Changes in lipid and protein constituents of rafts and caveolae in multidrug resistant cancer cells and their functional consequences

The carcinogenic process involves a complex series of genetic and biochemical changes that enables transformed cells to proliferate, migrate to secondary sites and, in some cases, acquire mechanisms that make cancer cells resistant to chemotherapy. This phenomenon in its most common form is known as multidrug resistance (MDR). It is usually mediated by overexpression of P-glycoprotein (P-gp) or other plasma membrane ATPases that export cytotoxic drugs used in chemotherapy, thereby reducing their efficacy. However, additional adaptive changes are likely to be required in order to confer a full MDR phenotype. Recent studies have shown that acquisition of MDR is accompanied by up-regulation of lipids and proteins that constitute lipid rafts and caveolar membranes, notably glucosylceramide and caveolin. These changes may be related to the fact that in MDR cells a significant fraction of cellular P-gp is associated with caveolin-rich membrane domains, they may be involved in drug transport and they could have an impact on drug-induced apoptosis and on the phenotypic transformation of MDR cancer cells.     

Reversal of P-glycoprotein (P-gp) mediated multidrug resistance in colon cancer cells by cryptotanshinone and dihydrotanshinone of Salvia miltiorrhiza

ObjectiveMultidrug resistance (MDR) of cancer cells to a broad spectrum of anticancer drugs is an obstacle to successful chemotherapy. Overexpression of P-glycoprotein (P-gp), an ATP-binding cassette (ABC) membrane transporter, can mediate the efflux of cytotoxic drugs out of cancer cells, leading to MDR and chemotherapy failure. Thus, development of safe and effective P-gp inhibitors plays an important role in circumvention of MDR. This study investigated the reversal of P-gp mediated multidrug resistance in colon cancer cells by five tanshinones including tanshinone I, tanshinone IIA, cryptotanshinone, dihydrotanshinone and miltirone isolated from Salvia miltiorrhiza (Danshen), known to be safe in traditional Chinese medicine.MethodsThe inhibitory effects of tanshinones on P-gp function were compared using digoxin bi-directional transport assay in Caco-2 cells. The potentiation of cytotoxicity of anticancer drugs by effective tanshinones were evaluated by MTT assay. Doxorubicin efflux assay by flow cytometry, P-gp protein expression by western blot analysis, immunofluorescence for P-gp by confocal microscopy, quantitative real-time PCR and P-gp ATPase activity assay were used to study the possible underlying mechanisms of action of effective tanshinones.ResultsBi-directional transport assay showed that only cryptotanshinone and dihydrotanshinone decreased digoxin efflux ratio in a concentration-dependent manner, indicating their inhibitory effects on P-gp function; whereas, tanshinone I, tanshinone IIA and miltirone had no inhibitory effects. Moreover, both cryptotanshinone and dihydrotanshinone could potentiate the cytotoxicity of doxorubicin and irinotecan in P-gp overexpressing SW620 Ad300 colon cancer cells. Results from mechanistic studies revealed that these two tanshinones increased intracellular accumulation of the P-gp substrate anticancer drugs, presumably by down-regulating P-gp mRNA and protein levels, and inhibiting P-gp ATPase activity.ConclusionsTaken together, these findings suggest that cryptotanshinone and dihydrotanshinone could be further developed for sensitizing resistant cancer cells and used as an adjuvant therapy together with anticancer drugs to improve their therapeutic efficacies for colon cancer.     

Phosphorylation of RNA helicase A by DNA-dependent protein kinase is indispensable for expression of the MDR1 gene product P-glycoprotein in multidrug-resistant human leukemia cells

