Cheng Jian, Dai Jing-Ying, Chen Bao-An, Cai Xiao-Hui, Wang Shuai, Gao Feng
Department of Hematology and Oncology, Southeast University, Nanjing, Jiangsu Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2012 Jun;20(3):558-63.
Both tetrandrine (Tet) and 5-bromotetrandrine (BrTet) can effectively reverse P-glycoprotein (P-gp)-mediated multidrug resistance (MDR). The structure of multidrug resistance associated protein 7 (MRP7) has its own specificity and difference compared with other members of the MRP family. This study was aimed to investigate whether Tet and BrTet can inhibit the expression level of MRP7 so as to further look into the mechanisms of the reversal effects of Tet and BrTet on MDR. The inhibitory effects of daunorubicin (DNR) used alone on the proliferation of K562 and K562/A02 cells were evaluated by MTT assay, the IC(50) of DNR and drug resistant folds were calculated. The mRNA level of MRP7 was tested by real-time PCR, and the protein levels of MRP7 and P-gp were tested by Western blot. The DNR accumulation was analyzed by flow cytometry (FCM). The results showed that the resistance of K562/A02 cells to DNR was 23.65-folds of that of K562 cells. After administration of 1 µmol/L Tet or 2 µmol/L BrTet, the mRNA level of MRP7 in the K562/A02 cells decreased to 2% and 12% respectively, and the protein level of MRP7 decreased by 53.2% and 83.7% respectively. The protein level of P-gp decreased by 58.47% and 52.20% in the 1 µmol/L Tet and 2 µmol/L BrTet groups. FCM detection showed that 1 µmol/L Tet and 2 µmol/L BrTet significantly increased the accumulation of DNR in K562/A02 cells by 94.32% and 271% respectively. It is concluded that Tet and BrTet both can reverse MDR in vitro. The mechanisms may be related to the inhibition of MRP7 overexpression and the increase of anticancer drug concentration in cells. At the same molar concentration, the effects of Tet and BrTet in inhibiting the protein level of MRP7 expression do not show significant difference.
汉防己甲素（Tet）和5-溴汉防己甲素（BrTet）均能有效逆转P-糖蛋白（P-gp）介导的多药耐药（MDR）。多药耐药相关蛋白7（MRP7）的结构与MRP家族的其他成员相比有其自身的特异性和差异。本研究旨在探讨Tet和BrTet是否能抑制MRP7的表达水平，从而进一步探究Tet和BrTet逆转MDR的作用机制。采用MTT法评估单独使用柔红霉素（DNR）对K562和K562/A02细胞增殖的抑制作用，计算DNR的半数抑制浓度（IC50）和耐药倍数。采用实时荧光定量PCR检测MRP7的mRNA水平，采用蛋白质印迹法检测MRP7和P-gp的蛋白水平。采用流式细胞术（FCM）分析DNR的蓄积情况。结果显示，K562/A02细胞对DNR的耐药性是K562细胞的23.65倍。给予1 μmol/L Tet或2 μmol/L BrTet后，K562/A02细胞中MRP7的mRNA水平分别降至2%和12%，MRP7的蛋白水平分别下降53.2%和83.7%。在1 μmol/L Tet组和2 μmol/L BrTet组中，P-gp的蛋白水平分别下降58.47%和52.20%。FCM检测显示，1 μmol/L Tet和2 μmol/L BrTet分别使K562/A02细胞中DNR的蓄积量显著增加94.32%和271%。结论：Tet和BrTet均能在体外逆转MDR。其机制可能与抑制MRP7过表达及增加细胞内抗癌药物浓度有关。在相同摩尔浓度下，Tet和BrTet对MRP7蛋白表达水平的抑制作用无显著差异。
