Iron N-(2-hydroxy acetophenone) glycinate (FeNG), a non-toxic glutathione depletor circumvents doxorubicin resistance in Ehrlich ascites carcinoma cells in vivo

Multidrug resistance-associated protein 1 (MRP1) reduces intracellular anticancer drug accumulation either by co transporting them with glutathione (GSH) or extruding drug-GSH conjugates outside of the cell. Thus, MRP1 confers multidrug resistance (MDR) and worsen successful chemotherapeutic treatment against cancer. Although the exact mechanism of MRP1 involved in MDR remains unknown, the elevated level of intracellular GSH is considered as a key factor responsible for MDR in cancer. Hence the quest for non-toxic molecules that are able to deplete intracellular GSH has profound importance to subdue MDR. The present preclinical study depicts the resistance reversal potentiality of an iron complex; viz. Ferrous N-(2-hydroxy acetophenone) glycinate (FeNG) developed by us in doxorubicin resistant Ehrlich ascites carcinoma (EAC/Dox) cells. FeNG potentiate cytotoxic effect of doxorubicin on EAC/Dox cells ex vivo and also increases the survivability EAC/Dox bearing Swiss albino mice in vivo as well. Moreover, in vivo administration of FeNG significantly depletes intracellular GSH with ensuant increase in doxorubicin concentration in EAC/Dox cells without alternation of MRP1 expression. In addition, intra-peritoneal (i.p.) application of FeNG in normal or EAC/Dox bearing mice does not cause any systemic toxicity in preliminary trials in mouse Ehrlich ascites carcinoma model. Therefore, the present report provides evidence that FeNG may be a promising new resistance modifying agent against drug resistant cancers.     

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．     

Cytotoxicity, intracellular distribution and uptake of doxorubicin and doxorubicin coupled to cell-penetrating peptides in different cell lines: A comparative study

One of the major obstacles which are opposed to the success of anticancer treatment is the cell resistance that generally develops after administration of commonly used drugs. In this study, we try to overcome the tumour cell resistance of doxorubicin (Dox) by developing a cell-penetrating peptide (CPP)-anticancer drug conjugate in aim to enhance its intracellular delivery and that its therapeutic effects. For this purpose, two cell-penetrating peptides, penetratin (pene) and tat, derived from the HIV-1 TAT protein, were chemically conjugated to Dox. The cytotoxicity, intracellular distribution and uptake were accessed in CHO cells (Chinese Hamster Ovarian carcinoma cells), HUVEC (Human Umbilical Vein Endothelial Cells), differentiated NG108.15 neuronal cell and breast cancer cells MCF7drug-sensitive or MDA-MB 231 drug-resistant cell lines. The conjugates showed different cell killing activity and intracellular distribution pattern by comparison to Dox as assessed respectively by MTT-based colorimetric cellular cytotoxicity assay, confocal fluorescence microscopy and FACS analysis. After treatment with 3 μM with Dox-CPPs for 2 h, pene increase the Dox cytotoxicity by 7.19-fold in CHO cells, by 11.53-fold in HUVEC cells and by 4.87-fold in MDA-MB 231 cells. However, cytotoxicity was decreased in NG108.15 cells and MCF7. Our CPPs-Dox conjugate proves the validity of CPPs for the cytoplasmic delivery of therapeutically useful molecules and also a valuable strategy to overcome drug resistance.     

Separate roles for the Golgi apparatus and lysosomes in the sequestration of drugs in the multidrug-resistant human leukemic cell line HL-60

The sequestration of drugs away from cellular target sites into cytoplasmic organelles of multidrug-resistant (MDR) cancer cells has been recently shown to be a cause for ineffective drug therapy. This process is poorly understood despite the fact that it has been observed in a large number of MDR cancer cell lines. Analysis of drug sequestration in these cells has traditionally been done using fluorescent anthracycline antibiotics (i.e. daunorubicin, doxorubicin). This narrow selection of substrates has resulted in a limited understanding of sequestration mechanisms and the intracellular compartments that are involved. To better characterize this phenotype, we chose to examine the sequestration of molecules having different acid/base properties in the MDR HL-60 human leukemic cell line. Here we show that weakly basic drug daunorubicin is sequestered into lysosomes according to a pH partitioning type mechanism, whereas sulforhodamime 101, a zwitterionic molecule, is sequestered into the Golgi apparatus through a drug transporter-mediated process. Quantitative intracellular pH measurements reveal that the lysosome-tocytosol pH gradient is expanded in the MDR line. Moreover, the MDR cells overexpress the multidrug resistance-related protein (MRP1), which is localized to the Golgi apparatus. These results demonstrate, for the first time, that two distinct mechanisms for intracellular compartmentalization are operational in a single MDR cell line.     

