Wang Yong-Zhong, Feng Zhen-Qing
Department of Pathology, Nanjing Medical University, Nanjing 210029, Jiangsu, China.
Biomed Environ Sci. 2006 Aug;19(4):285-91.
To investigate the apoptosis-inducing effect of endogenous nitric oxide (NO) suppression in gastric cancer cells and its mechanisms.
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.
Gastric cancer cells were significantly apoptotic after treatment with N(G)-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-253 phosphorylation. 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.
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.
探讨内源性一氧化氮（NO）抑制对胃癌细胞凋亡的诱导作用及其机制。
采用流式细胞术检测胃癌细胞凋亡情况。运用蛋白质免疫印迹法分析胃癌细胞中磷酸化FKHRL1（苏氨酸-32、丝氨酸-253）和FKHRL1的表达。进行免疫荧光分析以定位细胞内磷酸化FKHRL1（苏氨酸-32、丝氨酸-253）和FKHRL1。通过脂质体转染试剂转染FKHRL1-HA野生型和突变型FKHRL1-HA T32A构建体。采用格里斯反应测定NO生成量。
与对照组相比，用N(G)-单甲基-L-精氨酸（L-NMMA，一种一氧化氮合酶抑制剂）处理后胃癌细胞凋亡显著（P<0.01）。L-NMMA诱导的胃癌细胞凋亡呈剂量依赖性且与时间无关。然而，半胱天冬酶-3、6、7、8、10抑制剂Z-DEVD-fmk并不能阻止细胞凋亡。免疫荧光分析显示，当L-NMMA阻断内源性一氧化氮生成时，FKHRL1蛋白在SGC-7901细胞核中强烈表达，而磷酸化FKHRL1苏氨酸-32蛋白在细胞质中强烈表达，但FKHRL1丝氨酸-253磷酸化无变化。尽管如此，ROCK蛋白在磷酸化FKHRL1苏氨酸-32阳性的SGC-7901细胞中强烈表达。磷酸肌醇-3-羟基激酶（PI3K）抑制剂渥曼青霉素不能阻断L-NMMA诱导的磷酸化FKHRL1苏氨酸-32蛋白表达。然而，蛋白激酶ROCK的特异性抑制剂Y-27632显著阻断了由磷酸化FKHRL1苏氨酸-32介导的L-NMMA诱导的细胞凋亡（P<0.01）。当转染FKHRL1-HA野生型细胞导致FKHRL1苏氨酸-32磷酸化增加时，观察到NO生成量显著减少（P<0.01），细胞凋亡显著增加（P<0.01）。
L-NMMA可能通过促进FKHRL1苏氨酸-32磷酸化并启动FKHRL1至ROCK激酶的信号传导来触发胃癌细胞凋亡。这种凋亡信号传导过程不依赖于PI3K/Akt以及半胱天冬酶-3。
