Leukemia Cell Line HL-60 Research Articles

Thymoquinone induces cell proliferation inhibition and apoptosis in acute myeloid leukemia cells: role of apoptosis-related WT1 and BCL2 genes

Acute myeloid leukemia (AML) is an aggressive and heterogeneous disease characterized by an abnormal proliferation and impaired differentiation of the myeloid precursor cells. The outcome for most AML patients remains poor with high relapse rates and chemotherapy remains the first line treatment for AML. The Wilms tumor wt1 and the anti-apoptotic BCL2 genes are upregulated in AML and are known to be involved in apoptosis inhibition. In the present study we evaluated the molecular mechanisms underlie the anti-proliferative and pro-apoptotic activities exerted by thymoquinone (TQ), the major biologically active compound of the black seed oil on acute myeloid leukemia (AML) cell line-HL60. Cell proliferation was determined by WST-1 assay and apoptosis rate was assessed by flow cytometry using annexin-V/7AAD staining. The expression of target genes was analyzed by real-time RT–PCR analysis. TQ significantly reduced HL60 cell viability and induced apoptosis in a dose and time-dependent manner. In order to decipher the molecular mechanisms underlie the anti-cancer activities induced by TQ in AML cells, we investigated its effect on the expression of WT1 and BCL2 genes. TQ significantly decreased the expression of WT1 and BCL2 genes in a dose and time-dependent manner. In summary, these findings suggest that TQ induces cell proliferation inhibition and apoptosis in acute myeloid leukemia cells most likely through targeting the apoptosis-related WT1 and BCL2 genes and also suggest that TQ could be a promising strategy for AML therapy.

Loss of RAF kinase inhibitor protein is involved in myelomonocytic differentiation and aggravates RAS-driven myeloid leukemogenesis.

RAS-signaling mutations induce the myelomonocytic differentiation and proliferation of hematopoietic stem and progenitor cells. Moreover, they are important players in the development of myeloid neoplasias. RAF kinase inhibitor protein (RKIP) is a negative regulator of RAS-signaling. As RKIP loss has recently been described in RAS-mutated myelomonocytic acute myeloid leukemia, we now aimed to analyze its role in myelomonocytic differentiation and RAS-driven leukemogenesis. Therefore, we initially analyzed RKIP expression during human and murine hematopoietic differentiation and observed that it is high in hematopoietic stem and progenitor cells and lymphoid cells but decreases in cells belonging to the myeloid lineage. By employing short hairpin RNA knockdown experiments in CD34+ umbilical cord blood cells and the undifferentiated acute myeloid leukemia cell line HL-60, we show that RKIP loss is indeed functionally involved in myelomonocytic lineage commitment and drives the myelomonocytic differentiation of hematopoietic stem and progenitor cells. These results could be confirmed in vivo, where Rkip deletion induced a myelomonocytic differentiation bias in mice by amplifying the effects of granulocyte macrophage-colony-stimulating factor. We further show that RKIP is of relevance for RAS-driven myelomonocytic leukemogenesis by demonstrating that Rkip deletion aggravates the development of a myeloproliferative disease in NrasG12D-mutated mice. Mechanistically, we demonstrate that RKIP loss increases the activity of the RAS-MAPK/ERK signaling module. Finally, we prove the clinical relevance of these findings by showing that RKIP loss is a frequent event in chronic myelomonocytic leukemia, and that it co-occurs with RAS-signaling mutations. Taken together, these data establish RKIP as novel player in RAS-driven myeloid leukemogenesis.

