DADS Induce Autophagy Of The Leukaemia K562Cell Mediated By UVRAG

Objective: To observe the effect of UVRAG on autophagy of K562cell induced bydiallyl disulfide (DADS), and explore the mechanism of autophagy induced by DADSin K562cells. It will supply potential targets for the treatment of leukemia.Furthermore, it provides some theoretical and experimental basis for applying DADSinto the leukemia treatment in future.Methods: K562cells were cultured in K562cell culture systems. Theultrastructure of K562cells treated with DADS (10mg/L、20mg/L、40mg/L、80mg/L)for12hours was observed by transmission election microscopy (TEM).K562cellstreated with40mg/LDADS for indicated time (3hours、6hours、12hours、24hours)，the autophagic vacuoles(AV) were observed by fluorescence microscope throughacridine orange(AO) staining.Reverse transcription polymerse chain reaction(RT-PCR) was employed to detect the expression levels of UVRAG mRNA inducedby different concentrations of DADS in K562cells for12hours.Western blotting wasemployed to detect the expression levels of UVRAG protein induced by differentconcentrations of DADS(10mg/L、20mg/L、40mg/L、80mg/L) in K562cells for12hours.Results: The double membrane structure autophagosome can be observed in thecytoplasm after treatment with different concentrations of DADS (10mg/L、20mg/L、40mg/L、80mg/L) for12hours by TEM. Autophagosome was found most obvious in40mg/LDADS group, while it was absent in the blank reference group and the vehiclereference group.The autophagic vacuoles were found in K562cells treated with DADS,40mg/Lgroup, determined by AO staining fluorescence microscope. Its concentration wasfound to increase with treatment time, reached a peak value at12hours. A slightlydecrease was observed after24hours, which could be related with the degradation of acidic vesicle.Using RT-PCR, the expression levels of UVRAG mRNA were measured to be0.621±0.249,0.671±0.019,0.812±0.009and0.738±0.044, respectively,. for treatmentgroups which treated by DADS for12hours with concentrations of10,20,40and80mg/L while the blank reference group and vehicle reference group were0.554±0.018and0.572±0.018, respectively. The UVRAG mRNA expression levels of theK562cells of the treatment groups were higher than that of the reference groups (P<0.05).The Western Blotting result showed gray value of UVRAG Actin were0.731±0.044,0.812±0.069,1.151±0.175and0.880±0.071for treatment of12hours with differentDADS concentrations of10,20,40and80mg/L. while the blank reference groupand vehicle reference group were0.293±0.072and0.251±0.031.The UVRAG proteinexpression levels of the K562cells of the treatment groups were higher than that ofthe reference groups (P <0.05).Conclusion：DADS can induce autophagy of leukaemia k562cell; the mechanismmay be associated with the autophagy pathway of UVRAG mediated.