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.     

Distinct N-glycan glycosylation of P-glycoprotein isolated from the human uterine sarcoma cell line MES-SA/Dx5

The uterine sarcoma human cell line MES-SA/Dx5 overexpresses the MDR1 gene product, P-glycoprotein (Pgp). Pgp is a heavily glycosylated, ATP-dependent drug efflux pump expressed in many human cancers. There are more than 150 known isoforms of Pgp, which complicates the characterization of Pgp glycans because each isoform could present a different glycome. The contribution of these oligosaccharides to the structure and function of Pgp remains unclear. We identified distinct Pgp glycans recognized by the lectins in the digoxigenin (DIG) glycan differentiation kit from Roche Allied Science, all of which were N-glycans. Pgp was isolated using both slab and preparative gel elution. The monoclonal antibody C219 was used to identify the presence of Pgp and Pgp treated with PNGase F on our blots. Pgp isolated from MES-SA/Dx5 cells contains at least two different complex N-glycans--one high mannose tree, detected by GNA, and one branched hybrid oligosaccharide-capped with terminal sialic acids, detected by SNA and MAA. DSA, specific for biantennary oligosaccharides possessing beta(1-4)-N-acetyl-D-glucosamine residues, also recognized the blotted Pgp and is probably detecting the core Galbeta(1-4)-GlcNAc(x) component found in other Pgps.     

Proteomic analysis of proteins responsible for the development of doxorubicin resistance in human uterine cancer cells

Drug resistance is a common cause of failure in cancer chemotherapy treatments. In this study, we used a pair of uterine sarcoma cancer lines, MES-SA, and the doxorubicin-resistant MES-SA/Dx5 as a model system to examine resistance-dependent cellular responses and to identify potential therapeutic targets. We used two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) to examine the global protein expression changes induced by doxorubicin treatment and doxorubicin resistance. A proteomic study revealed that doxorubicin-exposure altered the expression of 87 proteins in MES-SA cells, while no significant response occurred in similarly treated MES-SA/Dx5 cells, associating these proteins with drug specific resistance. By contrast, 37 proteins showed differential expression between MES-SA and MES-SA/Dx5, indicating baseline resistance. Further studies have used RNA interference, cell viability analysis, and analysis of apoptosis against asparagine synthetase (ASNS) and membrane-associated progesterone receptor component 1 (mPR) proteins, to monitor and evaluate their potency on the formation of doxorubicin resistance. The proteomic approach allowed us to identify numerous proteins, including ASNS and mPR, involved in various drug-resistance-forming mechanisms. Our results provide useful diagnostic markers and therapeutic candidates for the treatment of doxorubicin-resistant uterine cancer.     

Lysosomal accumulation of drugs in drug-sensitive MES-SA but not multidrug-resistant MES-SA/Dx5 uterine sarcoma cells

Sequestration of drugs in intracellular vesicles has been associated with multidrug-resistance (MDR), but it is not clear why vesicular drug accumulation, which depends upon intracellular pH gradients, should be associated with MDR. Using a human uterine sarcoma cell line (MES-SA) and a doxorubicin (DOX)-resistant variant cell line (Dx-5), which expresses p-glycoprotein (PGP), we have addressed the relationship between multidrug resistance, vesicular acidification, and vesicular drug accumulation. Consistent with a pH-dependent mechanism of vesicular drug accumulation, studies of living cells vitally labeled with multiple probes indicate that DOX and daunorubicin (DNR) predominately accumulate in lysosomes, whose lumenal pH was measured at < 4.5, but are not detected in endosomes, whose pH was measured at 5.9. However, vesicular DOX accumulation is more pronounced in the drug-sensitive MES-SA cells and minimal in Dx5 cells even when cellular levels of DOX are increased by verapamil treatment. While lysosomal accumulation of DOX correlated well with pharmacologically induced differences in lysosome pH in MES-SA cells, lysosomal accumulation was minimal in Dx5 cells regardless of lysosomal pH. We found no differences in the pH of either endosomes or lysosomes between MES-SA and Dx5 cells, suggesting that, in contrast to other MDR cell systems, the drug-resistant Dx5 cells are refractory to pH-dependent vesicular drug accumulation. These studies demonstrate that altered endomembrane pH regulation is not a necessary consequence of cell transformation, and that vesicular sequestration of drugs is not a necessary characteristic of MDR.     

