| Objective: (1) To investigate the differences of mRNA and protein expressions of HSP90 and chk1 among normal liver, hepatocellular carcinoma and paired adjacent cancerous liver tissues, analyze the correlation of the level of expressions of HSP90 and chk1 and the clinicopathological parameters in liver cancer. (2) To observe the effects of geldanamycin (GA) on cell proliferation inhibition and apoptosis of human hepatoma cell line BEL7404. (3) To observe the changes of the mRNA and protein expression of chk1 before and after treatment with HSP90 inhibitor geldanamycin (GA), discuss the mechanism of HSP90 in apoptosis of human hepatoma cell line BEL7404 and provide new therapeutic targets for hepatocarcinoma.Methods: (1) The levels of protein expression of HSP90 and chk1 were assayed by immunohistochemistry (IHC) in 41 hepatocellular carcinoma, 41 paired adjacent cancerous liver tissues and 9 normal livers. (2) The levels of mRNA expression of HSP90 and chk1 were assayed by reverse transcription PCR (RT-PCR) among 46 hepatocellular carcinoma, 46 paired adjacent cancerous liver tissues and 9 normal livers. (3) Cultured human hepatoma cell line Bel-7404 in vitro and observed the impacts of HSP90 inhibitor geldanamycin (GA) on the growth of human hepatoma cell line Bel-7404 by methyl thiazolyl tetrazolium (MTT) method. Studied the influence of HSP90 inhibitor geldanamycin (GA) on apoptosis of human hepatoma cell line BEL7404 by electron microscope, acridine orange fluorescent staining and Annexin V-EGFP/PI double staining FCM assay. (4) The influence of HSP90 inhibitor geldanamycin (GA) on cell cycle of human hepatoma cell line Bel-7404 was assayed by FCM. (5) The changes of the mRNA and protein expression of chk1 before and after treatment with HSP90 inhibitor geldanamycin (GA) were assayed by RT-PCR and Western-blot.Results: (1) The positive protein expression rates of HSP90 were 85.37% (35/41),56.1% (23/41),22.22% (2/9) in hepatocellular carcinoma, paired adjacent cancerous liver tissues and normal liver respectively. The rate in hepatocellular was significantly higher than the other two groups. The positive expression of HSP90 was associated with clinical stages, differentiation degree of cancer cells and recurrence (P<0.05), and was irrelevant with age, gender, tumor size, tumor nodus, serum level of AFP and portal vein embolus(P>0.05). The positive protein expression rates of chk1 were 80.49 % (33/41),70.73 % (29/41),11.13 % (1/9) in three tissues respectively. The rate in hepatocellular was higher than that in paired adjacent cancerous liver tissues, and significantly higher than that in normal liver tissues. The positive expression of chk1 was associated with pathological classification (P<0.05), and was irrelevant with age, gender, tumor size, tumor nodus, portal vein embolus, serum level of AFP, hepatic cirrhosis and ascites (P>0.05). Rank correlation analysis showed the expression between HSP90 and chk1 in hepatocellular carcinoma had a positive relationship.(2) The mRNA expression level of HSP90 was 0.718±0.372, 0.437±0.207 and 0.136±0.104 in hepatocellular carcinoma, paired adjacent cancerous liver tissues and normal liver respectively, there are significantly differences among groups (P<0.01). The mRNA expression of HSP90 was associated with TNM stages,differentiation degree of cancer cells and recurrence (P<0.05). The mRNA expression level of chk1 was 0.320±0.146, 0.193±0.071 and 0.098±0.000 in three tissues respectively, and there are significant differences among groups (P<0.01). The mRNA expression of chk1 was associated with pathological classification (P<0.05).(3) HSP90 inhibitor geldanamycin (GA) inhibited BEL7404 cell line at the concentration of 1-20 umol/L with 10.1 %～76.7 % inhibitory ratio. The growth inhibition ratio increased gradually after treatment with GA to BEL7404 cells 24h, 48h, and 72h respectively. And this inhibitory effect showed remarkable time and dose dependence.(4) G0/G1 period cells increased while G2/M period cells decreased either with treatment of 5 umol/L GA , or 10 umol/L GA, or 15 umol/LGA.GA arrested cell cycle at phase G0/G1,which showed time and dose dependency.(5) GA induced apoptosis of BEL7404 cells with the apoptosis rate of 10.2±1.35 %, 21.3±1.30 %, and 38.70±1.43 %, while the rate in the control group was 6.31±0.82 % by Annexin V-EGFP/PI double staining FCM assay. This suggested that GA could induce apoptosis of hepatoma cells.(6) The proportion of apoptosis cells increased gradually along with the different concentration of GA by acridine orange fluorescent staining. Condensation of chromatin at margins of nuclei, disintegration of nucleolus, vacuoles in cytoplasm, even apoptotic body were observed in the GA group under transmission electron microscope (TEM). This suggested that the inhibitory effect of GA on the growth of tumor cells was probably related to its arresting cell cycle at G0/G1 phase and induced apoptosis of tumor cells.(7) After BEL7404 cells were treated with 10 uM GA for various time intervals, chk1 mRNA expression showed no significant difference before and after GA treatment by a semi-quantitative RT-PCR analysis. Chk1 protein showed an obviously decrease after 24h treatment with GA assayed with western blot. These results indicated that the inhibitory effect of GA was due to degradation of chk1 protein but not decreased synthesis.Conclusions: The mRNA and protein expressions of HSP90 and chk1 in hepatocellular carcinoma are higher than paired adjacent cancerous liver tissues and normal liver tissues, and are associated with clinicopathological classification. HSP90 inhibitor geldanamycin (GA) inhibits the growth of hepatoma cells in vitro, induces apoptosis and blocks cell cycle of hepatoma cells. The inhibition of HSP90 can decrease the expression of chk1 in human hepatoma cell line Bel-7404, without affecting the synthesis of mRNA of chk1. The mechanism of anti-tumor activity of HSP90 inhibitor GA may be related to the effect of blocking the chk1 pathway. HSP90 and chk1 are hopeful new therapeutic targets for hepatocarcinoma.
|
PDF Full Text Request