S100A8 and S100A9 in Hematologic Malignancies: From Development to Therapy.

S100A8 and S100A9 are multifunctional proteins that can initiate various signaling pathways and modulate cell function both inside and outside immune cells, depending on their receptors, mediators, and molecular environment. They have been reported as dysregulated genes and proteins in a wide range of cancers, including hematologic malignancies, from diagnosis to response to therapy. The role of S100A8 and S100A9 in hematologic malignancies is highlighted due to their ability to work together or as antagonists to modify cell phenotype, including viability, differentiation, chemosensitivity, trafficking, and transcription strategies, which can lead to an oncogenic phase or reduced symptoms. In this review article, we discuss the critical roles of S100A8, S100A9, and calprotectin (heterodimer or heterotetramer forms of S100A8 and S100A9) in forming and promoting the malignant bone marrow microenvironment. We also focus on their potential roles as biomarkers and therapeutic targets in various stages of hematologic malignancies from diagnosis to treatment.

Transformation of Hematopoietic Stem and Progenitor Cells by Leukemia Extracellular Vesicles: A Step Toward Leukemogenesis.

Regulation of hematopoietic stem and progenitor cells (HSPCs) self-renewal, expansion, and differentiation is an inevitable process for normal hematopoiesis in the bone marrow (BM) niche, where leukemia cells are born, proliferate and occupy the microenvironment. External mediators such as extracellular vesicles (EVs) shed from leukemia cells, are one of the most important cell to cell communicators, and leading to phenotype and genotype modification and subsequently, fate of the cell. This review highlights recent evidences about the possible roles of leukemia derived-EVs on maintenance, proliferation, and death of HSPCs in a same microenvironment as leukemia cells. In addition, it focuses on mechanisms involved in the transformation of BM niche in favor of leukemia microenvironment remodeling by leukemia derived-EVs.

Altered Expression of CD44, SIRT1, CXCR4, miR-21, miR-34a, and miR-451 Genes in MKN-45 Cell Line After Docetaxel Treatment.

PURPOSE: Today it is known that the gene expression profile of cancer stem cells differs from other cancer cells, which may lead to the resistance to routine treatments. The aim of this study was to investigate the effect of docetaxel (DOC) treatment on CD44+ cell frequency in human gastric cancer (GC) MKN-45 cell line and its effect on expression levels of SIRT1, CXCR4, microRNA (miR)-21, miR-451, and miR-34a that are closely correlated with the chemoresistance or self-renewal of cancer stem cells (CSCs). METHODS: The cytotoxic effect of DOC on MKN-45 cell line was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-assay. The frequency of CD44+ cells was measured by flow cytometry in the treated and control groups. The expression level of SIRT1, CXCR4, miR-21, miR-451, and miR-34a was assessed in DOC-treated and non-treated cells using quantitative real-time PCR. Data were analyzed using Statistical Package for the Social Sciences (SPSS) software. RESULTS: The half-maximal inhibitory concentration (IC50) of DOC was 10 µg/ml after 48 h. Flow cytometry showed a significant increase in CD44+ cells after treatment with DOC (94.3%) when compared with non-treated cells (84.6%) (P < 0.01). The expression of SIRT1, CXCR4, and miR-21 was up-regulated (1.4-fold, 6.7-fold, and 1.22-fold, respectively, P < 0.05) in DOC-treated cells relative to non-treated cells, while miR-451 and miR-34a were down-regulated (0.14-fold and 0.36-fold, respectively, P < 0.05). CONCLUSION: DOC treatment affected CD44+ cell frequency in MKN-45 cell line and induced significant changes in the expression of SIRT1, CXCR4, miR-21, miR-451, and miR-34a that are implicated in stemness and chemo-radioresistance, which might offer new insights for future GC therapies.

LY86, LRG1 and PDE9A genes overexpression in umbilical cord blood hematopoietic stem progenitor cells by acute myeloid leukemia (M3) microvesicles.

