Role of the highly structured 5'-end region of MDR1 mRNA in P-glycoprotein expression

Overexpression of P-glycoprotein, encoded by the MDR1 (multidrug resistance 1) gene, is often responsible for multidrug resistance in acute myeloid leukaemia. We have shown previously that MDR1 (P-glycoprotein) mRNA levels in K562 leukaemic cells exposed to cytotoxic drugs are up-regulated but P-glycoprotein expression is translationally blocked. In the present study we show that cytotoxic drugs down-regulate the Akt signalling pathway, leading to hypophosphorylation of the translational repressor 4E-BP [eIF (eukaryotic initiation factor) 4E-binding protein] and decreased eIF4E availability. The 5'-end of MDR1 mRNA adopts a highly-structured fold. Fusion of this structured 5'-region upstream of a reporter gene impeded its efficient translation, specifically under cytotoxic stress, by reducing its competitive ability for the translational machinery. The effect of cytotoxic stress could be mimicked in vivo by blocking the phosphorylation of 4E-BP by mTOR (mammalian target of rapamycin) using rapamycin or eIF4E siRNA (small interfering RNA), and relieved by overexpression of either eIF4E or constitutively-active Akt. Upon drug exposure MDR1 mRNA was up-regulated, apparently stochastically, in a small proportion of cells. Only in these cells could MDR1 mRNA compete successfully for the reduced amounts of eIF4E and translate P-glycoprotein. Consequent drug efflux and restoration of eIF4E availability results in a feed-forward relief from stress-induced translational repression and to the acquisition of drug resistance.     

Post-transcriptional regulation of P-glycoprotein expression in cancer cell lines

The present study of inhibitors shows that the histone deacetylase-induced increase in P-glycoprotein (Pgp) mRNA (MDR1 mRNA) does not parallel either an increase in Pgp protein or an increase in Pgp activity in several colon carcinoma cell lines. Furthermore, studying the polysome profile distribution, we show a translational control of Pgp in these cell lines. In addition, we show that the MDR1 mRNA produced in these cell lines is shorter in its 5' end that the MDR1 mRNA produced in the MCF-7/Adr (human breast carcinoma) and K562/Adr (human erythroleukemia) cell lines, both of them expressing Pgp. The different size of the MDR1 mRNA is due to the use of alternative promoters. Our data suggest that the translational blockade of MDR1 mRNA in the colon carcinoma cell lines and in wild-type K562 cells could be overcome by alterations in the 5' end of the MDR1 mRNA in the resistant variant of these cell lines, as in the case of the K562/Adr cell line. This is, to our knowledge, the first report demonstrating that the presence of an additional 5' untranslated fragment in the MDR1 mRNA improves the translational efficiency of this mRNA.     

Synthesis and evaluation of substituted dibenzo[c,e]azepine-5-ones as P-glycoprotein-mediated multidrug resistance reversal agents

A series of substituted dibenzo[c,e]azepine-5-ones (7a-h) were synthesized and evaluated as P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) reversal agents. The most potent compound 7h could significantly and selectively enhance the chemo-sensitivity of drug-resistant K562/A02 cells to the cytotoxic effect of adriamycin (ADR) in a dose-dependent manner. Further studies indicated that 7h could markedly increase intracellular accumulation of both rhodamine 123 and ADR in K562/A02 cells and inhibit their efflux from the cells. And 7h had little effect on the levels of P-gp mRNA and protein in K562/A02 cells. These results suggest that the anti-MDR effect of 7h might be attributed to the inhibition of drug efflux function of P-gp, leading to the increased drug accumulation in K562/A02 cells, and thus the compound could be served as a lead for developing P-gp-mediated MDR reversal agents.     

