Effect Of Alantolactone On Chronic Myelogenous Leukemia Drug-resistant K562/ADR Cells

Objective: Inula helenium （Compositae） is a perennial medical herb widelydistributed in Europe, North American, Xinjiang of our country and other regions．Preparations of its roots are used in medicine as invigorating stomach, expectorant, diuresis, helminthicide. Alantolactone, isoalantolactone, dihydroalantolactoneand dihydroisoalantolactone extracted from the root of Inula helenium possess different physiological activities such as anti-tumor, helminthicide, antimicrobial, anti-inflammatory, analgesis and hypoglycemic. In this sdudy, The human chronic myelogenous leukemia （CML） cell line K562and drug-resistant cell line K562/ADR were used to study the effect of alantolactone on inhibiting proliferation K562/ADR cells in vitro and further to explore its molecular machinism.Methods:1. CML cell line K562and drug-resistant cell line K562/ADR were choosed to study the effect of alantolactone on inhibiting proliferation of K562/ADR cells.2. K562/ADR cells were treated with different concentrations of alantolactone for different time points, and then cell viability was analyzed with trypan blue exclusion test and MTT assay.3. The percentage of apoptosis cell wasanalyzed by flow cytometry using Annexin V-FITC/PI staining.4. The effect ofalantolactone on the intracellular accumulation of Dox in K562/ADR cells wasmeasured by flow cytometry.5. The molecular character of apoptosis was analyzed by detecting Cleaved-caspase-9, Cleaved-caspase-3, Cleaved-PARP, Bax, Bcl-2,Cytochrome C protein expression using Western blot after treatment with alantolactone. Meanwhile, we also detected the expreesions of Bcr/Abl and P-gp protein.Results:1. We investigated the differences in P-gp and Bcr/Abl protein levels inK562cells and K562/ADR cells, and found remarkably increased expression of P-gp inK562/ADR cells, but almost no expression in K562cells. Furthermore, Bcr/Abl expression in K562/ADR cells was approximately3-fold more than in K562cells （p <0.05）. The results of trypan blue exclusion test and MTT showed that alantolactoneeffectively inhibited the proliferation of K562/ADR cells in dose-and time-dependentway, and the IC₅₀for24h was about4.7μM.2. Flow cytometric analysis displayed thatwith the increased concentration of alantolactone there were significant increase inapoptotic rate （from1.35%to16.91%,29.61%,46.26%, respectively）, and apoptosisrelated proteins were greatly changed （p <0.05）, alantolactone significantly decreasedBcl-2protein expression and increased Bax expression, and induced the release ofcytochrome C from the mitochondria to cytosol. This was followed by caspase-9andcaspase-3activation, and cleavage of PARP in a dose-dependent manner.3. Flowcytometric analysis showed that alantolactone significant inhibited the efflux ofanticancer drugs from tumor cells into the surrounding tissue. Alantolactone may partlyreduce the resistance.Conclusion:1. Alantolactone significantly inhibited K562and K562/ADR cell growth.2. Alantolactone induces apoptosis mainly via the mitochondrial （or intrinsic）apoptotic pathway.3. The possible molecular mechanisms, of which alantolactone significantlyinhibited K562/ADR cell growth and partially reveased drug resistance, are thedownregulation of Bcr/Abl and P-glycoprotein （P-gp）. All these results demonstrate thatPolyphyllin D may be a potent therapeutic agent against CML...