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

Abstract

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.

Matrine inhibits the growth of natural killer/T-cell lymphoma cells by modulating CaMKIIγ-c-Myc signaling pathway

“…Our previous study reported that matrine induced apoptosis in leukemia cells [ 16 ]. In this study, matrine induced apoptosis of NKTCL cell line NK92 cells in a dose dependent manner from 3.83 ± 0.78% to 62.9 ± 0.66% after matrine treatment at different concentrations for 48 h (Fig.…”

Matrine Reverses the Warburg Effect and Suppresses Colon Cancer Cell Growth via Negatively Regulating HIF-1α

“…The in vivo usage of matrine for cancer treatment in mice have been reported in several studies. The dose ranges from 25 to 100 mg/kg (Zhou et al, 2014; Wu et al, 2017; Cho et al, 2018; Shen et al, 2018; Wu et al, 2018). In our study, we use 80 mg/kg (0.32 mmol/kg) and the 7-days daily intraperitoneal administration shows distinct anticancer effect without significant side effects.…”

Autophagy: New Insights into Mechanisms of Action and Resistance of Treatment in Acute Promyelocytic leukemia

“…Interestingly, when differentiation was reestablished in maturation-resistant APL cells (by addition of 8-CPT-cAMP to ATRA treatment), autophagy activation and p62/SQSTM1 upregulation were both restored in these cells, underscoring the idea that autophagy and differentiation are interconnected processes. Furthermore, matrine, an alkaloid found in plants from the genus Sophora, was shown to improve the differentiation ability of ATRA-resistant APL cells through induction of autophagy and ubiquitin degradation of PML-RARα [74]. In another study, the addition of lithium (an autophagy inducer) to ATRA reactivated differentiation in ATRA maturation-resistant HL60-Diff-R AML cells, emphasizing the concept that autophagy modulation may represent a means to restore differentiation [12].…”