Reversion Of Multidrug Resistance In Human Leukemic Cell Line By Carnosic Acid And Its Mechanism

Objective;One common problem to successful chemotherapy is the development of multidrug resistance(MDR)by tumor cells to multiple chemotherapeutic agents.Generation of MDR is complex,and P-glycoprotein(P-gp) acts as an energized drug pump,reducing the intracellular concentration of drugs, even structurally unrelated ones.Modulators of P-gp function can restore the sensitivity of MDR cells to anticancer drugs.The emergence of acquired resistance results from host factors and genetic or epigenetic changes in the cancer cells. Otherwise,signal transduction pathway of cell apoptosis control and drug metabolism also can cause MDR,and these were paid more attention nowadays.The emergence of acquired resistance results from host factors and genetic or epigenetic changes in the cancer cells.Camosic acid(CA),antioxidant polyphenols present in Rosmarinus officinalis L,has many pharmacological effects,such as anti-oxidant,anti-tumor, anti-thrombotic and anti-inflammatory effect.CA also can reverse MDR of Staphylicoccus aureus,so we hypothesized if CA has the reversal effect on MDR of cancer.The purpose of this study was to study the effects of Carnosic acid(CA),on reversing the multidrug resistance(MDR)of human erythrocyte leukemia cell line K562/AO2,and to wxplore the mechanism of reversal of MDR of K562/AO2 cell line by CA.Method;Reversing effect of CA was evaluated by determining cellular proliferation with MTT assays.The intracellular adriamycin fluorescence intensity and P-gp fluorescence intensity were measured by flow cytometry(FCM).Subcellular distribution of adriamycin was detected via Laser Scanning Confocal Microscopy (LSCM).The mRNA expression of mdr1,bcl-2 and bax were detected via semiquantitative reverse transcription polymerase chain reaction(RT-PCR).P-gp protein was detected by Western blot using mAb anti-MDR1.The study was aimed to detect the gene expression profile changes between CA treated K562/AO2 cells and untreated K562/AO2 cells,and to investigate the underlying mechanisms of MDR by using RT² Profiler^TMPCR Array.Result;MTT assay showed that CA caused IC₅₀of ADM decreased from 16.31μg/mL to 1.35μg/mL,while VRP decreased IC₅₀to 0.86μg/mL,reversal multiple were 12.08 and 18.97,it had significant compared with untreated group (P＜0.01).The intracellular ADM concentration of K562/AO2 was 17.05 via FCM, after treated with CA or VRP,it increased to 60.53 and 46.16,it had significant compared with untreated group(P＜0.01).In living cells,the concentration of ADM was examined by LSCM.According to ADM concentration(x)and its corresponding fluorescence intensity(y),we got the linear equation;Y=8.47×10^-3X-1.34,then we got the intracellular ADM concentration by this formula.It increased from 4.86μg/mL to 15.56μg/mL and 10.99μg/mL respectively,after treated with CA or VRP.In K562 cells,ADM fluorescence mainly located in the nucleus and was diffusely present in the cytoplasm,while it was distributed in perinuclear and perimembrane chiefly in K562/AO2 cells.After treated with reversal agents,the diffused distribution of intracellular ADM was recovered in nuclear and cytoplasm,and aggregation of intracellular ADM was increased greatly.Mean fluorescence intensity of P-gp in K562/AO2 cells was 44.40.After added CA or VRP,it decreased to 23.8 and 22.84, there were significant differences between treated groups and untreated group (P＜0.05).RT-PCR assay showed that CA inhibited the expressions of mdr1 and bcl-2 mRNA in K562/AO2 cells,while bax mRNA was increased.We also observed that CA inhibited the expression of P-gp by western blot.We identified some genes about drug metabolism differentially expressed using genechip technology.Our expression analysis also identified 42 multidrug resistance candidate genes that were associated with resistance to the tested reversal agent,35 of them were up-regulated and 7 of them were down-regulated.Abnormal glycometabolism has closed relationship with many diseases,glycolysis is the mainly metabolism pathway of cancer cells.So the rate-limiting emzyme of glycolysis play an important role in diagnosis and treatment of leukemia,such as Hexokinaseâ…¡(HKâ…¡)and Pyruvate kinase(PK).CA could inhibit expression of the key emzyme of glycolysis pathway,stop the glycolysis of cancer cells and suppress energy metabolism.The cancer cells were induced to apoptosis because of lacking energy. CA could act on the primary controlling molecules of signal transduction pathway related with the MDR of the cancer.There are 25 genes that related with reversing MDR were up-regulated in signal transduction pathway microarray.The genes play important roles in many physiological courses of cell cycle control,cell differentiation and apoptosis,such as p300/CBP,IL-4R,IRF-1,TP5313,NFKIB and FOS.CA could cause the changes in the signal transduction pathway;p53 pathway,TGF-βsignal pathway and NF-κB signal pathway,which had closed relationship with cell proliferation,apoptosis and drug resistance.These genes were differentially expressed before and after treated with CA,which strongly suggesting that these genes might contribute to MDR in K562/AO2 cells.Conclusion;CA can reverse the MDR of K562/AO2 cells in vitro,which may be associated with down-regulating expression of mdr1 gene and inhibiting the expression of P-gp,which showed that CA could inhibit the function and suppress the expression of of P-gp.Moreover,CA can decrease expression of bcl-2,and increase expression of bax,which indicated that the reversal mechanism of CA also related with apoptosis induction.CA also caused changes of genes of signal transduction and drug metabolism gene chips.In signal transduction assay,the expressions of 25 genes up-regulated and it related with many signal pathway;p53 pathway,TGF-βsignal pathway,STAT pathway,NF-κB signal pathway and MAPK signal pathway.The results indicated that CA can serve as a novel,non-toxic modulator of MDR,and can reverse the MDR of K562/AO2 cells in vitro.