内质网应激对肝癌细胞硼替佐米耐药性的影响

Effect of endoplasmic reticulum stress on the resistance of hepatocellular carcinoma cells to bortezomib

目的探讨内质网应激对肝癌细胞硼替佐米耐药性的影响。方法将对数生长期HepG2和HuH7细胞分别分为0、5、10、15、20、30 nmol·L^(-1)硼替佐米组,采用细胞计数试剂盒(CCK-8)检测硼替佐米干预24 h时各组细胞存活率及干预24、48、72、96 h时细胞增殖率。将对数生长期HepG2和HuH7细胞分别分为0、10、15 nmol·L^(-1)硼替佐米组,采用Western blot法检测HepG2和HuH7细胞中凋亡相关蛋白切割片段的半胱氨酸蛋白酶-3(cleaved caspase-3)和内质网应激相关蛋白葡萄糖调节蛋白78(GRP78)、磷酸化真核翻译起始因子2α(p-eIF-2α)、激活转录因子4(ATF4)的表达。构建对硼替佐米耐药的HepG2细胞株(HepG2/Bort细胞),通过抑制生长曲线计算细胞的半抑制浓度(IC 50),并计算细胞耐药指数(RI)。将HepG2/Bort细胞分为对照组、硼替佐米组(给予15 nmol·L^(-1)硼替佐米干预)、硼替佐米+4-苯基丁酸(4-PBA)组(给予15 nmol·L^(-1)硼替佐米和8 mmol·L^(-1)4-PBA干预)、硼替佐米+牛磺脱氧胆酸(TUDCA)组(给予15 nmol·L^(-1)硼替佐米和1 mmol·L^(-1)TUDCA干预),药物作用24 h后,采用CCK-8法检测HepG2/Bort细胞存活率,采用Western blot法检测HepG2/Bort细胞中凋亡相关蛋白cleaved caspase-3和内质网应激相关蛋白GRP78、p-eIF-2α、ATF4的表达。结果硼替佐米干预24 h后,5、10、15、20、30 nmol·L^(-1)硼替佐米组HepG2和HuH7细胞存活率均显著低于0 nmol·L^(-1)硼替佐米组(P<0.05)。硼替佐米干预0、24 h时,5 nmol·L^(-1)硼替佐米组与0 nmol·L^(-1)硼替佐米组HepG2和HuH7细胞增殖率比较差异无统计学意义(P>0.05);硼替佐米干预48、72、96 h时,5 nmol·L^(-1)硼替佐米组HepG2和HuH7细胞增殖率显著低于0 nmol·L^(-1)硼替佐米组(P<0.05)。干预24 h后,10、15 nmol·L^(-1)硼替佐米组HepG2和HuH7细胞内cleaved caspase-3表达显著高于0 nmol·L^(-1)硼替佐米组(P<0.05)。HepG2/Bort细胞和HepG2细胞对硼替佐米的IC 50分别为122.36、13.23 nmol·L^(-1),HepG2/Bort细胞对硼替佐米的RI为9.25。HepG2/Bort细胞中GRP78、p-eIF-2α、ATF4蛋白表达量显著高于HepG2细胞(P<0.05)。硼替佐米组HepG2/Bort细胞存活率显著低于对照组(P<0.05),硼替佐米+4-PBA组和硼替佐米+TUDCA组HepG2/Bort细胞存活率显著低于硼替佐米组(P<0.05)。硼替佐米组HepG2/Bort细胞中cleaved caspase-3相对表达量显著高于对照组(P<0.05),硼替佐米+4-PBA组和硼替佐米+TUDCA组HepG2/Bort细胞中cleaved caspase-3相对表达量显著高于硼替佐米组(P<0.05)。结论硼替佐米可以抑制肝癌细胞增殖,并诱导其凋亡;内质网应激抑制剂可以提高HepG2/Bort细胞对硼替佐米的敏感性,本研究为临床克服肝癌耐药提供了新的治疗策略。

