Identification of several cyclosporine binding proteins in lymphoid and non-lymphoid cells in vivo.

The immunosuppressant cyclosporine A (CSA) has been shown to bind to the ubiquitous cellular protein, cyclophilin, and to inhibit its rotamase activity. In the present study, 3H-cyclosporine diazirine analogue was used to photolabel viable human cells of lymphoid and fibroblast origin in order to identify the intracellular targets for the drug. While cyclophilin was strongly labeled in situ, additional minor cyclosporine-protein complexes of 25, 40, 46 and 60 kDa were identified in the T cell leukemia cell line Jurkat. These proteins bound specifically, since only active CSA but not inactive CSH or FK506 competed for binding. Photolabeling of MRC5 cells, a CSA resistant human fibroblast cell line, revealed a 25 kDa complex as the major product, while the 46 and 60 kDa bands were not detectable and cyclophilin labeling was only faint, even though both MRC5 and Jurkat cells contain similar cyclophilin concentrations. Thus, our data suggest that the intracellular targets of CSA and/or the accessibility to cyclophilin varies considerably in drug sensitive and resistant cell types, which may contribute to explaining the lymphocyte selectivity of the drug.     

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).     

Evidence for Multidrug Resistance-1 P-Glycoprotein-dependent Regulation of Cellular ATP Permeability

The mechanisms responsible for regulating epithelial ATP permeability and purinergic signaling are not well defined. Based on the observations that members of the ATP-binding cassette (ABC)¹ family of proteins may contribute to ATP release, the purpose of these studies was to assess whether multidrug resistance-1 (MDR1) proteins are involved in ATP release from HTC hepatoma cells. Using a bioluminescence assay to detect extracellular ATP, increases in cell volume increased ATP release ∼3-fold. The MDR1 inhibitors cyclosporine A (10 μm) and verapramil (10 μm) inhibited ATP release by 69% and 62%, respectively (p < 0.001). Similarly, in whole-cell patch-clamp recordings, intracellular dialysis with C219 antibodies to inhibit MDR1 decreased ATP-dependent volume-sensitive Cl⁻ current density from −33.1 ± 12.5 pA/pF to −2.0 ± 0.3 pA/pF (−80 mV, p≤ 0.02). In contrast, overexpression of MDR1 in NIH 3T3 cells increased ATP release rates. Inhibition of ATP release by Gd³⁺ had no effect on transport of the MDR1 substrate rhodamine-123; and alteration of MDR1-substrate selectivity by mutation of G185 to V185 had no effect on ATP release. Since the effects of P-glycoproteins on ATP release can be dissociated from P-glycoprotein substrate transport, MDR1 is not likely to function as an ATP channel, but instead serves as a potent regulator of other cellular ATP transport pathways. Received: 20 November 2000/Revised: 25 May 2001     

Lymphokine-activated killer cell susceptibility and adhesion molecule expression of multidrug resistant breast carcinoma

Reports showing susceptibility of multidrug resistant (MDR) cancer cells to immune effectors, together with P-glycoprotein (P-gp) expression in immune effector subsets, including immature natural killer (NK) cells, and some activated T cells, suggest P-gp or some changes associated with it, have implications in immune-mediated mechanisms. A series of experiments were done to determine the nature of alterations associated with susceptibility to immune effector cells of MDR tumor cells. A cell line isolated from the malignant pleural effusion of a breast cancer patient was transfected with human and murine MDR1 genes, and four variants with different levels of MDR were obtained. Lymphokine-activated killer (LAK) activity was measured by a ⁵¹Chromium release, and conjugate formation assays. MDR1 transfectant P-gp⁺ breast carcinoma lines had increased LAK susceptibility compared to their parent line. Some part of the increased LAK susceptibility of drug-resistant cell lines was at the binding/recognition level as shown by conjugate formation assays. This suggests that differences may exist between paired cell lines with respect to the expression of cell adhesion molecules (CAMs). Monoclonal antibodies (mAbs) to CAMs and flow cytometry were used to quantitate these antigens. The CAMs studied were those previously found to be upregulated by stimulating NK cells with (interleukin-2) IL-2; ICAM-1 (CD54), LFA-3 (CD58), N-CAM (CD56), and the β chain of LFA-1 (CD18). Although no differences in these CAMs were found between the breast carcinoma line and its MDR1-transfected variants, the target susceptibility results given above suggest that IL-2 treatment could be effective in combination with current protocols using chemotherapeutics, monoclonal antibodies (mAbs) and stem cell transplantation.     