BACKGROUND: Microvesicles as a new device of cell-cell communication are potentially able to induce some phenotypes and genotypes of an origin cell in a target cell. We evaluate the role of leukemia microvesicles on the leukemia stem cells (LSCs)-specific genes expression in healthy hematopoietic stem progenitor cells (HSPCs). METHODS: HL-60 and NB-4 cell lines were selected for microvesicles isolation by ultracentrifugation. Healthy HSPCs were obtained by magnetic association cell sorting (MACS) and CD-34 micro-beads from umbilical cord blood samples and then, were treated with 20 and 40 µg/ml leukemia microvesicles for 10 days, respectively. LY86, LRG1 and PDE9A genes expression as LSC specific genes were analyzed by QRT-PCR. Surface CD-34 antigen as stemness marker was measured by flow cytometry technique. RESULTS: Healthy HSPCs showed a significant increase in LSC specific genes expression after treatment with both 20 and 40 µg/ml leukemia microvesicles at day 10. All studied groups showed more than 70% surface CD-34 antigen at the last day of experiment which proved HSPCs stemness. CONCLUSION: Our results suggest that healthy HSPCs can be transformed genetically by leukemia microvesicles to over express LSC specific genes. This may be further evidence of leukemia-like transformation by leukemia microvesicles.

MicroRNA-21 over expression in umbilical cord blood hematopoietic stem progenitor cells by leukemia microvesicles.

Microvesicles are able to induce the cell of origin's phenotype in a target cell. MicroRNA-21, as an oncomir, is up-regulated in almost all cancer types such as leukemia which results in cell proliferation. In this study, we examine the ability of leukemia microvesicles to induce proliferation in hematopoietic stem progenitor cells (HSPCs) via microRNA-21 dysregulation. Herein, leukemia microvesicles were isolated from HL-60 and NB-4 cell lines by ultracentrifugation, and then their protein content was measured. Normal HSPCs were isolated from umbilical cord blood samples by a CD-34 antibody. These cells were treated with 20 and 40 µg/mL leukemia microvesicles for 5 and 10 days, respectively. Cell count, CD-34 analysis, and a microRNA-21 gene expression assay were done at days 5 and 10. HSPCs showed a significant increase in both microRNA-21 gene expression and cell count after treating with leukemia microvesicles compared with the control group. CD-34 analysis as stemness proof did not show any difference among the studied groups. This data suggests that HSPC proliferation followed by microRNA-21 gene over expression can be another evidence of a leukemia-like phenotype induction in a healthy target cell by leukemia microvesicles.

Leukemia microvesicles affect healthy hematopoietic stem cells.

Microvesicles are released by different cell types and shuttle mRNAs and microRNAs which have the possibility to transfer genetic information to a target cell and alter its function. Acute myeloid leukemia is a malignant disorder, and leukemic cells occupy all the bone marrow microenvironment. In this study, we investigate the effect of leukemia microvesicles on healthy umbilical cord blood hematopoietic stem cells to find evidence of cell information transferring. Leukemia microvesicles were isolated from acute myeloid leukemia patients and were co-incubated with healthy hematopoietic stem cells. After 7 days, cell count, hematopoietic stem cell-specific cluster of differentiation (CD) markers, colony-forming unit assay, and some microRNA gene expressions were assessed. Data showed a higher number of hematopoietic stem cells after being treated with leukemia microvesicles compared with control (treated with no microvesicles) and normal (treated with normal microvesicles) groups. Also, increased levels of microRNA-21 and microRNA-29a genes were observed in this group, while colony-forming ability was still maintained and high ranges of CD34+, CD34+CD38-, CD90+, and CD117+ phenotypes were observed as stemness signs. Our results suggest that leukemia microvesicles are able to induce some effects on healthy hematopoietic stem cells such as promoting cell survival and some microRNAs deregulation, while stemness is maintained.

Leukemia cell microvesicles promote survival in umbilical cord blood hematopoietic stem cells.

Microvesicles can transfer their contents, proteins and RNA, to target cells and thereby transform them. This may induce apoptosis or survival depending on cell origin and the target cell. In this study, we investigate the effect of leukemic cell microvesicles on umbilical cord blood hematopoietic stem cells to seek evidence of apoptosis or cell survival. Microvesicles were isolated from both healthy donor bone marrow samples and Jurkat cells by ultra-centrifugation and were added to hematopoietic stem cells sorted from umbilical cord blood samples by magnetic associated cell sorting (MACS) technique. After 7 days, cell count, cell viability, flow cytometry analysis for hematopoietic stem cell markers and qPCR for P53 gene expression were performed. The results showed higher cell number, higher cell viability rate and lower P53 gene expression in leukemia group in comparison with normal and control groups. Also, CD34 expression as the most important hematopoietic stem cell marker, did not change during the treatment and lineage differentiation was not observed. In conclusion, this study showed anti-apoptotic effect of leukemia cell derived microvesicles on umbilical cord blood hematopoietic stem cells.

