Correlation Between ZNF217 And DDP Sensitivity Of Ovarian Cancer And Its Mechanism

BACKGROUNDOvarian cancer, a common gynecological tumor, has a high fatality in patients. Ovarian cancer is highly malignant, and easy to spread and transfer through blood and lymphatic system. The 5-year survival rate for advanced ovarian cancer patients is low, and it is mainly due to the chemotherapy drugs resistance. ZNF217 gene, a recently identified cancer gene, is located on chromosome 20ql3.2, the Kruppel-like transcription factors encoded by which is belong to the zinc finger protein family. ZNF217 gene is closely related to the occurrence and development of several tumors, such as ovarian cancer, mammary cancer, and lung cancer. We have previously found that the amplification degree of ZNF217 gene was correlated with clinical stages of ovarian cancer, the cancer cells'abilities of movement and invasion were apparently impaired after RNAi to ZNF217 gene, indicating that there is relationship between ZNF217 and the metastasis of ovarian cancer. Some investigator found that bZNF217 could decrease the apoptosis induced by adriacin, the adriacin sensitivity increase after inhibiting the ZNF217 expression by RNAi. But it is still not clear if there is some link between ZNF217 and chemotherapy drugs resistance.The chemotherapy drug resistance is an important reason for the failure of tumor therapy, and its main mechanism is the blocking-up of apoptosis pathway mediated by apoptotic genes. IAPs are a special anti-apoptosis family, including 8 members, XIAP, survivin, c-IAP1, livin, and so on. Most IAPs can block the apoptosis pathway through directly inhibiting the enzyme activity of caspase-3, caspase-7 and caspase-9, inducing the chemotherapy drug resistance. The over-expression of IAPs is related to the failure of tumor treatment. The expression of XIAP and survivin increased in ovarian cancer tissues, the over-expression of them can inhibit the apoptosis of ovarian cancer cells and is correlated with DDP and paclitaxel resistance. After the sense XIAP cDNA is transfected into DDP-sensitive ovarian cancer cells, the over-expression of XIAP can make Akt phosphorylate and then inactivate. The inactivation of Akt blocks the apoptosis induced by DDP. It is considered that the DDP sensitivity of ovarian cancer cells maybe related to the inhibition of caspase-3 activation by XIAP. Clinical data analysis shows that survivin is correlated with the paclitaxel resistance. The paclitaxel resistance increases 4-6 times after sense survivin cDNA is stably transfected into human ovarian cancer cell lines without survivin gene. It is found that the expression of survivin mRNA increase significantly, while the apoptosis rate does not increase correspondingly after stimulation by paclitaxel or carboplatin at low concentration, indicating ovarian cancer cells can enhance anti-apoptosis ability by increasing the expression of survivin, then improving the paclitaxel or carboplatin resistance. All above findings show both XIAP and survivin are important factors in the mechanism of chemotherapy drug resistance of ovarian cancer cells.Is there a close relationship between ZNF217 and clinical chemotherapy resistance? How does ZNF217 gene regulation affect on DDP sensitivity of ovarian cancer cells? And what is its mechanism? These questions have not been answered. The purposes of the study include:(1) determining if there is a close correlation between ZNF217 protein expression and chemotherapy sensitivity, clinical stages, pathological types and differentiation grades of clinical tissue samples of ovarian cancer by immunohistochemistry and detecting their DDP sensitivity by ATP method; (2) making clear if there is some difference in ZNF217 mRNA and ZNF217 protein expression between DDP-resistant and DDP- sensitive ovarian cancer cell lines; (3) identifying the effects of ZNF217 on the DDP sensitivity of ovarian cancer cells and its mechanism in the perspective of apoptosis pathway via RNAi and