Autophagy Gene Beclin 1 And PI3K/PKB Signaling Pathway In Epithelial Ovarian Cancer

Background & Objective Epithelial ovarian cancer is the first leading cause of gynecologic malignancy-related deaths. It is urgent to find effective treatment of ovarian carcinoma in clinical practice. Surgery, chemotherapy and radiation therapy are conventional treatment for ovarian carcinoma, and gene therapy is regard as a new treatment strategy for ovarian carcinoma.Autophagy plays a critical role in removing damaged or surplus organelles in order to maintain cellular homeostasis. For example, by removing damaged organelles, autophagy may limit the exposure of cellular DNA to genotoxic- stresses such as free radicals. The removal of damaged organelles through autophagic degradation would thus decrease the basal mutation rate and suppress oncogenesis.Beclin 1, the first identified mammalian autophagy gene product, is the first identified tumor suppressor protein that functions in the lysosomal degradation pathway of autophagy. Beclin1 is essential to formation of autophagosome. Some studies reported that Beclin1 expression was down-regulated in breast carcinoma, and loss of Beclin 1 would contribute to an increased incidence of cancer, such as heptocellular carcinoma, lung adenocarcinoma and lymphoma. It is unclear that correlation of autophagy Gene Beclinl to tumorigenesis and development of epithelial ovarian cancer.It was well known that apoptosis was regulated by PI3K/PKB signaling pathway, and this pathway is invovlved in ihe control of autophagy. Some studies reported the classⅠPI3K inhibit autophagy and the classⅢPI3K stimulates autophagy.This study was to investigate Beclinl expression and expression of ClassⅠPI3K (p110α), ClassⅢPI3K (hvps34) and p-PKB in PI3K/PKB signaling pathway in epithelial ovarian carcinoma., and to explore relationship between autophagy gene Beclinl and involved PI3K/PKB signaling pathway and the occurrence, development of epithelial ovarian carcinoma. We study the effect of Beclinl overexpression on the growth of ovarian carcinoma cell line SKOV3 in vitro and vivo, and on change of PI3K/PKB signaling pathway, which provide some theoretic basis for gene therapy that target at Beclinl.Methods 1. The expression of Beclin 1, ClassⅠPI3K (p110α), ClassⅢPI3K (hvps34) and p-PKB was detected by immunohistochemistry in 25 normal ovarian tissues, 25 benign neoplasia tissues, 19 borderline tissues, and 69 epithelial ovarian carcinoma tissues.2. The gene fragment coding for Beclinl was obtained from human normal ovarian tissue using RT-PCR, and inserted into the eukaryotic expression plasmid pcDNA3.1(+) to construct the recombinant plasmid pcDNA3.1/Beclin1.3. The vectors were transfected into SKOV3 cells by lipofectamine 2000. The expression levels of Beclin1, hvps34, p110αand p-PKB mRNA and protein were detected by real-time RT-PCR, western blot analysis after transfection. Expression of Beclinl in SKOV3 cells transfected with plasmid pcDNA3.1/Beclin1 was detected by immunohistochemistry. Autophagic vacuoles were observed by electron microscopy and fluorescence microscope. Cell cycles, apoptotic rates and autophagy were measured by flow cytometry (FCM). MTT was used to evaluate the effect of Beclinl overexpression on the inhibition of proliferation and growth of the transfected cells and SKOV3 cells,4. SKOV3 cells transfected with plasmid pcDNA3.1/Beclinl and pcDNA3.1 were seeded hypopercutaneously on nude mice. The carcinogenic and growth activities of cancer cell in vivo were observed. Beclinl protein expression in tumor tissues was detected by immunohistochemisty.Results 1. The higher expression level of Beclin 1 and hvps34 was found in normal and benign ovarian neoplasia tissues. The expression of Beclin 1 and hvps34 was reduced in the borderline lesions, and the lowest level was detected in the ovarian carcinoma tissues(P<0.05). The level of p110αand p-PKB expression increased slightly in the borderline ovarian tissues, and the expression of p110αand p-PKB was higher in the epithelial ovarian carcinoma tissues than that of the other three groups (P<0.05). Beclin 1 expressions in stageⅠ-Ⅱ, high and middle grade and negative lymph node metastasis epithelial ovarian cancer tissues were higher than that in stageⅢ-Ⅳ, low grade, positive lymph node metastasis ovarian cancer tissues, and the expressions of p110αand p-PKB in the former were lower than that in the later(P<0.05).The expression of Beclinl, p110α, hvps34 and p-PKB has no significant difference in normal and benign ovarian neoplasia tissues. The higher expression level of Beclinl and hvps34 was found in normal and benign ovarian neoplasia tissues. The expression of Beclinl and hvps34 was reduced in the borderline lesions, and the lowest level was detected in the ovarian carcinoma tissues(P<0.05). The level of pllOaand p-PKB expression increased slightly in the borderline ovarian tissues, and the expression of p110αand p-PKB was higher in the epithelial ovarian carcinoma tissues than that of the other three groups ( P<0.05). Beclin 1 expressions in stageⅠ- Ⅱ, high and middle grade and negative lymph node metastasis epithelial ovarian cancer tissues were higher than that in stageⅢ-Ⅳ, low grade, positive lymph node metastasis ovarian cancer tissues, and the expressions of p100αand p-PKB in the former were lower than that in the later(P<0.05).2.The eukaryotic expression vectors of pcDNA3.1/Beclinl were constructed successfully, and verified by PCR, restriction endonucleases digestion and DNA sequencing.3.Beclinl mRNA and protein expression were increased in SKOV3 cells transfected with pcDNA3.1/Beclin1. Hvps34 expression was up-regulated and the expression level of p100αand p-PKB was down-regulated after transfection with pcDNA3.1/Beclin1. A lot of Autophagic vacuoles were observed in SKOV3-Beclinl cells by electron microscopy. MDC staining positive cells were increased after transfection with pcDNA3.1/Beclin1. FCM investigation showed the apoptotic rate was (21.26±3.89)% in SKOV3 cells after transfection with pcDNA3.1/Beclin1, which was higher than that in SKOV3 cells transfected with pcDNA3.1 and SKOV3 cells (P<0.05). After transfected by vector pcDNA3.1/Beclin1, the ratios of G1 phase of SKOV3 cells were increased and the ratios of S phase were decreased significantly. FCM showed that fluorescent intensity of SKOV3 cells transfected with pcDNA3.1/Beclin1 was higher than higher than that of SKOV3 cells transfected with pcDNA3.1 and SKOV3 cells. MTT assay revealed the cell proliferations of SKOV3 cells was inhibited after transfection with pcDNA3.1/Beclin1, and cell inhibitory rate was 58.68% (P<0.05) .4. After transfected with vector pcDNA3.1/Beclin1, the carcinogenic activity of SKOV3 cells was decreased in nude mice, and the rate of inhibition was 50.27%. The expressions of Beclinl protein were increased in SKOV3- Beclin1 group. Conclusions Beclin 1 expression was down-regulated in epithelial ovarian cancer tissues, and the expression of p100α, hvps34 and p-PKB is abnormal in PI3K/PKB pathway, which may be correlated with the occurrence and development of ovarian carcinoma. Beclin 1 overexpression stimulated the expression of ClassⅢPI3K (hvps34) , and inhibited the expression level of p100αand p-PKB. Beclin 1 overexpression can induce autophagy and apoptosis in SKOV3 cells, and inhibit tumorigenesis of SKOV3 cells in vitro and vivo. So it might be one of the ideal strategies for gene therapy of ovarian carcinoma.