Horvitz HR, Golub TR (2005) MicroRNA expression profiles classify human cancers. Nature. 2005. 435, 834\u0026ndash;838\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRattanapan Y, Korkiatsakul V, Kongruang A, Siriboonpiputtana T, Rerkamnuaychoke B, Chareonsirisuthigul T (2020) MicroRNA expression profiling of epithelial ovarian cancer identifies new markers of tumor subtype. Microrna. 2020. 9, 289\u0026ndash;294\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReiter RJ, Sharma RN, Chuffa LG, Silva DGH, Rosales-Corral S (2024) Intrinsically synthesized melatonin in mitochondria and factors controlling its production. Histol Histopathol. 2024. 7, 18776\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang H-M, Zhang Y (2014) Melatonin: A well-documented antioxidant with conditional pro-oxidant actions. J Pineal Res. 2014. 57, 131\u0026ndash;146\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMortezaee K, Najafi M, Farhood B, Ahmadi A, Potes Y, Shabeeb D, Musa AE (2019) Modulation of apoptosis by melatonin for improving cancer treatment efficiency: An updated review. Life Sci. 2019. 228, 228\u0026ndash;241\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlorido J, Rodriguez-Santana C, Martinez-Ruiz L, L\u0026oacute;pez-Rodr\u0026iacute;guez A, Acu\u0026ntilde;a-Castroviejo D, Rusanova I, Escames G (2022) Understanding the mechanism of action of melatonin, which induces ROS production in cancer cells. Antioxidants (Basel). 2022. 11, 1621\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlorido J, Martinez-Ruiz L, Rodriguez-Santana C, L\u0026oacute;pez-Rodr\u0026iacute;guez A, Hidalgo-Guti\u0026eacute;rrez A, Cottet-Rousselle C, Lamarche F, Schlattner U, Guerra-Librero A, Aranda-Mart\u0026iacute;nez P et al (2022) Melatonin drives apoptosis in head and neck cancer by increasing mitochondrial ROS generated via reverse electron transport. J Pineal Res. 2022. 73, 12824\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang H-M, Zhang Y, Zhang B-X (2011) The role of mitochondrial complex III in melatonin-induced ROS production in cultured mesangial cells. J Pineal Res. 2011. 50, 78\u0026ndash;82\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGurer-Orhan H, Ince E, Konyar D, Saso L, Suzen S (2018) The role of oxidative stress modulators in breast cancer. Curr Med Chem. 2018. 25, 4084\u0026ndash;4101\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarklund S, Marklund G (1974) Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem. 1974. 47, 469\u0026ndash;474\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAebi H (1974) Catalase. In Bergmeyer, H.U., Ed., Methods of Enzymatic Analysis. 1974. Verlag Chemie/Academic Press Inc., Weinheim/NewYork, 673\u0026ndash;684\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahman I, Kode A, Biswas SK (2007) Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nature Protocols. 2007. 1, 3159\u0026ndash;3165\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarrara IM, Melo GP, Bernardes SS, Neto FS, Ramalho LNZ, Marinello PC, Luiz RC, Cecchini R, Cecchini AL (2019) Looking beyond the skin: cutaneous and systemic oxidative stress in UVB-induced squamous cell carcinoma in hairless mice. J Photochem Photobiol B. 2019. 195, 17\u0026ndash;26\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKeen JH, Habig WH, Jakoby WB (1976) Mechanism for the several activities of the glutathione S-transferases. J Biol Chem. 1976. 251, 6183\u0026ndash;6188\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerman HM, Battistuz T, Bhat TN, Bluhm WF, Bourne PE, Burkhardt K, Feng Z, Gilliland GL, Iype L, Jain S et al (2002) The Protein Data Bank. Acta Crystallogr D Biol Crystallogr. 2002. 58, 899\u0026ndash;907\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeng EC, Goddard TD, Pettersen EF, Couch GS, Pearson ZJ, Morris JH, Ferrin TE (2023) UCSF ChimeraX: Tools for structure building and analysis. Protein Sci. 2023. 32, 4792\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim S, Thiessen PA, Bolton EE, Chen J, Fu G, Gindulyte A, Han L, He J, He S, Shoemaker BA et al (2016) PubChem Substance and Compound databases. Nucleic Acids Res. 2016. 44, 1202\u0026ndash;1213\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAcu\u0026ntilde;a-Castroviejo D, Noguiera-Navarro MT, Reiter RJ, Escames G (2018) Melatonin actions in the heart; more than a hormone. Melatonin Res. 2018. 