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Characterisation of non-P-glycoprotein multidrug-resistant Ehrlich ascites tumour cells selected for resistance to
D Nielsen1, J Eriksen, C Maare
1Department of Oncology, Herlev Hospital, University of Copenhagen, Copenhagen, Denmark. dln@dadlnet.dk
Abstract:
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.
Insights
This study developed a mitoxantrone-resistant cancer cell line (EHR2/MITOX) exhibiting multidrug resistance. Resistance is linked to reduced topoisomerase II, increased multidrug resistance-associated protein (MRP) mRNA, and altered ATPase activity, suggesting a novel transport mechanism.
Area of Science:
- Cancer Biology
- Pharmacology
- Molecular Biology
Background:
- Acquired resistance to chemotherapy is a major challenge in cancer treatment.
- Understanding the mechanisms of drug resistance is crucial for developing effective therapies.
Purpose of the Study:
- To characterize the mechanisms of mitoxantrone resistance in a newly developed Ehrlich ascites tumour cell line (EHR2/MITOX).
- To investigate the role of drug transporters, topoisomerase II, and drug accumulation in acquired chemoresistance.
Main Methods:
- Development of a mitoxantrone-resistant cell line (EHR2/MITOX) through in vivo selection.
- Assessment of drug sensitivity, drug accumulation, and drug efflux.
- Analysis of P-glycoprotein expression, topoisomerase IIalpha and IIbeta levels, and multidrug resistance-associated protein (MRP) mRNA expression.
- Enzymatic assays of microsomal ATPase activity.
Main Results:
- EHR2/MITOX cells showed high resistance to mitoxantrone, daunorubicin, and etoposide, but remained sensitive to vincristine.
- A 13-fold increase in multidrug resistance-associated protein (MRP) mRNA was observed, while P-glycoprotein expression remained low.
- Topoisomerase IIalpha and IIbeta levels were significantly reduced in resistant cells.
- Reduced net drug accumulation and increased efflux of daunorubicin were observed in EHR2/MITOX cells.
- Microsomes from resistant cells exhibited significant basal ATPase activity, inhibited by verapamil, suggesting a novel transporter.
Conclusions:
- The acquired mitoxantrone resistance in EHR2/MITOX cells is multifactorial.
- Key mechanisms include reduced topoisomerase II, increased MRP mRNA expression, and decreased drug accumulation potentially due to a novel verapamil-sensitive transporter with ATPase activity.