Patients with acute myeloid leukemia can be subclassified based on the constitutive cytokine release of the leukemic cells; the possible clinical relevance and the importance of cellular iron metabolism

Objective: Acute myeloid leukaemia (AML) is a heterogeneous malignancy; we studied how the constitutive cytokine release by the AML cells varies among patients.

Methods: We investigated the constitutive release of 28 mediators during in vitro culture for 79 consecutive patients.

Results: Constitutive cytokine release profiles differed among patients, and hierarchical clustering identified three subsets with high, intermediate and low release, respectively. The high-release subset showed high levels of most mediators, usually monocytic differentiation as well as altered mRNA expression of proteins involved in intracellular iron homeostasis and molecular trafficking; this subset also included 4 out of 6 patients with inv(16). Spontaneous in vitro apoptosis did not differ among the subsets. For the high-release patients, cytokines were released both by CD34⁺ and CD34^? cells. The mRNA and released protein levels showed statistically significant correlations only for eleven of the cytokines. The overall survival after intensive anti-leukemic therapy was significantly higher for high-release compared with low-release patients. Pharmacological targeting of iron metabolism (iron chelation, transferrin receptor blocking) altered the cytokine release profile.

Conclusions: Subclassification of AML patients based on the constitutive cytokine release may be clinically relevant and a part of a low-risk (i.e. chemosensitive) AML cell phenotype.     

Inhibitors of LSD1 as a potential therapy for acute myeloid leukemia

Introduction: Epigenetic dysregulation plays a critical role in the pathogenesis of acute myeloid leukemia (AML). Alterations in histone methylation lead to aberrant silencing of expression of multiple genes involved in tumor suppression and cell cycling, resulting in myeloid maturation arrest and proliferation of early myeloid progenitors. One promising approach targeting chromatin regulatory proteins is inhibition of lysine specific demethylase-1 (LSD1), an enzyme responsible for demethylation of histone H3 as well as other functions

Areas covered: Available literature on LSD1 in normal and malignant hematopoiesis was identified in PubMed and reviewed. Areas addressed here include the biology of LSD1, pharmacologic inhibitors, and preclinical data supporting the rationale for LSD1 inhibition in AML therapy.

Expert opinion: LSD1 inhibitors represent a promising novel epigenetic approach for AML therapy. Preclinical studies have revealed that pharmacologic LD1 inhibitors function primarily by altering stem cell programs and restoring myeloid differentiation to AML cells. These effects are markedly enhanced in combination with trans-retinoic acid or histone deacetylase inhibitors with little toxicity. Currently, multiple oral LSD1 inhibitors are undergoing phase 1 investigation in patients with AML. The results of these clinical trials are eagerly awaited.     

Targeting the angiopoietin (Ang)/Tie-2 pathway in the crosstalk between acute myeloid leukaemia and endothelial cells: studies of Tie-2 blocking antibodies,exogenous Ang-2 and inhibition of constitutive agonistic Ang-1 release

Background: The Tie-2 receptor can bind its agonistic ligand Angiopoietin-1 (Ang-1) and the potential antagonist Ang-2. Tie-2 can be expressed both by primary human acute myeloid leukaemia (AML) cells and endothelial cells, and Tie-2-blocking antibodies are now being evaluated in clinical trials for cancer treatment. Design and methods: We investigated the effects of Tie-2-blocking antibodies, exogenous Ang-2 and pharmacological agents on AML cell proliferation and the release of angioregulatory mediators. Results: Tie-2-blocking antibodies had a growth inhibitory effect on human AML cells co-cultured with microvascular endothelial cells, but this inhibition was not observed when leukaemic cells were co-cultured with fibroblasts or osteoblasts. AML cell viability in co-cultures was not altered by anti-Tie-2. Furthermore, anti-Tie-2 decreased hepatocyte growth factor (HGF) levels and increased CXCL8 levels in co-cultures, whereas the levels of endocan (a proteoglycan released by endothelial cells) were not altered. The only significant effects of exogenous Ang-2 were decreased levels of HGF and endocan. Constitutive AML cell release of agonistic Ang-1 was decreased by the proteasomal inhibitor bortezomib and the specific IκB-kinase/NFκB inhibitor BMS-345541. Conclusion: We conclude that various strategies for inhibition of Tie-2-mediated signalling should be considered in AML therapy, possibly in combination with other antiangiogenic strategies.     