P-糖蛋白在多药耐药的K562细胞转运苯妥英纳与卡马西平中的作用

研究证实,多药转运体与难治性癫痫耐药机制密切相关,P-糖蛋白在其中起重要作用．主要研究P-糖蛋白拮抗剂维拉帕米对P-糖蛋白过表达的K562细胞耐药性及细胞内苯妥英纳与卡马西平浓度的影响．首先建立了P-糖蛋白高表达的K562/Dox(阿霉素诱导)耐药细胞株,比较耐药细胞株和P-糖蛋白表达阴性的K562细胞株对苯妥英纳和卡马西平的耐药性,并观察给予维拉帕米后,耐药细胞内抗癫痫药物的浓度变化．结果发现,苯妥英纳和卡马西平对K562/Dox细胞株的半数抑制浓度(IC₅₀)明显高于K562细胞株,加入维拉帕米后,苯妥英纳和卡马西平对K562/Dox 细胞的IC₅₀明显下降,逆转倍数分别为2.5和1.5．进一步研究发现,K562/Dox细胞内苯妥英纳和卡马西平的浓度均显著少于其药敏K562细胞,仅分别为正常K562细胞的23.6%和32.2%．当加入维拉帕米后,K562/Dox细胞内抗癫痫药物浓度明显升高(P < 0.05)．由此证明,高表达的P-糖蛋白参与了细胞的药物转运,在难治性癫痫的耐药机制中扮演重要角色．     

Drug sequestration in cytoplasmic organelles does not contribute to the diminished sensitivity of anthracyclines in multidrug resistant K562 cells.

Cells that acquire multidrug resistance (MDR) are characterized by a decreased accumulation of a variety of drugs. In addition, sequestration of drugs in intracellular vesicles has often been associated with MDR. However, the nature and role of intracellular vesicles in MDR are unclear. We addressed the relationship between MDR and vesicular anthracycline accumulation in the erythroleukemia cell line K562 and a drug-resistant counterpart K562/ADR that overexpresses P-glycoprotein. We used four anthracyclines (all of which are P-glycoprotein substrates): daunorubicin and idarubicin, which have good affinity for DNA and as weak bases can accumulate inside acidic compartments; hydroxyrubicin, which binds to DNA but is uncharged at physiological or acidic pH and thus cannot accumulate in acidic compartments; and WP900, an enantiomer of daunorubicin, which is a weak DNA binder but has the same pKa and lipophilicity as daunorubicin. The intrinsic fluorescence of anthracyclines allowed us to use macro- and micro-spectrofluorescence, flow cytometry, and confocal microscopy to characterize their nuclear or intravesicular accumulation in living cells. We found that vesicular accumulation of daunorubicin, WP900 and idarubicin, containing a basic 3'-amine was predominantly restricted to lysosomes in both cell lines, that pH regulation of acidic compartments was not defective in human K562 cells, and that vesicular drug accumulation was much more pronounced in the parental tumor cell line than in the multidrug-resistant cells. These results indicate that vesicular anthracycline sequestration does not contribute to the diminished sensitivity to anthracyclines in multidrug-resistant K562 cells.     

Assessment of multidrug resistance reversal using dielectrophoresis and flow cytometry

In cancer, multidrug resistance (MDR) is the simultaneous resistance of tumor cells to different natural product anticancer drugs that have no common structure. This is an impediment to the successful treatment of many human cancers. A common correlate of MDR is the overexpression of a membrane protein, P-glycoprotein. Many studies have shown that MDR can be reversed after the use of substrate analogs, called MDR modulators. However, our understanding of MDR modulation is incomplete. In this article, we examine the electrical properties of the human leukemic cells (K562) and its MDR counterpart (K562AR) using dielectrophoresis and flow cytometry (with a membrane potential sensitive dye, DIOC5), both before and after treatment with XR9576 (a P-glycoprotein-specific MDR-reversal agent). The results show significant differences in the cytoplasmic conductivity between the cell lines themselves, but indicate no significant changes after modulation therapy. We conclude that the process of MDR modulation is not associated with changes in the electrical properties of cancer cells. Moreover, the results demonstrate that using the flow cytometry method alone, with MDR cells, may produce artifactual results--whereas in combination with dielectrophoresis, the results show the role of MDR modulators in preventing drug efflux in MDR cells.     

