| ObjectiveThe chemotherapeutic treatments represent one of the principal clinical options, and may contribute to inhibiting disease progression and improving overall survival, quality of life for leukemia patients. Unfortunately, the resistance by tumor cells to chemotherapeutic drugs results in the failure of chemotherapy or disease relapse. Multidrug resistance (MDR) means that tumor cells after exposition to one specific drug may produce the cross-resistance to other structurally and functionally unrelated anti-cancer drugs, which is one of the major causes of the failure of chemotherapy or disease relapse. It is still a major challenge to overcome MDR in the clinical cure of leukemia patients. Generally using single reversal agent obtains unsatisfactory effect and more toxicity with high-dose application so that it is limited in the clinical application. It has been identified that both Cyclosporine-A (CSA) and Arsenic trioxide (AS2O3) have reversal effect on tumor cells, but the research about combination of CSA and AS2O3 to reverse MDR of tumor cells has not been reported at present. The study was planned to select the non-cytotoxic dosage of CSA and AS2O3 in K562/ADM cells by WST-1 assay, and observe the reversal effect of combination of CSA and AS2O3 on K562/ADM cells acquired the MDR phenotype, a adriamycin-resistant cell line of human leukemia, then detect the expression of MDR1mRNA, hMLH1mRNA by reverse transcriptase-Polymerase Chain Reaction (RT-PCR), and P - gp, hMLHl protein by immunohistochemical method, finally explore the mechanism of reversal effect of CSA and As2O3.The research would aim to provide the theoretical basis for reversing MDR in clinic, and new therapeutic strategies against relapsed/refractory tumor.Method1. Cell toxicity and reversal times of CSA and/or AS2O3 on K562 and K562/ADM cells were determined by WST-1 assay.2. The expression levels of MDR1mRNA and hMLH1mRNA were determined by RT-PCR, including these groups:K562 cell, K562/ADM cell, K562/ADM cell with the non-cytotoxic dosage of the combination of CSA and AS2O3 (K562/ADM+ CSA non-cytotoxic dosage +AS2O3 non-cytotoxic dosage).3. The expression levels of P-gp and hMLH1 protein were determined by immunohistochemical method, including these groups:K562 cell, K562/ADM cell, K562/ADM cell with the non-cytotoxic dosage of the combination of CSA and AS2O3 (K562/ADM+CSAnon-cytotoxic dosage +AS2O3 non-cytotoxic dosage,).4. Data analyzed:They were using software SPSS 11.5. The quantative data was presented as mean±standard difference. The mean of two groups was analyzed with the t test. The mean of three groups or more was analyzed with the one-way analysis of variance after test of homogeneity of variance.Taking a=0.05 as the significant standard of test.Results1. WST-1 results showed:IC50 of ADM was 32.47±0.012μg/ml in K562/ADM cell line and 1.06±0.019(μg/ml in K562 cell line, there was a significant difference (P<0.05). Drug resistance activity of K562/ADM cell line to ADM was 30.63 fold greater than that of K562 cell line. IC50 of CSA was 0.372+0.016μg/ml in K562/ADM cell line and 0.357±0.018μg/ml in K562 cell line, there was no significant difference (P>0.05). Drug resistance activity of K562/ADM cell line to ADM was 1.04 fold greater than that of K562 cell line. IC50 of As2O3 was 0.923±0.232μg/ml in K562/ADM cell line and 0.733±0.142μg/ml in K562 cell line, there was no significant difference (P>0.05). Drug resistance activity of K562/ADM cell line to ADM was 1.26 fold greater than that of K562 cell line. Non-cytotoxic dosage of CSA (0.0005μg/ml) and As2O3 (0.002μg/ml) alone decreased IC50 of ADM in K562/ADM cell line to 20.52±2.34μg/ml,21.62±1.81μg/ml, and fold reversal was 1.58,1.50 respectively.CSA (0.0005μg/ml)+As2O3 (0.002μg/ml) group decreased IC50 of ADM to 9.63±1.57μg/mland fold reversal was 3.37.2. RT - PCR results showed:Compared with the K562/ADM cell, the expression levels of MDR1mRNA in the K562 cell and K562/ADM+ CSAnon-cytotoxic dosage+As2O3non-cytotoxic dosagewere both reduced more (P<0.05). There was no significant difference (P>0.05) between the K562 cell and the K562/ADM+ CSAnon-cytotoxic dosage+As2O3non-cytotoxic dosage.Compared with the K562/ADM cell, the expression levels of hMLH1mRNA in the K562 cell and K562/ADM+ CSAnon-cytotoxic dosage+As2O3non-cytotoxic dosagewere both increased more (P<0.05). There was no significant difference (P>0.05) between the K562 cell and the K562/ADM+ CSAnon-cytotoxic dosage+As2O3non-cytotoxic dosage.3. Immunohistochemical method results showed:Compared with the K562/ADM cell, the expression levels of P-gp in the K562 cell and K562/ADM+ CSAnon-cytotoxic dosage+As2O3non-cytotoxic dosagewere both reduced more (P<0.05). There was no significant difference (P>0.05) between the K562 cell and the K562/ADM+ CSAnon-cytotoxic dosage+As2O3non-cytotoxic dosage.Compared with the K562/ADM cell, the expression levels of hMLH1 protein in the K562 cell and K562/ADM+ CSAnon-cytotoxic dosage+As2O3non-cytotoxic dosagewere both increased more (P<0.05). There was no significant difference (P>0.05) between the K562 cell and the K562/ADM+ CSAnon-cytotoxic dosage+As2O3non-cytotoxic dosage.Conclusion1. CSA and As2O3 were effective antitumor drugs with obvious inhibiting effect on K562/ADM cell proliferation. K562/ADM had no drug resistance to CSA and As2O3.2. There was no effect of overlaping toxicity at the non-cytotoxic dosage of the combination of the two drugs. The effect of combination of two agents was superior to that of single agent.3. The non-cytotoxic dosage of the combination of CSA and As2O3 may induce the upregulation of hMLHl gene/protein, and down-regulation of MDR-1 gene/protein in K562/ADM cell.These gene and protein may be involved in the formation of MDR in K562/ADM cell. |
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