Clinical Evaluation of Liposome Encapsulated Doxorubicin and the Modulation of Multidrug Resistance in Cancer Cells

Abstract

Doxorubicin is the cornerstone of some widely used combination chemotherapy regimens because of its high anticancer activity in a number of human neoplasms. However, its clinical use is highly compromised because of treatment-limiting acute and chronic toxicities of which cardiotoxicity has the most debilitating effect. Our laboratories have demonstrated that liposome encapsulated doxorubicin (LED) provides important advantages in regards to the attenuation of cardiotoxicity in rodents by altering pharmacokinetics and pharmacodynamics of the drug, provides effective protection from immunotoxicity and maintains full therapeutic activity of the drug in liposomes. A Phase I clinical trial of LED in cancer patients has establish the maximum tolerated dose of 90 mg/m² with granulocytopenia being the major treatment-limiting toxicity. We have performed a Phase II trial of LED in 20 recurrent breast cancer patients at a dose of 75 mg/m² as an intravenous infusion every three weeks. Objective responses were observed in 9/20 patients of which 5 demonstrated a complete response. Hematologic toxicity with LED consisted of only grade 1-2 granulocytopenia in some patients, whereas gastrointestinal toxicity, mucositis and venous sclerosis were markedly reduced. Alopecia was complete in all patients. Twelve patients received cumulative LED doses of more than 400 mg/m² and 8 of them received doses of over 500 mg/m². Five of these patients were followed by endomyocardial biopsies and 4 of them were found to be Billingham Grade 0 whereas one of them had Billingham Grade 1 toxicity (cumulative dose of 750 mg/m²). This Phase II trial demonstrates higher therapeutic efficacy of LED than free doxorubicin in recurrent breast cancer patients with strong indication of cardiotoxicity protection at doses of 500-800 mg/m².

The emergence of tumor cells resistant to major classes of cytotoxic agents is a predominant obstacle in cancer treatment. This resistance is frequently related to the expression of a plasma membrane P-glycoprotein (pgp) of 170 Kd that is encoded by a family of MDR genes. Support for the involvement of pgp in MDR has been shown by transaction of sensitive cells with an expression vector containing full length cDNA of the MDR1 gene, which results in the appearance of pgp and the sensitive cells convert to the drug-resistant phenotype. Our studies demonstrate that LED modulates very effectively the MDR phenotype in LZ cells, a Chinese hamster cell line made resistant to doxorubicin and the cellular drug uptake was 2 to 3 fold higher with LED exposure than with free drug. This modulation of drug resistance and enhanced cellular drug uptake is effected by the direct binding of liposomes to pgp on the surfaces of MDR phenotype cells. LED completely inhibited the photoaffinity labeling of pgp by azidopine in membrane vesicles of HL-60/VCR cells and in KB-GSV2 cells transfected with human MDR gene. These studies demonstrate that LED has unique effectiveness in overcoming MDR phenotype in cancer cells and appears to be a potentially attractive modality of treatment of human cancers.     

Detergents as intrinsic P-glycoprotein substrates and inhibitors

We assessed the interaction of three electrically neutral detergents (Triton X-100, C₁₂EO₈, and Tween 80) with P-glycoprotein (ABCB1, MDR1) and identified the molecular elements responsible for this interaction. To this purpose we titrated P-glycoprotein in inside-out plasma membrane vesicles of MDR1-transfected mouse embryo fibroblasts (NIH-MDR1-G185) with the detergents below their critical micelle concentration, CMC. The P-glycoprotein ATPase measured as a function of the detergent concentration yielded bell-shaped activity curves which were evaluated with a two-site binding model. The lipid-water partition coefficient and the transporter-water binding constant of the detergents were measured independently. Knowledge of these two parameters allowed assessment of the free energy of detergent binding to P-glycoprotein in the lipid membrane, ΔG_tl⁰, that reflects the direct detergent-transporter affinity. It increased as the number of ethoxyl groups increased, suggesting that these hydrogen bond acceptor groups are the key elements for the detergent-transporter interaction in the lipid membrane. The free energy of binding to P-glycoprotein per ethoxyl group (EO) was determined as approximately ΔG_EO⁰ = − 1.6 kJ/mol. The present findings moreover document that, depending on the concentration applied, detergents are intrinsic substrates for, or inhibitors of P-glycoprotein.     

