[LASS2 interacts with V-ATPase and inhibits cell growth of hepatocellular carcinoma].
Homo sapiens longevity assurance homologue 2 (LASS2) is a novel gene isolated from a human liver cDNA library by our laboratory, and it is a human homologue of the yeast longevity assurance gene LAG1 (Saccharomyces cerevisiae longevity assurance gene). According to our previous results, LASS2 could interact with subunit c of vacuolar type H(+)-ATPase (V-ATPase), and the overexpression of LASS2 could inhibit the cell growth of a human hepatocellular carcinoma (HCC) cell line, SMMC-7721. In order to understand the role of the interaction between LASS2 and V-ATPase in HCC cell growth, we transiently transfected plasmid pCMV-HA2-LASS2 into HCCLM3, a HCC cell line without the significant expression of endogenous LASS2. The pH-sensitive fluorescence probes, BCECF and BCECF-AM, were used to measure the intracellular and extracellular H(+) concentrations of HCCLM3 cells respectively. The effect of LASS2 gene on apoptosis was evaluated with Annexin-V/FITC and propidium iodide (PI) by flow cytometry. Western blot was used to detect cytochrome c (Cyt c) in the cytosol and mitochondria, as well as pro-caspase-3 in cytosol. The results showed that the cell growth of LASS2-transfected HCCLM3 cells was significantly inhibited compared with that of the mock control. LASS2 transfection increased intracellular H(+) concentration of HCCLM3 cells, while decreased extracellular H(+) concentration. Moreover, LASS2 transfection significantly enhanced the apoptosis of HCCLM3 cells. In LASS2-transfected cells, the amounts of Cyt c increased in the cytosol, while decreased in the mitochondria. Meanwhile, the expression of pro-caspase-3 in the cytosolic extracts was decreased. These results implicate that LASS2 gene might increase intracellular H(+) of HCC cells via the interaction with V-ATPase, thereby inducing cell apoptosis through mitochondrial pathway.
Tang
Ning
N
Shanghai Medical College, Fudan University, Shanghai, China.
Jin
Jie
J
Deng
Yun
Y
Ke
Rong-Hu
RH
Shen
Qiu-Jin
QJ
Fan
Shao-Hua
SH
Qin
Wen-Xin
WX
D017209:Apoptosis; D006528:Carcinoma, Hepatocellular / Q000473:pathology*; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D006801:Humans; D008113:Liver Neoplasms / Q000473:pathology*; D008565:Membrane Proteins / Q000378:metabolism*; D034741:RNA, Small Interfering; D051049:Sphingosine N-Acyltransferase / Q000378:metabolism*; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*
D008565:Membrane Proteins; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; C438311:CERS2 protein, human; D051049:Sphingosine N-Acyltransferase; C505407:CASP3 protein, human; D053148:Caspase 3; D025262:Vacuolar Proton-Translocating ATPases