Temporal expression of transporters and receptors in a rat primary co-culture blood–brain barrier model

Abstract

1.?The more relevant primary co-cultures of brain microvessel endothelial cells and astrocytes (BMEC) are less utilized for screening of potential CNS uptake when compared to intestinal and renal cell lines.

2.?In this study, we characterized the temporal mRNA expression of major CNS transporters and receptors, including the transporter regulators Pxr, Ahr and Car in a rat BMEC co-cultured model. Permeability was compared with the Madin–Darby canine kidney (MDCKII)-MDR1 cell line and rat brain in situ perfusion model.

3.?Our data demonstrated differential changes in expression of individual transporters and receptors over the culture period. Expression of ATP-binding cassette transporters was better retained than that of solute carrier transporters. The insulin receptor (IR) was best maintained among investigated receptors. AhR demonstrated high mRNA expression in rat brain capillaries and expression was better retained than Pxr or Car in culture. Mdr1b expression was up-regulated during primary culture, albeit Mdr1a mRNA levels were much higher. P-gp and Bcrp-1 were highly expressed and functional in this in vitro system.

4.?Permeability measurements with 18 CNS marketed drugs demonstrated weak correlation between rBMEC model and rat in situ permeability and moderate correlation with MDCKII-MDR1 cells.

5.?We have provided appropriate methodologies, as well as detailed and quantitative characterization data to facilitate improved understanding and rational use of this in vitro rat BBB model.     

Effects of a prostaglandin F2alpha derivative glaucoma drug on EGF expression and E-cadherin expression in a corneal epithelial cell line

Abstract

Purpose: We examined the effects of travoprost on cell proliferation-related signals and E-cadherin expression in vitro and in situ in order to obtain evidence to support the hypothesis that topical travoprost impairs the integrity of the corneal epithelium.

Methods: A human corneal epithelial cell culture was treated with travoprost (0.4?mg/ml) and/or PD168393 (an EGF receptor inhibitor, 10?μM). The culture was then processed for cell proliferation, an mRNA expression analysis of epidermal growth factor (EGF) and E-cadherin, and protein expression analysis of E-cadherin by immunocytochemistry and Western blotting. The eyes of C57/BL6 mice were incubated in serum-free medium plus travoprost (0.4?mg/ml) and/or PD168393 (10?μM). After being cultured for 24?h, the expression patterns of phospho-EGFR, phospho-ERK, E-cadherin, and Ki67 were immunohistochemically examined in paraffin sections.

Results: The addition of travoprost up-regulated EGF mRNA expression and cell proliferation in the corneal epithelial cell culture, and this was cancelled by the addition of PD168393. This FP agonist also decreased E-cadherin expression levels in the cell–cell contact zone, and this was cancelled by the addition of PD168393. In the organ culture, the addition of travoprost to the medium up-regulated the expression of phospho-EGFR and phospho-ERK as well as cell proliferation, and down-regulated the expression of E-cadherin in the corneal epithelium, particularly in basal cells, whereas PD168393 reversed these effects.

Conclusions: Travoprost activates epithelial cell proliferation by up-regulating an EGF-related signal in association with the suppression of E-cadherin localization in the cell–cell contact zone. Modulation of the EGF signal may be a strategy to minimize the negative impact of this mitogen on reformation of corneal barrier function during epithelial renewal.     

Effects of indirubin and isatin on cell viability,mutagenicity, genotoxicity and BAX/ERCC1 gene expression

Context: Indigofera suffruticosa Miller (Fabaceae) and I. truxillensis Kunth produce compounds, such as isatin (ISA) and indirubin (IRN), which possess antitumour properties. Their effects in mammalian cells are still not very well understood.

Objective: We evaluated the activities of ISA and/or IRN on cell viability and apoptosis in vitro, their genotoxic potentials in vitro and in vivo, and the IRN- and ISA-induced expression of ERCC1 or BAX genes.