Development of multidrug resistance (MDR) in cancer frequently involves overexpression of the MDR1 gene product P-glycoprotein (P-gp), a drug transporter which severely impedes the efficacy of chemotherapy. Because intensive efforts to identify therapeutics that reverse MDR by inhibiting the drug transport activity of P-gp have not yet met with success, we have focused on the alternative strategy of targeting MDR1 promoter activation to knockdown P-gp expression in cancer cells. We recently identified RNA helicase A (RHA) inhibition as a rational strategy to downregulate P-gp in leukemia cells by showing that RHA RNAi knockdown abrogated P-gp expression in MDR variants of human leukemia HL-60 cells. In that report, we also demonstrated that RHA activated the MDR1 promoter in the MDR variant cells but not in the drug-sensitive counterpart. This led us to hypothesize that P-gp induction by RHA required cooperation with another factor present only in the MDR variants. Here, we identify the RHA cooperating factor as DNA-PK catalytic subunit (cs), and we show that DNA-PKcs resides with RHA at the MDR1 promoter in a multiprotein complex. Furthermore, targeted DNA-PKcs inhibition abrogated P-gp expression in the MDR variant cells. We demonstrate that constitutive multisite RHA phosphorylation producing retarded migration in SDS-PAGE is catalyzed by DNA-PKcs in the MDR variants, and does not occur in the parental cells, which are DNA-PKcs deficient. The indispensable role played by DNA-PK in P-gp overexpression in MDR leukemia cells in this report identifies targeted DNA-PK inhibition as a rational strategy to reverse drug resistance in cancer.     

Different modalities of intercellular membrane exchanges mediate cell-to-cell p-glycoprotein transfers in MCF-7 breast cancer cells

Multi-drug resistance (MDR) is a phenomenon by which tumor cells exhibit resistance to a variety of chemically unrelated chemotherapeutic drugs. The classical form of multidrug resistance is connected to overexpression of membrane P-glycoprotein (P-gp), which acts as an energy dependent drug efflux pump. P-glycoprotein expression is known to be controlled by genetic and epigenetic mechanisms. Until now processes of P-gp gene up-regulation and resistant cell selection were considered sufficient to explain the emergence of MDR phenotype within a cell population. Recently, however, "non-genetic" acquisitions of MDR by cell-to-cell P-gp transfers have been pointed out. In the present study we show that intercellular transfers of functional P-gp occur by two different but complementary modalities through donor-recipient cells interactions in the absence of drug selection pressure. P-glycoprotein and drug efflux activity transfers were followed over 7 days by confocal microscopy and flow cytometry in drug-sensitive parental MCF-7 breast cancer cells co-cultured with P-gp overexpressing resistant variants. An early process of remote transfer was established based on the release and binding of P-gp-containing microparticles. Microparticle-mediated transfers were detected after only 4 h of incubation. We also identify an alternative mode of transfer by contact, consisting of cell-to-cell P-gp trafficking by tunneling nanotubes bridging neighboring cells. Our findings supply new mechanistic evidences for the extragenetic emergence of MDR in cancer cells and indicate that new treatment strategies designed to overcome MDR may include inhibition of both microparticles and Tunneling nanotube-mediated intercellular P-gp transfers.     

Induction of autoantibodies to murine P-glycoprotein: consequences on drug sensitivity in MDR cancer cells and on the expression of mdr genes in organs

Overexpression of the 170 kDa plasma membrane P-glycoprotein (P-gp) represents the most common MDR mechanism in chemotherapy. In this work, specific autoantibodies to fragments from extracellular loops 1, 2, and 4 of the murine MDR1 P-gp were elicited in mice using synthetic palmitoylated peptides reconstituted in liposomes and alum. The highest IgG level was observed after the third immunization and the immune response against lipopeptides was still detected more than 200 days after immunizations. Immunocytochemichal studies revealed that these antibodies were specific for P-gp. When incubated with P-gp-expressing MDR cell lines, serum from immunized mice restored sensitivity to either doxorubicin or vinblastine, or had no effect in a cell type specific manner, suggesting that several mechanisms may occur in the establishment of the MDR phenotype. The expression of mdr1 and mdr3 genes was unchanged in organs from mice immunized with palmitoylpeptides grafted on liposomes. These results suggest that the induction of autoantibodies to P-gp is a safe strategy to overcome MDR in cancer chemotherapy.     

