| 【Objective】（1）Evaluate kinesin Eg5expression in hepatocellular carcinoma(HCC) tissues，and its association with clinicopathological characteristics（.2）Analysisof Eg5mRNA and protein expression level in the different metastatic potential livercancer cell lines，and discusses the relationship between with tumor metastasis.（3）Research the anti-tumor effect and the mechanism of apoptosis in different transferefficiency of liver cancer cell lines by using S-Trityl-L-Cysteine(STLC).【Methods】（1）Immunohistochemistry was used to detect expression of Eg5inhepatocellular carcinoma (HCC) tissues. The correlation between Eg5expression andclinicopathologic features and metastasis were also analyzed.(2) Immunohistochemistryassay was used to determine the expression of Eg5in three HCC cell lines withvarious metastatic potentials.To examine the expression of kinesin Eg5mRNA ininflammatory pseudotumor liver tissue and without（PVTT）liver cancer tissue andwithin（PVTT）liver cancer tissue and the three HCC cell lines with various metastaticpotentials by reverse transcription polymerase chain reaction (RT-PCR) and Analysesthe expression of kinesin Eg5by Western blotting. The relationship its expressionbetween with tumor metastasis are also analyzed.（3）The HCC cell lines MHCC97Hwith various metastatic potentials was exposed to various concentrations of STLC (1、5、10、50、100μmol/L) and compared with the control group during24h,48hours,72hours.To detect the effect of apoptosis in the three HCC cell lines MHCC97H by MTT method.Annexin-v/PI immunofluorescence staining method was used to observe cell apoptosis. To investigate the expression of BIM mRNA and AIF mRNA in drug treatmentMHCC97H cells by reverse transcription polymerase chain reaction (RT-PCR).Immunofluorescence was used to analyze BIM and AIF the transfer between cytoplasmand nucleus. Colorimetric method was used to measure caspase3activity.【Results】Eg5is mainly expressed in the cytoplasm of liver cells.Eg5had not seen theobvious expression in normal liver tissues, The positive rate of Eg5expression inHCC tissues were62.3%(33/53), and the expression level was significantly correlatedwith tumor size（χ~2=10.162，P=0.002）and Edmondson grading (χ~2=4.414，P=0.038)and TNM stage（χ~2=13.061， P=0.000）and whether PVTT（χ~2=4.098，P=0.041）.No significant correlation was found with gender, age and whether tumor diolame ornot. Immunohistochemistry was show that Eg5is mainly expressed in the cytoplasm inthe three HCC cell lines with various metastatic potentials. The results from Westernblotting were consistent with the results of RT-PCR analysis of Eg5mRNA expression.Among the three cell lines, the expression level in MHCC97H, which with the highestmetastatic potential, was significantly higher than that with lower (P<0.05); while Eg5mRNA expression was not detected in inflammatory pseudotumor liver tissue.Expression level of Eg5was significantly increased in within（PVTT）liver cancer tissuecompared with without（PVTT）liver cancer tissue tissues (P<0.05).(3) MTT methodrevealed that STLC inhibited MHCC97H cells growth and induced its death. Thetypical phenomenon of mitotic catastrophe was revealed by immunofluorescencestaining, BIM and AIF the transfer between cytoplasm and nucleus was also observed.The results from RT-PCR analysis of BIM mRNA and AIF mRNA expression inMHCC97H cells were significant increased after STLC interface48h.(P<0.05) Therewas significant difference in caspase3activity when compared with the control group（P<0.05）.【Conclusion】 Eg5was not obviously expressed in normal liver tissues, high expression in hepatocellular carcinoma tissues and HCC cell lines，and its expressionlever was the correlation of it with metastasis of HCC.Eg5may act as a potential target of HCC chemotherapy. STLC as a targeted therapydrugs could induce the apoptosis to human HCC cell lines. BIM and AIF may besynergistic caspase3participated in the process of apoptosis of hepatocellular carcinomacells which was induced by STLC. |
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