Expression Of Drug Efflux Pumps Research Articles

W1960 Reactive Oxygen Species Induced By the Compound Bisphenol-a-Diglycidyl-Ether Cause Senescence and Apoptosis in Colorectal Cancer Cell Lines Regardless of MDR1 or p53 Status

Background: Reactive oxygen species (ROS) can be toxic. Tumors have elevated ROS, and the ability of tumor cells to accommodate additional ROS may be limited compared to normal cells, suggesting the possibility of “redox therapy”. ROS are critical to the mechanism of many cancer drugs, and experimental agents that affect ROS metabolism are being tested. However, this strategy has been difficult in colorectal cancer (CRC) due to high level expression of drug efflux pumps and loss of the stress-activated p53 pathway that normally controls expression of ROS scavenging enzymes. We show that ROS induced by the compound bisphenol-A-diglycidyl-ether (BADGE) cause senescence and apoptosis in CRC cell lines, including those with high levels of MDR1 efflux pump activity and loss of p53 function. Methods: ROS, Jnk kinase signaling, apoptosis, and senescence (irreversible cell cycle arrest) were measured in HT-29, SW620, HCT-116, and HCT-15 cells. ROS were measured using the redox-sensitive dye DCFDA. Jnk kinase activation was measured by Western blot, and apoptosis by annexinV staining. Markers of senescence included large cells with vacuolated cytoplasm, ability to maintain constant viable cell numbers over three weeks, and appearance of senescence-associated beta-galactosidase. Results: BADGE treatment caused immediate (<1h) dose-dependent increases in ROS that lasted for at least 12h. At later times, a further increase in ROS corresponding to apoptosis was observed. The consequences of BADGE treatment were proportional to the amount of ROS generated. 50μM BADGE caused senescence but not apoptosis, while higher doses (75-100μM) triggered ROS and Jnk kinasedependent apoptosis. Increasing glutathione-dependent ROS scavenging with N-acetylcysteine allowed greater doses of BADGE to be tolerated, while reducing glutathione with buthionine sulfoximine sensitized the cells to BADGE. BADGE triggered sustained Jnk kinase activation, and Jnk inhibitors decreased the pro-apoptotic effects of BADGE. The concentration of BADGE resulting in half-maximal apoptosis (IC50) was 75μM in all cell lines, regardless of MDR1 or p53 status. The MDR1 inhibitor verapamil, while reducing efflux of rhodamine 123, did not increase the IC50 of BADGE in HCT-15 cells. BADGE concentrations that caused ROS and apoptosis did not suppress PPARγ-dependent transcription, and PPARγ knockdown did not replicate or interfere with the effects of BADGE, suggesting that the effects of BADGE are PPARγ-independent. Conclusions: Depending on dose, ROS lead to senescence or apoptosis even in MDR1 high p53 mutant CRC cells. BADGE is a possible candidate for CRC redox therapy.

A nuclear receptor-like pathway regulating multidrug resistance in fungi

Multidrug resistance (MDR) is a serious complication during treatment of opportunistic fungal infections that frequently afflict immunocompromised individuals, such as transplant recipients and cancer patients undergoing cytotoxic chemotherapy. Improved knowledge of the molecular pathways controlling MDR in pathogenic fungi should facilitate the development of novel therapies to combat these intransigent infections. MDR is often caused by upregulation of drug efflux pumps by members of the fungal zinc-cluster transcription-factor family (for example Pdr1p orthologues). However, the molecular mechanisms are poorly understood. Here we show that Pdr1p family members in Saccharomyces cerevisiae and the human pathogen Candida glabrata directly bind to structurally diverse drugs and xenobiotics, resulting in stimulated expression of drug efflux pumps and induction of MDR. Notably, this is mechanistically similar to regulation of MDR in vertebrates by the PXR nuclear receptor, revealing an unexpected functional analogy of fungal and metazoan regulators of MDR. We have also uncovered a critical and specific role of the Gal11p/MED15 subunit of the Mediator co-activator and its activator-targeted KIX domain in antifungal/xenobiotic-dependent regulation of MDR. This detailed mechanistic understanding of a fungal nuclear receptor-like gene regulatory pathway provides novel therapeutic targets for the treatment of multidrug-resistant fungal infections.

