Apoptosis or programmed cell death is a critical physiological phenomenon playing an essential role in ovarian tissue homeostasis, the developmental process of organs, and deletion of potentially dangerous or defective cells . However, any fault in the control of apoptosis could give rise to pathological conditions like various type of ovarian cancer . Therefore, strategies targeting apoptosis-related molecules have a great value in the treatment of OC. Moreover, the failure of cells to undergo apoptosis may develop chemotherapy resistance, which is the main cause of treatment failure in OC [27, 28]. Inhibition of PI3K/Akt signaling and induction of oxidative stress have been known as two therapeutic strategies to sensitize the resistant tumoral cells to apoptosis . In the current study, we demonstrated that melatonin sensitized the OVCAR3 cells to cisplatin, as a well-known chemotherapy agent, via induction of oxidative stress and inhibition of PI3K/Akt signaling. Overexpression and activation of PI3K/Akt signaling, as a critical cell process regulator of cell proliferation, has been demonstrated in OC , which can develop cisplatin resistance . Hence, inhibition of PI3K/Akt signaling activation could be a therapeutic target for epithelial OC . Many previous studies have also demonstrated that the PI3K/Akt signaling regulates ROS production and aberrant PI3K/Akt signaling contributes to the overproduction of ROS [32, 33]. On the other hand, excessive ROS levels can potentiate the activation of PI3K/Akt signaling mainly through inhibition of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) . In this study, we used Akt siRNA to inhibit Akt protein expression. We observed that Akt knockdown was associated with decreased Akt phosphorylation, increased ROS generation, and apoptosis induction in the Akt siRNA group in comparison to the control siRNA. In line with our results, previous reports have also shown that inhibition of PI3K/Akt pathway is accompanied by excessive ROS production [33, 34]. It seems that Akt counteracts with ROS generation in oxidative stress pathway and consequently apoptosis in cancer cells. Several studies have been reported the beneficial effects of melatonin on the apoptosis of cancer cells by focusing on oxidative stress . ROS play an important role in the activation of apoptosis pathway, so that elevation in ROS generation leads to an alteration in mitochondrial membrane potential (MMP) and defects in the respiratory chain, consequently initiates the apoptotic process [36, 37]. Our data demonstrated that co-treatment of OVCAR3 cells with melatonin sensitized the cells to cisplatin treatment via increased ROS production in this cell line. In line with our result, several studies have established that the antitumor activity of melatonin on cancer cells is through ROS-dependent activation of apoptotic cell death [38, 39]. Likewise, we found that cisplatin as a clinically-proven drug to fight various cancer types caused an increase in ROS production, and this elevation in combination therapy with melatonin was much more prominent than cisplatin alone. Recently, many researchers reported that cisplatin-induced cytotoxicity is strongly associated with increased ROS generation . Moreover, we observed a significant decrease in the p-Akt levels in the melatonin, combination therapy, and Akt siRNA groups versus cisplatin-treated cells. It seems that the simultaneous reduction of Akt phosphorylation and ROS production are rationales to the sensitization of OVCAR3 cells to cisplatin. Moreover, activated Akt has the ability to phosphorylate one of its targets, GSK3β . Multiple consequences may occur in presence of activated (non-phosphorylated) or inactivated (phosphorylated) form of GSK3β. NF-kB activity is positively regulated by GSK3β, thus, aberrated Akt pathway may lead to high level of GSK3β, increased NF-kB function and finally OC proliferation . As expected, we observed decreased level of p-GSK3β in Akt siRNA treatment group as well as combination therapy group. Notably, our results demonstrated that neither cisplatin nor melatonin treatments could alter the expression level of GSK3β. In concordant with other reports, we demonstrated the OVCAR3 cells sensitized to cisplatin and melatonin by regulating the activity of GSK3β . Akt signaling pathway may be activated by integrin ligation which triggers integrin-linked kinase activation and subsequently leads to elevation of Hypoxia-Induced Factor (HIF)-1 and Vascular Endothelial Growth Factor (VEGF) expression . HIF1 is a crucial factor in angiogenesis which expressed in response to low oxygen concentration and contributes to survival and proliferation of cancer cells . Following the phosphorylation of Akt, HIF1 enters the nucleus, acts as a transcription factor and triggers the transcription of VEGF gene. This signaling pathway leads to angiogenesis and tumor proliferation . On the other hand, p53 can negatively modulates this pathway through expression induction of MDM2 which subsequently triggers the degradation of HIF1 by the proteasome pathway . Zhihong et.al have hypothesized that downregulating HIF-1 would be an efficient strategy for overcoming cisplatin resistance of human OC cells . They demonstrated that cisplatin has the ability to downregulates HIF1 expression, thus inhibits the proliferation of cancer cells by induction of ROS production . Accumulating studies revealed the potential of cisplatin to abolish the expression of HIF1 and VEGF in OC [49, 50]. Furthermore, increasing evidence indicated that melatonin potentiates to affects the angiogenesis by targeting HIF-1 under hypoxic conditions [51, 52]. These findings were in line with our results, in which we showed the combination therapy would significantly downregulates both HIF1 and VEGF expression level compared to untreated control group and even cisplatin alone and melatonin alone treatment. Furthermore, align with our results, p53 expression has been demonstrated to be associated with cisplatin sensitivity in OC . We showed that combination treatment of cisplatin and melatonin had a positive significant association with p53 expression level as compared to untreated control and cisplatin treated groups. In concordance to our results, several studies revealed that p53 facilitates the apoptosis by cisplatin treatment [54–56].
