The gene encoding vacuolar H(+)-ATPase subunit C is overexpressed in multidrug-resistant HL60 cells.

Previous studies have suggested that vacuolar H(+)-ATPase activity may play a role in modulating drug transport mechanism in multidrug resistant HL60 cells. In the present study we have used a cDNA of human vacuolar H(+)-ATPase subunit C (SC-H(+)-ATPase) to analyze expression of this gene in HL60 cells isolated for resistance to adriamycin or vincristine. The results demonstrate that development of resistance to either agent results in a major increase in the levels of SC-H(+)-ATPase mRNA. Furthermore in resistant cells which have partially reverted to drug sensitivity there is a parallel reduction in SC-H(+)-ATPase mRNA levels. Southern blot analysis shows that the SC-H(+)-ATPase gene is not amplified in the resistant cells. These results therefore demonstrate a correlation between the development of multidrug resistance and enhanced expression of the SC-H(+)-ATPase gene.     

Characterization of a membrane-associated protein kinase of multidrug-resistant HL60 cells which phosphorylates P-glycoprotein

Cells containing increased levels of the membrane phosphoprotein P-glycoprotein exhibit a multidrug-resistant phenotype. In the present study we have analyzed protein kinases capable of phosphorylating P-glycoprotein in membranes of HL60 cells isolated for resistance to vincristine. Analysis of this system demonstrates that in isolated membranes the protein kinase inhibitor staurosporine greatly reduces P-glycoprotein phosphorylation. In contrast, the kinase inhibitor H-7 does not affect this reaction. Fractionation of solubilized membrane proteins from sensitive and resistant cells on DEAE-cellulose reveals a major protein kinase (PK-1) which exhibits optimal activity in the presence of Mn2+ and histone H1. This enzyme fraction does not contain detectable levels of protein kinase C or cAMP-dependent protein kinase. PK-1 phosphorylation of two endogenous proteins is, however, greatly enhanced in the presence of phosphatidylserine or phosphatidyl-inositol. In reaction mixtures containing Mg2+ or Mn2+ in the absence of phospholipid, PK-1 from resistant cells phosphorylates an endogenous protein of 180 kilodaltons (P180), which exhibits an electrophoretic mobility identical to P-glycoprotein. In parallel experiments with PK-1 from sensitive cells there is no detectable phosphorylation of a P180 protein. P180 phosphorylated by PK-1 from resistant cells is immunoprecipitated by antibody against P-glycoprotein. Additional studies demonstrate that PK-1 is capable of phosphorylating specific synthetic peptides which correspond to the sequence of P-glycoprotein. Peptide phosphorylation occurs at both serine and threonine residues. These studies thus identify a novel membrane-associated protein kinase in HL60 cells which is capable of phosphorylating P-glycoprotein. This enzyme may have an important role in regulating levels of multidrug resistance.     

Multidrug resistant HOB1 lymphoma cells express P-glycoprotein that does not play the major role in the development of drug resistance to adriamycin.

Two cell lines resistant to 0.1 microM vincristine (VCR) and 2.0 microM adriamycin (ADR), respectively, (designated HOB1/VCR0.1 and HOB1/ADR2.0) were established from a human immunoblastic B lymphoma cell line. These cell lines showed the typical MDR phenotype with overexpression of P-glycoprotein and decreased [3H]VCR accumulation. The retention amounts of intracellular [3H]VCR in these two cell lines could be augmented by verapamil. However, in spite of the overproduction of P-glycoprotein, both HOB1/VCR1.0 and HOB1/ADR2.0 cells did not exhibit decreased accumulation of intracellular [14C]ADR. And the retention of [14C]ADR was not affected by verapamil. Our data support that P-glycoprotein is a drug transporter more important for the development of drug resistance to VCR than to ADR.     

Dibenzocyclooctadiene lignans: a class of novel inhibitors of multidrug resistance-associated protein 1

We recently reported that dibenzocyclooctadiene lignans were a novel class of P-glycoprotein (P-gp) inhibitors. In this study, we demonstrated that the lignans of this class were also effective inhibitors of multidrug resistance-associated protein 1 (MRP1). The activities of 5 dibenzocyclooctadiene lignans (schisandrin A, schisandrin B, schisantherin A, schisandrol A, and schisandrol B) to reverse MRP1-mediated drug resistance were tested using HL60/Adriamycin (ADR) and HL60/Multidrug resistance-associated protein (MRP), two human promyelocytic leukemia cell lines with overexpression of MRP1 but not P-gp. The five lignans could effectively reverse drug resistance of the two cell lines to vincristine, daunorubicin, and VP-16. This study, together with our previous reports, proves that dibenzocyclooctadiene lignans have multiple activities against cancer multidrug resistance, including inhibition of P-gp and MRP1, and enhancement of apoptosis. Considering that cancer multidrug resistance (MDR) is multifactorial, agents with broad activities are preferable to the use of combination of several specific modulators to prevent drug-drug interaction and cumulative toxicity.     

