Experimental Study On The Effection And Mechanism Of Puerarin Inhibites The Human Ovarian Cancer Cells And Reverses Drug Resistance

BackgroundOvarian cancer is one of common malignant tumors of female genital mutilation, ovarian cancer accounts for 23-27% of gynecologic malignancies, mortality was first in gynecological malignancies. After the incidence of cervical cancer, endometrial cancer, ranked third, second only to cervical cancer. Despite the continuous improvement and application of surgical methods and treatment methods,5 year survival rate of patients with ovarian cancer is still around 30%. Therefore, it is urgent to develop new and selective anti-cancer chemotherapy drugs to change the status quo, so as to improve the sensitivity or reverse drug resistance, improve the survival rate of ovarian cancer patients. MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression at post-transcriptional level by blocking of translation and/or degradation of target mRNAs.Tumor-related miRNAs may play a role in tumor cell differentiation proliferation and apoptosis. Aberrant expression or mutation of miRNAs has been detected in several tumors. The expression of miRNA has obvious tissue specificity, and the miRNA version is involved in the diagnosis and prognosis of many tumors. For example, Lee et al. found that miR-125b inhibited ovarian cancer cells through post-transcriptional inactivation of EIF4EBP1. In conclusion, a new strategy for the development of targeted therapy for miRNA failure recovery is possible.The structure and mechanism of a large number of cancer cells to resist chemotherapy drugs are different. For example, paclitaxel, doxorubicin, widely used in the treatment of ovarian cancer chemotherapy, not just because the tumor has lost the sensitivity of chemotherapy drugs, which is currently defined as multidrug resistance (MDR). MDR, originated from the cell membrane expression of multidrug resistance protein increased and glutathione s transfer enzyme activity change and over expression and DNA topoisomerase expression and its activity. The MDR1 (P-gp) and GST-π are regarded as an important symbol of PI prediction of tumor resistance. At present, the effect of P-gp mediated tumor multi drug resistance was confirmed by the screening of drug efflux pump function and inhibit the protein expression. Therefore, with the down-regulation of P-gp expression can be related with resistance of tumor multi drug produced reversal, and increase the efficacy of cancer to chemotherapy. The resistance mechanism is very complex, only P-gp for solving the problem of resistance is not scientific, seeking the reversal effect, reversal agent has low side effect in the field of cancer become another thorny issue. Therefore, how to design and discovery of multidrug resistance reversal agents with high efficacy and low toxicity is an arduous task in front of us. In addition, there are reports that miR-27a and miR-451 can regulate the expression of P-gp, but whether the natural compounds can regulate the expression of microRNA to inhibit the expression of P-gp has not been reported.Natural plant medicine is an important part of disease prevention, control and development. For example, paclitaxel, an important clinical first-line chemotherapy drugs, is a kind of from two terpenoid plant taxus. Puerarin (Puerarin) Department of kudzu extract from the dried roots of legumes, isolated. Puerarin contains six kinds of flavonoids. Since 1993 the Ministry of Health puerarin was approved for clinical use, because of its strong anti-cardiovascular ischemia and hypoxia activity, expansion of coronary and cerebral blood vessels, reducing myocardial oxygen consumption, improve myocardial contractility, microcirculation and improve learning and memory and so on. The main clinical medicine is used to relieve angina, the treatment of myocardial infarction, hyperviscosity, arrhythmia and cerebrovascular disease embolism, has become an important cardiovascular drugs. Kudzu extract with mice fed on ECS tumor, S180 sarcoma and Lewis lung tumors have a certain inhibitory effect, inhibition rate at 30% provisional boundaries. Also make normal mice or mice with phagocytic capacity of peritoneal macrophages increased. A number of studies showed that puerarin can inhibit the proliferation of tumor cells, inducing cell apoptosis and cell cycle arrest in human small cell lung cancer. Recent studies have found that puerarin can reverse human gastric cancer cell line SGC7901/VCR multidrug resistance. The mechanism may be related to prompted P-gp and MRP protein expression. We therefore hypothesized that puerarin could reverse the multidrug resistance effect.Based on the domestic and overseas progress in this field and our preliminary research data, in this study, we elucidated the effect and underlying mechanism of puerarin on proliferation and transformation of ovarian cancer in vitro, explored the role of miR-125b expression in this progress. Furthermore, this study provide on the reversal of multidrug resistance in cisplatin resistant ova Provide on the reversal of multidrug resistance in cisplatin resistant ovarian cancer cells to inhibit P-gp/MDR1 expression and function of Puerarin in the first test. The results of our study support tumorigenesis of this specific cancer and provides molecular foundation f Provide on the reversal of multidrug resistance in cisplatin resistant ovarian cancer cells to inhibit P-gp/MDR1 expression and function of Puerarin in the first test. The results of our study support or the early detection and therapy of ovarian cancer.Part I Anti-proliferation and apoptosis-inducing effect of puerarin on SKOV3 cellsObjective:To evaluate the effect of proliferation, apoptosis and migration of puerarin on SKOV3 cells.Methods:MTT assay with different concentrations of puerarin on SKOV3 growth inhibition; flow cytometry (FCM) to detect effects of puerarin on apoptosis-inducing