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

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

Identification of the Multidrug Resistance-Related Membrane Glycoprotein as an Acceptor for Cyclosporine

Abstract

The immunosuppressive agent cyclosporine A (CSA) has been shown to reverse multidrug resistance (MDR) in malignant cells. In the present study, a ³H-cyclosporine diazirine analogue (³H-PL-CS) was used to photolabel viable MDR cells. The 170 kDa membrane P-glycoprotein, which functions as a drug efflux pump, was strongly labeled. The binding of ³H-cyclosporine diazirine analogue to P-glycoprotein was competable by excess cyclosporine A and by the nonimmunosuppressive cyclosporine H. These results suggest that cyclosporine reverses the MDR phenotype by binding directly to P-glycoprotein and that this binding is not dependent on the immunosuppressive potential of the cyclosporine derivative. The identification of P-glycoprotein as a cyclosporine binding protein has obvious implications for cancer chemotherapy.     

P-糖蛋白线粒体转位与线粒体DNA缺失肿瘤细胞多药耐药产生的关系

线粒体DNA缺失细胞(ρ~0细胞)拮抗化疗药物诱导的凋亡,但其确切机制尚不明确。本研究探讨P-gp线粒体转位与人肝癌细胞(SK-Hepl)mtDNA缺失细胞(ρ~0SK-Hep1)多药耐药产生的关系。以SK-Hep1、ρ~0SK-Hep1和转线粒体细胞SK-Hep1Cyb为研究对象,CCK-8方法检测细胞对药物敏感性;AnnexinV/PI双染法及DAPI染色法检测细胞凋亡;Westernblot检测P-gp表达;激光共聚焦显微镜结合免疫荧光检测P-gP细胞内分布。结果显示,SK-Hep1、ρ~0SK-Hep1和SK-Hep1Cyb细胞对多柔比星(DOX)的IC_(50)分别为0.62±0.02μg/ml、4.93±0.17μg/ml和0.57±0.02μg/ml。SK-Hep1、ρ~0SK-Hep1和SK-Hep1Cyb细胞凋亡率分别为1 1.25%±1.36%、4.75%±0.98%和14.50%±1.57%,ρ~0SK-Hep1对细胞凋亡有明显抗性。Western blot检测发现ρ~0细胞内P-gP、Bax、Bcl-2表达增加,Bcl-2/Bax比值增加。免疫荧光共定位显示,ρ~0细胞线粒体内P-gP...     

Biogenesis of mitochondria: XXV. Studies on the mitochondrial genomes of petite mutants of yeast using ethidium bromide as a probe

This paper describes investigations into the effects of ethidium bromide on the mitochondrial genomes of a number of different petite mutants derived from one respiratory competent strain of Saccharomyces cerevisiae. It is shown that the mutagenic effects of ethidium bromide on petite mutants occur by a similar mechanism to that previously reported for the action of this dye on grande cells. The consequences of ethidium bromide action in both cases are inhibition of the replication of mitochondrial DNA, fragmentation of pre-existing mitochondrial DNA, and the induction, often in high frequency, of cells devoid of mitochondrial genetic information (ρ ° cells).The susceptibility of the mitochondrial genomes to these effects of ethidium bromide varies in the different clones studied. The inhibition of mitochondrial DNA replication requires higher concentrations of ethidium bromide in petite cells than in the parent grande strain. Furthermore, the susceptibility of mitochondrial DNA replication to inhibition by ethidium bromide varies in different petite clones.It is found that during ethidium bromide treatment of the suppressive petite clones, the over-all suppressiveness of the cultures is reduced in parallel with the reduction in the over-all cellular levels of mitochondrial DNA. Furthermore, ethidium bromide treatment of petite clones carrying mitochondrial erythromycin resistance genes (ρ^?ER^r) leads to the elimination of these genes from the cultures. The rates of elimination of these genes are different in two ρ^?ER^r clones, and in both the gene elimination rate is slower than in the parent ρ⁺ ER^r strain. It is proposed that the rate of elimination of erythromycin resistance genes by ethidium bromide is related to the absolute number of copies of these genes in different cell types. In general, the more copies of the gene in the starting cells, the slower is the rate of elimination by ethidium bromide. These concepts lead us to suggest that petite mutants provide a system for the biological purification of particular regions of yeast mitochondrial DNA and of particular relevance is the possible purification of erythromycin resistance genes.     