Reversal of multidrug resistance in cancer cells by Rhizoma Alismatis extract

Prolonged chemotherapy may lead to the selective proliferation of multidrug resistant (MDR) cancer cells. In MDR HepG2-DR and K562-DR cells that over-expressed P-glycoprotein (Pgp), the extract of the rhizomes of Alisma orientalis (Sam) Juzep. showed a synergistic growth inhibitory effect with cancer drugs that are Pgp substrates including actinomycin D, puromycin, paclitaxel, vinblastine and doxorubicin. At the same toxicity levels the herbal extract was more effective than verapamil, a standard Pgp inhibitor, in enhancing cellular doxorubicin accumulation and preventing the efflux of rhodamin-123 from the MDR cells. The extract restored the effect of vinblastine on the induction of G(2)/M arrest in MDR cells. Our data suggest that A. orientalis may contain components that are effective inhibitors of Pgp.     

Design, synthesis and antiproliferative activity of novel aminosubstituted benzothiopyranoisoindoles

The synthesis of a number of new benzothiopyrano[4,3,2-cd]isoindole aminoderivatives designed as structural analogues of the key metabolite of the anticancer agent Ledacrine (nitracrine) and their in vitro cytotoxic activity evaluation against HCT-116, MES-SA, and MES-SA/Dx cancer cell lines is reported. The majority of the derivatives possessed noticeable cytotoxicity in a low μM range indicating an interesting structure-activity relationship.     

Quinoline derivative KB3-1 potentiates paclitaxel induced cytotoxicity and cycle arrest via multidrug resistance reversal in MES-SA/DX5 cancer cells

AIMS: The resistance to chemotherapeutic drugs is a major problem for successful cancer treatment. Multidrug resistance (MDR) phenotype is characterized by over-expression of P-glycoprotein (P-gp) on the cancer cell plasma membrane that extrudes drugs out of the cells. Therefore, novel MDR reversal agents are desirable for combination therapy to reduce MDR and enhance anti-tumor activity. Thus, the present study was aimed to evaluate the potent efficacy of novel quinoline derivative KB3-1 as a potent MDR-reversing agent for combined therapy with TAX. MAIN METHODS: MDR reversing effect and TAX combined therapy were examined by Rhodamine accumulation and efflux assay and Confocal immunofluorescence microscopy, Western blotting, TUNEL assay, and cell cycle analysis. KEY FINDINGS: The discovery of quinoline-3-carboxylic acid [4-(2-[benzyl-3[-(3,4-dimethoxy-phenyl)-propionyl]-amino]-ethyl)-phenyl]-amide (KB3-1) as a novel MDR-reversal agent. KB3-1 significantly enhanced the accumulation and retention of a P-gp substrate, rhodamine-123 in the P-gp-expressing MES-SA/DX5 uterine sarcoma cells but not in the P-gp-negative MES-SA cells at non-toxic concentrations of 1 microM and 3 microM. Similarly, fluorescence microscopy observation revealed that KB3-1 reduced the effluxed rhodamine-123 expression on the membrane of MES-SA/DX5 cells. Consistent with decreased P-gp pumping activity, confocal microscopic observation revealed that KB3-1 effectively diminished the expression of P-gp in paclitaxel (TAX)-treated MES-SA/DX-5 cells. Furthermore, Western blotting confirmed that KB3-1 reduced P-gp expression and enhanced cytochrome C release and Bax expression in TAX treated MES-SA/DX-5 cells. In addition, KB3-1 enhanced TAX-induced apoptotic bodies in MES-SA/DX5 cells by TdT-mediated-dUTP nick-end labeling (TUNEL) staining assay aswell as potentiated TAX- induced cytotoxicity, G2/M phase arrest and sub-G1 apoptosis in MES-SA/DX5 cells but not in MES-SA cells. Interestingly, KB3-1 at 3 microM had comparable MDR-reversal activity to 10 microM verapamil, a well-known MDR- reversal agent. SIGNIFICANCE: KB3-1 can be a MDR-reversal drug candidate for combination chemotherapy with TAX.     

NSC23925, Identified in a High-Throughput Cell-Based Screen,Reverses Multidrug Resistance

Background

Multidrug resistance (MDR) is a major factor which contributes to the failure of cancer chemotherapy, and numerous efforts have been attempted to overcome MDR. To date, none of these attempts have yielded a tolerable and effective therapy to reverse MDR; thus, identification of new agents would be useful both clinically and scientifically.