Examination of CD302 as a potential therapeutic target for acute myeloid leukemia

Acute myeloid leukemia (AML) is the most common form of adult acute leukemia with ~20,000 new cases yearly. The disease develops in people of all ages, but is more prominent in the elderly, who due to limited treatment options, have poor overall survival rates. Monoclonal antibodies (mAb) targeting specific cell surface molecules have proven to be safe and effective in different haematological malignancies. However, AML target molecules are currently limited so discovery of new targets would be highly beneficial to patients. We examined the C-type lectin receptor CD302 as a potential therapeutic target for AML due to its selective expression in myeloid immune populations. In a cohort of 33 AML patients with varied morphological and karyotypic classifications, 88% were found to express CD302 on the surface of blasts and 80% on the surface of CD34+ CD38- population enriched with leukemic stem cells. A mAb targeting human CD302 was effective in mediating antibody dependent cell cytotoxicity and was internalised, making it amenable to toxin conjugation. Targeting CD302 with antibody limited in vivo engraftment of the leukemic cell line HL-60 in NOD/SCID mice. While CD302 was expressed in a hepatic cell line, HepG2, this molecule was not detected on the surface of HepG2, nor could HepG2 be killed using a CD302 antibody-drug conjugate. Expression was however found on the surface of haematopoietic stem cells suggesting that targeting CD302 would be most effective prior to haematopoietic transplantation. These studies provide the foundation for examining CD302 as a potential therapeutic target for AML.

Preliminary results indicate resveratrol affects proliferation and apoptosis of leukemia cells by regulating PTEN/PI3K/AKT pathway.

PTEN-PI3K/AKT signaling pathway is widely involved in the regulation of cell proliferation, cell cycle, apoptosis, and invasion. Resveratrol (Resv) is a natural botanical ingredient involved in several biological activities. It is still unclear in terms of whether Resv may exert anti-leukemia effects by regulating the PTEN-PI3K/AKT pathway. This study investigated the effect of Resv on leukemia cell proliferation and apoptosis by regulating PTEN-PI3K/AKT pathway. Human normal peripheral blood PBMC cells, and human acute promyelocytic leukemia (APL) cell line NB-4 and HL-60 cells were cultured in vitro. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect Phosphatase and tensin homolog (PTEN) mRNA expression. Western blot was adopted to test PTEN protein expression. HL-60 and NB-4 cells were treated with 0, 5, 10, and 20 μM Resv, respectively. Cell proliferation was analyzed by cell counting kit8 (CCK-8) assay. The level of caspase-3 was measured by Western blot. HL-60 cells were divided into control group, 20 μM Resv treatment group, and Resv+PTEN inhibitor SF1670 group. Cell apoptosis was determined by flow cytometry. Cell proliferation was assessed by EdU staining. Compared with peripheral blood mononuclear cell (PBMC), PTEN mRNA and protein levels were significantly decreased in NB-4 and HL-60 cells. Resv significantly inhibited the proliferation activity in HL-60 and NB-4 cells, and increased the activity of caspase-3. Resv treatment up-regulated the expression of PTEN and reduced the expression of p-AKT protein in HL-60 cells. However, Resv treatment markedly suppressed the proliferation of HL-60 and induced apoptosis. SF1670 treatment in the presence of Resv significantly antagonized the down-regulation of p-AKT protein expression induced by Resv, resulting in decreased apoptosis and enhanced cell proliferation. Resv can up-regulate PTEN expression and inhibit the activity of PI3K/AKT pathway to play an anti-leukemia effect through suppressing cell proliferation and inducing apoptosis.

USP39 regulates the cell cycle, survival, and growth of human leukemia cells.

Ubiquitin-specific peptidase 39 (USP39) is one member of the cysteine proteases of the USP family, which represents the largest group of DeUbiquitinases with more than 50 members in humans. The roles of USP39 in human cancer have been widely investigated. However, the roles of USP39 in human leukemia and the underlying mechanism remain unknown. Here we reported the function of USP39 in human leukemia. We observed that the expression of USP39 was up-regulated in human leukemia cells and the high expression of USP39 was correlated with poor survival of the patients with leukemia. Lentivirus-mediated knockdown of USP39 repressed the proliferation and colony formation of human leukemia cell lines HL-60 and Jurkat cells. Mechanism study showed that USP39 knockdown induced the arrest of cell cycle and apoptosis of leukemia cells. In addition, our microarray and bioinformatic analysis demonstrated that USP39 regulated diverse cellular signaling pathways that were involved in tumor biology, and several pivotal genes (IRF1, Caspase 8, and SP1) have been validated by quantitative real-time polymerase chain reaction. Knockdown or IRF1 partially restored the proliferation rate of leukemia cells with USP39 knockdown. Taken together, our findings implicate that USP39 promotes the development of human leukemia by regulating cell cycle, survival, and proliferation of the cells.