Mitochondrial alterations induced by serum amine oxidase and spermine on human multidrug resistant tumor cells

Summary. Multidrug resistance (MDR) has been studied extensively because it is one of major problems in cancer chemotherapy. The MDR phenotype is often due to overexpression of P-glycoprotein (P-gp), that acting as an energy-dependent drug efflux pump exports various anticancer drugs out of cells. The major goal of our investigation is to establish whether bovine serum amine oxidase (BSAO), which generates the products H₂O₂ and aldehyde(s), from the polyamine spermine, is able to overcome MDR of human cancer cells. The cytotoxicity of the products was evaluated in both drug-sensitive (LoVo WT) and drug-resistant (LoVo DX) colon adenocarcinoma cells. A clonogenic cell survival assay demonstrated that LoVo DX cells were more sensitive than LoVo WT cells. Exogenous catalase protected cells against cytotoxicity mainly due to the formation of H₂O₂. However, spermine-derived aldehyde(s) still induced some cytotoxicity. The cytotoxic effect was totally inhibited in the presence of both enzymes, catalase and NAD-dependent aldehyde dehydrogenase (ALDH). Transmission electron microscopy investigations showed that BSAO and spermine induced evident mitochondria alterations, more pronounced in MDR than in LoVo WT cells. The mitochondrial activity was checked by flow cytometry studies, labelling cells with the probe JC1, that displayed a basal hyperpolarized status of the mitochondria in multidrug-resistant cells. After treatment with amine oxidase in the presence of polyamine-spermine, the cells showed a marked increase in mitochondrial membrane depolarization higher in LoVo DX than in LoVo WT cells. Our findings suggest that toxic oxidation products formed from spermine and BSAO could be a powerful tool in the development of new anticancer treatments, mainly against MDR tumor cells.     

Genetic manipulation of a latent defect in yeast cytochrome biosynthesis utilizing cytoduction.

The effects of a mit^? mutation, oxi2, and the ρ° mutation on expression of a defective nuclear structural gene for δ-aminolevulinic acid synthase (cyd1) were compared. The technique of cytoduction was used to introduce oxi2 mitochondria into a cyd1ρ° recipient cell, thereby permitting comparison of isonuclear strains. Like ρ°, the oxi2 mutation caused an apparent unmasking of the cytochrome deficiency associated with the cyd1 mutation, provided cells were grown on glucose. When cyd1 strains with ρ⁺, ρ° or oxi2 mitochondrial genotypes were grown on galactose medium, substantial cytochrome formation occurred in each case. It is concluded that the exacerbation of the cyd1-dependent cytochrome deficiency by ρ° or oxi2 mutations depends upon glucose repression. However, derepression resulting from growth on galactose medium does not fully overcome the cyd1 defect, since both cyd1ρ°oxi2 strains require added δ-aminolevulinic acid for maximum cytochrome biosynthesis.     

Effects of hypotonic and hypoionic media on drug pumping by P-glycoprotein expressed in epithelial and nonepithelial cell lines

