Targeting PKC-mediated signal transduction pathways using enzastaurin to promote apoptosis in acute myeloid leukemia-derived cell lines and blast cells

Recent studies in acute myeloid leukemia (AML) suggest activation of pro-proliferative signaling cascades including those mediated by protein kinase C (PKC) represent a poor prognostic factor for patients. The classical PKC isoforms α and β generally support survival signaling and have emerged as important targets for anti-cancer therapy. Enzastaurin is a PKC β inhibitor and is in clinical trials for lymphomas, gliomas, and lung cancer. Presently, it is not known if enzastaurin could be effective against AML. In the current study, we found that high dose enzastaurin was found to promote apoptosis in the AML-derived cell lines and in blast cells from AML patients. The mechanism of cell death, however, likely does not involve PKC β as another PKC β inhibitor was not toxic to AML cell lines and did not promote enzastaurin-induced cell killing. While enzastaurin is fairly specific for PKC β, the agent can inhibit other PKC isoforms at higher concentrations. Enzastaurin was effective at inhibiting PKC α phosphorylation and membrane localization in the AML cell lines and suppressed phosphorylation of BCL2. Furthermore, enzastaurin suppressed activation of ERK (which can be activated by PKC α). Analysis of the serine/threonine phosphorylation profile in HL60 cells after enzastaurin treatment revealed that the drug inhibits the phosphorylation of a distinct set of proteins while promoting phosphorylation of another set of proteins. This suggests the drug may regulate multiple signaling pathways. Taken together, these findings suggest that enzastaurin could be effective in the therapy of AML.     

Annexin A1 mediates the anti‐adhesive effects of dexamethasone during the cell–cell interaction between the all‐trans retinoic acid‐treated acute promyelocytic leukemic cells and endothelial cells

Annexin A1 (AnxA1) is an important anti‐inflammatory mediator during granulocytic differentiation in all trans‐retinoic acid (ATRA) treated acute promyelocytic leukemic (APL) cells. Dexamethasone has been used successfully to prevent complications in ATRA‐treated APL patients, although its mechanism of action is still not clear. In the present study, we have examined the effect of dexamethasone on the modulation of AnxA1 in ATRA‐APL NB4 (ATRA‐NB4) cells, ATRA‐NB4 cells‐derived microparticles (MPs) and its role during cell–cell interaction between ATRA‐NB4 cells and endothelial cells. Our results have shown that dexamethasone can inhibit the percentage of ATRA‐NB4 cells expressing surface AnxA1 and its receptor FPR2/ALX in a time‐dependent manner based on flow cytometric analysis. However, dexamethasone treatment of ATRA‐NB4 cells has no significant effect on the level of AnxA1 mRNA, the total cellular level of AnxA1 protein or the release of AnxA1 from these cells, as determined by RT‐PCR, Western blotting, and ELISA, respectively. Further studies demonstrate that dexamethasone is able to significantly inhibit the adhesion of ATRA‐NB4 cells to endothelial cells, and this anti‐adhesive effect can be inhibited if the cells were pre‐treated with a neutralizing antibody specific for AnxA1. Finally, dexamethasone also enhances the release of AnxA1‐containing MPs from ATRA‐NB4 cells which can in turn prevent the adhesion of the ATRA‐NB4 cells to endothelial cells. We conclude that biologically active AnxA1 originating from dexamethasone‐treated ATRA‐APL cells and their MPs plays an anti‐adhesive effect and this contributes to inhibit the adhesion of ATRA‐APL cell to endothelial cells. J. Cell. Biochem. 114: 551–557, 2013. © 2012 Wiley Periodicals, Inc.     

Promyelocytic leukemia nuclear bodies support a late step in DNA double-strand break repair by homologous recombination

The PML protein and PML nuclear bodies (PML-NB) are implicated in multiple cellular functions relevant to tumor suppression, including DNA damage response. In most cases of acute promyelocytic leukemia, the PML and retinoic acid receptor alpha (RARA) genes are translocated, resulting in expression of oncogenic PML-RARα fusion proteins. PML-NB fail to form normally, and promyelocytes remain in an undifferentiated, abnormally proliferative state. We examined the involvement of PML protein and PML-NB in homologous recombinational repair (HRR) of chromosomal DNA double-strand breaks. Transient overexpression of wild-type PML protein isoforms produced hugely enlarged or aggregated PML-NB and reduced HRR by ~2-fold, suggesting that HRR depends to some extent upon normal PML-NB structure. Knockdown of PML by RNA interference sharply attenuated formation of PML-NB and reduced HRR by up to 20-fold. However, PML-knockdown cells showed apparently normal induction of H2AX phosphorylation and RAD51 foci after DNA damage by ionizing radiation. These findings indicate that early steps in HRR, including recognition of DNA double-strand breaks, initial processing of ends, and assembly of single-stranded DNA/RAD51 nucleoprotein filaments, do not depend upon PML-NB. The HRR deficit in PML-depleted cells thus reflects inhibition of later steps in the repair pathway. Expression of PML-RARα fusion proteins disrupted PML-NB structure and reduced HRR by up to 10-fold, raising the possibility that defective HRR and resulting genomic instability may figure in the pathogenesis, progression and relapse of acute promyelocytic leukemia.     

The thrombopoietin/MPL pathway in hematopoiesis and leukemogenesis

Hematopoietic stem cells (HSC) comprise a small percentage of total hematopoietic cells. Their ability to self-renewal is key to the continuous replenishment of the hematopoietic system with newly formed functional blood cell types while maintaining their multipotential capacity. Understanding the extrinsic signals that are essential to HSC maintenance will provide insights into the regulation of hematopoiesis at its most primitive stage, and with the knowledge applied, will potentially lead to improved clinical transplantation outcomes. In this review, we will summarize the current understanding of the role of the thrombopoietin/MPL signaling pathway in HSC maintenance during adult and fetal hematopoiesis. We will also speculate on the downstream key players in the pathway based on published data, and summarize the role of this pathway in leukemia.     

The Role of Serum High Mobility Group Box 1 and Interleukin‐6 Levels in Acute Pancreatitis: A Meta‐Analysis

The purpose of this meta‐analysis was to comprehensively investigate the correlation between high mobility group box 1 (HMGB1) and interleukin‐6 (IL‐6) in relation to acute pancreatitis. A highly regulated exploration of various electronic databases, supplemented by manual searching methods, was performed in an attempt to identify pertinent articles of a useful nature. Subsequently, high‐quality cohort studies that were deemed to comply with the arduous inclusion and exclusion criteria were selected for our meta‐analysis. The extensive data analyses reported in our meta‐analysis were conducted in connection with the Comprehensive Meta‐analysis 2.0 (CMA 2.0). A total of 395 studies (135 Chinese studies and 260 English studies) were initially retrieved. 27 of those studies were selected for our meta‐analysis, comprising of 896 cases of mild acute pancreatitis (MAP), 700 cases of severe acute pancreatitis (SAP) as well as 312 healthy controls. Pooled data suggested that serum HMGB1 and IL‐6 levels of SAP and MAP patients were higher than in healthy controls. Moreover, serum HMGB1 and IL‐6 levels of SAP patients exhibited significantly higher levels than in that of MAP patients. Based on the rigorous investigation of our meta‐analysis, it was concluded that serum HMGB1 and IL‐6 levels might be used as effective indicators for pancreatic lesions as well as the degree of inflammatory response, owing ultimately to the observations and data analyses, suggesting that serum HMGB1 and IL‐6 levels share a close correlation with the severity of pancreatitis. J. Cell. Biochem. 119: 616–624, 2018. © 2017 Wiley Periodicals, Inc.