Human Acute Promyelocytic Leukemia Cells Research Articles

In Vivo Analysis of PML-RARA in a Humanized Mouse Model

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML) characterized by the formation of a PML-RARa fusion protein, which leads to the accumulation of abnormal promyelocytes. Xenograft mouse models with human leukemic cells have advantages for analyzing the human leukemias in vivo, especially for genetic analyses. However, human primary APL cells are difficult to engraft even in very severely immunodeficient mice, such as NOD/shi-SCID IL2Rg-/- (NOG) mice. In order to understand the mechanisms involved in human APL leukemogenesis, we established a humanized in vivo APL model using the transplantation of PML-RARA-transduced CD34+ cells from human cord blood into NOG mice. The expression of PML-RARa in the CD34+ cells disrupted the nuclear bodies in vitro. The clonogenic assay showed that PML-RARa inhibited the total colony formation, but favored the growth of myeloid colonies. When CD34+ cells with PML-RARA were transplanted, they proliferated in the NOG mice for more than three to four months after transplantation (in 24 out of the 34 mice). All 16 mice with more than 3,000 PML-RARA-transduced CD34+ cells were engrafted, while the engraftment was only detected in eight out of 18 mice when the cell density used for transplantation was less than 3,000 cells. These cells possessed abundant azurophilic abnormal granules in the cytoplasm, and some of them had bundles of Auer rods. They expressed CD13, CD33 and CD117, but not HLA-DR or CD34. In addition, the gene expression analysis revealed that these cells and human primary APL were clustered together among various types of AML, suggesting that these induced APL cells well recapitulated human primary APL. Similar to human primary APL, the induced APL cells possessed the ability for myeloid differentiation after treatment with all-trans retinoic acid in vitro and in vivo, and a very low potential for re-transplantation, which was similarly observed in both unsorted induced APL cells and the CD34- fraction. When human cord blood was fractionated before the PML-RARA transduction, the CD34+/CD38+ cells and common myeloid progenitors (CMP) in the CD34+/CD38+ cells led to the efficient development of APL in vivo. These findings demonstrate that CMP is a target for PML-RARA in APL, whereas the resultant CD34- APL cells may share the ability to maintain the leukemia. DisclosuresNo relevant conflicts of interest to declare.

Mechanisms of resistance to apoptosis in the human acute promyelocytic leukemia cell line NB4.

Current frontline therapies have improved overall survival in acute promyelocytic leukemia (APL) patients to exceptional rates; however, relapse is still a problem among high-risk and old patients. Therefore, the development of better and safer therapies continues to be a goal in the treatment of this disease. In the present work, we examined three different pathways that hinder cell death in the APL cell line NB4, shedding light on the mechanisms that underlie resistance to apoptosis in these cells and that might help provide them with a proliferative advantage. We found that the proteasome inhibitor MG-132 specifically induces in NB4 cells an Nrf2-mediated antioxidant response which counteracts mitochondria-dependent apoptosis induced by the lipophilic cation dequalinium. More importantly, we also demonstrated that high basal autophagy levels and the gain-of-function of mutant p53 are intrinsic mechanisms of resistance to apoptosis in this cell line. According to our results, the pharmacological inhibition of autophagy and p53 mutants are useful tools to explore resistance to apoptosis in APL and other types of cancer and could be the bases of new therapeutic approaches that improve the efficiency and allow dose reduction of the current treatments.

