Low-level doxorubicin resistance in P-glycoprotein-negative human pancreatic tumour PSN1/ADR cells implicates a brefeldin A-sensitive mechanism of drug extrusion

Abstract

The human pancreatic tumour cell line PSN1/ADR, stepwise selected in 17-510 nM doxorubicin, displayed a multidrug resistance not conferred by P-glycoprotein (P-gp). Resistance to 17-51 nM doxorubicin was accompanied by overexpression of the vesicular marker lung resistance-related protein (LRP). Further selection in 170 nM doxorubicin led to the activation of multidrug resistance-associated protein (MRP) and to the development of drug accumulation/retention defects sensitive to verapamil. In addition, these defects were reversible by the vesicular traffic inhibitors brefeldin A, fluoroaluminate and nocodazole. In contrast, in human ovarian H134AD cells that are resistant to 1700 nM doxorubicin and used as P-gp-positive controls, the drug efflux was inhibited only by verapamil. The tyrosine kinase inhibitor genistein was a potent blocker of doxorubicin efflux in the PSN1/ADR cells but showed no activity in the H134 AD cells. The doxorubicin cytotoxicity in the PSN1/ADR cells was enhanced both by verapamil and brefeldin A, whereas in the parental PSN1 cells they demonstrated the opposite effects, being respectively sensitising and protecting. The P-gp-negative PSN1/ADR cells adapted to 510 nM doxorubicin retained brefeldin A-sensitive doxorubicin accumulation defects while MRP declined. The persistence of brefeldin A-responsive phenotype on the background of variable MRP expression suggests this agent as a useful functional probe for non-P-gp-mediated resistance to plasma-achievable doxorubicin concentrations. Images Figure 2

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.…”

Intrinsic radiosensitivity of human pancreatic tumour cells and the radiosensitising potency of the nitric oxide donor sodium nitroprusside

“…The last four cell lines display the highest degree of morphological and immunocytochemical differentiation (Maillet et al, 1993). PSN1/ADR is a multidrug-resistant subline established in our laboratory from PSN1 by selection in 170 nM doxorubicin (Verovski et al, 1996). The adherent cultures were maintained in RPMI 1640 medium (Gibco, Paisley, UK) supplemented with 10% bovine calf serum (HyClone Laboratories, Logan, UT, USA) at 37°C in 5% carbon dioxide/95% air.…”

Vault-related resistance to anticancer drugs determined by the expression of the major vault protein LRP

“…Overexpression of LRP/MVP is not only seen in highly resistant MDR cell lines, but it is also frequently observed at the early steps of resistance selection (Moran 1997;Verovski, 1996;Versantvoort, 1995;Wyler, 1997). The mechanism(s) of drug resistance operative in these LRP/MVP-overexpressing MDR sublines displaying low levels of drug resistance are probably more clinically relevant, pointing to LRP/MVP as a potential useful marker of clinical resistance.…”

“…Overexpression of LRP/MVP has been found in a large number of drug-selected MDR cell lines of various histogenetic origins and selected by different drugs (Moran, 1997;Scheper, 1993;Verovski, 1996), indicating that diverse cancer cells react by up-regulating the expression of the LRP gene after exposure to anti-cancer agents. Overexpression of LRP/MVP is not only seen in highly resistant MDR cell lines, but it is also frequently observed at the early steps of resistance selection (Moran 1997;Verovski, 1996;Versantvoort, 1995;Wyler, 1997).…”