Materials and methods: HeLa and/or CHO-K1 cell lines were tested (3 or 24?h) in the MTT, Trypan blue exclusion, acridine orange/ethidium bromide, cytokinesis-blocked micronucleus (CBMN) and comet (36, 24 and 72?h) tests after treatment with IRN (0.1 to 200?μM) or ISA (0.5 to 50?μM). Gene expression was measured by RT-qPCR in HeLa cells. Swiss albino mice received IRN (3, 4 or 24?h) by gavage (50, 100 and 150?mg/kg determined from the LD₅₀ – 1?g/kg b.w.) and submitted to comet assay in vivo.

Results: IRN reduced the viability of CHO-K1 (24?h; 5 to 200?μM) and HeLa cells (10 to 200?μM), and was antiproliferative in the CBMN test (CHO-K1: 0.5 to 10?μM; HeLa: 5 and 10?μM). The drug did not induce apoptosis, micronucleus neither altered gene expression. IRN and ISA were genotoxic for HeLa cells (3 and 24?h) at all doses tested. IRN (100 and 150?mg/kg) also induced genotoxicity in vivo (4?h).

Conclusion: IRN and ISA have properties that make them candidates as chemotherapeutics for further pharmacological investigations.     

Platelet lysate embedded scaffolds for skin regeneration

Introduction: The work presents the development of acellular scaffolds extemporaneously embedded with platelet lysate (PL), as an innovative approach in the field of tissue regeneration/reparation. PL embedded scaffolds should have a tridimensional architecture to support cell migration and growth, in order to restore skin integrity. For this reason, chondroitin sulfate (CS) was associated with sodium alginate (SA) to prepare highly porous systems.

Methods: The developed scaffolds were characterized for chemical stability to γ-radiation, morphology, hydration and mechanical properties. Moreover, the capability of fibroblasts and endothelial cells to populate the scaffold was evaluated by means of proliferation test 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and confocal laser scanning microscopy study. The scaffolds, not altered by sterilization, were characterized by limited swelling and high flexibility, by foam-like structure with bubbles that formed a high surface area and irregular texture suitable for cell adhesion.

Results: Cell growth and scaffold population were evident on the bubble surface, where the cells appeared anchored to the scaffold structure.

Conclusion: Scaffold network based on CS and SA demonstrated to be an effective support to enhance and to allow fibroblasts and endothelial cells (human umbilical vein endothelial cells, HUVEC) adhesion and proliferation. In particular, it could be hypothesized that cell adhesion was facilitated by the synergic effect of PL and CS. Although further in vivo evaluation is needed, on the basis of in vitro results, PL embedded scaffolds seem promising systems for skin wound healing.     

靶向干预SETDB1对急性髓系白血病的治疗作用

目的 探究组蛋白甲基转移酶SETDB1在急性髓系白血病(acute myeloid leukemia,AML)恶性进展中的作用及干扰SETDB1对AML的治疗效果。方法 基于TCGA、GTEx、TARGET数据库,分析SETDB1在不同肿瘤的表达情况,并对比分析在AML骨髓细胞和正常骨髓细胞中的表达情况,进一步分析SETDB1的表达与AML患者不同风险分层、不同FAB(French-American-British)分型及微小残留病灶(minimalresidualdisease,MRD)的相关性;对SETDB1高/低表达患者进行基因富集分析。通过RT-qPCR考察shRNA序列对SETDB1的沉默效率;通过台盼蓝染色法考察沉默SETDB1对AML细胞的增殖及活力的影响;通过检测细胞表面抗原CD11b、CD14的比例、细胞核形态和氯化硝基四氮唑蓝(NBT)还原能力考察沉默SETDB1对AML细胞的分化治疗作用。结果 与其他肿瘤相比,SETDB1在AML中显著高表达,且在AML中的表达水平显著高于正常骨髓细胞。SETDB1在AML高风险患者中、MRD残留患者中也显著高表达。沉默SETDB...     

