Christian Maare

Cellular Resistance to Cancer Chemotherapy

Publisher Summary One of the most challenging aspects of cancer chemotherapy is the problem of resistance to clinical drugs. The reasons for clinical resistance may include pharmacokinetic or cell kinetic factors. It is generally accepted that cellular drug resistance is one of the major reasons that treatment fails. The mechanisms of resistance depend on several factors and circumstances that have given rise to the following classifications: (1) natural ( de novo or intrinsic) versus acquired resistance, (2) experimental versus clinical resistance, (3) resistance developed in rodent versus in human cell lines, (4) in vitro versus in vivo resistance and ( 5 ) low versus high degree of resistance. In addition, the dose schedule may affect resistance. Basically, cellular resistance depends on the biological possibilities which are available for a mammalian cell to escape cellular injury from a cytotoxic drug. Consequently, the mechanisms of resistance found in one cellular system may also occur in another. Considering drug resistance from this point of view, all mechanisms described may be relevant. The chapter begins with a description of different mechanisms of resistance, even though they may have been documented in only one of the classes of resistance, and focuses on cellular resistance to cancer chemotherapy.

Expression of P-glycoprotein and multidrug resistance associated protein in Ehrlich ascites tumor cells after fractionated irradiation.

PURPOSE To characterize irradiated murine tumor cells with respect to drug resistance, drug kinetics, and ATPase activity, and to evaluate the possible role of P-glycoprotein (PGP) and murine multidrug resistance associated protein (Mrp1) in the drug-resistant phenotype of these cells. METHODS AND MATERIALS Sensitive Ehrlich ascites tumor cells (EHR2) were in vitro exposed to fractionated irradiation (60 Gy). Western blot analysis was performed for determination of PGP and Mrp1, reverse transcriptase-polymerase chain reaction (RT-PCR) for determination of mdr1a + b mRNA, and semiquantitative RT-PCR for Mrp1 mRNA. The clonogenic assay was applied to investigate sensitivity, whereas the steady-state drug accumulation of daunorubicin (DNR), 3H-vincristine (VCR), and 3H-etoposide (VP16) was measured by spectrofluorometry and scintillation counting, respectively. For determining of ATPase activity, the release of inorganic phosphate from ATP was quantified using a colorimetric method. RESULTS Compared with EHR2, the irradiated cell line EHR2/irr showed increased expression of PGP (threefold), Mrp1 (eightfold), and Mrp1 mRNA (sixfold), and a slight reduction of mdr1b mRNA, whereas mdr1a was present in EHR2 but could not be detected in EHR2/irr. EHR2/irr developed sixfold resistance to VP16, twofold resistance to vincristine, but remained sensitive to DNR. Addition of the PGP inhibitor, verapamil (VER) or depletion of glutathione by buthionine sulfoximine (BSO) partly reversed the resistance in EHR2/irr. In EHR2/irr, the steady-state accumulation of 3H-VCR and 3H-VP16 was significantly decreased as compared with EHR2, whereas the accumulation of DNR was unchanged. The ATPase activity of plasma membrane vesicles prepared from EHR2/irr cells was similar to that of wild-type EHR2 cells. The ATPase activity was neither stimulated by vinblastine nor VER. CONCLUSION Irradiation induced a multidrug-resistant phenotype in sensitive tumor cells. This phenotype was characterized by increased expression of Mrp1 mRNA, Mrp1, and PGP but decreased expression of mdr1a + b mRNA. The influence of irradiation on PGP and Mrp1 expression seemed to be different.

Influx of daunorubicin in multidrug resistant Ehrlich ascites tumour cells: correlation to expression of P-glycoprotein and efflux. Influence of verapamil

