褪黑素通过cAMP-PKA信号转导通路抑制黑色素瘤细胞VEGF表达

Melatonin inhibited the expression of vascular endothelial growth factor (VEGF) by blockage of cAMP-PKA pathway in melanoma cells

目的探讨褪黑素对人黑色素瘤细胞A375 VEGF表达的影响及其可能涉及的信号转导通路。方法用褪黑素、褪黑素受体拮抗剂、forskolin+褪黑素、8-brom-camp+褪黑素处理A375细胞,Real-time PCR、Western blot、ELISA等检测VEGF mRNA、蛋白表达的变化。结果 10μmol/L褪黑素处理A375细胞24 h后,VEGF mRNA的表达水平下降了48.7%(P<0.01)、细胞培养上清中VEGF蛋白表达水平下降了24.3%(P<0.01);10μmol/L褪黑素处理A375细胞6、12和24 h后,胞浆中VEGF蛋白的表达水平分别下降了14.2%、27.3%和41.8%(P<0.01);10μmol/L褪黑素受体拮抗剂处理A375细胞24 h后,胞浆、细胞培养上清中VEGF蛋白表达水平与对照组相比分别下降了32.3%和20.1%(P<0.01);30μmol/L PKA激动剂(8-brom-camp)、500μmol/L腺苷酸环化酶激动剂(forskolin)处理A375细胞24 h后,胞浆VEGF的表达水平分别是对照组的1.58和2.24倍,细胞培养上清中VEGF的表达水平分别为对照组的1.65和2.39,与褪黑素组相比均差异有显著性(P<0.01)。结论褪黑素可下调A375细胞VEGF的表达,其作用机制可能是通过褪黑素受体阻断cAMP-PKA信号转导通路所致。

[ Objective ] To explore the effect of melatonin on VEGF expression in human melanoma cells A375 and the possible mechanisms of signal transduction pathways. [Methods] A375 cells were treated with melatonin, melatonin receptor inhibitors, forskolin＋MT, 8-brom-camp＋PGE2. VEGF mRNA and protein expression were exam- ined by Real-time PCR, Western blot and ELISA in A375 cells. [ Results ] The expression of VEGF mRNA in A375 cells were decreased by 48.7% （P 〈0.01）, and the expression of VEGF in culture supernatant of A375 cells were de- creased by 24.0% （P 〈0.01） after treated with MT （10/xmol//L） for 24 h. The expression of VEGF in cytoplasm of A375 cells were decreased by 14.2%,27.3%,41.8% （P 〈0.01） after treated with MT （10lxmol/L） for 6 h, 12 h, 24 h respectively. The expression of VEGF in cytoplasm and culture supernatant of A375 cells were decreased by 32.3%, 20.1% （P 〈0.01） after treated with melatonin receptor inhibitors（101xmol/L） for 24 h respectively. After the treatment of 8-brom-camp （301xmol/L） and forskolin （5001xmol/L） for 24 h, the expression of VEGF in cytoplasm of A375 cells was increased by 58.0% and 24.0%, and the expression of VEGF in culture supernatant of A375 cells was increased by 65% and 39%, respectively, vs MT （P 〈0.01）. [ Conclusion] It suggested that MT might down-regulate the ex- pression of VEGF through MT receptor of A375 cells, which could be partly related to the cAMP-PKA signal con-duction pathway.