Methodology/Principal Findings

To identify small molecule compounds that can reverse chemoresistance, we developed a 96-well plate high-throughput cell-based screening assay in a paclitaxel resistant ovarian cancer cell line. Coincubating cells with a sublethal concentration of paclitaxel in combination with each of 2,000 small molecule compounds from the National Cancer Institute Diversity Set Library, we identified a previously uncharacterized molecule, NSC23925, that inhibits Pgp1 and reverses MDR1 (Pgp1) but does not inhibit MRP or BCRP-mediated MDR. The cytotoxic activity of NSC23925 was further evaluated using a panel of cancer cell lines expressing Pgp1, MRP, and BCRP. We found that at a concentration of >10 µM NSC23925 moderately inhibits the proliferation of both sensitive and resistant cell lines with almost equal activity, but its inhibitory effect was not altered by co-incubation with the Pgp1 inhibitor, verapamil, suggesting that NSC23925 itself is not a substrate of Pgp1. Additionally, NSC23925 increases the intracellular accumulation of Pgp1 substrates: calcein AM, Rhodamine-123, paclitaxel, mitoxantrone, and doxorubicin. Interestingly, we further observed that, although NSC23925 directly inhibits the function of Pgp1 in a dose-dependent manner without altering the total expression level of Pgp1, NSC23925 actually stimulates ATPase activity of Pgp, a phenomenon seen in other Pgp inhibitors.

Conclusions/Significance

The ability of NSC23925 to restore sensitivity to the cytotoxic effects of chemotherapy or to prevent resistance could significantly benefit cancer patients.     

Synthesis and anticancer evaluation of vitamin K(3) analogues

Novel vitamin K(3) analogues were synthesized and evaluated for their anticancer activity. Compound 6, 9, 10, 11, 14, and (+/-)15 demonstrated a strong inhibitory activity against the tumor cells of A-549, Hep G2, MCF7, MES-SA, MES-SA/Dx5, MKN45, SW-480, and TW-039. Compound (+/-)15 displayed potent tumor cell cytotoxicity, and compound 14 selectively affected MCF7, even though it did not influence normal cells Detroit551 and WI-38. Compound (+/-)15 inhibited MES-SA and MES-SA/Dx5, and this specific result shows that compound (+/-)15 may become a good anticancer drug candidate.     

Radiopharmaceuticals for assessment of multidrug resistance P-glycoprotein-mediated drug transport activity

Multidrug resistance (MDR) mediated by overexpression of MDR1 P-glycoprotein (Pgp) is one of the best characterized transporter-mediated barriers to successful chemotherapy in cancer patients. Thus, noninvasive interrogation of Pgp-mediated transport activity in vivo would be beneficial in guiding therapeutic choices. Both small organic medicinals as well as metal complexes characterized as transport substrates for Pgp are amenable to incorporation of PET or SPECT radionuclides and may enable noninvasive imaging of Pgp in cancer patients. Toward this objective, clinically approved agents, exemplified by (99m)Tc-Sestamibi and (99m)Tetrofosmin, have already shown promise for the functional evaluation of Pgp-mediated transport activity in human tumors in vivo. In addition, selected agents from an upcoming class of substituted Schiff-base gallium(III) complexes containing an N(4)O(2) donor core in their organic scaffold and capable of generating both SPECT and PET radiopharmaceuticals have also been shown to be promising for noninvasive assessment of Pgp activity in vitro and in vivo.     

Characterization of a novel 99mTc-carbonyl complex as a functional probe of MDR1 P-glycoprotein transport activity

Multidrug resistance (MDR) mediated by overexpression of MDR1 P-glycoprotein (Pgp) is one of the best characterized barriers to chemotherapy in cancer patients. Furthermore, the protective function of Pgp-mediated efflux of xenobiotics in various organs has a profound effect on the bioavailability of drugs in general. Thus, there is an expanding requirement to noninvasively interrogate Pgp transport activity in vivo. We herein report the Pgp recognition properties of a novel 99mTc(I)-tricarbonyl complex, [99mTc(CO)3(MIBI)3]+ (Tc-CO-MIBI). Tc-CO-MIBI showed 60-fold higher accumulation in drug-sensitive KB 3-1 cells compared to colchicine-selected drug-resistant KB 8-5 cells. In KB 8-5 cells, tracer enhancement was observed with the potent MDR modulator LY335979 (EC50 = 62 nM). Similar behavior was observed using drug-sensitive MCF-7 breast adenocarcinoma cells and MCF-7/MDR1 stable transfectants, confirming that Tc-CO-MIBI is specifically excluded by overexpression of MDR1 Pgp. By comparison, net accumulation in control H69 lung tumor cells was 9-fold higher than in MDR-associated protein (MRP1)-expressing H69AR cells, indicating only modest transport by MRP1. Biodistribution analysis following tail vein injection of Tc-CO-MIBI showed delayed liver clearance as well as enhanced brain uptake and retention in mdr1a/1b(-/-) gene deleted mice versus wild-type mice, directly demonstrating that Tc-CO-MIBI is a functional probe of Pgp transport activity in vivo.     

