Regulation of volume-sensitive Cl- channels in multi-drug resistant MCF7 cells

The P-glycoprotein (P-gp) is thought to be involved in the regulation of volume-sensitive chloride channels. In this study, the possible coupling between P-gp and swelling-activated chloride channels has been examined in MCF7 cells with sensitive (MDR-), resistant (MDR+), and reversed resistant (MDR(REV)) phenotypes. Western blot analysis showed that incubation of cells with doxorubicin induced P-gp expression in a reversible manner. Exposure of MDR+ cells to hypotonicity resulted in an inhibition of P-gp activity while hypotonic challenges induced swelling-activated chloride currents (I(Cl-swell)) in MDR-, MDR+, and MDR(REV) MCF7 cells. While verapamil inhibited I(Cl-swell) in all cell types, doxorubicin and vincristine rapidly and reversibly inhibited I(Cl-swell) uniquely in MDR+. Intracellular dialysis of MDR+ cells with C219 anti-P-gp antibody abolished the sensitivity of I(Cl-swell) to doxorubicin and led to a response pattern very close to that of MDR- cells. Taken together, these results strongly suggest that the P-glycoprotein regulates I(Cl-swell) in resistant MCF7.     

Agents which reverse multidrug-resistance are inhibitors of [3H]vinblastine transport by isolated vesicles.

Resistance of human cancer cells to multiple cytotoxic hydrophobic agents (multidrug resistance) is due to overexpression of the MDR1 gene whose product is the ATP-dependent multidrug transporter, P-glycoprotein. We have previously reported that plasma membrane vesicles partially purified from multidrug-resistant human KB carcinoma cells, but not from drug-sensitive cells, accumulated [3H]vinblastine in an ATP-dependent manner (Horio, M., Gottesman, M.M. and Pastan, I. (1988) Proc. Natl. Acad. Sci. USA 85, 3580-3584). Certain calcium-channel blockers, quinidine, and phenothiazines are able to overcome multidrug resistance in cultured cells. In this work, the effect of these reversing agents on ATP-dependent vinblastine (VBL) transport by vesicles from drug-resistant KB cells has been characterized. Azidopine was the most potent inhibitor of ATP-dependent VBL uptake tested (ID50: concentration of inhibitor such that the transport of vinblastine is inhibited by 50%, less than 1 microM). Verapamil, quinidine, and the tiapamil analogue RO-11-2933 were potent but less effective inhibitors (ID50 less than 5 microM). Diltiazem, nifedipine and trifluoperazine were even less effective. These agents had no effect on Na(+)-dependent and Na(+)-independent L-leucine uptake by the vesicles, indicating that the inhibition of ATP dependent VBL transport by these agents is not a non-specific effect, as might result from leaks in the vesicle membrane. Verapamil, quinidine, azidopine and trifluoperazine increased the apparent Km value of vinblastine transport, suggesting that these agents may be competitive inhibitors of vinblastine transport.     

Protein kinase C-independent correlation between P-glycoprotein expression and volume sensitivity of Cl− channel

The possible correlation between P-glycoprotein (PGP) and volume-sensitive Cl⁻ channel was examined in a pair of cell lines: a subline of the human epidermoid KB cell (KB-3-1) and the corresponding MDR1-transfected cell line (KB-G2). Western blot analysis and indirect immunofluorescence studies indicated that KB-G2, but not KB-3-1, exhibits the PGP expression. Patch-clamp whole-cell recordings showed that osmotic swelling activates Cl⁻ currents not only in PGP-expressing but also in PGP-lacking cells. The amplitude of the maximal current was indistinguishable between both cells. Activation of protein kinase C (PKC) or loading with a PKC inhibitor failed to affect the swelling-induced activation of the Cl⁻ currents in both cells. The relation between whole-cell Cl⁻ currents and cell size measured simultaneously showed that volume sensitivity of the Cl⁻ channel was augmented by the PGP expression irrespective of the activity of PKC on the plasma membrane. A similar increase in volume sensitivity of the Cl⁻ channel was also induced by the expression of the ATP hydrolysis-deficient PGP mutant, K433M. We conclude that P-glycoprotein does not represent the volume-sensitive Cl⁻ channel but that its expression modulates volume sensitivity of the Cl⁻ channel in a manner independent of its ATPase activity or of the protein kinase C activity. Received: 25 September 1996/Revised: 12 December 1996     

