Acid-sensing ion channel 1a mediates acid-induced pyroptosis through calpain-2/calcineurin pathway in rat articular chondrocytes.

The pyroptosis is a causative agent of rheumatoid arthritis, a systemic autoimmune disease merged with degenerative articular cartilage. Nevertheless, the precise mechanism of extracellular acidosis on chondrocyte pyroptosis is largely unclear. Acid-sensing ion channels (ASICs) belong to an extracellular H+ -activated cation channel family. Accumulating evidence has highlighted activation of ASICs induced by extracellular acidosis upregulate calpain and calcineurin expression in arthritis. In the present study, to investigate the expression and the role of acid-sensing ion channel 1a (ASIC1a), calpain, calcineurin, and NLRP3 inflammasome proteins in regulating acid-induced articular chondrocyte pyroptosis, primary rat articular chondrocytes were subjected to different pH, different time, and different treatments with or without ASIC1a, calpain-2, and calcineurin, respectively. Initially, the research results showed that extracellular acidosis-induced the protein expression of ASIC1a in a pH- and time-dependent manner, and the messenger RNA and protein expressions of calpain, calcineurin, NLRP3, apoptosis-associated speck-like protein, and caspase-1 were significantly increased in a time-dependent manner. Furthermore, the inhibition of ASIC1a, calpain-2, or calcineurin, respectively, could decrease the cell death accompanied with the decreased interleukin-1ß level, and the decreased expression of ASIC1a, calpain-2, calcineurin, and NLRP3 inflammasome proteins. Taken together, these results indicated the activation of ASIC1a induced by extracellular acidosis could trigger pyroptosis of rat articular chondrocytes, the mechanism of which might partly be involved with the activation of calpain-2/calcineurin pathway.

Targeting ferroptosis contributes to ATPR-induced AML differentiation via ROS-autophagy-lysosomal pathway.

Ferroptosis, a newly discovered form of non-apoptotic cell death, is induced by an excessive degree of iron-dependent lipid peroxide. ATPR, a novel all-trans retinoic acid (ATRA) derivative, has been extensively developed to show superior anticancer effect than ATRA in acute myeloid leukemia (AML). However, whether ferroptosis exists during ATPR treatment of AML remains unclear. Herein, we found that ferroptosis occurred in an AML xenograft mouse model of ATPR treatment. In vitro, ATPR was verified to induce ferroptosis in a dose-dependent manner by proferroptotic protein marker, lipid peroxidation, and lipid ROS, which could be significantly reversed by ferrostatin-1. Using lysosomal inhibitor chloroquine and iron chelator desferrioxamine, we further revealed that ATPR-induced ferroptosis was regulated by autophagy via iron homeostasis, especially Nrf2. Furthermore, targeting ferroptosis contributes to ATPR-induced AML differentiation. In conclusion, these results indicated that ferroptosis play an important role in ATPR-induced differentiation, and suggested that ATPR would provide a potential therapeutic value for AML treatment.

ATPR triggers acute myeloid leukaemia cells differentiation and cycle arrest via the RARα/LDHB/ERK-glycolysis signalling axis.

Acute myeloid leukaemia (AML) remains a therapeutic challenge and improvements in chemotherapy are needed. 4-Amino-2-trifluoromethyl-phenyl retinate (ATPR), a novel all-trans retinoic acid (ATRA) derivative designed and synthesized by our team, has been proven to show superior anticancer effect compared with ATRA on various cancers. However, its potential effect on AML remains largely unknown. Lactate dehydrogenase B (LDHB) is the key glycolytic enzyme that catalyses the interconversion between pyruvate and lactate. Currently, little is known about the role of LDHB in AML. In this study, we found that ATPR showed antileukaemic effects with RARα dependent in AML cells. LDHB was aberrantly overexpressed in human AML peripheral blood mononuclear cell (PBMC) and AML cell lines. A lentiviral vector expressing LDHB-targeting shRNA was constructed to generate a stable AML cells with low expression of LDHB. The effect of LDHB knockdown on differentiation and cycle arrest of AML cells was assessed in vitro and vivo, including involvement of Raf/MEK/ERK signalling. Finally, these data suggested that ATPR showed antileukaemic effects by RARα/LDHB/ ERK-glycolysis signalling axis. Further studies should focus on the underlying leukaemia-promoting mechanisms and investigate LDHB as a therapeutic target.