Zoledronic acid exerts antitumor effects in NB4 acute promyelocytic leukemia cells by inducing apoptosis and S phase arrest

The aim of this study was to investigate the antitumor effect of zoledronic acid (ZOL) in the NB4 human acute promyelocytic leukemia (APL) cell line and explore the potential mechanism of action of this compound. NB4 cells were exposed to various concentrations (0–200μM) of ZOL. Cell viability was measured by MTS assay. The extent of cell apoptosis and distribution of cells in the different phases of the cell cycle were analyzed with flow cytometry. The expression of apoptosis- and cell cycle-related proteins was assayed by Western blot. The combined effect of ZOL and arsenic trioxide (ATO) on the proliferation of NB4 cells was also determined. The results of this study indicate that ZOL inhibits cell proliferation in a time- and dose-dependent fashion and also induces apoptosis and S phase arrest in a dose-dependent manner. The Western blot analysis confirmed the induction of apoptosis and S phase arrest, revealing that the pro-apoptosis proteins Bax, Puma and activated caspase-9 were upregulated and the anti-apoptosis proteins Bcl-2 and Bcl-xL were downregulated. ZOL at a concentration of 50μM synergized with 0.5μM ATO on the growth inhibition of NB4 cells. Taken together, our data indicate that ZOL exerts a potent antitumor effect on NB4 cells by inducing apoptosis and cell cycle arrest, and that ZOL can synergize with the traditional chemotherapy drug ATO.

Epigenetic and molecular mechanisms underlying the antileukemic activity of the histone deacetylase inhibitor belinostat in human acute promyelocytic leukemia cells.

Therapeutic strategies targeting histone deacetylase (HDAC) inhibition have become promising in many human malignancies. Belinostat (PXD101) is a hydroxamate-type HDAC inhibitor tested in phase I and II clinical trials in solid tumors and hematological cancers. However, little is known about the use of belinostat for differentiation therapy against acute myelogenous leukemia. Here, we characterize the antileukemia activity of belinostat as a single drug and in combination with all-trans-retinoic acid (RA) in promyelocytic leukemia HL-60 and NB4 cells. Belinostat exerted dose-dependent growth-inhibitory or proapoptotic effects, promoting cell cycle arrest at the G0/G1 or the S transition. Apoptosis was accompanied by activation of caspase 3, degradation of PARP-1, and cell cycle-dependent changes in the expression of survivin, cyclin E1, and cyclin A2. Belinostat induced a dose-dependent reduction in the expression of EZH2 and SUZ12, HDAC-1, HDAC-2, and histone acetyltransferase PCAF (p300/CBP-associated factor). Belinostat increased acetylation of histone H4, H3 at K9 and H3 at K16 residues in a dose-dependent manner, but did not reduce trimethylation of H3 at K27 at proapoptotic doses. Combined treatment with belinostat and RA dose dependently accelerated and reinforced granulocytic differentiation, accompanied by changes in the expression of CD11b, C/EBPα (CCAAT/enhancer binding protein-α), and C/EBPε. Our results concluded the usefulness of belinostat, as an epigenetic drug, for antileukemia and differentiation therapy.

Paper-based electrochemical cyto-device for sensitive detection of cancer cells and in situ anticancer drug screening

In this work, using human acute promyelocytic leukemia cells (HL-60) as a model, a novel microfluidic paper-based electrochemical cyto-device (μ-PECD) was fabricated to demonstrate a facile, portable, and disposable approach for cancer cell detection and in situ screening of anticancer drugs in a high-throughput manner. In this μ-PECD, aptamers modified three-dimensional macroporous Au-paper electrode (Au-PE) was fabricated and employed as the working electrode for specific and efficient cancer cell capture as well as for sequential in-electrode 3D cell culture. This Au-PE showed enhanced capture capacity for cancer cells and good biocompatibility for preserving the activity of captured living cells. Sensitive cancer cell detection was achieved in this μ-PECD, which could respond down to four HL-60 cells in 10μL volume with a wide linear calibration range from 5.0×102 to 7.5×107 cellsmL−1 and exhibited good stability and reproducibility. Then, in situ anticancer drug screening was successfully implemented in this μ-PECD through monitoring of the apoptotic cancer cells after the in-electrode 3D cell culture with drug-containing culture medium, demonstrating its wide range of potential applications to facilitate effective clinical cancer diagnosis and treatment.

