The Effect Of T-cadherin On The Proliferation And Invasion Of Malignant Melanoma

Melanoma is a cancer that arises from melanocytes. Melanoma is the most aggressive form of skin cancer and advantages stages are inevitably resistant to conventional radiotherapeutic and chemotherapeutic agents and other external stimuli poses a selective advantage for tumor progression, metastasis formation. Not only is the incidence increasing,but the number of annual deaths from melanoma is also on the rise worldwide.In the United States, melanoma will be diagnosed in approximately40,000new patients each year and be responsible for about7000deaths. There is no satisfactory treatment for patients with metastatic melanoma that have an estimated5-year survival of6%.T-cadherin also known as cadherin13, H-cadherin (heart)(CDH13) is a unique member of cadherin superfamily because it lacks the transmembrane and cytoplasmic domains and is anchored to the cells membrane through the glycosyl-phosphatidylinositol (GPI) anchor. Our study was to research T-cadherin gene’s possible mechanisms of proliferation inhibition in B16F10cells,and this study maybe do some benefit to clinical gene therapy.Objective:This study was aimed to investigate the effect of T-cadherin on murine B16F10melanoma cells and to study its possible mechanisms of proliferation inhibition and induce apoptosis in B16F10cells. Finally we investigate the effect of T-cadherin on the invasiveness of malignant melanoma. So as to provide experimental base for treatment of T-cadherin.Methods:1.Cells used in this study:B16F10cells were used in this study. It is a high metastatic murine melanoma cell line originated from C57BL mouse.2.Transfecting B16F10:B16F10cells were cultured in vitro.When the confluence of B16F10cells were up to90%, the complex of pEGFP-N1-T-cadherin with LipofectamineTM2000was added and selected with G418.3.B16F10cells were cultured in vitro. The expression of T-cadherin mRNA and protein were measured by reverse transcription polymerase chain reaction (RT-PCR) and SP immunohistochemistry method.4.Proliferation effect of T-cadherin on B16F10cells was tested by MTT assay. Apoptosis rate was determined with AnnexinV-EGFP/PI double staining and cell cycle with PI staining was detected by flow cytometry.5.Cell invasiveness was determined by Matrigel Transwell in vitro invasion assay.6.The statistical analysis was performed with the SPSS11.5using one-way analysis of variance (ANOVA) and LSD-t test. P<0.05was considered statistically significant.Results:1.RT-PCR proved that there was transcription of T-cadherin in T-cadherin-transfected cells.2.immunohistochemical result showed that there was translation of T-cadherin in T-cadherin-transfected cells.3.The proliferation of B16F10cells:T-cadherin maybe inhibit the proliferation of B16F10cells. In the MTT assay, the OD value of cells was lower in T-cadherin-transfected cells(0.383±0.116) than that of un-transfected cells(1.142±0.053) and pEGFP-N1-transfected cells (1.250±0.055P<0.05); There was no statistical difference between un-transfected cells and pEGFP-N1-transfected cells (P>0.05).4.Flow cytometric analysis showed that T-cadherin induced B16F10cells apoptosis in vitro. The early apoptosis rate was higher in T-cadherin-transfected cells(28.157±3.692)%than that of un-transfected cells(2.893±0.454) and pEGFP-N1-transfected cells (2.350±0.578)%(P<0.05); There was no statistical difference between un-transfected cells and pEGFP-N1-transfected cells (P>0.05). 5.The frequency of hypoploid cells representing cells undergoing apoptosis reached (16.063±0.795)%, which was higher than that of un-transfected cells(0.523±0.140) and pEGFP-N1-transfected cells (0.417±0.087)%(P<0.05); There was no statistical difference between un-transfected cells and pEGFP-N1-transfected cells (P>0.05).6.Cell cycle analysis demonstrateed that T-cadherin caused an arrest at G2/M phase in B16F10cells. The percentages of cells in G2/M phase were significantly increased in T-cadherin-transfected cells(10.790±0.731)%than that of un-transfected cells(1.963±1.145)%and pEGFP-N1-transfected cells (1.263±0.459)%(P<0.05); There was no statistical difference between un-transfected cells and pEGFP-N1-transfected cells (P>0.05).7.The number of invasion cells was significantly lower in T-cadherin-transfected cells (2.2±0.490) compared with the un-transfected cells (16.8±1.393)and blank vector transfectants(15.0±1.265), with a significant difference(P<0.05). There was no statistical difference between un-transfected cells and blank vector transfectants(P>0.05).MTT assay showed that the OD value of invasion cells was obviously lower in T-cadherin-transfected cells (0.124±0.003) than that of un-transfected cells (0.263±0.006) and blank vector transfectants (0.247±0.012), with a significant difference(P<0.05). There was no statistical difference between un-transfected cells and blank vector transfectants(P>0.05).Conclusions1.T-cadherin could inhibit the proliferation of B16F10cells via G2/M arrest and induction of cell apoptosis in vitro.2.T-cadherin suppresses the invasive ability of malignant melanoma cells in vitro.