功劳木组分F_6逆转白血病MDR的作用和机制

目的:背景与目的从中药功劳木中提取、分离出来的异喹啉类生物碱组分(Fraction 6,F6),具有抗病毒、抗炎、降血压等生理活性。近年有文献报道异喹啉类生物碱具有一定的逆转肿瘤细胞耐药的作用。本文以白血病多药耐药细胞(K562/ADM)为对象来研究F6逆转肿瘤多药耐药性(Mu ltidrug resistance,MDR)的效果及作用机制,以寻找具有多药耐药逆转活性的新型中药。方法:采用MTT法检测F6及阿霉素(Adriamyc in,ADM)对K562/S和K562/ADM细胞增殖的抑制作用;RT-PCR法检测F6对耐药肿瘤细胞MDR1基因mRNA表达的影响;流式细胞仪分析细胞内罗丹明123(Rhodam ine123,Rh123)浓度,以检测F6对肿瘤细胞膜P糖蛋白(P-glycoprote in,P-gp)泵功能的影响;免疫组化方法检测F6对肿瘤细胞膜P-gp表达水平的影响;流式细胞仪检测F6对肿瘤细胞凋亡的作用。结果:10 mg/L为F6的无毒剂量;ADM对K562/S和K562/ADM细胞的IC50分别为(1.68±0.08)mg/L和(80.25±1.06)mg/L;无毒剂量F6与ADM联合应用后对K562/S和K562/ADM细胞的IC50分别为(1.09±0.07)mg/L和(16.68±0.72)mg/L,此剂量F6使K562/ADM细胞的IC50下降4.81倍;无毒剂量的F6应用前后,K562/ADM细胞MDR1基因表达水平和细胞膜P-gp表达水平无明显差异;Rh123蓄积试验中无毒剂量的F6应用后使K562/ADM细胞内Rh123浓度由F6应用前的29.21%升高到85.35%,Rh123外排试验中无毒剂量的F6应用后使K562/ADM细胞内Rh123浓度由F6应用前的27.19%升高到59.22%;凋亡检测结果显示无毒剂量的F6使K562/ADM细胞凋亡率升高3.82倍。结论:F6能有效逆转白血病细胞的MDR;F6逆转白血病MDR的机制为通过抑制肿瘤细胞膜上P-gp的药泵功能,增加耐药细胞内化疗药物浓度逆转耐药性,而不是抑制MDR1基因和P-gp表达。

Reversal effect and mechanism of F6 extracted from Caulis Mahoniae on leukemia multidrug resistance cells

Objective: Fraction 6(F6),an isoquinoline alkaloid extracted from Caulis Mahoniae,a traditional Chinese herb,possesses such biological activities as antivirus、anti-inflammation and decreasing blood pressure.Recently,the reversal tumor multidrug resistance(MDR) effect of isoquinoline alkaloids have been reported.This study is to examine the reversal effect and reversal mechanism of Fraction 6 on human leukemia multidrug resistance cells(K562/ADM) to empolder new reversal agent of tumor multidrug resistance.Methods: MTT assay was used to evaluate inhibitory effect of Fraction 6 alone、adriamycin(ADM) alone and Fraction 6 combined with adriamycin on proliferation of human leukemia cell lines,including K562/S and K562/ADM.MDR1 gene mRNA was determined with RT-PCR assay.Intracellular Rhodamine 123 concentrations and apoptosis of cells were analyzed by Flow cytometer.Immunity histochemistry assay was used to examine the expression of P-glycoprotein(P-gp) on membrane of tumor cells.Results: Inhibitory rates of 10 mg/L Fraction 6 for K562/S and K562/ADM cells were under 10%,so this concentration was regarded as innocuous dosage of Fraction 6.The IC50 values of adriamycin for K562/S and K562/ADM cells detected by MTT assay were(1.68±0.08) mg/L and(80.25±1.06) mg/L, respectively.The IC50 values of innocuous dosage of Fraction 6 combined with adriamycin for K562/S and K562/ADM cells were(1.09±0.07) mg/L and(16.68±0.72) mg/L,respectively.Fraction 6 at this concentration made IC50 value of K562/ADM cells decreased 4.81 fold.Expression levels of MDR1 gene and Pgp had not distinct difference between in Fraction 6-used groups with in Fraction 6 and unused group Incubation of K562/ADM cells with Fraction 6 caused a marked increase in accumulation and a notable decrease in efflux of Rh123.The apoptosis rates were much higher in Fraction 6 plus adriamycin groups than in adriamycin groups.Conclusion: Fraction 6 exhibits potent effects in vitro in the reversal of P-gp-mediated MDR,and its reversal mechanism is that it can increase intracellular chemotherapy drugs concentrations by inhibiting "bump"function of P-gp other than inhibiting MDR1 gene and P gp expression.