Synthesis and evaluation of Strychnos alkaloids as MDR reversal agents for cancer cell eradication

Natural products represent the fourth generation of multidrug resistance (MDR) reversal agents that resensitize MDR cancer cells overexpressing P-glycoprotein (Pgp) to cytotoxic agents. We have developed an effective synthetic route to prepare various Strychnos alkaloids and their derivatives. Molecular modeling of these alkaloids docked to a homology model of Pgp was employed to optimize ligand–protein interactions and design analogues with increased affinity to Pgp. Moreover, the compounds were evaluated for their (1) binding affinity to Pgp by fluorescence quenching, and (2) MDR reversal activity using a panel of in vitro and cell-based assays and compared to verapamil, a known inhibitor of Pgp activity. Compound 7 revealed the highest affinity to Pgp of all Strychnos congeners (K_d = 4.4 μM), the strongest inhibition of Pgp ATPase activity, and the strongest MDR reversal effect in two Pgp-expressing cell lines. Altogether, our findings suggest the clinical potential of these synthesized compounds as viable Pgp modulators justifies further investigation.     

Anti-AIDS agents 89. Identification of DCX derivatives as anti-HIV and chemosensitizing dual function agents to overcome P-gp-mediated drug resistance for AIDS therapy

In this study, 19 dicamphanoyl-dihydropyranochromone (DCP) and dicamphanoyl-dihydropyranoxanthone (DCX) derivatives, previously discovered as novel anti-HIV agents, were evaluated for their potential to reverse multi-drug resistance (MDR) in a cancer cell line over-expressing P-glycoprotein (P-gp). Seven compounds fully reversed resistance to vincristine (VCR) at 4 μM, a 20-fold enhancement compared to the first generation chemosensitizer, verapamil (4 μM). The mechanism of action of DCPs and DCXs was also resolved, since the most active compounds (3, 4, and 7) significantly increased intracellular drug accumulation due, in part, to inhibiting the P-gp mediated drug efflux from cells. We conclude that DCPs (3 and 4) and DCXs (7, 11, and 17) can exhibit polypharmacologic behavior by acting as dual inhibitors of HIV replication and chemoresistance mediated by P-gp. As such, they may be useful in combination therapy to overcome P-gp-associated drug resistance for AIDS treatment.     

Screening of deoxyribozyme with high reversal efficiency against multidrug resistance in breast carcinoma cells

Specific inhibition of P-glycoprotein (Pgp) expression, which is encoded by multidrug resistance gene-1 (MDR1), is considered a well-respected strategy to overcome multidrug resistance (MDR). Deoxyribozymes (DRz) are catalytic nucleic acids that could cleave a target RNA in sequence-specific manner. However, it is difficult to select an effective target site for DRz in living cells. In this study, target sites of DRz were screened according to MDR1 mRNA secondary structure by RNA structure analysis software. Twelve target sites on the surface of MDR1 mRNA were selected. Accordingly, 12 DRzs were synthesized and their suppression effect on the MDR phenotype in breast cancer cells was confirmed. The results showed that 4 (DRz 2, 3, 4, 9) of the 12 DRzs could, in a dose-dependent response, significantly suppress MDR1 mRNA expression and restore chemosensitivity in breast cancer cells with MDR phenotype. This was especially true of DRz 3, which targets the 141 site purine-pyrimidine dinucleotide. Compared with antisense oligonucleotide or anti-miR-27a inhibitor, DRz 3 was more efficient in suppressing MDR1 mRNA and Pgp protein expression or inhibiting Pgp function. The chemosensitivity assay also proved DRz 3 to be the best one to reverse the MDR phenotype. The present study suggests that screening targets of DRzs according to MDR1 mRNA secondary structure could be a useful method to obtain workable ones. We provide evidence that DRzs (DRz 2, 3, 4, 9) are highly efficient at reversing the MDR phenotype in breast carcinoma cells and restoring chemosensitivity.     

