Betulin, a Newly Characterized Compound in Acacia auriculiformis Bark, Is a Multi-Target Protein Kinase Inhibitor.

The purpose of this work is to investigate the protein kinase inhibitory activity of constituents from Acacia auriculiformis stem bark. Column chromatography and NMR spectroscopy were used to purify and characterize betulin from an ethyl acetate soluble fraction of acacia bark. Betulin, a known inducer of apoptosis, was screened against a panel of 16 disease-related protein kinases. Betulin was shown to inhibit Abelson murine leukemia viral oncogene homolog 1 (ABL1) kinase, casein kinase 1ε (CK1ε), glycogen synthase kinase 3α/ß (GSK-3 α/ß), Janus kinase 3 (JAK3), NIMA Related Kinase 6 (NEK6), and vascular endothelial growth factor receptor 2 kinase (VEGFR2) with activities in the micromolar range for each. The effect of betulin on the cell viability of doxorubicin-resistant K562R chronic myelogenous leukemia cells was then verified to investigate its putative use as an anti-cancer compound. Betulin was shown to modulate the mitogen-activated protein (MAP) kinase pathway, with activity similar to that of imatinib mesylate, a known ABL1 kinase inhibitor. The interaction of betulin and ABL1 was studied by molecular docking, revealing an interaction of the inhibitor with the ABL1 ATP binding pocket. Together, these data demonstrate that betulin is a multi-target inhibitor of protein kinases, an activity that can contribute to the anticancer properties of the natural compound and to potential treatments for leukemia.

Modulation of PTPN2/22 Function by Spermidine in CRISPR-Cas9-Edited T-Cells Associated with Crohn's Disease and Rheumatoid Arthritis.

Crohn's Disease (CD) and Rheumatoid Arthritis (RA) share some single nucleotide polymorphisms (SNPs) in protein tyrosine phosphatase non-receptor types 2 and 22 (PTPN2/22). Recently, we reported that clinical samples from CD and RA patients associated with PTPN2:rs478582 or PTPN22:rs2476601 genotypes were linked to overactive immune response and exacerbation of inflammation. Here, we investigated in vitro the effects of these SNPs in Jurkat T-cells using CRISPR-Cas9. All cells were evaluated for PTPN22/22 loss of function and effects on cell response. We measured gene expression via RT-qPCR and cytokines by ELISA. We also measured cell proliferation using a BrdU labeling proliferation ELISA, and T-cell activation using CD-25 fluorescent immunostaining. In PTPN2 SNP-edited cells, PTPN2 expression decreased by 3.2-fold, and proliferation increased by 10.2-fold compared to control. Likewise, expression of PTPN22 decreased by 2.4-fold and proliferation increased by 8.4-fold in PTPN22 SNP-edited cells. IFN-γ and TNF-α secretions increased in both edited cell lines. CD25 expression (cell activation) was 80.32% in PTPN2 SNP-edited cells and 85.82% in PTPN22 SNP-edited cells compared to 70.48% in unedited Jurkat T-cells. Treatment of PTPN2 and PTPN22-edited cells with a maximum 20 µM spermidine restored PTPN2/22 expression and cell response including cell proliferation, activation, and cytokines secretion. Most importantly, the effect of spermidine on edited cells restored normal expression and secretion of IFN-γ and TNF-α. The data clearly demonstrated that edited SNPs in PTPN2 or PTPN22 were associated with reduced gene expression, which resulted in an increase in cell proliferation and activation and overactive immune response. The data validated our earlier observations in CD and RA clinical samples. Surprisingly, spermidine restored PTPN2/22 expression in edited Jurkat T-cells and the consequent beneficial effect on cell response and inflammation. The study supports the use of polyamines dietary supplements for management of CD and in RA patients.

Whole transcriptome sequencing and integrated network analysis elucidates the effects of 3,8-Di-O-methylellagic acid 2-O-glucoside derived from Sanguisorba offcinalis L., a novel differentiation inducer on erythroleukemia cells.

