蛋白酶体抑制剂对肝癌HepG2细胞BAG3表达影响及其机制探讨

Influence of proteasome inhibitors on the expression of BAG3 in HepG2 cells and its mechanism

目的:探讨4种蛋白酶体抑制剂硼替佐米(bortezome,BZ)、环氧甲酮四肽(epoxomycin,Epox)、乳孢素(lactacystin,Lacta)和MG132单独作用或者联合JNK特异性抑制剂SP600125,对肝癌HepG2细胞内BAG3蛋白表达的影响及分子机制。方法:空白对照、100nmol/L BZ、100nmol/L Epox、500nmol/L Lacta和2μmol/L MG132单独或联合50μmol/L SP600125处理HepG2细胞;实时定量RT-PCR检测蛋白酶体抑制剂对肝癌HepG2细胞内BAG3基因mRNA表达水平影响,蛋白质印迹法检测蛋白酶体抑制剂单独或者联合JNK激酶特异性抑制剂对BAG3蛋白表达水平的影响;利用MTT试剂盒检测蛋白酶体抑制剂细胞存活率,Hoechest33258染色检测细胞凋亡率。结果:蛋白酶体抑制剂BZ、Epox、Lacta和MG132均不同程度上调BAG3基因的mRNA和蛋白表达水平,而SP600125显著抑制蛋白酶体抑制剂引起的BAG3表达上调。MTT结果显示,SP600125显著抑制HepG2细胞对蛋白酶体抑制剂的敏感性;进一步Hoechst33258染色结果显示,4种蛋白酶体抑制剂联合SP600125作用引起HepG2细胞的凋亡率分别为(49.2±3.2)%、(58.7±3.5)%、(56.8±3.4)%和(53.4±3.3)%,明显高于蛋白酶体抑制剂单独作用组的(7.2±2.8)%、(16.7±3.2)%、(12.3±2.9)%和(11.4±3.0)%,P<0.05。结论:蛋白酶体抑制剂通过JNK信号通路上调BAG3的表达,抑制JNK活性增加了HepG2细胞对蛋白酶体抑制剂的敏感性。

OBJECTIVE： To investigate the effect of Bortezome,epoxomycin,lactacystin and MG132 on the expres- sion of BAG3 in HepG2 cells and the role of JNK pathway. METHODS： HepG2 cells were treated with vehicle or 100 nmol/L Bortezome, 100 nmol/L epoxomycin, 500 nmol/L lactacystin, 2 gmol/L MG132 alone or pretreated with 50 μmol/L SP600125. The mRNA expression of BAG3 gene was determined by RT-PCR, and the protein level of BAG3 was determined by western blot. The survival ratio was measured by MTT assay, Hoechst33258 staining and fluorescence microscope. RESULTS： Proteasome inhibitors BZ, Epox, Lacta and MG132 can up-regulate the mRNA and protein level of BAG3, yet SP600125 can inhibit the level of BAG3 induced by proteasome inhibitors. The results of MTT demonstrated that SP600125 can inhibit the cell sensitivity to proteasome inhibitors of HepG2 cells. And subsequently the results of Hoechst 33258 staining proved that different proteasome inhibitors combined with SP600125 could respectively induce apoptosis with the apoptotic rate of （49.2 ±3.2） %, （ 58.7 ± 3.5） %, （56.8 ± 3.4）%, （ 53.4 ± 3.3） %, which had significantly higher effect in apoptosis than proteasome inhibitors alone with the apoptotic rate of （7.2 ±2.8） %, （16.7± 3.2）%,（12.3±2.9） %, （11.4±3.0）% （P〈0.05）. CONCLUSION： JNK signals play an important role in the upregulation of BAG3 induced by proteasome inhibition in HepG2 cells and inhibition of JNK can enhance the sensitivity of HepG2 cells with proteasome inhibitors.