The effects of anisotonic and anisoionic media on the drug-pumping function of P-glycoprotein (Pgp) were studied in epithelial and nonepithelial cells. We used HT-29 colon cells (HT-29/Pgp^?) induced to express Pgp and MDR phenotype (HT-29/Pgp⁺) and NIH3T3 (3T3/Pgp^?) cells which were stably transfected with human MDR1 DNA (3T3/Pgp⁺). Intracellular concentrations of rhodamine 123 (R-123) preloaded into cells were monitored as a function of time by fluorescence imaging microscopy, while cells were superfused with media of different tonicity and/or ionic strength. Efflux was analyzed by a single exponential decay function. In all media tested efflux was considerably higher in Pgp⁺ than Pgp^? cells. In both HT-29 and 3T3 cells loaded with dye in isotonic conditions, dye efflux was not significantly different whether it was measured in isoionic-isotonic (130 mM NaCI, 300 mOsm), hypoionic-isotonic (87 mM NaCI), or hypoionic-hypotonic (200, 150, or 100 mOsm) media throughout the entire experiment or whether the media were changed during the experiment. Similar results were obtained when cells were preincubated and preloaded with dye under hypotonic conditions. Under extreme hypotonic and hypoionic challenge (changing from 130 mM NaCI-300 mOsm to 43 mM NaCI-100 mOsm medium), 3T3 cells, but not HT-29 cells, underwent marked shape and size changes which reduced R-123 cell-associated fluorescence. The changes were most conspicuous in Pgp⁺ cells, possibly reflecting a Pgp effect on the osmotic or osmoregulatory properties of the cells. However, drug-pumping activity remained essentially unimpaired even under the most extreme hypotonic/hypoionic conditions. © 1995 Wiley-Liss, Inc.     

99mTc(N)-DBODC(5), a potential radiolabeled probe for SPECT of multidrug resistance: in vitro study

[^99mTc(N)(DBODC)(PNP5)]⁺ [DBODC is bis(N-ethoxyethyl)dithiocarbamato; PNP5 is bis(dimethoxypropylphosphinoethyl)ethoxyethylamine], abbreviated as ^99mTc(N)-DBODC(5), is a lipophilic cationic mixed compound investigated as a myocardial imaging agent. The findings that this tracer accumulates in mitochondrial structures through a mechanism mediated by the negative mitochondrial membrane potential and that the rapid efflux of ^99mTc(N)-DBODC(5) from nontarget tissues seems to be associated with the multidrug resistance (MDR) P-glycoprotein (P-gp) transport function open up the possibility to extend its clinical applications to tumor imaging and noninvasive MDR studies. The rate of uptake at 4 and 37 °C of ^99mTc(N)-DBODC(5) was evaluated in vitro in selected human cancer cell lines and in the corresponding sublines before and after P-gp and/or MDR-associated protein (MRP) modulator/inhibitor treatment using ^99mTc-sestamibi as a reference. The results indicated that (1) the uptake of both ^99mTc(N)-DBODC(5) and ^99mTc-sestamibi is correlated to metabolic activity of the cells and (2) the cellular accumulation is connected to the level of P-gp/MRP expression; in fact, an enhancement of uptake in resistant cells was observed after treatment with opportune MDR inhibitor/modulator, indicating that the selective blockade of P-gp/MRP prevented efflux of the tracers. This study provides a preliminary indication of the applicability of ^99mTc(N)-DBODC(5) in tumor imaging and in detecting P-gp/MRP-mediated drug resistance in human cancer. In addition, the possibility to control the hydrophobicity and pharmacological activity of this heterocomplex through the variation of the substituents on the ligands backbone without affecting the P₂S₂ coordinating sphere makes ^99mTc(N)-DBODC(5) a suitable scaffold for the preparation of a molecular probe for single photon emission computed tomography of MDR.     

