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17-DMAG disrupted the autophagy flux leading to the apoptosis of acute lymphoblastic leukemia cells by inducing heat shock cognate protein 70
Life Sciences ( IF 5.2 ) Pub Date : 2020-03-07 , DOI: 10.1016/j.lfs.2020.117532 Gang Xu 1 , Xiujuan Ma 2 , Fang Chen 3 , Di Wu 4 , Jianing Miao 4 , Yang Fan 4
Life Sciences ( IF 5.2 ) Pub Date : 2020-03-07 , DOI: 10.1016/j.lfs.2020.117532 Gang Xu 1 , Xiujuan Ma 2 , Fang Chen 3 , Di Wu 4 , Jianing Miao 4 , Yang Fan 4
Affiliation
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B-lineage acute lymphoblastic leukemia (B-ALL) is most common in children. We had reported heat shock protein 90 (Hsp90) over-expressed in high risk B-ALL children. 17-DMAG is a water soluble Hsp90 inhibitor, which was proved to be effective for advanced solid tumors and hematological malignancy. However, there is little research on its application in newly diagnosed B-ALL. And the detailed mechanism is seldom discussed. Primary blast cells from 24 newly diagnosed B-ALL pediatric patients and two B-ALL cell lines were used in this study. Cell viability was measured by MTS assay. Apoptosis was evaluated by flow cytometry after annexin V-PI double staining. Protein expression was detected by immunoblotting analysis and immunofluorescence imaging. Cyto-ID autophagy detection assay was performed to show the autophagosomes and LysoTracker labeling to show the lysosomes. Gene knockdown was performed by RNA interference, and mRNA expression was measured by RT-qPCR. We showed 17-DMAG induced apoptosis in newly diagnosed B-ALL blasts and cell lines effectively. 17-DMAG induced heat shock cognate protein 70 (Hsc70) expression significantly. High expressed Hsc70 inhibited cathepsin D post-transcriptionally to impede the autophagic flux, which lead to the cell death. Our work added new information towards understanding the molecular pharmacology of 17-DMAG, and suggested the newly diagnosed B-ALL pediatric patients might be benefited from 17-DMAG. Furthermore, we proved Hsc70 participated in the mechanism of cell death 17-DMAG leading in B-ALL.
中文翻译：
17-DMAG 通过诱导热休克同源蛋白 70 扰乱自噬流，导致急性淋巴细胞白血病细胞凋亡
B 系急性淋巴细胞白血病 (B-ALL) 在儿童中最常见。我们曾报道热休克蛋白 90 (Hsp90) 在高危 B-ALL 儿童中过度表达。 17-DMAG是一种水溶性Hsp90抑制剂，被证明对晚期实体瘤和血液恶性肿瘤有效。然而，其在初诊B-ALL中的应用研究甚少。并且详细的机制很少被讨论。本研究使用了来自 24 名新诊断的 B-ALL 儿科患者的原代母细胞和两种 B-ALL 细胞系。通过MTS测定来测量细胞活力。膜联蛋白V-PI双染色后通过流式细胞仪评估细胞凋亡。通过免疫印迹分析和免疫荧光成像检测蛋白质表达。进行 Cyto-ID 自噬检测分析以显示自噬体，并进行 LysoTracker 标记以显示溶酶体。通过RNA干扰进行基因敲除，并通过RT-qPCR测量mRNA表达。我们发现 17-DMAG 可有效诱导新诊断的 B-ALL 母细胞和细胞系的细胞凋亡。 17-DMAG 显着诱导热休克同源蛋白 70 (Hsc70) 表达。高表达的 Hsc70 会在转录后抑制组织蛋白酶 D，从而阻碍自噬流，从而导致细胞死亡。我们的工作为理解 17-DMAG 的分子药理学增加了新的信息，并表明新诊断的 B-ALL 儿科患者可能受益于 17-DMAG。此外，我们还证明Hsc70参与了B-ALL中17-DMAG导致的细胞死亡机制。
更新日期：2020-03-07
中文翻译：
17-DMAG 通过诱导热休克同源蛋白 70 扰乱自噬流，导致急性淋巴细胞白血病细胞凋亡
B 系急性淋巴细胞白血病 (B-ALL) 在儿童中最常见。我们曾报道热休克蛋白 90 (Hsp90) 在高危 B-ALL 儿童中过度表达。 17-DMAG是一种水溶性Hsp90抑制剂，被证明对晚期实体瘤和血液恶性肿瘤有效。然而，其在初诊B-ALL中的应用研究甚少。并且详细的机制很少被讨论。本研究使用了来自 24 名新诊断的 B-ALL 儿科患者的原代母细胞和两种 B-ALL 细胞系。通过MTS测定来测量细胞活力。膜联蛋白V-PI双染色后通过流式细胞仪评估细胞凋亡。通过免疫印迹分析和免疫荧光成像检测蛋白质表达。进行 Cyto-ID 自噬检测分析以显示自噬体，并进行 LysoTracker 标记以显示溶酶体。通过RNA干扰进行基因敲除，并通过RT-qPCR测量mRNA表达。我们发现 17-DMAG 可有效诱导新诊断的 B-ALL 母细胞和细胞系的细胞凋亡。 17-DMAG 显着诱导热休克同源蛋白 70 (Hsc70) 表达。高表达的 Hsc70 会在转录后抑制组织蛋白酶 D，从而阻碍自噬流，从而导致细胞死亡。我们的工作为理解 17-DMAG 的分子药理学增加了新的信息，并表明新诊断的 B-ALL 儿科患者可能受益于 17-DMAG。此外，我们还证明Hsc70参与了B-ALL中17-DMAG导致的细胞死亡机制。
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