| Background: Leukemia is one of the most common hematological malignances and it represents a large proportion of malignancies during childhood and adolescence. Furthermore, there is an increase in the incidence rate of leukemia in the aged and the death rate of leukemia is high. All above makes leukemia a big burden for families and societies. Combination chemotherapy is the most important strategy for the treatment of leukemia. However, the development of resistance to multiple chemotherapeutic drugs is a major obstacle in the treatment of leukemia. Although clinical drug resistance is a multi-factorial problem, the so-called multidrug resistance (MDR) is one of the most frequent causes for the failure of chemotherapy. MDR is characterized by the tumoral cells developing cross-resistance to different chemotherapeutic agents which are structurally and functionally dissimilar. There may be numerous and diverse mechanisms involved in the development of MDR, such as alterations in expressions and functions of intracellular proteins, enzymes and genes as well as that in the extracellularenviroment. As we know, the transport-based classical MDR mechanisms involve the overexpression of P-glycoprotein, multidrug resistance-associated protein, breast cancer resistance protein, anthracycline resistance-associated protein, transporter associated with antigen processing and lung resistance-related protein. The term non-classical MDR is used to describe non-transport based mechanisms that affect multiple drug classes. This type of resistance can be caused by altered activity of specific enzyme systems (such as glutathione S-transferase, topoisomerase, thymidylate synthase and dihydrofolate reductase), which can decrease the cytotoxicity of drugs in a manner independent of intracellular drug concentrations, which remain unaltered. Further research has revealed that anticancer drugs typically induce programmed cell death or apoptosis. The decision, whether a cell continues through cell cycle or undergoes apoptosis, is dependent upon a complex interplay of a team of genes and proteins that exert a regulatory role in cellular events. Resistance may, therefore, develop with loss of genes required for cell death or overexpression of genes that block cell death. What to be mentioned is that the resistance mechanisms are numerous and diverse and that the development of MDR is not a result of single cause but a result of multiple ones.MDR is a major obstacle in the treatment of leukemia. Much attention, therefore, has been devoted to the development of agents which can reverse MDR. Until now, a broad range of compounds have been identified that are able to reverse MDR by blocking P-glycoprotein activity in vitro. These compounds include the following types of agents: calcium channel blockers, calmodulin antagonists, cyclosporins, quinolines, anti-estrogens and so on. The two substances that have received the most attention in the clinic are verapamil and cyclosporin A. However, they have not been widely usedin the treatment of cancer patients because the dosage required to reverse MDR are either toxic or not clinically achievable. Therefore, some researchers began to screen for new MDR modulators, which are more active and less toxic, in the natural dietary compounds that are nontoxic in animals.Tea is the most widely consumed beverage in the world, second only to water. Evidence from epidemiologic studies indicates that frequent consumption of green tea is inversely associated with the risk of several types of human cancer, and studies with animal and in vitro cell culture models have revealed (-) epigallocatechin-3-gallate (EGCG) as a major chemopreventive ingredient of green tea. The major bioactivity of EGCG includes reducing plasma lipid, anti-oxidation, anti-thrombosis, anti-flammatory, anti-mutation, anti-cancer, and so on. Extensive research in the last few years has revealed that EGCG could suppress the processes of transformation, proliferation and metastasis of tumor. Moreover, EGCG can exhibit anti-leukemic activity by suppressing the proliferation as well as by inducing the differentiation and apoptosis of leukemic cells. In several recent studies, EGCG reversed the multidrug resistance of human solid tumors, such