Tetrandrine and fangchinoline,bisbenzylisoquinoline alkaloids from Stephania tetrandra can reverse multidrug resistance by inhibiting P-glycoprotein activity in multidrug resistant human cancer cells

The overexpression of ABC transporters is a common reason for multidrug resistance (MDR) in cancer cells. In this study, we found that the isoquinoline alkaloids tetrandrine and fangchinoline from Stephania tetrandra showed a significant synergistic cytotoxic effect in MDR Caco-2 and CEM/ADR5000 cancer cells in combination with doxorubicin, a common cancer chemotherapeutic agent. Furthermore, tetrandrine and fangchinoline increased the intracellular accumulation of the fluorescent P-glycoprotein (P-gp) substrate rhodamine 123 (Rho123) and inhibited its efflux in Caco-2 and CEM/ADR5000 cells. In addition, tetrandrine and fangchinoline significantly reduced P-gp expression in a concentration-dependent manner. These results suggest that tetrandrine and fangchinoline can reverse MDR by increasing the intracellular concentration of anticancer drugs, and thus they could serve as a lead for developing new drugs to overcome P-gp mediated drug resistance in clinic cancer therapy.     

Ascorbate promotes the cellular accumulation of doxorubicin and reverses the multidrug resistance in breast cancer cells by inducing ROS-dependent ATP depletion

Chemotherapy is the most effective strategy for the treatment of metastatic breast cancer. However, P-glycoprotein (P-gp)-mediated multidrug resistance severely limits the efficacy of chemotherapy and is a major cause of the failure during chemotherapeutic treatment. In this study, we investigated the anticancer effects of combining chemotherapeutic drugs with ascorbate (AA) in human breast cancer cells. We found that combined administration of AA can improve the sensitivity of both MCF-7 and doxorubicin (Dox)-resistant MCF-7/Adr cells to Dox in vitro and in vivo by a reactive oxygen species (ROS)-dependent mechanism. Further studies proved that AA can promote the accumulation of Dox in MCF-7/Adr cells when combined with doxorubicin. AA had no effect on the expression of P-gp at the mRNA and protein levels, but could decrease its activity as demonstrated by an obvious inhibition of efflux of P-gp substrate Rh 123. AA reduced ATP levels in both MCF-7 and MCF-7/Adr cells, and pretreating AA-stimulating cells with catalase completely rescued ATP levels. With ATP reduction, we observed an increased cellular calcium and the appearance of vacuoles and micropores on the cell surface, indicating the increased cell membrane permeability in AA-treated MCF-7/Adr cells. The above results suggest that AA could promote the cellular accumulation of doxorubicin by inducing ROS-dependent ATP depletion. Clinically, a combination of AA with doxorubicin would be a novel strategy for reversal of the multidrug resistance in human breast cancer cells during chemotherapy.     

Down-regulation of P-glycoprotein expression by sustained intracellular acidification in K562/Dox cells

We have investigated the involvement of intracellular pH (pH_i) in the regulation of P-glycoprotein (P-gp) in K562/DOX cells. The selective Na⁺/H⁺ exchanger1 (NHE1) inhibitor cariporide and the “high K⁺” buffer were used to induce the sustained intracellular acidification of the K562/DOX cells that exhibited more alkaline pH_i than the K562 cells. The acidification resulted in the decreased P-gp activity with increased Rhodamine 123 (Rh123) accumulation in K562/DOX cells, which could be blocked by the P-gp inhibitor verapamil. Moreover, the acidification decreased MDR1 mRNA and P-gp expression, and promoted the accumulation and distribution of doxorubicin into the cell nucleus. Interestingly, these processes were all pH_i and time-dependent. Furthermore, the change of the P-gp expression was reversible with the pH_i recovery. These data indicate that the tumor multidrug resistance (MDR) mediated by P-gp could be reversed by sustained intracellular acidification through down-regulating the P-gp expression and activity, and there is a regulative link between the pH_i and P-gp in K562/DOX cells.     

Nrf2/HO-1/HMGB1信号通路在白血病细胞K562/A02化疗耐药中的机制研究

目的:通过观察高迁移率族蛋白1(HMGB1)、转录因子NF-E2相关因子2(Nrf2)及血红素加氧酶1(HO-1)基因沉默对白血病化疗耐药细胞(K562/A02细胞株)的影响,探讨该信号通路在白血病化疗耐药中的作用及其可能机制。方法:将HMGB1基因、Nrf2基因及HO-1基因的特异性干扰RNA分别转染阿霉素耐药细胞株K562/A02,荧光实时定量(RT-PCR)方法检测HMGB1、Nrf2及HO-1的mRNA表达水平,Western blot方法检测HMGB1、Nrf2及HO-1的蛋白表达水平,免疫荧光方法检测Nrf2的蛋白表达,并使用CCK-8方法检测转染前后K562/A02细胞株的细胞活性。结果:HMGB1基因、Nrf2基因或HO-1基因沉默的K562/A02细胞活性皆显著低于对照组及空白组(P0.05),化疗敏感性恢复。结论:HMGB1高表达导致了白血病细胞株K562/A02对阿霉素的化疗耐药,Nrf2/HO-1信号通路参与了HMGB1诱导的K562/A02细胞的化疗耐药,其表达上调可恢复K562/A02细胞对阿霉素的敏感性。     

Suppression of multidrug resistance by treatment with TRAIL in human ovarian and breast cancer cells with high level of c-Myc