Acute erythroid leukemia (AEL) is a rare and aggressive hematologic malignancy with no specific treatment. Sanguisorba officinalis L. (S. officinalis), a well-known traditional Chinese medicine, possesses potent anticancer activity. However, the active components of S. officinalis against AEL and the associated molecular mechanisms remain unknown. In this study, we predicted the anti-AML effect of S. officinalis based on network pharmacology. Through the identification of active components of S. officinalis, we found that 3,8-Di-O-methylellagic acid 2-O-glucoside (DMAG) not only significantly inhibited the proliferation of erythroleukemic cell line HEL, but also induced their differentiation to megakaryocytes. Furthermore, we demonstrated that DMAG could prolong the survival of AEL mice model. Whole-transcriptome sequencing was performed to elucidate the underlying molecular mechanisms associated with anti-AEL effect of DMAG. The results showed that the total of 68 miRNAs, 595 lncRNAs, 4030 mRNAs and 35 circRNAs were significantly differentially expressed during DMAG induced proliferation inhibition and differentiation of HEL cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that the differentially expressed miRNAs, lncRNAs, mRNAs and circRNAs were mainly involved in metabolic, HIF-1, MAPK, Notch pathway and apoptosis. The co-expression networks showed that miR-23a-5p, miR-92a-1-5p, miR-146b and miR-760 regulatory networks were crucial for megakaryocyte differentiation induced by DMAG. In conclusion, our results suggest that DMAG, derived from S. officinalis might be a potent differentiation inducer of AEL cells and provide important information on the underlying mechanisms associated with its anti-AEL activity.

Curcumin as an Epigenetic Therapeutic Agent in Myelodysplastic Syndromes (MDS).

Growth Factor Independence 1 (GFI1) is a transcription factor with an important role in the regulation of development of myeloid and lymphoid cell lineages and was implicated in the development of myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). Reduced expression of GFI1 or presence of the GFI1-36N (serine replaced with asparagine) variant leads to epigenetic changes in human and murine AML blasts and accelerated the development of leukaemia in a murine model of human MDS and AML. We and other groups previously showed that the GFI1-36N allele or reduced expression of GFI1 in human AML blasts is associated with an inferior prognosis. Using GFI1-36S, -36N -KD, NUP98-HOXD13-tg mice and curcumin (a natural histone acetyltransferase inhibitor (HATi)), we now demonstrate that expansion of GFI1-36N or -KD, NUP98-HODXD13 leukaemic cells can be delayed. Curcumin treatment significantly reduced AML progression in GFI1-36N or -KD mice and prolonged AML-free survival. Of note, curcumin treatment had no effect in GFI1-36S, NUP98-HODXD13 expressing mice. On a molecular level, curcumin treatment negatively affected open chromatin structure in the GFI1-36N or -KD haematopoietic cells but not GFI1-36S cells. Taken together, our study thus identified a therapeutic role for curcumin treatment in the treatment of AML patients (homo or heterozygous for GFI1-36N or reduced GFI1 expression) and possibly improved therapy outcome.

A novel dipeptide type inhibitor of the Wnt/ß-catenin pathway suppresses proliferation of acute myelogenous leukemia cells.

The Wnt/ß-catenin pathway is an attractive target for the treatment of acute myelogenous leukemia (AML), since aberrant activation of the Wnt/ß-catenin pathway contributes to carcinogenesis in various types of cancers including AML. Screening of an in-house compound library, constructed at Kyoto Pharmaceutical University, identified a novel compound designated "31" that was found to be an inhibitor of the Wnt/ß-catenin pathway. The compound inhibited T-cell factor (TCF) activity in a TCF firefly luciferase-reporter assay and suppressed the proliferation of several human AML cell lines in a dose-dependent manner. Compound 31 arrested the cell cycle of AML cells at the G1 stage and induced apoptosis. Decrease in protein and mRNA expression level of Wnt pathway-related molecules was confirmed by the analyses of western blotting and quantitative reverse transcription-polymerase chain reaction. In addition, compound 31 combined with idarubicin synergistically inhibited the proliferation of AML cells. In conclusion, these results strongly suggest that compound 31 has potential as a novel anti-AML agent targeting the Wnt/ß-catenin signaling pathway.