Multidrug resistant tumour cells shed more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart cells

BackgroundMultidrug resistance (MDR) is a serious impediment to cancer treatment, with overexpression of drug efflux pumps such as P-glycoprotein (P-gp) playing a significant role. In spite of being a major clinical challenge, to date there is no simple, minimally invasive and clinically validated method for diagnosis of the MDR phenotype using non-tumour biological samples. Recently, P-gp has been found in extracellular vesicles (EVs) shed by MDR cancer cells. This study aimed to compare the EVs shed by MDR cells and their drug-sensitive cellular counterparts, in order to identify biomarkers of MDR.MethodsTwo pairs of MDR and drug-sensitive counterpart tumour cell lines were studied as models. EVs were characterized in terms of size and molecular markers and their protein content was investigated by proteomic analysis and Western blot.ResultsWe found that MDR cells produced more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart. EVs from MDR cells contained P-gp and presented a different content of proteins known to be involved in the biogenesis of EVs, particularly in the biogenesis of exosomes.ConclusionsThe determination of the size and of this particular protein content of EVs shed by tumour cells may allow the development of a minimally-invasive simple method of detecting and predicting MDR.General significanceThis work describes for the first time that cancer multidrug resistant cells shed more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart cells, carrying a specific content of proteins involved in EV biogenesis that could be further studied as biomarkers of MDR.     

Hyaluronan substratum induces multidrug resistance in human mesenchymal stem cells via CD44 signaling

Little information is available concerning multidrug resistance (MDR) in mesenchymal stem cells, although several studies have reported that MDR is associated with hyaluronan in neoplastic cells. We have evaluated whether a hyaluronan-coated surface modulates MDR in placenta-derived human mesenchymal stem cells (PDMSCs). We have found that PDMSCs cultured on a tissue-culture polystyrene surface coated with 30 μg/cm² hyaluronan are more resistant than control PDMSCs to doxorubicin. Inhibiting PI3K/Akt signaling has shown that the PI3K/Akt pathway modulates both P-glycoprotein activity and doxorubicin resistance. In addition, 10 μM verapamil dramatically suppresses the doxorubicin resistance induced by the hyaluronan-coated surface, indicating that P-glycoprotein activity is necessary for MDR. We have further found that PDMSCs treated with CD44 small interfering RNA (siRNA) and grown on a polystyrene surface coated with 30 μg/cm² hyaluronan have fewer P-glycoprotein⁺ cells and lower CD44 expression levels (less than 60% in both cases) than PDMSCs not treated with CD44 siRNA and grown on the hyaluronan-coated surface. Moreover, treatment with CD44 siRNA suppresses the hyaluronan-substratum-induced doxorubicin resistance. We conclude that a hyaluronan substratum induces MDR in PDMSCs through CD44 signaling. This work was supported by research grant NSC95–2745-B-006–003-MY2 from the National Science Council, Taiwan, and by Landmark Project Grant A25, funded by the Taiwan Ministry of Education, from National Cheng Kung University.     

Upregulation of P-glycoprotein in rat hepatoma ρ° cells: Implications for drug–DNA interactions

Rat hepatoma cells lacking mitochondrial DNA (ρ° cells) were used as a model system to examine the possible roles of mitochondrial DNA as a target for the DNA-acting anticancer drug Adriamycin (doxorubicin). The ρ° cells were 45-fold less sensitive to Adriamycin than the parental ρ⁺ cells containing mitochondrial DNA. Other non-DNA-acting drugs also exhibited similar behaviour, and this was shown to be due to a multidrug resistance (MDR) phenotype in the ρ° cells. This was indicated by confocal microscopy where ρ⁺ cells exhibited thirteenfold higher cellular levels of Adriamycin than ρ° cells. Upregulation (tenfold) of P-glycoprotein in ρ° cells was also confirmed by Northern dot blot analysis. Since the MDR phenotype is present in ρ° cells and upregulation of P-glycoprotein is maintained in these cells, ρ° cells are not a good model system for drug-DNA studies (where the drug is susceptible to extrusion by P-glycoprotein), and any such results obtained with this system must be treated with considerable caution. J. Cell. Biochem. 69:463–469, 1998. © 1998 Wiley-Liss, Inc.     