Up-regulation of P-glycoprotein expression by catalase via JNK activation in HepG2 cells

Overexpression of the MDR1 gene is one of the reasons for multidrug resistance (MDR). Some studies suggested that antioxidants could down-regulate MDR1 expression as a possible cancer treatment. In this report, we try to determine the effects of antioxidants (catalase or N-acetylcysteine [NAC]) on the regulation of intrinsic MDR1 overexpression in HepG2 cells. Adding catalase or N-acetylcysteine to the HepG2 culture led to a significant increase of MDR1 mRNA and P-glycoprotein drug transporter activity. After catalase or NAC treatment, a reduced intracellular reactive oxygen species (ROS) was observed. The JNK inhibitor SP600125 abolished the positive effects of catalase on drug transporter activity in a dose-dependent manner. Furthermore, the up-regulation of P-glycoprotein functions by catalase was only observed in HepG2 cells but not in other cell lines tested (MCF-7, A549, A431). These data suggested that catalase can up-regulate P-glycoprotein expression in HepG2 cells via reducing intracellular ROS, and JNK may mediate this process.     

mdr-1和bcl-2基因在K562/ADM多药耐药细胞中的共表达

为探讨肿瘤细胞多药耐药(MDR)形成的分子机理,本文观察了mdr-1、bcl-2和bax基因及其编码蛋白在人红白血病细胞株K562/ADM中的可能共表达。结果显示,在K562/ADM细胞中,在以mdr-1及P-gp过度表达为 特征的MDR形成时,其bcl-2及产物Bcl-2也过度表达,其中Bcl-2的表达阳性率约为相应敏感株K562的11倍;而Bax在二种细胞中均呈阳性表达,但无显著差异(P>0.05),提示bcl-2基因在mRNA和蛋白水平上的过度表达可能是K562/ADM细胞MDR形成时细胞凋亡耐受的分子基础。     

The AU-rich 3' untranslated region of human MDR1 mRNA is an inefficient mRNA destabilizer.

The human multidrug resistance gene MDR1 encodes a membrane-bound protein, referred to as P-glycoprotein, that acts as a pump to extrude toxins from cells. The 3' untranslated region (3'UTR) of the human MDR1 mRNA is very AU-rich (70%) and contains AU-rich sequences similar to those shown to confer rapid decay on c-myc, c-fos, and lymphokine mRNAs. We tested the ability of the MDR1 3'UTR to act as an mRNA destabilizing element in the human hepatoma cell line HepG2. The MDR1 mRNA has an intermediate half-life of 8 h in HepG2 cells compared to a half-life of 30 min for c-myc mRNA. The MDR1 mRNA half-life was prolonged to >20 h upon treatment with the protein synthesis inhibitor cycloheximide. We constructed expression vectors containing the human beta-globin coding region with the 3'UTR from either MDR1 or c-myc. The c-myc 3'UTR increased the decay of the chimeric mRNA, but the MDR1 3'UTR had no effect. We tested the ability of MDR1 3'UTR sequences to compete for interaction with AU-binding proteins in cell extracts; MDR1 RNA probes had a fivefold lower affinity for AU-binding proteins that interact with the c-myc AU-rich 3'UTR. Overall, our data suggest that the MDR1 3'UTR does not behave as an active destabilizing element in HepG2 cells.     

