Human Acute Myeloid Leukemia HL-60 Research Articles

Novel indole retinoid derivative induces apoptosis and cell cycle arrest and modulates AKT and ERK signaling in HL-60 cells.

Chemotherapy with targeted drugs is the first line therapy option for acute and chronic myeloid leukemia. However, hematopoietic stem cell transplantation may be used in high-risk patients or patients with failed responses to chemo drugs. Discovery and development of more effective new agents with lower side effects is the main aim of leukemia treatment. In this study, a novel retinoid compound with tetrahydronaphthalene ring was synthesized and evaluated for anticancer activity in human chronic and acute myeloid leukemia cell lines K562 and HL-60. Novel N-(1H-indol-1-yl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-carboxamide was synthesized based on molecular hybridization of the two different bioactive structures retinoid head and indole. The effects of the synthesized carboxamide compound, which was referred to as compound 5, were determined in K562 chronic myeloid leukemia and HL-60 acute myeloid leukemia cell lines and L929 fibroblast cell line, which served as a control. Colorimetric MTT and caspase3 activity tests, flow cytometry, western blot, and microscopic examinations were used to evaluate biological activity. Compound 5 more effectively induced cell death in HL60 cells in comparison to K562 cells and L929 fibroblast cells. Therefore, further mechanism of cell death was investigated in HL60 cell line. It was found that compound 5 induced remarkable cytotoxicity, caspase3 activation, and PARP fragmentation in HL60 cells. Flow cytometric staining showed that the percentage of cells arrested in G0/G1 was also increased with compound 5 treatment. Important modulator proteins of cell proliferation p-ERK, p-AKT, and p-m-TOR were also found to be inhibited with compound 5 treatment. Collectively, our results reveal compound 5, which is a novel indole retinoid compound as a potential active agent for the treatment of acute promyelocytic leukemia.

Green synthesis of copper oxide spindle like nanostructure using Hibiscus cannabinus flower extract for antibacterial and anticancer activity applications

In this study, CuO nanospindle synthesized using petal extract of Hibiscus cannabinus (H. cannabinus) was attempted to have better antibacterial activity against Gram-positive and Gram-negative bacteria. Cytotoxic activity was found to be 70 % against human acute myeloid leukemia HL60 cell line at 500 µg/ml. Morphological features of CuO was analyzed by scanning electron microscopy (SEM). Structural properties were analyzed by X-ray diffraction (XRD) pattern and the Fourier transform infrared (FTIR) spectrum of CuO provides additional evidence for the formation of monoclinic crystal structured CuO. Energy dispersive X-ray spectroscopy (EDAX) analysis exposed the pristine properties of CuO nanospindles.

Anti-leukemic activity of Cyperus rotundus L. on human acute myeloid leukemia HL-60 cells in vitro

Context: Acute myeloid leukemia (AML) is the most common form of acute leukemia. Currently, many people use medicinal herbs for the treatment of cancers. Nut grass (Cyperus rotundus L.) is a medicinal plant widely used in conventional medicine due to its role as an anti-cancer. Aims: To evaluate the effect of Cyperus rotundus tuber (CRT) ethanolic extract on cell proliferation, differentiation, cell cycle, and apoptotic of HL-60 cells. Methods: HL-60 cells line as a model for AML was cultured under the influence of CRT extract concentrations (35.4, 354, and 3540 µg/mL) for 48 h. Furthermore, the cell was subjected to carboxyfluorescein succinimidyl ester staining for proliferation, using a CD117 marker for differentiation. Annexin V-FITC and PI for cell cycle and apoptosis. The effect of this herb was studied by flow cytometry. The data were statistically analyzed with one-way ANOVA (p≤0.05) and the Tukey test using SPSS version 16 for Windows. Results: The results showed that the CRT inhibited proliferation activity, differentiation, cell cycle arrest, and apoptosis. Conclusions: The data clearly showed the potential anti-cancer activity of CRT on HL-60 cells and suggested that it could help develop promising therapeutic agents for AML treatments.