Mitochondrial membranes and mutagenesis by ethidium bromide

The conversion of wild type (ρ⁺) to cytoplasmic petites (ρ^?) in Saccharomyces cerevisiae, à mutation in mitochondrial DNA, can be brought about with high efficiency by low concentrations of ethidium bromide (EB). The rate and extent of mutagenesis and its expression can be influenced, and even reversed, by a number of genetic lesions, agents or treatments affecting mitochondrial structure and metabolism. Among them are incubation at 45°, exposure to Antimycin A, growth on different carbon sources and the presence or absence of 2 different gene products previously implicated in the repair of UV induced lesions in mitochondrial DNA. Based on these observations a model for EB mutagenesis is advanced which postulates a complex between mitochondrial DNA and the inner membrane as the target susceptible to modification by EB. This model predicts that altered membranes should lead to changes in the susceptibility of cells to the mutagenic action of EB. This prediction has been verified by comparing cells that contain one of 2 structurally quite distinct monounsaturated C₁₈ fatty acids in their mitochondrial phospholipids: greater resistance to mutagenesis and ease of thermal protection is exhibited when cells – and mitochondria – contain oleic (Δ⁹cis, m.p. < 5°) rather than petroselinic (Δ⁶cis, m.p. 28°) acid in their phospholipids. As a corollary, studies on EB mutagenesis and mitochondrial DNA may be used as probes for the mitochondrial inner membrane to reveal some perhaps novel functions.     

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.     

Activation of K-Cl Cotransport by Mild Warming in Guinea Pig Red Cells

Unidirectional, ouabain-insensitive K⁺ influx rose steeply with warming at temperatures above 37°C in guinea pig erythrocytes incubated in isotonic medium. The only component of ouabain-insensitive K⁺ influx to show the same steep rise was K-Cl cotransport (Q₁₀ of 10 between 37 and 41°C); Na-K-Cl cotransport remained constant or declined and residual K⁺ influx in hypertonic medium with ouabain and bumetanide rose only gradually. Similar results were obtained for unidirectional K⁺ efflux. Thermal activation of K-Cl cotransport-mediated K⁺ influx was fully dependent on the presence of chloride in the medium; none occurred with nitrate replacing chloride. The increase of K⁺ influx through K-Cl cotransport from 37 to 41°C was blocked by calyculin A, a phosphatase inhibitor. The Q₁₀ of K-Cl cotransport fully activated by hydroxylamine and hypotonicity was about 2. The time course of K⁺ entry showed an immediate transition to a higher rate when cells were instantly warmed from 37 to 41°C, but there was a 7-min time lag in returning to a lower rate when cells were cooled from 41 to 37°C. These results indicate that the steepness of the response of K-Cl cotransport to mild warming is due to altered regulation of the transporter. Total unidirectional K⁺ influx was equal to total unidirectional K⁺ efflux at 37–45°C, but K⁺ influx exceeded K⁺ efflux at 41°C when K-Cl cotransport was inhibited by calyculin or prevented by hypertonic incubation. The net loss of K⁺ that results from the thermal activation of isosomotic K-Cl cotransport reported here would offset a tendency for cell swelling that could arise with warming through an imbalance of pump and leak for Na⁺ or for K⁺. Received: 1 November 1997/Revised: 5 March 1998     

The S?/?M checkpoint at 37°C and the recovery of viability of the mutant pol δ ts3 require the crb2 + /rhp9 + gene in fission yeast

We have isolated a mutant in fission yeast, in which mitosis is uncoupled from completion of DNA replication when DNA synthesis is impaired by a thermosensitive mutation in the gene encoding the catalytic subunit of DNA polymerase δ. By functional complementation, we cloned the wild-type gene and identified it as the recently cloned checkpoint gene crb2 ⁺ /rhp9 ⁺ . This gene has been implicated in the DNA damage checkpoint and acts in the Chk1 pathway. Unlike the deleted strain dcrb2, cells bearing the crb2-1 allele were not affected in the DNA repair checkpoint after UV or MMS treatment at 30° C, but were defective in this checkpoint function when treated with MMS at 37° C. We analysed the involvement of Crb2 in the S/M checkpoint by blocking DNA replication with hydroxyurea, by using S phase cdc mutants, or by overexpression of the mutant PCNA L68S. Both crb2 mutants were unable to maintain the S/M checkpoint at 37° C. Furthermore, the crb2 ⁺ gene was required, together with the cds1 ⁺ gene, for the S/M checkpoint at 30° C. Finally, both the crb2 deletion and the crb2-1 allele induced a rapid death phenotype in the polδts3 background at both 30° C and 37° C. The rapid death phenotype was independent of the checkpoint functions. Received: 25 May 1998 / Accepted: 21 September 1998     

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.     