Influence of I2PP2A gene silencing by RNA interference on proliferation and apoptosis of human acute promyelocytic leukemia cell line NB4-R1

To explore the effect of RNA interference of human I2PP2A gene on the proliferation and apoptosis of retinoic acid-resistant human acute promyelocytic leukemia (APL) cell line NB4-R1. Designed and constructed a RNA interference lentiviral vector I2PP2A-shRNA which targeted against I2PP2A gene, then transfected it into NB4-R1 via polybrene mediation. The I2PP2A expression levels before and after transfection were detected by qRT-PCR and Western blot, respectively. Meanwhile, the proliferation and apoptosis rates of each group were determined by CCK-8 and flow cytometry assay. The protein expressions of caspase-8 and PARP were detected by Western blot. Both qRT-PCR and Western blot data showed the I2PP2A expression level was significantly downregulated in the transfection group. The I2PP2A mRNA expression level decreased by (70.0 ± 9.6)% and (64.0 ± 6.2)% respectively, compared with blank control and negative control group, and the I2PP2A protein expression level showed a consistent trend. CCK-8 proliferation assay indicated the NB4-R1 cell proliferation rate in I2PP2A-shRNA transfection group significantly reduced compared to blank control group (P<0.05). Flow cytometry results showed that NB4-R1 apoptosis rate in I2PP2A-shRNA transfection group increased by (6.30 ± 0.67) times and (6.04 ± 0.56) times, respectively (P<0.01). After inhibition of I2PP2A, the total caspase-8 and total PARP expressions decreased by (44.0 ± 3.1)% and (57.0 ± 4.0)%, respectively; Meanwhile, the cleaved caspase-8 (p43) and cleaved PARP (p89) increased by (36.0 ± 2.5)% and (45.0 ± 4.8)%, respectively compared with blank control group (P<0.05). I2PP2A gene silenced by RNA interference could inhibit the proliferation and promote the apoptosis of NB4-R1, which may be regulated through caspase-8-induced exogenous apoptosis pathway.

Endocan, a potential prognostic and diagnostic biomarker of acute leukemia.

Recent evidence indicated that endocan may be a potential cell marker and a new target for cancers including acute leukemia since the serum endocan level in patients with acute leukemia was associated with the status of the disease, i.e., endocan was higly expressed in untreated acute leukemia, but decreased after chemotherapy and increased again during bone marrow regeneration. The present study showed that there was high level expression of endocan in cytoplasm of bone marrow blasts of patients with acute myeloid leukemia or acute lymphoblastic leukemia. The expression level of endocan was significantly decreased when the patients underwent remission after chemotherapy and re-bounces back when the acute leukemia relapsed. No obvious change in expression of endocan was observed before and after chemotherapy if the patients showed no remission after chemotherapy. (N-(4-Hydroxyphenyl) retinamide), a potent anti-angiogenic agent, could not only down-regulate the expression of vascular epithelial growth factor, but also decrease endocan transcription and expression in NB4 cells, a human acute promyelocytic leukemia cell line. These observations suggest that endocan could act as a predictor for the severity and the prognosis of acute leukemia. The findings could be used as the basis for future targeted therapy directed against bone marrow angiogenesis in acute leukemia treatment.

Prohibitin 2 represents a novel nuclear AKT substrate during all‐ trans retinoic acid–induced differentiation of acute promyelocytic leukemia cells

