A relationship between multidrug resistance and growth-state dependent cytotoxicity of the lysosomotropic detergent N-dodecylimidazole.

Multidrug resistance (MDR) in cultured cells and tumors is associated with overproduction of P-glycoprotein, a plasma membrane efflux pump normally present at very low levels. The cytotoxic action of N-dodecylimidazole (C12-Im), a lysosomotropic detergent, on cultured cells was previously shown to be strongly dependent on growth state, with rapidly growing cells being most sensitive and confluent cells most resistant. We show here that this may be due to a growth dependent increase in cellular P-glycoprotein activity. Both verapamil and nifedipine, structurally unrelated P-glycoprotein inhibitors, increased markedly the sensitivity of CHO fibroblasts to killing by C12-Im; the increase was greater in confluent than in growing cells. Also, verapamil inhibitable 3H-daunomycin efflux was more efficient from confluent than from subconfluent cells. The MDR cell line CH(R)C5 differed from all cell lines previously examined in that it did not show a growth-dependent decrease in C12-Im sensitivity, and sensitivity was not increased by verapamil or nifedipine. We suggest that a growth-dependent increase in MDR activity is a general property of cultured cells, except for those specifically overexpressing P-glycoprotein.     

A mechanism for P-glycoprotein-mediated apoptosis as revealed by verapamil hypersensitivity

Selection of tumor cell lines with anticancer drugs has led to the appearance of multidrug-resistant (MDR) subclones with P-glycoprotein 1 (P-gp1) expression. These cells are cross-resistant to several structurally and functionally dissimilar drugs. Interestingly, in the process of gaining resistance, MDR cells become hypersensitive or collaterally sensitive to membrane-active agents, such as calcium channel blockers, steroids, and local anaesthetics. In this report, hypersensitivity to the calcium channel blocker, verapamil, was analyzed in sensitive and resistant CHO cell lines. Our results show that treatment with verapamil preferentially induced apoptosis in MDR cells compared to drug-sensitive cells. This effect was independent of p53 activity and could be inhibited by overexpression of the Bcl-2 gene. The induction of apoptosis by verapamil had a biphasic trend in which maximum cell death occurred at 10 microM, followed by improved cell survival at higher concentrations (50 microM). We correlated this effect to a similar biphasic trend in P-gp1 ATPase activation by verapamil in which low concentrations of verapamil (10 microM) activated ATPase, followed by inhibition at higher concentrations. To confirm the relationship between apoptosis and ATPase activity, we used two inhibitors of P-gp1 ATPase, PSC 833 and ivermectin. These ATPase inhibitors reduced hypersensitivity to verapamil in MDR cells. In addition, low concentrations of verapamil resulted in the production of reactive oxygen species (ROS) in MDR cells. Taken together, these results show that apoptosis was preferentially induced by P-gp1 expressing cells exposed to verapamil, an effect that was mediated by ROS, produced in response the high ATP demand by P-gp1.     

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.     

Multidrug resistance p-glycoprotein inhibits antiapoptotic action of mitochondria-targeted antioxidant SkQR1

Mitochondria-targeted antioxidants of the SkQ family that accumulate in energized mitochondria protect cells from oxidative stress by increasing the level of reduced glutathione and decreasing cell damage induced by hydrogen peroxide. The exposure of various human transformed cell lines to SkQR1, a fluorescent member of the SkQ family, showed that SkQR1 was pumped out of the chemotherapy resistant cells by P-glycoprotein, one of the main transport proteins that determines multidrug resistance typical for many neoplastic cells. It was also shown that SkQR1 pumping is neutralized by P-glycoprotein inhibitors (verapamil and pluronic L61). In experiments on K-562 cells, it was found that the subline sensitive to chemotherapy is protected by SkQR1 from apoptosis induced by hydrogen peroxide. The protection of resistant subline cells is only evident after the inhibition of P-glycoprotein.     

Verapamil hypersensitivity of vincristine resistant Chinese hamster ovary cell lines

Vincristine resistant CHO cell lines, obtained by prolonged selection in semi-inhibitory drug concentrations show considerable hypersensitivity to verapamil. Their D10 values are around 0.2 micrograms/ml compared to 23 micrograms/ml for unselected controls. Reversion of vincristine resistance during growth in vincristine free medium is correlated with reversal of verapamil sensitivity indicating that the two aspects of the cells' phenotype have a common underlying cause. The rate of uptake of calcium in the absence and presence of verapamil is similar in the vincristine resistant cells and the controls. The correlation of verapamil sensitivity with vincristine resistance is not a universal feature of CHO cell lines resistant to antimicrotubular drugs, since it was found that other resistant cell lines which have been selected by short term exposure to high drug concentrations were not verapamil hypersensitive.     