1, 21\u0026ndash;26\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCucielo MS, Ces\u0026aacute;rio RC, Silveira HS, Gaiotte LB, Dos Santos SAA, de Campos Zuccari DAP, Seiva FRF, Reiter RJ, de Almeida Chuffa LG (2022) Melatonin reverses the Warburg-type metabolism and reduces mitochondrial membrane potential of ovarian cancer cells independent of MT1 receptor activation. Molecules. 2022. 27, 4350\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChuffa LGA, Reiter RJ, Lupi LA (2017) Melatonin as a promising agent to treat ovarian cancer: Molecular mechanisms. Carcinogenesis. 2017. 38, 945\u0026ndash;952\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen X, Hao B, Li D, Reiter RJ, Bai Y, Abay B, Chen G, Lin S, Zheng T, Ren Y et al (2021) Melatonin inhibits lung cancer development by reversing the Warburg effect via stimulating the SIRT3/PDH axis. J Pineal Res. 2021. 71, 12755\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Lima Mota A, Victorasso Jardim-Perassi B, Bergamin De Castro T, Colombo J, Sonehara NM, Gomes Nishiyama VK, Gon\u0026ccedil;alves Pierri VA, Aparecida D, De Campos Zuccari P (2019) Melatonin modifies tumor hypoxia and metabolism by inhibiting HIF-1α and energy metabolic pathway in the in vitro and in vivo models of breast cancer. Melatonin Res. 2019. 2, 83\u0026ndash;98\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAkbarzadeh M, Movassaghpour AA, Ghanbari H, Kheirandish M, Fathi Maroufi N, Rahbarghazi R, Nouri M, Samadi N (2017) The potential therapeutic effect of melatonin on human ovarian cancer by inhibition of invasion and migration of cancer stem cells. Sci Rep. 2017, 7, 1\u0026ndash;11\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBu S, Wang Q, Sun J, Li X, Gu T, Lai D (2020) Melatonin suppresses chronic restraint stress-mediated metastasis of epithelial ovarian cancer via NE/AKT/β-catenin/SLUG axis. Cell Death Dis. 2020, 11, 1\u0026ndash;17\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCucielo MS, Freire PP, Em\u0026iacute;lio-Silva MT, Romagnoli GG, Carvalho RF, Kaneno R, Hiruma-Lima CA, Delella FK, Reiter RJ, de Chuffa LG (2023) A. Melatonin enhances cell death and suppresses the metastatic capacity of ovarian cancer cells by attenuating the signaling of multiple kinases. Pathol Res Pract. 2023, 248,154637\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao Y, Xiao X, Zhang C, Yu W, Guo W, Zhang Z, Li Z, Feng X, Hao J, Zhang K, Xiao B, Chen M, Huang W, Xiong S, Wu X, Deng W (2017) Melatonin synergizes the chemotherapeutic effect of 5-fluorouracil in colon cancer by suppressing PI3K/AKT and NF-κB/iNOS signaling pathways. J Pineal Res. 2017. 62, 12380\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMao L, Yuan L, Slakey LM, Jones FE, Burow ME, Hill SM (2010) Inhibition of breast cancer cell invasion by melatonin is mediated through regulation of the p38 mitogen-activated protein kinase signaling pathway. Breast Cancer Res. 2010. 12, 107\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReiter RJ, Sharma R, Rosales-Corral S, Manucha W, Chuffa LG, de Zuccari A (2021) C Melatonin and pathological cell interactions: mitochondrial glucose processing in cancer cells. Int J Mol Sci. 2021. 22, 12494\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaucher E, Mykoliuk I, Fediuk M, Smolle-Juettner FM (2022) Autophagy, oxidative stress, and cancer development. Cancers (Basel). 2022. 14, 1637\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReuter S, Gupta SC, Chaturvedi MM, Aggarwal BB (2010) Oxidative stress, inflammation, and cancer: How are they linked? Free Radic Biol Med. 2010. 49, 1603\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCruz EMS, Concato VM, de Morais JMB, Silva TF, Inoue FSR, de Souza Cremer M, Bid\u0026oacute;ia DL, Machado RRB, de Almeida Chuffa LG, Mantovani MS, Panis C, Pavanelli WR, Seiva FRF (2023) Melatonin modulates the Warburg effect and alters the morphology of hepatocellular carcinoma cell line resulting in reduced viability and migratory potential. Life Sci. 2023. 319, 121530\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrieto-Dom\u0026iacute;nguez N, Ord\u0026oacute;nez R, Fern\u0026aacute;ndez A, M\u0026eacute;ndez-Blanco C, Baulies A, Garcia-Ruiz C, Fern\u0026aacute;ndez-Checa JC, Mauriz JL, Gonz\u0026aacute;lez-Gallego J (2016) Melatonin-induced increase in sensitivity of human hepatocellular carcinoma cells to sorafenib is associated with reactive oxygen species production and mitophagy. J Pineal Res. 2016. 