Biological Effects Of Tumor Associated Antigen CHP2 In Ovarian Cancer Cell

[Background and Objective] Ovarian carcinomas is the leading cause of death by gynecological cancer in women. Metastatic spread of self-regulated growth ovarian cancer cells via invasion to distant tissues is the primary cause of treatment failure and subsequent death in cancer patients. Calcineurin B homologous protein isoform 2 (CHP2) was identified to be expressed in various malignant tissue and cell lines, but not in the normal tissue counterpart. The biological function of CHP2 related to cancer progression is still unknown. Thus, the aims of this study are to analysis the biological effects of CHP2 on ovarian tumor cell.[Methods]1. The Expression of CHP2 in ovarian cancer tissues: RT-PCR analysis was used to detect the expression of CHP2 mRNA in 20 specimens of normal human ovarian tissues and ovarian cancer tissues respectively.2. Plasmid construction and transfection and production of stable clones: The entire open reading frame without termination codon was obtained by RT-PCR from total RNA of hepatocellular carcinoma tissues and then cloned into the pMD18-T Simple vector. The vector pEGFP-N1 was prepared to create pEGFP-Nl-CHP2 with insertion of the CHP2 gene. Cells were transfected by lipofectamine 2000 mixtures. After incubated 48 hours, fluorescent images were acquired with fluorescence biomicroscope for examination of the cellular localization of the encoded protein. Approximately 2 weeks later, independent clones were selected for G418 resistance and detected by fluorescence biomicroscope.3. RT-PCR analysis of CHP2 and NHE1 expression: The expression of CHP2 and NHE1 was identified by RT-PCR analysis. And fluorescence biomicroscope was also amplified to study the expression.4. Cell proliferation assay: Cells were plated at the same density. At 12, 24, 36, 48, 60, and 72 h, cells were counted by using viability analyzer (according to trypan blue dye exclusion method).5. Adhesion assay: Cells were plated onto the fibronectin-coated 6-well plates at the same density, and incubated for 15 min, 30 min, 60 min, or 90min. At the end of incubation, the number of adherent cells was determined by using automated cell viability analyzer.6. Wound healing assay: Cells were plated on fibronectin pre-coated 6-well plates at the same density, and artificial wounds were created by scraping with a pipette tip. Wound closure was monitored at 0 hour, and 36 hour after wounding. The distance of the wound areas were measured by Image-Pro software, set at 100% for 0 h, and the mean percentage of the total distances of the wound areas were calculated.7. Invasion assay: Transwell chambers were coated with Matrigel. Conditioned medium from NIH3T3 cells was used as chemoattractant in the lower chamber. Cells inoculated in the upper chambers at the same doses. After 12 hours incubation the cells that had penetrated through to the bottom of the chamber were counted with microscope (40x field).8. Statistical analysis: Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Neuman-Keuls analysis as a post hoc test, using SPSS 11.5 for Windows. In all statistical comparisons, ap value of <0.05 was considered to be statistically significant.[Results] CHP2 mRNA was expressed in 1 of 20 normal ovarian cases and in 17 of 20 ovarian cancer cases. The positive rate between two group was significant(X~2=25.86, p<0.05) . With RT-PCR analysis, CHP2-transfected OVCAR3/CHP2 cancer cells showed high CHP2 gene expression, whereas none transfected clones did not produce detectable CHP2 mRNA. Strong NHE1 expression was seen in the parental OVCAR3 cell line; no obvious change was observed after CHP2 transfection. With the green fluorescent protein (GFP) reporter gene, fluorescence microscopy confirmed that most of the EGFP/CHP2 fusion protein was localized in the plasma membrane of OVCAR3 cell while GFP was uniformly distributed over whole cells of empty vector transfected OVCAR3 cells. CHP2-transfected OVCAR3/CHP2 cells showed increased proliferation rates and exhibited increased activities of cell adhesion, migration, and invasion (p<0.05) .[Conclusion] Our results suggest that expression of the CHP2 gene increases activities of cell proliferation, adhesion, migration, and invasion. We propose that an intact CHP2-NHE1 pathway is essential for the altered biological behavior of ovarian cancer cell. It is also suggested that CHP2 may be a new target of biological anticancer therapy for CHP2 positive tumors.