Swelling-activated chloride channels in multidrug-sensitive and - resistant cells

Resistance to chemotherapeutic agents in neoplastic cells is often mediated by expression of P-glycoprotein, which functions as a drug- efflux pump for a broad range of substrates. We have used a combination of patch clamp and video-imaging techniques to examine the expression and drug-efflux function of P-glycoprotein and to determine the possible correlation with swelling-activated chloride channels in drug- sensitive and -resistant cell lines. Two pairs of cell lines were used in these experiments: (a) control NIH-3T3 cells and a corresponding MDR1-transfectant; and (b) control 8226 myeloma cells and a derivative cell line selected for resistance to chemotherapeutic agents. Control cells lacked detectable P-glycoprotein expression based on Western blotting, immunofluorescence staining with a specific monoclonal antibody, and a functional assay of rhodamine-123 (R123) efflux. Resistant cells expressed P-glycoprotein at high levels and rapidly exported R123. During whole-cell recording using either hyperosmotic pipette solution or hypoosmotic Ringer solution, cell swelling was accompanied by Cl- channel opening in all four cell lines. The rates of induction, biophysical properties and magnitudes of Cl conductance (gCl) were indistinguishable between control and corresponding multidrug-resistant cells: gCl reached 0.96 +/- 0.31 (n = 14) and 0.83 +/- 0.31 nS/pF (mean +/- SD; n = 31) in NIH-3T3 and NIH-3T3/MDR cells, respectively; and 0.31 +/- 0.20 (n = 9) and 0.37 +/- 0.22 nS/pF (n = 7) in 8226 and 8226/Dox40 cells, respectively. gCl exhibited moderate outward rectification in symmetrical Cl- solutions, with a rectification ratio of 1.4 at +/- 50 mV. Cl- channels slowly closed during strong depolarization beyond +60 mV. Using video-imaging techniques with SPQ as a fluorescent probe, we monitored Cl(-)-channel opening in intact drug-sensitive and -resistant cells. gCl, measured either with whole-cell recording or SPQ imaging, was blocked by DIDS (voltage-dependent Kd < 50 microM at +40 mV), NPPB (Kd approximately 30 microM), and tamoxifen (complete and irreversible block approximately 10 microM). None of these blockers inhibited R123 efflux. NPPB accelerated R123 efflux, an effect that was mimicked by CCP, a mitochondrial uncoupler. In contrast, verapamil selectively blocked R123 efflux (Kd = 0.3 to 0.5 microM); 10 microM left gCl unaltered. Induction of gCl was not affected by vincristine or doxorubicin in the pipette solution. Moreover, the rate of R123 efflux did not change during cell swelling. We conclude that P-glycoprotein and swelling- activated chloride channels function independently and are separable by expression and by pharmacological sensitivities.     

Clitocine Reversal of P-Glycoprotein Associated Multi-Drug Resistance through Down-Regulation of Transcription Factor NF-κB in R-HepG2 Cell Line