situations SKOV3 cells; and using transwell migration assay and Matrigel invasion assay effects of Puerarin ovarian cancer cell invasion ability change; Western blot to detect the expression of SKOV3 cells MDR-1, P-gp protein.Results:1. MTT assay to detect different concentrations of puerarin role in the growth of ovarian cancer cells SKOV3 24 hours, respectively, showed that different concentrations of puerarin (40μg/ml,80μg/ml,160ug/ml) compared with the control group, tumor cell growth inhibition are subject to significant and dose-dependent manner, the growth of the concentration of the tumor cells was inhibited more significantly (P <0.001). Puerarin different culture time observed puerarin stimulate longer, inhibiting the growth of tumor cells was significantly (P <0.001).2. Application of FACS assay with different concentrations of puerarin were acting on apoptosis of ovarian cancer cell SKOV3 24 hours later, it showed different concentrations of puerarin (40μg/ml,80μg/ml, 160μg/ml) compared with the control group, a significant increase in apoptosis of tumor cells, and dose-dependent manner, the higher the concentration of apoptosis of tumor cells more obvious (P<0.001).3. Applications western blot assay with different concentrations of puerarin were acting on ovarian cancer cell apoptosis related protein caspase3 SKOV3 24 hours later, caspase9 and survivin protein expression showed that different concentrations of puerarin (40p.g/ml, 80μg/ml,160μg/ml) compared with the control group, caspase3, caspase9 protein expression was up-regulated and dose-dependent manner, the higher the concentration of apoptosis of tumor cells more obvious (P<0.001). Survivin protein expression is just the opposite.4. Using transwell migration assay and Matrigel invasion assay can be found in the role after puerarin, compared with the control group, invasion and migration showed a significant decrease in ovarian cancer cell SKOV3 in a dose-dependent manner, the higher the concentration of tumor cells, the invasion and migration capacity of puerarin decreased more significantly (P<0.01).Conclusion:Puerarin has anti-proliferation and apoptosis-inducing effect on SKOV3 cells, and inhibits the invasion and migration of SKOV3 cells.Part Ⅱ Puerarin inhibits proliferation via up-regulation of microRNA-125b expression in ovarian cancer cell linesObjective:This study aimed to test whether puerarin could inhibit growth of ovarian cancer cells and reveal its underlying molecular mechanism.Methods:Cells viability was evaluated using MMT assay in SKOV-3 and OVCAR-3 cells. Apoptosis of ovarian cancer cells was analyzed by flow cytometry. Bcl-2 levels were analyzed by Western blot and microRNAs levels were determined by real-time RT-PCR.Results:Puerarin induced apoptosis by promoting expression of miR-125b, and then inhibiting expression of Bcl-2, a known target for miR-125b. Moreover, knockdown of miR-125b could reverse the reduction of cell viability induced by puerarin in SKOV-3 and OVCAR-3 cells.Conclusion:Puerarin inhibits proliferation via up-regulation of microRNA-125b expression in ovarian cancer cell lines.Part Ⅲ Experimental study on puerarin injection reverse multidrug resistance of human ovarian cancerObjective:To observe the effect of puerarin injection on multidrug resistance of human ovarian cancer cell line SKOV3/DDP in vitro, and to explore its mechanism.Methods:Using MTT drug sensitivity test, the cytotoxicity of puerarin and its reverse SKOV3/DDP to 5-FU DDP and MMC multi drug resistance were observed. Cell apoptosis rate was detected using flow cytometry before and after puerarin injection. Western blot was used to detect the different concentrations of Puerarin were in SKOV3 ovarian cancer cells after 24 hours of apoptosis related proteins Bcl-2 and Bax protein expression changes and drug resistant cell line SKOV3/DDP MRD-1 and P-gp expression.Results:1. SKOV3/DDP cells showed strong resistance to DDP, resistance to multiples of 18.63; for 5-FU, MMC show weak resistance. MTT assay to detect different concentrations of puerarin role in ovarian cancer cell SKOV3 and resistant cell growth rates SKOV3/DDP24 hours, respectively, showed that different concentrations of puerarin (40μg/ml,80μg/ml, 160μg/ml) and compared to the control group, the growth of tumor cells was significantly inhibited by, and dose-dependent manner, the higher the concentration of tumor cell growth by inhibiting the more significant (P <0.001). Inhibiting the growth of resistant cells more sensitive cells weaker.2. Applications western blot assay with different concentrations of puerarin were acting on ovarian cancer cell SKOV3 and SKOV3/DDP after 24 hours resistance-associated protein MRD-1 and P-gp protein expression showed that different concentrations of puerarin (40μg/ml, 80μg/ml,160μg/ml) compared with the control group, MRD-1 and P-gp protein expression decreased, and dose-dependent manner, the higher the resistance-associated protein puerarin concentration of tumor cells to reduce the more obvious (P<0.05).3. Applications western blot assay with different concentrations of puerarin were acting on ovarian cancer cell SKOV3 and SKOV3/DDP 24 hours after the apoptosis-related protein Bcl-2 and Bax protein expression showed that different concentrations of puerarin (40μg/ml,80μg/ml, 160μg/ml) compared with the control group, Bcl-2 protein expression decreased, and dose-dependent manner, the higher the resistance-associated protein puerarin concentration of tumor cells to reduce the more obvious (P<0.05). The protein expression of Bax on the contrary, Bcl-2/Bax ratio increased with puerarin concentration decreased, apoptosis increased.Conclusion:Puerarin can induce apoptosis in ovarian cancer cell SKOV3 and reversal of drug resistance, the mechanism MDR-1, P-gp protein expression by reducing, inhibiting the expression be 1-2 while enhancing the expression of bax, induce tumor cell apoptosis.