The Study On The Mechanism Of Hsp90 Inhibitor 17-DMAG Inducing Apoptosis Of Acute Lymphoblastic Leukemia Cells Via Autophagy-lysosome Pathway

Objective: Acute B lymphocytic leukemia(B-ALL)is one of the most common hematologic malignancy in children.The main treatment of B-ALL is the risk-adapted chemotherapy.Due to the serious toxic effects of chemotherapy drugs and the chemotherapy resistance,new anti-leukemia agents have become a hot spot in B-ALL treatment.According to our previous proteomics study in children with B-ALL,the expression of heat shock protein 90(Hsp90)in the high-risk group was significantly higher than that in the low-risk group in children with B-ALL.The high expression of Hsp90 was reported to be closely related to the progressing and the poor prognosis in acute leukemia.Hsp90 inhibitor has become a new target drug for tumor therapy due to its high selectivity for cancer cells.17-DMAG is a Hsp90 inhibitor with lysosomotropic property.It has entered the clinical phase I trial in adult advanced solid tumor,lymphoma,acute myeloid leukemia and chronic lymphocytic leukemia.However 17-DMAG has been rarely reported in therapeutic studies of B-ALL therapy and its the mechanism of action remains unclear.Hence,we focused in the effect of 17-DMAG on proliferation and apoptosis of B-ALL cells,and the underlying mechanism.Methods: 1.Cell viability assays(MTS).The cell proliferation was detected after 24 h,48h and 72 h by MTS assay at different concentrations of 17-DMAG(0.01,0.1,1,10,100μM)treatments of in vitro B-ALL Nalm6 and Reh cells.2.Apoptosis detection(Annexin V-FITC/PI double staining).Cell apoptosis was detected by flow cytometry after 17-DMAG treatment with different concentrations(1μM,10μM).3.Autophagic level detection: Macroautophagy levels and chaperone-mediated autophagy(CMA)levels were evaluated at 17-DMAG(1μM)treatment time points(2h,4h,6h,8h,12 h,24h)in Nalm6 and Reh cells.Western blotting was used to detect the expression changes of macroautophagy markers LC3 B II and P62,as well as the expression level of Hsc70 and receptor Lamp2 a mediated by the chaperone.The accumulation of autophagosomes in cells was detected by autophagosomes staining.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to analyze the mRNA expression levels of Hsc70 and Lamp2 a.4.Lysosomal quantity and function assay.17-DMAG(1μM)treatment of Nalm6 and Reh cells(2h,12 h,24h)followed by Lyso-Brite staining.The effect of 17-DMAG on the number of lysosomes in cells was observed under fluorescence microscope.Western blotting was used to detect the protein expression level of the acid hydrolase cathepsin D in lysosome after 17-DMAG treatment of cells(24h).5.Cell transfection.In Nalm6 and Reh cells,Hsc70 gene expression(Electroporation)was knockdown by siRNA.The Real-time quantitative polymerase chain reaction(RT-qPCR)and confocal microscopy imaging to analyze of Hsc70 and lysosomal acid hydrolase cathepsin D in the expression levels of genes and protein levels,respectively.Western blotting was used to detect the effect of 17-DMAG on the expression of cell lysosomal acid hydrolase cathepsin D protein after blocking the ubiquitin-proteasome degradation process.Results: 1.17-DMAG inhibited the growth of B-ALL cells: 17-DMAG inhibited the proliferation of Nalm6 and Reh cells in a time-dose-dependent manner.2.17-DMAG induced the apoptosis of B-ALL cells: 17-DMAG induced the apoptosis of Nalm6 and Reh cells,and the apoptotic rate was positively correlated with the dose administrated(P <0.01).3.17-DMAG disturbed the autophagic flux of B-ALL cells: In the early stage(0h-4h)of treatment,macroautophagy was inhibited.The expression of LC3 B II and P62 protein increased synchronously with the prolongation of treatment time,suggesting the autophagic flux blocked in the late stage(4h-24h)of 17-DMAG treatment.The autophagosomes did not change in quality significantly in the early stage of 17-DMAG treatment(2h),but increased notablely in the late stage(24h).The expression of Hsc70 gene and protein was up-regulated,and gradually increased with the prolongation of treating time,reaching the peak of expression at 24h(P <0.01).However,the expression of the gene and protein Lamp2 a was not detected at the same time,suggesting that the CMA level is unchanged.4.17-DMAG disturbed the lysosomal degradation pathway in B-ALL cells: There was no significant change in lysosomal staining after 17-DMAG treatment,suggesting that 17-DMAG induced autophagosome accumulation without altering the lysosome quality.Western blotting showed that the expression of the lysosomal acid hydrolase cathepsin D was down-regulated after 17-DMAG treatment,indicating that the degradation function of lysosomes was impaired.5.17-DMAG inhibited cathepsin D expression by upregulatting Hsc70 expression: At the mRNA expression level,17-DMAG-induced high expression of Hsc70 down-regulated the expression of cathepsin D in Nalm6 cells(P <0.05),but Reh cells were unaffected.Hsc70 knockdown and 17-DMAG treatment after knockdown Hsc70 did not change the expression of cathepsin D.At the protein level,17-DMAG induced high expression of Hsc70 down-regulated the expression of cathepsin D in Nalm6(P <0.01)and Reh cells(P <0.05).17-DMAG-induced down-regulation of cathepsin D expression restored after inhibition of ubiquitin-proteasome degradation,indicating that high expression after Hsc70 induction increased the protein degradation of cathepsin D.It is suggested that Hsc70 was involved in the post-translational regulation of cathepsin D in both cells.Conclusions: This study confirmed that 17-DMAG induced apoptosis of B-ALL cells in vitro.17-DMAG inhibited cell macroautophagy levels in the early stages of administration.In the late stage of administration,17-DMAG inhibited the expression of lysosomal acid hydrolase cathepsin D,leading to the blockage of autophagic flux and triggering apoptosis.17-DMAG induced high expression of Hsc70,which might be involved in ubiquitin-proteasome degradation of lysosomal acid hydrolase cathepsin D protein.