| Ovarian cancer is the most lethal gynecological malignancy. Patients usually have poor prognosis because of late diagnosis, relapse and chemoresistance. Therefore, the death rate of ovarian cancer is the highest of all gynecological malignancy, with the overall 5-year survival is only 30%. This severly influence the health of women. Debulking surgery followed by platinum-taxane based chemotherapy is the standard of care for patients with advanced stage ovarian cancer. However, multidrug resistance and toxicity limit the application of chemotherapy for ovarian cancer patients. It is pressing to seek more effective and lower cytotoxicity anti-tumor agents from natural products for the treatment of ovarian cancer.Neferine is a bisbenzylisoquinoline alkaloid isolated from the embryos of lotus (Nelumbo nucifera). Studies have shown that neferine possesses anti-oxidant, anti-platelet aggregation, anti-arrhythmic, anti-inflammation and anti-hypertensive effects. Recently, neferine has been found to be a potent anti-tumor agent on ovarious tumors in vitro by inducing cell cycle arrest, autophagy and apoptosis. Autophagy is one of the key mechanisms to degrade and recycle damaged and harmful cellular components to mediate metabolic adaptation and maintain energy homeostasis. A number of studies have shown that autophagy itself fulfils a dual role, having either pro-death or pro-survival role depending on the cell type and strength of specific stimuli. On the one hand, autophagy plays a pro-survival role through contributing to cytoprotective events that help cells to survive and avoid cell death. On the other hand, autophagy can stimulate a pro-death signal pathway and ultimately result in cell death in cancers. Autophagy has been reported to be induced by some anticancer agents, such as temozolomide, dexamethasone,6-thioguanine, and camptothecin, as well as to ionising radiation.In our previous study, we have evaluated the inhibitory effects of neferine on U20S, Saos-2 and MCF-7 cells. Neferine obviously inhibited the proliferation of these cell lines. However, it is unknown whether neferine possesses anti-tumor effect on human ovarian cancer. In the present study, we evaluated the growth-inhibitory effect of neferine on human ovarian cancer cell lines and characterized the specific mechanisms underlying the growth-inhibitory effect of neferine as follows.1. The growth-inhibitory effects of neferine on the proliferation of human ovarian cancer cells and normal fallopian epithelial cells.We examined the growth-inhibitory effects of neferine on human ovarian cancer cells A2780, HO8910, SKOV3 and normal fallopian epithelial cell by MTT assay and clonogenic assay, respectively. MTT assay reveals that neferine obviously inhibited growth of various ovarian cancer cells in a dose-and time-dependent manner. The result of clonogenic assay indicates that the colony-forming abilities of ovarian cancer cells were greatly inhibited by neferine in a dose-dependent manner, compared with control. Neferine is less effective on non-malignant fallopian tube epithelial cells than ovarian cancer cells.2. Cell cycle analysis of human ovarian cancer cells in response to neferine treatmentInduction of cell cycle arrest is one of the mechanisms of anti-tumor agents. In this part, flow cytometry and Western Blotting were uessed to examined the effects of neferine on cell cycle distribution and the underlying molecular mechanism.(1) A2780 cells was analyzed cell cycle distribution by flow cytometry treated with neferine. Results have shown that neferine induced an obvious G1 cell cycle arrest in a dose-dependent manner in A2780 cells (Control,55.1%; 5μM,56.6%; 10μM, 79.5%)(2) The expression of G1/S transition related proteins level were examined by western blotting. G1 cycle arrest induced by neferine accompanied with an increase of p21 and p27 and a decrease of cyclinDl. There is no significant change of the protein level of cyclinE with neferine treatment.3. Induction of autophagy and apoptosis in human ovarian cancer cells by neferineAutophagy and apoptosis have also been reported to be responsible for the inhibition of cancer cell proliferation. A number of studies have reported that MAPK (Mitogen-actived protein kinase) and mTOR (The mammalian target of rapamycin) are involved in the process of autophagy. In this part, we first investigated autophagy induced by neferine through fluorescent microscopy, Western Blotting and Transmission Electron Microscope (TEM). Furthermore, we examined the signal pathways related to autophagy. Finally, we used Annexin V-PI assay to examine apoptosis induced by neferine.(1) Neferine induced autophagy in human ovarian cancer cells.1) A2780 cells were stablely transfected with an LC3-GFP expression vector and treated with neferine to monitor autophagosome formation through GFP-LC3 expression.24h later, neferine induced a significant increase in GFP-LC3 puncta formation in A2780 cells when compared to untreated cells.2) Western Blotting results revealed that the protein level of LC3II and Atg7 increased in a dose-and time-dependent manner after neferine treatment. However, Beclinl and p62 were not affected significantly by neferine.(2) Autophagy induced by neferine is relavant to mTOR pathway. The phosophorylation levels of p70S6K and 4EBP1, which are responsible for control of cell growth and act as the downstream target of mTOR were examined. Results showed that the phosophorylation levels of p70S6K and 4EBP1 were both decreased in a dose- and time-dependent manner, suggesting that autophagy induced by neferine is relavant to mTOR pathway in ovarian cancer cells.(3) Both p38 MAPK and JNK1/2 pathways are required for neferine-induced autophagy. We treated A2780 and HO8910 (another ovarian cancer cell line) with 10μM neferine under the same conditions. The results showed that increased LC3II and phosophorylation levels of p38 and JNK1/2 in both ovarian cancer cell lines. To further prove whether p38 MAPK and JNK1/2 activation are involved in neferine-induced autophagy in A2780 cells, we used SB203580 (p38 inhibitor) and SP600125 (JNK1/2 inhibitor) to block p38 MAPK and JNK1/2 activity, respectively. Western Blotting analysis showed that pretreatment with either JNK1/2 or p38 MAPK specific inhibitor attenuated neferine-induced LC3II accumulation. These results suggest that p38 MAPK and JNK1/2 signaling pathways were activated by neferine treatment and contributed to the induction of autophagy in ovarian cancer cells.(4) Neferine induced apoptosis in human ovarian cncer cells. Apoptotic cells were determined by Annexin V-PI staining. Neferine treatment dose-dependently increased the percentages of apoptotic cells, from 2.6% in the control to 7.9%(5 μM),9.8%(10 μM) and 47.8%(15 μM).In conclusion our findings indicate that whereas neferine possesses a potent growth-inhibitory effect on ovarian cancer cells, it is less effective on non-malignant fallopian tube epithelial cells; neferine induces G1 cell cycle arrest through elevation of p21 and p27 and the depression of cyclinDl; more importantly, we found that neferine could induce ovarian cancer cells autophagy and apoptosis, autophagy induced by neferine is relavant to mTOR, it also depends on the activation of p38 MAPK and JNK1/2. |
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