Kohji Takara

An Update on Overcoming MDR1-Mediated Multidrug Resistance in Cancer Chemotherapy

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.

Simvastatin and lovastatin, but not pravastatin, interact with MDR1

The 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase inhibitor, pravastatin, was compared with simvastatin and lovastatin from the viewpoint of susceptibility to interaction with or via the multidrug transporter, MDR1 (P‐glycoprotein). This was carried out using the MDR1‐overexpressing cell line LLC‐GA5‐COL150, established by transfection of MDR1 cDNA into porcine kidney epithelial LLC‐PK1 cells, and [3H]digoxin, which is a well‐documented substrate for MDR1. Pravastatin, at 25–100 μM, had no effect on the transcellular transport of [3H]digoxin whereas simvastatin and lovastatin suppressed the basal‐to‐apical transport of [3H]digoxin and increased the apical‐to‐basal transport. It was suggested that recognition by MDR1 was due to the hydrophobicity. In conclusion, simvastatin and lovastatin are susceptible to interaction with or via MDR1, but pravastatin is not. This is important information when selecting the HMG‐CoA reductase inhibitors for patients taking drugs that are MDR1 substrates.

Interaction of docetaxel ('Taxotere') with human P-glycoprotein

The interaction of docetaxel (“Taxotere”) with P‐glycoprotein (P‐gp) was examined using porcine kidney epithelial LLC‐PK1 and LLC‐GA5‐COL150 cells, overexpressing human P‐gp selectively on the apical plasma membrane by transfection of human MDR1cDNA into the LLC‐PK1 cells. The basal‐to‐apical transport of [14C]docetaxel in LLC‐GA5‐COL150 cells significantly exceeded that in LLC‐PK1 cells, but the apical‐to‐basal transport was decreased in LLC‐GA5‐COL150 cells. The intracellular accumulation after its basal or apical application to LLC‐GA5‐COL150 cells was 4‐ to 20‐fold lower than that of LLC‐PK1 cells. Multidrug resistance (MDR) modulators, i.e., cyclosporin A and SDZ PSC 833, inhibited the basal‐to‐apical transport and increased the apical‐to‐basal transport of [14C] docetaxel in LLC‐GA5‐COL150 cells, but verapamil affected only apical‐to‐basal transport. The intracellular accumulation after basal or apical application to LLC‐GA5‐COL150 cells was also increased by these three MDR modulators. These observations demonstrated that docetaxel is a substrate for human P‐gp, suggesting that docetaxel‐drug interactions occur via P‐gp. The inhibition of [14C]docetaxel transport by the MDR modulators, as well as daunorubicin and vinblastine, was also found in LLC‐PK1 cells, which endogenously express P‐gp at lower levels, and concentrations showing similar levels of inhibition were lower than those in the case of LLC‐GA5‐COL150 cells. These observations indicate that it is necessary to consider the Pharmacokinetic and pharmacodynamic interactions of docetaxel via P‐gp.

Effects of 12 Ca2+ antagonists on multidrug resistance, MDR1-mediated transport and MDR1 mRNA expression

The effects of 12 Ca(2+) antagonists on MDR1 were examined by two independent models: the inhibitory effect on MDR1-mediated transport of [(3)H]digoxin using MDR1-overexpressing LLC-GA5-COL150 cell monolayers and the reversal effect on cytotoxicity of vinblastine or paclitaxel using MDR1-overexpressing Hvr100-6 cells. The inhibitory effects on [(3)H]digoxin transport were assessed as the 50% inhibitory concentration during 4 h exposure, and the values were the lowest for nicardipine (4.54 microM), manidipine (4.65 microM) and benidipine (4.96 microM), followed by bepridil (10.6 microM), barnidipine (12.6 microM), efonidipine (13.0 microM), verapamil (13.2 microM) and nilvadipine (18.0 microM). The reversal effect on cytotoxicity was assessed by the 50% growth inhibitory concentration after 3 days exposure, and the resistance to vinblastine or paclitaxel in Hvr100-6 cells was reversed by manidipine, verapamil, benidipine, barnidipine, and nicardipine, in that order. Bepridil, barnidipine, efonidipine, verapamil and nilvadipine showed similar inhibitory effects on [(3)H]digoxin transport, but barnidipine and verapamil showed a stronger effect in reversal of cytotoxicity. Real-time quantitative RT-PCR assay indicated a decrease in MDR1 mRNA expression by barnidipine and verapamil. It is concluded that Ca(2+) antagonists cannot only be direct inhibitors of MDR1 but that some may at the same time act as inhibitors of expression of MDR1 via down-regulation of MDR1 mRNA.

