低氘水对人黑色素瘤A375细胞增殖、迁移、凋亡的调控作用及其机制探讨

Effects of deuterium-depleted water on proliferation,migration,and apoptosis of human melanoma A375 cells

目的观察低氘水(DDW)对人黑色素瘤A375细胞增殖、迁移、凋亡的调控作用,并探讨相关机制。方法A375细胞与人角质形成细胞HaCaT分别用氘体积分数为0.0025%、0.0050%、0.0100%的DDW和超纯水(氘体积分数0.0150%)配成的高糖DMEM培养基培养。采用细胞增殖实验和平板克隆形成实验检测A375细胞和Ha⁃CaT细胞的增殖能力,采用划痕实验检测两种细胞迁移能力,DAPI染色观察两种细胞形态变化。采用流式细胞仪检测A375细胞凋亡和细胞周期分布情况,ATP酶测试盒检测A375细胞Na^(+)-K^(+)-ATP酶、Ca^(2+)-Mg^(2+)-ATP酶、总ATP酶活性,采用Westernblotting法检测A375细胞中的凋亡相关蛋白(Bcl-2、Bax)及Caspase通路蛋白(Caspase-3、cleaved Caspase-3、Caspase-9、cleavedCaspase-9、PARP、cleavedPARP)。结果与超纯水处理相比,0.0100%、0.0050%、0.0025%氘体积分数的DDW处理的A375细胞增殖抑制率依次增高,克隆形成率和细胞迁移率依次降低(P均<0.05);DDW处理的A375细胞出现核染色质固缩、凝聚、核碎裂等现象,细胞核中凋亡小体增多。DDW对HaCat细胞的增殖能力、集落形成能力、细胞迁移能力及细胞形态没有明显影响。与超纯水处理相比,0.0100%、0.0050%、0.0025%氘体积分数的DDW处理的A375细胞凋亡率及G_(0)/G_(1)期细胞比例依次增高,G_(2)/M期细胞比例依次降低(P均<0.05)。与超纯水处理相比,0.0100%、0.0050%、0.0025%氘体积分数的DDW处理的A375细胞Na^(+)-K^(+)-ATP酶、Ca^(2+)-Mg^(2+)-ATP酶、总ATP酶活性及Bcl-2、Caspase-9、PARP蛋白表达依次降低,Bax、cleaved Caspase-3/9、cleavedPARP蛋白表达依次增高(P均<0.05)。结论低氘水能选择性地抑制A375细胞增殖、迁移并诱导细胞凋亡,呈剂量依赖性,而对人角质形成细胞无明显影响;低氘水的作用机制可能与抑制ATP酶活性、激活Caspase信号通路有关。

Objective To investigate the effects of deuterium-depleted water(DDW)on proliferation,migration, and apoptosis of human melanoma A375 cells and the possible molecular mechanism. Methods A375 cells and keratinocytes(HaCaT cells)were treated with high-glucose DMEM media containing DDW with deuterium volume fraction of 0. 002 5%,0. 005 0% and 0. 010 0% and ultra pure water(deuterium volume fraction is 0. 015 0%). The proliferation of A375 cells and HaCaT cells was detected by cell proliferation experiment and plate clone formation experiment. Scratch assay was used to detect the migration ability of A375 cells and HaCaT cells;DAPI staining was used to observe the morphological characteristics of cells;flow cytometry was used to detect the apoptosis and cell cycle distribution of A375 cells. The ATPase test kit was used to detect the activities of Na-+K^(+)-ATPasee,Ca^(2+)-Mg^(2+)-ATPase and total ATPase in A375 cells. Western blotting was used to detect apoptosis-related proteins(Bcl-2,Bax)and Caspase pathway proteins(Caspase- 3,cleaved Caspase-3,Caspase-9,cleaved Caspase-9,PARP,and cleaved PARP)in A375 cells. Results Compared with ultra pure water treatment,the proliferation inhibition rate of A375 cells treated with DDW with deuterium volume fraction of 0. 010 0%,0. 005 0%,and 0. 002 5% increased sequentially,while the colony formation rate and cell migration rate decreased sequentially(all P<0. 05). DDW-treated A375 cells showed nuclear chromatin pyknosis,condensation and nuclear fragmentation,and the number of apoptotic bodies in the nucleus increased. DDW had no significant effect on proliferation,colony formation,migration and cell morphology of HaCat cells. Compared with ultra pure water treatment,DDW treatment with deuterium volume fraction of 0. 010 0%,0. 005 0%,and 0. 002 5% increased the apoptosis rate and the proportion of cells in the G_(0)/G_(1) phase,but decreased the proportion of cells in the G_(2)/M phase of A375 cells(all P<0. 05). Compared with ultra pure water treatment,the activities of Na^(+)-K^(+)-ATPase,Ca^(2+)-Mg^(2+)-ATPase and T-ATPase and the protein expression levels of Bcl- 2,Caspase-9 and PARP of A375 cells treated by DDW with deuterium volume fraction of 0. 010 0%,0. 005 0%,and 0. 002 5%;Bax,Cleaved Caspase-3/9,and Cleaved PARP protein expression levels increased sequentially(all P<0. 05). Conclusions DDW can selectively inhibit the proliferation,migration and induce apoptosis of A375 cells,but has no significant effect on HaCaT cells. The mechanism of DDW may be related to the inhibition of ATPase activity and activation of Caspase signaling pathway.