Cyclic AMP-induced p53 destabilization is independent of EPAC in pre-B acute lymphoblastic leukemia cells in vitro.

CONTEXT: Activation of the tumor suppressor protein p53 facilitates the cellular response to genotoxic stress. Thus, releasing the wild-type p53 from indirect suppression would be crucial to successful killing of cancer cells by DNA-damaging therapeutic agents. OBJECTIVE: The aim of this study was to investigate the inhibitory role of cyclic adenosine monophosphate (cAMP) levels on p53 protein in acute lymphoblastic leukemia (ALL) cells. More importantly, we were interested to show through which receptor cAMP acts to promote p53 degradation. MATERIALS AND METHODS: In cell cultures, we investigated the effects of forskolin/3-isobutyl-1-methylxanthine (IBMX) on stimulated p53 of ALL cell lines. Western blotting analysis was performed to detect the expression of p53, phospho-p53, acetylated-p53, phospho-cAMP response element-binding protein (CREB), and Mdm2 proteins. Flow cytometry was applied to analyze apoptosis. The gene expression of p53 and its target genes was examined by real-time polymerase chain reaction. RESULTS: We show that elevation of cAMP levels in ALL cells exposed to DNA damage attenuates p53 accumulation. Inhibition of proteosome function with MG-132 reversed the inhibitory effect of cAMP on p53. However, targeting the p53-Mdm2 interaction did not rescue accumulated p53 from the destabilizing signal of cAMP. The specific agonist of the cAMP receptor exchange protein activated by cAMP had no effect on p53 expression in doxorubicin-treated NALM-6 cells, whereas PKA activators decreased p53 accumulation. DISCUSSION AND CONCLUSION: Our studies demonstrate that cAMP-PKA pathway regulates the sensitivity toward DNA-damaging agents via inhibition of a p53-dependent pathway in B-cell precursor ALL (BCP-ALL) cells.

Association of FXII 5'UTR 46C>T polymorphism with FXII activity and risk of thrombotic disease.

OBJECTIVE: Thrombotic diseases are caused by genetic and environmental factors. There are a number of well-characterized genetic defects that lead to increased risk of thrombosis. Results from previous studies have indicated that FXII is involved in pathogenesis of thrombophilic diseases. However, the results in this regard are highly controversial. Plasma FXII activity levels are strongly determined by a 46CγT polymorphism in the FXII gene. In the present study, the risk of thrombophilic diseases related to this polymorphism was investigated in a case-control study. METHODS: One hundred and sixty subjects were studied: 120 patients diagnosed with thrombophilia (96 venous thromboembolism, 24 arterial thrombosis), and 40 age-gender-matched controls. For each subject, FXII activity level was measured by a one-step clotting assay with FXII-deficient plasma, and 46CγT polymorphism was genotyped using a restriction fragment length polymorphism (RFLP) method. RESULTS: In this study, the previous observation that individuals with different genotypes for the 46 CγT polymorphism showed significant differences in Factor XII activity levels was confirmed. Most importantly, FXII activity ≤ 68% was associated with an increased risk of venous thrombosis with an adjusted OR of 4.7 (95%CI= 1.03-21.1, P=0.04). However, it was not a risk factor for arterial thrombosis with adjusted OR of 5 (95%CI= 0.91-27.1, P=0.09). In CT and TT genotype the adjusted ORs were respectively 2 (95%CI=0.9-4.4, P=0.11) and 2.3 (95%CI=0.45-11, P=0.48) for patients with venous thrombosis compared with the controls. Similarly, the adjusted ORs in arterial thrombosis were 1.2 (95%CI=0.4-3.6, P=0.76) for CT and 1.8 (95%CI=0.2-14.9, P=0.59) for TT genotype. Thus, we did not find any association of the mutated T allele in the heterozygous or homozygous state with an increased risk of both venous and arterial thrombosis. CONCLUSION: Lower FXII activity is not a risk factor; rather, it simply represents a risk marker for thrombosis.