| Ovarian cancer is the second most common gynecological malignancy behind uterine cancer, and is the leading cause of gynecologic cancer-related death. Cisplatin-based combination chemotherapy is very important for the treatment of ovarian cancer. However, the resistance developed by ovarian cancer cells to cisplatin limits its clinical application and results in relapse, metastases and high mortality.Therefore, exploring effective resistance reversal agents or chemosensitizer play a key role in improving ovarian cancer chemotherapy.Genistein has been identified as the predominat isoflavone in soybearn enriched food. It has a close similarity in structure to estrogens and so is labeled as a phytoestrogen. It has widely biological activities, including anti-cancer, anti-oxidant, anti-inflammatory and improving cardiovascular function and so on. Genistein is a potential agent in caner prevention and therapy. Recent studies show that Genistein can enhance the chemosensitivity of drug-resistant cancer cells by promoting apoptosis, but its mechanism remains unclear.Genistein has been well known as a natural anti-oxidant and a scavenger of ROS, and many studies have focused on it. Yet in fact, Genistein is not only an anti-oxidant but also a pro-oxidant, and the pro-oxidative action may be more important for its anti-tumor and inducing apoptosis properties. Genistein may act as a pro-oxidative agent, stimulating ROS production by oxidative stress. ROS is closely related to apoptosis, and it can mediate apoptosis by DNA fragmentation.Several anti-tumor drugs induced apoptosis is mediated by ROS, such as cisplatin. Cancer cells have a lower ability of scavenging ROS than that of normal cells, and is sensitive to ROS. Therefore, Genistein-mediated oxidative stress could situmulate ROS generation in cancer cells, change the redox homeostasis and induce apoptosis eventually. It may be one of the mechanisms of its anti-tumor activity. According to the above-mentioned, we investigate the relationship between Genistein-mediated oxidative stress and cisplatin sensitivity of ovarian cancer cells, and its possible mechanism.Objective1. To investigate the different level of ROS between ovarian cancer A2780 and CP70 cell lines, and analysis the relationship beween the difference and the level of ROS.2. To investigate the effects of ROS in the process of Genistein induced apoptosis in drug-resistant ovarian cancer cell CP70. 3. To investigate the effect and mechanism of Genistein-mediated oxidative stress in cisplatin-induced apoptosis in drug-resistant ovarian cancer cell line CP70.Methods1. The level of ROS in A2780 and CP70cells was measured by Flow cytometry.2. MTT assay was used to detect the proliferation inhibitory effect of different drugs on CP70 cells.3. Flow cytometry was used to analyze the level of ROS, mitochondrial membrance potential and apoptotic rate in CP70 cells.4. SP immunohistochemical technique and Western blot were used to detect the expression of Bcl-2 and Caspasce-3 proteins in CP70 cells.5. Apoptotic rate, mitochondrial membrance potential, the level of ROS and the expression of Bcl-2 and Caspasce-3 proteins in CP70 cells was detected after the pretreatment of NAC.Results1. The level of ROS in A2780 cells was higher than that in CP70 cells.2. Genistein inhibited the proliferation and induced the apoptosis of CP70 cells in a dose-and time-dependent manner.3. The level of ROS and apoptotic rate were increased obviously, while mitochondrial membrance potential was decreased after CP70 cells treated with different concentrations of Genistein. 4. After co-treatment of genistein and cisplatin, the level of ROS and apoptotic rate were significantly raised, and mitochondrial membrance potential was decreased ,as compared with cisplatin alone.5. Bcl-2 and Caspasce-3 proteins localized in both in cytoplasmic and nucleus in CP70 cells. The expression of Bcl-2 was decreased and Caspase-3 was increased after treated with Genistein.6. Pretreatment with antioxidant NAC, the level of ROS was effectively inhibited, while mitochondrial membrance potential and apoptotic rate was increased, the expression of Bcl-2 was increased and Caspase-3 was decreased.Conclusion1. The level of ROS is different between A2780 and CP70 cells, and this may be one of the mechanisms of drug-resistance in CP70 cells.2. Genistein could inhibit the proliferation and induce apoptosis of CP70 cells in a time-and dose-dependent manner, and ROS plays an important role in the process.3. Genistein-mediated oxidative stress increases cisplatin sensitivity of drug-resistant ovarian cancer cell CP70 by reducing the expression of Bcl-2 and increasing the expression of Caspase-3, this may be a mechanism of its anticancer activity. |
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