MDR-positive Cells Research Articles

Nanoparticle-Peptide-Drug Bioconjugates for Unassisted Defeat of Multidrug Resistance in a Model Cancer Cell Line.

Multidrug resistance (MDR) is a significant challenge in the treatment of many types of cancers as membrane-associated transporters actively pump drugs out of the cell, limiting therapeutic efficacy. While nanoparticle (NP)-based therapeutics have emerged as a mechanism for overcoming MDR, they often rely on the delivery of multiple anticancer drugs, nucleic acid hybrids, or MDR pump inhibitors. The effectiveness of these strategies, however, can be limited by their off-target toxicity or the need for genetic transfection. In this paper, we describe a NP-peptide-drug bioconjugate that achieves significant cell killing in MDR-positive cancer cells without the need for additional drugs. We use a quantum dot (QD) as a central scaffold to append two species of peptide, a cell-uptake peptide to facilitate endocytic internalization and a peptide-drug conjugate that is susceptible to cleavage by esterases found within the endocytic pathway. This approach relies on spatiotemporal control over drug release, where endosomes traffic drug away from membrane-resident pumps and release it closer to the nucleus. Cellular internalization studies showed high uptake of the NP-drug complex and nuclear localization of the drug after 48 h in MDR-positive cells. Additionally, cellular proliferation assays demonstrated a 40% decrease in cell viability for the NP-drug bioconjugate compared to free drug, confirming the utility of this system in overcoming MDR in cancer cells.

Microparticles induce multifactorial resistance through oncogenic pathways independently of cancer cell type.

Multidrug resistance (MDR) is considered a multifactorial event that favors cancer cells becoming resistant to several chemotherapeutic agents. Numerous mechanisms contribute to MDR, such as P-glycoprotein (Pgp/ABCB1) activity that promotes drug efflux, overexpression of inhibitors of apoptosis proteins (IAP) that contribute to evasion of apoptosis, and oncogenic pathway activation that favors cancer cell survival. MDR molecules have been identified in membrane microparticles (MP) and can be transferred to sensitive cancer cells. By co-culturing MP derived from MDR-positive cells with recipient cells, we showed that sensitive cells accumulated Pgp, IAP proteins and mRNA. In addition, MP promoted microRNA transfer and NFκB and Yb-1 activation. Therefore, our results indicate that MP can induce a multifactorial phenotype in sensitive cancer cells.

Discovery of Novel 2‐N‐Aryl‐Substituted Benzenesulfonamidoacetamides: Orally Bioavailable Tubulin Polymerization Inhibitors with Marked Antitumor Activities

The discovery and optimization of a series of 2-N-aryl-substituted benzenesulfonamidoacetamides as novel tubulin polymerization inhibitors are described. Pharmacophore exploration of hit compound AH-487 identified the optimal structure of N-heteroaryl-2-(4-methoxy-N-(3-(trifluoromethyl)phenyl)phenylsulfonamido)acetamide as a potent antimitotic agent. Subsequent lead compounds 4b and 4c, with N-4-aminophenyl and N-1H-indol-5-yl substitutions at the acetamide position, respectively, were shown to induce cell-cycle arrest at the G(2) /M phase and lead to an accumulation of HeLa cells in the sub-G(1) phase. More significantly, these lead compounds (3c, 4b, and 4c) exhibit impressive cytotoxicity against a panel of cancer cells including P-glycoprotein-overexpressing MDR-positive cells, with potency greater than or equal to clinically studied benzenesulfonamide E7010. Mechanistic studies demonstrated that derivatives of AH-487 disrupt mitotic spindles by inhibiting microtubule polymerization and induce apoptosis via induction of Bcl-2 phosphorylation in tumor cells. The optimized leads 4b and 4c strongly inhibited the growth of human hepatocellular carcinoma cells in a mouse xenograft model.

