Tumor Conditioned Medium Induces Vascular Smooth Muscle Cell Apoptosis

Objective: Tumor growth and metastasis depend on angiogenesis, tumor vessels often lack the smooth muscle layer, and the instability of tumor vessels is conducive to tumor invasion and metastasis. Recently, study on tumor angiogenesis focused on the interaction between tumor cells and endothelial cells. The impact of tumorigenesis on vascular smooth muscle cells(VSMCs) characteristics has not been reported. In the present study, we used tumor conditioned medium(TCM) to in vitro mimic the tumor microenvironment and to elucidate the effect and the molecular mechanism of TCM on VSMC proliferation.Methods and Results:1 TCM inhibits VSMC proliferationMTT assay and cell counts showed that the proliferation activity and cell number of VSMC were significantly inhibited in a concentration-dependent manner after TCM stimulation for 24 h, compared with the control group(P<0.05).2 The effect of TCM on PCNA expression in VSMCsWestern blot assay showed that PCNA protein levels in VSMCs were not changed after TCM stimulation. Therefore, we speculated that the decreased VSMC proliferation activity and cell number may result from apoptosis.3 TCM induces VSMC apoptosisBoth early and late cell apoptosis were detected by flow cytometry and fluorescence microscopy after Annexin-V-FITC/PI double staining. The results showed that both early and late cell apoptotic rate of TCM group were higher than that of the control group(P<0.01).JC-1 Mitochondrial Membrane Potential Assay Kit was used to reflect the situation of early apoptotic cells by detecting the change of cell membrane potential. Fluorescence microscopy results showed that early apoptotic cells of TCM group were higher than that of the control group.TUNEL FITC Apoptosis Detection Kit was used to detect DNA break that is a late marker of cell apoptosis. Fluorescence microscopy results showed that late apoptotic cells in TCM group were higher than that of the control group.4 TCM induces the activation of Caspase-3 in VSMCsCaspase-3 activation is a critical determinant of apoptosis. Pro-caspase-3(32KD) is activated into two large subunits(17KD) and two small subunit(12KD) in the early stages of apoptosis. Caspase-3 activity was significantly decreased in late apoptotic and dead cells. Western blot assay showed that pro-Caspase-3 protein level in VSMCs was down-regulated after 24-hour TCM treatment(P<0.05). However, cleaved-Caspase-3 was up-regulated under the same conditions(P<0.01). The results indicated that the apoptosis induced by TCM may involve the caspase cascade.5 TCM increases the expression of Bax, and inhibits the expression of Bcl-2 in VSMCsThe expression and regulation of Bcl-2 family is one of the key factors affecting apoptosis, and plays an important role in apoptosis signal transduction pathway. Bcl-2 and Bax are the most representative antiapoptotic and proapoptotic genes of Bcl-2 family. Western blot assay showed that Bcl-2 protein level in VSMCs was down-regulated after 24-hour TCM treatment. However, Bax was up-regulated under the same conditions, and the ratio of Bax to Bcl-2 was increased(P<0.01). The results indicated that TCM may induce apoptosis of VSMCs through the mitochondrial pathway.Conclusions:1 TCM-induced apoptosis of VSMC is one of the mechanisms of tumor vascular structural abnormalities.2 TCM-induced apoptosis of VSMC involves the caspase cascade and mitochondria-mediated apoptosis.