Ellen Friche

Different modes of anthracycline interaction with topoisomerase II. Separate structures critical for DNA-cleavage, and for overcoming topoisomerase II-related drug resistance.

In contrast to the classic anthracyclines (doxorubicin and daunorubicin), aclarubicin (ACLA) does not stimulate topoisomerase II (topo II) mediated DNA-cleavage. This distinction may be important with respect to topo II-related drug resistance, and the aim of this study was to clarify drug-structures responsible for this difference. Various ACLA analogs were tested for: (a) interaction with purified topo II, (b) induction of DNA cleavage in cells, (c) cellular uptake and (d) cytotoxicity. A remarkable distinction was seen between analogs containing the chromophore aklavinone (AKV) (e.g. ACLA) which have a carboxymethyl group (COOCH3) at C-10 and drugs with a beta-rhodomycinone (RMN) chromophore with hydroxyl groups at C-10 and at C-11. Thus, RMN-containing analogs, including the aglycone RMN itself, effectively stimulated topo II-mediated DNA cleavage. In contrast, AKV-containing drugs inhibited DNA cleavage and antagonized cytotoxicity mediated by RMN-containing drugs. In OC-NYH/VM cells, exhibiting multidrug resistance due to an altered topo II phenotype (at-MDR), cross-resistance was only seen to the RMN-containing drugs whereas no cross-resistance was seen to the non-DNA cleaving AKV-containing compounds. Thus, our data show that one domain in the anthracycline is of particular importance for the interaction with topo II, namely the positions C-10 and C-11 in the chromophore, and further that at-MDR was circumvented by a COOCH3 substitution at position C-10. These findings may provide guidance for the synthesis and development of new analogs with activity in at-MDR cells.

Comparison of cyclosporin A and SDZ PSC833 as multidrug-resistance modulators in a daunorubicin-resistant Ehrlich ascites tumor.

SummaryRecent studies by Boesch et al. have demonstrated that a nonimmunosuppressive cyclosporin analog, SDZ PSC 833 (an analog of cyclosporin D), is an active multidrug-resistance modifier that is at least 10 times more potent than cyclosporin A. In vitro accumulation and cytotoxicity experiments using daunorubicin (DNR) and vincristine (VCR) under the influence of SDZ PSC 833 and cyclosporin A were performed in wild-type (EHR2) and the corresponding highly DNR-resistant (about 80-fold) Ehrlich ascites tumor cells (EHR2/DNR+). In accumulation experiments, both SDZ PSC 833 and cyclosporin A were found to reverse the multidrug-resistant (MDR) phenotype, but to the same degree at equimolar concentrations. Thus, in EHR2/DNR+ cells, both cyclosporins at 5 μg/ml enhanced DNR and VCR accumulation to sensitive levels, but only a negligible effect on DNR accumulation in the drug-sensitive cells was seen. In the clonogenic assay, the cytotoxicity of the two modulators was equal. The lethal dose for 50% of the cell population (LD50) was approx. 7 μg/ml for both compounds, and no toxicity was observed at concentrations below 2 μg/ml. At nontoxic doses, both cyclosporins effectively increased the cytotoxicity of DNR and VCR in a concentration-dependent manner. The dose-response curves were nearly identical and did not demonstrate differences in modulator potency. These data permit the conclusion that cyclosporin A and SDZ PSC 833 do raise the intracellular accumulation of DNR and VCR to the same levels and that SDZPSC 833 does not potentiate cytotoxicity better than cyclosporin A in EHR2/DNR+ cells. However, since the new compound is nonimmunosuppressive and causes less organ toxicity, clinical studies of its MDR modulating effect seem highly relevant.

