The Effect And Anti-Apoptosic Mechanism Of Lysophosphatidic Acid On Cisplatin-Induced Apoptosis In Human Ovarian Cancer Cells

Ovarian cancer is the common female malignancy and is the leading cause of death from gynecological malignancies. In 2006, 20,180 women had been newly diagnosed, and 15,310 had been died from ovarian cancer in the United States. The majority of patients are diagnosed with advanced epithelial ovarian cancer with widespread metastatic disease. The dismal outcome for ovarian cancer results from an inability to detect the tumor at an early curable stage. As 90% of stageⅠA and 70% of stageⅡtumors can be cured by current management, ovarian cancer diagnosed at an early stage has a good prognosis. The most likely way to identify ovarian cancer at an early, curable stage and to develop new, effective therapies for advanced ovarian cancers is to improve our understanding of the processes leading to the initiation and progression of this disease. Lysophosphatidic acid (LPA) levels are also significantly elevated in ascites and plasma from 90% of patients with ovarian cancer regardless of stage. LPA levels are elevated in plasma from 90% stageⅠ, 100% stageⅡ~Ⅳand recurrent ovarian cancer. This suggests that LPA in plasma might be a marker for diagnosis of ovarian cancer, judging prognosis, or monitoring response to therapy.Studies demonstrated that LPA stimulated the expression of a number of genes that involved in the promotion of proliferation, survival, angiogenesis and metastasis, highlighting the possibility that LPA may contribute to cancer development and progression through regulation of gene expression. LPA showed to have growth-factor-like activities and LPA biological actions are mediated by G protein-coupled receptors (GRCP) that are specific for LPA. LPA stimulates cell migration, proliferation, survival, adhesion, and secretion of angiogenic factors though LPA receptors. At least four GRCPs have been identified so far (LPA1/Edg-2, LPA2/Edg-4, LPA3/Edg-7, and the recently identified GPR23/p2y9/LPA4). Normal ovarian epithelial cells express low levels of mRNA for the LPA2/Edg-4 and LPA3/Edg-7, and mRNA levels for the LPA2/Edg-4 and LPA3/Edg-7 are markedly elevated in ovarian cancer cells. The expression of these receptors may mediate LPA-induced LPA production by ovarian cancer cells. LPA1/Edg-2 appears to function as a negative receptor for LPA in ovarian cancer cells. LPA receptors might provide a clue for the development of new therapeutic agents.The lethality of ovarian cancer is primarily attributable to the advanced stage of the disease at the time of initial diagnosis, approximately 70%~80% of patients present with disease that has spread beyond the ovaries. The 5-year survival rate is only around 30%. Platinum-based chemotherapy is the most active ovarian cancer chemotherapy and standard treatment for nearly all woman diagnosed with ovarian cancer. Although most patients initially respond to this treatment, few are cured. Resistance to cisplatin is the major cause of treatment failure. The major mechanism of cisplatin-induced tumor killing is via induction of apoptosis. As a chemotherapy drug, Cisplatin performs apoptotic effect in a lot of tumor cells including ovarian cancer cell. It has been reported that LPA can promote either survival or apoptosis in different cell types, and it may also have dual effects in the same cell type under different conditions. So we wondered that if LPA could cooperate with Cisplatin or antagonize Cisplatin in ovarian cancer therapy. We found that LPA had a strong antiapoptotic effect in the human ovarian cancer cell lines SKOV3 and 3AO and prevented Cisplatin-induced apoptosis. We presumed that LPA was able to antagonize the effect of Cisplatin. Despite the fact that LPA has been found to be a strong antiapoptotic agent in a number of cell lines, the mechanism of the protective effect has not been elucidated. This study is also about the antiapoptotic mechanisms of LPA in the human