Correlation Between Reversing Effect Of Cepharanthine (CEP) On Multidrug Resistance And GST-π Or DNA TopoⅡ In MCF-7/ADR Cells

Background and objectiveMultidrug resistance (MDR) is the major failure cause in the chemotherapy of human malignant cancers. The term of MDR is the capability of tumor cells exposed to single drug developing resistance as well as to a broad range of structurally and functionally unrelated drugs. A number of studies have demonstrated that the Glutathione S-transferases pi(GST-π) is involved in the development of tumor cells MDR by the biotransformation and detoxification of xenobiotic and endobiotic compounds, while the decrease of cleavable complex formation that results from the quantity or quality change of DNA Topoi-somerase II(Topo II) leads to the production of MDR. Cepharanthine (CEP), which has various stronger biological activities is a bisbenzylisoquinoline alkaloid isolated from the tubers of Stephanie delavayi Diels. And its reversing effect on multidrug resistance has been proved in foreign studies. However, the correlation between the its possible mechanism and GST-π or DNA TopoII activity has not been reported. This study aims at the methods of cytology and molecular biology to determine the reversing effects of CEP and further investigate MDR in its molecular mechanism. MethodsThe multidrug-resistant human breast cancer cell line MCF-7/ADR resistant to adriamycin (ADR) was adopted in the experiments to compare with its parental sensitive cell line MCF-7. MTT(3-(4,-5-dimethylthiazol)-2,-5-diphenylte-2H- tetrazolium bromide) assay was used to determine the cytotoxity of ADR alone and ADR combined with CEP or verapamil(VER) in MCF-7 and MCF-7/ADR cell lines. Immunohistochemistry(IHC) technique was used to determine the expression of GST-π in MCF-7 and MCF-7/ADR cell lines as well as its expression that treated with drugs. DNA TopoII, extracted from MCF-7 and MCF-7/ADR cells, in which the amount of protein was determined by the Coomassie blue G-250 dye. In addition, TopoII activity and the effect of drugs on it weremeasured by the ATP-dependent relaxation of supercoiled pBR322 DNA. The aims of this study were not only to elucidate the relationship between drug resistance of MCF-7/ADR to ADR and GST- or DNA TopoII, but also to investigate the mechanism through which CEP reverses drug resistance. Results1. Cytotoxity of ADR in MCF-7 and MCF-7/ADR cell linesThe IC50 of ADR in MCF-7 and MCF-7/ADR cell lines determined by MTT were 0.50±0.03μmol·L-1 and 27.37±0.83μmol·L-1 respectively. There was significant difference between them(P<0.05) and resistance fold was 54.7.2. Cytotoxity of ADR combined with CEP or VER in MCF-7 and MCF-7/ADR cell linesCytotoxity of CEP or VER alone in MCF-7 and MCF-7/ADR cell lines was weak. The inhibitory rates (IR) of MCF-7 and MCF-7/ADR cells treated with 8μmol·L-1 CEP were(10.27±1.25)% and (9.86±2.03)% respectively, (5.10±0.82)% and (4.75±0.58)% if treated with 4μmol· L-1 CEP, (2.34±0.71)% and (2.15±0.47)% with 2 μmol· L-1 CEP respectively. The IR of MCF-7 and MCF-7/ADR cells treated with 8 μmol ·L-1 VER were(4.96±0.39)% and (4.38±1.24)%, respectively. The IC50 of ADR combined with 4μ mol· L-1 CEP or 8 μ mol · L-1 VER in MCF-7 cells were 0.49±0.02μ mol · L-1 or 0.51± 0.03μ mol· L-1, without significant difference compared with that of ADR alone(P>0.05). The IC50 in MCF-7/ADR cells were 2.03±0.09 μ mol·L-1 or 3.76±0.11μ mol·I-1, which showed significant difference(P<0.05) compared with the IC50 of ADR alone in MCF-7/ADR cells. The reversing fold was 13.5 and 7.3 respectively. The reversal effect of CEP was higher than that of VER, the difference between combined with CEP and combined with VER was significant (P<0.05).3. Effect of ADR combined with drugs on expression of GST- in MCF-7 and MCF-7/ADR cellsThe expression of GST-μ in plasma of cell was measured with IHC and the results were analyzed with HIPAS-1000 computer-assisted image analyzing system(IAS) to achieve semi-quantitative data. The GST- value in control group of MCF-7 cell line was 3.43±1.91. The value of 0. 17 μ mol · L-1 ADR alone was...