Modulation of mitochondrial permeability transition pore affects multidrug resistance in human hepatocellular carcinoma cells

Multidrug resistance (MDR) is a critical problem in the chemotherapy of cancers. Human hepatocellular carcinoma (HCC) responds poorly to chemotherapy owing to its potent MDR. Chemotherapeutic drugs primarily act by inducing apoptosis of cancer cells, and defects in apoptosis may result in MDR. Mitochondrial permeability transition (mPT) is implicated as an important event in the control of cell death or survival and mPT represents a target for the development of cytotoxic drugs. This study aimed to investigate the effects of selective opener (Atractyloside glycoside, ATR) and inhibitor (Cyclosporine A, CsA) of mitochondrial permeability transition pore (mPTP) on a CDDP-resistant HCC cell line (SK-Hep1 cells). In this study, a stable MDR phenotype characterization of SK-Hep1 cell line (SK-Hep1/CDDP cells) was established and used to investigate the role of mPTP in MDR. Results suggested that ATR accelerated the decrease of mitochondrial membrane potential (ΔΨm), reduced the Bax activity, and increased the apoptosis of SK-Hep1/CDDP cells; while CsA inhibited mPTP opening, reduced and delayed the decline of mitochondrial membrane potential, and increased the Bax activity, leading to increased tolerance of SK-Hep1/CDDP cells to apoptosis induction. However, mPTP activity had no effect on the expression of MDR1 in cells,meanwhile the P-gp translocation to mitochondria was increased, and functionally activated. In conclusion, selective modulation of mPTP can affect MDR in human HCC cells. Therefore, activation of mPTP may provide a new strategy to sensitize cancer cells to chemotherapeutic drugs and to reverse the MDR in cancer cells.     

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.     

Ultrastructural effects of colchicine,vinblastine, and taxol in drug-sensitive and multidrug-resistant J 774.2 cells

Summary Cell lines derived from the murine macrophage-like cell J 774.2 are resistant to the cytotoxic effects of colchicine, vinblastine, and taxol. These multidrug-resistant (MDR) cells overproduce a family of 130–150 kDa P-glycoproteins (P-gp) associated with the plasma membrane region and display other typical features of the MDR phenotype. Ultrastructural analysis of drug-treated cells indicated that although hallmark structural effects engendered by each drug at efficacious doses were profound in the drug-sensitive J 774.2 cells, they were not evident in the similarly treated MDR cell lines. Thus, MDR phenotypic expression involved maintaining drug levels at subthreshold values so as to preclude the advent of these morphologic changes, and allowed vital tubulin-associated cellular processes, including replication, to occur. Using a polyclonal antibody specific for the P-gp, electron microscopic immunocytochemical evidence is presented for substantial association of P-gp with the plasma membrane/cell surface in the resistant cells which was not demonstrable in the drug-sensitive J 774.2 cells. This key cell surface localization of P-gp is germane to the postulated transport and related mechanisms whereby P-gp may play a pivotal role in endowing cells with multidrug resistance.     

Acquisition of MDR phenotype by leukemic cells is associated with increased caspase-3 activity and a collateral sensitivity to cold stress

The acquisition of a multidrug-resistant (MDR) phenotype by tumor cells that renders them unsusceptible to anti-neoplasic agents is one of the main causes of chemotherapy failure in human malignancies. The increased expression of P-glycoprotein (MDR1, P-gp, ABCB1) in tumor cells contributes to drug resistance by extruding chemotherapeutic agents or by regulating programmed cell death. In a study of MDR cell survival under cold stress conditions, it was found that resistant leukemic cells with P-gp over-expression, but not their sensitive counterparts, are hypersensitive to cold-induced cell death when exposed to temperatures below 4 °C. The transfection of parental cells with a P-gp-expressing plasmid makes these cells sensitive to cold stress, demonstrating an association between P-gp expression and cell death at low temperatures. Furthermore, we observed increased basal expression and activity of effector caspase-3 at physiological temperature (37 °C) in MDR cells compared with their parental cell line. Treatment with a caspase-3 inhibitor partially rescues MDR leukemic cells from cold-induced apoptosis, which suggests that the cell death mechanism may require caspase-3 activity. Taken together, these findings demonstrate that P-gp expression plays a role in MDR cell survival, and is accompanied by a collateral sensitivity to death induced by cold stress. These findings may assist in the design of specific therapeutic strategies to complement current chemotherapy treatment against cancer.     