Modulation of function of three ABC drug transporters, P-glycoprotein (ABCB1), mitoxantrone resistance protein (ABCG2) and multidrug resistance protein 1 (ABCC1) by tetrahydrocurcumin, a major metabolite of curcumin

Many studies have been performed with the aim of developing effective resistance modulators to overcome the multidrug resistance (MDR) of human cancers. Potent MDR modulators are being investigated in clinical trials. Many current studies are focused on dietary herbs due to the fact that these have been used for centuries without producing any harmful side effects. In this study, the effect of tetrahydrocurcumin (THC) on three ABC drug transporter proteins, P-glycoprotein (P-gp or ABCB1), mitoxantrone resistance protein (MXR or ABCG2) and multidrug resistance protein 1 (MRP1 or ABCC1) was investigated, to assess whether an ultimate metabolite form of curcuminoids (THC) is able to modulate MDR in cancer cells. Two different types of cell lines were used for P-gp study, human cervical carcinoma KB-3-1 (wild type) and KB-V-1 and human breast cancer MCF-7 (wild type) and MCF-7 MDR, whereas, pcDNA3.1 and pcDNA3.1-MRP1 transfected HEK 293 and MXR overexpressing MCF7AdrVp3000 or MCF7FL1000 and its parental MCF-7 were used for MRP1 and MXR study, respectively. We report here for the first time that THC is able to inhibit the function of P-gp, MXR and MRP1. The results of flow cytometry assay indicated that THC is able to inhibit the function of P-gp and thereby significantly increase the accumulation of rhodamine and calcein AM in KB-V-1 cells. The result was confirmed by the effect of THC on [³H]-vinblastine accumulation and efflux in MCF-7 and MCF-7MDR. THC significantly increased the accumulation and inhibited the efflux of [³H]-vinblastine in MCF-7 MDR in a concentration-dependent manner. This effect was not found in wild type MCF-7 cell line. The interaction of THC with the P-gp molecule was clearly indicated by ATPase assay and photoaffinity labeling of P-gp with transport substrate. THC stimulated P-gp ATPase activity and inhibited the incorporation of [¹²⁵I]-iodoarylazidoprazosin (IAAP) into P-gp in a concentration-dependent manner. The binding of [¹²⁵I]-IAAP to MXR was also inhibited by THC suggesting that THC interacted with drug binding site of the transporter. THC dose dependently inhibited the efflux of mitoxantrone and pheophorbide A from MXR expressing cells (MCF7AdrVp3000 and MCF7FL1000). Similarly with MRP1, the efflux of a fluorescent substrate calcein AM was inhibited effectively by THC thereby the accumulation of calcein was increased in MRP1-HEK 293 and not its parental pcDNA3.1-HEK 293 cells. The MDR reversing properties of THC on P-gp, MRP1, and MXR were determined by MTT assay. THC significantly increased the sensitivity of vinblastine, mitoxantrone and etoposide in drug resistance KB-V-1, MCF7AdrVp3000 and MRP1-HEK 293 cells, respectively. This effect was not found in respective drug sensitive parental cell lines. Taken together, this study clearly showed that THC inhibits the efflux function of P-gp, MXR and MRP1 and it is able to extend the MDR reversing activity of curcuminoids in vivo.     

A 2′←5′-Adenylate Trimer With a Positively Charged 2′(3′)-Terminal 8-(4-Aminobunl)Aminoadenosine Residue: Synthesis,Conformation and RNase L Binding

Abstract

An analogue of the 2-5A core trimer containing an 8-(4-aminobutyl)-aminoadenosine (1; A) residue at the 2′(3′)-terminus [2; (2′,5′)A₂A^?] was synthesized. The conformation of (2′,5′)A₂A^? was studied by ₁H, ¹³C-NMR, and CD spectroscopy. The (2′,5′)A₂A^? exhibits very low binding ability to the RNase L of mouse L cells, but slightly enhanced resistance to digestion by SVPD compared to the parent trimer.     

Interaction of forskolin with the P-glycoprotein multidrug transporter.

