[Effect of MELK Inhibitor OTSSP167 on Diffuse Large B-Cell Lymphoma].

OBJECTIVE: To investigate the effect of MELK inhibitor OTSSP167 against diffuse large B-cell lymphoma (DLBCL). METHODS: The effect of OTSSP167 on activity, proliferation, and apoptosis of DLBCL cell line (SUDHL2 and HBL1) was detected by CCK-8 assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, and Annexin V-FITC/PI double staining, respectively. DLBCL cells were inoculated into nude mice, after 4 weeks of OTSSP167 treatment, the effect of OTSSP167 on DLBCL growth in vivo was detected. Caspase-GloTM 3/7 enzyme activity assay kit was used to detect the effect of OTSSP167 on Caspase 3/7 enzyme activity of DLBCL cells. The expression levels of apoptosis and cycle-related proteins were detected by Western blot. RESULTS: OTSSP167 significantly inhibited the activity of SUDHL2 and HBL1 cells in a dose-dependent manner (r =-0.61, r =-0.52). EdU staining showed that OTSSP167 could significantly inhibit the proliferation of SUDHL2 and HBL1 cells. Annexin V-FITC/PI result showed that OTSSP167 could significantly promote the apoptosis of SUDHL2 and HBL1 cells (P <0.001). The result of in vivo experiment showed that OTSSP167 could inhibit the growth of SUDHL2 cells in nude mice. The result of TUNEL staining of tumor further confirmed that OTSSP167 could promote the apoptosis of SUDHL2 cells. Caspase 3/7 enzyme activity test demonstrated that OTSSP167 could significantly increase caspase activity in SUDHL2 and HBL1 cells (r =0.98, r =0.87). Western blot showed that OTSSP167 could dose-dependently inhibit the expression of PARP, Bcl-xL, and Bcl-2 in apoptosis signaling pathway (r =-0.93, r =-0.66, r =-0.87), while p53 protein was significantly up-regulated (r =0.82). The expression of cell cycle-related proteins cdc2, Cyclin E1, Cyclin A2, and Cyclin B1 also showed a dose-dependent down-regulation (r =-0.89, r =-0.83, r =-0.61, r =-0.93). CONCLUSION: The MELK inhibitor OTSSP167 can inhibit the proliferation and promote the apoptosis of DLBCL cells by inhibiting the expression of cycle-related proteins and anti-apoptosis-related proteins.

[Effect of BBG on Acute Graft-Versus-Host Disease in Mice after Allogeneic Hematopoietic Stem Cell Transplantation].

OBJECTIVE: To investigate the effect of P2X7R antagonist brilliant blue G (BBG) on aGVDH of mice after allo-HSCT. METHODS: aGVHD mouse model after HSCT was established and treated with the P2X7R antagonist BBG of different dosages (50 mg/kg and 75 mg/kg). After treatment, the survival, body weight, pathological and liver function of aGVDH mice were abserved, and the expression levels of P2X7, NLRP3, caspase-1, IL-1ß, IL-18 mRNA and protein were evaluated by real-time PCR and Western blot. RESULTS: The allo-HSCT aGVHD mouse model was successfully established, the intraperitoneal injection of BBG alleviated the aGVHD clinical manifestations including roachback, ruffled fur, skin peeling and weight loss of recipient mice, decreased P2X7R and IL-1ß expression and reduced the mRNA levels of P2X7R, NLRP3, Caspase-1, IL-1ß and IL-18. Furthermore, GVHD group receiving 75 mg/kg BBG showed most significant difference of these indexes. CONCLUSION: BBG alleviates liver inflammatory damage induced by aGVHD after allo-HSCT, and decreases the expression of proinflammatory cytokines. Moreover, the protective effect of that of BBG 75 mg/kg group is better than that of BBG 50 mg/kg group.

Cytotoxic T Lymphocyte Antigen-4 Down-Regulates T Helper 1 Cells by Increasing Expression of Signal Transducer and Activator of Transcription 3 in Acute Graft-versus-Host Disease.

Numerous previous studies have suggested that cytotoxic T lymphocyte antigen-4 (CTLA-4) plays an important role in acute graft-versus-host disease (GVHD). How CTLA-4 acts in regulating acute GVHD remains unknown, however. In the present study, we found that, compared with healthy controls, CTLA-4 plasma and relative mRNA levels in patients with acute GVHD were initially decreased and then markedly elevated after 28 days of treatment. CTLA-4 levels were higher in patients with grade I-II acute GVHD compared with those with grade III-IV acute GVHD both before and after treatment. Up-regulation of CTLA-4 significantly increased the luciferase activity and degree of phosphorylation of signal transducer and activator of transcription 3 (STAT3). Meanwhile, T cell activation was significantly inhibited, and levels of IFN-γ, IL-17, and IL-22 decreased. These findings suggest that CTLA-4 might be involved in the pathogenesis of acute GVHD, and may down-regulate T helper 1 cells by increasing STAT3 expression in acute GVHD.

Antithymocyte globulin combined with cyclosporine A down-regulates T helper 1 cells by modulating T cell immune response cDNA 7 in aplastic anemia.

Antithymocyte globulin (ATG) combined with cyclosporine A (CsA) has been widely used as a standard regimen in the treatment of aplastic anemia (AA), especially in severe aplastic anemia (SAA). Abnormally activated T cells might be the immune pathogenesis of AA. T cell immune response cDNA 7 (TIRC7) has been demonstrated its essential role in T cell activation; however, little is known about the role of TIRC7 in AA. In this study, we documented that TIRC7 levels in CsA group were higher than that in ATG + CsA (AC) group only in the follow-up phase (P < 0.05; P < 0.05); nevertheless, TIRC7 levels in SAA group were elevated than non severe aplastic anemia group not only in the treatment phase (P < 0.05; P < 0.05) but also in the follow-up phase (P < 0.05; P < 0.01). The trend of changes of T helper (Th) 1, Th17 and Th22 levels before and after treatment was similar to the changes of TIRC7 levels in either AC group or CsA group. Thus, TIRC7 might be involved in the pathogenesis of AA and AC might down-regulate Th1 cells by modulating the expression of TIRC7 in AA.

Wwp2, an E3 ubiquitin ligase that targets transcription factor Oct-4 for ubiquitination.

The POU transcription factor Oct-4 is a master regulator affecting the fate of pluripotent embryonic stem cells. However, the precise mechanisms by which the activation and expression of Oct-4 are regulated still remain to be elucidated. We describe here a novel murine ubiquitin ligase, Wwp2, that specifically interacts with Oct-4 and promotes its ubiquitination both in vivo and in vitro. Remarkably, the expression of a catalytically inactive point mutant of Wwp2 abolishes Oct-4 ubiquitination. Moreover, Wwp2 promotes Oct-4 degradation in the presence of overexpressed ubiquitin. The degradation is blocked by treatment with proteasome inhibitor. Fusion of a single ubiquitin to Oct-4 inactivates its transcriptional activity in a heterologous Oct-4-driven reporter system. Furthermore, overexpression of Wwp2 in embryonic stem cells significantly reduces the Oct-4-transcriptional activities. Collectively, we demonstrate for the first time that Oct-4 can be post-translationally modified by ubiquitination and that this modification dramatically suppresses its transcriptional activity. These results reveal that the functional status of Oct-4, in addition to its expression level, dictates its transcriptional activity, and the results open up a new avenue to understand how Oct-4 defines the fate of embryonic stem cells.