| Objective: Multidrug resistance (MDR) means cancer cells exposure to a single anticancer drug lead to cross-resistance to many other structurally and functionally unrelated anticancer drugs, which is the major cause of failure of chemotherapy. As a result, greatly reduced the chemotherapy effect of anticancer drugs. Reversal of MDR is a clinical important problem urgently to be resolved. The mechanism of MDR is multiplicity. One cancer cells always relates to several mechanisms, results in one drug reversing MDR less effectiveness. So combined two or more drugs aiming at different mechanism, better chemotherapy effect maybe can obtained. There are investigates show that Arsenic trioxide (As2O3) and Tetrandrine (TTD) can reverse MDR alone, but combined the two are not reported. Our department compound As2O3 and TTD to reverse MDR on K562/A02 cell line. Investigate the curative effects and mechanisms. To provide theoretically and experimental bases for clinical use.Methods: Cytotoxicity was determined by MTT assay. After incubated K562, K562/A02 cell line and K562/A02 cell line which were treated with As2O3,TTD,As2O3+TTD for 1 hour, then all incubated in ADM for 2 hours, cellular ADM concentration was examined by flow cytometry. The above-mentioned index were used to value the reversal effect of As203and TTD along with ADM on K562/A02 singlely or combined used. mdrl gene,GST-πand Topo II expressed in K562, K562/A02 cell line and K562/A02 cell line treated with As2O3 2uM/L,TTD 1.5625ug/ml,As2O3 2uM/L+TTD 1.5625ug/ml for 2 days was determined by RT-PCR and immunohistochemical technique respectively.Results: 1.①IC50 of ADM was 74.35ug/ml in K562/A02 cell line and 1.13ug/ml in K562 cell line. Drug resistance activity of K562/A02 cell line to ADM was 65.77 fold greater than that of K562 cell line.②As2O3 0.5,1,2 uM/L decreased IC50 of ADR in K562/A02 cell line, and fold reversal was 1.09,1.23,1.45 respectively, the inhibition activity was enhanced as the doses of the drugs increased. We choose 2uM/L as the best reversal concentration.③TTD 0.4,0.8,1.5625ug/ml decreased IC50 of ADM in K562/A02 cell line, and fold reversal was 1.36,1.72,2.87 respectively. Fold reversal was increased as the dose of ADM increased, and we choose 1.5625ug/ml as the best reversal concentration.④As2O3 2uM/L+TTD 1.5625ug/ml group fold reversal was 8.83. Fold reversal was higher than that of As2O3 or TTD alone.⑤After incubated two cell lines in ADM for 2 hours, cellular ADM concentration in K562 cell line (97.14±1.66) was higher than that in K562/A02 cell line (6.85±0.42) (P<0.001).⑥After incubated in ADM for 2 hours, cellular ADM concentration of K562/A02 cell line treated with As2O3 2uM/L (9.32±0.40) was higher than that in K562/A02 cell line without treated with drug (P<0.001).⑦Cellular ADM concentration of K562/A02 cell line treated with TTD 1.5625ug/ml (58.00±1.33) was higher than that in K562/A02 cell line without treated with drug (P<0.001).⑧ADM concentration of K562/A02 cell line treated with As2O3 2uM/L+TTD 1.5625ug/ml (64.34±1.33) was higher than that in K562/A02 cell line without treated with drug (P<0.001), and higher than that in K562/A02 cell line treated with As2O3 2uM/L or TTD 1.5625ug/ml alone (P<0.001). 2. Analysis of mdrl gene expression was based on value of mdrl/β-actin.①mdr1 gene expression was increased in K562/A02 cell line (3.5285±0.2929) than that in K562 cell line (0.7996±0.0655) (P<0.01);②After treated with As2O3,TTD,As2O3+TTD for 2 days, mdr1 gene expression was redused in K562/A02 cell line (excepted treated with As2O3) than that in K562/A02 cell line without drugs (P<0.01);③mdr1 gene expression in K562/A02 cell line treated with As2O3+TTD (1.0072±0.0677) for 2 days was lower than that in K562/A02 cell line treated with TTDalone (1.5489±0.0778) (P<0.01). 3. Immunohistochemical results showed:①The K562 cell line was light brownish red color, the expression of GST-πwas poor; K562/A02 cell line and K562/A02 cell line treated with TTD 1.5625ug/ml was colored with brownish red color, the expression of GST-πwas masculine; K562/A02 cell line treated with As2O3 2uM/L,As2O3 2uM/L+TTD 1.5625ug/ml was colored with lighter brownish red color, the expression of GST-πwas poorer; There were no difference between K562/A02 cell line treated with As2O3 2uM/L and As2O3 2uM/L+TTD 1.5625ug/ml.②We can see the K562 cell line was brownish red color, the expression of TopoⅡwas masculine; K562/A02 cell line and K562/A02 cell line treated with TTD 1.5625ug/ml was colored with light brownish red color, the expression of TopoⅡwas poor; K562/A02 cell line treated with As2O3 2uM/L,As2O3 2uM/L+TTD 1.5625ug/ml was colored with darker brownish red color, the expression of GST-πwas stronger; There were no difference between K562/A02 cell line treated with As203 2uM/L and As2O3 2uM/L+TTD 1.5625ug/ml.Conclution:①As2O3 and TTD were effective antitumor drugs with obvious inhibiting effect on tumor cells;②Non cytotoxic dosage of As2O3 and TTD could partly reverse the MDR of K562/A02. The effect of combination of two agents were superior to that of single uses;③Overexpression of mdrl and GST-π, lessexpression of TopoⅡwere related to mechanism of MDR;④The reversal mechanisms of As2O3 and TTD were different, As2O3 can reversed the MDR of K562/A02 cell line by downregulating mdr1 expression,reducing GST-πquantity and increasing Topo II quantity. But, the action time and dose of drug were too less to sure whether there were relation between As2O3 and mdrl gene. It need to be investigate deeply; As2O3 reversed the MDR of K562/A02 cell line by downregulating mdr1 expression. |
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