Systems biology drug screening identifies statins as enhancers of current therapies in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a B lymphoid malignancy highly dependent on the microenvironment. Despite new targeted therapies such as ibrutinib and venetoclax, disease progression and relapse remain an issue. CLL cell interactions with the supportive tissue microenvironment play a critical role in disease pathogenesis. We used a platform for drug discovery based on systems biology and artificial intelligence, to identify drugs targeting key proteins described to have a role in the microenvironment. The selected compounds were screened in CLL cell lines in the presence of stromal cells to mimic the microenvironment and validated the best candidates in primary CLL cells. Our results showed that the commercial drug simvastatin was the most effective and selective out of the tested compounds. Simvastatin decreased CLL cell survival and proliferation as well as cell adhesion. Importantly, this drug enhanced the antitumor effect of venetoclax and ibrutinib. We proposed that systems biology approaches combined with pharmacological screening could help to find new drugs for CLL treatment and to predict new combinations with current therapies. Our results highlight the possibility of repurposing widely used drugs such as statins to target the microenvironment and to improve the efficacy of ibrutinib or venetoclax in CLL cells.

Fatty acid synthase, a novel poor prognostic factor for acute lymphoblastic leukemia which can be targeted by ginger extract.

Altered metabolism of fatty acid synthesis is considered a hallmark characteristic of several malignancies, including acute lymphoblastic leukemia (ALL). To evaluate the impact of fatty acid synthase (FASN) on drug resistant ALL, bone marrow samples were collected from 65 pediatric ALLs, including 40 de novo and 25 relapsed patients. 22 non-cancer individuals were chosen as controls. Quantitative RT-PCR showed increased expression levels of FASN in drug resistant patients compared with the therapy responders. Single and combined treatment of malignant cells were analyzed using Annexin-V/PI double staining and MTT assays. Incubation of resistant primary cells with ginger showed simultaneous increased apoptosis rates and reduced FASN expression levels. Furthermore, docking studies demonstrated high affinity bindings between ginger derivatives and FASN thioesterase and ketosynthase domains, compared with their known inhibitors, fenofibrate and morin, respectively. Finally, combined treatment of in-house multidrug resistant T-ALL subline with ginger and dexamethasone induced drug sensitivity and down regulation of FASN expression, accordingly. To the best of our knowledge, this is the first study that introduces FASN upregulation as a poor prognostic factor for drug resistant childhood ALL. Moreover, it was revealed that FASN inhibition may be applied by ginger phytochemicals and overcome dexamethasone resistance, subsequently.

Novel therapeutic strategies for MLL-rearranged leukemias.

MLL rearrangement is one of the key drivers and generally regarded as an independent poor prognostic marker in acute leukemias. The standard of care for MLL-rearranged (MLL-r) leukemias has remained largely unchanged for the past 50 years despite unsatisfying clinical outcomes, so there is an urgent need for novel therapeutic strategies. An increasing body of evidence demonstrates that a vast number of epigenetic regulators are directly or indirectly involved in MLL-r leukemia, and they are responsible for supporting the aberrant gene expression program mediated by MLL-fusions. Unlike genetic mutations, epigenetic modifications can be reversed by pharmacologic targeting of the responsible epigenetic regulators. This leads to significant interest in developing epigenetic therapies for MLL-r leukemia. Intriguingly, many of the epigenetic enzymes also involve in DNA damage response (DDR), which can be potential targets for synthetic lethality-induced therapies. In this review, we will summarize some of the recent advances in the development of epigenetic and DDR therapeutics by targeting epigenetic regulators or protein complexes that mediate MLL-r leukemia gene expression program and key players in DDR that safeguard essential genome integrity. The rationale and molecular mechanisms underpinning the therapeutic effects will also be discussed with a focus on how these treatments can disrupt MLL-fusion mediated transcriptional programs and impair DDR, which may help overcome treatment resistance.

An Extract of Transgenic Senna obtusifolia L. Hairy Roots with Overexpression of PgSS1 Gene in Combination with Chemotherapeutic Agent Induces Apoptosis in the Leukemia Cell Line.