Clustering of endocytic organelles in parental and drug-resistant myeloid leukaemia cell lines lacking centrosomally organised microtubule arrays

Spatial organisation and trafficking of endocytic organelles in mammalian cells is tightly regulated and dependent on cytoskeletal networks. The dynamics of endocytic pathways is modified in a number of diseases, including cancer, and notably in multidrug resistant (MDR) cells that are refractory to the effects of several anti-cancer agents. These cells often upregulate expression of drug-efflux pumps but this may be synergistic with alternative resistance mechanisms including increased acidification of endocytic organelles that enhances vesicular sequestration of weak-base anti-cancer drugs such as daunorubicin away from their nuclear target. Here, we characterised the distribution of sequestered daunorubicin in commonly used leukaemia cell lines, HL-60, K562, KG1a and the multidrug resistant HL-60/ADR line, and related this to the spatial distribution of their endocytic organelles and microtubule networks. HL-60 and KG1a cells contained microtubule arrays emanating from organising centres, and their endocytic organelles and daunorubicin labelled vesicles were scattered throughout the cytoplasm. HL-60/ADR and K562 cells showed extensive clustering of early and recycling endosomes, late endosomes, lysosomes and daunorubicin to a juxtanuclear region but these cells lacked microtubule arrays. Microtubular organisation within these clustered regions was however, required for spatial tethering of endocytic organelles and the Golgi, as treatment with nocodazole and paclitaxel had major effects on their distribution. HL-60 and HL-60/ADR cells had similar lysosomal pH of <5.0 and overall these findings suggests a general relationship between the absence of microtubule arrays and the propensity of leukaemia cell lines to cluster endocytic organelles and daunorubicin into the juxtanuclear region.

Targeting cancer stem cells

Recent evidence has demonstrated the existence of a small subset of the tumour mass that is wholly responsible for the sustained growth and propagation of the tumour. This cancer stem cell (CSC) compartment is also likely to be responsible both for disease relapse and the resistance to therapy that often accompanies relapse. The evidence for CSCs in various malignancies is presented. The failure of existing therapeutics to eradicate CSCs suggests that they are relatively resistant to present cancer treatments. This resistance may reflect the preservation of normal stem cell protective mechanisms, such as an increased expression of drug efflux pumps or alterations in apoptotic, cell cycle and DNA repair mechanisms. Targeting these mechanisms, and taking advantage of potential differences in the biology of normal stem cells and CSCs, such as differences in surface phenotype, self renewal/quiescence and stem cell–niche interactions are discussed and preliminary preclinical or clinical data are presented. Finally, the authors give their opinion of the direction in which one must travel to successfully target the CSC and improve treatment outcomes in malignant disease.

Biofilm resistance

Device-related infections in most nosocomial diseases can be traced to the formation of biofilms (microbial communities encased within polysaccharide-rich extracellular matrix) by pathogens on surfaces of these devices. Candida species are the most common fungi isolated from these infections, and biofilms formed by these fungal organisms are associated with drastically enhanced resistance against most antimicrobial agents. This enhanced resistance contributes to the persistence of this fungus despite antifungal therapy. Candida biofilms exhibit enhanced resistance against most antifungal agents, except echinocandins and lipid formulations of AmB. The expression of drug efflux pumps during the early phase of biofilm formation and alterations in membrane sterol composition contribute to resistance of these biofilms against azoles. Metabolic dormancy and ECM do not appear to contribute to resistance, although in a mixed-species biofilm, ECM does retard the diffusion of drugs across biofilm. These multifactorial mechanisms of resistance in fungal biofilms constitute a broad-spectrum defense that is effective against many types of antifungal agents, and represent a common theme present across microbial biofilms.