Novel benzo chromene derivatives: design, synthesis, molecular docking, cell cycle arrest, and apoptosis induction in human acute myeloid leukemia HL-60 cells

A series of benzo[h]chromenes, benzo[f]chromenes, and benzo[h]chromeno[2,3-d]pyrimidines were prepared. All the newly synthesised compounds were selected by National Cancer Institute for single-dose testing against 60 cell lines. Benzo[h]chromenes 5a and 6a showed promising anti-cancer activity and selected for the five-dose testing. Compounds 5a and 6a suppressed cell growth in HL-60 by the induction of cell cycle arrest, which was confirmed using flow cytometry and Annexin V-FITC/PI assays showed at the G1/S phase by regulating the expression of CDK-2/CyclinD1, triggering cell apoptosis by activating both the extrinsic (Fas/Caspase 8) and intrinsic (Bcl-2/Caspase 3) apoptosis pathways, which were determined by the western blot. Benzo[h]chromenes 5a and 6a decreased the protein expression levels of Bcl-2, CDK-2, and CyclinD1 and increased the expression of caspase 3, caspase 8, and Fas. In silico molecular analysis of compounds 5a and 6a in CDK-2 and Bcl-2 was performed.

Antiproliferative Activity and Apoptotic Potential of Curcumin on Human Acute Myeloid Leukemia Cell Line (HL-60)

Curcuma Longa or also known as turmeric is a prominent natural remedy in Asia that has been utilized to heal diseases for decades. It has been demonstrated to have substantial biological activities, including anticarcinogenic, antimutagenic, antioxidant, anti-inflammatory, anti-infectious, hypocholesterolemic, and chemopreventive properties. Curcumin is a therapeutic substance extracted from Curcuma Longa that is thought to have anti-cancer properties against a variety of cancer types. However, its anti-cancer potential on acute myeloid leukemia (AML) remains uncertain [1,2]. 
 
 The objective of this intervention study was to examine the anti-proliferative and apoptotic effects of curcumin on the HL-60 cell line of acute myeloid leukemia. A stock solution of pure curcumin compound was obtained by dissolving it in dimethyl sulfide (DMSO). HL-60 cells were treated with six different concentrations which were 100, 50, 25, 12.5, 6.25, 3.125 µg/ml and incubated at three distinct incubation period (24, 48 and 72 hours). The in vitro cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and quantified using a microplate reader at 570 nm. Acridine orange (AO) and propidium iodide (PI) staining were employed to evaluate morphological changes. 
 
 MTT assays results show that curcumin has a high cytotoxicity effect on HL-60 cells. The maximal half inhibitory concentration (IC50) value of curcumin was consistently decreased when the incubation time increased. The IC50 of curcumin on HL-60 cells were reported to be 13, 8, and 3.5 µg/ml at 24, 48 and 72 hours of incubation, respectively. There was a significantly different IC50 value (p<0.05) of curcumin between the incubation periods (Figure 1). The morphological alteration was shown in AO/PI staining by the appearance of early apoptosis, late apoptosis, and necrotic cells.
 These findings illustrated that curcumin revealed an effective anti-cancer action on human acute myeloid leukemia HL-60 cell line. Further research is required to determine the mechanism of cell death in HL-60 cells treated with pure curcumin compound.
 
 In conclusion, the study revealed the role of putative targeted genes in the biological mechanism in gout pathogenesis has provides insight of the potential biomarkers in developing the personalized medicine better treatment for gout patient in ensuring better patient healthcare.

Effects of SIDT2 on the miR-25/NOX4/HuR axis and SIRT3 mRNA stability lead to ROS-mediated TNF-α expression in hydroquinone-treated leukemia cells.