Multiple drug-resistance in variant of a human non-small cell lung carcinoma cell line,DLKP-A

A 300-fold adriamycin resistant variant (DLKP-A) of the human lung squamous cell carcinoma line DLKP was established by stepwise selection in increasing concentrations of adriamycin. Different levels of cross-resistance were observed towards VP-16, VM-26, colchicine, vincristine and, somewhat unexpectedly, cis-platin. Resistance was stable for at least 3 months in culture in the absence of drug. P-glycoprotein overexpression was detected by immunofluorescence and Western Blotting, and a direct causal role for P-glycoprotein overexpression in the resistant phenotype was established by transfection with an mdr1 specific antisense oligonucleotide. A modified cryopreservation procedure was necessary for the resistant variant line. The resistant population displays clonal heterogeneity with respect to resistance level. A higher frequency of double minute chromosomes was observed in DLKP-A when compared with the parental cell line.Abbreviations ADR adriamycin - COLH colchicine - C-PT cis-platin - MDR multidrug resistance - NSCLC non-small cell lung carcinoma - VCR vincristine - VP-16 etoposide - VM-26 tenoposide     

Analysis of P-glycoprotein phosphorylation in HL60 cells isolated for resistance to vincristine.

In the present study we have analyzed the involvement of phosphorylation in the function of P-glycoprotein and have also examined sites of phosphorylation along the P-glycoprotein polypeptide chain. The results show that in HL60 cells isolated for resistance to vincristine the protein kinase inhibitor staurosporine induces a major inhibition in the phosphorylation of P-glycoprotein. Further studies show that under the same conditions in which staurosporine inhibits P-glycoprotein phosphorylation there is a concomitant increase in cellular drug accumulation and a major inhibition in drug efflux. Additional studies using pulse-chase experiments show that the P-glycoprotein phosphate groups are metabolically active and that the protein undergoes rapid cycles of phosphorylation and dephosphorylation in the cell. Structural analyses demonstrate that cleavage of 32P-labeled P-glycoprotein at Asp-Pro linkages with formic acid results in the formation of a major phosphorylated peptide of 35 kDa and a minor peptide of 42 kDa. Western blot analysis using site-specific anti-sera against P-glycoprotein suggests that P35 represents a phosphorylated fragment containing P-glycoprotein amino acids 446-744. Analysis of tryptic peptides using site-specific antisera identifies a second major phosphorylated region of P-glycoprotein which contains amino acids 745-1088. These studies thus suggest that phosphorylation plays an important role in the biological activity of P-glycoprotein. The results also indicate that two adjacent internal regions are highly phosphorylated in the P-glycoprotein molecule.     

Regulation of ribosomal protein S25 in HL60 cells isolated for resistance to adriamycin.

The ribosomal protein S25 gene is highly overexpressed in HL60 cells isolated for resistance to adriamycin. In contrast there is no overexpression of 3 other ribosomal genes which code for proteins S14, S17 and S24. Studies with an antibody against a synthetic peptide of the S25 protein show that although the S25 gene is overexpressed in resistant cells there is no corresponding increase in the levels of S25 protein. These results suggest that the r-protein levels are highly regulated by translational controls or protein turnover.     

TCP1 increases drug resistance in acute myeloid leukemia by suppressing autophagy via activating AKT/mTOR signaling