Thanatin activity on multidrug resistant clinical isolates of Enterobacter aerogenes and Klebsiella pneumoniae

Efflux pumps protect bacterial cells by ejecting intracellular toxic molecules such as antibiotics, detergents and defensins that have penetrated the cell envelope. Some of these efflux pumps recognise structurally unrelated compounds (mdr pump) and account for the resistance of some organisms to two or more agents. It would be of interest to identify molecules that are able to circumvent the problems created by multidrug resistance phenotypes during antibiotic therapy. We have studied the activity of thanatin, a 21-residue cationic antimicrobial peptide produced by an insect, against three bacterial species. The antibacterial effect depended on the size of lipopolysaccharide side chains. In clinically resistant isolates of Enterobacter aerogenes and Klebsiella pneumoniae, the biological activity of thanatin is independent of the membrane permeability, possibly controlled by one or more porins, and/or the expression of drug efflux pumps, two mechanisms which confer high level antibiotic resistance. In addition, thanatin was able to improve the activity of structurally unrelated antibiotics (norfloxacin, chloramphenicol, tetracycline) on a multidrug- resistant E. aerogenes clinical isolate.

Regulation of biliary drug efflux pump expression by hormones and xenobiotics

Biliary elimination of endogenous compounds and xenobiotics usually requires carrier-mediated systems allowing movement across the canalicular membrane of hepatocytes. The major systems implicated belong to the ATP binding cassette transporter family: P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2), principally mediate the passage into the bile of cationic and anionic compounds, respectively, whereas the bile salt export pump (BSEP) handles biliary acids and also some anticancer drugs. Expression of these canalicular proteins can be altered in response to various hormones and structurally unrelated xenobiotics. Indeed, glucocorticoids up-regulate expression of both MRP2 and BSEP in rat hepatocytes, whereas insulin induces P-gp. P-gp expression is also up-regulated by numerous chemical carcinogens, such as polycyclic aromatic hydrocarbons and 2-acetylaminofluorene and by some anticancer drugs, such as anthracyclins. 2-Acetylaminofluorene also induces MRP2; in addition, expression of this transporter in liver cells is increased in response to various drugs, such as the barbiturate phenobarbital, the chemopreventive agent, oltipraz and the anticancer drug, cisplatin. Most of the chemical inducers acting on canalicular transporter levels are well-known to up-regulate some hepatic drug metabolizing enzymes, suggesting a coordinate regulation of liver detoxifying proteins in response to these compounds.

Ascorbic acid enhances arsenic trioxide–induced cytotoxicity in multiple myeloma cells

AbstractMultiple myeloma (MM) is a clonal B-cell malignancy characterized by slow-growing plasma cells in the bone marrow (BM). Patients with MM typically respond to initial chemotherapies; however, essentially all progress to a chemoresistant state. Factors that contribute to the chemorefractory phenotype include modulation of free radical scavenging, increased expression of drug efflux pumps, and changes in gene expression that allow escape from apoptotic signaling. Recent data indicate that arsenic trioxide (As2O3) induces remission of refractory acute promyelocytic leukemia and apoptosis of cell lines overexpressing Bcl-2 family members; therefore, it was hypothesized that chemorefractory MM cells would be sensitive to As2O3. As2O3 induced apoptosis in 4 human MM cell lines: 8226/S, 8226/Dox40, U266, and U266/Bcl-xL. The addition of interleukin-6 had no effect on cell death. Glutathione (GSH) has been implicated as an inhibitor of As2O3-induced cell death either through conjugating As2O3 or by sequestering reactive oxygen induced by As2O3. Consistent with this possibility, increasing GSH levels with N-acetylcysteine attenuated As2O3 cytotoxicity. Decreases in GSH have been associated with ascorbic acid (AA) metabolism. Clinically relevant doses of AA decreased GSH levels and potentiated As2O3-mediated cell death of all 4 MM cell lines. Similar results were obtained in freshly isolated human MM cells. In contrast, normal BM cells displayed little sensitivity to As2O3 alone or in combination with AA. Together, these data suggest that As2O3 and AA may be effective antineoplastic agents in refractory MM and that AA might be a useful adjuvant in GSH-sensitive therapies.