异硫氰酸苯乙酯对K562／A02细胞阿霉素耐药影响的研究

Effect of Phenylhexyl Isothiocyanate on Adriamycin Resistance of K562/A02 Cell Line

本实验研究探讨异硫氰酸苯乙酯(phenylhexyl isothiocyanate,PHI)对K562/A02细胞的阿霉素(ADM)耐药性与敏感性的影响,并研究其可能的相关机制。运用MTT法分别检测阿霉素(ADM)以及PHI+ADM对K562/A02细胞的生长抑制率,计算其耐药倍数;用流式细胞仪检测PHI作用前后细胞的凋亡、细胞内ADM浓度的变化和MRP1蛋白变化;分光光度仪测定细胞内还原性谷胱目肽(GSH)的含量;RT-PCR半定量检测PHI作用前后MRP1mRNA的水平。结果显示,随着PHI浓度的增加,细胞生存率降低;两药联合应用时K562/A02细胞凋亡率均增加;当PHI≥20μmol/L时其耐药倍数有显著差异(p<0.05),两种细胞内ADM浓度增加亦有显著差异(p<0.05)。单独运用1μg/ml ADM时K562/A02细胞内的GSH含量下降5%,单独运用PHI时K562/A02细胞内的GSH含量随着PHI浓度的增加先是轻度升高尔后下降,GSH含量下降点约在10μmol/L;当PHI≥20μmol/L与1μg/mlADM联合应用时,K562/A02细胞内的GSH含量随着PHI浓度的升高而进行性下降;而不同浓度的PHI的作用前后,两种细胞的MRP1的表达无论是蛋白水平还是基因水平都无显著差异(p>0.05)。结论:PHI对K562/A02细胞的细胞毒作用与细胞内GSH耗竭无直接关系,PHI不但可以增强K562/A02细胞对ADM的敏感性,而且通过耗竭细胞内GSH部分地逆转K562/A02细胞对ADM的耐药。联合使用PHI可减少ADM的用量,从而降低其毒副作用。

This study was to investigate the effect of phenylhexyl isothiocyanate（PHI） on drug resistance and sensitivity on K562/A02 cell line to adriamycin （ADM） and to elucidate the possible mechanisms. The inhibition rates of ADM and PHI ＋ ADM on growth of K562/A02 cell line were measured by MTT assay, and K562/A02 cell resistant multiple was calculated. The apoptosis rate of K562/A02 cell line, the changes of intracellular ADM concentrations and MRP1 protein level were detected by flow cytometry （FCM）. Intracellular deoxidized GSH level was determined by spectrometric enzyme assay and MRP1 mRNA was assayed by semiquantitative RT-PCR before and after using PHI. The results indicated that the survival rate of K562/A02 cell line decreased with the increasing concentration of PHI. Apoptosis rate increased after treatment in combination with two above drugs, the changes of drug resistance multiple and intracellular ADM level had statistical significance between K562/A02 and K562 cells （p 〈 0.05 ）, when the concentration of PHI was more than 20μmol/L. Intracellular GSH level in K562/A02 cell line reduced 5% when 1 μg/ml ADM was used alone, and it increased slightly at first, then decreased when more than 10μmol/L PHI was used. When more than 20 pLmoL/L PHI was used in combination with 1μg/ml ADM, intracellular GSH level in K562/A02 cell line decreased progressively with increasing the concentration of PHI. The expressions of MRP1 mRNA and protein had no statistical significance between K562/A02 and K562 cells （p 〉 0.05 ） after or before PHI was used. It is concluded that the cytotoxicity of PHI to K562/A02 cell line does not associate with the depletion of the intracellular GSH. PHI not only enhances the sensitivity of K562/A02 cell line to ADM, but also partially reverses effect of K562/A02 cell resistance to ADM. ADM combined with PHI can diminish side effect and dosage of ADM.