Reversal of P-glycoprotein-mediated multidrug resistance by cholesterol derived from low density lipoprotein in a vinblastine-resistant human lymphoblastic leukemia cell line.

P-glycoprotein (P-gp) is believed to be one of the most common causes of multidrug resistance (MDR) in chemotherapy. Studies have shown that the biosynthesis of cholesterol and cholesterol esters interfere with the function of P-gp. Since low density lipoprotein (LDL) carries a large amount of cholesterol, we investigated the effect of cholesterol derived from LDL on a line of human lymphoblastic leukemia MDR cells, CEM/VLB. Our results demonstrated that, in addition to increased cytotoxicity, the uptake of vinblastine in CEM/VLB cells increased, and LDL subsequently increased the intracellular vinblastine concentrations retained by CEM/VLB cells. The cholesterol levels in the membrane of the MDR cells were restored, while LDL significantly decreased the P-gp-associated ATPase activity. Current studies have shown that LDL leads to the resensitization of CEM/VLB cells to cytotoxic agents, likely through the restoration of cholesterol and reduction of P-gp-associated ATPase in the cell membrane.     

Carotenoids reverse multidrug resistance in cancer cells by interfering with ABC-transporters

Proteins of the ATP-binding cassette superfamily, mainly P-glycoprotein (P-gp; MDR1), play an important role in the development of multidrug resistance (MDR) in cancer cells and thus in the potential failure of chemotherapy. A selection of carotenoids (β-carotene, crocin, retinoic acid, canthaxanthin, and fucoxanthin) was investigated whether they are substrates of P-gp, and if they can reverse MDR in resistant Caco-2 and CEM/ADR5000 cells as compared to the sensitive parent cell line CCRF-CEM. The activity of ABC transporter was determined in resistant and sensitive cells by spectrofluorometry and flow cytometry using the substrates doxorubicin, rhodamine 123, and calcein as fluorescent probes. The carotenoids increased accumulation of these P-gp substrates in a dose-dependent manner indicating that they themselves also function as substrates. Fucoxanthin and canthaxanthin (50-100μM) produced a 3-5-fold higher retention of the fluorescent probes than the known competitive inhibitor verapamil. Carotenoids showed a low cytotoxicity in cells with MDR with IC(50) values between 100 and 200μM. The combination of carotenoids with eight structurally different cytotoxic agents synergistically enhanced their cytotoxicity in Caco-2 cells, probably by inhibiting the function of the ABC transporters. For example, fucoxanthin synergistically enhanced the cytotoxicity of 5-FU 53.37-fold, of vinblastine 51.01-fold, and of etoposide 12.47-fold. RT-PCR was applied to evaluate the mRNA levels of P-gp in Caco-2 cells after treatment with carotenoids. Fucoxanthin and canthaxanthin significantly decreased P-gp levels to 12% and 24%, respectively as compared to untreated control levels (p<0.001). This study implies that carotenoids may be utilised as chemosensitisers, especially as adjuvants in chemotherapy.     

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.     

The small GTPases Rab5 and RalA regulate intracellular traffic of P-glycoprotein

P-glycoprotein (P-gp) is a plasma membrane glycoprotein that can cause multidrug resistance (MDR) of cancer cells by acting as an ATP-dependent drug efflux pump. The regulatory effects of the small GTPases Rab5 and RalA on the intracellular trafficking of P-gp were investigated in HeLa cells. As expected, overexpressed enhanced green fluorescent protein (EGFP)-tagged P-gp (P-gp-EGFP) is mainly localised to the plasma membrane. However, upon cotransfection of either dominant negative Rab5 (Rab5-S34N) or constitutively active RalA (RalA-G23V) the intracellular P-gp-EGFP levels increased approximately 9 and 13 fold, respectively, compared to control P-gp-EGFP cells. These results suggest that Rab5 and RalA regulate P-gp trafficking between the plasma membrane and an intracellular compartment. In contrast, coexpression of constitutively active Rab5 (Rab5-Q79L) or dominant negative RalA (RalA-S28N) had no effect on the localisation of P-gp-EGFP. Furthermore, the intracellular accumulation of daunorubicin, a substrate for P-gp, increased significantly with an increased intracellular localisation of P-gp-EGFP. These results imply that it may be possible to overcome MDR by controlling the plasma membrane localisation of P-gp.     