Multidrug resistance(MDR)is one of the major reasons for failure in cancer chemotherapy and its suppression may increase the efficacy of therapy. The human multidrug resistance 1 (MDR1) gene encodes the plasma membrane P-glycoprotein (P-gp) that pumps various anti-cancer agents out of the cancer cell. R-HepG2 and MES-SA/Dx5 cells are doxorubicin induced P-gp over-expressed MDR sublines of human hepatocellular carcinoma HepG2 cells and human uterine carcinoma MES-SA cells respectively. Herein, we observed that clitocine, a natural compound extracted from Leucopaxillus giganteus, presented similar cytotoxicity in multidrug resistant cell lines compared with their parental cell lines and significantly suppressed the expression of P-gp in R-HepG2 and MES-SA/Dx5 cells. Further study showed that the clitocine increased the sensitivity and intracellular accumulation of doxorubicin in R-HepG2 cells accompanying down-regulated MDR1 mRNA level and promoter activity, indicating the reversal effect of MDR by clitocine. A 5'-serial truncation analysis of the MDR1 promoter defined a region from position -450 to -193 to be critical for clitocine suppression of MDR1. Mutation of a consensus NF-κB binding site in the defined region and overexpression of NF-κB p65 could offset the suppression effect of clitocine on MDR1 promoter. By immunohistochemistry, clitocine was confirmed to suppress the protein levels of both P-gp and NF-κB p65 in R-HepG2 cells and tumors. Clitocine also inhibited the expression of NF-κB p65 in MES-SA/Dx5. More importantly, clitocine could suppress the NF-κB activation even in presence of doxorubicin. Taken together; our results suggested that clitocine could reverse P-gp associated MDR via down-regulation of NF-κB.     

Increased ABCB1 Expression in TP-110-Resistant RPMI-8226 Cells

TP-110, a novel proteasome inhibitor, has been found to possess potent growth inhibition in human multiple myeloma cells. To enhance its therapeutic effects, we established TP-110-resistant RPMI-8226 (RPMI-8226/TP-110) cells and elucidated their resistance mechanisms. The IC₅₀ value for TP-110 cytotoxicity in the RPMI-8226/TP-110 cells was about 10-fold higher than that of the parental sensitive cells. The RPMI-8226/TP-110 cells exhibited distinct drug resistance to other proteasome inhibitors. Furthermore, they showed high cross-resistance to the cytotoxic effects of doxorubicin, etoposide, taxol, and vincristine. P-glycoprotein (MDR1), encoded by ABCB1, was elevated in the RPMI-8226/TP-110 cells, and the MDR1 inhibitor verapamil overcame their resistance to TP-110. The results of DNA microarray and RT-PCR suggested that the expression of ABCB1 is significantly elevated in RPMI-8226/TP-110 cells. This indicates that resistance in RPMI-8226/TP-110 cells is involved in the expression of P-glycoprotein, a drug-efflux pump.     

Analysis of the steady-state and initial rate of doxorubicin efflux from a series of multidrug-resistant cells expressing different levels of P-glycoprotein.

Continuous monitoring of fluorescence (CMF) has been used to examine doxorubicin efflux from intact human myeloma cells. The time resolution of these measurements has enabled detailed comparison of the initial rates of efflux for the drug-sensitive myeloma line RPMI 8226 and a series of sequentially derived multidrug-resistant (MDR) lines expressing different amounts of human MDR protein (P-glycoprotein). Cells that are 3-, 10-, 60-, or 120-fold resistant to doxorubicin export approximately 10, 20, 30, or 33% more doxorubicin than the parental sensitive cells, respectively, when all are preloaded to the same level of total intracellular drug. Remarkably, however, when cells are loaded to the same level of exchangeable drug the initial rates of efflux are found to be virtually identical. This agreement between rates is apparently not dependent on the drug concentration. Approximately 50% of the increase in the steady-state level of doxorubicin efflux for the resistant cells is abolished upon glucose starvation. However, surprisingly, the apparent initial rates of efflux from the treated and untreated cells are found to be virtually the same. Pretreatment of the resistant cells with verapamil reduces the steady-state level of efflux but increases the apparent initial rate at some concentrations. Conversely, vincristine does not alter steady state but slows the initial rate of efflux from both sensitive and resistant cells by approximately the same extent. Finally, quite interestingly, a nearly linear relationship between pHi and relative steady state of efflux is found for the series of cell lines. These data are interpreted in terms of existing models for MDR.     