61, 396\u0026ndash;407\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReiter RJ, Mayo JC, Tan DX, Sainz RM, Alatorre-Jimenez M, Qin L (2016) Melatonin as an antioxidant: under promises but over delivers. J Pineal Res. 2016. 61, 253\u0026ndash;278\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZare H, Shafabakhsh R, Reiter RJ, Asemi Z (2019) Melatonin is a potential inhibitor of ovarian cancer: molecular aspects. J Ovarian Res. 2019. 12, 1\u0026ndash;8\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReiter RJ, Sharma R, Rosales-Corral S, de Campos Zuccari DAP, de Almeida Chuffa LG (2022) Melatonin: A mitochondrial resident with a diverse skill set. Life Sci. 2022. 301, 120612\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMehrzadi S, Safa M, Kamrava SK, Darabi R, Hayat P, Motevalian M (2017) Protective mechanisms of melatonin against hydrogen-peroxide-induced toxicity in human bone-marrow-derived mesenchymal stem cells. Can J Physiol Pharmacol. 2017. 95, 773\u0026ndash;786\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReiter RJ, Sharma R, Tan DX, Huang G, de Almeida Chuffa LG, Anderson G (2023) Melatonin modulates tumor metabolism and mitigates metastasis. Expert Rev Endocrinol Metab. 2023. 18, 321\u0026ndash;336\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMihanfar A, Yousefi B, Darband SG, Sadighparvar S, Kaviani M, Majidinia M (2021) Melatonin increases 5-fluorouracil-mediated apoptosis of colorectal cancer cells through enhancing oxidative stress and downregulating survivin and XIAP. Bioimpacts. 2021. 11, 253\u0026ndash;261\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmmar OA, El-Missiry MA, Othman AI, Amer ME (2022) Melatonin is a potential oncostatic agent to inhibit HepG2 cell proliferation through multiple pathways. Heliyon. 2022. 8, 08837\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKopustinskiene DM, Bernatoniene J (2021) Molecular mechanisms of melatonin-mediated cell protection and signaling in health and disease. Pharmaceutics. 2021. 13, 1\u0026ndash;19\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalendiran GK, Dabur R, Fraser D (2004) The role of glutathione in cancer. Cell Biochem Funct. 2004. 22, 343\u0026ndash;352\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOsseni RA, Rat P, Bogdan A, Warnet JM, Touitou Y (2000) Evidence of prooxidant and antioxidant action of melatonin on human liver cell line HepG2. Life Sci. 2000. 68, 387\u0026ndash;399\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlbertini MC, Radogna F, Accorsi A, Uguccioni F, Paternoster L, Cerella C, De Nicola M, D\u0026rsquo;Alessio M, Bergamaschi A, Magrini A, Ghibelli L (2006) Intracellular pro-oxidant activity of melatonin deprives U937 cells of reduced glutathione without affecting glutathione peroxidase activity. Ann N Y Acad Sci. 2006. 1091, 10\u0026ndash;16\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSau A, Pellizzari Tregno F, Valentino F, Federici G, Caccuri AM (2010) Glutathione transferases and development of new principles to overcome drug resistance. Arch Biochem Biophys. 2010. 500, 116\u0026ndash;122\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAudemard-Verger A, Silva NM, Verstuyft C, Costedoat-Chalumeau N, Hummel A, Le Guern V, Sacr\u0026eacute; K, Meyer O, Daugas E, Goujard C, Sultan A, Lobbedez T, Galicier L, Pourrat J, Le Hello C, Godin M, Morello R, Lambert M, Hachulla E, Vanhille P, Queffeulou G, Potier J, Dion JJ, Bataille P, Chauveau D, Moulis G, Farge-Bancel D, Duhaut P, Saint-Marcoux B, Deroux A, Manuzak J, Franc\u0026egrave;s C, Aumaitre O, Bezanahary H, Becquemont L, Bienvenu B (2016) Glutathione S Transferases Polymorphisms Are Independent Prognostic Factors in Lupus Nephritis Treated with Cyclophosphamide. PLoS One. 2016, 11, 0151696\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi S, Li C, Jin S, Liu J, Xue X, Eltahan AS, Sun J, Tan J, Dong J, Liang XJ (2017) Overcoming Resistance to Cisplatin by Inhibition of Glutathione S-Transferases (GSTs) with Ethacraplatin Micelles in Vitro and in Vivo. Biomaterials 2017, 144, 119\u0026ndash;129\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTownsend DM, Tew KD (2003) The role of glutathione-S-transferase in anti-cancer drug resistance. Oncogene 22:7369\u0026ndash;7375\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDa Silva RF, Cardozo DM, Rodrigues GOL, Souza-Ara\u0026uacute;jo CN, Migita NA, Andrade LAL, Derchain S, Yunes JA, Guimar\u0026atilde;es F (2017) CAISMOV24, a new human low-grade serous ovarian carcinoma cell line. BMC Cancer. 