SD大鼠神经干细胞评价药物神经毒性模型研究

目的：应用SD大鼠神经干细胞评价药物的神经毒性,为新药早期筛选和临床前安全性评价提供体外替代方法。方法：体外培养SD大鼠神经干细胞,传代后得到稳定的第二代神经球。以已知具有神经毒性的长春新碱、顺铂、瑞芬太尼、丙泊酚注射液、丙戊酸钠、苯妥英钠、丙烯酰胺、乙醇、氧化铁纳米粒子作为神经毒性阳性物质,以培养基作为神经毒性空白对照品;以没有神经毒性且具有促进神经细胞生长的神经生长因子作为检测模型的敏感性;以验证SD大鼠神经干细胞模型对神经毒性药物的检出能力。结果：长春新碱、顺铂、丙泊酚注射液、苯妥英钠、丙烯酰胺、氧化铁纳米粒子可引起全部或部分神经球解离破碎,神经干细胞坏死。顺铂、丙戊酸钠和苯妥英钠可见显著性的抑制神经球聚集。长春新碱、顺铂、瑞芬太尼、氧化铁纳米粒子、丙泊酚注射液、丙戊酸钠、苯妥英钠、丙烯酰胺、乙醇均表现剂量相关性的神经干细胞增殖毒性作用。神经生长因子可见促进神经球聚集及神经干细胞增殖。结论：本文以SD大鼠神经干细胞模型,以神经干细胞体外生长发育指标,验证了已知神经毒性抗肿瘤药物、麻醉剂、抗癫痫药物等的神经毒性特征。评价结果与这些药物已知的神经毒性作用特点一致,该评价方法可作为药物神经毒性临床前安全性评价研究的体外替代试验。     

In vitro and in vivo models of celiac disease

ABSTRACT

Introduction: Human in vitro blood-brain barrier (BBB) models could be important tools for studying BBB development, maintenance and regulation. However, our capacity to obtain information from these models is still limited in part because only in recent years have (i) these models been derived from non-brain cell sources (e.g. stem cells), (ii) microfluidic systems been developed to recapitulate aspects of BBB physiology and (iii) new insights into the molecular and cellular mechanisms of BBB diseases (e.g. Huntington´s, Allan-Herndon-Dudley Syndrome) been described.

Area covered: This article reviews the technological advances in the derivation of human cells from the neurovascular unit using stem cells and the creation of personalized BBB models generated from patients with neurodegenerative diseases. It also reviews the scientific advances generated from in vitro BBB models.

Expert opinion: The recent technological advances in the derivation of human cells from the neurovascular unit from stem cells as well as in the generation of BBB-on-a-chip that recapitulate in vitro part of the BBB physiology are significant to generate more robust BBB models; however, a considerable effort is still needed to validate the potential of these models to recapitulate the in vivo cellular and molecular mechanisms, in particular regarding BBB function in health and disease.     

The acute exposure effects of inhaled nickel nanoparticles on murine endothelial progenitor cells

Abstract

Introduction: The discovery of endothelial progenitor cells (EPCs) may help to explain observed cardiovascular effects associated with inhaled nickel nanoparticle exposures, such as increases in vascular inflammation, generation of reactive oxygen species, altered vasomotor tone and potentiated atherosclerosis in murine species.

Methods: Following an acute whole body inhalation exposure to 500?µg/m³ of nickel nanoparticles for 5?h, bone marrow EPCs from C57BL/6 mice were isolated. EPCs were harvested for their RNA or used in a variety of assays including chemotaxis, tube formation and proliferation. Gene expression was assessed for important receptors involved in EPC mobilization and homing using RT-PCR methods. EPCs, circulating endothelial progenitor cells (CEPCs), circulating endothelial cells (CECs) and endothelial microparticles (EMPs) were quantified on a BD FACSCalibur to examine endothelial damage and repair associated with the exposure.

Results and conclusions: Acute exposure to inhaled nickel nanoparticles significantly increased both bone marrow EPCs as well as their levels in circulation (CEPCs). CECs were significantly elevated indicating that endothelial damage occurred due to the exposure. There was no significant difference in EMPs between the two groups. Tube formation and chemotaxis, but not proliferation, of bone marrow EPCs was impaired in the nickel nanoparticle exposed group. These results coincided with a decrease in the mRNA of receptors involved in EPC mobilization and homing. These data provide new insight into how an acute nickel nanoparticle exposure to half of the current Occupational Safety & Health Administration (OSHA) permissible exposure limit may adversely affect EPCs and exacerbate cardiovascular disease states.     

