| Chemotherapy is one of the major therapies for leukemia. However, its therapeutic effects are often affected by the presence of multidrug resistance (MDR) in the tumor cells. Reversal of MDR, consequently, remains to be much researched as a hotspot in this field. Recent researches have demonstrated that many factors may contribute to MDR, such as the increased drugs efflux, which can be induced by P-glycoprotein (Pgp) and multidrug-associated protein(MRP) etc, the reduced topoisomerase- II activity, the enhanced metabolic detoxification, the inhibited apoptosis, the enhanced DNA repair, etc.Curcumin (Cur) is a kind of phenolics abstracted from the rhizome of the curcuma longa L or turmeric plant, and it is now known to possess an antioxidant and anti-inflammatory property, and an anti-carcinogenic and hypoglycemic effect. According to Hofsli et al. in 1989, erythromycin (EM) also has a sound effect on the reversal of drug resistance in fibrosarcoma. Verapamil (Ver) has long been reported to be able to reverse MDR in previous clinical researches.The present study was designed to explore the reversal mechanism of MDRin K562/A02 cell line, a human erythroleukemia cell line, which has a typical phenotype of MDR with a biological property of high mdrl mRNA level and high Pgp expression, treated with Cur, EM or Cur combined with EM respectively with Ver as a control. MTT assay, flow cytometry, immuno-histochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) was employed in this study.[Objective]To investigate respectively the effects of Cur or Cur combined with EM on MDR reversal in vitro and their mechanism.[Methods] First part:1. MTT assay was employed to determine the sensitivity of Cur-treated K562/A02 cells to anticancer drugs;2. Flow cytometry was used to measure intracellular mean fluorescence intensity (MFI) of DNR;3. Pgp expression in K562, K562/A02 cell line and K562/A02 cell line treated with Cur was determined by immunohistochemistry. RT-PCR technique was used to examine the mdrl mRNA level.Second part:1. Experiments of MDR reversal were conducted based on the chosen non-cellular-toxic doses of Cur, EM, Cur plus EM, and Ver;2. The MDR reversing effects of Cur combined with EM were explored with Ver as a control, using the same method mentioned in the first part.[ Results ] 1. Reversing MDR of K562/A02 cells with Cur(1) IC50s of chemotherapy agents, adriamycin (ADM), daunorubicin(DNR), vincristine (VCR) and Vepeside (VP-16) in K562/A02 cell line were remarkably higher than those in K562 cell line, and the resistant folds (RsF) to the four chemotherapy agents were 41.0, 25.2, 723.4, and 14.3 respectively.(2)Cur was observed to exert a concentration-dependent (1.25-20mg/L) inhibition on the proliferation of both K562 cell line and K562/A02 cell line. At the concentration below 2.5 mg/L, Cur had little affect on proliferation of the two cell lines and the tumor cells' survival rate exceeded 90%.(3)The sensitivity of the K562/A02 cell lines to ADM, DNR, VCR, VP-16 was increased when treated with Cur (2.5mg/L). The reversal folds (RsF) were 4.9, 4.8, 7.9, and 3.3 respectively and the relative reversal rates (RRR) were 81.7%, 82.4%, 87.4%, and 74.6% respectively.(4)The DNR MFI in K562/A02 cells was significantly increased when treated with Cur (2.5mg/L) (P<0.01). Cur (2.5mg/L) did not affect the efflux of DNR of the wild-type drug-sensitive K562 cells (P>0.05).(5)Quantitative analysis of Pgp expression based on gray scale numerus (GSN) of Pgp.It was found that the more Pgp expressed,the deeper cellular membrane was dyed,and the lower GSN was. Immunohistochemistry showed that Pgp expression in K562/A02 cells was significantly higher than that in K562 cells (P<0.01). Pgp expression in K562/A02 cells was significantly depressed when treated with Cur (2.5mg/L) for 72h (P<0.01), but was still higher than that in K562 cells (P<0.01).