Molecular Study of P-Glycoprotein in Multidrug Resistance Using Surface Plasmon Resonance

P-Glycoprotein is an integral membrane protein which mediates the energy-dependent efflux of various antitumor agents from multidrug-resistant cancer cells. Surface plasmon resonance was used for the detection of P-glycoprotein after solubilization from drug-resistant and drug-sensitive Chinese hamster ovary cells and for the analysis of its interaction with cyclosporin A, a competitive inhibitor of drug efflux. Detection of P-glycoprotein relied on its binding to the monoclonal antibody C219 which was immobilized on a sensor chip. Binding of Zwittergent 3-14-solubilized P-glycoprotein to the antibody was concentration-dependent and reflected the relative abundance of P-glycoprotein in both cell lines. It was abolished when C219 was omitted or replaced by a rabbit anti-mouse IgG antibody and considerably reduced after precipitation of P-glycoprotein with wheat germ agglutinin. Preincubation of solubilized proteins with cyclosporin A increased the amount of protein bound to the antibody by approximately 30%. These results indicate that surface plasmon resonance is well suited to the detection of P-glycoprotein from biological samples and shows promise as a tool for the study of its interaction with different drugs.     

Multidrug resistance P-glycoprotein inhibits the antiapoptotic action of mitochondria-targeted antioxidant SkQR1

Mitochondria-targeted antioxidants of the SkQR1 family, being accumulated in energized mitochondria, protect cells from oxidative stress by increasing the level of reduced glutathione and decreasing the cell-damaging effect induced by hydrogen peroxide. Using various human transformed cell lines and SkQR1 (a fluorescent member of the SkQ family), we show that SkQRI is ejected from chemotherapy-resistant cells by P-glycoprotein - one of the main transport proteins determining multidrug resistance typical for many neoplastic cells. It is also shown that SkQR1 ejection is neutralized by P-glycoprotein inhibitors (verapamil and pluronic L61). In experiments on K562 cells, it was found that the subline sensitive to chemotherapy is protected by SkQRI from apoptotic action of hydrogen peroxide. Protection of the resistant subline occurs only after inhibition of P-glycoprotein.     

MDR3 P-glycoprotein, a phosphatidylcholine translocase, transports several cytotoxic drugs and directly interacts with drugs as judged by interference with nucleotide trapping

The human MDR3 gene is a member of the multidrug resistance (MDR) gene family. The MDR3 P-glycoprotein is a transmembrane protein that translocates phosphatidylcholine. The MDR1 P-glycoprotein related transports cytotoxic drugs. Its overexpression can make cells resistant to a variety of drugs. Attempts to show that MDR3 P-glycoprotein can cause MDR have been unsuccessful thus far. Here, we report an increased directional transport of several MDR1 P-glycoprotein substrates, such as digoxin, paclitaxel, and vinblastine, through polarized monolayers of MDR3-transfected cells. Transport of other good MDR1 P-glycoprotein substrates, including cyclosporin A and dexamethasone, was not detectably increased. MDR3 P-glycoprotein-dependent transport of a short-chain phosphatidylcholine analog and drugs was inhibited by several MDR reversal agents and other drugs, indicating an interaction between these compounds and MDR3 P-gp. Insect cell membranes from Sf9 cells overexpressing MDR3 showed specific MgATP binding and a vanadate-dependent, N-ethylmaleimide-sensitive nucleotide trapping activity, visualized by covalent binding with [alpha-(32)P]8-azido-ATP. Nucleotide trapping was (nearly) abolished by paclitaxel, vinblastine, and the MDR reversal agents verapamil, cyclosporin A, and PSC 833. We conclude that MDR3 P-glycoprotein can bind and transport a subset of MDR1 P-glycoprotein substrates. The rate of MDR3 P-glycoprotein-mediated transport is low for most drugs, explaining why this protein is not detectably involved in multidrug resistance. It remains possible, however, that drug binding to MDR3 P-glycoprotein could adversely affect phospholipid or toxin secretion under conditions of stress (e.g. in pregnant heterozygotes with one MDR3 null allele).     