In line with our finding, melatonin has been reported to reduce phosphorylation of Akt in different cancer cells such as hepatoma cells , lung cancer , and breast cancer [59, 60]. Activation of caspase 3, as an irreversible step of apoptosis, plays an important role in the apoptosis process and any defect in its function or downregulation of its expression may lead to development of carcinogenesis . Moreover, it has been shown that the expression of caspase-3 is decreased or undetectable in OC cells, which is one of the causes of resistance to chemotherapy agents . However, its expression is increased in response to anti-cancer therapy leading to apoptosis in cancer cells . As shown in Henkels et al. study , we also found that cisplatin increased the cleavage of caspase 3 in OVCAR3 cells and combination therapy with melatonin also elevated caspase 3 activation, indicating that melatonin sensitized OVCAR3 cell to cytotoxic effect of cisplatin and promoted cell death. According to the results of MTT (Fig. 1C), 24 h treatment with cisplatin along with melatonin lowered the IC50 of cisplatin more than 50% in compare to cisplatin alone (4.1 µM vs. 12.8 µM). XIAP, a direct inhibitor of caspase 3 and 7, and Survivin proteins are inhibitors of apoptosis which can promote cell cycle progression . Evidence shows that cancer cells have elevated expression levels of apoptosis inhibitor proteins promoting cell survival and tumor growth and consequently chemoresistance . Therefore, therapies targeting inhibitors of apoptosis protein in cancer may improve the sensitivity of cancer cells to chemotherapies and hence potentiate cell death. In this study, we observed a slight reduction, but not significantly, in XIAP protein expression in the cisplatin and combined therapy with melatonin. Surprisingly, melatonin-treated cells displayed elevated expression of XIAP compared to the treatment with cisplatin alone. Elevated expression of XIAP along with elevated caspase 3 activation is an inconsistent point in our results which should be further investigated to be clarified. Asselin et al. have reported that cisplatin decreases XIAP content at protein levels which in turn induces the cleavage of pro-caspase 9, pro-caspase 3, as well as Akt and therefore apoptosis in ovarian cancer cells . Conversely, overexpression of XIAP prevents cisplatin-induced Akt cleavage and increases p-Akt content, which in turns protects OC cells by inhibiting apoptosis .
Survivin is another apoptosis inhibitor protein that controls cell proliferation and suppresses apoptosis-induced cell death . Accumulating studies have shown that cancer cells have a higher expression level of Survivin [68, 69]. Overexpression of Survivin in normal and tumor cells has prevented cell death that promotes by apoptotic stimuli, such as caspases and anti-cancer drugs. Importantly, down-regulation of Survivin is accompanied by overexpression of caspase 3 which could have therapeutic benefits in OC cells . The blockade of Survivin expression has also been reported to improve the antitumor activity of chemotherapeutic drugs in OC . In this study, unexpectedly, melatonin had no effect on the protein expression of Survivin. Moreover, only a reduction in Survivin expression was observed in the Akt siRNA group, indicating the critical role of Akt in cell proliferation. In contrast to our results, Fan et al. have demonstrated that melatonin reverses apoptosis resistance in human hepatocellular carcinoma by inhibition of Survivin and XIAP . However, many previous studies have confirmed pro-apoptotic, and oncostatic properties of melatonin . Pro-apoptotic effect of melatonin in cancer cells is mainly through modulation of the release of pro-apoptotic (BAX, caspase 3, and cytochrome c) and anti-apoptotic (Bcl-2) proteins, as well as oxidative stress [72–74]. Yun et al. also found that melatonin attenuated phosphorylation of Akt and induced caspase 3 activity and hence increased apoptosis in lung cancer cells . These discrepancies highlight the need to more elucidating investigations in future.