Dihydroartemisinin-Bile Acid Hybridization as an Effective Approach to Enhance Dihydroartemisinin Anticancer Activity.

A series of hybrid compounds based on natural products-bile acids and dihydroartemisinin-were prepared by different synthetic methodologies and investigated for their in vitro biological activity against HL-60 leukemia and HepG2 hepatocellular carcinoma cell lines. Most of these hybrids presented significantly improved antiproliferative activities with respect to dihydroartemisinin and the parent bile acid. The two most potent hybrids of the series exhibited a 10.5- and 15.4-fold increase in cytotoxic activity respect to dihydroartemisinin alone in HL-60 and HepG2 cells, respectively. Strong evidence that an ursodeoxycholic acid hybrid induced apoptosis was obtained by flow cytometric analysis and western blot analysis.

Cytotoxic Activity of Methoxy-4’amino Chalcone Derivatives Against Leukemia Cell Lines

Background: Chemotherapy is a common treatment for leukemia as well as in other cancer treatment. The lack of tumor selectivity and development of multi-drug resistance by chemotherapy caused the development of new strategy in cancer treatment become a pressing need. This study was performed to evaluate the anticancer activity and selectivity of seven derivatives of chalcones against K562 and HL-60 leukemia cell lines. Materials and Methods: The cytotoxicity of chalcone’s seven derivatives (compound 1-7) was tested by using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyme-thoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) method. The percentage of cell mortality data was calculated then the IC50 was analyzed using probit analysis (SPSS 17). The selectivity index (SI) then calculated from IC50 ratio of normal lymphocyte cells and cancerous cells line (HL-60 and K562).Results: The IC50 of almost all seven tested compounds were lower in HL-60 cell lines than K562 cell lines, except for Compound 7. The number and position of methoxy groups in chalcone derivatives influenced the anticancer and cancer selectivity of chalcone derivatives.Conclusion: The results revealed that the number and position of methoxy groups in chalcone derivatives influenced the anticancer and cancer selectivity of chalcone derivatives.Keywords: anticancer, chalcone derivatives, methoxy-4’-amino chalcone, leukemia, cytotoxic, selectivity

Green synthesis of hematite (α-Fe2O3) nanoparticles using Rhus punjabensis extract and their biomedical prospect in pathogenic diseases and cancer

Hematite nanoparticles (NPs) are chemically and thermodynamically more stable among iron oxide NPs therefore these are considered highly suitable for biomedical applications. This work explores Rhus punjabensis extract potential as both reducing and capping agent for phytomediated synthesis of NPs from ferric chloride precursor. The structural, morphological and compositional investigations were carried out with XRD, SEM, TEM, EDS, and FFT. The synthesized NPs were hematite (Fe2O3) with rhombohedral crystal structure of an approximate size of 41.5 ± 5 nm. TEM, FTIR, and TG/DTA indicated phase-pure Fe2O3 formation with controlled size through use of capping agent. Biological activities depict that synthesized NPs had free radical scavenging, antioxidative potential and reducing power activities. These activities are due to phenolic and flavonoid like properties by attachment of different functional groups on the surface of NPs. The NPs exhibited significant Antileishmanial, antibacterial and protein kinase inhibition activities as compared with the corresponding plant extract. NPs depicted cytotoxic effect against HL-60 leukemic and DU-145 prostate cancer cell lines with ED50 values of 11.9 and 12.79 μg/ml, respectively. The inhibitory effect observed in the NF-κB assay suggested apoptotic potential of hematite NPs to cancerous cells. The green chemistry route reported in this paper for Fe2O3 NP synthesis can be safely and efficiently explored for treatment of cancer and as theranostic agent.