Isolation and characterization of an adriamycin-resistant breast tumor cell line

Summary An adriamycin-resistant human breast tumor cell line MDA-A1 ^R was generated by step-wise selection in increasing concentrations of drug from the parent cell line MDA-MB-231. MDA-A1 ^R cells grow as loosely attached cell aggregates with a doubling time of 28–32 h; the MDA-MB-231 parent cell line grows as a standard monolayer culture with a 20-h doubling time. The MDA-A1 ^R cell line is highly resistant to adriamycin compared to the parent cell line, and is cross-resistant to velban and colchicine suggestive of a multidrug resistance (MDR) phenotype. MDA-A1 ^R cells exhibit reduced net adriamycin conent as compared to the parent cell line. The MDR-associated P-glycoprotein gene is amplified approximately 10-to 30-fold in MDA-A1 ^R cells. P-glycoprotein sequences are overexpressed in the resistant cells and are stable for up to 13 wk after drug removal. Moreover, MDA-A1 ^R cells show the presence of very high levels of P-glycoprotein. MDA-A1 ^R is thus an in vitro model system to study the mechanism of MDR in human breast cancer. This work was supported in part by grant C30195 from the National Institute of health, Bethesda, MD. Portion of this study appeared as a poster presentation at the Tissue Culture Association meeting, Las Vegas, 1988.     

Starvation-induced autophagy is regulated by mitochondrial reactive oxygen species leading to AMPK activation

Starvation is the most extensively studied condition that induces autophagy. Previous studies have demonstrated that starvation-induced autophagy is regulated by reactive oxygen species (ROS) such as superoxide (O₂^?) but the source for ROS under starvation conditions and the downstream signaling pathways regulating autophagy are unclear. In this study, a cervical cancer HeLa cell line was generated that was deficient in mitochondrial electron transport chain (mETC) (HeLa ρ° cells). This resulted in endogenous levels of O₂^? being significantly reduced and failed to be induced under starvation of glucose, L-glutamine, pyruvate, and serum (GP) or of amino acids and serum (AA) compared to wild type (wt) HeLa cells. In contrast, H₂O₂ production failed to increase under GP starvation in both wild type and ρ° cells whereas it increased in wt cells but not in ρ° cells under AA starvation. GP or AA starvation induced autophagy was blocked in ρ° cells as determined by the amount of autophagosomes and autolysosomes. Autophagy is regulated by 5′ adenosine monophosphate-activated protein kinase (AMPK) activation and AMPK is activated under starvation conditions. We demonstrate that ρ° cells and HeLa cells over expressing manganese-superoxide dismutase 2 (SOD2) cells fail to activate AMPK activation following starvation. This indicates that mitochondrial ROS might regulate AMPK activation. In addition, inhibiting AMPK activation either by siRNA or compound C resulted in reduced autophagy during starvation. Using a ROS scavenger NAC, AMPK activation is reduced under starvation condition and mTOR signaling is increased. Taken together, mitochondria-generated ROS induces autophagy mediated by the AMPK pathway under starvation conditions.     

Mitochondria of a human multidrug‐resistant hepatocellular carcinoma cell line constitutively express inducible nitric oxide synthase in the inner membrane

