| Background and Aims: The incidence and mortality of colorectal cancer have been increasing over the last two decades. Tumor cell metastasis to lymph node or liver is responsible for most cancer-related deaths. PRL-3, a recently-identified protein tyrosine phosphatase associated with metastasis of colorectal cancers, is known to play a role in cell migration, tumor invasion and metastasis. However, analysis of PRL-3 expression in human colorectal cancer with its clinical significance has not been fully established. The aims of this study are to investigate the significance of PRL-3 expression in tumor progression and metastasis of colorectal canceras, and to elucidate the effects of SoV, an inhibitor of PRL-3 phosphatase, on cellular proliferation, cell cycle and motility through blocking the expression of PRL-3.Methods: Forty-six colorectal cancers and six colorectal adenomas, together with their matched normal mucosa, metastatic lymph nodes and livers, were employed in the study. The expression of PRL-3 mRNA and protein were detected by semi-quantitative reverse transcription polymerase chain reaction and immunohistochemistry, respectively, and their relationships with clinicopathological parameters were analyzed. The levels of PRL-3 expression in 7 colorectal cancer cell lines were examined byWestern blotting method, in order to screen out which cell line expressed the highest level of PRL-3 protein. Then this cell line was employed in the next experiments of drug intervention. MTT assay was used to analyze the effects of SoV on cancer cellular proliferation after the cells were exposed to SoV at various concentrations of 0.1, 0.2, 0.3, 0.4, 0.5 umol/L, respectively, for 1 to 7 days. We chose the optimal concentration and time to test the effects of SoV on the cancer cell cycle and apoptosis by flow cytometry, and that on the cancer cell motility and migrating velocity by wound healing assay. Finally, we analyzed the expression of PRL-3 mRNA in the cells of SoV group using in situ hybridization. In the present study, the effects of FuDR were also investigated.Results: The expression of PRL-3 was hardly detected in colorectal normal epithelium and adenoma, compared to the higher level expression in primary colorectal cancers (P<0.05) and the highest level in metastatic lymph nodes and livers (P<0.01). The expression level or incidence of positive immunoreactivity of PRL-3 in the primary cancers with metastatic lymph nodes was significantly higher than that without nodal metastasis (P<0.01, = 0.001, respectively). PRL-3 expression was closely associated with TNM stage (P=0.019) and nodal metastasis (P=0.026). Moreover, PRL-3 mRNA expression level was correlated with tumor invasive depth (P<0.05) and liver metastasis (PO.01). The expression of PRL-3 was clearly detected in 5 of 7 (71.4%) colorectal cancer cell lines by Western blotting. Then we chose the colonic cancer cell line, Colo-320, which expressed the highest level of PRL-3 as the target cell. MTT assay showed that SoV inhibited the cell proliferation in dose- and time-dependent manners. After exposure to SoV atconcentration of 0.5 umol/L for 48 hrs, the proportion of cells in S-phase significantly decreased, compared to that in control cells (PO.01), and as the result, the cell division was blocked in S-phase. No obvious signs of apoptosis were observed. Under this condition, SoV significantly reduced the cancer cell migration-promoting activity. More exact velocity measurement of 20 cells motility was made, with 12.84 ± 6.78/xm/h vs. 39.12 ± lO.lljun/h in SoV group and control one, respectively (P<0.00001). The results of in situ hybridization presented that strong PRL-3 mRNA expression was detected in control cells, median expression in FuDR cells and weak or no signals in SoV cells. FuDR at various concentrations (2-8 umol/L) reduced the cell proliferation as well as SoV group in 96hrs. However, SoV showed more intensive restraint from 96 to 120 hrs. The rate of cell DNA synthesis in FuDR group dropped down remarkably and the cell cycle was blocked in G2/M-phase, showing a typical apoptosis wave.Conclusions: More intensive PRL-3 phosphatase is located in the cytoplasmic membranes. The distribution pattern of PRL-3 may correlate with the invasive and metastatic ability of the colorectal cancers. The high expression of PRL-3 in advanced colorectal cancers and metastatic lesions suggests that PRL-3 play a causative role in tumor progression and metastasis. PRL-3 gene mutation may account for one of the metastatic mechanisms. Therefore, PRL-3 could be regarded as a novel molecular marker for tumor stage and metastasis. SoV can effectively inhibit the Colo-320 cell proliferation and migration ability through restraining PRL-3 expression in the cells, which may be yielded through down-regulation of PRL-3 mRNA and catalytical inactivity of PRL-3 phosphatase. SoV shows the same goodefficacy to colonic cancer cells as the traditional drug, FuDR. Overall, the results might serve a laboratorial foundation to support clinical appliance of SoV in the future.
|
PDF Full Text Request