Forskolin and 1,9-dideoxyforskolin, an analogue that does not activate adenylyl cyclase, were tested for their ability to enhance the cytotoxic effects of adriamycin in human ovarian carcinoma cells, SKOV3, which are sensitive to adriamycin and express low levels of P-glycoprotein, and a variant cell line, SKVLB, which overexpresses the P-glycoprotein and has the multidrug resistance (MDR) phenotype. Forskolin and 1,9-dideoxyforskolin both increased the cytotoxic effects of adriamycin in SKVLB cells, yet had no effect on SKOV3 cells. Two photoactive derivatives of forskolin have been synthesized, 7-O-[[2-[3-(4-azido-3- [125I]iodophenyl)propionamido]ethyl] carbamyl]-7-deacetylforskolin, 125I-7-AIPP-Fsk, and 6-O-[[2-[3-(4-azido-3- [125I]iodophenyl)propionamido]ethyl]carbamyl]forskolin, 125I-6-AIPP-Fsk, which exhibit specificity for labeling the glucose transporter and adenylyl cyclase, respectively (Morris et al., 1991). Both photolabels identified a 140-kDa protein in membranes from SKVLB cells whose labeling was inhibited by forskolin and 1,9-dideoxyforskolin. There was no specific labeling of proteins in membranes from the SKOV3 cells. The overexpressed 140-kDa protein in SKVLB membranes was identified as the P-glycoprotein by immunoblot analysis and immunoprecipitation using anti-P-glycoprotein antiserum. Total inhibition of photolabeling of the P-glycoprotein was observed with verapamil, nifedipine, diltiazem, and vinbalastine, and partial inhibition was observed with colchicine and cytochalasin B. Forskolin was less effective at inhibiting the photolabeling of the P-glycoprotein than 1,9-dideoxyforskolin or a lipophilic derivative of forskolin. The data are consistent with forskolin binding to the P-glycoprotein analogous to that of other chemosensitizing drugs that have been shown to partially reverse MDR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rhamnetin induces sensitization of hepatocellular carcinoma cells to a small molecular kinase inhibitor or chemotherapeutic agents

BackgroundThe rapid development of multi-drug resistance (MDR) process has hindered the effectiveness of advanced hepatocellular carcinoma (HCC) treatments. Notch-1 pathway, which mediates the stress-response, promotes cell survival, EMT (epithelial–mesenchymal transition) process and induces anti-apoptosis in cancer cells, would be a potential target for overcoming MDR process. This study investigated the potential application of rhamnetin, a specific inhibitor of Notch-1 pathway, in anti-tumor drug sensitization of HCC treatment.MethodsThe expression of miR-34a, proteins belonging to Notch-1 signaling pathway or MDR-related proteins was detected by quantitative polymerase chain reaction (qPCR) and western blot assay. To identify whether rhamnetin induces the chemotherapeutic sensitization in HCC cells, the MTT-assays, flow cytometry, soft agar, trans-well and nude mice assays were performed.ResultsThe endogenous expression of miR-34a was significantly increased and the expression of Notch-1 and Survivin was downregulated after rhamnetin treatment. Treatment of rhamnetin also reduced the expression of MDR related proteins P-GP (P-glycoprotein) and BCRP (breast cancer resistance protein). Rhamnetin increased the susceptibility of HCC cells and especially HepG2/ADR, a MDR HCC cell line, to a small molecular kinase inhibitor sorafenib or chemotherapeutic drugs etoposide and paclitaxel. The IC₅₀ value of those drugs correspondingly decreased.ConclusionsTogether, our findings suggest that rhamnetin treatment may attenuate the MDR process in HCC cells. These findings may contribute to more effective strategies for HCC therapy.General significanceRhamnetin acts as a promising sensitizer to chemotherapy and may be a novel approach to overcome the MDR process of HCC.     

Chloride channel-3 mediates multidrug resistance of cancer by upregulating P-glycoprotein expression