Classic multidrug resistance is characterized by a decrease in the intracellular concentration of drugs in resistant cells as compared to sensitive cells. This is correlated with the presence of P-glycoprotein in the membrane. P-glycoprotein is responsible for an active efflux of drug. In this study we investigated the correlation between P-glycoprotein and influx of daunorubicin. Four Ehrlich ascites tumour cell lines selected in vivo for resistance to daunorubicin were investigated. The sublines EHR2/0.1, EHR2/0.2, passage no. 12 of EHR2/0.8, EHR2/0.4, and passage no. 72 of EHR2/0.8 were 6-, 6-, 5-, 33-, and 35-fold resistant to daunorubicin, respectively. All sublines overexpressed P-glycoprotein as determined with Western blot. Influx was measured over 40 sec. In glucose-enriched medium influx was significantly decreased in all but one of the resistant sublines. A correlation between P-glycoprotein, degrees of resistance, and influx was demonstrated in four sublines. Comparing influx experiments with efflux experiments (Nielsen et al., Biochem Pharmacol 1994, 47, 2125-2135) we found a linear relationship between influx and efflux in the resistant sublines (r = 0.97). Verapamil (5.5 microM, 11.0 microM) increased influx significantly in all resistant sublines, whereas the drug had no effect on sensitive cells. Verapamil (3.3 microM) increased influx in the EHR2/0.8 (passage no. 72) subline to the level of sensitive cells. Comparing this result with efflux experiments, verapamil was found to increase influx preferentially. Depletion of energy (medium without glucose including Na(+)-azide) increased influx in all resistant sublines. In EHR2/0.4 and EHR2/0.8 (passage no. 72) the influx, however, was still significantly decreased after depletion of energy. In these cells further addition of verapamil increased influx to the level of EHR2. These data were consistent with the hypothesis that P-glycoprotein effluxes drug directly from the plasma membrane.

Kinetics of daunorubicin transport in Ehrlich ascites tumor cells with different expression of P-glycoprotein: Influence of verapamil

The classical multidrug resistance (MDR) phenotype is characterized by a decrease in the intracellular drug concentration in resistant cells as compared to sensitive cells. P-glycoprotein (P-gp) is thought to be responsible for an active efflux of lipophilic drugs. Four Ehrlich ascites tumor cell lines selected in vivo for resistance to daunorubicin (DNR) and their sensitive counterpart were investigated. The resistant sublines EHR2/0.1, EHR2/0.2, EHR2/0.4, and EHR2/0.8 were developed by treatment of tumor bearing mice with DNR 0.1, 0.2, 0.4, and 0.8 mg/kg x 4 weekly, respectively. One passage from EHR2/0.1, EHR2/0.2, and EHR2/0.4 and two passages from EHR2/0.8 were investigated. Western blot analysis showed significantly different amounts of P-gp (a 6-fold variation). Efflux of DNR in a drug free medium was investigated with and without presence of verapamil (VER). Efflux from sensitive and resistant cells was described by mono- and bi-exponential kinetics, respectively. In all cases but one, a correlation between resistance, expression of P-gp, P-gp mediated efflux capacity and effect of VER was established. In passage No. 12 of EHR2/0.8, however, a high expression of P-gp was found in spite of a low degree of resistance and a low efflux capacity. In this subline the effect of VER did not correlate to the expression of P-gp. Active efflux seemed to be saturable and was suggested to constitute the major route of efflux in MDR cells. A dose-response relationship was established for the effect of VER on efflux. In conclusion, the results support that P-gp acts as a drug efflux pump. No simple correlation, however, could be established between P-gp and drug transport in all the investigated cell lines. Other factors which might influence transmembranous transportation of DNR are suggested. The active efflux capacity of the cell lines seemed to determine the degree of resistance and the sensitivity to circumvention by VER.

Characterisation of Non-P-Glycoprotein multidrug-resistant Ehrlich ascites tumour cells selected for resistance to mitoxantrone

An Ehrlich ascites tumour cell line (EHR2) was selected in vivo for resistance to mitoxantrone (MITOX). The resistant cell line (EHR2/MITOX) was 6123-, 33-, and 30-fold-resistant to mitoxantrone, daunorubicin, and etoposide, respectively, but retained sensitivity to vincristine. The resistant cells showed moderate sensitisation to mitoxantrone on treatment with verapamil or cyclosporin A. Compared with EHR2, the multidrug resistance-associated protein mRNA was increased 13-fold in EHR2/MITOX. Western blot analysis showed an unchanged, weak expression of P-glycoprotein. Topoisomerase IIalpha was reduced to one-third in EHR2/MITOX relative to EHR2 cells, whereas topoisomerase IIbeta was present in EHR2 but could not be detected in EHR2/MITOX. In the resistant subline, net accumulation of MITOX (120 min) and daunorubicin (60 min) was reduced by 43% and 27%, respectively, as compared with EHR2. The efflux of daunorubicin from preloaded EHR2/MITOX cells was significantly increased. EHR2/MITOX microsomes had a significant basal unstimulated ATPase activity. The apparent K(i) value for vanadate inhibition of the ATPase activity in EHR2/MITOX microsomes was not significantly different from the K(i) value for P-glycoprotein-positive cells. However, whereas verapamil (50 microM) inhibited the ATPase activity of EHR2/MITOX microsomes, it stimulated the ATPase activity of microsomes derived from P-glycoprotein-positive cells. In conclusion, the resistance in EHR2/MITOX was multifactorial and appeared to be associated with: 1) a quantitative reduction in topoisomerase IIalpha and beta protein; 2) reduced drug accumulation, probably as a result of increased expression of a novel transport protein with ATPase activity; and 3) increased expression of MRP mRNA.