Reversal Effect of ST6GAL 1 on Multidrug Resistance in Human Leukemia by Regulating the PI3K/Akt Pathway and the Expression of P-gp and MRP1

β-galactoside α2, 6-sialyltransferse gene (ST6GAL) family has two members, which encode corresponding enzymes ST6Gal I and ST6Gal II. The present atudy was to investigate whether and how ST6GAL family involved in multidrug resistance (MDR) in human leukemia cell lines and bone marrow mononuclear cells (BMMC) of leukemia patients. Real-time PCR showed a high expression level of ST6GAL1 gene in both MDR cells and BMMCs (*P<0.05). Alternation of ST6GAL1 levels had a significant impact on drug-resistant phenotype changing of K562 and K562/ADR cells both in vitro and in vivo. However, no significant changes were observed of ST6GAL2 gene. Further data revealed that manipulation of ST6GAL1 modulated the activity of phosphoinositide 3 kinase (PI3K)/Akt signaling and consequently regulated the expression of P-glycoprotein (P-gp, *P<0.05) and multidrug resistance related protein 1 (MRP1, *P<0.05), which are both known to be associated with MDR. Therefore we postulate that ST6GAL1 is responsible for the development of MDR in human leukemia cells probably through medicating the activity of PI3K/Akt signaling and the expression of P-gp and MRP1.     

Overexpression of sorcin in multidrug resistant human leukemia cells and its role in regulating cell apoptosis

In an attempt to identify novel proteins involved in the emergence of multidrug resistance (MDR) in leukemia cells, we adopted a proteomics approach to analyze protein expression patterns in leukemia cell lines, K562, and its MDR counterpart, K562/A02. Combining high-resolution two-dimensional gel electrophoresis and mass spectrometry, we compared the protein expression profiles between K562 and K562/A02. A total number of 22 protein spots with altered abundances of more than 2-fold were detected and 14 proteins were successfully identified. Consistent with our previous observations by cDNA microarray, sorcin, a 22-kDa calcium-binding protein, was also identified by this proteomic approach with a 10.4-fold up-regulation in K562/A02 cells. Overexpression of sorcin protein in K562 cells by gene transfection led to significantly reduced cytosolic calcium level and increased resistance to cell apoptosis. Further, leukemia cell lines over-expressing sorcin also showed up-regulation of Bcl-2, along with decreased level of Bax. Taken together, our results suggest that sorcin plays an important role in the emergence of MDR in leukemia cells via regulating cell apoptosis pathways, thus may represent both a new MDR marker for prognosis and a good target for anti-MDR drug development.     

Multidrug resistance increment in a human colon carcinoma cell line by colchicine

The most important mechanism in drug resistance is the multidrug resistance (MDR) phenomenon. It is possible to select MDR cells by in vitro exposure to cytotoxic agents. The resistance is due to the hyperexpression of the P-glycoprotein (P-Gp) that take drugs out from the cells. In this study, a colchicine resistant subline (HCA-2/1cch) was selected from a human colon adenocarcinoma after a short period of drug exposure, as an in vitro model of drug resistance selection. These cells showed cross-resistance to other drugs, which were not present in the medium during selection. The relative resistance was 3.32 for colchicine, 3.15 for vinblastine, 2.62 for vincristine and 5.22 for mitomycin C. P-glycoprotein levels were assayed by flow cytometry. It was found that a significant increase of 2.35 and 1.59 had occurred in the peak and mean channel of fluorescence, respectively, indicating an increment of P-glycoprotein expression in relation to the parental line. Moreover, verapamil (10 microg/ml) produced a partial reversion of multidrug resistance. The sensitisation rates were 7.41 for colchicine, 1.25 for vinblastine, 2.36 for vincristine and 1.17 for mitomycin C. The data obtained suggest that colchicine exposure period (10 weeks) and dose (0.5 microg/ml) assayed were sufficient to produce an increment in multidrug resistance. This resistance could be due to higher level of P-Gp expression.     