Many biologically-active plant-derived compounds have therapeutic or chemopreventive effects. The use of plant in vitro cultures in conjunction with modern genetic engineering techniques allows greater amounts of valuable secondary metabolites to be obtained without interfering with the natural environment. This work presents the first findings concerning the acquisition of transgenic hairy roots of Senna obtusifolia overexpressing the gene encoding squalene synthase 1 from Panax ginseng (PgSS1) (SOPSS hairy loot lines) involved in terpenoid biosynthesis. Our results confirm that one of PgSS1-overexpressing hairy root line extracts (SOPSS2) possess a high cytotoxic effect against a human acute lymphoblastic leukemia (NALM6) cell line. Further analysis of the cell cycle, the expression of apoptosis-related genes (TP53, PUMA, NOXA, BAX) and the observed decrease in mitochondrial membrane potential also confirmed that the SOPSS2 hairy root extract displays the highest effects; similar results were also obtained for this extract combined with doxorubicin. The high cytotoxic activity, observed both alone or in combination with doxorubicin, may be due to the higher content of betulinic acid as determined by HPLC analysis. Our results suggest synergistic effects of tested extract (betulinic acid in greater amount) with doxorubicin which may be used in the future to develop new effective strategies of cancer chemosensitization.

Dissecting the role of the CXCL12/CXCR4 axis in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) is the most common adult acute leukaemia with the lowest survival rate. It is characterised by a build-up of immature myeloid cells anchored in the protective niche of the bone marrow (BM) microenvironment. The CXCL12/CXCR4 axis is central to the pathogenesis of AML as it has fundamental control over AML cell adhesion into the protective BM niche, adaptation to the hypoxic environment, cellular migration and survival. High levels of CXCR4 expression are associated with poor relapse-free and overall survival. The CXCR4 ligand, CXCL12 (SDF-1), is expressed by multiple cells types in the BM, facilitating the adhesion and survival of the malignant clone. Blocking the CXCL12/CXCR4 axis is an attractive therapeutic strategy providing a 'multi-hit' therapy that both prevents essential survival signals and releases the AML cells from the BM into the circulation. Once out of the protective niche of the BM they would be more susceptible to destruction by conventional chemotherapeutic drugs. In this review, we disentangle the diverse roles of the CXCL12/CXCR4 axis in AML. We then describe multiple CXCR4 inhibitors, including small molecules, peptides, or monoclonal antibodies, which have been developed to date and their progress in pre-clinical and clinical trials. Finally, the review leads us to the conclusion that there is a need for further investigation into the development of a 'multi-hit' therapy that targets several signalling pathways related to AML cell adhesion and maintenance in the BM.

Huai Qi Huang Potentiates Dexamethasone-Mediated Lethality in Acute Lymphoblastic Leukemia Cells by Upregulating Glucocorticoid Receptor α.

BACKGROUND Glucocorticoids are important components of a number of chemotherapeutic regimens used to treat pediatric acute lymphoblastic leukemia (ALL). A primary cause of treatment failure of ALL is acquired resistance to glucocorticoids. Recently, traditional Chinese medicines were effectively used to treat solid tumors. Thus, the aim of this study was to investigate whether Huai Qi Huang (HQH), a traditional Chinese medicine, increased the efficacy of glucocorticoids in the treatment of ALL, and if so, to determine the underlying mechanism. MATERIAL AND METHODS Various concentrations of HQH were used to treat Jurkat and Nalm-6 cells for 24 to 72 hours. Subsequently, cells were co-treated with HQH and the glucocorticoid receptor agonist, dexamethasone (DEX), or a MEK inhibitor (PD98059) to verify the synergistic effects on apoptosis in Jurkat and Nalm-6 cells for 24 hours. Cell Counting Kit-8 assay and flow cytometry were used to measure cell viability and apoptosis, respectively. Protein and mRNA expression levels were assessed using western blotting and quantitative polymerase chain reaction. RESULTS The results revealed that cell survival was reduced and apoptosis was increased as the HQH concentration was increased, and this was accompanied with increases in the levels of BAX, cleaved-caspase-3 and glucocorticoid receptor alpha (GRalpha) and decreases in the levels of Bcl-2 and phospho-ERK (pERK). Glucocorticoid receptor ß (GRß) and total ERK (t-ERK) had no significant changes. Combined treatment with HQH and DEX or PD98059 increased apoptosis in Jurkat and Nalm-6 cells, and concurrently increased BAX, cleaved-caspase-3, GILZ, NFKBIA, and GRalpha and decreased Bcl-2 and pERK. CONCLUSIONS HQH enhanced the sensitivity of ALL cells to glucocorticoids by increasing the expression of GRalpha and inhibiting the MEK/ERK pathway, thus providing a rational foundation for the treatment of ALL with HQH.

Fetal liver Mll-AF4+ hematopoietic stem and progenitor cells respond directly to poly(I:C), but not to a single maternal immune activation.

