苦参碱逆转人结肠癌干细胞化疗耐药作用及相关机制研究

Reversal effect of matrine on chemotherapy resistance of human colon cancer stem cells and its underlying mechanism

为探讨苦参碱(matrine,MT)逆转人结肠癌HCT116/5-Fu细胞化疗耐药作用及调控磷酸肌醇3激酶(phosphatidylinositol-3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)/雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路的机制,采用MTT法检测HCT116/5-Fu细胞对5-氟尿嘧啶(5-fuorouracil,5-Fu)的耐药性并确定MT对HCT116/5-Fu细胞的最大无毒浓度(maximum non-toxic concentration,TC0)(抑制率<10%)。将TC0倍比稀释3个浓度,分别为MT高、中、低组,观察MT作用前后HCT116/5-Fu细胞耐药性的变化。采用流式细胞术检测各组细胞的凋亡情况,进一步采用实时荧光定量PCR及Western blotting分别检测ABCB1基因及磷酸化PI3K(phospho-PI3K,p-PI3K)、磷酸化Akt(phospho-Akt,p-Akt)、磷酸化mTOR(phospho-mTOR,p-mTOR)、P-糖蛋白(P-glycoprotein,P-gp)的表达。结果显示,HCT116/5-Fu细胞对5-Fu的耐药指数为14.53,MT高、中、低组对HCT116/5-Fu细胞化疗耐药逆转指数分别为3.01、1.84、1.27,且MT可改变化疗药5-Fu中HCT116/5-Fu细胞的生存状态;MT各组HCT116/5-Fu细胞的凋亡率分别为(42.41±1.43)%、(23.76±0.47)%、(12.29±0.18)%,显著高于耐药组(P<0.01);MT可显著降低ABCB1耐药基因和P-gp的表达,并下调p-PI3K、p-Akt、p-mTOR蛋白表达(P<0.05)。提示MT可逆转人结肠癌HCT116/5-Fu细胞的化疗耐药,这可能与其降低ABCB1耐药基因和P-gp表达,抑制PI3K/Akt/mTOR信号通路,促进细胞凋亡有关。

We aimed to explore the effect of matrine(MT) on the chemotherapy resistance of human colon cancer HCT116/5-Fu stem cells and its relation to phosphatidylinositol-3-kinase(PI3 K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)pathways. MTT assay was used to detect the resistance of HCT116/5-Fu cells towards 5-fuorouracil(5-Fu) and to determine the maximum non-toxic concentration(TC0) of MT for HCT116/5-Fu cells(inhibition rate <10%). The TC0 of MT was diluted by double ratio dilution method into MT high, middle and low concentration solutions. The changes of HCT116/5-Fu cell resistance after the treatment with the three concentration of MT were investigated. The effect of MT on cell status was observed. The apoptosis of each group was detected by flow cytometry. The expression of ABCB1 gene was examined by real time fluorescent quantification PCR and phospho-PI3 K(p-PI3 K),phospho-Akt(p-Akt),phospho-mTOR(p-mTOR) and P-glycoprotein(P-gp) proteins were detected by Western blotting. The results showed that the resistance index of HCT116/5-Fu cells towards 5-Fu was 14.53, and the resistance index of HCT116/5-Fu cells in MT high, middle and low dose groups were 3.01, 1.84, and 1.27 respectively. MT changed the survival state of HCT116/-Fu cells in 5-Fu. The apoptosis rates of HCT116/5-Fu cells in the three MT groups were(42.41±1.43)%,(23.76±0.47)% and(12.29±0.18)% respectively, which were significantly higher than that in the drug resistant group(P<0.01);In addition, MT significantly reduced the expression of drug-resistant gene ABCB1 and P-gp, and down-regulated the expression of p-PI3 K, p-Akt, p-mTOR proteins(P<0.05). In conclusion, MT could reverse the chemotherapy resistance of human colon cancer HCT116/5-Fu cells, which may be related to the reduction of drug-resistant gene ABCB1 and P-gp expression,and the increase of apoptosis via PI3 K/Akt/mTOR signaling pathway.