Steroidal glycosides from Ornithogalum dubium Houtt

The phytochemical study of Ornithogalum dubium Houtt. (Asparagaceae) led to the isolation of five undescribed steroidal glycosides together with two known ones. Their structures were established by using NMR analysis and mass spectrometry as (25R)-3β-hydroxyspirost-5-en-1β-yl O-α-L-arabinopyranosyl-(1 → 2)-α-L-rhamnopyranoside, (25S)-3β-hydroxyspirost-5-en-1β-yl O-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranoside, (22S)-16β-[(α-L-rhamnopyranosyl)oxy]-22-hydroxycholest-5-en-3β-yl O-β-D-glucopyranosyl-(1 → 4)-β-D-glucopyranoside, (22S,23S)-1β,3β,11α,16β,23-pentahydroxy-5α-cholest-24-en-22β-yl β-D-glucopyranoside, (22S,23S)-3β-[(β-D-glucopyranosyl)oxy]-22,23-dihydroxy-5α-cholest-24-en-16β-yl O-α-L-rhamnopyranosyl)-(1 → 4)-β-D-glucopyranoside. Their cytotoxic activities against two human cells, a lung carcinoma A-549 and a promyelocytic leukemia HL-60 cell lines, were evaluated by using the XTT method. The results showed no significant cytotoxicity on the tested cells. The influence of the potentiation of cisplatin cytotoxicity in A-549 cells was also investigated and a slight effect was observed only for the (25R) spirostane-type derivative.

Determination of anti-cancer and apoptotic effects of Tricholoma terreum, Tricholoma arvernense and Tricholoma imbricatum species

Macrofungi has been used for food and therapeutic purposes for centuries. In terms of their medicinal properties, the fungi contain a variety of polysaccharides, polysaccharide-protein complexes and secondary metabolites. The antimicrobial, antioxidant and anticancer effects are associated with these ingredients. As a cancer-associated fungus, Ganoderma lucidum is the best known in the world. On the other hand, anticancer activity and contents of many mushrooms were investigated. In the genus Tricholoma, T. matsutake is well known in Japan. The medicinal properties of this mushroom have been demonstrated. In this study Tricholoma terreum, Tricholoma arvernense and Tricholoma imbricatum from Turkey have been investigated for anticancer activity and apoptotic effects. Derived extracts from collected fungi were tested against HL 60 and MCF-7 human cancer cell lines. Antiproliferative effect were determined by using MTT method and determination of apoptotic cells was performed using Hoechst/Propidium iodide (HO/PI) double staining method. Different extracts from T.terreum, T. arvernense and T.imbricatum species inhibited 50% of the cells in the concentration range of 15-20 mg / ml, especially in the HL-60 leukemia cell line and in apoptosis research with these extracts T. imbricatum methanol extract caused about %60 apoptotic cell death at the 40 mg/ml concentration. Keywords : Apoptozis, Tricholoma, Cell culture DOI : 10.7176/JSTR/5-2-03

Correlation between IL-7 genomic protein methylation level and acute myeloid leukemia.

To detect Interleukin-7 (IL-7) gene methylation status and transcription level in leukemia cells of peripheral blood of patients with Acute Myelocytic Leukemia (AML) and in the cell lines (HL-60, HL-60/ADM, SKM-1) of AML and myelodysplastic syndrome (MDS), and explore its relationship with the pathogenesis of AML. A total of 55 AML patients (AML group) and 30 healthy adults (Healthy group) from June 2015 to June 2018 were enrolled in this study. The genomic DNA of leukemia cells in peripheral blood was extracted. The methylation-specific PCR (MSP) method was used to detect the methylation rate of the IL-7 gene in peripheral blood of AML group and Health group. Meanwhile, the methylation level of the IL-7 gene leukemia cell lines HL-60/ADM, HL-60, and MV4-11 and SKM-1 were detected in vitro. At the same time, the expression level of IL-7 in peripheral blood was detected by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) kit. The methylation rate of IL-7 gene in peripheral blood of the AML group and Healthy group was 72.7% (40/55) vs. 3.3% (1/30) (p<0.01); IL-7 gene methylation occurred in HL-60/ADM, HL-60, MV4-11 and SKM-1 cell lines. IL-7 gene methylation appears in peripheral blood leukemia cells and AML and MDS cell lines of AML patients. The expression of IL-7 in peripheral blood of patients with AML is significantly decreased, suggesting that this phenomenon is related to the pathogenesis of AML.

