The Mechanisms Of Tropomyosin-1 Promotes Cancer Cell Apoptosis Via The P53-mediated Mitochondrial Pathway In Renal Cell Carcinoma

Objectives:Tropomyosin-1(TPM1),a widely expressed actin-binding protein,is down-regulated in many tumors and associated with cancer progression.A previous study from our group suggested that TPM1 could be involved in renal cell carcinoma(RCC)apoptosis,but the mechanisms and details remained unknown.The aim of the present study was to reveal in more detail the molecular mechanisms by which TPM1 expression affects tumor cell apoptosis and proliferation in RCC.These effects were assessed by TPM1 over-expression and knockdown in two different RCC cell lines.Methods:1.Transfect transiently the plasmid containing five different TPM1 expression vectors into renal cancer cells 786-O and ACHN.2.Cell viability assay: MTT experiment to detect the effect of TPM1 expression on the proliferation of RCC.3.DAPI staining to detect the changes in RCC nucleus.4.Single cell gel electrophoresis(comet assay)to detect the damage of DNA.5.Flow cytometry(Annexin V-FITC and PI double staining)to measure the extent of 786-O and ACHN cell apoptosis.6.Gel electrophoresis and western blot analysis to analyze the expression of key proteins in the mitochondrial apoptosis pathway.Results:1.The level of TPM1 protein in the pEX4-TPM1 over-expression group(OE)was markedly increased compared with its negative control pEX4-NC group(OE-NC),TPM1 protein levels in the pGPU6/GFP/Neo-shTPM1 knockdown group(KD)were decreased compared with its negative control pGPU6/GFP/Neo-shNC group(KD-NC)in 786-O cells,while there is no significant difference in OE-NC,KD-NC and mock groups.2.MTT assay showed that over expression of TPM1 in OE group reduced 786-O cell line cell proliferation,but had no significant effect in other groups and in ACHN cell line.3.DAPI staining showed OE groups had significantly higher apoptosis rates compared with their negative control groups in both 786-O and ACHN cell lines.No significant difference was observed between the KD groups and its negative control group in both cell lines.4.Flow cytometry results after PI and Annexin-V-FITC double staining showed that TPM1 up-regulation could promote apoptosis of both cell lines,while TPM1 knockdown did not have any significant effect.5.Single cell gel electrophoresis(comet assay)showed over expression of TPM1 significantly induced an increase in tail DNA%,tail length and tail moment in 786-O cells and some ACHN cells.6.Western blot showed up-regulation of TPM1 significantly reduced the expression level of the anti-apoptotic factor Bcl-2,and it increased the expression levels of the pro-apoptotic factors Bax,P53 and caspase-3.Conclusin:1.TPM1 up-regulation inhibits 786-O cell proliferation.2.TPM-1 promotes cancer cell apoptosis via the P53-mediated mitochondrial pathway in renal cell carcinoma.