T-complex protein 1 (TCP1) is one of the subunits of chaperonin-containing T complex (CCT), which is involved in protein folding, cell proliferation, apoptosis, cell cycle regulation, and drug resistance. Investigations have demonstrated that TCP1 is a factor being responsible for drug resistance in breast and ovarian cancer. However, the TCP1 role in acute myeloid leukemia (AML) remains elusive. In the present study, we discovered that the TCP1 expression was elevated in AML patients and high TCP1 expression was associated with low complete response rate along with poor overall survival. TCP1 showed higher expression in the adriamycin-resistant leukemia cell line HL60/A and K562/A, comparing to their respective parent cells HL60 and K562 cells. TCP1 inhibition suppressed drug resistance in HL60/A and K562/A cells, whereas TCP1 overexpression in HL60 cells incremented drug resistance, both in vitro and in vivo. Mechanistic investigations revealed that TCP1 inhibited autophagy and adriamycin-induced cell apoptosis, and TCP1-mediated autophagy inhibition conferred resistance to adriamycin-induced cell apoptosis. Furthermore, TCP1 interacted with AKT and mTOR to activate AKT/mTOR signaling, which negatively regulates apoptosis and autophagy. Pharmacological inhibition of AKT/mTOR signal particularly activated autophagy and resensitized TCP1-overexpressing HL60 cells to adriamycin. These findings identify a novel role of TCP1 regarding drug resistance in AML, which advise a new strategy for overcoming drug resistance in AML through targeting TCP1/AKT/mTOR signaling pathway.Subject terms: Prognostic markers, Acute myeloid leukaemia     

Dimethylsulfoxide, retinoic acid and 12-O-tetradecanoylphorbol-13-acetate induce a selective decrease in the phosphorylation of P150, a surface membrane phosphoprotein of HL60 cells resistant to adriamycin

Studies have been carried out to analyze protein phosphorylation in membranes isolated from adriamycin resistant HL60 cells which have been grown for various time periods in the presence of dimethylsulfoxide (DMSO), retinoic acid (RA) or 12-O-tetradecanoylphorbol-13-acetate (TPA). The results show that membranes isolated from cells treated with these agents are defective in the phosphorylation of P150, a membrane phosphoprotein associated with drug resistance in HL60 cells. This response is highly selective since only a few membrane proteins show decreased phosphorylation levels under these conditions. Magnesium dependent protein kinase activity in membranes from cells treated with DMSO, RA or TPA is not altered relative to untreated membranes under conditions where there is a major decrease in P150 phosphorylation. Additional studies also show that treatment of resistant cells with TPA results in a major decrease in the in vivo phosphorylation of P150. These results thus demonstrate that agents capable of inducing differentiation in HL60 cells can selectively modulate the phosphorylation of P150. This system should be of value in clarifying mechanisms involved in the phosphorylation of this protein.     

Cyclohexyl-linked tricyclic isoxazoles are potent and selective modulators of the multidrug resistance protein (MRP1)

Structure–activity relationship (SAR) studies on the tricyclic isoxazole series of MRP1 modulators have resulted in the identification of potent and selective inhibitors containing cyclohexyl-based linkers. These studies ultimately identified compound 21b, which reverses drug resistance to MRP1 substrates, such as doxorubicin, in HeLa-T5 cells (EC₅₀ = 0.093 μM), while showing no inherent cytotoxicity. Additionally, 21b inhibits ATP-dependent, MRP1-mediated LTC₄ uptake into membrane vesicles prepared from the MRP1-overexpressing HeLa-T5 cells (EC₅₀ = 0.064 μM) and shows selectivity (1115-fold) against the related transporter, P-glycoprotein, in HL60/Adr and HL60/Vinc cells. Finally, when dosed in combination with the oncolytic MRP1 substrate vincristine, 21b showed tumor regression and growth delay in MRP1-overexpressing tumors in vivo.     

Tricyclic isoxazoles are novel inhibitors of the multidrug resistance protein (MRP1)

Tricyclic isoxazoles were identified from a screen as a novel class of selective multidrug resistance protein (MRP1) inhibitors. From a screen lead, SAR efforts resulted in the preparation of LY 402913 (9h), which inhibits MRP1 and reverses drug resistance to MRP1 substrates, such as doxorubicin, in HeLa-T5 cells (EC(50)=0.90 microM), while showing no inherent cytotoxicity. Additionally, LY 402913 inhibits ATP-dependent, MRP1-mediated LTC(4) uptake into membrane vesicles prepared from the MRP1-overexpressing HeLa-T5 cells (EC(50)=1.8 microM). LY 402913 also shows selectivity ( approximately 22-fold) against the related transporter, P-glycoprotein, in HL60/Adr and HL60/Vinc cells. Finally, when dosed in combination with the oncolytic MRP1 substrate vincristine, LY 402913 delays the growth of MRP1-overexpressing tumors in vivo.     