Regulation of MDR1 gene expression in multidrug-resistant cancer cells is independent from YB-1

The MDR1 gene encoded transmembrane ABC-transporter MDR1/P-glycoprotein can mediate the phenotype of multidrug resistance (MDR), a major obstacle in the clinical management of cancer patients. It was hypothesized that YB-1 is a fundamental regulatory factor of the MDR1 gene in tumor cells and can therewith enhance drug resistance. To analyze the potential impact of YB-1 in MDR cancer cells, two specific anti-YB-1 small interfering RNAs (siRNAs) were designed for transient triggering the gene-silencing RNA interference (RNAi) pathway in the MDR cell lines EPG85-257RDB and EPP85-181RDB as well as in their drug-sensitive counterparts EPG85-257P and EPP85-181P. Since both siRNAs showed biological activity, for stable inhibition of YB-1 corresponding tetracycline-inducible short hairpin RNA (shRNA)-encoding expression vectors were designed. By treatment of the cancer cells with these constructs, the expression of the targeted YB-1 encoding mRNA and protein was completely inhibited following tetracycline exposure. These gene-silencing effects were not accompanied by modulation of the MDR1 expression or by reversal of the drug-resistant phenotype. In conclusion, the data demonstrate the utility of the analyzed RNAs as powerful laboratory tools and indicate that YB-1 is not involved in the regulation of the MDR1 gene or the development of the drug-resistant phenotype in MDR cancer cells.     

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.     

Changes in the Expression of miR-381 and miR-495 Are Inversely Associated with the Expression of the MDR1 Gene and Development of Multi-Drug Resistance

Multidrug resistance (MDR) frequently develops in cancer patients exposed to chemotherapeutic agents and is usually brought about by over-expression of P-glycoprotein (P-gp) which acts as a drug efflux pump to reduce the intracellular concentration of the drug(s). Thus, inhibiting P-gp expression might assist in overcoming MDR in cancer chemotherapy. MiRNAome profiling using next-generation sequencing identified differentially expressed microRNAs (miRs) between parental K562 cells and MDR K562 cells (K562/ADM) induced by adriamycin treatment. Two miRs, miR-381 and miR-495, that were strongly down-regulated in K562/ADM cells, are validated to target the 3’-UTR of the MDR1 gene. These miRs are located within a miR cluster located at chromosome region 14q32.31, and all miRs in this cluster appear to be down-regulated in K562/ADM cells. Functional analysis indicated that restoring expression of miR-381 or miR-495 in K562/ADM cells was correlated with reduced expression of the MDR1 gene and its protein product, P-gp, and increased drug uptake by the cells. Thus, we have demonstrated that changing the levels of certain miR species modulates the MDR phenotype in leukemia cells, and propose further exploration of the use of miR-based therapies to overcome MDR.     

Identification of residues in the first cytoplasmic loop of P-glycoprotein involved in the function of chimeric human MDR1-MDR2 transporters.

The human MDR1 gene encodes the multidrug transporter (P-glycoprotein), a multidrug efflux pump. The highly homologous MDR2 gene product does not appear to be a functional multidrug pump. We have constructed a chimeric protein in which the first intracytoplasmic loop and the third and fourth transmembrane domains of the MDR1 protein were replaced by the analogous region of MDR2. Substitution of the MDR2 sequences encompassing amino acid residues 140 to 229 resulted in 17 amino acid changes, 10 in the intracytoplasmic loop (amino acids 141-188) and 7 in the transmembrane regions. This chimeric protein was expressed on the surface of NIH 3T3 cells where it bound [3H]azidopine but did not confer drug resistance. When only 4 residues, 165, 166, 168, and 169, were changed back to MDR1 amino acids, a functional drug transporter was recovered. When residues 165, 166, 168, and 169 from MDR2 were substituted into a functional MDR1 cDNA, the resulting construction was not able to confer drug resistance. These results indicate that the major functional differences between MDR1 and MDR2 in this region of P-glycoprotein reside in a small segment of the first intracytoplasmic loop. We also independently analyzed the effect of replacing Asn183 of MDR1 with Ser which occurs in MDR2. Substitution of Ser at position 183 in combination with Val at position 185 in P-glycoprotein resulted in a relative increase in resistance to actinomycin D, vinblastine, and doxorubicin in transfected NIH 3T3 cells. These results emphasize the importance of the first intracytoplasmic loop in P-glycoprotein in determining function and relative drug specificity of the transporter.