T(4;11) MLL-AF4 acute leukemia is one of the most aggressive malignancies in infant and pediatric populations. Epidemiological and functional studies have highlighted the influence of an overstimulation of the immune system on leukemia development. This study aimed at assessing if the cell-of-origin of t(4;11) MLL-AF4 acute leukemia is sensitive to a viral or bacterial mimic and if maternal immune activation can lead to a full-blown leukemia. To answer this, we used the Mll-AF4 pre-leukemia mouse model that initiates the expression of Mll-AF4 in the first definitive hematopoietic cells formed during embryonic development. We observed an increase in proliferation upon hematopoietic differentiation of fetal liver Mll-AF4+ Lineage-Sca1+ckit+ (LSK) cells exposed to the immune stimulants, poly(I:C) or LPS/lipopolysaccharide. This was accompanied by increased expression of a subset of MLL-AF4 signature genes and members of the Toll-like receptor signaling pathways in fetal liver Mll-AF4+ LSK exposed to poly(I:C), suggesting that the cell-of-origin responds to inflammatory stimuli. Maternal immune activation using a single dose of poly(I:C) did not lead to the development of leukemia in Mll-AF4+ and control offspring. Instead, aging MLL-AF4+ mice showed an increased proportion of T-lymphoid cells in the spleen, lost their B-lymphoid bias, and had decreased frequencies of hematopoietic stem and multipotent progenitor cells. Overall, this study suggests that the fetal liver Mll-AF4+ LSK cells are sensitive to direct exposure to inflammatory stimuli, especially poly(I:C); however, maternal immune activation induced by a single exposure to poly(I:C) is not sufficient to initiate MLL-AF4 leukemogenesis.

Epigenetic Modifiers in Myeloid Malignancies: The Role of Histone Deacetylase Inhibitors.

Myeloid hematological malignancies are clonal bone marrow neoplasms, comprising of acute myeloid leukemia (AML), the myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML), the myeloproliferative neoplasms (MPN) and systemic mastocytosis (SM). The field of epigenetic regulation of normal and malignant hematopoiesis is rapidly growing. In recent years, heterozygous somatic mutations in genes encoding epigenetic regulators have been found in all subtypes of myeloid malignancies, supporting the rationale for treatment with epigenetic modifiers. Histone deacetylase inhibitors (HDACi) are epigenetic modifiers that, in vitro, have been shown to induce growth arrest, apoptotic or autophagic cell death, and terminal differentiation of myeloid tumor cells. These effects were observed both at the bulk tumor level and in the most immature CD34⁺38- cell compartments containing the leukemic stem cells. Thus, there is a strong rationale supporting HDACi therapy in myeloid malignancies. However, despite initial promising results in phase I trials, HDACi in monotherapy as well as in combination with other drugs, have failed to improve responses or survival. This review provides an overview of the rationale for HDACi in myeloid malignancies, clinical results and speculations on why clinical trials have thus far not met the expectations, and how this may be improved in the future.

A Systems Biology-Based Approach to Uncovering Molecular Mechanisms Underlying Effects of Traditional Chinese Medicine Qingdai in Chronic Myelogenous Leukemia, Involving Integration of Network Pharmacology and Molecular Docking Technology.

BACKGROUND The method of multiple targets overall control is increasingly used to predict the main active ingredient and potential target group of Chinese traditional medicines and to determine the mechanisms involved in their curative effects. Qingdai is the main traditional Chinese medicine used in the treatment of chronic myelogenous leukemia (CML), but the complex active ingredients and antitumor targets in treatment of CML have not been clearly defined in previous studies. MATERIAL AND METHODS We constructed a protein-protein interaction network diagram of CML with 638 nodes (proteins) and 1830 edges, based on the biological function of chronic myelocytic leukemia by use of Cytoscape, and we determined 19 key gene nodes in the CML molecule by network topological properties analysis in a data bank. Then, we used the Surflex-dock plugin in SYBYL7.3 docking and acquired the protein crystal structures of key genes involved in CML from the chemical composition of the traditional Chinese medicine Qingdai with key proteins in CML networks. RESULTS According to the score and the spatial structure, the pharmacodynamically active ingredients of Qingdai are Isdirubin, Isoindigo, N-phenyl-2-naphthylamine, and Isatin, among which Isdirubin is the most important. We further screened the most effective activity key protein structures of CML to find the best pharmacodynamically active ingredients of Qingdai, according to the binding interactions of the inhibitors at the catalytic site performed in best docking combinations. CONCLUSIONS The results suggest that Isdirubin plays a role in resistance to CML by altering the expressions of PIK3CA, MYC, JAK2, and TP53 target proteins. Network pharmacology and molecular docking technology can be used to search for possible reactive molecules in traditional chinese medicines (TCM) and to elucidate their molecular mechanisms.