Interaction of digoxin with antihypertensive drugs via MDR1

The multidrug transporter MDR1 (P-glycoprotein)-mediated interaction between digoxin and 29 antihypertensive drugs of various types was examined by using the MDR1 overexpressing LLC-GA5-COL150 cells, which were established by transfecting MDR1 cDNA into porcine kidney epithelial LLC-PK1 cells. These cells construct monolayers with tight junctions, and enable the evaluation of transcellular transport. The MDR1 was highly expressed on the apical membrane (urine side). The basal-to-apical and apical-to-basal transcellular transport of [3H]digoxin in LLC-GA5-COL150 cells was time- and temperature-dependent. The basal-to-apical transport of [3H]digoxin was markedly increased, whereas the apical-to-basal transport was decreased in LLC-GA5-COL150 cells, compared with the host LLC-PK1 cells, suggesting that [3H]digoxin was a substrate for MDR1. Most of the Ca2+ channel blockers used here markedly inhibited basal-to-apical transport and increased apical-to-basal transport. Exceptions were diltiazem, nifedipine and nitrendipine, which hardly showed inhibitory effects on transcellular transport of [3H]digoxin. Alpha-blocker doxazosin and beta-blocker carvedilol also inhibited transcellular transport of [3H]digoxin, but none of the angiotensin converting enzyme inhibitors and AT1 angiotensin II receptor antagonists used here were active. These observations will promote understanding of the digoxin-drug interactions resulting from their actions on MDR1, and which may aid in avoiding these unexpected effects of digoxin.

Inhibitory effects of a cyclosporin derivative, SDZ PSC 833, on transport of doxorubicin and vinblastine via human P-glycoprotein

The inhibitory effects of SDZ PSC 833 (PSC833), a non‐immunosuppressive cyclosporin derivative, on the P‐glycoprotein (P‐gp)‐mediated transport of doxorubicin and vinblastine were compared with those of cyclosporin A (Cs‐A). The transcellular transport of the anticancer drugs and PSC833 across a monolayer of LLC‐GA5‐COL150 cells, which overexpress human P‐gp, was measured. Both PSC833 and Cs‐A inhibited P‐gp‐mediated transport of doxorubicin and vinblastine in a concentration‐dependent manner and increased the intracellular accumulation of doxorubicin and vinblastine in LLC‐GA5‐COL150 cells. The values of the 50%‐inhibitory concentration (IC50) of PSC833 and Cs‐A for doxorubicin transport were 0.29 and 3.66 μM, respectively, and those for vinblastine transport were 1.06 and 5.10 μM, respectively. The IC50 of PSC833 for doxorubicin transport was about 4‐fold less than that for vinblastine transport, suggesting that the combination of PSC833 and doxorubicin might be effective. PSC833 itself was not transported by P‐gp and had higher lipophilicity than Cs‐A. These results indicated that the inhibitory effect of PSC833 on P‐gp‐mediated transport was 5‐ to 10‐fold more potent than that of Cs‐A, and this higher inhibitory effect of PSC833 may be related to the absence of PSC833 transport by P‐gp and to the higher lipophilicity of PSC833.

Molecular changes to HeLa cells on continuous exposure to cisplatin or paclitaxel.

AbstractObjective: To achieve a reversal of multidrug resistance (MDR) in cancer chemotherapy, it is crucial to clarify the characteristics of MDR cells generated by various types of chemotherapeutic agents and to find novel targets. Methods: Cisplatin- and paclitaxel-resistant HeLa sublines (HeLa/CDDP and HeLa/TXL, respectively) were established by continuous exposure and their cellular changes were examined based on growth inhibition assays, the transport activity of P-glycoprotein/MDR1, and a RT-PCR analysis of MDR-related factors. Results: HeLa/CDDP cells showed cross-resistance to platinum derivatives, whereas HeLa/TXL cells were resistant to a variety of MDR1 substrates. Transport activity of MDR1 was reduced in HeLa/CDDP cells and the expression of MDR1 was significantly accelerated in HeLa/TXL cells, compared with HeLa cells. In addition, the expression levels of MDR-related transporters (MRP1–5 or BCRP), βtubulin which is a target for taxanes, and apoptosis-regulated factors were comparable among the three cell lines. On the other hand, the mRNA levels of γ-glutamyl transferase, but not γ-glutamyl cysteine synthetase, were higher in HeLa/CDDP cells than in HeLa and HeLa/TXL cells. Conclusions: HeLa/CDDP cells showed decreased activity and expression of MDR1 and overexpression of

Transport mechanism of anthracycline derivatives in human leukemia cell lines: uptake and efflux of pirarubicin in HL60 and pirarubicin-resistant HL60 cells

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