Changes in subcellular doxorubicin distribution and cellular accumulation alone can largely account for doxorubicin resistance in SW-1573 lung cancer and MCF-7 breast cancer multidrug resistant tumour cells

Abstract

Summary Doxorubicin accumulation defects in multidrug reistant tumour cells are generally small in comparison to the resistance factors. Therefore additional mechanisms must be operative. In this paper we show by a quantitative approach that doxorubicin resistance in several P-glycoprotein-positive non-small cell lung cancer and breast cancer multidrug resistant cell lines can be explained by a summation of accumulation defect and alterations in the efficacy of the drug once present in the cell. This alteration of efficacy was partly due to changes in intracellular drug localisation, characterised by decreased nuclear/cytoplasmic doxorubicin fluorescence ratios (N/C-ratios). N/C-ratios were 2.8-3.6 in sensitive cells, 0.1-0.4 in cells with high (>70-fold) levels of doxorubicin resistance and 1.2 and 1.9 in cells with low or intermediate (7.5 and 24-fold, respectively) levels of doxorubicin resistance. The change of drug efficacy was reflected by an increase in the total amount of doxorubicin present in the cell at equitoxic (IC50) concentrations. N/C ratios in highly resistant P-glycoprotein-containing cells could be increased with the resistance modifier verapamil to values of 1.3-2.7, a process that was paralleled by a decrease of the cellular doxorubicin amounts present at IC50. At the low to moderate residual levels of resistance, obtained with different concentrations of verapamil, a linear relationship between IC50 and cellular doxorubicin amounts determined at IC50 was found. This shows that at this stage of residual resistance, extra reversal by verapamil should be explained by further increase of drug efficacy rather than by increase of cellular drug accumulation. A similar relationship was found for Pglycoprotein-negative MDR cells with low levels of resistance. Since in these cells N/C ratios could not be altered, verapamil-induced decrease of IC50 must be due to increased drug efficacy by action on as yet unidentified targets. Although the IC50 of sensitive human cells cannot be reached with resistance modifiers, when using these relationships it can be shown by extrapolation that cellular and nuclear doxorubicin amounts at IC50 at complete reversal of resistance were the same as in sensitive cells. It is concluded that doxorubicin resistance factors for multidrug resistant cells can for a large part, and in the case of P-glycoprotein-containing cells probably fully, be accounted for by decreased amounts of drug at nuclear targets, which in turn is characterised by two processes only: decreased cellular accumulation and a shift in the ratio nuclear drug/cytoplasmic drug.

Efflux Kinetics and Intracellular Distribution of Daunorubicin Are Not Affected by Major Vault Protein/Lung Resistance-Related Protein (Vault) Expression

“…The sequestration of anthracyclines in cytoplasmic structures/vesicles has been described in several drug-resistant cell lines and has been associated with the expression of P-gp, MRP1, and/or MVP (32)(33)(34)(35)(36)(37)(38)(39)(40). However, much is still unclear about the sequestration process and its role in MDR.…”

Intracellular distribution of anthracyclines in drug resistant cells

“…The shift in the ratio nuclear drug/cytoplasmic drug has been reported to be related to resistance to doxorubicin (2.8-3.6 in sensitive cells vs. 0.1-0.4 in cells with 70-fold higher level of resistance) (Schurrhuis et al, 1993). Biophysical studies (Frezard and Garnier-Suillerot, 1991) demonstrated that the intercalation between the base pairs of DNA induced the partial quenching of DOX fluorescence.…”

Decreased sensitivity of carcinoembryonic antigen cDNA-transfected cells to adriamycin

“…One possible explanation would involve increased plasma membrane traffic and endosomal compartment as defined by endosomal number, volume and membrane area (Sehested et al, 1987). Alternatively, subcellular compartmentalization, such as a shift of ADR from the nucleus to the cytoplasm (Schuurhuis et al, 1993), may be implied, since ADR in the cytoplasm is rapidly excreted whereas it remains for a long time in the nucleus (Schuurhuis et al, 1993).…”