c-MYC and reactive oxygen species play roles in tetrandrine-induced leukemia differentiation.

Tetrandrine is a broadly used bisbenzylisoquinoline alkaloid component of traditional Chinese medicine that has antitumor effects in some cancer types. In this study, we investigated the effects of tetrandrine on leukemia in vitro and in vivo. The results showed that tetrandrine effectively induced differentiation and autophagy in leukemia cells. In addition, tetrandrine treatment activated the accumulation of reactive oxygen species (ROS) and inhibited c-MYC protein expression. Further, we found that treatment with the ROS scavengers N-acetyl-L-cysteine (NAC) and Tiron as well as overexpression of c-MYC reduced tetrandrine-induced autophagy and differentiation. Moreover, a small molecular c-MYC inhibitor, 10058-F4, enhanced the tetrandrine-induced differentiation of leukemia cells. These results suggest that ROS generation and c-MYC suppression play important roles in tetrandrine-induced autophagy and differentiation, and the results from in vivo experiments were consistent with those from in vitro studies. Therefore, our data suggest that tetrandrine may be a promising agent for the treatment of leukemia.

Green tea polyphenol EGCG causes anti-cancerous epigenetic modulations in acute promyelocytic leukemia cells.

Green tea (Camellia sinensis) catechin epigallocatechin-3-gallate (EGCG) has been shown to possess diverse anti-cancerous properties. We demonstrated EGCG ability to inhibit acute promyelocytic leukemia (APL) cell proliferation and cause apoptosis. In addition, quantitative real-time polymerase chain reaction (RT-qPCR) analysis revealed elevated expression of genes associated with cell cycle arrest and differentiation (p27, PCAF, C/EBPα, and C/EBPɛ). Furthermore, EGCG caused anti-cancerous epigenetic changes: downregulation of epigenetic modifiers DNMT1, HDAC1, HDAC2, and G9a was observed by RT-qPCR analysis. Reduced amount of H3K9me2 after treatment with EGCG confirmed G9a downregulation. Polycomb repressive complex 2 (PRC2) core components were also shown to be downregulated in gene and protein level. Chromatin immunoprecipitation (ChIP) analysis revealed that EGCG treatment enhanced hyperacetylated H4 and acetylated H3K14 histones binding to the promoter regions of p27, PCAF, C/EBPα, and C/EBPɛ and reduced binding effect to PRC2 core component genes EZH2, SUZ12, and EED. Our results indicate that EGCG, as cell proliferation inhibitor and epigenetic modifier, might be useful for APL treatment.

Personalizing Chinese medicine by integrating molecular features of diseases and herb ingredient information: application to acute myeloid leukemia.

Traditional Chinese Medicine (TCM) has been widely used as a complementary medicine in Acute Myeloid Leukemia (AML) treatment. In this study, we proposed a new classification of Chinese Medicines (CMs) by integrating the latest discoveries in disease molecular mechanisms and traditional medicine theory. We screened out a set of chemical compounds on basis of AML differential expression genes and chemical-protein interactions and then mapped them to Traditional Chinese Medicine Integrated Database. 415 CMs contain those compounds and they were categorized into 8 groups according to the Traditional Chinese Pharmacology. Pathway analysis and synthetic lethality gene pairs were applied to analyze the dissimilarity, generality and intergroup relations of different groups. We defined hub CM pairs and alternative CM groups based on the analysis result and finally proposed a formula to form an effective anti-AML prescription which combined the hub CM pairs with alternative CMs according to patients' molecular features. Our method of formulating CMs based on patients' stratification provides novel insights into the new usage of conventional CMs and will promote TCM modernization.

Effects of bufalin on up-regulating methylation of Wilm's tumor 1 gene in human erythroid leukemic cells.