The AKT/PKB kinase is essential for cell survival, proliferation, and differentiation; however, aberrant AKT activation leads to the aggressiveness and drug resistance of many human neoplasias. In the human acute promyelocytic leukemia cell line NB4, nuclear AKT activity increases during all-trans retinoic acid (ATRA)-mediated differentiation. As nuclear AKT activity is associated with differentiation, we sought to identify the nuclear substrates of AKT that were phosphorylated after ATRA treatment. A proteomics-based search for nuclear substrates of AKT in ATRA-treated NB4 cells was undertaken by using 2D-electrophoresis/mass spectrometry (MS) in combination with an anti-AKT phospho-substrate antibody. Western blot analysis, an in vitro kinase assay, and/or site-directed mutagenesis were performed to further characterize the MS findings. MS analysis revealed prohibitin (PHB)-2, a multifunctional protein involved in cell cycle progression and the suppression of oxidative stress, to be a putative nuclear substrate of AKT. Follow-up studies confirmed that AKT phosphorylates PHB2 on Ser-91 and that forced expression of the PHB2(S91A) mutant results in a rapid loss of viability and apoptotic cell death. Activation of nuclear AKT during ATRA-mediated differentiation results in the phosphorylation of several proteins, including PHB2, which may serve to coordinate nuclear-mitochondrial events during differentiation.

Ascorbic Acid Synergistically Potentiates Phloxine B‐induced Photocytotoxicity in Human Acute Promyelocytic Leukemia Cells

Ascorbic acid (AsA) is known as an antioxidant but concomitantly possesses a pro-oxidant property. Because the impact of AsA on photodynamic therapy response is unclear, we investigated the effect of AsA on photocytotoxicity induced by phloxine B in human acute promyelocytic leukemia HL-60 cells. AsA synergistically enhanced phloxine B-induced photocytotoxic effects, including inhibition of cell proliferation, DNA ladder formation, and caspase-3 activation, whereas AsA itself showed no photocytotoxicity. AsA also enhanced the consumption of the reduced glutathione level compared with the cells treated with phloxine B alone under the light condition. Combination of AsA with phloxine B under the light condition enhanced the phosphorylation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase (MAPK). These effects were completely cancelled by catalase. These results suggest that AsA synergistically enhances phloxine B-induced photocytotoxicity, possibly through the extracellular oxidative stress-dependent MAPK pathway activation.

The Novel Arsenical Darinaparsin Is Transported by Cystine Importing Systems

Darinaparsin (Dar; ZIO-101; S-dimethylarsino-glutathione) is a promising novel organic arsenical currently undergoing clinical studies in various malignancies. Dar consists of dimethylarsenic conjugated to glutathione (GSH). Dar induces more intracellular arsenic accumulation and more cell death than the FDA-approved arsenic trioxide (ATO) in vitro, but exhibits less systemic toxicity. Here, we propose a mechanism for Dar import that might explain these characteristics. Structural analysis of Dar suggests a putative breakdown product: dimethylarsino-cysteine (DMAC). We show that DMAC is very similar to Dar in terms of intracellular accumulation of arsenic, cell cycle arrest, and cell death. We found that inhibition of γ-glutamyl-transpeptidase (γ-GT) protects human acute promyelocytic leukemia cells (NB4) from Dar, but not from DMAC, suggesting a role for γ-GT in the processing of Dar. Overall, our data support a model where Dar, a GSH S-conjugate, is processed at the cell surface by γ-GT, leading to formation of DMAC, which is imported via xCT, xAG, or potentially other cystine/cysteine importing systems. Further, we propose that Dar induces its own import via increased xCT expression. These mechanisms may explain the enhanced toxicity of Dar toward cancer cells compared with ATO.

Synthesis and biological evaluation of novel indoline-2,3-dione derivatives as antitumor agents.

A new series of 1,5-disubstituted indolin-2,3-diones was synthesized and their inhibition of the growth of a human acute promyelocytic leukemia (HL-60) cell line was evaluated. These compounds had promising inhibition of HL-60 cell growth in vitro. Results indicated that compounds with a benzyl substituent at the N-1 position on the indolin-2,3-dione ring had more potent antiproliferative activity than those with a (4-fluorobenzyl) amino-2-oxoethyl substituent at the N-1 position. Among the compounds synthesized, compound 8l inhibited half of cell growth at a concentration of 0.07 μM and compound 8p did so at a concentration of 0.14 μM. These compounds may serve as lead compounds for further optimization in order to develop novel anticancer agents.