P-gp localization in mitochondria and its functional characterization in multiple drug-resistant cell lines

Multidrug resistance (MDR) phenotype is characterized by the over-expression of P-glycoprotein (P-gp) on cell plasma membranes that extrudes several drugs out of cells. Cells that express the MDR phenotype are resistant to the mitochondrial related apoptosis and to several anticancer drugs. This study assessed the presence of P-gp in mitochondria and its role in parental drug-sensitive (P5) and in P5-derived MDR1 cells P1(0.5) hepatocellular carcinoma (HCC) cell lines and in drug-sensitive (PSI-2) and mdr1-transfected (PN1A) NIH/3T3 cells. By using Western blot analysis, confocal laser microscopy, measurements of Rhodamine 123 transport across mitochondrial membranes, MDR1 small interfering RNA and flow cytometry analysis, experiments indicate that P-gp is expressed in mitochondria of P1(0.5) and PN1A cells and it is functionally active. Rho 123 accumulation was largely reduced in mitochondria of P1(0.5) cells as compared to those of P5 cells; the reduced uptake of fluorescence in mitochondria of MDR cells was due to P-gp-mediated Rho 123 efflux. In conclusion, these data demonstrate that functionally active P-gp is expressed in the mitochondrial membrane of MDR-positive cells and pumps out anticancer drugs from mitochondria into cytosol. Therefore, P-gp could be involved in the protection of mitochondrial DNA from damage due to antiproliferative drugs.

Biphasic accumulation kinetics of [ 99mTc]-hexakis-2-methoxyisobutyl isonitrile in tumour cells and its modulation by lipophilic P-glycoprotein ligands

Aim: To study the accumulation and washout kinetics of [ 99mTc]-hexakis-2-methoxyisobutyl isonitrile ( 99mTc-MIBI) in MDR positive and MDR negative tumour cells and how this is modified by lipophilic P-glycoprotein ligands. Methods: The tumour cells were incubated in the presence and absence of the ligands and the uptakes of 99mTc-MIBI, rhodamine 123 and 2-[ 18F]fluoro-2-deoxy- d-glucose ( 18FDG) were measured. Results: The accumulation of 99mTc-MIBI in the tumour cells followed biphasic kinetics. Verapamil and cyclosporin A increased the membrane fluidity and significantly enhanced the 99mTc-MIBI uptake of the MDR negative cells, while the rhodamine 123 uptake was not affected. Verapamil significantly increased the uptake of rhodamine 123 and 18FDG but did not modify that of 99mTc-MIBI in the MDR positive cells. Cyclosporin A significantly increased the 18FDG uptake of the MDR positive and negative tumour cells; these effects were ouabain-sensitive. Depolarization of the cytoplasmic membrane, acidification of the extracellular medium and the administration of CCCP decreased the accumulation of 99mTc-MIBI and rhodamine 123 uptake in the tumour cells. Conclusions: Lipophilic P-glycoprotein ligands modified the biphasic accumulation kinetics of the 99mTc-MIBI uptakes of MDR negative and positive tumour cells in different and complex ways and could therefore mask the P-glycoprotein pump-dependent changes in tracer accumulation.

N-methylation of anthracyclines modulates their cytotoxicity and pharmacokinetic in wild type and multidrug resistant cells

Anthracyclines are the most commonly used classes of anticancer agents in chemotherapy. Development of resistance to these molecules is one of the major reasons for treatment failure. The overexpression of the membrane transporter P-glycoprotein (P-gp) is among the principal mechanisms involved in this phenomenon. This pump, which is responsible for the multidrug resistance (MDR) phenotype, decreases the toxicity of a wide range of unrelated anticancer drugs by increasing their cellular efflux. Structure-activity relationship experiments have shown that the positively charged amino group of the anthracyclines could be responsible for their transport by P-gp. Here, we used three new anthracyclines that shared the same chromophore but differed by the degree of N-methylation of their sugar moiety. Oxaunomycin (OXN) possessed a non-methylated amino group, while LB-1 was monomethylated and β-clamycin T (BCT) was dimethylated. In sensitive cells (FLC), reduced cytotoxicity was related to the level of N-methylation; whereas in resistant cells (DOX-RFLC 1 and DOX-RFLC 2) overexpressing different levels of P-gp, increased N-methylation enhanced anthracycline cytotoxicity. Decreased resistance in DOX-RFLCs was associated with an increased drug accumulation due to a reduced cellular efflux. As expected, the MDR modulator verapamil decreased resistance to these anthracyclines by increasing the cellular accumulation. These results suggest that N-methylation of anthracyclines circumvents resistance by diminishing drug transport by P-gp in MDR-positive cells. These observations could be the consequence of the steric hindrance created by the methyl group(s) which may impair the interaction between the positively charged amino group and the active site of P-gp.