OBJECTIVE: To explore the effects of bufalin on inhibiting proliferation, up-regulating methylation of Wilm' tumor 1 gene (WT1) as well as its possible mechanisms in human erythroid leukemic (HEL) cells. METHODS: The HEL cells were treated with bufalin at various concentrations to observe cellular morphology, proliferation assay and cell cycle. The mRNA and protein expression levels of WT1 were detected by reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry, DNA methylation of WT1 and protein expression levels of DNA methyltransferase 3a (DNMT3a) and DNMT3b were analyzed by methylation-specific PCR, and Western blot respectively. RESULTS: The bufalin was effective to inhibit proliferation of HEL cells in a dose-dependent manner, their suppression rates were from 23.4%±2.1% to 87.2%±5.4% with an half maximal inhibit concentration (IC50) of 0.046 µmol/L. Typical apoptosis morphology was observed in bufalin-treated HEL cells. The proliferation index of cell cycle decreased from 76.4%±1.9% to 49.7%±1.3%. The expression levels of WT1 mRNA and its protein reduced gradually with increasing doses of bufalin, meanwhile, the methylation status of WT1 gene changed from unmethylated into partially or totally methylated. While, the expression levels of DNMT3a and DNMT3b protein gradually increased by bufalin treatment in a dose-dependent manner. CONCLUSIONS: Bufalin can not only significantly inhibit the proliferation of HEL cells and arrest cell cycle at G0/G1 phase, but also induce cellular apoptosis and down-regulate the expression level of WT1. Our results provide the evidence of bufalin for anti-leukemia, its mechanism may involve in increasing WT1 methylation status which is related to the up-regulation of DNMT3a and DNMT3b proteins in erythroid leukemic HEL cells.

Application of vitamin D and vitamin D analogs in acute myelogenous leukemia.

Acute myeloid leukemia (AML) is characterized by the accumulation of malignant, transformed immature hematopoietic myeloid precursors that have lost their ability to differentiate and proliferate normally. Current treatment for AML requires intensive cytotoxic chemotherapy and results in significant morbidity and mortality, especially in older patients. Effective and better-tolerated treatment is urgently needed. Studies have shown that 1α,25-dihydroxyvitamin D3 (1,25-D3, active VD3) or vitamin D analogs (VDAs) can potently differentiate AML cells in vitro and ex vivo, which led to early clinical trials in AML and myelodysplastic syndrome patients. However, one major limiting factor in the clinical application of active VD3 or VDAs is the supraphysiologic dose required, which results in systemic hypercalcemia. Several important questions (i.e., dosage, method of delivery, metabolism of 1,25-D3 in situ, systemic hypercalcemia, and mechanisms of action of combination treatment) have to be addressed before vitamin D treatment can be applied to the clinical setting. This review focuses on 1,25-D3's mechanism of action in AML, preclinical data, and clinical trial outcomes, with an emphasis on major roadblocks to successful trials and suggestions for future directions.

Extracellular ATP induces apoptosis through P2X7R activation in acute myeloid leukemia cells but not in normal hematopoietic stem cells.

Recent studies have shown that high ATP levels exhibit direct cytotoxic effects on several cancer cells types. Among the receptors engaged by ATP, P2X7R is the most consistently expressed by tumors. P2X7R is an ATP-gated ion channel that could drive the opening of a non-selective pore, triggering cell-death signal. We previously demonstrated that acute myeloid leukemia (AML) cells express high level of P2X7R. Here, we show that P2X7R activation with high dose ATP induces AML blast cells apoptosis. Moreover, P2X7R is also expressed on leukemic stem/progenitor cells (LSCs) which are sensitive to ATP-mediated cytotoxicity. Conversely, this cytotoxic effect was not observed on normal hematopoietic stem/progenitor cells (HSCs). Notably, the antileukemic activity of ATP was also observed in presence of bone marrow stromal cells and its addition to the culture medium enhanced cytosine arabinoside cytotoxicity despite stroma-induced chemoresistance. Xenotransplant experiments confirmed ATP antineoplastic activity in vivo.Overall, our results demonstrate that P2X7R stimulation by ATP induced a therapeutic response in AML at the LSC level while the normal stem cell compartment was not affected. These results provide evidence that ATP would be promising for developing innovative therapy for AML.