Time-lapse live cell imaging and flow analysis of multidrug resistance reversal by verapamil in bladder cancer cell lines.

OBJECTIVES: To examine the effects of verapamil on the intracellular drug pharmacokinetics of epirubicin using alternative dosing schedules. The results might inform the choices for optimizing clinical chemotherapy. METHODS: Sensitive parental (MGH-U1) and multidrug resistant (MDR) (MGH-U1R and MGH-U1-MMC) bladder cancer cell lines were used. Fluorescence time-lapsed studies were performed on cells incubated with epirubicin alone or combined with verapamil. Flow cytometry was performed after the alternative dosing regimens. RESULTS: Verapamil reversed the epirubicin localization patterns in MDR cells. Time-lapse imaging showed that nuclear epirubicin accumulation in MDR cells with verapamil followed the parental curve. The maximal reversal took >60 minutes. Flow cytometry showed increased epirubicin uptake in MDR cells co-incubated with verapamil. Preincubation was not as effective as co-incubation. CONCLUSIONS: The results of our model indicate that longer exposure to MDR-class drugs, exemplified by epirubicin, increases uptake and the MDR reversing action of co-treatment with verapamil. The present results highlight the need for additional clinical trials of drug dosing and scheduling for combination intravesical chemotherapy regimens.

Multidrug resistance in a urothelial cancer cell line after 3, 1-hour exposures to mitomycin C.

PURPOSE: The development of multidrug resistance is a problem in chemotherapy for many tumors. In vitro models of multidrug resistance require adapted cell strains that are conventionally produced from parental lines by chronic low dose drug exposure. Because adjunctive intravesical chemotherapy for superficial bladder cancer uses short courses of high dose treatment, we investigated whether such exposure of the RT112 cell line (Catalogue No. ACC 418, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) to mitomycin C, which is a common intravesical agent, would elicit multidrug resistance. MATERIALS AND METHODS: Three 1-hour exposures to graded concentrations were done at 3-week intervals. The highest mitomycin C concentrations permitting recovery in cultures and, therefore, available for examination were 3.13 and 1.06 microg/ml. Cross-resistance to epirubicin in surviving cultures was visualized by confocal microscopy and quantified by MTT residual viable biomass assay. Spheroids were made by the agarose technique and exposed to high dose mitomycin C to assess the probability that the relevant concentrations might be found clinically in some cell layers of a superficial lesion. RESULTS: Resistance was induced by 3 short drug exposures. The evidence for this was functional (MTT assay) and by intracellular localization. Toxicity to an alternative multidrug resistance class drug was lowered in surviving clones and nuclear exclusion of the drug was noted. Spheroid experiments showed sharp gradients of incorporated drug across the outermost layers of cells, suggesting that a proportion of cells in clinical superficial bladder cancer would be exposed to drug at concentrations that generated the resistant clones in these experiments. CONCLUSIONS: We report multidrug resistance induction using 2 independent methodologies. The results have implications for the development of experimental models and the likelihood of resistance resulting from clinical regimens. Brief exposure can elicit detectable resistance. It is arguable that selective rather than instructive mechanisms are involved, and the levels of drug required are likely to exist in a superficial transitional cell carcinoma frond exposed at its surface to high drug concentrations.

The nuclear membrane in multidrug resistance: microinjection of epirubicin into bladder cancer cell lines.

OBJECTIVE: To assess whether microinjecting epirubicin into cells showing multidrug resistance (MDR, common to many cancers, including bladder cancer, with resistance to, e.g. anthracyclines and mitomycin C) spares the nucleus, as when these drugs accumulate, distribution in MDR cells characteristically spares the nucleus, suggesting that the nuclear membrane is responsible for excluding cytotoxic drugs from MDR nuclei. MATERIALS AND METHODS: Nuclear exclusion of drugs is an important feature of resistance in MDR cells, as many MDR-susceptible drugs have cytotoxic actions within the nucleus. Drug accumulation in 'classical' P-glycoprotein-mediated MDR cells is greatly reduced by efflux. Microinjection of epirubicin into the cytoplasm of MDR cells bypasses the P-glycoprotein efflux pump on the plasma membrane. Nuclear sparing would directly implicate the nuclear membrane in this phenomenon. Because of their fluorescence properties, which allow study by confocal microscopy and flow cytometry, anthracyclines have also been used extensively to investigate MDR. Thus sensitive (MGH-U1 and RT112) and MDR (MGH-U1R and MGH-U1-MMC) bladder cancer cell lines were used. Adherent cells from each cell line were individually microinjected with epirubicin (0.5 mg/mL) and a 77 kDa fluorescein isothiocyanate (FITC)-dextran (0.5 mg/mL). The pattern of nuclear epirubicin uptake in injected cells was then evaluated by confocal microscopy. The 77 kDa FITC-dextran allowed easier identification of injected cells and was also excluded from their nuclei. RESULTS: Sensitive bladder cancer cell lines all showed a nuclear accumulation pattern of epirubicin, consistent with their normal uptake after exposure to epirubicin. The MDR cell lines showed the characteristic nuclear-sparing pattern of epirubicin uptake, similar to the normal uptake pattern after epirubicin exposure. The 77 kDa FITC-dextran showed clearly which cells had been microinjected, and was excluded from the nuclei of all injected cells. Cell viability was confirmed by acridine-orange staining after initial visualization of injected cells. CONCLUSION: The nuclear membrane is responsible for the nuclear exclusion of epirubicin in MDR cells. Further work is necessary to determine the mechanisms involved.