Objective To investigate the effect of endoplasmic reticulum stress on the resistance of hepatocellular carcinoma cells to bortezomib.Methods The HepG2 and Huh7 cells in logarithmic growth phase were divided into 0,5,10,15,20 and 30 nmol·L^(-1) bortezomib groups.The cell survival rate after 24 hours of bortezomib intervention and the cell proliferation rate after 24,48,72 and 96 hours of bortezomib intervention were detected by cell counting kit-8(CCK-8).The HepG2 and Huh7 cells in logarithmic growth phase were divided into 0,10 and 15 nmol·L^(-1) bortezomib groups.The expressions of apoptosis related protein cleaved cysteinyl aspartate specific proteinase(cleaved caspase-3)and endoplasmic reticulum stress related proteins glucose regulated protein 78(GRP78),phosphorylated eukaryotic initiation factor-2α(p-eIF-2α),activating transcription factor 4(ATF4)in HepG2 and Huh7 cells were detected by Western blot method.The bortezomib resistant HepG2 cell line(HepG2/Bort cells)were constructed,the 50%inhibiting concentration(IC 50)and the resistance index(RI)were calculated by inhibition growth curve.The HepG2/Bort cells were divided into control group,bortezomib group(The cells were intervened with 15 nmol·L^(-1) bortezomib),bortezomib+4-phenylbutyric acid(4-PBA)group(The cells were intervened with 15 nmol·L^(-1) bortezomib and 8 mmol·L^(-1)4-PBA),bortezomib+tauroursodeoxy cholic acid(TUDCA)group(The cells were intervened with 15 nmol·L^(-1) bortezomib and 1 mmol·L^(-1) TUDCA).After 24 hours of drug treatment,the survival rate of HepG2/Bort cells was detected by CCK-8,and the expression of apoptosis related protein cleaved caspase-3 and endoplasmic reticulum stress-related protein GRP78,p-eIF-2αand ATF4 in HepG2/Bort cells were detected by Western blot method.Results The survival rates of HepG2 and Huh7 cells in 5,10,15,20 and 30 nmol·L^(-1) bortezomib group were significantly lower than those in 0 nmol·L^(-1) bortezomib group after 24 hours of bortezomib intervention(P<0.05).There was no significant difference in the proliferation rate of HepG2 and Huh7 cells between 0 nmol·L^(-1) bortezomib group and 5 nmol·L^(-1) bortezomib group after 0 and 24 hours of bortezomib intervention(P>0.05).After 48,72 and 96 hours of bortezomib intervention,the proliferation rates of HepG2 and Huh7 cells in 5 nmol·L^(-1) bortezomib group were significantly lower than those in 0 nmol·L^(-1) bortezomib group(P<0.05).After 24 hours of intervention,the expression of cleaved caspase-3 in HepG2 and Huh7 cells in 10 and 15 nmol·L^(-1) bortezomib groups was significantly higher than that in 0 nmol·L^(-1) bortezomib group(P<0.05).The IC 50 of HepG2/Bort cells and HepG2 cells to bortezomib was 122.36 and 13.23 nmol·L^(-1),respectively,and the RI of HepG2/Bort cells to bortezomib was 9.25.The expressions of GRP78,p-eIF-2αand ATF4 protein in HepG2/Bort cells were significantly higher than those in HepG2 cells(P<0.05).The survival rate of HepG2/Bort cells in the bortezomib group was significantly lower than that in the control group(P<0.05),and the survival rate of HepG2/Bort cells in the bortezomib+4-PBA group and bortezomib+TUDCA group was significantly lower than that in the bortezomib group(P<0.05).The relative expression of cleaved caspase-3 in HepG2/Bort cells in the bortezomib group was significantly higher than that in the control group(P<0.05),and the relative expression of cleaved caspase-3 in HepG2/Bort cells in the bortezomib+4-PBA group and bortezomib+TUDCA group was significantly higher than that in the bortezomib group(P<0.05).Conclusion Bortezomib can inhibit the proliferation and induce the apoptosis of hepatoma carcinoma cells.Endoplasmic reticulum stress inhibitor can improve the sensitivity of HepG2/Bort cells to bortezomib.This study provides a new treatment strategy for overcoming drug resistance of liver cancer.