as hepatocellular carcinoma and colon carcinoma in vitro. However, nothing is known about whether EGCG could reverse the multidrug resistance of human leukemic cells or not.Objective: To investigate the reversal effect of EGCG, alone or in combination with CsA or VRP, on a multidrug resistant human leukemic cell line K562/A02 and its possible mechanisms.Materials and Methods: 1. Cell lines and cell culture: Parental human K562 leukemic cell line was from stocks held by the Institute of Hematology, Qilu Hospital, Shandong University. Drug-resistant K562/A02 cell line was purchased from the Institute of Hematology ofCAMS. Both cell lines were maintained in RPMI 1640 medium supplemented with 10% calf serum, 100U/ml penicillin and 100mg/L streptomycin at 37℃ in a humidified atmosphere of 95% air and 5% carbon dioxide. 2. MTT cytotoxicity assay: 2.1 Detected the cytotoxicity of EGCG, CsA and VRP to K562/A02 cells, respectively. The dosage required for 10% inhibition of cell growth (IC10) was calculated from linear plots of percentage cell inhibition versus dosage. Concentrations less than IC10 were used to test their reversal effects. Meanwhile, the growth inhibition of K562/A02 cells by EGCG combined with CsA or VRP as well as CsA combined with VRP were measured respectively; 2.2 Detected the sensitivity of K562 and K562/A02 cells to doxorubicin respectively and calculated the resistance factor; 2.3 Detected the reversal of MDR in K562/A02 cells by EGCG, CsA and VRP respectively, as well as by any two of the three. 3. The intracellular concentration of doxorubicin was detected by flow cytometry. 4. The expression of mdr1 mRNA was detected by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). 5. The expression of P-glycoprotein was detected by western blot. 6. The expressions of Bcl-2 and Bax, both are apoptosis-related proteins, were analyzed by immunocytochemistry. 7. Statistical analysis: All of the experiments were performed at least three times. Data were presented as means± SD. The One-Way ANOVA was used to compare the differences between groups. The Two-Way ANOVA was used to analyze the factor interactions. The SPSS 10.0 software was used to perform the above analysis. P values < 0.05 were considered significant.Results: 1. The IC10 of EGCG, CsA and VRP on K562/A02 cells were 103.2μmol/L, 3.7μmol/L and 4.2μmol/L, respectively. So, 40μmol/L, 60μmol/L and 80μmol/L EGCG, 1μmol/L CsA and 1μmol/LVRP were used to perform the following reversal experiments. Meanwhile, percentages of growth inhibition on K562/A02 cells were no more than 10% by combinations of any two of 80μmol/L EGCG, CsA 1μmol/L and 1μmol/L VRP. Thereby we detected the reversal effect of MDR by above combinations in the following experiments. 2. The IC50 for doxorubicin on K562/A02 and K562 cells were 113.34mg/L and 1.12mg/L, respectively. Thus the resistance factor was 101.20-fold. 3. Sensitivity of K562/A02 cells to doxorubicin increased somewhat in the presence of 40μmol/L, 60μmol/L and 80μmol/L EGCG, the IC50 decreased to 9.66mg/L, 7.67mg/L and 4.68mg/L, as well as the modulating factors were 11.73-, 14.78- and 24.22-fold, respectively. In the presence of 1 μmol/L CsA and 1μmol/L VRP, the IC50 decreased to 7.47mg/L and 8.50mg/L, and the modulating factors were 15.17- and 13.33-fold, respectively. Meanwhile, the combinations could decrease the IC50 significantly as compared with any of them alone, and the modulating factors increased accordingly. 4. The mean fluorescent intensities of doxorubicin in K562/A02 and K562 cells without treatment were (6.33 ± 0.30) and (25.01 ±0.13) respectively. The intracellular concentrations of doxorubicin in K562/A02 cells increased when cells were incubated with 40μmol/L, 60μmol/L and 80μmol/L EGCG, and their mean fluorescent intensities were (9.40 ± 0.30), (12.88 ±0.10) and (16.62 ± 0.15), respectively. In addition, there were statistical differences between any two of the three groups. In the presence of 1μmol/L CsA and 1μmol/L VRP, the intracellular concentrations of doxorubicin in K562/A02 cells increased and their mean fluorescent intensities were (16.59 ± 0.15) and (10.56 ± 0.06) respectively. Moreover, the intracellular concentrations of doxorubicin in K562/A02 cells were much higher when incubated with combinations of any two of EGCG, CsA and VRP, than incubated with any one ofthem alone. Meanwhile, there were synergistic actions for combinations of any two of EGCG, CsA and VRP in the aspect of increasing the intracellular concentrations of doxorubicin. 