Purification and reconstitution of functional human P-glycoprotein

The overexpression of the P-glycoprotein, theMDR1 gene product, has been linked to the development of resistance to multiple cytotoxic natural product anticancer drugs in certain cancers and cell lines derived from tumors. P-glycoprotein, a member of the ATP-binding cassette (ABC) superfamily of transporters, is believed to function as an ATP-dependent drug efflux pump with broad specificity for chemically unrelated hydrophobic compounds. We review here recent studies on the purification and reconstitution of P-glycoprotein to elucidate the mechanism of drug transport. P-glycoprotein from the human carcinoma multidrug resistant cell line, KB-V1, was purified by sequential chromatography on anion exchange followed by a lectin (wheat germ agglutinin) column. Proteoliposomes reconstituted with pure protein exhibited high levels of drug-stimulated ATPase activity as well as ATP-dependent [³H]vinblastine accumulation. Both the ATPase and vinblastine transport activities of the reconstituted P-glycoprotein were inhibited by vanadate. In addition, the vinblastine transport was inhibited by verapamil and daunorubicin. These studies provide strong evidence that the human P-glycoprotein functions as an ATP-dependent drug transporter. The development of the reconstitution system and the availability of recombinant protein in large amounts due to recent advances in overexpression of P-glycoprotein in a heterologous expression system should facilitate a better understanding of the function of this novel protein.     

Functionally active homodimer of P-glycoprotein in multidrug-resistant tumor cells.

P-glycoprotein plays a key role in multidrug resistance of tumor cells. In order to elucidate the possible quarternary structure/function relationship of P-glycoprotein, we treated multidrug-resistant human leukemia K562/ADM cells with the crosslinking reagent, disuccinimidyl suberate. In addition to 180K P-glycoprotein, a 340K protein was immunoprecipitated with an anti-P-glycoprotein monoclonal antibody, MRK-16. The 340K protein is most probably a dimeric P-glycoprotein, since only the 180K P-glycoprotein was immunoprecipitated with MRK-16 when K562/ADM cells were treated with the cleavable crosslinking reagent, dithiobis(succinimidylpropionate), and analysed under reduced conditions. The dimeric P-glycoprotein was photolabeled with [3H]azidopine like the 180K monomeric P-glycoprotein and the photolabeling was inhibited by excess amount of vincristine and verapamil. The dimeric P-glycoprotein could be a functionally active form of the protein involved in the transport of antitumor agents.     

The over-expression of P-glycoprotein in K-562 and DAUDI cells, is associated with a high susceptibility to NK and LAK cells.

We evaluated the susceptibility to natural killer (NK) or lymphokine activated killer (LAK) cell-mediated cytolysis of two pairs of drug sensitive/resistant tumor cell lines which were extensively characterized at phenotypic and genotypic level. In the DAUDI cell system, the acquired capability of tumor cell variants to grow in the presence of a relatively high concentration of vinblastine (VBL) is associated with a marked increase to NK and LAK susceptibility. In contrast in the K-562 cell system, no correlation between drug-resistance, P-glycoprotein expression and susceptibility to NK or LAK activity seems to occur.     

CCN3 suppresses mitogenic signalling and reinstates growth control mechanisms in Chronic Myeloid Leukaemia

CCN3, a tumour suppressor gene, is down-regulated as a result of BCR-ABL tyrosine kinase activity in Chronic Myeloid Leukaemia (CML). We have established a stable CCN3 expression model in the human K562 CML cell line and have further validated the role for CCN3 in the leukaemogenic process. K562 cells stably transfected with CCN3 (K562/CCN3; 2.25 × 10⁶ copies per 50 ng cDNA) demonstrated over 50% reduction in cell growth in comparison to cells stably transfected with empty vector (K562/control; p = 0.005). K562/CCN3 cells had reduced colony formation capacity (reduced by 29.7%, p = 0.03) and reduced mitogenic signalling in comparison to K562/control cells (reduced by 29.5% (p = 0.002) and 37.4% (p = 0.017) for phosphorylation levels of ERK and AKT respectively). K562/CCN3 cells showed an accumulation of events within the subG₀ phase of the cell cycle and increased apoptosis was confirmed by a three-fold increase in annexin V binding (p < 0.05). K562/CCN3 cells exposed to Imatinib (1 μM and 5 μM) showed an increase in events within the subG₀ phase of cell cycle over 96 h and mirrored the enhanced cell kill demonstrated by Annexin staining. Wild type K562 cells treated with recombinant human Ccn3 (10 nM) in combination with Imatinib (5 μM) also displayed enhanced cell kill (p = 0.008). K562/CCN3 cells displayed increased adhesion to matrigel™ (2.92 ± 0.52 fold increase compared to K562/control) which was commensurate with increased expression of the alpha 6 and beta 4 integrins (6.53 ± 0.47 and 1.94 ± 0.07 fold increase in gene expression respectively (n = 3, p < 0.05)). CCN3 restores cellular growth regulatory properties that are absent in CML and sensitises CML cells to imatinib induced apoptosis. CCN3 may provide novel avenues for the development of alternate therapeutic strategies.     