Enhancement of differentiation induction and upregulation of CCAAT/enhancer-binding proteins and PU.1 in NB4 cells treated with combination of ATRA and valproic acid

The effects of all-trans retinoic acid (ATRA) and valproic acid (VPA), alone and in combination, on the human acute promyelocytic leukemia (APL) cell line NB4 were investigated in view of differentiation induction and growth inhibition. After 48h of treatment, not only ATRA but also VPA induced differentiation in NB4 cells, and their combination further augmented the differentiation activity. Furthermore, the upregulation of transcription factors including CCAAT/enhancer-binding proteins (CEBPα, β, ε) and PU.1, which are known to be critical factors for normal myelopoiesis, granulocytic maturation and being repressed in APL, concurred with the differentiation induction. A significant cell growth inhibition was observed after the treatment with VPA, which was further strengthened by the addition of ATRA. Given the importance of C/EBPs and PU.1 in myeloid development, these results, thus, suggest that restoration of the normal function of the myeloid cell transcriptional machinery is a major molecular mechanism underlying the differentiation induction in NB4. Therefore, these results may provide novel insights into a possible combinational therapeutic approach for APL patients.

Oxidation Of Peroxiredoxins By Thioredoxin Inactivation Is Mediated In Arsenic Trioxide-Induced Reactive Oxygen Species Formation and Its Cytotoxicity In Acute Promyelocytic Leukemia Cells

BackgroundsThe Arsenic trioxide (ATO) is an effective cancer therapeutic drug for acute promyelocytic leukemia (APL). ATO exerts its effect mainly raising oxidative stress. However, not only the mechanisms of reactive oxygen species (ROS) generation by ATO but involvement of redox enzymes including peroxiredoxin (PRX) and thioredoxin (TRX) remains elusive. Aim of current study is to elucidate the mechanism of redox enzymes to elevate ROS during ATO-induced apoptosis in APL-derived NB4 cells. MethodsNB4 cell line, which is one of the human acute promyelocytic leukemia cell lines, was cultured in RPMI-1640 medium supplemented with 10% FBS in CO2 humidified atmosphere at 37°C. NB4 cells were cultured with 2 μM arsenic trioxide to induce apoptosis for 16-48 hours. Apoptosis was measured by staining with 7-amino-actinomycin D (7-AAD) with flow cytometry. 2, 7-dichlrodihydro-fluorescein-diacetate (H2DCF-DA) and MitoSOX Red were used to detect cellular and mitochondrial ROS. SO2 form for PRX I, PRX II, and PRX III was detected by western blot assay using PRX SO2 form-specific antibody. Monomer/Dimer assay for PRX I, PRX II, PRX III, and TRX I was performed by western blot using non-reducing gel. ResultsIntracellular ROS of NB4 cells was increased significantly after 16 hour of ATO but decreased after 24 hour of ATO. Mitochondrial ROS of NB4 cells was increased significantly after 39 hour of ATO. Apoptosis of NB4 cell after ATO treatment was increased as time elapsed (24% on 16hr, 26% on 24hr, 48% on 39hr, and 60% on 48hr). Monomer, indicated active and reduced form, of peroxiredoxins was decreased and cysteine sulfinic acid (CP–SO2H) peroxiredoxins, indicated inactive and oxidized peroxiredoxins, was increased in NB4 cells after ATO treatment as time goes by. Similarily, monomer of thioredoxin-1 (active thioredoxin) was decreased and multimer of thioredoxin-1 (inactive thioredoxin) was increased in NB4 cells after ATO treatment as time elapsed. ConclusionsOur data showed inactivation of peroxiredoxins by oxidation was developed during ATO-induced ROS generation and APL cell apoptosis. These peroxiredoxins oxidation was probably due to increment of reduced thioredoxin in NB4 cells after ATO treatment. These findings suggest ATO-induced anti-leukemic activity is more likely due to a TRX system-mediated cellular redox changes. Our study may provide the insights for finding novel targets in the development of new therapies, which potentiate ATO-induced apoptosis in APL cells. Disclosures:No relevant conflicts of interest to declare.