Influence of temperature rise by 2.5°C on the increase of apoptosis of HL-60 cells treated with busulfan.

Hyperthermia is one of the new and still poorly known methods used in cancer treatment. It consists of raising the patient's body temperature for therapeutic purposes. The article presents the results of in vitro studies describing the effect of an elevated temperature of 39.5°C, the busulfan cytostatic and their combination on the level of apoptosis of human leukemia HL-60 cells. During the experiments, the influence of a 2.5°C temperature increase on the behavior of the population of 2 groups of HL-60 cells, with busulfan cytostatic and without the cytostatic, was investigated. The control group consisted of 2 groups of HL-60 cells incubated at 37.0°C with the cytostatic and without the cytostatic. Two questions were asked: 1. Is low-temperature hyperthermia likely to have an effect on the effectiveness of busulfan cytostatic? 2. Does the increase in temperature by 2.5°C have an effect on the level of apoptosis in the unsaturated HL-60 cell line? Human promyelocytic leukemia cell line HL-60 was used in the experiments to examine the influence of temperature on apoptosis HL-60 in 2 separated incubators set to 37.0°C and 39.5°C for 3 h. Apoptosis was assessed with flow cytometry using Annexin V. An increase in mortality of HL-60 cells was found in the case of simultaneous exposure to elevated temperature and busulfan in comparison to the group of cells treated with the cytostatic alone. There was no observed effect of an elevated temperature of 39.5°C alone on the level of HL-60 cell apoptosis. Analysis of the study results indicates that low-temperature hyperthermia may be used to increase the effectiveness of busulfan treatment. No effect of an elevated temperature of 39.5°C on the level of apoptosis in HL-60 cells that were not treated with busulfan was observed. There is a need to test the efficacy of other cytostatic agents at elevated temperatures.

Synergistic activity of parthenolide and cytarabine in acute promyelocytic leukemia HL-60 cell line

Journal of Unexplored Medical Data is an open access journal, which considers well-conducted medical science studies, including clinical trials, pilot studies, research reports, original articles, reviews and short communications in the field of medical science.

RA-XXV and RA-XXVI, Bicyclic Hexapeptides from Rubia cordifolia L.: Structure, Synthesis, and Conformation.

Two RA-series bicyclic hexapeptides, RA-XXV (4) and RA-XXVI (5), which have no N-methyl group at Tyr-5, were isolated from the roots of Rubia cordifolia L. Their amino acid compositions and sequences were determined by interpretation of MS, and 1D and 2D NMR data and their relative structures were elucidated by XRD analysis of 4 and RA-XXVI acetate (6). The absolute stereochemistry of 4 was established by the total synthesis of 4, and that of 5, by the chemical correlation with 4. Peptides 4 and 5 exhibited cytotoxicity toward human promyelocytic leukemia HL-60 (IC50 =0.062 and 0.066 μm, respectively) and human colonic carcinoma HCT-116 (IC50 =0.028 and 0.051 μm, respectively) cell lines. Analysis of the conformational structures of 4 and 6 in the crystalline state and those of 4 and 5 in solution revealed that the N-methyl group at Tyr-5 functions to make this series of peptides preferentially adopt the active conformation.