(16)As shown by RT-PCR, the mdrl mRNA level in K562/A02 cell line was decreased by Cur (1.0-2.5mg/L) in a concentration-dependant way. The mdrl mRNA level was significantly depressed when treated with Cur (2.5mg/L) for 72h (P<0.01).2. Reversing MDR of K562/A02 cells with Cur combined with EM(1)When treated with EM (≤120 mg/L), Ver(≤5.0 mg/L ) or Cur (2.5 mg/L) combined with EM (120mg/L), the cell survival rate in both K562 and K562/A02 cell lines all exceeded 90%. Cur (2.5 mg/L), EM (120 mg/L), Ver (5.0 mg/L ) and Cur (2.5 mg/L) combined with EM (120mg/L) were determined to be employed in the following reversal experiment of MDR;(2)IC50 of ADM in K562/A02 cells was decreased, treated with Cur (2.5 mg/L), EM (120 mg/L), Ver (5.0 mg/L), or Cur (2.5 mg/L) combined with EM (120mg/L), from 57.186±2.638 to 11.631 ±0.763, 15.436±0.740, 10.983 ± 0.658 and 5.061±0.599 respectively. And the RsF was 4.9, 3.7, 5.2, and 11.3 respectively. The RvF, when treated with Cur (2.5 mg/L) combined with EM (120mg/L) was higher than that when treated with Cur (2.5 mg/L), EM (120 mg/L) or Ver (5.0mg/L) alone.(3)The DNR MFI in K562/A02 cells treated with DNR for 180min was a little higher than that for 90min with no significant difference (P>0.05). The DNR MFI in K562 cells treated with DNR for 180min was lower than that for 90min(P<0.01).The DNR MFI in K562/A02 cells was increased significantly when treated with Cur (2.5 mg/L), EM (120 mg/L), Ver (5.0 mg/L), or Cur (2.5 mg/L) combined with EM (120mg/L). The DNR MFI in K562/A02 cells when treated with Cur (2.5 mg/L), EM (120 mg/L), or Cur (2.5 mg/L) combined with EM (120mg/L) for 180 min was higher than that for 90 min. The DNR MFI in K562/A02 cells treated with Cur (2.5 mg/L) combined with EM (120mg/L) was higher than that in K562/A02 cells treated by Cur (2.5 mg/L), EM (120 mg/L), or Ver (5.0 mg/L) alone (P<0.01). There was no significant difference between DNR MFI of K562/A02 cells treated with Cur (2.5 mg/L) and that of those treated with EM (120 mg/L), though the former was a little higher than the latter(P>0.05).(4)Pgp expression was reduced in K562/A02 cells treated with each group of drugs than that in K562/A02 cells untreated (P<0.01), but was still higher than that in K562 cells (P<0.01). Pgp expression in K562/A02 cells treated with each group of drugs for 5 days were lower than their counterparts treated for 3 days (P<0.01). Pgp expression in K562/A02 cells treated with Cur (2.5 mg/L) and EM (120 mg/L) alone were higher than that treated with Ver (5.0 mg/L) (P<0.05). When the former two drugs were used together, Pgp expression in K562/A02 cells were lower than that in the latter (P<0.01). There was no significant difference in Pgp expression between K562/A02 cells treated with Cur (2.5 mg/L) and those treated with EM (120 mg/L) (P>0.05).(5) It was shown by RT-PCR that mdrl mRNA level in K562/A02 cells was reduced when the cells were treated with each group of drugs (P<0.01). Those treated with each group of drugs for 5 days had lower mdrl mRNA level than their counterparts treated for 3 days, but had a higher mdrl mRNA level than K562 cells (P<0.01). The mdrl mRNA level in K562/A02 cells treated with Cur (2.5 mg/L) and EM (120 mg/L) respectively alone were higher than in K562/A02 cells treated with Ver (5.0mg/L) (P<0.05). The mdrl mRNA level in K562/A02 cells treated with Cur (2.5 mg/L) combining with EM (120 mg/L) was lower than that in K562/A02 cells treated with Ver (5.0mg/L) (P<0.01). The mdrl mRNA level in K562/A02 cells treated with Cur (2.5mg/L) for 3 days was a little lower than that in K562/A02 cells treated with EM (120 mg/L) for 3 days with no significant difference between them (P>0.05), the mdrl mRNA level in K562/A02 cells treated with Cur (2.5mg/L) for 5 days was lower than that in K562/A02 cells treated with EM120 mg/L for 5 days (P<0.01). [Conclusions]... |
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