Purification of the 170- to 180-kilodalton membrane glycoprotein associated with multidrug resistance. 170- to 180-kilodalton membrane glycoprotein is an ATPase

170-180-kDa membrane glycoprotein (P-glycoprotein) associated with multidrug resistance is involved in drug transport mechanisms across the plasma membrane of resistant cells. From sequence analysis of cDNAs of the P-glycoprotein gene, it is postulated that the active drug-efflux pump function may be attributable to the protein. However, purification of the P-glycoprotein while preserving its enzymatic activity has not been reported. In this study, we have purified the P-glycoprotein from the human myelogenous leukemia K562 cell line resistant to adriamycin (K562/ADM) by means of one-step immunoaffinity chromatography using a monoclonal antibody against P-glycoprotein. The procedure was simple and efficiently yielded an electrophoretically homogeneous P-glycoprotein sample. By solubilization with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, the purified P-glycoprotein was found to have ATPase activity. This ATP hydrolysis may be coupled with the active efflux of anticancer drugs across the plasma membrane of multidrug-resistant cells.     

Glycoprotein 170 induces platelet-activating factor receptor membrane expression and confers tumor cell hypersensitivity to NK-dependent cell lysis

Multidrug resistance (MDR) confers resistance to anticancer drugs and reduces therapeutic efficiency. It is often characterized by the expression of the MDR1 gene product P-glycoprotein (or gp170) at the membrane of tumor cells. To further propose a potential complementary tool in cancer treatment, the sensitivity of gp170 tumor cells to NK-dependent lysis was investigated. Two kinds of cells were generated from wild-type K562 erythroleukemic cells: the first were derived from Taxol-selected cells and cloned, whereas the second were retrovirally transduced by the cDNA of the MDR1 gene. The last process was also applied to the human embryonal carcinoma cells called Tera-2 cells. First, both cloned and MDR-1 K562 cells appeared highly susceptible to naive NK cell killing. Interestingly, in addition, Tera-2 cells that were not sensitive to NK lysis could be killed when they expressed gp170 at their membranes. In previous data, we demonstrated that NK cell release of bimolecular complexes composed of perforin and platelet-activating factor (PAF) interacting with the PAF-R, which has to be expressed on the target cell membranes, were components of NK tumor cell killing. In the present study, we show that gp170 has the capacity to drive constitutive PAF-R expression on tumor cells, which could be responsible for hypersensitivity to NK lysis and accelerated cell death.     

Down-regulation of P-glycoprotein expression by sustained intracellular acidification in K562/Dox cells

We have investigated the involvement of intracellular pH (pH_i) in the regulation of P-glycoprotein (P-gp) in K562/DOX cells. The selective Na⁺/H⁺ exchanger1 (NHE1) inhibitor cariporide and the “high K⁺” buffer were used to induce the sustained intracellular acidification of the K562/DOX cells that exhibited more alkaline pH_i than the K562 cells. The acidification resulted in the decreased P-gp activity with increased Rhodamine 123 (Rh123) accumulation in K562/DOX cells, which could be blocked by the P-gp inhibitor verapamil. Moreover, the acidification decreased MDR1 mRNA and P-gp expression, and promoted the accumulation and distribution of doxorubicin into the cell nucleus. Interestingly, these processes were all pH_i and time-dependent. Furthermore, the change of the P-gp expression was reversible with the pH_i recovery. These data indicate that the tumor multidrug resistance (MDR) mediated by P-gp could be reversed by sustained intracellular acidification through down-regulating the P-gp expression and activity, and there is a regulative link between the pH_i and P-gp in K562/DOX cells.     