Thrombin Generationand Fibrinolytic Activity in Microparticles In Acute Promyelocytic Leukemia

IntroductionThe coagulopathy in acute promyelocytic leukemia (APL) is unique among the leukemias and accounts for the major complications of hemorrhage and thrombosis. The pathogenesis is in large part the result of increased expression by the APL promyelocytes of the procoagulant TF, and the fibrinolytic components of tPA , PAI-1 and Annexin A2. Recently, microparticles derived from plasma of APL patients and from culture medium of the human APL cell line NB4 have been shown to contain antigens of procoagulant tissue factor (TF), and the fibrinolytic components of tPA , Annexin A2 and PAI-1 (Kwaan HC, Rego EM. Role of microparticles in the hemostatic dysfunction in acute promyelocytic leukemia. Seminars in thrombosis and hemostasis 2010;36:917-24;Kwaan et al:Microparticles in acute promyelocytic leukemia. 53rdASH Annual Meeting; Blood 2011 Abstract No.3346). Results of functional activities of these components and thrombin generation in the microparticles are presented in the present study. Material and MethodsBlood samples were collected in citrate vacutainers from 23 patients at the time of diagnosis and after induction therapy with ATRA at one month when there was molecular remission, under the approval of the respective institutional review boards. Control samples were collected from 37 healthy subjects. Preparation of Microparticlessodium citrated blood is centrifuged at 1500g for 20 minutes. The plasma portion is collected and centrifuged a second time at 3000g for 3 minutes to eliminate any extraneous platelets. 250µl of plasma is then centrifuged at 20,000g for 15 minutes at 4C. The resultant pellet contains the microparticles. [Standardization of platelet-derived microparticle enumeration by flow cytometry with calibrated beads: results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop. Lacroix R. Robert S. Poncelet P. Kasthuri RS. Key NS. Dignat-George F. ISTH SSC Workshop. Journal of Thrombosis & Haemostasis. 8(11):2571-4, 2010 Nov. ResultsConclusion Microparticles bearing TF have been shown to play a thrombogenic role in cancer. We had previously shown that there is an increase of microparticles containing procoagulants and fibrinolytic factors in APL and that these microparticles are mostly derived from CD33 positive (myeloid) cells. Present findings show that procoagulant and fibrinolytic factors are functional. The TF activity, thrombin generation are increased in the APL microparticles. It is noteworthy that thrombin generation is increased in the APL microparticles. In contrast, there is a complete absence of thrombin generation in the control microparticles. This suggests that the TF in microparticles, which are components of cell membranes, are not activated in healthy subjects but in APL. Disclosures:No relevant conflicts of interest to declare.

Coumarins and Lignans from Zanthoxylum schinifolium and Their Anticancer Activities

Zanthoxylum schinifolium is an aromatic shrub, and its pericarp and leaves are widely used in culinary applications in East Asian countries. It has also long been used in traditional Oriental medicine for treating the common cold, stomach ache, diarrhea, and jaundice. In this study, we identified two new compounds, zanthoxyloside (1) and schinifolisatin A (13), along with 23 known coumarins (2-12) and lignans (14-25), from a methanol extract of the stems of Z. schinifolium . The chemical structures of the compounds were determined by mass, 1D-, and 2D NMR spectroscopy. The anticancer effects of the isolated compounds were examined in three human cancer cell lines. Compounds 10-12 significantly reduced the proliferation of HL-60 human acute promyelocytic leukemia cells with IC50 values of 4.62-5.12 μM. Treatment of PC-3 prostate cancer cells and SNU-C5 colorectal cancer cells with compound 10 resulted in potent antiproliferative activity, with IC50 values of 4.39 and 6.26 μM, respectively. Also, compounds 10-12 induced the apoptosis of three cancer cells. Furthermore, the induction of apoptosis was accompanied by down-regulation of p-ERK1/2 MAPK, p-AKT, and c-myc levels, in a time-dependent manner. These data suggested that compounds 10-12 from Z. schinifolium have potential in cancer treatment.

