Autophagy and Ubiquitin-Mediated Proteolytic Degradation of PML/Rarα Fusion Protein in Matrine-Induced Differentiation Sensitivity Recovery of ATRA-Resistant APL (NB4-LR1) Cells: in Vitro and in Vivo Studies

• Published in: Cellular physiology and biochemistry Vol. 48; no. 6; pp. 2286 - 2301
• Main Authors: Wu, Dijiong , Shao, Keding, Zhou, Qihao, Sun, Jie, Wang, Ziqi, Yan, Fei, Liu, Tingting, Wu, Xiangping, Ye, Baodong, Huang, He, Zhou, Yuhong
• Format: Journal Article
• Language: English
• Published: Basel, Switzerland S. Karger AG 01.08.2018; Cell Physiol Biochem Press GmbH & Co KG
• Subjects: Acids; Acute promyelocytic leukemia; Alkaloids - pharmacology; Animals; Antineoplastic Agents - pharmacology; Apoptosis; ATRA-resistant; Autophagy; Autophagy - drug effects; CCAAT-Enhancer-Binding Proteins - genetics; CCAAT-Enhancer-Binding Proteins - metabolism; Cell Differentiation - drug effects; Cell Line, Tumor; Differentiation; Drug Resistance, Neoplasm - drug effects; Humans; Kinases; Leukemia; Leukemia, Promyelocytic, Acute - pathology; Male; Matrine; Mice; Mice, Inbred BALB C; Mice, Nude; Oncogene Proteins, Fusion - genetics; Oncogene Proteins, Fusion - metabolism; Original Paper; PML-RARα fusion protein; Proteins; Proto-Oncogene Proteins c-myc - antagonists & inhibitors; Proto-Oncogene Proteins c-myc - metabolism; Quinolizines - pharmacology; Recovery (Medical); STAT1 Transcription Factor - genetics; STAT1 Transcription Factor - metabolism; Tretinoin - pharmacology; Ubiquitin; Ubiquitin - metabolism; Ubiquitination - drug effects; United States > US; China; Ubiquitin; ATRA-resistant; Acute promyelocytic leukemia; Matrine; PML-RARα fusion protein; Differentiation; Autophagy
• ISSN: 1015-8987; 1421-9778
• DOI: 10.1159/000492646

Background/Aims: Although the cure rate of acute promyelocytic leukemia (APL) has exceeded 90%, the relapse/refractory APL that resistant to all-trans retinoic acid (ATRA) or ATO was still serious concern. Matrine (MAT) could improve the differentiation ability of ATRA-resistant APL cells. This study aimed to explore how the APL-specific fusion protein was degraded in ATRA-resistant APL with the application of MAT and ATRA. Methods: ATRA-sensitive (NB4) and ATRA-resistant (NB4-LR1) cell lines were used. Nitroblue tetrazolium reduction assay and flow cytometry were used to detect the differentiation ability. The activity of ubiquitin-proteasome and autophagy-mediated pathways in both cells treated with ATRA with or without MAT were compared in protein and mRNA level (Western blot analysis, qRT-PCR), the Fluorescent substrate Suc-LLVY-AMC detection was used to detect the activity of proteasome, and electron microscope for observing autophagosome. MG 132(proteasome inhibitor), rapamycin (autophagy activator), hydroxychloroquine (lysosomal inhibitor) and STI571 [retinoic acid receptor alpha (RARα) ubiquitin stabilizer] were used as positive controls. The effect of MAT was observed in vivo using xenografts. Results: MAT improved the sensitivity of NB4-LR1cells to ATRA treatment, which was consistent with the expression of PML-RARα fusion protein. MAT promoted the ubiquitylation level in NB4-LR1. MG 132 induced the decrease in RARα in both cell lines, and hampered the differentiation of NB4 cells. MAT also promoted the autophagy in NB4-LR1 cells, with an increase in microtubule-associated protein 1 light chain3 (LC3)-II and LC3-II/LC3-I ratio and exhaustion of P62. The expression of LC3II increased significantly in the MAT and ATRA + MAT groups in combination with lysosomal inhibitors. A similar phenomenon was observed in mouse xenografts. MAT induced apoptosis and differentiation. Conclusions: Autophagy and ubiquitin-mediated proteolytic degradation of PML/RARα fusion protein are crucial in MAT-induced differentiation sensitivity recovery of NB4-LR1 cells.