| AimChemotherapy is an important mean to treat malignant cancers, but it s a problem that tumor cells have resistance especially multidrug resistance(MDR) to unticancerdrugs. In recent years, a number of studies have demonstrated that the B-cell lymphoma/leukemia-2(Bcl-2) and p210bcr/abl are involved in the development of tumor cells drug resistance. Cepharanthine Hydrochlorid (CH), 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 Bcl-2 or P210bcr/abl has not been reported. This study aims at the methods of cytology and molecular biology to determine the reversing effects of CH and further investigate MDR in its molecular mechanism. Methods1. The multidrug-resistant human chronic leukaemia cell line K562/ADR resistant to adriamycin (ADR) was adopted in the experiments to compare with its parental sensitive cell line K562. 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 CH or verapamil(VER) in K562 and K562/ADR cell lines. Immunohistochemistry(IHC) technique was used to determine the expression of Bcl-2 in K562 and K562/ADR cell lines as well as its expression that treated with drugs. Theamount of total protein, extracted from K562 and K562/ADR cells, was determined by the Coomassie blue G-250 dye. Western blot was used to determine the expression of p2jQbcr/abi jn K562 and K562/ADR cell line as well as its expression that treated with drugs. The aims of this study were not only to elucidate the relationship between drug resistance of K562/ADR to ADR and Bcl-2 or P210bcr/abl, but also to investigate themechanism through which CH reverses drug resistance. Results1. Cytotoxity of ADR in K562 and K562/ADR cell linesThe IC50 of ADR in K562 and K562/ADR cell lines determined by MTT were 0.42 ± 0.02 u mol ? L"1 and 12.15 ±0.19 u mol ? L"1 respectively. There was significant difference between them(P<0.05) and resistance fold was 28.26.2. Cytotoxity of ADR combined with CH or VER in K562 and K562/ADR cell linesCytotoxity of CH or VER alone in K562 and K562/ADR cell lines was weak. The inhibitory rates (IR) of K562 and K562/ADR cells treated with 4 n mol ? L'1 CH were(3.36 ±0.92)% and (4.41 ±1.13)% respectively. The IR of K562 and K562/ADR cells treated with 4 u mol ? I/1 VER were(7.16±0.24)% and (7.11 ±0.74)%, respectively. The IC50 of ADR combined with 4 u mol ? L/1 CH or 4 u mol ? L"1 VER in K562 cells were 0.42 ±0.02 u mol ? L"' or 0.43 ±0.01 y. mol ? L"1,without significant difference compared with that of ADR alone(P>0.05). The IC50 in K562/ADR cells were 1.79 ±0.04 u mol ? L'1 or 6.36 ± 0.16 V- mol ? L"1, which showed significant difference(P<0.05) compared with the IC50 of ADR alone in K562/ADR cells. The reversing fold was 6.79 and 1.91 respectively. The reversal effect of CH was higher than that of VER, the difference between combined with CH and combined with VER was significant (PO.05).3. Effect of ADR combined with drugs on expression of Bcl-2 in K562 and K562/ADR cellsThe expression of Bcl-2 was measured with IHC and the results were analyzed with Shanghai shanfu computer-assisted image analyzing system(IAS) to achieve semi-quantitative data. The Bcl-2 value in control group of K562 cell line was (4.54 ± 0.91)%. The value of 0.2 u mol ? L/lADR alone was (3.93 ±0.82)%.And (3.64+0.38)% if combined with 4 u mol ? L"1 CH. Compared with control group, there was no significant difference in each group (P>0.05). The value was (28.74±5.15)% in control group ofK562/ADR cell line and (28.30 + 3.46)% in 2 n mol ? L/'ADR alone.And if combined with 4 u mol ? L"'CH the value was (11.57 + 2.33)%. The expression of Bcl-2 of control group of K562/ADR cell line was significantly higher than that in control group of K562 cell line(P<0.05). While there was no significant difference between group of ADR alone and control group(P>0.05). Compared with control group and ADR group, the expression of Bcl-2 of ADR combined CH group declined sharply (P<0.05), but it was higher than that in corresponding K562 cells(P<0.05). 4. Effect of drugs on the expression of P210bcr/abl in K562 and K562/ADR cell linesWestern blotting was used to determine the expression of p210bcr/abl and semi-quantitative analyzed by Gene Genius gel image analyzing system. No significant difference showed in the expression of P210bcr/abl in K562 cell line treated with 0.2 u mol ?!/' ADR, ADR combined 4 u mol ?L/'CH compared with control group(P>0.05). The OD ratio was 2.13+0.06, 2.12 + 0.08, 2.18 + 0.05 respectively. The expression of p210bcr/abi jn K562/ADR cell line treated with 2 u mol ? L"*ADR was also not significantly different from control group(P>0.05), while the group of ADR combined with 4 u mol 'L'1 CH could decrease the expression of P210bcr/abl . The OD ratio was 2.73 + 0.12, 2.75 + 0.10, 2.41+0.04 respectively. There was significant difference compared with group of ADR alone and control group(P<0.05). The differences between the expression of p21obcr/abi in K562 and K562/ADR cells were clearly (PO.05).Conclusion1. CH reverses drug resistance of K562/ADR cell line partly. The reversal effect of CH is stronger than that of VER.2. The higher expression of Bcl-2 and p210bcr/abl are one of mechanisms through which K562/ADR cell line resisted to drugs.3. CH reduce the expression of Bcl-2 and P210bcr/abl in K562/ADR cell line, which is related to the reversal effect of drug resistance. |
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