Characterisation of multidrug-resistant Ehrlich ascites tumour cells selected in vivo for resistance to etoposide

An Ehrlich ascites tumour cell line (EHR2) was selected for resistance to etoposide (VP16) by in vivo exposure to this agent. The resulting cell line (EHR2/VP16) was 114.3-, 5.7-, and 4.0-fold resistant to VP16, daunorubicin, and vincristine, respectively. The amount of salt-extractable immunoreactive topoisomerase IIalpha and beta in EHR2/VP16 was reduced by 30-40% relative to that in EHR2. The multidrug resistance-associated protein (MRP) mRNA was increased 20-fold in EHR2/VP16 as compared with EHR2, whereas the expression of P-glycoprotein was unchanged. In EHR2/VP16, the steady-state accumulation of [(3)H]VP16 and daunorubicin was reduced by 64% and 17%, respectively, as compared with EHR2. Deprivation of energy by addition of sodium azide increased the accumulation of both drugs to the level of sensitive cells. When glycolysis was restored by the addition of glucose to EHR2/VP16 cells loaded with drug in the presence of sodium azide, extrusion of [(3)H]VP16 and daunorubicin was induced. Addition of verapamil (25 microM) decreased the efflux of daunorubicin to the level of sensitive cells, but had only a moderate effect on the efflux of [(3)H]VP16. The resistant cells showed moderate sensitisation to VP16 on treatment with verapamil, whereas cyclosporin A had no effect. Compared with that of sensitive cells, the ATPase activity of plasma membrane vesicles prepared from EHR2/VP16 cells was very low. Vanadate inhibited the ATPase activity of EHR2/VP16 microsomes with a K(i) value of 30 microM. ATPase activity was slightly stimulated by daunorubicin, whereas vinblastine, verapamil, and cyclosporin A had no effect. In conclusion, development of resistance to VP16 in EHR2 is accompanied by a significant reduction in topoisomerase II (alpha and beta) and by increased expression of MRP mRNA (20-fold). MRP displays several points of resemblance to P-glycoprotein in its mode of action: 1) like P-glycoprotein, MRP causes resistance to a range of hydrophobic drugs; 2) MRP decreases drug accumulation in the cells and this decrease is abolished by omission of energy; and 3) MRP increases efflux of drug from cells. However, compared with that of P-glycoprotein-positive cells, the ATPase activity of MRP-positive cells is found to be low and not able to be stimulated by verapamil.

Influence of chemosensitizers on resistance mechanisms in daunorubicin-resistant Ehrlich ascites tumour cells

Abstract.Aim: To determine whether treatment with chemosensitizers influences the development of the drug-resistant phenotype. Methods: Three sublines were developed from the sensitive Ehrlich ascites tumour cell line (EHR2) and six sublines from the EHR2/DNR cell line positive for P-glycoprotein (PGP) by treatment with daunorubicin (DNR), a combination of DNR and verapamil (VER), or a combination of DNR and cyclosporin A (CsA). A clonogenic assay was used to determine resistance, the expression of PGP, the multidrug resistance associated protein (Mrp1) and topoisomerase IIα and β were measured by Western blotting, and reverse transcriptase-polymerase chain reaction was used for determination of mdr1a and b, and Mrp1 mRNA. Results: Compared with the EHR2 cell line, the amounts of mdr1a mRNA increased significantly in all sublines except EHR2/DNR, whereas mdr1b mRNA levels were unchanged. Compared with the EHR2 subline selected in DNR alone, the levels of mdr1a mRNA and PGP were significantly lower in the EHR2 sublines selected in the presence of chemosensitizer. Furthermore, mdr1a mRNA and PGP were unchanged in all cotreated sublines selected from the PGP-positive EHR2/DNR cell line. The mRNA and protein levels of Mrp1 did not change significantly in any of the cell lines. Only one DNR plus VER-selected subline showed a decrease in topoisomerase IIα (one-third as compared with EHR2). All DNR plus CsA-selected sublines showed significantly less resistance than the corresponding DNR- and DNR plus VER-selected sublines. The effect of VER and CsA on cytotoxicity was retained in all cell lines treated with chemosensitizer. Conclusions: Selection in chemosensitizer resulted in a decrease in the expression of mdr1a and PGP. These chemosensitizers do not seem to influence Mrp1 expression or topoisomerase II. Selection in CsA may retard the development of resistance.