Volume-Activated Chloride Currents in Fetal Human Nasopharyngeal Epithelial Cells

Volume-activated chloride channels have been studied by us extensively in human nasopharyngeal carcinoma cells. However, the chloride channels in the counterpart of the carcinoma cells have not been investigated. In this study, volume-activated chloride currents (I_cl,vol) were characterized in normal fetal human nasopharyngeal epithelial cells using the whole-cell patch-clamp technique. Under isotonic conditions, nasopharyngeal epithelial cells displayed only a weak background current. Exposure to 47% hypotonic solution activated a volume-sensitive current. The reversal potential of the current was close to the calculated equilibrium potential for Cl⁻. The peak values of the hypotonicity-activated current at +80 mV ranged from 0.82 to 2.71 nA in 23 cells. Further analysis indicated that the density of the hypotonicity-activated current in most cells (18/23) was smaller than 60 pA/pF. Only five cells presented a current larger than 60 pA/pF. The hypotonicity-activated current was independent of the exogenous ATP. Chloride channel inhibitors ATP, tamoxifen and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), inhibited the current dramatically. The anion permeability of the hypotonicity-activated chloride channels was I⁻ > Br⁻ > Cl⁻ > gluconate. Unexpectedly, in isotonic conditions, ATP (10 mM) activated an inward-rectified current, which had not been observed in the nasopharyngeal carcinoma cells. These results suggest that, under hypotonic challenges, fetal human nasopharyngeal epithelial cells can produce I_cl,vol, which might be involved in cell volume regulation.     

Molecular and functional identification of sodium channels in K562 cells

Modulations of ion channel activity underlie rapid changes in membrane transport of cations in various nonexcitable cells. Previously, in smooth muscle cells, macrophages, lymphocytes, carcinoma and leukemia cell lines, non-voltage-gated sodium (NVGS) channels have been found. The activity of NVGS channels was shown to be critically dependent on the organization of actin cytoskeleton. The molecular identity of NVGS channels remains unclear. The present work is focused on molecular and functional identification of NVGS channels in human myeloid leukemia K562 cells. Degenerin/epithelial Na⁺ channels (DEG/ENaC) can be considered as possible molecular correlates. By using RT-PCR, expression of ??-, ??-, and ??-hENaC subunits in the K562 cells was detected. Various modes of the patch-clamp method were used to examine functional properties of sodium channels??specifically, to test the effect of amiloride on single channel and integral currents. The biophysical characteristics of the NVSG channels were close to those of ENaC; the channels have unitary conductance of 12 pS (145 mM Na⁺) and were impermeable to divalent cations (Ca²⁺ and Mg²⁺). We found that amiloride did not inhibit NVGS channels. Importantly, no amiloride-blockable sodium current was detected in the plasma membrane of K562 cells. Taken together, our observations suggest that amiloride-insensitive sodium channels in the K562 cells belong to the ENaC family.     

Increased vinblastine binding to membrane vesicles from multidrug-resistant KB cells

Human KB carcinoma cells resistant to high levels of colchicine, vinblastine, vincristine, adriamycin, and actinomycin D exhibit reduced accumulation of these structurally unrelated chemotherapeutic agents (Akiyama, S.-I., Fojo, A., Hanover, J. A., Pastan, I., and Gottesman, M. M. (1985) Somatic Cell Mol. Genet. 11, 117-126; Fojo, A., Akiyama, S.-I., Gottesman, M. M., and Pastan, I. (1985) Cancer Res. 45, 3002-3007). To examine the mechanism of reduced drug accumulation in these cells, we measured [3H]vinblastine ([3H]VBL) binding to membrane vesicles made from drug-sensitive (KB-3-1), drug-resistant (KB-C4), and revertant (KB-R1) cells. Membrane vesicles from KB-C4 cells bound up to 8-fold more [3H]VBL than vesicles from the parental KB-3-1 or revertant KB-R1 cell lines. No difference in binding of [3H]dexamethasone, to which the cells are equally sensitive, was observed. The difference in [3H]VBL binding by vesicles from resistant and sensitive cells was eliminated by the addition of 10 micrograms/ml verapamil, which is known to reverse the multidrug-resistance phenotype. Drug binding by KB-C4 vesicles was osmotically insensitive, temperature-dependent, and trypsin-sensitive. Binding of [3H]VBL by KB-C4 vesicles was inhibited by vinblastine, vincristine, and daunomycin (in decreasing order). Dexamethasone at 100 microM, colchicine at 100 microM, and actinomycin D at 100 microM did not significantly inhibit [3H]VBL accumulation. No significant differences in tubulin content were detected among vesicles from sensitive and resistant cells. These data demonstrate that membrane vesicles from multiply drug-resistant cells bind increased amounts of vinblastine.     

