| Objective:To select the new acridone compound LZY13 as a reversal agent and investigate the reversal effect on multidrug resistance of tumor cells, and explore its possible reversal mechanism of action in vitro.Methods:The human leukemia K562 cells and adriamycin-resistant human leukemia K562/ADM cells were used to detect the inhibitory effect of cell proliferation about LZY13 and adriamycin by MTT assay, and to measure the drug resistance fold of K562/ADM cells on ADM. MTT assay was used to screen the innocuous dosage of LZY13 for K562 and K562/ADM cells, and to detect the reversal effect on multidrug resistance of tumor cells in vitro. The effects of LZY13 on intracellular accumulation of adriamycin and rhodamine-123 in K562 and K562/ADM cells were detected by the high content screening(HCS). In order to further observe the potential signaling pathway of LZY13, the effect of cell proliferation about LZY13 combined with inhibitors in K562/ADM cells was measured by MTT assay. The effects of LZY13 on ADM induced K562/ADM cell apoptosis, autophagy and reactive oxygen species(ROS) level were analyzed by flow cytometry. The effects of LZY13 on the expression of P-gp and tumor suppressor gene p53 in drug-resistant human leukemia K562/ADM cells were detected by Western Blot method.Results:MTT assay results showed that the IC50 values of adriamycin in K562 and K562/ADM cells were(1.06 ± 0.12) mg/L and(74.15 ± 6.52) mg/L respectively, and the drug resistance fold of K562/ADM cell for ADM was 70 times.The results of MTT assay indicated that when the concentration of LZY13 was 1 ?mol/L and the following dosage, the inhibitory ratio of two cells was less than 10%, which had no significant effect on cell proliferation. Therefore, the concentration of 1 ?mol/L was regarded as innocuous dosage of LZY13.MTT assay showed that LZY13(0.1 to 1 μmol / L) could be related to the concentration to reduce the IC50 value of adriamycin in drug-resistant K562/ADM cell, successfully reversed the adriamycin’s tolerance of drug-resistant human leukemia K562/ADM cell in vitro, and the reversal fold increased in a dose-dependent manner. Under the same concentration(1 ?mol/L), the reversal effect of LZY13 was stronger 59.88 times than the positive control drug verapamil, its reversal effect was obviously better than the latter.The intracellular accumulation of adriamycin and rhodamine-123 demonstrated that LZY13 could increase the accumulation of ADM and Rh123 in the multidrug resistant K562/ADM cells in a concentration dependent manner.From the results of flow cytometry and MTT assay, we could see that the number of apoptotic cells of multidrug resistance human leukemia K562/ADM cell was increased significantly under LZY13 combined with adriamycin. Moreover, the apoptosis ratio and cell inhibition rate of LZY13 and ADM combination group were both decreased significantly after cell treatment with caspase family inhibitor Z-VAD-FMK.MDC staining and MTT assay results showed that LZY13 could promote ADM induced autophagy in K562/ADM cells. After LZY13 combined with adriamycin, MDC staining fluorescence intensity was enhanced and the autophagy ratio of K562/ADM cell was increased significantly, and accordingly its inhibition rate was also increased obviously; after adding 3-MA, the autophagy ratio of LZY13 and ADM combination group and cell inhibition rate were significantly reduced.Intracellular ROS level test results demonstrated that DCF staining positive cells obviously increased namely intracellular ROS content increased significantly after LZY13 combined with adriamycin, while its cell growth inhibition rate also increased obviously; after adding antioxidants catalase treated cells, the ROS content of K562/ADM cell and its cell inhibition rate were both significantly decreased.From Western Blot analysis, we found that P-gp protein was showed a high expression level in drug-resistant K562/ADM cell, and the expression of P-gp protein was significantly decreased after LZY13 combined with adriamycin treatment. Furthermore, the expression of anti oncogene p53 was increased after LZY13 combined with adriamycin; and then adding Pifithrin-α treated cells, the expression of p53 was obviously down-regulated.Conclusions:Acridone compound LZY13 could effectively reverse the tolerance of drug-resistant human leukemia K562/ADM cell in vitro, and was able to restore the sensitivity of chemotherapeutic drugs efficiently. The mechanism of action of its reversal effect could be as follows: LZY13 could inhibit the expression of P-gp protein and the drug efflux function, increase intracellular concentration of chemotherapy drugs, and promote apoptosis inducing effect of chemotherapy drugs against multidrug resistant cell, prompting chemotherapeutic drugs induced drug-resistant cells autophagy, causing elevated levels of ROS in the resistant cells and up-regulated the protein expression of tumor suppressor gene p53. |
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