The synergistic anti-tumour activity of ICI 182,780 in combination with docetaxel is mediated by P-glycoprotein inhibition

Docetaxel is a semi-synthetic drug that has been shown to be effective in refractory advanced breast cancer. Its main mechanism of action seems to be to block microtubule depolymerisation. In this study we investigated the interaction between docetaxel and the pure anti-oestrogen ICI 182,780 on different oestrogen receptor-negative cancer cells, some of which express the classical multi-drug resistance (MDR) phenotype. ICI 182,780 potentiated the anti-proliferative effect of docetaxel only in the MDRpositive cells. Isobolic analysis demonstrated that this chemosensitising effect was a synergism. In the same conditions where synergism was detected, cell cycle analysis demonstrated an augmentation of cells blocked at the G2/M phase of the cell cycle, suggesting that ICI 182,780 increases the activity of docetaxel. In order to test this hypothesis, we performed bcl-2 western blot analysis and demonstrated that the addition of ICI 182,780 to docetaxel induced bcl-2 phosphorylation only in MDR-positive cells. The functional inactivation of bcl-2 is probably responsible for the commitment to apoptosis, since the combined docetaxel/ICI 182,780 treatment was able to foster a massive apoptosis in MDR-bearing cells as demonstrated by morphological analysis. Our results suggest that the synergism between docetaxel and ICI 182,870 is due to a block in Pglycoprotein activity, thus determining cell cycle block, bcl-2 inactivation and apoptosis induced by

Reversal of doxorubicin, etoposide, vinblastine, and taxol resistance in multidrug resistant human sarcoma cells by a polymer of spermine.

We have previously described the synthesis of a cytotoxic polymeric conjugate of spermine (Poly-SPM) which is able to inhibit the transport of polyamines (spermine, spermidine, and putrescine) into normal and malignant cells. Recent studies examining the toxicity of Poly-SPM in parental and multidrug resistant (MDR) cancer cells have revealed a cross-resistance in the MDR variant Dx5 to the toxic effects of the conjugate in the MDR-positive cells. There were also differences in spermine and putrescine uptake rates between parental and MDR-positive with the MDR-positive cells having a lower Vmax and a higher Km. The ability of this Poly-SPM to reverse MDR was examined in MDR variants (Dx5 cells) of the human sarcoma cell line MES-SA. The cells express high levels of the mdr1 gene product, P-glycoprotein, and are 25-to 60-fold resistant to doxorubicin (DOX), etoposide (VP-16), vinblastine (VBL), and taxol (TAX). Cytotoxicity was measured by the MTT [3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Poly-SPM (50 microM) lowered the drug concentration IC50 values in the Dx5 cells by 37-fold with VBL, 42-fold with DOX, 29-fold with VP-16, and 25-fold with TAX when compared to the control IC50 values without Poly-SPM. This reversal of resistance was concentration dependent, decreasing 17-fold with DOX, 6.1-fold with VBL, 19-fold with VP-16, and 5-fold with TAX when 25 microM Poly-SPM was used. No modulation was observed in the parental cell line MES-SA, which does not express the mdr1 gene. Poly-SPM had no influence on the IC50 of non-MDR chemotherapeutic agents such as cisplatin. The modulation studies correlated with the ability of Poly-SPM to reverse the cellular accumulation defect of [3H]-VBL and [3H]-TAX in the Dx5 but not MES-SA cells. Pretreatment of the Dx5 cell with alpha-difluoromethylornithine (DFMO at 2 and 5 microM) for 24 h increased the function of the MDR transporter to further decrease the cellular accumulation of VBL and TAX when compared to untreated cells. DFMO pretreatment is known to upregulate the polyamine transporter(s). These findings show that, in addition to inhibiting polyamine transport, Poly-SPM reverses MDR in Dx5 cells, suggesting a potential relationship between the polyamine influx transporter and the MDR efflux pump. This potential functional link between the polyamine influx transporter(s) and the MDR efflux transporter (P-glycoprotein) offers a novel approach to inhibiting this form of drug resistance.