Design,synthesis and biological evaluation of LBM-A5 derivatives as potent P-glycoprotein-mediated multidrug resistance inhibitors

A novel series of P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) inhibitors with triazol-N-phenethyl-tetrahydroisoquinoline or triazol-N-ethyl-tetrahydroisoquinoline scaffold were designed and synthesized via click chemistry. Most of the synthesized compounds showed higher reversal activity than verapamil (VRP). Among them, the most potent compound 4 showed a comparable activity with the known potent P-gp inhibitor WK-X-34 with lower cytotoxicity toward K562 cells (IC₅₀ >100 μM). Compared with VRP, compound 4 exhibited more potency in increasing drug accumulation in K562/A02 MDR cells. Moreover, compound 4 could significantly reverse MDR in a dose-dependent manner and also persist longer chemo-sensitizing effect than VRP with reversibility. Further mechanism studies revealed that compound 4 could remarkably increase the intracellular accumulation of Adriamycin (ADM) in K562/A02 cells as well as inhibit rhodamine-123 (Rh123) efflux from the cells. These results suggested that compound 4 may represent a promising candidate for developing P-gp-mediated MDR inhibitors.     

Combretastatin-like chalcones as inhibitors of microtubule polymerization. Part 1: Synthesis and biological evaluation of antivascular activity

The α-methyl chalcone SD400 is a potent inhibitor of tubulin assembly and possesses potent anticancer activity. Various chalcone analogues were synthesized and evaluated for their cell growth inhibitory properties against the K562 human chronic myelogenous leukemia cell line (SD400, IC₅₀ 0.21 nM; combretastatin A4 CA4, IC₅₀ 2.0 nM). Cell cycle analysis by flow cytometry indicated that these agents are antimitotic (SD400, 83% of the cells are in G₂/M phase; CA4 90%). They inhibit tubulin assembly at low concentration (SD400, IC₅₀ 0.46 μM; CA4, 0.10 μM) and compete with [³H]colchicine for binding to tubulin (8% [³H]colchicine remained bound to tubulin after competition with SD400 or CA4). Upon treatment with SD400, remarkable cell shape changes were elicited in HUVEC cells, consistent with vasculature damaging activity.     

Synthesis and antiproliferative evaluation of certain iminonaphtho[2,3-b]furan derivatives

Certain iminonaphtho[2,3-b]furan derivatives were synthesized from their respective carbonyl precursors in the regiospecific and the stereospecific manners. These compounds were evaluated for their antiproliferative effects against four human carcinoma cells (MCF7, NCI-H460, SF-268, and K562) and the normal fibroblast cell line (Detroit 551). Among them, (Z)-4-(hydroxyimino)naphtho[2,3-b]furan-9(4H)-one (8) and (Z)-4-methoxy-iminonaphtho[2,3-b]furan-9(4H)-one (9) exhibited GI₅₀ values of 0.82 and 0.60 μM, respectively, against the growth of K562 cells and were inactive against the normal fibroblast Detroit 551. The selectivity index (SI) on K562 cell for 8 and 9 was >121.95 and >166.67, respectively, which is comparable to daunorubicin (SI = 239) and is more favorable than camptothecin (SI = 16.5). The cell cycle analysis on K562 indicated that these compounds arrest the cell cycle at the G2/M phase. The morphological assessment and DNA fragmentation analysis indicated that 9-induced cell apoptosis in K562 cells. The apoptotic induction may through caspase-3 activity and cleavage of PARP.     

Partial purification of an IGF-II receptor/binding protein from the erythroleukemia cell line K562

We previously reported that insulin-like growth factor II (IGF-11) stimulated clonal growth of an erythroleukemia cell line, K562, in semi-solid agar, an effect not mimicked by insulin-like growth factor I (IGF-1), as IGF-I receptors are generally not expressed in this cell line. Affinity crosslinking of intact K562 cells with ¹²⁵I-IGF-II revealed that the labeled hormone predominantly bound to a protein with a molecular weight of approximately 75 K. We report here the partial purification of the 75 K IGF-II binding protein from K562 cells. Triton X-100-solubilized K562 cells were subjected to Sephacryl-400, followed by Sephacryl-200 chromatography. Fractions of interest were collected and applied to a Sepharose-IGF-II column or an immunoaffinity column. The immuno-affinity column was prepared using an antiserum against placental membrane-derived material eluted from the Sephacryl-400 column in the elution volume, corresponding to the IGF-II binding protein from K562 cells. An affi-gel 10 affinity column, prepared with a protein A purified IgG fraction of this antiserum (antibody-29), retarded proteins showing binding specificity for IGF-II, with apparent molecular weights of 76 K, 87 K, and 70 K under reducing conditions. These protein bands were similar to the proteins retarded in the IGF-II affinity column, when evaluated by affinity crosslinking and SDS-PAGE. Fractionation of the purified material from the antibody-29 affinity column on Superose 12 revealed 6 protein peaks. Affinity crosslinking of the peak fractions from FPLC resulted in single bands with a molecular weight of 75 K under reducing conditions with variable specificity for IGF-II.     

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).