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OBJECTIVE@#To investigate the effects of LASS2/TMSG1 gene overexpression on proliferation and apoptosis of human lung cancer A549 cells and explore the possible mechanism.@*METHODS@#We examined LASS2/TMSG1 expression level in a previously constructed A549 cell line overexpressing LASS2/TMSG1 using Western blotting. The proliferation and apoptosis of the cells were detected using colony-forming assay, CCK-8 assay, Hoechst/PI double staining and flow cytometry. Fourteen nude mice were randomized into 2 groups (n=7) to receive subcutaneous injection of A549 cells with or without LASS2/TMSG1 overexpression on the back of the neck, and the cell proliferation in vivo was observed. The expression levels of p38 MAPK protein and p-p38 MAPK protein in the xenografts were detected with Western blotting. ELISA was used to detect the levels of ceramide and p38 MAPK protein in cultured A549 cell supernatants and the xenografts in nude mice.@*RESULTS@#Compared with the negative control cells, A549 cells with LASS2/TMSG1 overexpression had significantly lowered proliferation ability in vitro with increased early apoptosis rate (P < 0.05), and showed obvious growth inhibition after inoculation in nude mice(P < 0.05). Western blotting showed that in both cultured A549 cells and the xenografts in nude mice, LASS2/TMSG1 gene overexpression significantly increased the expression levels of p38 MAPK protein and p-p38 MAPK protein (P < 0.05); the results of ELISA also revealed significantly increased levels of ceramide and p38 MAPK protein in the cell supernatant andxenografts as well (P < 0.05).@*CONCLUSION@#Overexpression of LASS2/TMSG1 gene can significantly inhibit the proliferation and promote early apoptosis of human lung cancer A549 cells both in vitro and in vivo possibly by upregulating the expressions of ceramide and p38 MAPK protein to activate a signal transduction cascade.
Subject(s)
Animals , Humans , Mice , A549 Cells , Apoptosis , Cell Line, Tumor , Cell Proliferation , Lung Neoplasms , Membrane Proteins/metabolism , Mice, Nude , p38 Mitogen-Activated Protein Kinases/metabolism , Signal Transduction , Tumor Suppressor Proteins/metabolismABSTRACT
Purpose To investigate the expression of tumor metastasis suppressor gene-1 (TMSG-1) in colorectal cancer tissues and liver metastases,and to analyses the relationship between expression of TMSG-1 and clinicopathologic characteristics. Methods Immunohistochemical SP methods was used to detect the expression of TMSG-1 protein in 200 cases of colorectal cancer and 52 cases of liver metastases. Results The ratio of high,moderate and negative of TMSG-1 expression in primary colorectal carcinoma were 42. 5％ (85/200) ,29. 5％ (59/200) and 28. 0％ (56/200) respectively. The expression of TMSG-1 was significantly associated with tumor differentiation, invasion depth,lymph node metastasis,distant metastasis and TNM stages (P < 0. 05) ,but unrelate to age,gender and tumor size. The high, moderate and negative expression ratio of TMSG-1 in liver metastases were 17. 3％ (9/52) ,50. 0％ (26/52) ,32. 7％(17/52) ,respectively. The expression of TMSG-1 in liver metastases was significantly lower than that in primary lesion (P < 0. 05) . The expression of TMSG-1 in liver metastases, and there was no significant correlation between the expression of TMSG-1 in liver metastases and clinicopathologic characteristics. Conclusion The expression of TMSG-1 is significantly down-regulated in the liver metastases,which is associated with the development and progression of colorectal cancer. TMSG-1 will be used as a new tumor marker for predicting the prognosis of colorectal cancer.