Chloride channel-3 (ClC-3), a member of the ClC family of voltage-gated Cl⁻ channels, is involved in the resistance of tumor cells to chemotherapeutic drugs. Here, we report a new mechanism for ClC-3 in mediating multidrug resistance (MDR). ClC-3 was highly expressed in the P-glycoprotein (P-gp)-dependent human lung adenocarcinoma cell line (A549)/paclitaxel (PTX) and the human breast carcinoma cell line (MCF-7)/doxorubicin (DOX) resistant cells. Changes in the ClC-3 expression resulted in the development of drug resistance in formerly drug-sensitive A549 or MCF-7 cells, and drug sensitivity in formerly drug-resistant A549/Taxol and MCF-7/DOX cells. Double transgenic MMTV-PyMT/CLCN3 mice with spontaneous mammary cancer and ClC-3 overexpression demonstrated drug resistance to PTX and DOX. ClC-3 expression upregulated the expression of MDR1 messenger RNA and P-gp by activating the nuclear factor-κB (NF-κB)-signaling pathway. These data suggest that ClC-3 expression in cancer cells induces MDR by upregulating NF-κB-signaling-dependent P-gp expression involving another new mechanism for ClC-3 in the development of drug resistance of cancers.     

A potato cDNA encoding a homologue of mammalian multidrug resistant P-glycoprotein

A homologue of the multidrug resistance (MDR) gene was obtained while screening a potato stolon tip cDNA expression library with³⁵S-labeled calmodulin. The mammalian MDR gene codes for a membrane-bound P-glycoprotein (170–180 kDa) which imparts multidrug resistance to cancerous cells. The potato cDNA (PMDR1) codes for a polypeptide of 1313 amino acid residues (ca. 144 kDa) and its structural features are very similar to the MDR P-glycoprotein. The N-terminal half of the PMDR1-encoded protein shares striking homology with its C-terminal half, and each half contains a conserved ATP-binding site and six putative transmembrane domains. Southern blot analysis indicated that potato has one or two MDR-like genes. PMDR1 mRNA is constitutively expressed in all organs studied with higher expression in the stem and stolon tip. The PMDR1 expression was highest during tuber initiation and decreased during tuber development.     

Kuguacin J isolated from Momordica charantia leaves inhibits P-glycoprotein (ABCB1)-mediated multidrug resistance

Multidrug resistance (MDR) is a major factor in the failure of chemotherapy in cancer patients. Resistance to chemotherapy has been correlated to the overexpression of ABC drug transporters including P-glycoprotein (P-gp) that actively efflux chemotherapeutic drugs from cancer cells. Our previous study showed that bitter melon (Momordica charantia) leaf extract (BMLE) was able to reverse the MDR phenotype by increasing the intracellular accumulation of chemotherapeutic drugs. In the present study, bioguided fractionation was used to identify the active component(s) of BMLE that is able to modulate the function of P-gp and the MDR phenotype in a human cervical carcinoma cell line (KB-V1). We found that kuguacin J, one of the active components in BMLE, increased sensitivity to vinblastine and paclitaxel in KB-V1 cells. A flow cytometry assay indicated that kuguacin J inhibits the transport function of P-gp and thereby significantly increases the accumulation of rhodamine 123 and calcein AM in the cells. These results were confirmed by [³H]-vinblastine transport assay. Kuguacin J significantly increases intracellular [³H]-vinblastine accumulation and decreased the [³H]-vinblastine efflux in the cells. Kuguacin J also inhibited the incorporation of [¹²⁵I]-iodoarylazidoprazosin into P-gp in a concentration-dependent manner, indicating that kuguacin J directly interacts with the drug-substrate-binding site on P-gp. These results indicate that kuguacin J modulates the function of P-gp by directly interacting at the drug-substrate-binding site, and it appears to be an effective inhibitor of P-gp activity in vitro and thus could be developed as an effective chemosensitizer to treat multidrug-resistant cancers.     

3-(Trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine: Synthesis and chemical characterization of a heterobifunctional carbene-generating crosslinking reagent

A new hydrophobic heterobifunctional photocrosslinking reagent 3-(trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine (TRIMID), a carbene precursor, and its radioiodinated analogue [¹²⁵I]TRIMID, have been synthesized and chemically characterized. The reagents were applied for membrane protein modification in human erythrocyte membranes and purple membranes fromHalobacterium halobium. Covalent labeling of the anion transport protein (band 3) via the isothiocyanate function was confirmed. Radiolabeled TRIMID was detected in at least two thermolysin-generated transmembrane fragments of the anion transport protein, and half-maximal inhibition of the erythrocyte anion transport activity was attained with 2.2 mM reagent. In bacteriorhodopsin (BR), a common binding site for the monofunctional phenylisothiocyanate and the bifunctional crosslinking reagent was identified: preincubation of purple membranes with TRIMID suppressed phenylisothio-[¹⁴C]-cyanate binding to BR. [¹²⁵I]TRIMID was recovered in V-1, the N-terminal segment of BR, which includes the phenylisothiocyanate binding site Lys-41. Light-induced intramolecular crosslinking of band 3-derived thermolytic fragments was not observed, although the carbene was generatedin situ and photocrosslinking of the protease V8 fragments of BR was not detected. Chemical and physicochemical characteristics of the new reagent are discussed with regard to limitations imposed for photoinduced site-directed crosslink formation.     

