三氧化二砷逆转低浓度索拉非尼对人肝癌细胞促迁移作用及其机制

Inhibitory effect and mechanism of arsenic trioxide on human hepatocellular carcinoma cells migration induced by low dose of sorafenib

目的探讨三氧化二砷逆转低浓度索拉非尼对人肝癌细胞促进迁移作用及其机制。方法取对数生长期的人肝癌细胞MHCC97H,分别以三氧化二砷2μmol/L(三氧化二砷组)、索拉非尼3μmol/L(索拉非尼组)、两者联合(联合组)、LY294002 50μmol/L(LY组)及索拉非尼3μmol/L+LY294002 50μmol/L(LY+索拉非尼组)作用于细胞,并以仅含二甲基亚砜(DMSO)作为对照组。细胞划痕实验和Transwell迁移实验分别检测细胞横向和纵向迁移能力。Western blot实验检测p-Akt、E-cadherin、Vimentin、Snail蛋白表达。实验数据比较采用单因素方差分析和Bonferroni法。结果细胞划痕实验检测索拉非尼、三氧化二砷、联合组细胞横向迁移速率分别为对照组的(1.59±0.14)、(0.39±0.08)、(0.58±0.12)倍(t=7.20,-12.58,-6.62;P<0.05)。Transwell迁移实验检测索拉非尼、三氧化二砷、联合组及对照组细胞数分别为(285±26)、(169±18)、(194±19)、(228±9)个。与对照组相比,索拉非尼组细胞数明显增多(t=3.48,P<0.05),三氧化二砷组明显减少(t=-3.80,P<0.05),且联合组较索拉非尼组明显减少(t=-5.67,P<0.05)。Western blot检测显示,与对照组比,索拉非尼组p-Akt、Snail、Vimentin的表达增强,E-cadherin的表达减弱。三氧化二砷、联合组、LY组及LY+索拉非尼组p-Akt、Snail、Vimentin的表达减弱,E-cadherin的表达增强。结论三氧化二砷可通过抑制PI3K/Akt/Snail通路的激活,阻止人肝癌细胞上皮间质化,逆转低浓度索拉非尼对人肝癌细胞促迁移作用。

Objective To investigate the inhibitory effect and mechanism of arsenic trioxide on hepatocellular carcinoma (HCC) cells migration induced by low dose of sorafenib. Methods Human HCC cells MHCC97H in logarithmic phase were treated with 2μmol/L arsenic trioxide (arsenic trioxide group), 3μmol/L sorafenib (sorafenib group), 2μmol/L arsenic trioxide+3μmol/L sorafenib (combination group), 50 μmol/L LY294002(LY group) and 3 μmol/L sorafenib + 50 μmol/L LY294002 (LY+ sorafenib group) respectively. Dimethyl sulfoxide (DMSO) was used in the control group. Wound healing assay and Transwell migration assay were used to detect the ability of horizontal and vertical cell migration. The expression of p-Akt, E-cadherin, Vimentin and Snail proteins was measured by Western blot. The experiment data were compared using one-way ANOVA and Bonferroni test. Results Wound healing assay revealed that the horizontal migration speed in the sorafenib, arsenic trioxide and combination groups was (1.59±0.14), (0.39±0.08) and (0.58±0.12) times of that in the control group (t=7.20,-12.58,-6.62;P<0.05). Transwell migration assay revealed that the number of cells in the sorafenib, arsenic trioxide, combination and control groups was 285±26, 169±18, 194±19 and 228±9 respectively. Compared with the control group, the number of cells was signiifcantly increased in the sorafenib group (t=3.48, P<0.05), whereas signiifcantly decreased in the arsenic trioxide group (t=-3.80, P<0.05). The number of cells in the combination group was signiifcantly decreased than that in the sorafenib group (t=-5.67, P<0.05). Western blot revealed that the expression of p-Akt, Snail and Vimentin proteins was up-regulated, whereas the expression of E-cadherin protein was down-regulated in the sorafenib group compared with those in the control group. Compared with the control group, the expression of p-Akt, Snail and Vimentin proteins was down-regulated whereas the expression of E-cadherin protein was up-regulated in the arsenic trioxide, combination, LY and LY+sorafenib groups. Conclusion Arsenic trioxide can inhibit the epithelial-mesenchymal transition and reverse the promoting effect of low-dose sorafenib upon MHCC97H cell migration through suppressing the activation of PI3K/Akt/Snail signaling pathway.