A novel all-trans retinoic acid derivative inhibits proliferation and induces apoptosis of myelodysplastic syndromes cell line SKM-1 cells via up-regulating p53.

Myelodysplastic syndromes (MDS) are a varied set of hematologic neoplasms and a high risk of progression to acute myeloid leukemia (AML). 4-Amino-2-trifluoromethyl-phenyl retinate (ATPR), a novel all-trans retinoic acid (ATRA) derivative, play an important role in various types of cancer cells as a tumor inhibitor. However, little is known concerning its antitumor effect on MDS. The cell viability and the percentage of apoptotic cells were used to measure MTT, Flow Cytometry and Hoechst 33342/PI staining. In addition, real-time quantitative RT-PCR (qRT-PCR) and western blotting were used to analyzed the expression of p53, as well as the levels of BNIP3, apoptosis proteins of Caspase-3, BAX and BCL-2. After SKM-1 cells were incubated with DAC, ATRA and ATPR, the viability of the SKM-1 cells was inhibited in a dose- and time-dependent manner. Both Hoechst staining and flow cytometry showed the apoptosis of SKM-1 cells was increased. Moreover, SKM-1 cells treated with ATPR unveiled elevated mRNA and protein levels of p53, BNIP3, BAX and Caspase-3 expression and decreased BCL-2 expression. However, silencing p53 suppressed the pro-apoptosis function of ATPR. Consequently, these data provide the first evidence for ATPR increased apoptosis in SKM-1 cells by p53 that is mutually dependent on and obligatorily linked to BNIP3 gene activation.

Necrostatin-1 ameliorates adjuvant arthritis rat articular chondrocyte injury via inhibiting ASIC1a-mediated necroptosis.

Necroptosis, a necrotic cell death pathway regulated by receptor interacting protein (RIP) 1 and 3, plays a key role in pathophysiological processes, including rheumatoid arthritis (RA). However, whether necroptosis is involved in RA articular cartilage damage processes remain unclear. The aim of present study was to investigate the dynamic changes in arthritic chondrocyte necroptosis and the effect of RIP1 inhibitor necrostatin-1 (Nec-1) and acid-sensing ion channels (ASICs) inhibitor amiloride on arthritic cartilage injury and acid-induced chondrocyte necroptosis. Our results demonstrated that the expression of RIP1, RIP3 and mixed lineage kinase domain-like protein phosphorylation (p-MLKL) were increased in adjuvant arthritis (AA) rat articular cartilage in vivo and acid-induced chondrocytes in vitro. High co-expression of ASIC1a and RIP1 showed in AA rat articular cartilage. Moreover, Nec-1 and amiloride could reduce articular cartilage damage and necroinflammation in AA rats. In addition, acid-induced increase in necroptosis markers RIP1/RIP3 were inhibited by Nec-1, ASIC1a-specific blocker psalmotoxin-1 (PcTx-1) or ASIC1a-short hairpin RNA respectively, which revealed that necroptosis is triggered in acid-induced chondrocytes and mediated by ASIC1a. These findings indicated that blocking ASIC1a-mediated chondrocyte necroptosis may provide potential therapeutic strategies for RA treatment.

4-Amino-2-Trifluoromethyl-Phenyl Retinate induced leukemia cell differentiation by decreasing eIF6.

4-Amino-2-Trifluoromethyl-Phenyl Retinate (ATPR), an all-trans retinoic acid (ATRA) derivative, possesses the ability to relief several carcinoma. Here, we explored the potential molecular mechanism of eukaryotic translation initiation factor 6 (eIF6) in ATPR-induced leukemia cell differentiation. Our research showed that ATPR could inhibit cell proliferation and promote cell differentiation in several leukemia cell lines. Besides, ATPR remarkably reduced the expression of eIF6 in vitro. Interestingly, the reduction of eIF6 contributed to restraining proliferation of K562 cells by inhibiting CyclinD1, C-myc and blocking cell cycle, as well as promoting differentiation of K562 cells by increasing the expression of C/EBPε, cell surface antigen CD11b and inducing renal-shrinkage of nuclear. Furthermore, the over-expression of eIF6 restrained the effects of ATPR on cell proliferation and maturation in K562 cells. In Addition, Notch1/CBF-1 signal activated by Chrysin could increase expression of eIF6 and restrain the differentiation in ATPR-induced K562 cells. Taken together, all above results indicated that ATPR induced differentiation of leukemia cells by decreasing eIF6 through Notch1/CBF-1 signal, which might exert an innovative treatment for leukemia.