2017. 17, 756\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSilveira HS, Ces\u0026aacute;rio RC, V\u0026iacute;garo RA, Gaiotte LB, Cucielo MS, Guimar\u0026atilde;es F, Seiva FRF, Zuccari DAPC, Reiter RJ, Chuffa LGA (2024) Melatonin changes energy metabolism and reduces oncogenic signaling in ovarian cancer cells. Mol Cell Endocrinol. 2024. 592, 112296\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePutnam CD, Arvai AS, Bourne Y, Tainer JA (2000) Active and inhibited human catalase structures: ligand and NADPH binding and catalytic mechanism. J Mol Biol. 2000. 296, 295\u0026ndash;309\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYekta R, Dehghan G, Rashtbari S, Ghadari R, Moosavi-Movahedi AA (2018) The inhibitory effect of farnesiferol C against catalase; Kinetics, interaction mechanism and molecular docking simulation. Int J Biol Macromol. 2018. 113, 1258\u0026ndash;1265\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShahraki S, Saeidifar M, Delarami HS, Kazemzadeh H (2020) Molecular docking and inhibitory effects of a novel cytotoxic agent with bovine liver catalase. J Mol Struct. 2020. 1205, 127590\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"84c11a1d-d9a1-454d-8cf2-59d8b5057433","identifier":"10.13039/501100001807","name":"Fundação de Amparo à Pesquisa do Estado de São Paulo","awardNumber":"2021/12971-7 ","order_by":0},{"identity":"7fe73db3-fa60-4653-a596-25996a853737","identifier":"10.13039/501100003593","name":"Conselho Nacional de Desenvolvimento Científico e Tecnológico","awardNumber":"304108/2020-0 and 306117/2023-1 ","order_by":1},{"identity":"53bc22e1-7322-474a-9966-e509f31c687e","identifier":"10.13039/501100002322","name":"Coordenação de Aperfeiçoamento de Pessoal de Nível Superior","awardNumber":"88887.482443/2020-00","order_by":2}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"UNESP","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, Melatonin, Oxidative stress, Antioxidant defense, Catalase, Cell invasion and migration","lastPublishedDoi":"10.21203/rs.3.rs-5924048/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5924048/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOvarian cancer (OC), a highly recurrent and fatal tumor, poses diagnostic challenges due to generic symptoms and chemoresistance. Melatonin (Mel) is an indoleamine acting against tumor progression and exhibiting pro-oxidative actions in tumor cells. This study explores the impact of Mel on antioxidant defenses of OC cells (SKOV-3 and CAISMOV-24 lines), focusing on its receptor-dependent and -independent effects. Cell viability was assessed using the MTT method and the antioxidant system was analyzed by preparing supernatants for assessing glutathione (GS), reduced glutathione (GSH), oxidized glutathione (GSSG), catalase (CAT), glutathione S-transferase (GST), and superoxide dismutase (SOD). Mel stimulated its own intracellular levels, reducing cell viability in both cell lines. Notably, Mel independently of its membrane receptors, inhibited migration and invasion, thus showing its anti-tumoral potential. By investigating melatonin\u0026rsquo;s actions, we observed an impact on the antioxidant system primarily through the reduced activity of CAT and the GS axis. The modulation of these antioxidants by Mel demonstrates its multifaceted role in OC, emphasizing its therapeutic potential. We also demonstrated, for the first time, the theoretical ability of Mel to bind to CAT, which may be responsible for the reduction in enzyme activity. This study contributes with novel insights into Mel's receptor-independent actions, providing a foundation for further research in OC therapy.\u003c/p\u003e","manuscriptTitle":"Melatonin reduces cell motility and antioxidant defenses in ovarian cancer cell lines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-31 02:27:44","doi":"10.21203/rs.3.rs-5924048/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":"bdebc633-e258-49a4-8c87-ddad460f2497","owner":[],"postedDate":"January 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-31T02:27:44+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-31 02:27:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5924048","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5924048","identity":"rs-5924048","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}