Detection of P-glycoprotein in the nuclear envelope of multidrug resistant cells

P-glycoprotein is a plasma membrane efflux pump which is responsible for multidrug resistance of many cancer cell lines. A number of studies have demonstrated the presence of P-glycoprotein molecules, besides on the plasma membrane, also in intracellular sites, such as the Golgi apparatus and the nucleus. In this study, the presence and function of P-glycoprotein in the nuclear membranes of human breast cancer cells (MCF-7 WT) and their multidrug resistant variants (MCF-7 DX) were investigated. Electron and confocal microscopy immunolabelling experiments demonstrated the presence of P-glycoprotein molecules in the nuclear membranes of MCF-7 DX cells. Moreover, the labelling pattern was strongly dependent on pH values of the incubation buffer. At physiological pH (7.2), a strong labelling was detected in the cytoplasm and the nuclear matrix in both sensitive and resistant MCF-7 cells. By raising the pH to 8.0, the P-glycoprotein molecules were easily detected in the cytoplasm (transport vesicles and Golgi apparatus), plasma and nuclear membranes exclusively in MCF-7 DX cells. Furthermore, drug uptake and efflux studies, performed by flow cytometry on isolated nuclei in the presence of the P-glycoprotein inhibitor cyclosporin A, suggested the presence of a functional P-glycoprotein in the nuclear membrane, but not in the nuclear matrix, of drug resistant cells. Therefore, P-glycoprotein in the nuclear envelope seems to represent a further defense mechanism developed by resistant cells against antineoplastic agents.     

Multidrug resistance p-glycoprotein inhibits the antiapoptotic action of mitochondria-targeted antioxidant SkQR1

Mitochondria-targeted antioxidants of the SkQRI family, being accumulated in energized mitochondria, protect cells from oxidative stress by increasing the level of reduced glutathione and decreasing the cell-damaging effect induced by hydrogen peroxide. Using various human transformed cell lines and SkQR1 (a fluorescent member of the SkQ family), we show that SkQR1 is ejected from chemotherapy-resistant cells by P-glycoprotein--one of the main transport proteins determining multidrug resistance typical for many neoplastic cells. It is also shown that SkQR1 ejection is neutralized by P-glycoprotein inhibitors (verapamil and pluronic L61). In experiments on K562 cells, it was found that the subline sensitive to chemotherapy is protected by SkQR1 from apoptotic action of hydrogen peroxide. Protection of the resistant subline occurs only after inhibition of P-glycoprotein.     

Microfluorometric Evaluation of Calcein Acetoxymethyl Ester as a Probe for P-Glycoprotein-Mediated Resistance: Effects of Cyclosporin A and Its Nonimmunosuppressive Analogue SDZ PSC 833

A microtiter plate-based fluorometric assay for functional measurement of 170-kDa P-glycoprotein (Pgp)-mediated transport using fluorescent calcein as a probe is described. The myeloma RPMI 8226 cell line and two of its doxorubicin-resistant Pgp-expressing sublines, dox40 (high expression) and dox6 (low expression), were used as models. Nonfluorescent calcein acetoxymethyl ester (calcein/AM) was added to the cells and subsequent accumulation of calcein was measured in a 96-well scanning fluorometer after 30 min. There was an inverse relationship between Pgp expression and calcein/AM accumulation, which increased dose-dependently in the presence of cyclosporin A (CsA) and the nonimmunosuppressive analogue SDZ PSC 833 (PSC) in the Pgp-expressing cell lines. PSC appeared to restore uptake more effectively than CsA at low concentrations. Calcein accumulation was also increased in Pgp-expressing cells by the addition of the Pgp substrate vincristine and the metabolic inhibitor potassium cyanide, KCN. No effect was observed in parental cell lines. When parental and dox40 cells were mixed, 10% of dox40 cells could reproducibly be detected. The results indicate that microtiter-plate determination of calcein accumulation is a simple and sensitive method for functional determination of Pgp-mediated drug transport. The method may become useful, not only for preclinical screening for novel and improved resistance modifiers, but also for determination of Pgp activity in individual clinical tumor samples.     

Selection for high-level chloroquine resistance results in deamplification of the pfmdr1 gene and increased sensitivity to mefloquine in Plasmodium falciparum.

