ABSTRACT
BACKGROUND: Treatment of malignancies is still a major challenge in human and canine cancer, mostly due to the emergence of multidrug resistance (MDR). One of the main contributors of MDR is the overexpression P-glycoprotein (Pgp), which recognizes and extrudes various chemotherapeutics from cancer cells. METHODS: To study mechanisms underlying the development of drug resistance, we established an in vitro treatment protocol to rapidly induce Pgp-mediated MDR in cancer cells. Based on a clinical observation showing that a 33-day-long, unplanned drug holiday can reverse the MDR phenotype of a canine diffuse large B-cell lymphoma patient, our aim was to use the established assay to prevent the emergence of drug resistance in the early stages of treatment. RESULTS: We showed that an in vitro drug holiday results in the decrease of Pgp expression in MDR cell lines. Surprisingly, celecoxib, a known COX-2 inhibitor, prevented the emergence of drug-induced MDR in murine and canine lymphoma cell lines. CONCLUSIONS: Our findings suggest that celecoxib could significantly improve the efficiency of chemotherapy by preventing the development of MDR in B-cell lymphoma.
ABSTRACT
Membrane transporters play an important role in the absorption, distribution, metabolism and excretion of drugs. The cellular accumulation of many drugs is the result of the net function of efflux and influx transporters. Efflux transporters such as P-glycoprotein/ABCB1 have been shown to confer multidrug resistance in cancer. Although expression of uptake transporters has been confirmed in cancer cells, their role in chemotherapy response has not been systematically investigated. In the present study we have adapted a fluorescence-based cytotoxic assay to characterize the influence of key drug-transporters on the toxicity of approved anticancer drugs. Co-cultures of fluorescently labeled parental and transporter-expressing cells (expressing ABCB1, ABCG2 or OATP2B1) were screened against 101 FDA-approved anticancer drugs, using a novel, automated, triple fluorescence-based cytotoxicity assay. By measuring the survival of parental and transporter-expressing cells in co-cultures, we identify those FDA-approved anticancer drugs, whose toxicity is influenced by ABCB1, ABCG2 or OATP2B1. In addition to confirming known substrates of ABCB1 and ABCG2, the fluorescence-based cytotoxicity assays identified anticancer agents whose toxicity was increased in OATP2B1 expressing cells. Interaction of these compounds with OATP2B1 was verified in dedicated transport assays using cell-impermeant fluorescent substrates. Understanding drug-transporter interactions is needed to increase the efficacy of chemotherapeutic agents. Our results highlight the potential of the fluorescence-based HT screening system for identifying transporter substrates, opening the way for the design of therapeutic approaches based on the inhibition or even the exploitation of transporters in cancer cells.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Screening Assays, Antitumor/methods , Luminescent Proteins/genetics , Organic Anion Transporters/metabolism , Antineoplastic Agents/pharmacokinetics , Cell Line, Tumor , Cell Survival/drug effects , Coculture Techniques , Computer Simulation , Flow Cytometry , Fluorescent Dyes , Green Fluorescent Proteins/genetics , Humans , Organic Anion Transporters/genetics , Substrate Specificity , Transduction, Genetic , Red Fluorescent ProteinABSTRACT
BACKGROUND: The most common mechanism that reduces the efficacy of anticancer agents is overexpression of ATP-binding cassette (ABC) drug transporters. Phenothiazines and structurally-related compounds can sensitize multidrug-resistant (MDR) cells to chemotherapeutics. MATERIALS AND METHODS: Phenothiazine derivatives were investigated regarding their anticancer and MDR-reversing effect on colonic adenocarcinoma cells. The anti-proliferative and cytotoxic effects of the derivatives were assessed by the thiazolyl blue tetrazolium bromide (MTT) method, the modulation of the ABCB1 activity was measured by rhodamine 123 accumulation assay using flow cytometry. RESULTS: All phenothiazines exhibited potent cytotoxic effect on the sensitive and MDR colon adenocarcinoma cell lines. The inhibition of the ABCB1 transporter was greater in the presence of the phenothiazine derivatives than for the known ABCB1 inhibitor verapamil. CONCLUSION: It can be concluded that these derivatives show synergism in the presence of doxorubicin and could have potential as ABCB1 inhibitors.
