Correlation Between Expression Of PRL-3 And Metastasis In Human Epithelial Ovarian Cancer And Its Possible Inhibitor Treatment

Ovarian cancer is the leading cause of gynecologic cancer death worldwide, and its mortality rate exceeds that for all other gynaecological malignancy combined. The tumor metastasis is a major cause of higher-death among ovarian cancer patients, but it is very difficult to predict precisely the early metastasis of this disease. So, it might be expected that detecting the biomarker for the prediction of early metastasis and identifying the novel drugs for treatment will help the better detecting early stage lesion and enhancing overall survival.Tumor metastasis is a multiple genes, many factors and multi-step complicated process. protein tyrosine phosphatases(PTP) can adjust cells to grow, differentiation, signaling and cell cycle, and closely related with tumor. The phosphatases of regenerating livers (PRL-3) belongs to a small class of tyrosine phosphatases, and has been reported as a metastasis-associated phosphatase gene. Recently, the relationship between PLR-3 and tumor has been extensive explored. Mounting evidences have demonstrated that PRL-3 could be regarded as novel biomarkers and therapeutic targets of tumors. In 2001, Saha et al first confirmed the correlation between PRL-3 and metastasis in colorectal tumor. Furthermore, the correlation has been reported in various tumor types, but the study on the relationship of PRL-3 and ovarian tumor is still rare. Zeng et al found that Chinese hamster ovary cells stable expressing PRL-3 could significantly not only increase cell migration and invasion capacity in vitro, but also enhance the tumor formation and metastasis capacity in nude mice. Polato first confirmed the correlation between the PRL-3 and the development of ovarian cancer in 2005 and identified that the PRL-3 could become a novel therapeutic target in malignant tumor.Through clarifying the role of PRL-3 in tumor metastasis, Targeting PRL-3 will provide a useful strategy for tumor treatment. In our study, we aimed to investigate the possibility that exogenous drugs were used to treat ovary cancer by inhibition of PRL-3 expression.Mounting evidences showed that flavonoid compounds could inhibit tumor proliferation in vitro. Jaceosidin belongs to the anthocyanidins family. It has been demonstrated that Jaceosidin could induce cell apoptosis by blocking protein kinase phosphorylation pathway. Therefore, we presumed that Jaceosidin might inhibit the metastasis of ovarian cancer by targeting PRL-3.In addition, Pathak reported that pentamidine, as anti-parasitic medicine, has also the antitumor function in melanoma nude mice model by inhibition of PRL-3. Therefore, we also investigated whether pentamidine might treat ovarian cancer by blocking PRL-3 expression. Part I Correlative study between gene expression of phosphatase of regenerating liver-3 and metastasis in human epithelial tumors of ovaryObjective:To evaluate the relative expression of phosphatase of regenerating liver-3(PRL-3) mRNA in human epithelial ovarian cancer and its metastatic lesions, and investigate the relationship between PRL-3 expression and clinicopathologic parameters.Methods:The expression of PRL-3 mRNA was examined by semi-quantitatively PCR in the samples from the surgically resected epithelial ovary cancer (84 cases of primary tumors and 58 cases of metastasis lesions) and paired paratumor normal tissues (22 cases). We compared the expression of PRL-3 mRNA in primary cancer and its metastatic lesions with those in paired paratumor normal tissues, and investigate the relationship between PRL-3 expression and clinicopathological parameters of epithelial ovarian cancer.Results:1. The relative level of PRL-3 mRNA expression in primary ovarian cancer, its metastases and normal ovarian tissues:The PRL-3 mRNA level was increased in 84 primary tumor tissues as compared with paired paratumor normal tissues.The PRL-3 mRNA levels of metastases in 58 cases were significantly higher compared with the matched primary cancers.2. The relationship between PRL-3 mRNA expression and clinicopathological parameters of epithelial ovarian cancer:We investigated the relationship between PRL-3 mRNA expression and clinicopathologic parameters of epithelial ovarian cancer in 84 cases of patients, and found that reduced PRL-3 expression was correlated with 3-year overall survival (P=0.02) and cancer metastasis (P=0.00), but not with age (P=0.67), FIGO stages (P=0.43), and histologic grade (P=0.92).Conclusion: Our results showed that PRL-3 could contribute to