Upregulation of miR-142-3p Improves Drug Sensitivity of Acute Myelogenous Leukemia through Reducing P-Glycoprotein and Repressing Autophagy by Targeting HMGB1

miR-142-3p was reported to be downregulated in acute myelogenous leukemia (AML) and acted as a novel diagnostic marker. However, the regulatory effect of miR-142-3p on drug resistance of AML cells and its underlying mechanism have not been elucidated. Here, we found that miR-142-3p was significantly downregulated and high mobility group box 1 (HMGB1) was dramatically upregulated in AML samples and cells, as well as drug-resistant AML cells. P-gp level and autophagy were markedly enhanced in HL-60/ADR and HL-60/ATRA cells. miR-142-3p overexpression improved drug sensitivity of AML cells by inhibiting cell viability and promoting apoptosis, and inhibited P-gp level and autophagy in drug-resistant AML cells, whereas HMGB1 overexpression obviously reversed these effect. HMGB1 was demonstrated to be a target of miR-142-3p, and miR-142-3p negatively regulated HMGB1 expression. In conclusion, our study elucidated that upregulation of miR-142-3p improves drug sensitivity of AML through reducing P-glycoprotein and repressing autophagy by targeting HMGB1, contributing to better understanding the molecular mechanism of drug resistance in AML.     

Modulation of drug resistance in homoharringtonine-resistant C-1300 neuroblastoma cells with cyclosporine A and dipyridamole.

The development of resistance accounts for therapy failure in the majority of advanced cases of neuroblastoma in children. A new transplantable murine C-1300 neuroblastoma cell line was developed in vitro, by repeated exposure of a sensitive cell line to increasing, but sublethal, doses of Homoharringtonine (HHT). The ED50 of the highly resistant cells for HHT, using a standard agar colony assay, is 480 ng/ml, compared with 13 ng/ml for the sensitive parental line. The resistant cells have cross-resistance to a number of other agents, including adriamycin, vinca alkaloids, melphalan, and CCNU. Western blot analysis revealed progressive increases in P-glycoprotein, parallel to the graded development of resistance with a 29-fold elevation in the highest resistant cells. High-performance liquid chromatography (HPLC) indicated that resistant cells have a significantly lower uptake of HHT than parental sensitive cells. cyclosporine A (CsA) and dipyridamole (DPM) could modulate the acquired resistance and completely restore the cytotoxic effects of HHT and adriamycin as determined by the clonogenic assay. The reversal of resistance by CsA and DPM was dose dependent. With the relative low toxicity of dipyridamole and CsA in doses required for modulation of resistance, these agents may be candidates for clinical utilization in chemotherapy of resistant neuroblastoma.     

A novel role for Bsd2 in the resistance of yeast to adriamycin

In a search for undiscovered mechanisms of resistance to adriamycin, we screened a genomic library derived from Saccharomyces cerevisiae for genes related to adriamycin resistance. To our surprise, we found that overexpression of BSD2 rendered yeast cells resistant to adriamycin. Downregulation of the metal transporters Smf1 and Smf2 is the only activity of Bsd2 reported to date, and Bsd2 deficiency increases intracellular levels of Smf1 and Smf2. SMF2-disrupted cells exhibited significantly greater resistance to adriamycin, whereas the resistance of SMF1-disrupted cells was only slightly improved. The sensitivity of the SMF1- and SMF2-disrupted yeast cell line overexpressing BSD2 was almost the same as that of the BSD2-overexpressing parental yeast cell. Thus the overexpression of BSD2 and the disruption of SMF1 and SMF2 might be involved in the same mechanism that confers resistance to adriamycin. Although both SMF1- and SMF2-disrupted cells were very sensitive to EGTA, overexpression of BSD2 had little or no effect on sensitivity to EGTA. However, a partial decrease in the intracellular level of FLAG-Smf2 was observed by overexpression of BSD2. Thus, the resistance to adriamycin acquired by overexpression of BSD2 might be partially explained by down-regulation of Smf2, but in addition to Smf2, other as of yet unidentified targets of Bsd2 must also be responsible for the resistance.     