5'-Deoxy-5-fluorouridine increases daunorubicin uptake in multidrug-resistant cells and its activity is related with P-gp 170 expression.

Most anticancer agents fail to induce clear responses in the treatment of colorectal cancer. This can be explained by involvement of overexpression of the membrane glycoprotein, P‐gp 170, which is associated with multidrug resistance (MDR), and/or with involvement of ras. Fluoropyrimidines are amongst the few options in the chemotherapeutic treatment of colorectal cancers. 5′‐Deoxy‐5‐fluorouridine (dFUrd) needs intracellular activation via 5‐fluorouracil into 5‐fluoro‐2′‐deoxyuridine‐5′‐monophosphate and 5‐fluorouridine‐S′‐triphosphate. In the present study, the cytotoxic activity of dFUrd in vitro and dFUrd combined with daunorubicin (DNR) was assessed with the (3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium) bromide assay in cells with increased P‐gp 170 expression versus controls. Surprisingly, dFUrd was most active in cells with high P‐gp 170 expression, a finding which can not be explained by intracellular metabolic activity only. The results also show that dFUrd improves the DNR uptake in MDR‐positive cells, and this is related with increased cyto‐toxicity of the anthracycline.

Overexpression of multidrug resistance-associated p170-glycoprotein in acute non-lymphocytic leukemia.

Resistance to several cytotoxic agents, including anthracyclines, vinca alkaloids and epipodophylline derivatives (multidrug resistance, or MDR) can develop in tumor cells by overexpression of a 170-kd glycoprotein (p170) which is an essential component of a membrane transport system leading to increased drug efflux and decreased intracellular drug concentration. By means of a p170-directed monoclonal antibody (MRK-16) and immunocytochemistry (alkaline phosphatase anti-alkaline phosphatase technique), we investigated the expression of p170 in marrow blast cells of 59 cases (38 at diagnosis and 21 in relapse) of acute-non-lymphocytic leukemia (ANLL). The proportion of strongly MDR-positive cells was higher in relapse that at diagnosis (median 15.5% vs 1.5%). Out of 31 patients who were evaluable for the results of first remission induction, failure of first-line treatment (including Daunorubicin, standard-dose and high-dose Arabinosyl Cytosine, and sometimes also Mitoxantrone) occurred in 8/22 MDR-positive cases and in 1/9 MDR-negative ones (p = 0.21). Failure of first-line treatment was always associated with a progressive increase of p170 expression. Total failures (no remission plus early relapse) were more frequent (p = 0.001) among MDR-positive cases (16/22) than among the others (2/9). These data show that MDR is very frequent in ANLL also at diagnosis and suggest that MDR can contribute to early failure of standard treatment.

A Combined Approach for Purging Multidrug-Resistant Leukemic Cell Lines in Bone Marrow Using a Monoclonal Antibody and Chemotherapy

Selective removal of malignant cells (purging) from bone marrow (BM) is a concern in autologous BM transplantation (ABMT). Use of vincristine, etoposide, or doxorubicin for purging could be rendered ineffective by the presence of multidrug-resistant (MDR) tumor cells. To circumvent this particular problem, we investigated whether 17F9, a monoclonal IgG2b antibody directed against the cell surface product of the MDR gene, P-glycoprotein, is effective in selective removal of MDR cells from BM when used with rabbit complement (C'). Using two different cell lines we have demonstrated that 17F9 + C' selectively lyses MDR-positive cells. Three rounds of antibody + C' resulted in 96.4% +/- 3.6% kill of K562/DOX and 100% +/- 0% of CEM/VLB cells. Mixtures of malignant cells and normal BM resulted in 99.85% removal of K562/DOX and 99.91% removal of CEM/VLB clonogenic cells. This treatment did not affect normal committed precursors compared with C' alone. The addition of the cytotoxic agent etoposide (VP-16) following antibody purging results in a 4.6 log reduction of malignant cells. Furthermore, this antibody was effective when used against patients' leukemic blasts. These results suggest the use of 17F9 + C' is effective and selective for removal of MDR cells from BM before ABMT and the addition of VP-16 enhances the purging efficacy.