ABSTRACT
@#[Abstract] Objective: To investigate the effect of RG108 on the proliferation and apoptosis of human non-small cell lung cancer (NSCLC) cell lines (A549, H1299) and explore its molecular mechanism. Methods: A549 and H1299 cells were cultured in vitro and treated with different concentrations of RG108. The cell proliferation, cell cycle and apoptosis were detected by MTT assay and Flow cytometry, respectively. qPCR and Western blotting (WB) were used to detect the TFPI-2 mRNA and protein expressions as well as the expression of TMPRSS4 in cells. Meanwhile, the methylation status and degree of TFPI-2 promoter in cells were detected with Methylation-specific PCR (MSP) and colorimetry. Finally, siRNA-TFPI-2 and pcDNA3.0-TMPRSS4 plasmids were used to silence TFPI-2 or overexpress TMPRSS4, and then the changes in cell proliferation and apoptosis were detected. Results: After treatment with RG108, the proliferation rate of A549 and H1299 cells were significantly decreased (all P<0.05), while the apoptosis rate were significantly increased(P<0.05), the cell cycle were arrested in G1/S phase (P<0.05), and the intracellular mRNA and protein expressions of TFPI-2 were significantly increased (P<0.01 or P<0.05). Meanwhile, the methylase degree in TFPI-2 promoter region and the expression of TMPRSS4 in cells were all significanly decreased ( all P<0.05). After TFPI-2 silence, the proliferation levels of A549 and H1299 cells were significantly increased(all P<0.05); however, the apoptosis rate of A549 and H1299 cells were significantly reduced after transfection with pcDNA3.0-TMPRSS4(all P<0.05). Conclusion: RG108 can inhibit proliferation of A549 and H1299 cells and promote apoptosis by inhibiting the methylation of TFPI-2 and negatively regulates the expression of TMPRSS4.
ABSTRACT
Objective To investigate the role and mechanism of TMPRSS4 in radiation induced metastasis of hepatocellular carcinoma (HCC).Methods Metastatic model of human HCC was established by orthotopic implantation of histologically intact human HCC tissue into the liver of nude mice.Mice bearing xenografts in liver were killed after radiation and the residual tumors were resected and reimplanted into the liver of normal nude mice.At the end of sixth week,the mice were killed and the histopathological features,tumor volume,intrahepatic and lung metastasis were evaluated.Expression of epithelial-mesenchymal transition (EMT) related genes including N-cadherin,Vimentin,SIP1 and TMPRSS4 were measured by Western blotting and RT-PCR.Results The tumor volume and frequency of lung metastasis of control group was 2.25±0.52 cm3 and 66.7％,respectively.Compared to control group,tumor diameter (1.61±0.51 cm3,P＜0.05) and lung metastasis (12.5％,P＜0.05) were significantly inhibited 2 days after radiation.Whereas,30 days after radiation,tumor growth recovered (2.60±0.61 cm3,P＞0.05) and lung metastasis was enhanced (100％,P＜0.05).There were no intrahepatic metastasis in the control group and in the group of reimplantation of HCC 2 days after radiation,while the tumors from those 30 days after radiation showed enhanced intrahepatic metastasis (18 ± 8.05,P＜ 0.01 ),with overexpression of SIP1,N-cadherin,Vimentin and TMPRSS4,and reduced expression of E-cadherin.Conclusion The metastasis potential of residual HCC after radiation was first inhibited and then promoted.Overexpression of TMPRSS4 plays a critical role in radiation induced long-term metastasis of HCC by facilitating EMT.