蛇床子素诱导内质网应激抑制SMMC7721人肝癌细胞增殖的机制研究

Study on Mechanism of Osthole Inhibiting Human SMMC7721 Hepatoma Cell Proliferation Through Triggering Endoplasmic Reticulum Stress

目的研究蛇床子素抑制肝癌细胞增殖的机制。方法采用20-100μM蛇床子素干预SMMC7721人肝癌细胞24 h、48 h和72 h,运用MTT方法检测细胞增殖;采用20-80μM蛇床子素干预SMMC7721肝癌细胞24 h,运用流式细胞术检测细胞凋亡及Western blot检测Bax和Bcl-2蛋白表达;采用60μM蛇床子素干预SMMC7721肝癌细胞48 h,运用流式细胞术检测细胞周期,以实时荧光定量PCR技术检测GRP78、DDIT3、GADD34、ATF4、EIF2A、GDF15、CDKN1B、FOXO3、RICTOR、SGK1基因表达,Western blot方法检测CyclinD1、CyclinE1、p27Kip1和CDK4细胞周期蛋白及GRP78、GRP94、PERK、XBP-1s、CHOP、peIF2α(Ser51)内质网应激蛋白表达。各实验均以0.1%DMSO为对照组,并以0.1μM毒胡萝卜素为内质网应激诱导剂组。结果与对照组比较,100μM蛇床子素干预24 h显著抑制SMMC7721肝癌细胞增殖,其抑制率为36.76%(P<0.05);60-100μM蛇床子素干预48 h后显著抑制SMMC7721肝癌细胞增殖,抑制率为30.84%-52.49%(P<0.05);40-100μM蛇床子素干预72 h后显著抑制SMMC7721肝癌细胞增殖,抑制率为32.7%-73.15%(P<0.05)。大于60μM蛇床子素可诱导SMMC7721肝癌细胞晚期凋亡且显著抑制促凋亡蛋白Bax表达(P<0.05)。60μM蛇床子素干预SMMC7721肝癌细胞48 h后,G1期细胞数增加、G2期和S期减少,且CyclinD1、CyclinE1和p27Kip1蛋白表达显著被抑制(P<0.05)。20-80μM蛇床子素处理SMMC7721肝癌细胞24 h后,均显著促进GRP78、DDIT3(CHOP)、ATF4、FOXO3、RICTOR和SGK1基因表达(P<0.05);20μM蛇床子素显著促进GADD34和EIF2A基因表达(P<0.05);80μM蛇床子素显著促进GDF15基因表达(P<0.05),且CHOP、XBP-1s蛋白表达显著增强。结论蛇床子素通过诱导内质网应激反应以抑制SMMC7721肝癌细胞增殖,从而发挥抗肝癌活性。

Objective To study the mechanism of osthole inhibiting proliferation of hepatoma cells. Methods The human SMMC7721 hepatoma cells were treated with 20-100 μMosthole for 24 h, 48 h and 72 h.Then, the cell proliferation was tested using the MTT assay. The SMMC7721 hepatoma cells were treated with 20-80 μM osthole for24 h. After that, and apoptosis was detected by flow cytometry and Bax and Bcl-2 protein expressions were detected by Western blot. The SMMC7721 hepatoma cells were treated with 60μMosthole for 48 h. And cell cycle was detected by flow cytometry;GRP78, DDIT3, GADD34, ATF4, EIF2 A, GDF15, CDKN1 B, FOXO3, RICTOR and SGK1 mRNA expressions were monitored by quantitative real-time polymerase chain reaction(qRT-PCR);and expressions of cyclinsincluding CyclinD1, CyclinE1, p27 Kip1 and CDK4, as well as expressions of endoplasmic reticulum stress proteins including GRP78, GRP94, PERK, XBP-1 s, CHOP and p-eIF2α(Ser51) were detected by Western blot. In addition,0.1% DMSO is a control group in all the experiments, and 0.1 μM thapsigargin is endoplasmic reticulum stress agonist group.Results Compared with the control group, the cell proliferation was inhibited significantly by 100 μM osthole treatment for 24 h and the inhibition ratio was 36.76%(P < 0.05);the inhibition ratio was from 30.84% to 52.49%with60-100 μM osthole treatment for 48 h(P < 0.05);and the inhibition ratio was from 32.7% to 73.15%with 40-100 μM osthole treatment for 72 h(P < 0.05). The result showed that osthole of more than60 μM can induce late apoptotic of SMMC7721 hepatoma cells and significantly inhibit the Bax protein expression(P < 0.05). The cell number of G1 phase was increased, and those of G2 phase and S phase were decreased in SMMC7721 hepatoma cells treated with 60 μM osthole for 48 h. And CyclinD1, CyclinE1 and p27 Kip1 protein expressions were inhibited significantly(P < 0.05). The GRP78, DDIT3(CHOP), ATF4, FOXO3, RICTOR and SGK1 mRNA expressionsin SMMC7721 hepatoma cells were increased significantly by 20-80 μM osthole treatment for 24 h(P < 0.05);the GADD34 and EIF2 A mRNA expressions were increased significantly by 20 μM osthole(P < 0.05);and the GDF15 mRNA expression was increased significantly by 80 μM osthole(P < 0.05);moreover, CHOP and XBP-1 s protein expressions were significantly increased.Conclusions Osthole can inhibit the proliferation of SMMC7721 hepatoma cells through triggering endoplasmic reticulum stress, thereby exerting anti-hepatomaactivity.