In this study, we investigated the role of c-Myc in overcoming multidrug resistance (MDR) in human ovarian and breast cancer cells by TRAIL. We showed that P-gp expressing MDR variants (Hey A8-MDR and MCF7-MDR cells) with high level of c-Myc were highly susceptible to TRAIL treatment when compared to their drug-sensitive parental human ovarian cancer Hey A8 and breast MCF-7 cells, respectively. Up-regulation of DR5 TRAIL receptor and down-regulation of c-FLIP and the promotion of caspase-dependent cell death, which contribute to TRAIL sensitization of MDR cells, were regulated by the over-expressed c-Myc in the MDR cells. After targeted inhibition of c-Myc with specific siRNA, these responses to TRAIL disappeared and TRAIL-induced apoptosis was also suppressed in MCF7-MDR cells. Treatment with TRAIL significantly reduced P-glycoprotein (P-gp)-mediated efflux of rhodamine123 in both Hey A8-MDR and MCF7-MDR cells. Furthermore, TRAIL significantly potentiated the cytotoxicity of vinblastine, vincristine, doxorubicin and VP-16 that are P-gp substrate anticancer drugs in both MDR cells, which resulted in the reversal effect of TRAIL on the MDR phenotype. The present study shows for the first time that elevated c-Myc expression in the MDR cells plays a critical role in overcoming MDR by TRAIL that can act as a specific sensitizer for P-gp substrate anticancer drug.     

Single amino acid (482) variants of the ABCG2 multidrug transporter: major differences in transport capacity and substrate recognition

The human ABCG2 protein is an ATP binding cassette half-transporter, which protects our cells and tissues against various xenobiotics, while overexpression of ABCG2 in tumor cells confers multidrug resistance. It has been documented that single amino acid changes at position 482 resulted in altered drug resistance and transport capacity. In this study, we have generated nine Arg-482 mutants (G, I, M, S, T, D, N, K, Y) of ABCG2, and expressed them in insect cells. All ABCG2 variants showed cell surface expression and, in isolated membranes, an ABCG2-specific ATPase activity. When methotrexate accumulation was measured in inside-out membrane vesicles, this transport was supported only by the wild-type ABCG2. In intact cells, mitoxantrone was transported by all ABCG2 variants, except by R482K. Rhodamine 123 was extruded by most of the mutants, except by R482K, Y and by wild-type ABCG2. Hoechst 33342 was pumped out from cells expressing the wild-type and all Arg-482 variants, but not from those expressing R482K and Y. Our study demonstrates that the substrate specificity of the Arg (wild-type) form is unique and that amino acid replacements at position 482 induce major alterations in both the transport activity and substrate specificity of this protein.     

The hydroxyl group of S685 in Walker A motif and the carboxyl group of D792 in Walker B motif of NBD1 play a crucial role for multidrug resistance protein folding and function

Structural analysis of MRP1-NBD1 revealed that the Walker A S685 forms hydrogen-bond with the Walker B D792 and interacts with magnesium and the β-phosphate of the bound ATP. We have found that substitution of the D792 with leucine resulted in misfolding of the protein. In this report we tested whether substitution of the S685 with residues that prevent formation of this hydrogen-bond would also cause misfolding. Indeed, substitution of the S685 with residues potentially preventing formation of this hydrogen-bond resulted in misfolding of the protein. In addition, some substitutions that might form hydrogen-bond with D792 also yielded immature protein. All these mutants are temperature-sensitive variants. However, these complex-glycosylated mature mutants prepared from the cells grown at 27 °C still significantly affect ATP binding and ATP-dependent solute transport. In contrast, substitution of the S685 with threonine yielded complex-glycosylated mature protein that is more active than the wild-type MRP1, indicating that the interaction between the hydroxyl group of 685 residue and the carboxyl group of D792 plays a crucial role for the protein folding and the interactions of the hydroxyl group at 685 with magnesium and the β-phosphate of the bound ATP play an important role for ATP-binding and ATP-dependent solute transport.     

Hydrogen-bond formation of the residue in H-loop of the nucleotide binding domain 2 with the ATP in this site and/or other residues of multidrug resistance protein MRP1 plays a crucial role during ATP-dependent solute transport

MRP1 couples ATP binding/hydrolysis to solute transport. We have shown that ATP binding to nucleotide-binding-domain 1 (NBD1) plays a regulatory role whereas ATP hydrolysis at NBD2 plays a crucial role in ATP-dependent solute transport. However, how ATP is hydrolyzed at NBD2 is not well elucidated. To partially address this question, we have mutated the histidine residue in H-loop of MRP1 to either a residue that prevents the formation of hydrogen-bonds with ATP and other residues in MRP1 or a residue that may potentially form these hydrogen-bonds. Interestingly, substitution of H827 in NBD1 with residues that prevented formation of these hydrogen-bonds had no effect on the ATP-dependent solute transport whereas corresponding mutations in NBD2 almost abolished the ATP-dependent solute transport completely. In contrast, substitutions of H1486 in H-loop of NBD2 with residues that might potentially form these hydrogen-bonds exerted either full function or partial function, implying that hydrogen-bond formation between the residue at 1486 and the γ-phosphate of the bound ATP and/or other residues, such as putative catalytic base E1455, together with S769, G771, T1329 and K1333, etc., holds all the components necessary for ATP binding/hydrolysis firmly so that the activated water molecule can efficiently hydrolyze the bound ATP at NBD2.