A chloroquine resistant cloned isolate of Plasmodium falciparum, FAC8, which carries an amplification in the pfmdr1 gene was selected for high-level chloroquine resistance, resulting in a cell line resistant to a 10-fold higher concentration of chloroquine. These cells were found to have lost the amplification in pfmdr1 and to no longer over-produce the protein product termed P-glycoprotein homologue 1 (Pgh1). The pfmdr1 gene from this highly resistant cell line was not found to encode any amino acid changes that would account for increased resistance. Verapamil, which reverses chloroquine resistance in FAC8, also reversed high-level chloroquine resistance. Furthermore, verapamil caused a biphasic reversal of chloroquine resistance as the high-level resistance was very sensitive to low amounts of verapamil. These data suggest that over-expression of the P-glycoprotein homologue is incompatible with high levels of chloroquine resistance. In order to show that these results were applicable to other chloroquine selected lines, two additional mutants were selected for resistance to high levels of chloroquine. In both cases they were found to deamplify pfmdr1. Interestingly, while the level of chloroquine resistance of these mutants increased, they became more sensitive to mefloquine. This suggests a linkage between the copy number of the pfmdr1 gene and the level of chloroquine and mefloquine resistance.     

Relationship between multidrug resistance,hypersensitivity to resistance modifiers and cell volume in Chinese hamster ovary cells.

We have previously shown that very high levels of hypersensitivity to several resistance modifiers are correlated with increasing multidrug resistance in a series of Chinese hamster ovary cell lines. We have now selected a new member of the series which is an exception to this correlation in that although it is almost twice as multidrug resistant as the cell line from which it was derived, it shows much less hypersensitivity to resistance modifiers. Level of resistance modifier hypersensitivity correlated with the level of reduction of verapamil accumulation in these cells, and with the density of P-glycoprotein, but since the selection of this cell line has involved a doubling of cell volume, it was not correlated with total amount of P-glycoprotein.     

P-glycoprotein mediates the collateral sensitivity of multidrug resistant cells to steroid hormones

The overexpression of P-glycoprotein (P-gp, ABCB1) in cancer cells often leads to multidrug resistance (MDR) through reduced drug accumulation. However, certain P-gp-positive cells display hypersensitivity, or collateral sensitivity, to certain compounds that are believed to induce Pgp-dependent oxidative stress. We have previously reported that MDR P-gp-positive CHO cells are collaterally sensitive to verapamil (VRP; Laberge et al. (2009) [1]). In this report we extend our previous findings and show that drug resistant CHO cells are also collaterally sensitive to physiologic levels of progesterone (PRO) and deoxycorticosterone (DOC). Both PRO and DOC collateral sensitivities in CH^RC5 cells are dependent on P-gp-expression and ATPase, as knockdown of P-gp expression with siRNA or inhibition of P-gp-ATPase with PSC833 reverses PRO- and DOC-induced collateral sensitivity. Moreover, the mitochondrial complexes I and III inhibitors (antimycin-A and rotenone, respectively) synergize with PRO and DOC-induced collateral sensitivity. We also show that VRP inhibits PRO and DOC collateral sensitivity, consistent with earlier findings relating to the VRP’s modulation of PRO and DOC-stimulation of P-gp ATPase. The findings of this study demonstrate a P-gp-dependent collateral sensitivity of MDR cells in the presence of physiologically achievable concentrations of progesterone and deoxycorticosterone.     

Detection of a P-glycoprotein related pump in Chironomus larvae and its inhibition by verapamil and cyclosporin A

A membrane associated ATP-dependent efflux pump, similar in function to mammalian P-glycoprotein, was detected in anal papillae of Chironomus riparius larvae. Immunohistochemical analysis of larval tissues, using monoclonal antibodies against P-glycoprotein, was supplemented by functional in vivo and in vitro assays which confirmed the existence of a mechanism for transporting xenobiotic substances. The in vitro ATPase activity of homogenate fractions increased in the presence of typical P-glycoprotein substrates (vinblastine, actinomycin D or ivermectin). This increase was unaffected by inhibitors of other membrane ATPases (sodium azide, EGTA, ouabain), but sensitive to vanadate, cyclosporin A and verapamil which inhibit mammalian P-glycoprotein mediated ATP-consumption. Sublethal concentrations of specific P-glycoprotein-inhibitors such as verapamil or cyclosporin A synergistically enhanced the mortality of C. riparius towards ivermectin. Although cyclosporin A originates from entomopathogenic fungi, its mode of action in insects and its function during infection are not understood. Our results lend some credit to the hypothesis that this compound is possibly released to promote poisoning of the infected host by xenobiotics which are normally removed by a P-glycoprotein related pump. The putative role of insect P-glycoprotein homologues in the context of multiple resistance towards insecticides is discussed.     