Effect of verapamil on daunorubicin accumulation in Ehrlich ascites tumor cells

SummaryPrevious studies have demonstrated that verapamil may overcome resistance to anthracyclines. In vitro and in vivo experiments were performed on wild-type and resistant Ehrlich ascites tumor cells.Verapamil in concentrations of 25–50 μM enhances the accumulation of daunorubicin (DNR) in resistant cells to the same level as in wild-type cells. No significant effect of verapamil on influx or nuclear binding could be demonstrated, indicating that verapamil enhances DNR uptake by blocking active drug extrusion. Exposure of cells to a high concentration of Ca2+ did not influence the effect of verapamil on DNR accumulation, suggesting a different mode of verapamil action apart from the Ca2+-blocking effect. Attempts to circumvent acquired resistance to DNR in vivo with verapamil showed that the combination of the two drugs was more toxic than DNR given alone. The LD10 of DNR was determined as 3 mg/kg and the LD10 of the combination, as 2.5 mg/kg. The therapeutic effect of verapamil at a dose of 50 mg/kg and DNR of 2.5 mg/kg increased the life span of the mice by 50%. No difference was seen in the wild-type tumor in vivo.These data lead us to conclude that verapamil can reverse DNR resistance completely, but that verapamil at non-toxic dosage only reduces DNR resistance by 50% in vivo.

In vitro circumvention of anthracycline-resistance in ehrlich ascites tumour by anthracycline analogues

In previously reported studies, acquired experimental resistance and cross resistance to anthracyclines are related to decreased drug accumulation and retention. The decreased accumulation seems to depend on a cellular mechanism for active drug efflux. N-Acetyl-daunorubicin (N-acetyl-DNR) has demonstrated the ability to increase drug accumulation and to overcome experimental resistance to daunorubicin (DNR) in resistant cells. In the present in vitro study 25 different anthracycline analogues were tested for their influence on [3H]DNR accumulation in resistant cells. At equimolar concentrations (5 microM) four of the analogues enhanced [3H]DNR accumulation more than 200%. Increasing the concentration of the analogues 3-20-fold, 12 of the compounds could enhance [3H]DNR accumulation above 200%. No specific structural changes separated those 12 compounds from the 13 analogues with no or minor effect. The lipid solubility of the 25 analogues was examined by measuring the partition coefficient in octanol/phosphate and pentanol/phosphate buffer (pH 7.45). A good correlation was demonstrated between increased lipid solubility of the analogues and their effect on [3H]DNR accumulation in resistant cells. Further studies demonstrated that N,N-dibenzyl-DNR was able to potentiate cytotoxicity of DNR in resistant cells. It is concluded that several anthracycline analogues are able to reverse resistance, but it is not possible from the chemical structure to predict which analogue results in enhanced [3H]DNR accumulation in resistant cells.

Altered DNA topoisomerase II in multidrug resistance

The characteristic feature of multidrug resistance (MDR) associated with drugs that interact with DNA topoisomerase II (topo II) is alterations in topo II activity or amount (at-MDR). We have characterized the at-MDR phenotype in human leukemic CEM cells selected for resistance to the topo II inhibitor, VM-26. Compared to drug-sensitive cells, the key findings are that at-MDR cells exhibit (i) decreased topo II activity; (ii) decreased drug sensitivity, activity and amount of nuclear matrix topo II; (iii) increased ATP requirement of topo II; (iv) a single base mutation in topo II resulting in a change of Arg to Gln at position 449, at the start of the motif B/nucleotide binding site; and (v) decreased topo II phosphorylation, suggesting decreased kinase or increased phosphatase activities. Recent results using single-stranded conformational polymorphism analysis reveals the presence of a mutation in the motif B/nucleotide binding site of the topo IIα gene in CEM at-MDR cells and in another leukemic cell line selected for resistance to m-AMSA. Finally, we have observed marked changes in the nuclear distribution of topo II in cells treated with anti-topo II drugs and have also found these changes to be attenuated in drug-resistant cells. We postulate that traditional inhibitors of topo II alter the equilibrium of the strand-passing reaction such that the number of enzyme-DNA covalent complexes increases. We further suggest that when the enzyme is bound to DNA it is protected from proteolysis, thus allowing more topo II molecules to be detected. We propose that MDR associated with alterations in topo II may have clinical consequences, and our current efforts involve exploiting these biochemical and molecular observations in the development of probes that may be useful to identify such drug resistant cells in the tumors of patients.

