ABSTRACT
Objective To analyze the relationship between the JAK2V617F mutation and the expressions of phosphorylated Janus kinase 2 (p-JAK2), suppressor of cytokine signaling 1 (SOCS1), and SH2-containing protein tyrosine phosphatase 1 (SHP1) in JAK2V617F mutation positive myeloproliferative neoplasm (MPN) tissues, and to investigate the effects of JAK2 inhibitor ruxolitinib on regulating the proliferation of JAK2V617F mutation positive human erythroleukemia cell lines HEL and the expressions of SOCS1 and SHP1 in HEL cells. Methods A total of 48 patients with JAK2V617F mutation positive MPN (MPN group) and 24 patients with anemia (control group) in Hebei General Hospital and The First People’s Hospital of Baoding from Jul. 2012 to Aug. 2016 were enrolled in this study. The protein expressions of p-JAK2, SOCS1 and SHP1 in bone marrow biopsies (BMBs) were detected by immunohistochemistry. The correlations between JAK2V617F mutation level and the protein expressions of SOCS1 and SHP1 were analyzed by Spearman rank correlation analysis. HEL cells were treated with ruxolitinib at different concentrations (50, 100, 250, 500 and 1 000 nmol/L), and the viability of cells was determined by CCK-8 assay. The JAK2V617F mutation levels in MPN tissues and HEL cells and the mRNA expressions of JAK2, SOCS1 and SHP1 in HEL cells were detected by qPCR. The protein expressions of JAK2, SOCS1 and SHP1 in HEL cells were detected by Western blotting analysis. Results The ratio of JAK2V617F/JAK2 was (57.33±20.82)% in the MPN group and was zero in the control group. The protein expressions of p-JAK2, SOCS1 and SHP1 in BMBs of MPN patients were significantly different from those in the control group (all P0.01). The protein expressions of SOCS1 and SHP1 were negatively correlated with the mutation level of JAK2V617F (r＝－0.648, －0.692; P0.05). The expressions of SOCS1 and SHP1 in MPN patients with JAK2V617F/JAK250% were significantly higher than those in MPN patients with JAK2V617F/JAK2≥50% (P0.01), while the expression of p-JAK2 was significantly lower than that in MPN patients with JAK2V617F/JAK2≥50% (P0.01). After treatment with 250 nmol/L ruxolitinib for 24 h, 48 h, and 72 h, the viabilities of HEL cells were (60.06±3.87)%, (52.05±2.88)%, and (36.43±2.01)%, respectively. With the increase of ruxolitinib concentrations, the mRNA and protein expressions of JAK2 and the protein expression of p-JAK2 were gradually decreased (P0.01, P0.05), while the mRNA and protein expressions of SOCS1 and SHP1 were gradually increased (all P0.01). Conclusion Ruxolitinib can inhibit the expressions of JAK and the phosphorylation of JAK in HEL cells, enhance the expressions of SOCS1 and SHP1, and reduce the viability of HEL cells.
ABSTRACT
Objective: To investigate whether fusion protein SD-HA could regulate its downstream signaling molecule activity by competing with the phospho-BCR-ABL Y177 site, and its mechanisms to inhibit proliferation and induce apoptosis of K562 cells. Methods: Co-immunoprecipitation interaction technology analysis of fusion protein SD-HA functioned by potently binding to the phospho-BCR-ABL Y177 site, Ras, MAPK and Akt activities were observed in the Ad5F35-SD-HA-treated cells. Western blot analyses of SD-HA fusion protein on cell membrane receptor pathway to death cascade caspase-8, caspase-3 and PRAP were performed. Results: Exploration into the underlying mechanisms revealed that Ad5F35-SD-HA infection functioned by binding to the phospho-BCR-ABL Y177 site, which lead to a complex with Grb2. competitively disrupted the Grb2 SH2-phospho-BCR-ABL Y177 formation. The fusion protein SD-HA could reduce the activation of Ras and phosphorylation of MAPK (p-MAPK) and the expression level of p-ELK, inhibition of Ras-MAPK signaling pathway; SD-HA fusion protein could reduce p-Akt and Akt substrate p-GSK with inhibition of PI3K-Akt signaling pathway, thereby inhibiting the proliferation of K562 cells. Caspases-8-induced apoptosis signal could be activated by DED protein binding to DED domain of precursor caspases-8. Conclusions: The strategy of fusion protein SD-HA inhibiting-Y177 BCR-ABL and Grb2 binding could be used as a novel entry point for the treatment of chronic myeloid leukemia.
Subject(s)
Humans , Adenoviridae , Apoptosis , Cell Proliferation , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Oncogene Proteins, Fusion , Phosphatidylinositol 3-KinasesABSTRACT
Objective@#To investigate whether fusion protein SD-HA could regulate its downstream signaling molecule activity by competing with the phospho-BCR-ABL Y177 site, and its mechanisms to inhibit proliferation and induce apoptosis of K562 cells.@*Methods@#Co-immunoprecipitation interaction technology analysis of fusion protein SD-HA functioned by potently binding to the phospho-BCR-ABL Y177 site, Ras, MAPK and Akt activities were observed in the Ad5F35-SD-HA-treated cells. Western blot analyses of SD-HA fusion protein on cell membrane receptor pathway to death cascade caspase-8, caspase-3 and PRAP were performed.@*Results@#Exploration into the underlying mechanisms revealed that Ad5F35-SD-HA infection functioned by binding to the phospho-BCR-ABL Y177 site, which lead to a complex with Grb2. competitively disrupted the Grb2 SH2-phospho-BCR-ABL Y177 formation. The fusion protein SD-HA could reduce the activation of Ras and phosphorylation of MAPK (p-MAPK) and the expression level of p-ELK, inhibition of Ras-MAPK signaling pathway; SD-HA fusion protein could reduce p-Akt and Akt substrate p-GSK with inhibition of PI3K-Akt signaling pathway, thereby inhibiting the proliferation of K562 cells. Caspases-8-induced apoptosis signal could be activated by DED protein binding to DED domain of precursor caspases-8.@*Conclusions@#The strategy of fusion protein SD-HA inhibiting-Y177 BCR-ABL and Grb2 binding could be used as a novel entry point for the treatment of chronic myeloid leukemia.