Korean red ginseng extract induces proliferation to differentiation transition of human acute promyelocytic leukemia cells via MYC-SKP2-CDKN1B axis

Ethnopharmacological relevanceKorean red ginseng has been used as traditional medicine in East Asia. Recent scientific research revealed multiple effects of Korean red ginseng, including anticancer activity. To evaluate the effect of Korean red ginseng extract (KRGE) in acute promyelocytic leukemia (APL) and elucidate its molecular mechanism. Materials and methodsNB4 cells were treated with 1mg/ml KRGE for 48h and examined for cell proliferation and differentiation. Cell cycle distribution of KRGE-treated cells was analyzed and the expression level of G1 phase regulators was determined. MYC was overexpressed by retroviral transduction and its effect on SKP2 and CDKN1B gene expression, cell proliferation, cell cycle and differentiation was evaluated in KRGE-treated cells. ResultsKRGE alone was sufficient to induce granulocytic differentiation accompanied with growth inhibition. KRGE treatment resulted in cell cycle arrest at the G1 phase with augmented Cdkn1b proteins without changes in transcript levels. Cycloheximide treatment revealed reduced degradation of Cdkn1b protein by KRGE. In addition, KRGE treatment reduced expression of MYC and SKP2 genes, both at mRNA and protein levels. Upon ectopic expression of MYC, the effect of KRGE was reversed with lesser reduction and induction of SKP2 gene and Cdkn1b protein, respectively. Taken together, these results suggest a sequential molecular mechanism from MYC reduction, SKP2 reduction, Cdkn1b protein stabilization, G1 phase arrest to granulocytic differentiation by KRGE in human APL. ConclusionsKRGE induces leukemic proliferation to differentiation transition in APL through modulation of the MYC-SKP2-CDKN1B axis.

Induction of cancer cell death by apoptosis and slow release of 5-fluoracil from metal-organic frameworks Cu-BTC

This study aimed to evaluate the mechanism associated with cytotoxic activity displayed by the drug 5-fluorouracil incorporated in Cu-BTC MOF and its slow delivery from the Cu-BTC MOF. Structural characterization encompasses elemental analysis (CHNS), differential scanning calorimetry (DSC), thermogravimetric analysis (TG/DTG), Fournier transform infrared (FIT-IR) and X-ray diffraction (XRD) was performed to verify the process of association between the drug 5-FU and Cu-BTC MOF. Flow cytometry was done to indicate that apoptosis is the mechanism responsible for the cell death. The release profile of the drug 5-FU from Cu-BTC MOF for 48hours was obeisant. Also, the anti-inflammatory activity was evaluated by the peritonitis testing and the production of nitric oxide and pro-inflammatory cytokines were measured. The chemical characterization of the material indicated the presence of drug associated with the coordination network in a proportion of 0.82g 5-FU per 1.0g of Cu-BTC MOF. The cytotoxic tests were carried out against four cell lines: NCI-H292, MCF-7, HT29 and HL60. The Cu-BTC MOF associated drug was extremely cytotoxic against the human breast cancer adenocarcinoma (MCF-7) cell line and against human acute promyelocytic leukemia cells (HL60), cancer cells were killed by apoptosis mechanisms. The drug demonstrated a slow release profile where 82% of the drug was released in 48hours. The results indicated that the drug incorporated in Cu-BTC MOF decreased significantly the number of leukocytes in the peritoneal cavity of rodents as well as reduced levels of cytokines and nitric oxide production.

