Effect Of N,N-BIS(2-Chloroethyl)DOCOS-13-Enamide On The MDR Reversal Of BEL-7404 And Related Mechanism

Objective:Using human hepatoma adriamycin resistant BEL-7404/ADM cells for the study, to evaluate the effects of N, N-bis(2-chloroethyl)docos-13-enamide on BEL-7404/ADM cells and the associated mechanisms.Methods:1. Establishment of an Adriamycin resistant human hepatoma BEL-7404/ADM cells.The resistant cell line BEL-7404/ADM was induced by impacting with high dose of ADM for short time and gradually increasing concentration of ADM, the resistance index(RI) and drug sensitivity were measured by MTT colorimetric assay. Expressions of ABCB1/Pg-p, ABCC1/MRP1 and GST-π were measured quantitatively by RT-PCR.2. Study of the reversal mechanisms of N, N-bis(2-chloroethyl)docos-13-enamide on BEL-7404/ADM cells(1) N, N-bis(2-chloroethyl)docos-13-enamide alone or combined with ADM were used to treat with BEL-7404/ADM cells, the growth inhibitions were detected by MTT assays, the combination effect of N, N-bis(2-chloroethyl)docos-13-enamide with ADM on BEL-7404/ADM cells was calculated by reverse fold(RF).(2) The protein expressions of MRP and GST were detected by immunochemical assay.(3) The gene expressions of ABCB1/Pg-p, ABCC1/MRP 1 and GST-π of BEL-7404/ADM cell line were investigated by RT-PCR.(4) Expressions of GST-π was studied by Western blot.Result:1. The RI of BEL-7404/ADM was 15.4091 to ADM. The gene expressions of ABCB1/Pg-p, ABCC1/MRP1 and GST-π were enhanced in resistant cells(p<0.05, vs BEL-7404).2. (1) The IC50 value of ADM in BEL-7404/ADM cells was(34.7880±3.83)μg/ml, it decreased to(0.8238±0.27) μg/ml after combined with N, N-bis(2-chloroethyl)docos-13-enamide, the reverse index was 3.114.(2) N, N-bis(2-chloroethyl)docos-13-enamide attenuated the expressions of and GST-π at protein levels. The expressions of GST-α and ABCC1/MRP1 were not changed obviously after treated by N, N-bis(2-chloroethyl)docos-13-enamide.(3) N, N-bis(2-chloroethyl)docos-13-enamide attenuated the expressions of GST-π at mRNA levels. The mRNA expressions of ABCB1/Pg-p and ABCC 1/MRP 1 were not changed obviously after treated by N, N-bis(2-chloroethyl)docos-13-enamide. RT-PCR results showed that the mRNA expression level of ABCB 1/Pg-p and ABCC1/MRP1 was not changed obviously in the N, N-bis(2-chloroethyl)docos-13-enamide and ADM group,compared to the N, N-bis(2-chloroethyl)docos-13-enamide group and ADM group. (P<0.05).(4) N, N-bis(2-chloroethyl)docos-13-enamide can attenuate the expression of GST-π at protein level. Western blot results showed that the protein expression level of GST-π was downregulated in the N, N-bis(2-chloroethyl)docos-13-enamide group, compared to the BEL-7404 group and BEL-7404/ADM group. (P<0.05).Conclusion:1.Using high dose of Adriamycin for short time and gradually increasing concentration of Adriamycin from the BEL-7404 can establish the Adriamycin resistant human hepatoma cell line BEL-7404/ADM, the drug-resistance mechanisms may relate to overexpressions of ABCBl/Pg-p and ABCC1/MRP1.2. N, N-bis(2-chloroethyl)docos-13-enamide may not inhibit the growth of BEL-7404/ADM cells, and it has a synergistic effect with ADM, increase the sensitivity of BEL-7404/ADM cells to ADM.The mechanism is related to down regulate the expression of GST-π. The drug resistance mechanism of N, N-bis(2-chloroethyl)docos-13-enamide is irrelevant to increase the accumulation effect of ADM in the cancer cells.