西奥罗尼杀伤FLT3-ITD阳性急性髓系白血病细胞的作用及机制研究

目的 研究多靶点激酶抑制剂西奥罗尼对FLT3-ITD阳性急性髓系白血病（acute myeloid leukemia,AML）细胞的杀伤作用及其机制。方法 CCK8和集落形成试验检测不同浓度西奥罗尼对FLT3-ITD阳性AML细胞增殖和活力的影响;Annexin V/PI双染法检测西奥罗尼对FLT3-ITD阳性AML细胞株和原代细胞的凋亡诱导情况;Click-iT^® EdU试剂盒评估细胞周期的分布情况;裸鼠皮下瘤模型用于评估西奥罗尼对FLT3-ITD阳性AML细胞体内的杀伤作用;Western blotting探讨西奥罗尼杀伤FLT3-ITD阳性AML的潜在作用机制。结果 在FLT3-ITD阳性AML细胞株模型上,西奥罗尼具有降低细胞的活力、抑制细胞的集落形成、阻滞细胞周期和诱导凋亡的作用;通过8例原代FLT3-ITD阳性AML细胞研究发现,西奥罗尼对原代细胞同样具有诱导凋亡的潜能;裸鼠皮下瘤试验表明西奥罗尼降低FLT3-ITD阳性AML细胞体内增殖能力,延缓肿瘤的生长,而无明显不良反应;机制研究显示西奥罗尼降低VEGFR2的磷酸化水平,且下调其下游的MEK/Erk通路的磷酸化作用。结论 本研究明确了西奥罗尼在体外和体内具有杀伤FLT3-ITD阳性AML细胞的的潜能,抑制VEGFR2/MEK/Erk通路的活性可能是西奥罗尼发挥杀伤FLT3-ITD阳性AML细胞的潜在作用机制。     

Therapy of acute myeloid leukemia: therapeutic targeting of tyrosine kinases

Introduction: Tyrosine kinases (TKs) drive cell survival and proliferation in many normal and malignant cell types. TKs are frequently mutated in acute myeloid leukemia (AML) and hence are increasingly targeted. The management of AML has dramatically improved because of TKI-targeted treatment.

Areas Covered: This review provides a biological background for TK inhibitors (TKIs) in AML and reviews their use in the clinic. TK expression and mutation in AML are explored with a focus on TKs associated with specific AML subsets and treatment outcomes. TKIs that specifically target FLT3, c-Kit, and Jak2 are discussed. TKI targeting of specific genes mutated in individual cases and general ‘untargeted’ use of these agents are highlighted. Lastly, the mechanisms TKI drug resistance in AML are explored.

Expert Opinion: The use of TKIs in the clinic is improving outcomes for many patients. An improved understanding of tyrosine kinase biology and the expanding use of TKIs are likely to dramatically improve outcomes in the coming decade. TKIs and other targeted agents could gradually supplant the use of cytotoxic chemotherapy for AML.     

Vosaroxin is a novel topoisomerase-II inhibitor with efficacy in relapsed and refractory acute myeloid leukaemia

Introduction: Vosaroxin is a first-in-class anti-cancer quinolone that inhibits topoisomerase-II leading to cell cycle arrest and apoptosis. It has shown efficacy in a range of solid organ and haematopoietic tumours in vitro, and several clinical trials are underway or completed in the field of Acute Myeloid Leukaemia (AML). The treatment of relapsed and refractory AML is a clinical challenge, where long-term survival is rare without allogeneic haematopoietic stem cell transplantation.

Areas covered: We review the data from the published clinical trials of vosaroxin, including the recently presented Phase III VALOR study. In combination with intermediate dose cytarabine, vosaroxin almost doubled complete response (CR) rates in relapsed and refractory AML compared with cytarabine alone, and prolonged median survival by 1.4 months.

Expert opinion: Vosaroxin is a promising new agent in the treatment of AML, with the potential to improve CR rates in a high-risk group of patients with relapsed and refractory AML. However, higher CR rates have been associated with higher rates of treatment-related morbidity and mortality, especially in elderly/unfit patients. Maximising the potential of vosaroxin will therefore require the identification of patients most likely to benefit from vosaroxin-containing combination regimens.     