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Objective:Vacuolar ATPase (V-ATPase) was found within the membranes and internal organelles of a vast array of eukaryotic cells,and was related to various kinds of highly metastatic tumors.LASS2/TMSG1 gene was a novel tumor metastasis suppressor gene cloned from human prostate cancer cell line PC-3M in 1999 by our laboratory.It was found out that protein encoded by LASS2/TMSG1 could interact with the c subunit of V-ATPase (ATP6V0C).In this study,To use RNA interference to suppress the expression of ATP6V0C and try to further investigate the molecular mechanism of ATP6V0C in tumor metastasis and its relationship with LASS2/TMSG1 gene.Methods and Results:The expression level of ATP6V0C mRNA and protein in high metastatic potential prostate cancer cell lines (PC-3M-1E8 and PC-3M) was significantly higher than that in low metastatic potential prostate cancer cell lines (PC-3M-2B4 and PC-3),the expression level in PC-3M-1E8 being the highest.Follow-up tests selected PC-3M-1E8 cells for gene silencing.The expression and secretion of MMP-2 and the expression of MMP-9 in ATP6V0C siRNA transfected PC-3M-1E8 cells displayed no obvious change,but the activity of secreted MMP-9 was abated noticeably compared with the controls (P ＜ 0.05).Extracellular hydrogen ion concentration and V-ATPase activity in interference group were both reduced significantly compared with the controls (P ＜ 0.05).The migration and invasion capacity of ATP6V0C siRNA interfered cells in vitro were diminished significantly compared with the controls (P ＜ 0.05).Furthermore,a dramatic reduction of LASS2/TMSG1 mRNA and protein level after transfection of siRNA in PC-3M-1 E8 cells was discovered (P ＜ 0.05).Confocal immunofluorescence showed a vast co-localization of ATP6V0C protein and LASS2/TMSG1 protein in plasma and membrane.The co-localization signals of control group were much stronger than those of interference group.Conclusion:Specific siRNA silencing of ATP6V0C gene inhibits the invasion of human prostate cancer cells in vitro by mechanism of inhibiting V-ATPase activity and then reducing the extracellular hydrogen ion concentration,inhibiting MMP-9 activation and affecting ECM degradation and reconstruction.Meanwhile,ATP6V0C and LASS2/TMSG1 have interaction and it is likely that ATP6V0C functions as a feedback regulator of LASS2/TMSG1.
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Objective To study the expression and significance of tumor metastasis suppressor gene-1(TMSG1) in esophageal squamous cell carcinoma (ESCC) and EC109 cells.Methods Immunohistochemistry S-P method was used to examine the expression of TMSG-1 protein in 136 cases of ESCC and 37 cases of normal esophageal mucosa.We analyzed the relationship between TMSG-1 and clinicopathological data of ESCC patients.EC109 cells were treated with 3 μg/mL of cisplatin (CDDP) in vitro for 24 h (the intervention group) and the control group was set up at the same time.The proliferation-inhibitory capability was analyzed with MTT assay.RT-PCR was used to examine the expression of TMSG-1 in the intervention group and the control group.Results The positive rate of TMSG-1 in ESCC and normal esophageal mucosa was 52.2％ (71/136) and 94.6％ (35/37),respectively.The expression of TMSG-1 in ESCC was significantly lower than that in normal esophageal mucosa (P＜0.05).The expression of TMSG-1 was related to TNM stage,differentiation degree and lymph node metastasis (P＜0.05).After EC 109 cells were treated with CDDP for 24 h,the proliferation inhibition rate was increased significantly compared with the control group (P＜0.01).RT-PCR results showed that the expression of TMSG-1 in the cells of the intervention group was significantly higher than that in the control group (P＜ 0.01).Conclusion The abnormal expression of TMSG-1 may play a role in the development and metastasis of ESCC.Examination of TMSG-1 may be useful for making diagnosis and guiding clinical therapy of ESCC.
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Objective To study TMSG-1 and Cyclin D1 expressions in esophageal squamous cell carcinoma (ESCC) and their relevance to the clinicopathological data and prognosis. Methods Immunohistochemistry S-P method was used to examine the expressions of TMSG-1 and Cyclin D1 in pathological specimens of 136 cases of ESCC and 13 cases of normal esophageal mucosa. Contrast study among immunohistochemistry, clinicopathological data and prognosis was also analyzed. Results (1)The positive expression rates of TMSG-1 and Cyclin D1 in ESCC were 52.2% and 65.4%, respectively. The expressions of TMSG-1 and Cyclin D1 in ESCC were significantly higher than that in normal esophageal mucosa (P<0.05). (2)The expressions of TMSG-1 and Cyclin D1 were all related to clinical stage , differentiation degree and lymph node metastasis (P < 0.05). (3)The expression of TMSG-1 was negatively correlated to the expression of Cyclin D1 in ESCC (r=-0.386,P=0.000). (4)The expression levels of TMSG-1 and Cyclin D1 were independent risk factors in patients with ESCC (P < 0.05). Conclusions The abnormal expressions of TMSG-1 and Cyclin D1 may cooperatively play a role in initiation and development of ESCC. Co-examination of TMSG-1 and Cyclin D1 expressions in primary tumors may be useful for predicting prognosis of ESCC.