the invasion and metastasis of epithelial ovarian cancer, and might be associated with the initiation and development of ovarian cancer.Part II Jaceosidin induced cell apoptosis in human ovarian cancer cell line SKOV3 and Caov-3 through mitochondrial pathway, but no PRL-3-dependent pathwayObjective:To investigate the effect and mechanism of Jaceosidin (4',5,7- trihydroxy-3', 6-dimethoxyflavone) on cell growth and apoptosis in human ovarian cancer cell lines SKOV3 and Caov3 in vitro.Methods:1. We examined the anti-proliferation effect of Jaceosidin at different concentration (0,20,40 and 80 uM) on human cancer cell lines SKOV3 and Caov3 using MTT assay.2. After SKOV3 and Caov3 cells were treated with different concentration of Jaceosidin for 24 hours, Flow cytometry was used to measure the apoptosis rates of cells by annexin-V-FITC and PI staing.3. Mitochondrial membrane potential was measured using JC-1 staining by Flow cytometr. Results.4. The expression of PRL-3 mRNA was examined in SKOV3 and Caov3 cells treated with different concentrations of Jaceosidin for 24 hour.Results:1. Jaceosidin significantly reduced the proliferation of Caov-3 and Skov-3 cells in a concentration-dependent manner.2. Jaceosidin induced cell apoptosis in human ovary cancer cells SKOV3 in higher-concentration.3. Jaceosidin decreased mitochondrial membrane potential of SKOV3 in a concentration-dependent manner.4. SKOV3 cells presented PRL-3 expression.5. There was no difference of PRL-3 mRNA expression in SKOV3 cells treated with different concentrations of Jaceosidin (0,10,20 and 40?M.), compared with that of control group.Conclusion:1. Jaceosidin at a particular concentration could inhibit the growth of ovarian cancer cells SKOV3, and induce cell apoptosis through mitochondrial pathway.2. SKOV3 cells presented endogenous PRL-3 mRNA expression.3. Jaceosidin at each concentration could not affect endogenous PRL-3 mRNA expression in SKOV3 cell. Part III Inhibition of pentamidine on ovarian cancer cell line SKOV3 in vitro and PRL-3 expression in inoculated nude mice using in vivo pentamidine injectionObjective:To study the inhibitory effect of pentamidine on ovarian cancer SKOV3 cell line in vitro and PRL-3 expression in vivo.Methods:Growth of SKOV3 cells treated by different concentrations pentamidine (0.10?mol/L,1.00?mol/L?10.00?mol/L?100.00?mol/L) was evaluated by 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Amresco) assay,Furthermore, protein extracts from treatment of SKOV3 was prepared to detect the PRL-3 expression by western-blot. We then established a subcutaneously inoculated nude mice model with human ovarian cancer SKOV3 cells, and 32 mice were randomly divided into four groups according to different dose of pentamidine:?Hige-dose(10mg/kg);?Moderate-dose(5mg/kg);?low-dose(2.5mg/kg); while the same amount of PBS was intratumorally injected as blank control. Tumor growth was observed and tumor volume was continuously blindly measured by periodic caliper. Tumor size was calculated by the following formula:tumor volume (mm3)= (ab2)/2, where a = length in mm and b = width in mm, then the growth inhibition ratio was acquired. The PRL-3 expression in excised tumor was detected by RT-PCR.Results:1. Pentamidine inhibit on cell proliferation in ovarian cancer cells.2. The expression of PRL-3 proteins in human Skov-3 cells treated by different concentration of pentamidine. 3. The growth of xenograft tumor in nude mice treated with medium-dosage and large-dosage groups of pentamidine was significantly slower than that in control group, once daily for 5 consecutive, and had significant difference.4. The inhibitor rate of tumor was 11.1% in small-dosage group of pentamidine, 33.3% in medium-dosage group,55.6% in large-dosage group. The significant difference was observed in 3 groups, compared with that of control group.5. The treatment of Pentamidine in medium-dosage and large-dosage groups could significantly reduce the PRL-3 expression of xenograft tumor in a dose-dependent manner, with stronger effect in higher dose.6. During the experiment, the physiological status was normal in all nude mice, and not one animal died; more importantly, the treatment of pentamidine could not affect the weight of nude mice.Conclusions:1/ Pentamidine inhibited cell proliferation in SKOV3 cells in vitro, and lead to xenograft tumor retardation in SKOV3 inoculated nude mice.2/ Pentamidine could significantly reduce the PRL-3 expression in SKOV3 cells and in SKOV3 xenograft tumor.3/ Different dosage of pentamidine injection had no obvious toxic effect on SKOV3 inoculated nude mice...