5. An mdr1 specific PCR product was detected as an intense band in K562/A02 cells. This band was completely absent in K562 cells. EGCG decreased the expressions of mdrl mRNA in a dose-dependent manner, while CsA and VRP, alone or in combination, had no inhibitory effect on it. The combinations of EGCG with CsA or VRP decreased the expressions of mdrl mRNA in K562/A02 cells, but there were no differences when compared with EGCG alone. 6. The expressions of P-glycoprotein'were consistent with that of mdrl mRNA. 7. Both Bcl-2 and Bax were expressed in both K562 and K562/A02 cells. But in K562/A02 cells, the ratios of Bax/Bcl-2 were much less than that in K562 cells whether analyzed by positive rate or by integra (P<0.001). When 40μmol/L, 60μmol/L and 80μmol/L EGCG were present, the ratios of Bax/Bcl-2 in K562/A02 cells increased, and there were statistical differences when compared with untreated cells. However, there were no differences in the ratios of positive rate between any two of three groups, and significant differences were only observed between 80μmol/L EGCG and the other two groups when analyzed by integra. In the presence of 1μmol/L CsA and 1μmol/L VRP, alone or in combination, the ratios of Bax/Bcl-2 in K562/A02 cells increased whether analyzed by positive rate or by integra, and there were significant differences when compared with untreated cells. The differences were also observed when compared the combinations with either CsA or VRP alone. In the presence of EGCG combined with CsA or VRP, the ratios of Bax/Bcl-2 in K562/A02 cells increased, and there were significant differences when compared with untreated cells. Meanwhile, there were synergistic actions for EGCG combined with CsA or VRP in the aspect of increasing the ratios ofBax/Bcl-2.Conclusions: 1. EGCG was capable of reversing the multidrug resistance of K562/A02 cells in vitro. The reversal effect of combinations of any two of EGCG, CsA and VRP increased, and the EGCG combining with CsA group exhibited the strongest reversal effect. 2. To increase the accumulation of doxorubicin in K562/A02 cells was one of the possible mechanisms for EGCG, CsA and VRP, alone or in combination of any two of them, to reverse its drug resistance. EGCG acted in a dose-dependent manner. Co-incubation of any two of EGCG, CsA and VRP resulted in a significant increase in the intracellular concentrations of doxorubicin and the group of EGCG combined with CsA exhibited the strongest effect. 3. EGCG decreased the expressions of mdrl mRNA and P-glycoprotein in a dose-dependent manner, and it was one of the possible mechanisms for it to reverse the drug resistance on K562/A02 cells. On the other hand, 1μmol/L CsA and 1μmol/L VRP, alone or in combination, had no inhibitory effects on it. 4. The decrease in the ratios of Bax/Bcl-2 is one of the mechanisms involved in the development of MDR in K562/A02 cells. EGCG, CsA and VRP, alone or in combination of any two of them, exerted their reversal effects through causing an increase in the ratios. Combinations of any two of EGCG, CsA and VRP showed synergistic actions in increasing the ratios.Significance: To our knowledge, this is the first report showing that EGCG, a plant flavonoid derived from green tea, can reverse the multidrug resistance of human leukemic K562/A02 cells in vitro. Meanwhile, it is confirmed in our study that the reversal effect of EGCG increased when combined with CsA or VRP, which presents experimental evidences for combination reversal strategies. Furthermore, our study reveals that EGCG, alone or in combinationwith CsA or VRP, exerted its reversal effects through at least three pathways, including increasing the intracellular accumulation of chemotherapeutic drugs, decreasing expressions ofmdrl/P-glycoprotein and increasing the ratios of Bax/Bcl-2. Therefore, our study provides the theoretical base for the combination strategy in the practice of clinical reversal of MDR.
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