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Aim: Combination chemotherapy for treating cancer often is superior in clinical efficacy to monotherapy. The aim of this study was to investigate the schedule-dependent effect of 5-fluorouracil (5-FU) and platinum derivatives (cisplatin or oxaliplatin) in colorectal cancer (CRC) cell lines, and to explore factors affecting it., Methods: Two human CRC-derived cell lines, DLD-1 and HCT116, were used. Three treatment schedules were tested, and growth inhibitory effects were evaluated with a WST-1 assay. Combined effects were assessed with isobolograms and a combination index. Cellular accumulation and DNA-binding of platinum were measured with inductively coupled plasma mass spectrometry., Results: Exposure to 5-FU followed by cisplatin produced synergistic effects in DLD-1 cells, and the amount of platinum bound to DNA was substantially increased as compared with that for other schedules. 5-FU and oxaliplatin also tended to be synergistic when 5-FU was given first, but no significant change in the cellular kinetics of platinum was observed. On the other hand, in HCT116 cells, the combined effects of 5-FU and platinum derivatives were comparable among the three schedules., Conclusion: Exposure to 5-FU followed by cisplatin had a synergistic effect in DLD-1 cells, suggesting that the amount of platinum bound to DNA contributes to this result. Also, the effect was dependent on the type of platinum derivative and cell., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Previously, we have adapted a recently developed three-dimensional chemosensitivity test, the collagen gel droplet embedded culture drug sensitivity test (CD-DST), for evaluation of chemosensitivity of 12 anticancer drugs against colorectal adenocarcinoma, and surprisingly, it was found that tumor growth enhancement was occasionally observed even after exposure to anticancer drugs. In this study, the CD-DST was applied for human cervical carcinoma cell line HeLa-Ohio (HeLa) cells and its MDR1/P-glycoprotein-overexpressing subline, Hvr100-6 cells, and 12 anticancer drugs were assessed in terms of chemosensitivity and deterioration of tumor, and the results were compared with those by two-dimensional WST-1 assay. Growth enhancement was observed in Hvr100-6 cells, not in HeLa cells, for mitomycin C with the ratio of total volume of colonies in the treated group to that in the untreated group (T/C%) of 135.0%, doxorubicin with T/C% of 162.5% and cyclophosphamide with T/C% of 122.0%, and this was not observed in WST-1 assay. Multidrug resistance was detected both for CD-DST and WST-1 assay. The values of T/C% in CD-DST were comparable with or higher than those of the survival fraction (%) in WST-1 assay, and modification of WST-1 assay procedure gave similar results, suggesting a higher resistance in three-dimensional than in two-dimensional culture. Further investigations should be addressed to the association of MDR1/P-glycoprotein with tumor growth enhancement.

The intrinsic or acquired resistance to anticancer drugs remains one of the most significant factors impeding the progress of cancer chemotherapy. This phenomenon often involves simultaneous resistance to other anticancer drugs that differ in their chemical structure and mode of action and are not even used in chemotherapy. This phenotype has been called multidrug resistance (MDR). Although the cellular basis underlying MDR is not fully understood, several factors mediating therapy resistance in tumors have been proposed. One of the mechanisms leading to chemoresistance of tumor cells is the increased activity of transporter proteins. The best-characterized transporter protein is MDR1/P-glycoprotein, and a number of clinical investigations have suggested that its intrinsic or acquired overexpression resulted in a poor clinical outcome of chemotherapy. Various types of compounds and techniques for the reversal of MDR1/P-glycoprotein-mediated MDR have been developed, and efforts have concentrated on the inhibition of function and suppression of expression. This review summarizes the current state of knowledge of MDR1/P-glycoprotein and the modulation of MDR by targeting MDR1/P-glycoprotein.

The reversing effects of carvedilol and other beta-adrenoceptor antagonists on multidrug resistance (MDR) were assessed in HeLa cells and the MDR1-overexpressing derivative Hvr100-6 cells, established by stepwise increases of vinblastine concentration in the culture medium. The inhibitory effects on the transcellular transport and intracellular accumulation of [3H]vinblastine and [3H]daunorubicin were also assessed using LLC-GA5-COL150 cell monolayers, established by transfection of human MDR1 cDNA into porcine kidney epithelial LLC-PK1 cells. The cytotoxic effects of vinblastine, paclitaxel, doxorubicin and daunorubicin in Hvr100-6 were reversed 1.4- to 7.1-fold by carvedilol at the realistic clinical concentration of 1 microM, whereas other beta-adrenoceptor antagonists had weaker or no such effects. Transport experiments using LLC-GA5-COL150 cell monolayers demonstrated that this effect of carvedilol was due to the inhibition of MDR1-mediated transport of vinblastine, paclitaxel, doxorubicin and daunorubicin. These MDR1-mediated reversing effects of carvedilol were similar to those of 1 microM verapamil, suggesting that carvedilol could be a candidate modulator of MDR in clinical use. Since other beta-adrenoceptor antagonists had no inhibitory effect on transport, the effects of carvedilol were not related to beta-adrenoceptors and might have been due to antioxidant activity.

