热休克诱导人肝癌HepG2细胞HSP70和P-gp的表达及槲皮素对二者的抑制作用

背景与目的:研究提示热休克蛋白70(heatshockprotein70,HSP70)可抑制细胞凋亡,降低肿瘤热疗、化疗的疗效,但其与细胞化疗耐受性的内在关系尚不甚明确;P-糖蛋白(P-glycoprotein,P-gp)是一种耐药蛋白,在肿瘤耐药中起重要作用。本研究拟通过热休克诱导人肝癌HepG2细胞表达HSP70和P-gp,并观察槲皮素(quercetin,Qu)对二者表达的抑制效应。方法:恒温水浴法,42℃热休克HepG2细胞90min,分别采用免疫组化、流式细胞术检测热休克前及热休克后2h、4h、8h、12h、24h时HSP70和P-gp的表达情况;并于热休克前1h应用不同浓度槲皮素作用于HepG2细胞,观察其热休克后4h二者的表达。结果:(1)热休克诱导后,免疫组化显示细胞内HSP70表达增多,“核内迁移”;流式细胞术示HSP70阳性细胞数升高,4h达到最高(11.47%),较对照组(6.64%)升高近一倍(P<0.01),24h后回落到低值;P-gp阳性细胞数随HSP70升高后升高,12h达到最高值(96.31%),较对照组(33.95%)升高近两倍(P<0.01),24h后回落到低值。(2)热休克前应用槲皮素能有效抑制热休克诱导HSP70和P-gp的过量表达,以100～200μmol/L槲皮素作用明显(P<0.01),呈浓度依赖性。结论:热休克可诱导HepG2细胞HSP70和P-gp的过量表达,P-gp的表达可能与HSP70的表达有关;槲皮素可阻断热休克诱导HepG2细胞HSP7

Expression of HSP70 and P-glycoprotein (P-gp) in human hepatocarcinoma HepG2 cells induced by heat shock and inhibiting effect of quercetin on them

BACKGROUND & OBJECTIVE: Heat shock protein 70 (HSP70) has been thought to inhibit apoptosis and reduce the effectiveness of hyperthermal therapy and chemotherapy on cancer. However, the relationship between HSP70 and the drug resistance to chemotherapy has not been definited. P-glycoprotein (P-gp) was a kind of protein that could decrease the effectiveness of chemotherapy. In order to explore the relationship between HSP70 and P-gp, the human hepatocarcinoma line HepG2 cells was induced by heat shock in vitro, and the inhibiting effect of quercetin on them was observed at the same time to seek the method increasing the effectiveness of hyperthermal therapy on hepatocarcinoma. METHODS: HepG2 cells were bathed in water at 42 degrees C stably for 90 minutes. The expression of HSP70 and P-gp were detected at 0, 2, 4, 8, 12, and 24 hours after 42 degrees C heat shock using immunocytochemistry and flow cytometry (FCM). Furthermore, at 4 hours later after heat shock, they were also detected in HepG2 cells which had been pretreated by quercetin in different concentrations within an hour before 42 degrees C heat shock. RESULTS: (1) Immunocytochemistry showed that HSP70 overexpressed in the cells and "moved" from cytoplasm to nucleus and nucleolus after heat shock. The percentage of HSP70 positive cells came to the climax (11.47%) at 4th hour and increased about 2 folds compared with that of the control group (6.64%) (P< 0.01). At the 24th hour after heat shock, it declined to a low level. The percentage of cells with P-gp positive went up following HSP70 after heat shock. It came to the climax(96.31%) at the 12th hour. And it increased about 3 folds compared with that of the control group(33.95%) (P< 0.01). (2) The overexpression of HSP70 and P-gp induced by heat shock could be inhibited effectively by quercetin in a dose-dependent manner, especially by quercetin at the concentrations of 100- 200 micromol/L(P< 0.01). CONCLUSION: (1) The overexpression of HSP70 and P-gp in HepG2 cells can be induced by heat shock and P-gp maybe has a correlation with HSP70. (2) The overexpression of HSP70 and P-gp in HepG2 cells induced by heat shock can be inhibited by quercetin.