gene transfection. The study is aimed to clarify the mechanism of chemotherapy resistance in ovarian cancer, and to probably find a new therapeutic target point for ovarian cancer.MATERIALS AND METHODS1. The ovarian cancer tissues were taken from peking university Shenzhen hospital, Shenzhen people's Hospital, and Shenzhen futian people's hospital during 2008.8 to 2009.12. There were total 60 ovarian cancer samples, including 36 serous cystadenocarcinomas,9 mucous cystadenocarcinomas,8 clear-cell carcinomas and 7 endometrioid carcinomas. Two contrast groups respectively consisted of 15 benign ovarian tumors and 12 normal ovary tissues.2. The ovarian cancer tissues were made into cancer cell suspension, and the DDP sensitivity was determined with ATP method; the ovarian tissue paraffin sections were used to perform immunohistochemistry stain.3. Six ovarian cancer cell lines, including 3 DDP-sensitive lines (A2780, SKOV-3 and COC1) and 3 DDP-resistant lines (A2780-DDP-R, SKOV-3-DDP-R and COC1-DDP-R) were cultured. The resistance indexes (RI) of DDP-resistant cell lines were determined with ATP method. Intracellular localization of ZNF217 protein in the 6 ovarian tumor cell lines was detected by immunofluorescent cytochemistry. The expressions of ZNF217 mRNA and ZNF217 protein were determined by RT-PCR and Western blotting, respectively. 4. pGenesil-ZNF217-siRNA and pEGFP-N1-ZNF217 eukaryotic expression plasmids were constructed and transfected into A2780-DDP-R and A2780 cells, respectively, with cationic liposome. Under G418 selection, monoclones were selected and proliferated to establish stably-transfected cell lines.5. In 4 ovarian cancer cell lines, A2780, pEGFP-N1-ZNF217/A2780, A2780-DDP-R, pGenesil-ZNF217-siRNA/ A2780-DDP-R, growth inhibition rate were measured by ATP method, the apoptosis rate by flow cytometry, two apoptosis inhibitory proteins, XIAP and survivin by Western blotting, and the activity of two apoptosis effector enzymes, caspase3 and caspase9 by spectrophotography.6. All data were statistically analyzed with SPSS 13.0 software. Kruskal-Wallis test was used to compare nonparametric data, and Spearman correlation was used to analyze the correlation between rank data. Measurement data are shown as means±SE. One-way ANOVA test and LSD test were applied to compare data of more than two groups, and Independent samples t test to compare data of two groups. Concentration-inhibition curve was fitted by nonlinear regression model.RESULTS1. The DDP-sensitivity and the expression of ZNF217 protein in ovarian cancer tissueZNF217 protein was present in DDP-resistant, partially DDP-sensitive, and highly DDP-sensitive ovarian cancer tissues in different degrees, the difference among three groups was significant (x2=9.211, P=0.010), and a negative correlation was found between ZNF217 protein expression and the DDP-sensitivity (r=-0.394, P=0.002). The difference between I-II stages andⅢ-Ⅳstages was significant (x2=9.610, P=0.002), and there was a positive correlation between ZNF217 protein expression and clinical stages (r=0.404, P=0.001). ZNF217 protein expression was not significantly different in serous cystadenocarcinomas, mucous cystadenocarcinomas, clear-cell carcinomas and endometrioid carcinomas (x2=1.925, P=0.588), and there was no correlation between ZNF217 protein expression and pathological types (r=0.056, P=0.670). The difference of ZNF217 protein expression among normal ovarian tissues, benign ovarian tumours and ovarian cancer was significant (x2=20.469, P=0.000), and the ZNF217 protein expression was closely related to the occurrence of ovarian cancer (r=0.487, P=0.000)。ZNF217 protein was expressed in ovarian cancer tissues of different differentiation grades, the difference among three groups was significant (x2=9.934, P=0.007), and the ZNF217 protein expression was significantly related to the occurrence of ovarian cancer (r=-0.410, P=0.001)。