ovarian cancer cell line SKOV3. The actions of LPA are mediated by G protein-coupled receptors (GRCPs) that are specific for LPA. Before this study, the expression of LPA receptors and its correlation with chemotherapy resistance and prognosis were detected in human epithelial ovarian neoplasms tissue.PartⅠThe Lysophosphatidic acid (LPA) receptors expression and clinic significance in epithelial ovarian neoplasmsObjective: To investigate the Lysophosphatidic acid (LPA) receptors expression and their clinic significance in human epithelial ovarian neoplasms. The expression of LPA receptors and their correlation with chemotherapy resistance were analyzed in human epithelial ovarian carcinoma.Methods: The immunohistochemical staining technique was employed to measure the expression levels of Edg-2, Edg-4, and Edg-7 protein in human epithelial ovarian neoplasma.Results: The immunohistochemical staining technique revealed that the positive rates (90.0%; 83.3%; 45.6%) of Edg-2 protein in normal ovarian tissue, benign tumor and ovarian cancer tissue were significantly different (χ~2=11.29, P<0.01). The expression of Edg-2 protein was significantly higher in normal ovarian tissue and benign tumor compared with that in ovarian cancer tissue (P=0.001). Edg-2 protein expression was not significantly different in both benign tumor and normal ovarian tissue (P=0.57). The positive rate of Edg-4 protein (92.6%) was significantly higher in ovarian cancer compared with that in benign tumor (33.3%) and normal ovarian tissue (30.0%)(χ2=27.83, P<0.01); Edg-4 protein positive rate was not significantly different in both benign tumor and normal ovarian tissue (P>0.05). Edg-7 protein positive rate (89.7%) was significantly higher in ovarian cancer compared with that in benign tumor (41.7%) and in normal ovarian tissue (40.0%)(χ2=22.91, P<0.01), Edg-7 protein positive rate was not significantly different in both benign tumor and normal ovarian tissue (P>0.05). Expression of Edg-2 protein was significantly decreased compared with that of Edg-4 and Edg-7 in ovarian cancer (χ2=10.89, P<0.01); Correlation of clinic-pathological parameters showed Edg-4 and Edg-7 protein expression were significantly associated with FIGO stages and histological grades, except pathologic types and age. The expression of Edg-4 and Edg-7 in stageⅢandⅣwere significantly higher than that in stageⅠandⅡepithelial ovarian cancer (P<0.05). The protein expression of pathologic grade G2~3 was significantly higher compared with grade G1 (P<0.05). The expression of LPA receptors and their correlation with chemotherapy resistance: high-expression of Edg-4 and Edg-7 in ovarian cancer was correlated with chemotherapy resistance (P<0.01).Conclusion: Edg-2, Edg-4 and Edg-7 protein were expressed widely in human epithelial ovarian neoplasmas and normal ovarian tissue. Edg-4 and Edg-7 may be involved in the development and progression of human ovarian cancer. There was a significant correlation between Edg-4, Edg-7 and invasion and metastasis of epithelial ovarian cancer. Edg-4 and Edg-7 may be prognostic indicators in patients with epithelial ovarian cancer. High-expression of Edg-4 and Edg-7 were correlated with chemotherapy resistance based on platinum.PartⅡThe effect of Lysophosphatidic acid on Cisplatin-induced apoptosis in human ovarian cancer SKOV3 and 3AO cellsObjective: To investigate the effect of LPA on the apoptosis of human ovarian cancer cell line SKOV3 and 3AO cells treated with Cisplatin.Methods: Human ovarian cancer cell line SKOV3 and 3AO cells were cultured in intro, and Methyl thiazolyl tetrazolium (MTT) assay was used to examine the proliferative effect of LPA on SKOV3 and 3AO cells. Flow Cytometry (FCM) was used to examine the effect of LPA on the apoptosis of SKOV3 and 3AO cells treated with Cisplatin (DDP). Apoptosis was also detected by morphological analysis and DNA fragmentation.Results: First of all, we observed the effect of LPA on cisplatin-induced apoptosis by flow cytometry assay. Cells