| Multidrug resistance (MDR) is defined as the ability of tumor cells exposed to single drug to develop resistance to a broad range of structurally and functionally unrelated drugs. Recent years a number of experimental data indicate that expression of antiapoptotic gene regulated by NF- K B is involved in the development of MDR. Cepharanthine (CEP) is a bisbenzylisoquinoline alkaloid isolated from the tubers of Stephanie delavayi Diels which has various stronger biological activity. Recent years function of its reversing MDR was reported by foreign researcher. However, the correlationship between the possible mechanism by which it reverses MDR and nuclear factor-K B activity has not been reported. In this paper the MDR cell line EAC/ADR resistant to adriamycin (ADR) was compared with their parental sensitive cell line EAC. MTT (3-(4, 5-dimethylthiazol)-2, 5-diphenyltetr-azolium bromide) assy was used to determined the cytotoxity of ADR alone and ADR combined with CEP or verapamil (VER) in EAC/ADR and EAC cell lines. Effect of drugs on survival time of animal models was studied on the animal model of mice bearing transplantable EAC/ADR in vivo. The constitutive activity and activation of nuclear transcription factor NF- K B by drugs was measured by Dot-Enzyme-linked Immune sorbent assay (Dot-ELISA). The reversal effect of CEP on MDR cell line EAC/ADR in vitro and in vivo was studied. The aim of this study is not only to elucidate the relationship between drug resistance of EAC/ADR cells to ADR and the constitutive activity and activation of NF- K B in EAC/ADR cells, but also investigate the mechanism through which CEP reverses drug resistance, and we want to provide theoretical basis for clinical circumvention of drug resistance. The results are follows:1 .Cytotoxity of ADR in EAC and EAC/ADR cell clines Cytotoxity of ADR was measured by MTT assay. The IC of ADR in EAC and EAC/ADR cell lines were 1.10 + 0.08 u mol L-1, 24.35 + 0.56 umol L-1, with significant difference compared with each other (PO.05). Resistance fold was 22 folds.2. Cytotoxity of ADR combined with CEP or VER in EAC and EAC/ADR cell LinesCytotoxity of CEP or VER in EAC and EAC/ADR cell lineswas weak. The Inhibitory rate(IR)of EAC and EAC/ADR cells treated with 8 u mol-L-1 CEP were 18.43 + 0.96%, 13.49 + 1.27%. Thattreated with 4 u- mol -L-1 CEP 2 u mol -L-1 CEP were 7.75+1.51%, 6.03+1.57% and 5.51+1.24% ,4.69+1.92% , respectively. The IR of EAC and EAC/ADR cells treated with 8 u mol L-1 VER was 2.69 +0.77%, 2.25+0.39%. The IC50 of ADR combined with 4 u mol -I/1 CEP or 8 u mol L-1 VER in EAC cells was 1.20+0.08 u mol L-1 , 1.09 +0.06 u mol L-1 , without significant difference compared with that of ADR alone (1.10 +0.08 11 mol L-1) (P>0.05) . That in EAC/ADR cells was 1.92+0.11 u mol L-1, 2.66+0.28 n mol L-1. There was significant difference(P<0.05) compared with the IC50 of ADR alone in EAC/ADR cells (24.35+0.56 u mol L-1). The reversing fold was 13 folds and 9 folds respectively. The reversal effect of CEP was higher than that of VER (P<0.05).3. Effect of ADR combined with CEP on survival time of animal model of mice bearing transplantable EAC/ADR in vivoSurvival time of animal models treated with ADR or CEP were 12.6 + 3.32days, 14.4+2.27days, with no significant difference compared with control group (13.4+2.22days)(p>0.05). That treated with 1.72 y. mol kg-1 d-1 ADR combined with 29.43 n mol kg-1 d-1 CEP was 23.5+3.03days. There was significant difference compared with control group (p<0.05). Increase in life span over control was 75.37%.4.Effect of drugs on the constitutive activity and activation of NF- K B in EAC and EAC/ADR cell linesDot-ELISA was used to measure nuclear NF- K B content. Dot blot signals were scanned by a thin layer chromatograph scanner. The scanning value represents NF- K B activity. The scanning values of NF-icB in EAC and EAC/ADR cell lines were 1580.95+142.81, 2712.58 + 103.94. While treated with 0.77 u mol L-1 TPA(12-0-tetradecanoyl phorbol-13-aletate) 21.55 u mol L-1 ADR 4 u-mol L... |
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