2. ZNF217 expression in DDP-resistant and DDP-sensitive ovarian cancer cell linesThe IC50 values of DDP to A2780 and A2780-DDP-R were 18.1 mg/L and 47.9 mg/L (P< 0.01), respectively, that to SKOV-3 and SKOV-3-DDP-R were 9.6mg/L and 40.1 mg/L (P< 0.01), respectively, and that to COC1 and COC1-DDP-R were 4.8 mg/L and 15.3 mg/L (P< 0.01), respectively. The RIs of A2780-DDP-R, SKOV-3-DDP-R and COC1-DDP-R to DDP were 2.6,4.2 and 3.2, respectively. Laser scanning confocal microscopy showed that ZNF217 protein was mainly located in the cytoplasm of the DDP-sensitive cell lines, but it was in the nuclei of the DDP-resistant cell lines. RT-PCR and Western blotting showed that the expressions of both ZNF217 mRNA and ZNF217 protein were significantly higher in DDP-resistant cell lines than in DDP-sensitive cell lines.3. The ZNF217 gene regulation of ovarian cancer cell lines.Through amplifying the encoding sequence of ZNF217 gene and constructing it into the pEGFP-N1 plasmid, we successfully obtained the pEGFP-N1-ZNF217 eukaryotic expression plasmid. In addition, siRNA sequence targeting ZNF217 was also designed and cloned into the pGenesil-1 plasmid to construct the pGenesil-ZNF217-siRNA plasmid. The pEGFP-N1-ZNF217 and pGenesil-ZNF217 plasmids were transfected into A2780 and A2780-DDP-R cell lines, respectively. Then G418 was used to select stably-transfected cells. Stable transfection was certified via RT-PCR and Western blotting.4. The effects of ZNF217 gene regulation on the DDP-sensitivity of ovarian cancer cell lines and its mechanismThe growth inhibition rate was statistically different in four ovarian cancer cell lines (F=125.441, P=0.000). It was lower in pEGFP-N1-ZNF217/A2780 than in A2780, indicating ZNF217 over-expression can decrease DDP sensitivity. It was higher in pGenesil-ZNF217-siRNA/A2780-DDP-R than in A2780-DDP-R, indicating ZNF217 RNAi can increase DDP sensitivity. The results suggest ZNF217 expression level is a key factor to affect DDP sensitivity of ovarian cancer cells.The apoptosis rate of each cell lines was detected after DDP stimulation. The result showed that, the apoptosis rate was statistically different in four cell lines (F=78.688, P=0.000). It was lower in pEGFP-N1-ZNF217/A2780 than in A2780, indicating ZNF217 over-expression can decrease DDP-induced apoptosis. It was higher in pGenesil-ZNF217-siRNA/A2780-DDP-R than in A2780-DDP-R, indicating ZNF217 RNAi can increase DDP-induced apoptosis.Western blotting analysis was applied to detect the expression levels of XIAP and survivin, two apoptosis inhibitory proteins, and the result showed that, in ovarian cancer cell lines with higher levels of ZNF217, the expression level of XIAP and survivin increased significantly (F=63.981, P=0.000) (F=79.008, P=0.000).The activities of caspase3 and caspase9 were measured by spectrophotography. It was found that, in ovarian cancer cell lines with higher levels of ZNF217, the activities of both caspase 3 and caspase 9 decreased significantly, while in ovarian cancer cell lines with lower levels of ZNF217, their activities increased significantl (F=16.664, P=0.001) (F=28.320, P=0.000). These result suggested that, ZNF217 induced DDP-resistance in ovarian cancer cells through inhibiting apoptosis pathway, and XIAP, survivin, caspase3 and caspase9 were the key elements regulated by ZNF217 in the apoptosis pathway.CONCLUSIONS1. There is a negative correlation between ZNF217 protein expression level and the DDP sensitivity of ovarian cancer tissues.2. ZNF217 protein is mainly located in the cytoplasm of the DDP-sensitive cell lines, while in the nuclei of the DDP-resistant cell lines. The expressions of both ZNF217 mRNA and protein were significantly higher in DDP-resistant cell lines than in DDP-sensitive cell lines.3. The ZNF217 expression level can influence the DDP resistance of ovarian cancer cells. The higher is the ZNF217 expression level, the lower is the DDP sensitivity.4. ZNF217 induces the DDP resistance in ovarian cancer cells through up-regulating the expression of XIAP and survivin and down-regulating the activities of caspase3 and caspase9.