were treated for 48h with cisplatin in the presence or absence of LPA. DDP could inhibit the growth of SKOV3 and 3AO cells effectively, in a dose-dependent pattern. The apoptosis rates of cisplatin(2.5μg/ml)alone treatment were 13.18士1.24% and 13.02士1.18%, whereas decreased to 4.89士0.76% and 4.99士0.85% (P<0.01), when combined with LPA(10μmol/L)treatment. LPA treatment alone had no effect on apoptosis of cells. This finding indicated that LPA inhibited cisplatin-apoptosis in SKOV3 and 3AO. Apoptosis was also detected by morphological analysis of Hochest 33258-stained under fluorescence microscopy and light microscopy. Nuclear condensation and fragmentation was considered to be typical morphological alteration for apoptosis cells in DDP group, but lower nuclear fragmentation and condensation were observed in SKOV3 and 3AO when combined with LPA treatment. DDP incubation induced typical nucleosome spacing ladders in SKOV3 and 3AO cells, but the DNA ladder was diminished significantly when combined with LPA treatment compared to the DDP group. We also found that LPA did not alter the proliferative rate of SKOV3 and 3AO cells in the presence of 10% FBS by MTT assay.Conclusion: DDP could inhibit the growth of SKOV3 and 3AO cells effectively, in a dose-dependent pattern. LPA could protect the apoptosis induced by DDP in SKOV3 and 3AO cells. LPA likely directly prevented apoptosis induced by DDP rather than increased the proliferative rate of the cells.PartⅢThe mechanism of Lysophosphatidic acid anti-Cisplatin-induced apoptosis in human ovarian cancer SKOV3 cellsObjective: To investigate the mechanism of Lysophosphatidic acid anti-Cisplatin-induced apoptosis in human ovarian cancer SKOV3 cells.Methods: The expression of Bcl-2 family was analyzed by semi-quantitive RT-PCR. Western blot was used to examine the appearance of active fragments of caspase-9 and -3 in SKOV3 cells. Flow Cytometry (FCM) and Western Blot were used to examine the activation of the PI3-K/Akt pathway by LPA. PI3-K/Akt signaling pathway and PTX-sensitive Gi were detected in the anti-apoptotic effect of LPA.Results: Stimulation with DDP resulted in decrease in Bcl-2 mRNA level. LPA were found to up-regulate Bcl-2 mRNA and down-regulate Bax mRNA (P<0.01, P<0.01), but LPA had no effect on the expression of Bcl-XL mRNA and Bad mRNA. Caspase activity is known to be one of the major events in apoptotic tumor cells. Western blot analyses confirmed the appearance of active fragments of caspase-9 and -3 in the DDP-treated cells, and this activation was suppressed by cotreatment with LPA. Bax and Bcl-2 are located in mitochondria membrane and Caspase-9 and -3 are cleaved through mitochondrial pathway, so we considered that the mitochondrial pathway was involved in the anti-apoptotic effect of LPA.The PI3-K/Akt signaling pathway can be activated after LPA binding with GPCR, then perform a pro-survival/anti-apoptotic action in SKOV3 cell. We detected the phosphorylation of Akt with phosphorylated antibody of Akt by Western Blot. The result showed that LPA could activate Akt through inducing phosphorylation of Akt. Further more, we blocked PI3-K with its inhibitor, LY294002, the phosphorylation of Akt and the anti-apoptotic effect of LPA were suppressed by it. We considered that PI3-K/Akt signaling pathway was involved in anti-apoptotic action of LPA. Phosphorylation of Akt by LPA was inhibited by pretreatment of SKOV3 cells with pertussis toxin (PTX). These data indicated that the PTX-sensitive Gi coupled to LPA specificG-protein-coupled receptors may be involved in LPA mediated activation of the PI3-K/Akt pathway. Further more, the anti-apoptotic effect of LPA was also suppressed by PTX.Conclusion: LPA could protect the apoptosis through mitochondrial pathway. The PI3-K/Akt signaling pathway can be activated by LPA, then perform an anti-apoptotic action. Phosphorylation of Akt by LPA was inhibited by pertussis toxin (PTX). Further more, the anti-apoptotic effect of LPA was also suppressed by PTX.