Functional Expression of P-Glycoprotein in an Immortalised Cell Line of Rat Brain Endothelial Cells, RBE4

Abstract: The presence of P-glycoprotein in the cell plasma membrane limits the penetration of many cytotoxic substances into cells that express the gene product. There is considerable evidence also to indicate that P-glycoprotein is expressed as part of the normal blood-brain barrier in the luminal membranes of the cerebral capillary endothelial cells, where it presumably performs a protective function for the brain. This report describes the functional expression of P-glycoprotein in an immortalised cell line, RBE4, derived from rat cerebral capillary endothelial cells. The expression of P-glycoprotein is demonstrated by western immunoblotting and by immunogold and fluorescent staining with monoclonal antibodies. The cellular accumulation of [³H]colchicine and [³H]vinblastine is investigated and shown to be enhanced by the presence of azidothymidine, chlorpromazine, verapamil, cyclosporin A, and PSC 833 ([3'-keto-Bmt¹]-[Val²]-cyclosporin) at 50 or 100 µ M concentration. It is concluded that the RBE4 cell line is a valuable tool for investigating the mechanisms of P-glycoprotein activity both in the blood-brain barrier and in multidrug resistance in general.     

In archaebacteria, there is a doxorubicin efflux pump similar to mammalian P-glycoprotein.

We selected for study an anthracycline-resistant mutant from the archaebacteria Haloferax volcanii. This resistance was reversed by a Ca(2+)-channel antagonist, nifedipine (NDP). This resistance and its reversal by NDP suggest P-glycoprotein (Pgp) to be responsible for maintaining an anticancer drug concentration below the cytotoxic level. Using rhodamine 123 (RH123) as a substrate for Pgp, we then examined whether the resistance to anthracyclines in this bacteria might involve a Pgp-like anthracycline efflux pump. RH123 accumulation by the bacteria was determined with flow cytometry. A steady-state RH123 accumulation by the resistant cells revealed approx. one-fifteenth of that by the wild-type cells, which could be remarkably enhanced by NDP. The other modulators of Pgp, diltiazem and verapamil, also enhanced RH123 accumulation in resistant cells. The uncoupler FCCP completely restored RH123 accumulation in resistant cells to the wild-type cell level. RH123 unidirectional efflux from resistant cells after its preloading revealed much greater than that from wild-type cells, which was remarkably inhibited by FCCP. These confirmed that RH123 low accumulation involves its active efflux mechanism. Taken together, the present study indicated that lower evolutionary archaebacteria might also express a Pgp-like protein very similar to mammalian Pgp.     

Role of P-glycoprotein in the renal transport of dideoxynucleoside analog drugs.

P-glycoprotein (P-gp), the MDR1 multidrug transporter, is known to be expressed in several human organs and tissues, including the apical membrane of the renal proximal tubular cells. It has been reported that human immunodeficiency virus 1 (HIV-1) can trigger the expression of P-gp in cultured cells (i.e., H9, a T-lymphocyte cell line, and U937, a monocyte cell line), which may render the cells resistant to antiretrovirals. Since multiple membrane transport systems (i.e., organic cation, organic anion, and nucleoside systems) can be involved in the renal tubular transport of dideoxynucleoside analog drugs (DADs) (i.e., zidovudine and zalcitabine), we have questioned if P-gp is involved in the renal transport of DADs. Chinese hamster ovary colchicine-resistant cells (CH(R)C5), a cell line that is well known to highly express P-gp, and continuous renal epithelial cell lines (LLC-PK1 and OK), which have also been shown to express P-gp, were used. The accumulation of [3H]vinblastine (20 nM), an established P-gp substrate, by the monolayer cells was significantly enhanced in the presence of two P-gp inhibitors (i.e., verapamil and cyclosporin A) and nucleoside transport inhibitors (i.e., dipyridamole and dilazep). In contrast, DADs (i.e., zidovudine, lamivudine, didanosine, and zalcitabine) did not significantly affect vinblastine accumulation by these cell lines. These data suggest that P-gp does not play a significant role in the renal tubular transport of DADs. Dipyridamole and dilazep, two nucleoside membrane transport inhibitors, appear to be P-gp inhibitors.