CD34+ Derived Microparticles Regulate Apoptosis in Normal and Acute Myeloid Leukemia Cells

Introduction Microparticles (MPs) are small vesicles 100nm-1μm derived from the apoptotic or stimulated cells. The mechanism of their production is distinctive from exosomes or apoptotic bodies. MPs have been detected in the blood in many pathological conditions associated mainly with endothelial injury, thrombosis and inflammation. Our previous study showed that MPs originated also from CD34+ cells of umbilical cord blood, which is an alternative source for hemopoeitic stem cell transplantation. MPs considered as markers of cell activation, as well as apoptosis. Apoptosis is a complex interaction network regulated either through death receptor like FAS or through the internal pathway of Bcl2, Bax. The two pathways activate the executor of cell death program, caspases. The aim of this study is to elucidate the role of MPs in apoptosis of hemopoietic cells.Methods Umbilical cord blood units (UCB) were collected after informed consent. The units that were used in this study, were rejected as not appropriate for transplantation due to low volume. The HL60 promyelocytic leukemia cell line was cultured in RPMI (Life Technologies) supplemented with 10% fetal calf serum (FCS) and 1% penicillin-strepromycin. CD34+ MPs were isolated from the plasma of UCBs after centrifugation and magnetic bead MACS purification (Miltenyi Biotec). The number of CD34+ MPs was estimated by flow cytometry using CD34-PE and Annexin V-FITC Abs. Mononuclear cells (MNC) were collected after density gradient centrifugation on lymphoprep (Fresenius) and were cultured for 3 and 6 days in the presence of CD34+ MPs. Viability assays were performed using 7-AAD in flow cytometry. In another set of experiments different numbers of CD34+ MPs were used in MNC cultures. RNA was extracted from MNC using Qiagen RNA extraction kit and reverse transcribed into cDNA using Superscript II reverse transcriptase (Invitrogen) with random primers (Promega). RT-PCR was performed using Platinum Pfx polymerase (Invitrogen). The primers for FAS, BCL2, BAX, caspase 3, survivin and GAPDH genes were used. The PCR products were analysed in an agarose gel electrophoresis.Results Cell viability increased in UCB derived MNC (UCB-MNC) incubated with CD34+ MPs (800 /ml) after 3 day vs. 6 days of culture. The UCB-MNC viability was higher using 800/ml CD34+ MPs vs. 400/ml. In contrast, CD34+ MPs (800 /ml vs. 400/ml) did not affect the viability in one day MNC culture. Purified CD34+ MPs were applied to UCB-MNC cultures and by RT-PCR was shown increased expression of BCL2 gene as well as FAS and caspase-3 genes. The promyelocytc cell line HL60 has been used in order to analyze the effect of CD34+ MPs in leukemic cells. The expression of Bcl2 was decreased in HL60 cells co-incubated with CD34+ MPs. This result shows an opposite effect of CD34+ MPs in the apoptotic gene Bcl2 for the HL60 cells indicating that there are different mechanisms of MP function in various cell types.Conclusions In this study we have identified and monitored the time- and dose-dependent effect of CD34-derived microparticles in the viability of UCB mononuclear cells. Additionally, CD34+ MPs function is accosiated with the high expression of the pro- and anti-apoptotic Bcl2 and apoptotic FAS. In contrast CD34+ MPs decreases the expression of Bcl2 in the promyelocytic leukemia cell line HL60. Therefore the stem cell derived microparticles might serve as a potential regulator of apoptosis in normal and malignant hematopoietic cells. DisclosuresNo relevant conflicts of interest to declare.

The Crucial Role of NR4A1 Dependent Apoptosis Induced By Fenretinide in Acute Myeloid Leukemia