Roles of GINS2 in K562 human chronic myelogenous leukemia and NB4 acute promyelocytic leukemia cells

GINS2 has been found to be closely related to cell proliferation, survival and maintenance of genomic integrity in addition to the induction of polyploidy. However, the molecular mechanisms of GINS2-mediated tumor regression have not been fully elucidated. Our study showed that GINS2 interacted with PML-C (a structural domain of PML, which is a cutting product of PML-RARα) as demonstrated by yeast two-hybrid assay and co-immunoprecipitation, and PML (NLS-) also interacted with GINS2 by co-immunoprecipitation. Following transfection with plasmids expressing GINS2 siRNA, the apoptosis and cell cycle distribution were determined by flow cytometry. The results revealed that K562 chronic myelocytic leukemia cells and NB4 acute promyelocytic leukemia cells were arrested in the G2phase. Western blot assay indicated that the expression of cyclinA, cyclinD1 and cyclinB1 was decreased, and cyclinB1 was localized in the cytoplasm after GINS2 knockdown as demonstrated by immunofluorescence staining. The expression of the apoptosis-related protein Bcl-2 was decreased but that of Bax was increased in the cells transfected with GINS2 siRNA. The percentage of apoptotic cells was increased as detected by flow cytometry. Our findings demonstrated that GINS2 plays an important role in apoptosis and may function via the p38MAPK signaling pathway.

Notch signaling in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is initiated by the PML-RARA fusion oncogene and has a characteristic expression profile that includes high levels of the Notch ligand JAG1. In this study, we used a series of bioinformatic, in vitro, and in vivo assays to assess the role of Notch signaling in human APL samples, and in a PML-RARA knockin mouse model of APL (Ctsg-PML-RARA). We identified a Notch expression signature in both human primary APL cells and in Kit+Lin−Sca1+ (KLS) cells from pre-leukemic Ctsg-PML-RARA mice. Both genetic and pharmacologic inhibition of Notch signaling abrogated the enhanced self-renewal seen in hematopoietic stem/progenitor cells (HSPCs) from pre-leukemic Ctsg-PML-RARA mice, but had no influence on cells from age-matched wildtype mice. In addition, 6 of 9 murine APL tumors tested displayed diminished growth in vitro when Notch signaling was inhibited pharmacologically. Finally, we found that genetic inhibition of Notch signaling with a dominant negative MAML protein reduced APL growth in vivo in a subset of tumors. These findings expand the role of Notch signaling in hematopoietic diseases, and further define the mechanistic events important for PML-RARA-mediated leukemogenesis.

Green synthesis, antimicrobial and cytotoxic effects of silver nanoparticles using Eucalyptus chapmaniana leaves extract

ObjectiveTo synthesize silver nanopaticles from leaves extract of Eucalyptus chapmaniana (E. chapmaniana) and test the antimicrobial of the nanoparticles against different pathogenic bacteria, yeast and its toxicity against human acute promyelocytic leukemia (HL-60) cell line. MethodsTen milliliter of leaves extract was mixed with 90 mL of 0.01 mmol/mL or 0.02 mmol/mL aqueous AgNO3 and exposed to sun light for 1 h. A change from yellowish to reddish brown color was observed. Characterization using UV-vis spectrophotometery and X-ray diffraction analysis were performed. Antimicrobial activity against six microorganisms was tested using well diffusion method and cytoxicity test using 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, a yellow tetrazole was obtained on the human leukemia cell line (HL-60). ResultsUV-vis spectral analysis showed silver surface plasmon resonance band at 413 nm. X-ray diffraction showed that the particles were crystalline in nature with face centered cubic structure of the bulk silver with broad beaks at 38.50° and 44.76°. The synthesized silver nanoparticles efficiently inhibited various pathogenic organisms and reduced viability of the HL-60 cells in a dose-dependent manner. ConclusionsIt has been demonstrated that the extract of E. chapmaniana leaves are capable of producing silver nanoparticles extracellularly and the Ag nanoparticles are quite stable in solution. Further studies are needed to fully characterize the toxicity and the mechanisms involved with the antimicrobial and anticancer activity of these particles.