Multidrug-resistance (MDR) phenotype of human osteosarcoma cells evaluated by quantitative morphological and electron microscopy analyses

Summary— Multidrug-resistant (MDR) variants of a human osteosarcoma cell line (U-2 OS) have been recently obtained by continuous exposure to doxorubicin (DX). The growth and phenotypic characteristics of these cell lines have been demonstrated to be related to the level of expression of P-glycoprotein. In this work, the morphological changes associated with MDR have been evaluated by quantitative image analysis and transmission electron microscopy. Resistant cells present morphological changes with respect to sensitive cells at both cytoplasmic and nuclear level. Some of these changes appear to be related to the degree of resistance but not to the direct presence of DX, since deprived cells maintain some modified characters, while others are partly lost. These findings suggest that DX exposure affects cell metabolism causing progressive changes of the cell morphotype.     

Reversal of cisplatin and multidrug resistance by ribozyme-mediated glutathione suppression

gamma-Glutamylcysteine synthetase (gamma-GCS) is a key enzyme in glutathione (GSH) synthesis, and is thought to play a significant role in intracellular detoxification, especially of anticancer drugs. Increased levels of GSH are commonly found in the drug-resistant human cancer cells. We designed a hammerhead ribozyme against gamma-GCS mRNA (anti-gamma-GCS Rz), which specifically down-regulated gamma-GCS gene expression in the HCT-8 human colon cancer cell line. The aim of this study was to reverse the cisplatin and multidrug resistance for anticancer drugs. The cisplatin-resistant HCT-8 cells (HCT-8DDP cells) overexpressed MRP and MDR1 genes, and showed resistance to not only cisplatin (CDDP), but also doxorubicin (DOX) and etoposide (VP-16). We transfected a vector expressing anti-gamma-GCS Rz into the HCT-8DDP cells (HCT-8DDP/Rz). The anti-gamma-GCS Rz significantly suppressed MRP and MDR, and altered anticancer drug resistance. The HCT-8DDP/Rz cells were more sensitive to CDDP, DOX and VP-16 by 1.8-, 4.9-, and 1.5-fold, respectively, compared to HCT-8DDP cells. The anti-gamma-GCS Rz significantly down-regulated gamma-GCS gene expression as well as MRP/MDR1 expression, and reversed resistance to CDDP, DOX and VP-16. These results suggested that gamma-GCS plays an important role in both cisplatin and multidrug resistance in human cancer cells.     

Inhibition of ABC-transporter(s)' function in non-small cell lung cancer cells by platinum drugs]

With an account of the literature data that platinum drugs react with many cellular targets, including ATP and proteins, the authors suggested that disturbance of the function of energy-dependent ABC-transporters (markers of multidrug resistance, MDR) under the effect of platinum drugs could be a cause of increased efficacy of MDR agents (agents, MDR to which is developed by the classical mechanism) when used in combination with platinum drugs even in the treatment of multidrug resistant lung cancer. The cisplatin and carboplatin effect on accumulation of MDR doxorubicin in cells of non-small cell cancer was studied by flow cytometry with the use of biopsy specimens. The MDR phenotype of the tumors was determined by a change in doxorubicin intracellular accumulation under the action of the ABC-transporter(s)' inhibitors: verapamil and genistein (specific inhibitors of Pgp and MRP respectively) and sodium azide (an inhibitor of all energy-dependent ABC-transporters). The MDR phenotypes, i.e. Pgp-MRP+ or Pgp+MRP+, were detected in all the tumors investigated. Two types of changes in doxorubicin intracellular accumulation under the action of the inhibitors and the platinum drugs were shown: (a) an increase in doxorubicin cytoplasmic accumulation and (b) a change in subcellular distribution of the anthracycline (increased accumulation of doxorubicin in the cell nucleus and its higher binding to DNA). Cisplatin and carboplatin had an inhibitory effect on ABC-transporter(s) in all the tumors investigated but the effect of carboplatin was less pronounced. It was concluded that cisplatin and carboplatin stimulation of doxorubicin intracellular accumulation, as well as a change in subcellular distribution of the anthracycline under the action of the platinum drugs (increased doxorubicin accumulation in the cell nucleus) in multidrug resistant lung tumors could be at least partly explained by inhibition of the MDR transporter(s)' function. The results could provide a basis for the use of the sequential combination cisplatin (or carboplatin)-->doxorubicin in the treatment of multidrug resistant lung cancer.     