Tryptanthrin inhibits MDR1 and reverses doxorubicin resistance in breast cancer cells

Development of agents to overcome multidrug resistance (MDR) is important in cancer chemotherapy. Up to date, few chemicals have been reported to down-regulate MDR1 gene expression. We evaluated the effect of tryptanthrin on P-glycoprotein (P-gp)-mediated MDR in a breast cancer cell line MCF-7. Tryptanthrin could depress overexpression of MDR1 gene. We observed reduction of P-gp protein in parallel with decreases in mRNA in MCF-7/adr cells treated with tryptanthrin. Tryptanthrin suppressed the activity of MDR1 gene promoter. Tryptanthrin also enhanced interaction of the nuclear proteins with the negatively regulatory CAAT region of MDR1 gene promoter in MCF-7/adr. It might result in suppression of MDR1 gene. In addition, tryptanthrin decreased the amount of mutant p53 protein with decreasing mutant p53 protein stability. It might contribute to negative regulation of MDR1 gene. In conclusion, tryptanthrin exhibited MDR reversing effect by down-regulation of MDR1 gene and might be a new adjuvant agent for chemotherapy.     

Sulforaphane and erucin increase MRP1 and MRP2 in human carcinoma cell lines

Multidrug resistance (MDR) transporters have been termed the Phase III detoxification system because they not only export endogenous metabolites but provide protection from xenobiotic insult by actively secreting foreign compounds and their metabolites from tissues. However, MDR overexpression in tumors can lead to drug resistance, a major obstacle in the treatment of many cancers, including lung cancer. Isothiocyanates from cruciferous vegetables, such as sulforaphane (SF) and erucin (ER), are known to enhance the expression of Phase II detoxification enzymes. Here we evaluated the ability of SF and ER to modulate MDR mRNA and protein expressions, as well as transporter activity. The expression of P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1) and multidrug resistance protein 2 (MRP2) in liver (HepG2), colon (Caco-2) and lung (A549) cancer cells treated with ER or SF was analyzed by Western blotting. Neither SF nor ER affected P-gp expression in any of the cell lines tested. Both SF and ER increased the protein levels of MRP1 and MRP2 in HepG2 cells and of MRP2 in Caco-2 cells in a dose-dependent manner. In A549 lung cancer cells, SF increased MRP1 and MRP2 mRNA and protein levels; ER caused a similar yet smaller increase in MRP1 and MRP2 mRNA. In addition, SF and ER increased MRP1-dependent efflux of 5-carboxyfluorescein diacetate in A549 cells, although again the effect of SF was substantially greater than that of ER. The implication of these findings is that dietary components that modulate detoxification systems should be studied carefully before being recommended for use during chemotherapy, as these compounds may have additional influences on the disposition of chemotherapeutic drugs.     

Designed P-glycoprotein inhibitors with triazol-tetrahydroisoquinoline-core increase doxorubicin-induced mortality in multidrug resistant K562/A02 cells

Multidrug resistance (MDR) refers to the cross-resistance of cancer cells to one drug, accompanied by other drugs with different mechanisms and structures, which is one of the main obstacles of clinical chemotherapy. Overexpression of P-glycoprotein (P-gp) was an extensively studied cause of MDR. Therefore, inhibiting P-gp have become an important strategy to reverse MDR. In this study, two series of triazole-tetrahydroisoquinoline-core P-gp inhibitors were designed and synthesized. Among them, compound I-5 had a remarkable reversal activity of MDR activity and the preliminary mechanism study was also carried out. All the results proved that compound I-5 was considered as a promising P-gp-mediated MDR reversal candidate.