Expression of multidrug resistance genes and proteins and effect of selenite in anthracycline-resistant human tumor cell lines

Sammanfattning: Treatment failure due to development of drug resistance is a serious complication in cancer treatment and the major cause of death in these patients. This thesis has focused on to gain knowledge about the mechanisms of multidrug resistance and to establish in vitro methods to characterize the resistant tumor cell phenotype. As a new agent in the context of multidrug resistance, selenite was investigated. The ATP bioluminescence assay was developed for cytotoxicity studies and compared to a differential staining cytotoxicity assay (DiSC) by evaluation of tumor cells from 32 patients with acute myelocytic leukemia. The ATP assay correlated to the DiSC assay (r=0.8). The resistance of the doxorubicin resistant HL-60-R cells correlated with mRNA expression and amplification of the multidrug resistance gene (MDR-1). The MDR-1 gene amplification correlated also to the expression levels of the P-glycoprotein. In the doxorubicin resistant U-1285dox900 cells, the resistance correlated with the MRP1 gene and the protein expression whereas the MDR-1 gene or P-glycoprotein expression was not detectable. The cross-resistance profile in U-1285dox900 or HL-60-R did not include melphalane or CdA implying inability of P-gp and MRP1 to transport these drugs. A low cross-resistance to idarubicin compared to daunorubicin in U-1285dox900 cells was associated with a higher accumulation due to a slower outward transport of idarubicin. A low crossresistance to idarubicin suggests that it could be an effective drug for treatment of leukemias that overexpress MRP1. The resistance modifier verapamil significantly restored the intracellular uptake and enhanced the cytotoxic effect of anthracyclines in both MDR-1 and MRP1 overexpressing sublines, whereas co-incubation of daunorubicin or idarubicin with the gluthatione synthetase inhibitor, buthionine sulphoximine only restored sensitivity in the MRP1 expressing U-1285dox900 cells but not in the MDR-1 expressing HL-60-R sublines. The two doxorubicin-resistant lung carcinoma cell lines U-1285dox900 and GLC4/Adr were 3- and 4-fold respectively more sensitive to selenite toxicity than wild type cells. Necrosis was seen in the U-1285 after exposure to high selenite concentrations. Lower selenite concentrations induced massive apoptosis in the doxorubicin resistant-U-1285dox900 cells. The apoptosis was caspase-3 independent. Selenite exposure did not significantly affect the expression of the multi-drug resistant proteins. The activity of thioredoxin reductase (TrxR) was higher (50 and 25% respectively) in the drug-resistant U-1285dox900 and GLC4/Adr cell lines compared to wild type cells and was upon selenite exposure increased only 30% in drugresistant compared to a 4-fold increase in sensitive U-1285 cells. The activity of glutathione reductase increased 4-fold after selenite exposure of the U-1285 cells, but did not increase in the drug-resistant subline. Analysis of TrxR enzymatic activities and protein levels in these cells revealed a co-augmentation with selenite concentration. Maximum increase of TrxR was seen up to 1 µM in both sublines. A break point was noted at 10 µM selenite were the sensitive cells could increase the activity of TrxR whereas the doxorubicin-resistant U- 1285dox900 cells decreased their TrxR activity. Our results demonstrate pronounced selective selenium-mediated apoptosis in therapy-resistant cells and suggest that redox regulation through the thioredoxin system can be a target for cancer therapy.