Effects of P-glycoprotein expression on cyclic AMP and volume-activated ion fluxes and conductances in HT-29 colon adenocarcinoma cells

The tissue distribution of P-glycoprotein (Pgp) and the structurally related cystic fibrosis transmembrane conductance regulator (CFTR) is apparently mutually exclusive, particularly in epithelia; where one protein is expressed the other is not. To study the possible function(s) of Pgp and its potential effects on CFTR expression in epithelia, HT-29 colon adenocarcinoma cells, which constitutively express CFTR, were pharmacologically adapted to express the classical multidrug resistance (MDR) phenotype (Pgp⁺). Concomitant with the appearance of Pgp and MDR phenotype (drug resistance, reduced drug accumulation and increased drug efflux), CFTR levels and cAMP-stimulated Cl conductances were markedly decreased compared to wild-type HT-29 (Pgp^?) cells (as shown using the whole cell patch clamp technique). Removal of drug pressure led to the gradual decrease in Pgp levels and MDR phenotype, as evidenced by increased rhodamine 123 accumulation (Pgp-Rev). Concomitantly, CFTR levels and cAMP-stimulated Cl^? conductances incresed. The cell responses of Pgp/Rev cells were heterogeneous with respect to both Pgp and CFTR functions. We also studied the possible contribution of Pgp to hypotonically activated (HCS) ion conductances. K⁺ and Cl^? effluxes from Pgp^? cells were markedly increased by HCS. This increase was twice as high as that induced by the cation ionophore gramicidin; it was blocked by the Cl^? channel blocker DIDS (4,4′-disothiocyano-2,2′-disulfonic stilbene) and required extracellular Ca²⁺. In Pgp⁺ cells, the HCS-induced fluxes were not significantly different from those of Pgp^? cells. Verapamil (10 μM), which caused 80% reversal of Pgp-associated drug extrusion, failed to inhibit the HCS-evoked Cl^? efflux of Pgp⁺ cells. Similarly, HCS increased Cl^? conductance to the same extent in Pgp^?, Pgp⁺ and Pgp-Rev cells. Verapamil (100 μM), but not 1,9-dideoxyforskolin (50 and 100 μM), partially inhibited the HCS-evoked whole cell current (WCC) in all three lines. Since the inhibition by verapamil was not detected in the presence of the K⁺ channel blocker Ba²⁺ (3 mM), it is suggested that verapamil affects K⁺ and not Cl^? conductance. We conclude that hypotonically activated Cl^? and K⁺ conductances are similar in HT-29 cells irrespective of Pgp expression. Expression of high levels of Pgp in HT-29 cells confers no physiologically significant capacity for cell volume regulation. © 1994 Wiley-Liss, Inc.     

Microplate screening of the differential effects of test agents on Hoechst 33342, rhodamine 123, and rhodamine 6G accumulation in breast cancer cells that overexpress P-glycoprotein

A microplate screening method has been developed to evaluate the effects of test agents on the accumulation of the fluorescent P-glycoprotein (Pgp) substrates Hoechst 33342, rhodamine 123, and rhodamine 6G in multidrug-resistant (MDR) breast cancer cells that overexpress Pgp. All three substrates exhibit substantially higher accumulation in MCF7 non-MDR cells versus NCI/ADR-RES MDR cells, while incubation with 50 microM reserpine significantly reduces or eliminates these differences. Rhodamine 123 shows the lowest substrate accumulation efficiency in non-MDR cells relative to the substrate incubation level. The effects of several chemosensitizing agents and a series of paclitaxel analogs on the accumulation of each fluorescent substrate suggest that there are distinct differences in the substrate interaction profiles exhibited by these different agents. The described methods may be useful in Pgp-related research in the areas of cancer MDR, oral drug absorption, the blood-brain barrier, renal/hepatic transport processes, and drug-drug interactions.     

Synthesis and evaluation of daunorubicin-paclitaxel dimers

Bifunctional, heterodimeric compounds were synthesized to test their ability to create polyvalent arrays between DNA and microtubules in cells. Each dimer was examined for the capacity to bind to microtubules and for cytotoxicity against MES-SA and MES-SA/Dx5 cell lines.     

miR‐495 sensitizes MDR cancer cells to the combination of doxorubicin and taxol by inhibiting MDR1 expression

MDR1 is highly expressed in MDR A2780DX5 ovarian cancer cells, MDR SGC7901R gastric cancer cells and recurrent tumours. It pumps cytoplasmic agents out of cells, leading to decreased drug accumulation in cells and making cancer cells susceptible to multidrug resistance. Here, we identified that miR‐495 was predicted to target ABCB1, which encodes protein MDR1. To reduce the drug efflux and reverse MDR in cancer cells, we overexpressed a miR‐495 mimic in SGC7901R and A2780DX cells and in transplanted MDR ovarian tumours in vivo. The results indicated that the expression of MDR1 in the above cells or tumours was suppressed and that subsequently the drug accumulation in the MDR cells was decreased, cell death was increased, and tumour growth was inhibited after treatment with taxol‐doxorubicin, demonstrating increased drug sensitivity. This study suggests that pre‐treatment with miR‐495 before chemotherapy could improve the curative effect on MDR1‐based MDR cancer.