OBJECTIVENR4A1 is a member of the orphan nuclear receptor family, which is involved in biological processes such as cell proliferation, apoptosis, metabolism, and differentiation. The expression of NR4A1 is increased in a variety of solid tumors, which promotes oncogenesis and enhances the viability of tumor cells. However, in hematological malignancies, its expression is significantly lower than normal. Studies have shown that clearance of NR4A1 can lead to the progression of mice to acute myeloid leukemia, reducing the amount of NR4A gene expression can make mice progress to MDS / MPN, and even acute myeloid leukemia. This indicates that NR4A1 is an important tumor suppressor in acute myeloid leukemia. The present study was to investigate the effect of drug-induced NR4A1 expression on apoptosis in acute myeloid leukemia and its related mechanisms. The results suggested that NR4A1 may become a new target for the treatment of acute myeloid leukemia. METHODS: In vitro, on the one hand, bone marrow mononuclear cells from patients with newly diagnosed or relapsed acute myeloid leukemia were extracted as samples, and the expression of NR4A1 mRNA and protein was detected by Real-time PCR and Western blot. Acute myeloid leukemia cell lines HL60 and Kasumi-1 were tested for fenretinide activity and apoptosis, as well as NR4A1 and mitochondrial apoptosis pathway-related protein expression. We used siNR4A1 to knockdown NR4A1 expression and LMB to inhibit nuclear export, the apoptosis rate and apoptosis protein were detected. The mechanism of anti-apoptotic effect of NR4A1 was verified by western blot, immunofluorescence and co-immunoprecipitation. AML mice model were established and fenretinide was injected into the tail veins to observe the effects of NR4A1 expression on survival time, blood routine, bone marrow and important organs. Results: 1. NR4A1 expression in patients with acute myeloid leukemia was significantly higher than normal. 2. The expression of NR4A1 increased in a time- and concentration-dependent manner under the action of fenretinide. 3. After siNR4A1, the apoptosis of acute myeloid leukemia cells was significantly decreased, and the expression of apoptotic proteins was decreased. 4. The expression of apoptotic proteins was decreased after fenretinide combined with leptomycin treatment. After fenretinide treatment, the expression of NR4A1 was decreased in nuclear extracts, and the expression of mitochondrial extract was increased. After drug treatment, NR4A1 interacted with Bcl-2, and the bcl-2 BH3 domain in cytoplasm was exposed to play an anti-apoptotic effect. After successful modeling of AML mice, the survival rate of the fenretinide treatment group was significantly prolonged compared with the control group. The peripheral blood leukocytes, hemoglobin and platelet values were significantly different. The liver and spleen volume of the treatment group were significantly smaller than that of the control group. The mRNA and protein levels of NR4A1 in the spleen were significantly higher than those in the control group. Conclusion: The effect of fenretinide on acute myeloid leukemia cells induces NR4A1 expression and plays a pro-apoptotic effect. This apoptosis effect depends on the nuclear export of NR4A1. NR4A1 is located from the nucleus to the mitochondria, and binds to the Bcl-2 BH3 domain.Key words: NR4A1, fenretinide, acute myeloid leukemia, apoptosis, nuclear export DisclosuresNo relevant conflicts of interest to declare.

Anti-leukemia activity of 4-amino-2-aryl-6,9-dichlorobenzo[g]pteridines

Pteridines are bicyclic heterocyclic compounds with a pyrazino[2,3-d]pyrimidine nucleus that have shown a wide range of therapeutic utilities. Concretely, 4-aminopteridine derivatives have demonstrated both anti-inflammatory and anti-cancer properties, and some of them, such as methotrexate, are profusely used in medical practice. We have recently synthesized and tested the biological activity of a novel series of 4-amino-2-aryl-6,9-dichlorobenzo[g]pteridines, finding that they present anti-inflammatory properties, as they were able to inhibit in vitro the production of pro-inflammatory cytokines TNF-α and IL-6. Now, we have evaluated the anti-tumor potential of these compounds on HL-60 and K562 leukemia cell lines. Cells growing at exponential rate were exposed to decreasing doses of each compound, from 50 to 0.39μM, for 24, 48, and 72h. Cell viability was tested by MTT assay and cell death fashion determined by annexin V/propidium iodide assay. The cytotoxicity of the compounds was determined in differentiated macrophage-like HL-60 cells and in human peripheral blood mononuclear cells to evaluate the potential side effects on quiescent tumor cells and normal cells, respectively. Among the series, compounds 1a, 1b, 1g, 1j, and 1k showed anti-proliferative activity. Compounds 1j and 1k were active against both HL-60 and K562 cells, with a lower IC50 against HL-60 cells. Compounds 1a, 1b, and 1g had a great cytotoxic activity against HL-60, but they were far less potent against K562 cells. None had side effects in differentiated tumor cells or in human peripheral blood mononuclear cells. In conclusion, our results demonstrate that some compounds of this series of 4-amino-2-aryl-6,9-dichlorobenzo[g]pteridines have anti-cancer properties in vitro.