Antitumor activity of the two epipodophyllotoxin derivatives VP-16 and VM-26 in preclinical systems : a comparison of in vitro and in vivo drug evaluation

SummaryThe epipodophyllotoxines VP-16 and VM-26 are chemically closely related. VM-26 has been found to be considerably more potent than VP-16 in vitro in a number of investigations. Although the drugs have been known for >20 years, they have not been compared at clearly defined equitoxic doses on an optimal schedule in vivo and it has not been clarified as to whether a therapeutic difference exists between them. A prolonged schedule is optimal for both drugs; accordingly we determined the toxicity in mice using a 5-day schedule. The dose killing 10% of the mice (LD10) was 9.4 mg/kg daily (95% confidence limits, 7.4–11.8) for VP-16 and 3.4 (2.5–4.5) mg/kg daily for VM-26. In vitro, we found VM-26 to be 6–10 times more potent than VP-16 in a clonogenic assay on murine tumors P388 and L1210 leukemia and Ehrlich ascites. This pattern was also demonstrated in a multidrug-resistant subline of Ehrlich selected for resistance to daunorubicin (Ehrlich/DNR+), as it was 30 times less sensitive than Ehrlich cells to both VP-16 and VM-26. Using 90%, 45%, and 22% of the LD10 on the same murine tumors in vivo, we found that the effect of the two drugs was equal as evaluated by both the increase in life span and the number of cures. The drugs were also compared in nude mice inoculated with human small-cell lung cancer lines OC-TOL and CPH-SCCL-123; however, they were more toxic to the nude mice and only a limited therapeutic effect was observed. In conclusion, the complete cross-resistance between the two drugs suggests that they have an identical antineoplastic spectrum. VM-26 was more potent than VP-16 in vitro; however, this was not correlated to a therapeutic advantage for VM-26 over VP-16 in vivo

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.

Cytosolic free Ca2+ in daunorubicin and vincristine resistant ehrlich ascites tumor cells: Drug accumulation is independent of intracellular Ca2+ changes

The possible role of intracellular calcium on daunorubicin (DNR) accumulation in wild-type (EHR2) and multi-drug resistant (MDR) Ehrlich ascites tumor cell subline was investigated. DNR accumulation was not enhanced either by increasing the concentration of cellular calcium with the calcium ionophore ionomycin nor by chelating the cytosolic free Ca2+ by the membrane permeable Ca2(+)-buffering agents BAPTA or MAPTAM. No effect was observed in the presence of extremely low extracellular calcium concentration that prevent transmembrane calcium influx or when the cells were calcium depleted using EGTA and ionomycin. Using the fluorescent Ca2+ indicator fura-2 it is further shown that both drug-resistant daunorubicin (EHR2/DNR+) and vincristine (EHR/VCR+) sublines had lower (50-80 nM) concentration of cytosolic free calcium ([Ca2+]i) compared to their corresponding wild-type parenteral tumors (140-180 nM). In calcium free medium, however, no significant difference was found, all cell lines having a [Ca2+]i of 60-80 nM. Furthermore, the total amount of Ca2+ released to the cytosol with 10 microM ionomycin and 5 mM EGTA was 3-4-fold higher in EHR2 than in EHR2/DNR+ or EHR2/VCR+. Mobilization of Ca2+ with 1 microM ionomycin was almost identical in the presence and absence of Ca2+ in the extracellular medium in EHR2 as well as in EHR2/DNR+ suggesting that the increase in [Ca2+]i is mainly due to discharge of Ca2+ from intracellular stores. Furthermore, the total cell calcium [Ca2+]t concentration was slightly higher in EHR2/DNR+ and EHR2/VCR+ cells compared to EHR2. Incubation of the cells with the Ca2(+)-channel blocker verapamil or the intracellular Ca2(+)-antagonist TMB-8 causes depression of the Ca2(+)-response in terms of rise in [Ca2+]i caused by ionomycin. Sorcin, a major calcium-binding protein (Mr 22 kDa), is shown to be overproduced in EHR2/DNR+ cells. The overproduction of this protein in resistant cells may be related to the difference in the intracellular calcium observed in this study. Thus, though handling of Ca2+ is different in wild-type and MDR cell lines, our data suggest that calcium is not involved directly in drug transport processes and the level of Ca2+ per se have no influence on drug accumulation.

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.