Inhibition of P-glycoprotein expression and reversal of drug resistance of human hepatoma HepG2 cells by multidrug resistance gene (mdr1) antisense RNA

The development of multiple drug resistance in tumor cells is a significant problem in cancer therapy. In human, one of the reasons causing the resistance is due to the overexpression of the mdr1 gene product, P-glycoprotein. In our study, we had developed multiple drug resistant HepG2 cell line (HepG2/DR). To reverse the resistance, HepG2-DR cells were treated with antisense RNA against mdr1 gene. Total RNA and protein were extracted from the transfected cells. Northern analysis showed that mRNA level of mdr1 was decreased whereas a reduction in P-glycoprotein was detected by Western blot. By using flow cytometry, the ability of intracellular doxorubicin retention increased and drug efflux decreased in the treated cells. The result also showed that the cellular sensitivity to doxorubicin, vincristine and methotrexate measured in IC50 increased 83.3% 84.6% and 50% respectively. All these findings suggested that the expression of p-glycoprotein was successfully inhibited by antisense RNA and the drug resistance was reduced.     

Down-regulated miR-331-5p and miR-27a are associated with chemotherapy resistance and relapse in leukaemia

Multidrug resistance (MDR) and disease relapse are challenging clinical problems in the treatment of leukaemia. Relapsed disease is frequently refractory to chemotherapy and exhibits multiple drug resistance. Therefore, it is important to identify the mechanism by which cancer cells develop resistance. In this study, we used microRNA (miRNA) microarray and qRT-PCR approaches to investigate the expression of miRNAs in three leukaemia cell lines with different degrees of resistance to doxorubicin (DOX) compared with their parent cell line, K562. The expression of miR-331-5p and miR-27a was inversely correlated with the expression of a drug-resistant factor, P-glycoprotein (P-gp), in leukaemia cell lines with gradually increasing resistance. The development of drug resistance is regulated by the expression of the P-gp. Transfection of the K562 and, a human promyelocytic cell line (HL) HL60 DOX-resistant cells with miR-331-5p and miR-27a, separately or in combination, resulted in the increased sensitivity of cells to DOX, suggesting that correction of altered expression of miRNAs may be used for therapeutic strategies to overcome leukaemia cell resistance. Importantly, miR-331-5p and miR-27a were also expressed at lower levels in a panel of relapse patients compared with primary patients at diagnosis, further illustrating that leukaemia relapse might be a consequence of deregulation of miR-331-5p and miR-27a.     

Glutathione S-transferase does not play a role in multidrug resistance of L1210/VCR cell line

Multidrug resistance of cancer cells is often accompanied by the (over)expression of integral plasma membrane P-glycoprotein, an ATP-dependent transport pump for diverse unrelated compounds. The glutathione detoxification system represents another mechanism that may be involved in multidrug resistance. In the multidrug-resistant L1210/VCR cell line obtained by long-term adaptation of parental L1210 cells to vincristine, an increased expression of P-glycoprotein has previously been established. In this paper, we investigated if the glutathione detoxification system is also involved in the multidrug resistance of these cells. L1210/VCR cells with resistance induced by adaptation to vincristine were also found to be cross-resistant to vinblastine, actinomycin D, mitomycin C, doxorubicin and cyclophosphamide. The resistance of the above cells to vincristine and doxorubicin was accompanied by a depression of drug accumulation (which has not yet been established for other drug). L1210/VCR cells are able to survive better than sensitive cells under conditions when glutathione was depleted by L-buthionine sulfoximine. Nevertheless, L-buthionine sulfoximine did not influence the resistance of L1210/VCR cells to vincristine. Moreover, the presence of sublethal concentrations of cytostatics neither changed the IC50 value of resistant cells to L-buthionine sulfoximine nor the cytoplasmic activity of glutathione S-transferase, the crucial enzyme of glutathione detoxification system. All the above findings indicate that the glutathione detoxification system is not involved in the mechanisms that ensure the multidrug resistance phenotype of L1210/VCR cells.     

Overexpression of P-glycoprotein in human lung carcinoma xenografts after fractionated irradiation in vivo.

In vivo exposure of a human epidermoid lung carcinoma xenograft to seven irradiation treatments of 10 Gy in consecutive passages resulted in expression of resistance to vincristine. This about threefold drug resistance was detectable with a single dose of 1 mg/kg vincristine. Characterization of the radiation-pretreated subline showed that overexpression of P-glycoprotein, as determined by immunofluorescence and Mabs C219 and 265/F4, occurred in this tumor. After six X-ray fractions, only single positive cells were observed, whereas seven fractions produced an intense immunofluorescent reaction with both antibodies. Southern blot analyses indicated that no gene amplification had occurred. This result shows that irradiation can influence expression of P-glycoprotein and in this way influences drug resistance.