P-glycoprotein mediates resistance to A3 adenosine receptor agonist 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-n-methyluronamide in human leukemia cells

We studied effects of 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA) on apoptosis induction in the K562/Dox cell line, which overexpressed P-glycoprotein (P-gp, ABCB1, MDR1). We found that the K562/Dox cell line was significantly more resistant to Cl-IB-MECA than the maternal cell line K562, which did not express P-gp. Although both cell lines expressed the A3 adenosine receptor (A3AR), cytotoxic effects of Cl-IB-MECA were not prevented by its selective antagonist MRS1523 (3-propyl-6-ethyl-5-[(ethylthio)carbonyl]-2 phenyl-4-propyl-3-pyridine carboxylate). Analysis of cell extracts revealed that the intracellular level of Cl-IB-MECA was significantly lower in the K562/Dox cell line than in the maternal cell line K562. The downregulation of P-gp expression using shRNA targeting ABCB1 gene led to increased intracellular level of Cl-IB-MECA and restored cell sensitivity to this drug. Similarly, valspodar (PSC-833), a specific inhibitor of P-gp, restored sensitivity of the K562/Dox cell line to Cl-IB-MECA with concomitant increase of intracellular level of Cl-IB-MECA in the resistant cell line, while it affected cytotoxicity of Cl-IB-MECA in the sensitive cell line only marginally. An enzyme based assay provided evidence for interaction of P-gp with Cl-IB-MECA. We further observed that cytotoxic effects of Cl-IB-MECA could be augmented by activation of extrinsic cell death pathway by Apo-2L (TRAIL) but not FasL or TNF-α. Our results revealed that Cl-IB-MECA induced an increase in expression of TRAIL receptors in K562 cells, which could sensitize cells to apoptosis induction via an extrinsic cell death pathway. Importantly, these effects were inversely related to P-gp expression. In addition, MRS1523 did not affect Cl-IB-MECA induced expression of TRAIL receptors.     

Resveratrol enhances chemosensitivity of doxorubicin in multidrug-resistant human breast cancer cells via increased cellular influx of doxorubicin

Background

Multidrug resistance is a major problem in the treatment of breast cancer, and a number of studies have attempted to find an efficient strategy with which to overcome it. In this study, we investigate the synergistic anticancer effects of resveratrol (RSV) and doxorubicin (Dox) against human breast cancer cell lines.

Methods

The synergistic effects of RSV on chemosensitivity were examined in Dox-resistant breast cancer (MCF-7/adr) and MDA-MB-231 cells. In vivo experiments were performed using a nude mouse xenograft model to investigate the combined sensitization effect of RSV and Dox.

Results and conclusion

RSV markedly enhanced Dox-induced cytotoxicity in MCF-7/adr and MDA-MB-231 cells. Treatment with a combination of RSV and Dox significantly increased the cellular accumulation of Dox by down-regulating the expression levels of ATP-binding cassette (ABC) transporter genes, MDR1, and MRP1. Further in vivo experiments in the xenograft model revealed that treatment with a combination of RSV and Dox significantly inhibited tumor volume by 60%, relative to the control group.

General significance

These results suggest that treatment with a combination of RSV and Dox would be a helpful strategy for increasing the efficacy of Dox by promoting an intracellular accumulation of Dox and decreasing multi-drug resistance in human breast cancer cells.     

Multidrug-resistance phenotype of a subpopulation of T-lymphocytes without drug selection

Multidrug-resistant (MDR) cells demonstrate the increased activity of the membrane transport system performing efflux of diverse lipophylic drugs and fluorescent dyes from the cells. In order to detect MDR cells we have developed a simple test consisting of three steps: staining of the cells with fluorescent dye rhodamine 123, incubation in the dye-free medium and, finally, detection by fluorescence microscopy of the cells that have lost accumulated dye. The experiments with B-lymphoma cell lines with different degrees of MDR have shown that the cell fluorescence after the poststaining incubation is indeed inversely proportional to the degree of resistance. Application of this testing procedure to normal human or mouse leukocytes revealed the presence of the cells rapidly losing the dye in these populations. Cell fractionation experiments have shown that there are T-lymphocytes (most T-killers/suppressors and a part of T-helpers) that demonstrate rapid efflux of rhodamine 123. This characteristic was detected also in T-killer clones and cell line and in some T-lymphomas. The inhibitors of the MDR transport system, reserpine and verapamil, blocked the efflux of the dye from these cells. Rhodamine-losing T-lymphoma contained large amounts of the mRNA coding P-glycoprotein, the MDR efflux pump, and demonstrated increased resistance to rhodamine 123, gramicidin D, colchicine, and vincristine, the drugs belonging to the cross-resistance group for the MDR cells. The role of the increased activity of the MDR membrane transport system in T-lymphocytes is discussed.     