Halogenated Meroditerpenoids from a South Pacific Collection of the Red Alga Callophycus serratus.

A detailed examination of the red alga Callophycus serratus collected in Tonga led to the isolation of six new halogenated meroditerpenoids: callophycol C (1), callophycoic acid I (2), iodocallophycols E (3) and F (4), iodocallophycoic acid B (5), and callophycoic acid J (6). Of these, compounds 3-5 are new iodinated additions to the growing family of Callophycus meroditerpenoids. The relative configurations of compounds 1-6 were deduced by analyses of 1D NOE data and 1H-1H scalar coupling constants, and 3-6 are proposed to differ from the closely related compounds reported in the literature, iodocallophycoic acid A and iodocallophycols A-D. Iodocallophycol E (3) exhibited moderate cytotoxicity against the promyelocytic leukemia cell line HL-60 with an IC50 value of 6.0 μM.

Pyronaridine exerts potent cytotoxicity on human breast and hematological cancer cells through induction of apoptosis.

The potent antimalarial drug pyronaridine (PND) was tested for its potential as an anticancer drug. After exposing cancerous (17) and non-cancerous (2) cells to PND for 72 hr, PND was found to exhibit consistent and potent cytotoxic activity at low micromolar (μM) concentrations that ranged from 1.6 μM to 9.4 μM. Moreover, PND exerted a significant selective cytotoxicity index (SCI) on five out of seven breast cancer cell lines tested, with favorable values of 2.5 to 4.4, as compared with the non-cancerous breast MCF-10A cell line. By using the same comparison, PND exhibited a significant SCI on three out of four leukemia/lymphoma cell lines with promising values of 3.3 to 3.5. One breast cancer and one leukemia cell line were tested further in order to determine the likely mode of action of PND. PND was found to consistently elicit phosphatidylserine externalization, mitochondrial depolarization, and DNA fragmentation, in both the triple negative MDA-MB-231 breast cancer and HL-60 leukemia cell lines. In addition, PND treatment altered cell cycle progression in both cancer cells. Subsequent DNA mobility-shift assays, UV-Visible spectroscopic titrations, and circular dichroism (CD) experiments revealed that PND intercalates with DNA. The findings presented in this study indicates that PND induces apoptosis and interfered with cell cycle progression of cancer cell lines and these results indicate that this drug has the potential as a repurposed drug for cancer therapy.

Enhanced neutrophil autophagy and increased concentrations of IL-6, IL-8, IL-10 and MCP-1 in rheumatoid arthritis

Rheumatoid arthritis (RA) is a high morbidity and disability disease with numerous inflammatory cells infiltrating in interstitial of articular cartilages and bones. As the most abundant inflammatory cells, neutrophil has been reported that their apoptosis changed gradually in the circumstance of RA. Apoptosis, one modality of programmed cell death (PCD), is closely associated with autophagy, which indicates neutrophil autophagy may also alter in RA. Flow cytometry, western blotting, immunohistochemistry, immunofluorescence, transmission electron microscope and multiplex antibody microarray were used to comparative investigate the status of neutrophil autophagy in patients with RA and in vitro. The results showed that the expression of autophagy related LC3 protein was up-regulated with lower lysosomal pH in neutrophils from synovial fluid of RA and changed under stimulation of CQ and small RNA interferences (siRNAs) Atg5 transfection, which proved in acute promyelocytic leukemia HL-60 cell lines, predominantly a neutrophilic promyelocyte, treated by plasma and synovial fluid from RA. We further found out the concentration of IL-6, IL-8, IL-10 and MCP-1 was higher in their synovial fluid which may mediate neutrophil autophagy in RA via cytokine-cytokine receptor interaction and IL-17 signaling pathway. Our results indicate that neutrophil autophagy may be a novel perspective to understand the pathology which may provide a new maker to diagnose RA and IL-8, IL-10, MCP-1 specific antagonists and neutrophil autophagy target inhibitors may improve the therapeutic effect of RA someday.