小檗胺对K562细胞体外及体内作用的实验研究

为了探讨小檗胺(berbamine,BER)在体外及体内抗人白血病细胞株K562细胞的作用及其可能的机制,用MTr法测定K562细胞的增殖抑制,用流式细胞术检测凋亡细胞,用RT-PCR方法检测bcr/abl基因表达,用Westem blot方法检测BCR/ABL蛋白质水平表达,利用K562细胞荷瘤裸鼠模型观察小檗胺治疗后瘤体大小、组织病理及bcr/abl基因表达的改变.结果表明小檗胺对K562细胞有明显的增殖抑制作用,呈时间-浓度依赖关系.经8.0μg/ml BER作用24、48、72小时,对K562细胞的抑制率分别为(26.63±3.57)%,(61.84±4.74)%,(75.32±1.95)%,与对照组相比,P＜0.01.IC50值(72小时)为(5.227±1.307)μg/ml.与空白对照组相比,经8.0μg/ml小檗胺处理72小时后,K562细胞凋亡率明显增加[(61.77±4.35)%vs(0.50±0.14)%,P＜0.01].小檗胺能下调K562细胞bcr/abl mRNA表达和P210水平,且呈浓度-时间依赖效应,并与细胞凋亡率有一定的相关性.经8.0μg/ml BER处理后,与同浓度点对照组相比,72小时组的表达明显地减弱(0.97±0.01vs1.38±0.02,P＜0.01).4.0-16.0μg/ml BER处理24小时后,P210的相对表达量由未加药对照组的1.04±0.02降至16.0μg/ml组的0.63±0.01(P＜0.01),与所设的对照组药物酪氨酸蛋白激酶抑制剂STI571的作用结果相似.在K562荷瘤裸鼠模型,小檗胺治疗组瘤重较未治疗对照组的明显减少[(1.46±0.43)g vs(2.90±0.94)g,P＜0.01],抑瘤率为49.66%.瘤体细胞的bcr/abl mRNA的表达水平明显下调.结论小檗胺在体外对K562细胞具有明显的增殖抑制作用及明确的诱导凋亡作用,并呈时间-浓度依赖关系;小檗胺诱导细胞凋亡可能与其下调bcr/abl基因和(或)P210的表达水平有关;在荷瘤裸鼠体内,小檗胺同样具有显著的抑制K562细胞生长的作用,尤其能下调瘤体组织细胞bcr/abl mRNA的表达水平.

Effects of Berbamine on K562 Cells and Its Mechanisms in vitro and in vivo

To elucidate the antileukemi effects of berbamine and the possible molecular mechanisms in vitro and in vivo, MTT method was used to examine the effect of berbamine on K562 cell growth. The apoptosis rate was measured by flow cytometry. The mRNA expression level of BCR/ABL gene (semiquantity value) was determined by RT-PCR and the BCR/ABL protein (P210) level was detected by Western blot. The K562-bearing mice were used to reveal the therapeutic effect in vivo. The results showed that a significant time- and concentration-dependent inhibition of cell growth was found in the cells treated with berbamine. After the cells were exposed to 8.0 microg/ml berbamine for 24, 48 and 72 hours, the percentage of growth inhibition of K562 cells progressively increased by (26.63 +/- 3.57)%, (61.84 +/- 4.74)%, (75.32 +/- 1.95)%, respectively (compared with control, P < 0.01). The IC(50) (72 hours) value was 5.227 +/- 1.307 microg/ml. The apoptosis rate of K562 cells treated with 8.0 microg/ml berbamine for 24 and 72 hours increased from (29.20 +/- 3.82)% to (61.77 +/- 4.35)% (P < 0.01). Berbamine down-regulated the expression levels of bcr/abl gene and P210 in K562 cells in a time- and concentration-dependent manner. The bcr/abl expression decreased from (1.38 +/- 0.02) to (0.97 +/- 0.01) after exposure of the cells to 8.0 microg/ml berbamine for 0 and 72 hours (P < 0.01). When the cells were treated with 4.0 - 16.0 microg/ml berbamine for 24 hours, the level of P210 decreased from (0.95 +/- 0.03) to (0.63 +/- 0.01) (P < 0.01). In vivo, after treatment for 4 weeks, the tumor weight of berbamine-treated group was also lower than that of untreated group [(1.46 +/- 0.43) g vs (2.90 +/- 0.94) g, P < 0.01] and the inhibition rate was 49.66%, moreover, berbamine down-regulated the expression level of bcr/abl gene of tumor cells. It is concluded that berbamine can obviously inhibit the cell proliferation and induce apoptosis in K562 cell lines in a time- and concentration-dependent manner in vitro. The mechanisms of berbamine-induced apoptosis may be involved in down-regulation of bcr/abl gene expression and P210 level. In vivo, berbamine can aslo display a better antileukemic effect and down-regulate expression of bcr/abl gene. Berbamine extracted from Chinese herb may be a promising candidate of new drug for clinical anticancer treatment, especially for bcr-abl(+) diseases.