Mitochondria play a crucial role in pathways of stress conditions. They can be transported from one cell to another, bringing their features to the cell where they are transported. It has been shown in cancer cells overexpressing multidrug resistance (MDR) that mitochondria express proteins involved in drug resistance such as P‐glycoprotein (P‐gp), breast cancer resistant protein and multiple resistance protein‐1. The MDR phenotype is associated with the constitutive expression of COX‐2 and iNOS, whereas celecoxib, a specific inhibitor of COX‐2 activity, reverses drug resistance of MDR cells by releasing cytochrome c from mitochondria. It is possible that COX‐2 and iNOS are also expressed in mitochondria of cancer cells overexpressing the MDR phenotype. This study involved experiments using the human HCC PLC/PRF/5 cell line with and without MDR phenotype and melanoma A375 cells that do not express the MDR1 phenotype but they do iNOS. Western blot analysis, confocal immunofluorescence and immune electron microscopy showed that iNOS is localized in mitochondria of MDR1‐positive cells, whereas COX‐2 is not. Low and moderate concentrations of celecoxib modulate the expression of iNOS and P‐gp in mitochondria of MDR cancer cells independently from inhibition of COX‐2 activity. However, A375 cells that express iNOS also in mitochondria, were not MDR1 positive. In conclusion, iNOS can be localized in mitochondria of HCC cells overexpressing MDR1 phenotype, however this phenomenon appears independent from the MDR1 phenotype occurrence. The presence of iNOS in mitochondria of human HCC cells phenotype probably concurs to a more aggressive behaviour of cancer cells.     

Targeting Mitochondrial Cell Death Pathway to Overcome Drug Resistance with a Newly Developed Iron Chelate

Background

Multi drug resistance (MDR) or cross-resistance to multiple classes of chemotherapeutic agents is a major obstacle to successful application of chemotherapy and a basic problem in cancer biology. The multidrug resistance gene, MDR1, and its gene product P-glycoprotein (P-gp) are an important determinant of MDR. Therefore, there is an urgent need for development of novel compounds that are not substrates of P-glycoprotein and are effective against drug-resistant cancer.

Methodology/Principal Findings

In this present study, we have synthesized a novel, redox active Fe (II) complex (chelate), iron N- (2-hydroxy acetophenone) glycinate (FeNG). The structure of the complex has been determined by spectroscopic means. To evaluate the cytotoxic effect of FeNG we used doxorubicin resistant and/or sensitive T lymphoblastic leukemia cells and show that FeNG kills both the cell types irrespective of their MDR phenotype. Moreover, FeNG induces apoptosis in doxorubicin resistance T lymphoblastic leukemia cell through mitochondrial pathway via generation reactive oxygen species (ROS). This is substantiated by the fact that the antioxidant N-acetyle-cysteine (NAC) could completely block ROS generation and, subsequently, abrogated FeNG induced apoptosis. Therefore, FeNG induces the doxorubicin resistant T lymphoblastic leukemia cells to undergo apoptosis and thus overcome MDR.

Conclusion/Significance

Our study provides evidence that FeNG, a redox active metal chelate may be a promising new therapeutic agent against drug resistance cancers.     

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.     

Detection of Adriamycin–DNA adducts by accelerator mass spectrometry at clinically relevant Adriamycin concentrations

Limited sensitivity of existing assays has prevented investigation of whether Adriamycin–DNA adducts are involved in the anti-tumour potential of Adriamycin. Previous detection has achieved a sensitivity of a few Adriamycin–DNA adducts/10⁴ bp DNA, but has required the use of supra-clinical drug concentrations. This work sought to measure Adriamycin–DNA adducts at sub-micromolar doses using accelerator mass spectrometry (AMS), a technique with origins in geochemistry for radiocarbon dating. We have used conditions previously validated (by less sensitive decay counting) to extract [¹⁴C]Adriamycin–DNA adducts from cells and adapted the methodology to AMS detection. Here we show the first direct evidence of Adriamycin–DNA adducts at clinically-relevant Adriamycin concentrations. [¹⁴C]Adriamycin treatment (25 nM) resulted in 4.4 ± 1.0 adducts/10⁷ bp (~1300 adducts/cell) in MCF-7 breast cancer cells, representing the best sensitivity and precision reported to date for the covalent binding of Adriamycin to DNA. The exceedingly sensitive nature of AMS has enabled over three orders of magnitude increased sensitivity of Adriamycin–DNA adduct detection and revealed adduct formation within an hour of drug treatment. This method has been shown to be highly reproducible for the measurement of Adriamycin–DNA adducts in tumour cells in culture and can now be applied to the detection of these adducts in human tissues.