A novel flavonoid from Fissistigma cupreonitens, 5‑hydroxy‑7,8‑dimethoxyflavanone,competitively inhibited the efflux function of human P-glycoprotein and reversed cancer multi-drug resistance

BackgroundP-glycoprotein (P-gp) over-expression plays a vital role in not only systemic drug bioavailability but also cancer multi-drug resistance (MDR). Develop functional inhibitors of P-gp can conquer both problems.Purpose and study designThe aim of the present study was to research the P-gp modulating effects and MDR reversing ability of a novel flavonoid from Fissistigma cupreonitens, the underlying inhibitory mechanisms were further elucidated as well.MethodsCalcein-AM, rhodamine 123, and doxorubicin were fluorescent substrates for the evaluation of P-gp inhibitory function and detailed drug binding modes. Docking simulation was performed to reveal the in silico molecular bonding. ATPase assay and MDR1 shift assay were adopted to reveal the ATP consumption and conformational change of P-gp. The MDR reversing effects were demonstrated through cytotoxicity, cell cycle, and apoptosis analyses.Results5‑hydroxy‑7,8‑dimethoxyflavanone inhibited the efflux of rhodamine 123 and doxorubicin in a competitive manner, and increased the intracellular fluorescence of calcein at a concentration as low as 2.5 μg/ml. 5‑hydroxy‑7,8‑dimethoxyflavanone slightly changed P-gp's conformation and only stimulated ATPase at very high concentration (100 μg/ml). The docking results showed that 5‑hydroxy‑7,8‑dimethoxyflavanone and verapamil exhibited similar binding affinity to P-gp. The MDR reversing effects were prominent in the vincristine group, the reversal folds were 23.01 and 13.03 when combined with 10 μg/ml 5‑hydroxy‑7,8‑dimethoxyflavanone in the P-gp over-expressing cell line (ABCB1/Flp-In™-293) and MDR cancer cell line (KB/VIN), respectively.ConclusionThe present study demonstrated that 5‑hydroxy‑7,8‑dimethoxyflavanone was a novel effective flavonoid in the P-gp efflux inhibition and in vitro cancer MDR reversion.     

Chloride channel-3 mediates multidrug resistance of cancer by upregulating P-glycoprotein expression

Chloride channel-3 (ClC-3), a member of the ClC family of voltage-gated Cl⁻ channels, is involved in the resistance of tumor cells to chemotherapeutic drugs. Here, we report a new mechanism for ClC-3 in mediating multidrug resistance (MDR). ClC-3 was highly expressed in the P-glycoprotein (P-gp)-dependent human lung adenocarcinoma cell line (A549)/paclitaxel (PTX) and the human breast carcinoma cell line (MCF-7)/doxorubicin (DOX) resistant cells. Changes in the ClC-3 expression resulted in the development of drug resistance in formerly drug-sensitive A549 or MCF-7 cells, and drug sensitivity in formerly drug-resistant A549/Taxol and MCF-7/DOX cells. Double transgenic MMTV-PyMT/CLCN3 mice with spontaneous mammary cancer and ClC-3 overexpression demonstrated drug resistance to PTX and DOX. ClC-3 expression upregulated the expression of MDR1 messenger RNA and P-gp by activating the nuclear factor-κB (NF-κB)-signaling pathway. These data suggest that ClC-3 expression in cancer cells induces MDR by upregulating NF-κB-signaling-dependent P-gp expression involving another new mechanism for ClC-3 in the development of drug resistance of cancers.