Azidopine noncompetitively interacts with vinblastine and cyclosporin A binding to P-glycoprotein in multidrug resistant cells.

It is believed that P-glycoprotein (P-gp) is an energy-dependent drug efflux pump responsible for decreased drug accumulation in multidrug resistant (MDR) cells. In this study, we investigated whether azidopine, a photoactive dihydropyridine calcium channel blocker, is transported by P-gp in MDR Chinese hamster lung cells, DC-3F/VCRd-5L, and whether its binding site(s) on P-gp are distinct from those of Vinca alkaloids and cyclosporins. The efflux of azidopine from MDR cells was energy-dependent and inhibited by the cytotoxic agent vinblastine (VBL). Cyclosporin A (CsA), a modulator of MDR, also increased azidopine accumulation in MDR cells by decreasing the energy-dependent efflux of azidopine. P-gp in these cells was the only protein specifically bound to [3H]azidopine in photoaffinity experiments. The specific photoaffinity labeling of P-gp by [3H]azidopine was inhibited by CsA, SDZ 33-243, nonradioactive azidopine, and VBL with median concentrations (IC50) of 0.5, 0.62, 1.7, and 25 microM, respectively. The equilibrium binding of azidopine to plasma membranes of MDR variant DC-3F/VCRd-5L cells showed a single class of specific binding sites having a dissociation constant of 1.20 microM and a maximum binding capacity of 4.47 nmol/mg of protein. Kinetic analysis indicated that the inhibitory effect of VBL and CsA on azidopine binding to plasma membranes of MDR cells was noncompetitive, indicating that azidopine binds to P-gp at a binding site(s) different from the binding site(s) of these drugs.     

A novel flavonoid from Fissistigma cupreonitens, 5‑hydroxy‑7,8‑dimethoxyflavanone,competitively inhibited the efflux function of human P-glycoprotein and reversed cancer multi-drug resistance

BackgroundP-glycoprotein (P-gp) over-expression plays a vital role in not only systemic drug bioavailability but also cancer multi-drug resistance (MDR). Develop functional inhibitors of P-gp can conquer both problems.Purpose and study designThe aim of the present study was to research the P-gp modulating effects and MDR reversing ability of a novel flavonoid from Fissistigma cupreonitens, the underlying inhibitory mechanisms were further elucidated as well.MethodsCalcein-AM, rhodamine 123, and doxorubicin were fluorescent substrates for the evaluation of P-gp inhibitory function and detailed drug binding modes. Docking simulation was performed to reveal the in silico molecular bonding. ATPase assay and MDR1 shift assay were adopted to reveal the ATP consumption and conformational change of P-gp. The MDR reversing effects were demonstrated through cytotoxicity, cell cycle, and apoptosis analyses.Results5‑hydroxy‑7,8‑dimethoxyflavanone inhibited the efflux of rhodamine 123 and doxorubicin in a competitive manner, and increased the intracellular fluorescence of calcein at a concentration as low as 2.5 μg/ml. 5‑hydroxy‑7,8‑dimethoxyflavanone slightly changed P-gp's conformation and only stimulated ATPase at very high concentration (100 μg/ml). The docking results showed that 5‑hydroxy‑7,8‑dimethoxyflavanone and verapamil exhibited similar binding affinity to P-gp. The MDR reversing effects were prominent in the vincristine group, the reversal folds were 23.01 and 13.03 when combined with 10 μg/ml 5‑hydroxy‑7,8‑dimethoxyflavanone in the P-gp over-expressing cell line (ABCB1/Flp-In™-293) and MDR cancer cell line (KB/VIN), respectively.ConclusionThe present study demonstrated that 5‑hydroxy‑7,8‑dimethoxyflavanone was a novel effective flavonoid in the P-gp efflux inhibition and in vitro cancer MDR reversion.