Carbonic anhydrase IX inhibition is an effective strategy for osteosarcoma treatment

Objective: Hypoxia-inducible factor 1, a regulator of CA IX activity, is often overexpressed in human osteosarcoma (OS) but not in normal tissues, and its expression levels correlate with prognosis. In this study, we investigated the therapeutic potential of newly synthesized CA IX sulfonamide inhibitors in OS.

Methods: CA IX expression was evaluated in OS cell lines and bone marrow stromal cells (BMSC). After treatment with CA IX inhibitors, cell proliferation, apoptosis, cell cycle, extracellular and cytosolic pH changes were evaluated both in vitro and in mouse OS xenografts.

Results: CA IX expression levels were significantly higher in OS than in BMSC. Accordingly, CA IX inhibitor 3 induced remarkable cytotoxicity on OS cells without affecting BMSC proliferation. This activity was increased under hypoxia, and was mediated by cell cycle arrest and by the modulation of cytosolic and extracellular pH. In vivo, CA IX inhibitor 3 reduced tumor growth by inducing significant necrosis.

Conclusions: Our results provide a strong rationale for the clinical use of the newly synthesized CA IX inhibitor 3 in human OS.     

Therapeutic targeting of leukemic stem cells in acute myeloid leukemia – the biological background for possible strategies

Introduction: Acute myeloid leukemia (AML) is an aggressive malignancy, caused by the accumulation of immature leukemic blasts in blood and bone marrow. There is a relatively high risk of chemoresistant relapse even for the younger patients who can receive the most intensive antileukemic treatment. Treatment directed against the remaining leukemic and preleukemic stem cells will most likely reduce the risk of later relapse.

Areas covered: Relevant publications were identified through literature searches. The authors searched for original articles and recent reviews describing (i) the characteristics of leukemic/preleukemic stem cells; (ii) the importance of the bone marrow stem cell niches in leukemogenesis; and (iii) possible therapeutic strategies to target the preleukemic/leukemic stem cells.

Expert opinion: Leukemia relapse/progression seems to be derived from residual chemoresistant leukemic or preleukemic stem cells, and a more effective treatment directed against these cells will likely be important to improve survival both for patients receiving intensive treatment and leukemia-stabilizing therapy. Several possible strategies are now considered, including the targeting of the epigenetic regulation of gene expression, proapoptotic intracellular signaling, cell metabolism, telomere activity and the AML-supporting effects by neighboring stromal cells. Due to disease heterogeneity, the most effective stem cell-directed therapy will probably differ between individual patients.     

In vitro effect of IL-2 in combination with pazopanib or sunitinib on lymphocytes function and apoptosis of RCC cells

Objective: Combination of immunotherapy with tyrosine kinase inhibitors (TKIs) has been used with some success for the treatment of metastatic renal cell carcinoma. Herein we evaluate the in vitro effect of high-dose interleukin-2 (HDIL-2) and pazopanib or sunitinib on the lymphocyte function and on induction of apoptosis in renal cell carcinoma (RCC) cell lines.

Methods: Peripheral blood mononuclear cells (PBMCs) isolated from healthy donors or RCC patients were treated with different HDIL-2/TKI combinations. Effects of different combinations on proliferation and cytotoxic activity of PBMCs were evaluated, in addition to their effect on apoptosis of three different RCC cell lines.

Results: While sunitinib did not inhibit the proliferation of various immune cells induced by HDIL-2, pazopanib appeared to inhibit the HDIL-2–induced proliferation of these cells. Interestingly, none of the HDIL-2/TKI combinations appeared to compromise the functional properties of these cells. Additionally, significant proportion of RCC cell lines treated with pazopanib alone underwent apoptosis, while the proportions of apoptotic cells post-HDIL-2 or sunitinib were not different from the background. Furthermore, the combination of HDIL-2/pazopanib did not inhibit the pazopanib-induced RCC apoptosis.

Conclusion: The combination of HDIL-2 with either pazopanib or sunitinib exerts different anticancer mechanisms that could enhance the treatment efficacy.