Volume 19 Issue 4
Nov.  2019
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YONG-ZHONG WANG, ZHEN-QING FENG. Induction of Apoptosis by L-NMMA, via FKHRL1/ROCK Pathway in Human Gastric Cancer Cells[J]. Biomedical and Environmental Sciences, 2006, 19(4): 285-291.
Citation: YONG-ZHONG WANG, ZHEN-QING FENG. Induction of Apoptosis by L-NMMA, via FKHRL1/ROCK Pathway in Human Gastric Cancer Cells[J]. Biomedical and Environmental Sciences, 2006, 19(4): 285-291.

Induction of Apoptosis by L-NMMA, via FKHRL1/ROCK Pathway in Human Gastric Cancer Cells

  • To investigate the apoptosis-inducing effect of endogenous nitric oxide (NO) suppression in gastric cancer cells and its mechanisms. Methods Apoptosis of gastric cancer cells was detected by flow cytometry. Expression of phosphorylated FKHRL1 (thr-32, ser-253) and FKHRL1 in gastric cancer cells was analyzed using Western blotting.Immunofluorescence assay was performed to localize the intracellular phosphorylated FKHRL1 (thr-32, ser-253) and FKHRL1.Transfection of FKHRL1-HA wild type and mutant FKHRL1-HA T32A constructs was performed by lipofectamine plus reagent. NO generation was determined by Griess reaction. Results Gastric cancer cells were significantly apoptotic after treatment with NG-monomethyl-L-arginine (L-NMMA, a nitric oxide synthase inhibitor), compared with the control (P<0.01).The apoptosis of gastric cancer cells induced by L-NMMA was dose-dependent and time-independent. However, the Z-DEVD-fmk, a caspase-3, 6, 7, 8, 10 inhibitor, did not prevent the apoptosis. The immunofluorescence assays showed that FKHRL1 protein was strongly expressed in the nucleu and p-FKHRL1 thr-32 protein was strongly expressed in the cytoplasm of SGC-7901 cells when endogenous nitric oxide generation was blocked by L-NMMA, but no change in FKHRL1 ser-253phosphorylation. Nevertheless, ROCK protein was strongly expressed in p-FKHRL1 thr-32-positive SGC-7901 cells. The wortmannin, an inhibitor of phosphoinositol-3-OH kinase (PI3K), did not block the phosphorylated FKHRL1 thr-32 protein induced by L-NMMA. However, Y-27632, a specific inhibitor of the protein kinase ROCK, significantly blocked apoptosis induced by phosphorylated FKHRL1 thr-32 (P<0.01), which was mediated by L-NMMA. A significant decrease in NO generation (P<0.01) and a significant increase in apoptosis (P<0.01) were observed when FKHRL1-HA wild-type cells were transfected, which caused increased FKHRL1 thr-32 phosphorylation. Conclusions L-NMMA triggers gastric carcinoma cell apoptosis, possibly by promoting FKHRL1 thr-32 phosphorylation and initiating signal of FKHRL1 to ROCK kinase. This apoptotic signaling process is PI3K/Akt as well as caspase-3 independent.
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Induction of Apoptosis by L-NMMA, via FKHRL1/ROCK Pathway in Human Gastric Cancer Cells

Abstract: To investigate the apoptosis-inducing effect of endogenous nitric oxide (NO) suppression in gastric cancer cells and its mechanisms. Methods Apoptosis of gastric cancer cells was detected by flow cytometry. Expression of phosphorylated FKHRL1 (thr-32, ser-253) and FKHRL1 in gastric cancer cells was analyzed using Western blotting.Immunofluorescence assay was performed to localize the intracellular phosphorylated FKHRL1 (thr-32, ser-253) and FKHRL1.Transfection of FKHRL1-HA wild type and mutant FKHRL1-HA T32A constructs was performed by lipofectamine plus reagent. NO generation was determined by Griess reaction. Results Gastric cancer cells were significantly apoptotic after treatment with NG-monomethyl-L-arginine (L-NMMA, a nitric oxide synthase inhibitor), compared with the control (P<0.01).The apoptosis of gastric cancer cells induced by L-NMMA was dose-dependent and time-independent. However, the Z-DEVD-fmk, a caspase-3, 6, 7, 8, 10 inhibitor, did not prevent the apoptosis. The immunofluorescence assays showed that FKHRL1 protein was strongly expressed in the nucleu and p-FKHRL1 thr-32 protein was strongly expressed in the cytoplasm of SGC-7901 cells when endogenous nitric oxide generation was blocked by L-NMMA, but no change in FKHRL1 ser-253phosphorylation. Nevertheless, ROCK protein was strongly expressed in p-FKHRL1 thr-32-positive SGC-7901 cells. The wortmannin, an inhibitor of phosphoinositol-3-OH kinase (PI3K), did not block the phosphorylated FKHRL1 thr-32 protein induced by L-NMMA. However, Y-27632, a specific inhibitor of the protein kinase ROCK, significantly blocked apoptosis induced by phosphorylated FKHRL1 thr-32 (P<0.01), which was mediated by L-NMMA. A significant decrease in NO generation (P<0.01) and a significant increase in apoptosis (P<0.01) were observed when FKHRL1-HA wild-type cells were transfected, which caused increased FKHRL1 thr-32 phosphorylation. Conclusions L-NMMA triggers gastric carcinoma cell apoptosis, possibly by promoting FKHRL1 thr-32 phosphorylation and initiating signal of FKHRL1 to ROCK kinase. This apoptotic signaling process is PI3K/Akt as well as caspase-3 independent.

YONG-ZHONG WANG, ZHEN-QING FENG. Induction of Apoptosis by L-NMMA, via FKHRL1/ROCK Pathway in Human Gastric Cancer Cells[J]. Biomedical and Environmental Sciences, 2006, 19(4): 285-291.
Citation: YONG-ZHONG WANG, ZHEN-QING FENG. Induction of Apoptosis by L-NMMA, via FKHRL1/ROCK Pathway in Human Gastric Cancer Cells[J]. Biomedical and Environmental Sciences, 2006, 19(4): 285-291.

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