The multidrug resistant transporter MDR1/P-glycoprotein, the gene product of MDR1, is a glycosylated membrane protein of 170 kDa, belonging to the ATP-binding cassette superfamily of membrane transporters. MDR1 acts as an energy-dependent efflux pump that exports its substrates out of cells. MDR1 was originally isolated from resistant tumor cells as part of the mechanism of multidrug resistance, but over the last decade, it has been elucidated that human MDR1 is also expressed throughout the body to confer intrinsic resistance to the tissues by exporting unnecessary or toxic exogeneous substances or metabolites. A number of structurally unrelated drugs are substrates for MDR1, and MDR1 and other transporters are recognized as an important class of proteins for regulating pharmacokinetics and pharmacodynamics. In 2000, Hoffmeyer et al. performed a systemic screening for MDR1 polymorphisms and detected 15 single nucleotide polymorphisms (SNPs). They also indicated that a polymorphism in exon 26 at position 3435 (C3435T), a silent mutation, affected the expression level of MDR1 protein in duodenum, and thereby the intestinal absorption of digoxin. To date, the genotype frequencies of C3435T have been investigated extensively using a larger population and interethnic difference has been elucidated, and a total of 28 SNPs have been found at 27 positions on the MDR1 gene. Clinical studies on MDR1 genotype-related MDR1 expression and pharmacokinetics have also been performed around the world; however, results were not always consistent with Hoffmeyer's report. In this review, published reports are summarized for the future individualization of pharmacotherapy based on MDR1 genotyping. In addition, recent investigations have raised the possibility that MDR1 and related transporters play a fundamental role in regulating apoptosis and immunology, and in fact, there are reports of MDR1-related susceptibility to inflammatory bowel disease, HIV infection and renal cell carcinoma. Herein, these issues are also summarized, and the current status of the knowledge in the area of pharmacogenomics of other transporters is briefly introduced.

The cytotoxic effects of 27 anticancer drugs including amrubicin, vinorelbine, paclitaxel, docetaxel, gemcitabine, and irinotecan were evaluated in human cervical carcinoma HeLa cells, and drug-resistant HeLa-derived Hvrl-1, HvrlO-6, and Hvr100-6 cells, which were newly established by stepwise exposure to vinblastine. FACS and RT-PCR analysis indicated that MDR1 (P-glycoprotein) was induced without any alterations in expression of its related transporters. Hvrl00-6 cells showed 2- to 200-fold higher resistance to anthracyclines than HeLa cells, and unexpectedly showed slight resistance to idarubicin and amrubicin. The relative resistance to vinca-alkaloids was 300- to 600,000-fold, and HvrlOO-6 cells showed the highest relative resistance to vinorelbine. HvrlOO-6 cells also showed 4000- and 60000-fold resistance to the taxanes paclitaxel and docetaxel, respectively. Hvr100-6 cells were also resistant to 6-mercaptopurine, actinomycin D, etoposide, and mitomycin C, with relative resistance of 8-, 45000-, 12-, and 9-fold, respectively. In contrast, HvrlOO-6 cells showed no or slight resistance to platinum derivatives, pyrimidine analogues, and alkylating agents or to irinotecan and its active form, or tamoxifen. The cytotoxicity of anthracyclines, vinca-alkaloids, taxanes, actinomycin D, and etoposide was extensively reversed by cyclosporin A. Cyclosporin A had no effect on the cytotoxicity of 6-mercaptopurine or mitomycin C, suggesting that resistance to these drugs was not mediated via MDR1. The alterations in cytotoxicity by overexpression of MDR1 and effects of cyclosporin A could be also qualitatively explained by [3H]vinblastine uptake experiments. The 27 anticancer drugs analyzed here could be classified into substrates and nonsubstrates for MDR1. This will be useful for designing effective regimens for chemotherapy.

Methotrexate is administered in high doses to treat childhood acute lymphoblastic leukemia and malignant lymphoma. Hepatotoxicity and bone marrow suppression often limit its use, however. The objective of this study was to determine the genetic polymorphisms associated with the hepatotoxicity and elimination of methotrexate. Genetic polymorphisms of glutathione S-transferase (GST) genes including GSTT1 positive/null, GSTM1 positive/null, and GSTP1 A313G, and genes for reduced folate carrier 1 G80A ( RFC1 G80A), methylenetetrahydrofolate reductase C677T ( MTHFR C677T), and breast cancer resistant protein C421A ( BCRP C421A) were determined for 26 patients by the polymerase chain reaction (PCR) method or by direct sequencing. A high frequency of hepatotoxicity ( P = 0.035) was observed for patients with GSTM1 positive and RFC1 AA80, and serum concentrations of methotrexate 48 h after the start of infusion were higher for patients with the TT677 genotype of MTHFR ( P = 0.028). In conclusion, GSTM1 positive/null and RFC1 G80A polymorphisms could be predictors for hepatotoxicity, and the MTHFR C677T polymorphism is associated with elimination of methotrexate. [ABSTRACT FROM AUTHOR]

Purpose. To evaluate chemosensitivity and its correlation with expression levels of the multidrug resistant transporter (MDR1) and the multidrug resistance-associated proteins 1 and 2 (MRP1, MRP2) mRNA in human colorectal adenocarcinomas. Methods. Colorectal adenocarcinomas were obtained as surgical samples from 25 patients. The chemosensitivity of 12 anticancer drugs was assessed by the collagen gel droplet embedded culture drug sensitivity test (CD-DST). The expression levels of MDR1, MRP1, and MRP2 mRNA in colorectal adenocarcinomas were also evaluated by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). Results. The chemosensitivity was successfully evaluated for 16 of 25 patients, and the anticancer drugs were effective against the samples showing a relatively high growth rate. Gemcitabine hydrochloride was found to be more promising than those often prescribed for the treatment of colorectal adenocarcinoma. There was no correlation of the mRNA expression levels of MDR1 and MRP1 with the chemosensitivity of any anticancer drugs tested, but mitomycin C was found to be more effective for the colorectal adenocarcinoma with relatively high expression of MRP2 mRNA. [ABSTRACT FROM AUTHOR]