Our previous studies indicated that the benzene metabolite hydroquinone (HQ) evokes the ROS/p38 MAPK/protein phosphatase 2A/tristetraprolin axis, leading to increased TNF-α expression in human acute myeloid leukemia cell lines U937 and HL-60. In this study, we aimed to identify the upstream pathway involved in ROS-mediated TNF-α expression. HQ treatment increased SIDT2 expression, which subsequently decreased miR-25 and SIRT3 expression in U937 cells. Notably, miR-25 downregulation promoted SIDT2 expression in HQ-treated U937 cells. SIDT2 induced lysosomal degradation of SIRT3 mRNA, but inhibited miR-25 expression through a lysosome-independent pathway. MiR-25 inhibition reduced NOX4 mRNA turnover, resulting in increased NOX4 protein levels. NOX4 induces mitochondrial ROS production and HuR downregulation. Restoration of HuR expression increased SIRT3 expression, suggesting that NOX4-mediated HuR downregulation promotes SIDT2-mediated degradation of SIRT3 mRNA. Inhibition of NOX4 or SIRT3 overexpression abolished HQ-induced ROS production, thereby abolishing TNF-α upregulation. Overall, these results indicate that SIDT2 regulates the miR-25/NOX4/HuR axis and SIRT3 mRNA destabilization, leading to ROS-mediated TNF-α upregulation in HQ-treated U937 cells. HQ-induced increase in TNF-α expression in HL-60 cells was also mediated through a similar pathway.

Xanthohumol Induces ROS through NADPH Oxidase, Causes Cell Cycle Arrest and Apoptosis.

Reactive oxygen species (ROS) are either toxic in excess or essential for redox signalling at the physiological level, which is closely related to the site of generation. Xanthohumol (XN) is an important natural product of hops (Humulus lupulus L.) and was reported to induce ROS in mitochondria. While in the present study, our data indicate that NADPH oxidase (NOX) is another site. In human acute myeloid leukemia HL-60 cells, we first identified that cell proliferation was inhibited by XN without affecting viability, and this could be alleviated by the antioxidant N-acetyl-L-cysteine (NAC); cell cycles were blocked at G1 phase, apoptosis was induced in a dose-dependent manner, and malondialdehyde (MDA) content was upregulated. XN-induced ROS generation was detected by flow cytometry, which can be inhibited by diphenyleneiodonium chloride (DPI, a NOX inhibitor), while not by NG-methyl-L-arginine acetate (L-NMMA, a nitric oxide synthase inhibitor). The involvement of NOX in XN-induced ROS generation was further evaluated: immunofluorescence assay indicated subunits assembled in the membrane, and gp91phox knockdown with siRNA decreased XN-induced ROS. Human red blood cells (with NOX, without mitochondria) were further selected as a cell model, and the XN-induced ROS and DPI inhibiting effects were found again. In conclusion, our results indicate that XN exhibits antiproliferation effects through ROS-related mechanisms, and NOX is a source of XN-induced ROS. As NOX-sourced ROS are critical for phagocytosis, our findings may contribute to the anti-infection and anti-inflammatory effect of XN.

Grape seed proanthocyanidin extract induces apoptosis of HL-60/ADR cells via the Bax/Bcl-2 caspase-3/9 signaling pathway.

BackgroundOur previous study detailed the direct induction of apoptosis by grape seed proanthocyanidin extract (GSPE) in a multidrug resistant human acute myeloid leukemia (AML) HL-60/adriamycin (HL-60/ADR) cell line, although the mechanism of this effect was not detailed. This study aims to elucidate the mechanism underlying GSPE-induced cell apoptosis in HL-60/ADR cells.MethodsHL-60/ADR cells were studied to evaluate effects of GSPE (0–100 µg/mL); a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was employed to identify the cytotoxic effect of varying GSPE concentrations. Trypan blue staining was used to observe changes in cell viability; flow cytometry assays were used to verify apoptosis. Expression of Bax and Bcl-2 mRNA was analyzed using real-time polymerase chain reaction (PCR). Activity of caspase-3 and caspase-9 was also detected.ResultsHere, GSPE was found to inhibit HL-60/ADR cell growth and induce cell apoptosis in a dose-dependent manner. Real-time PCR findings revealed that GSPE concentrations above 75 µg/mL significantly increase expression of Bax mRNA (P<0.001). GSPE concentrations above 25 µg/mL were found to significantly decrease expression of Bcl-2 mRNA (P<0.01), while concentrations above 50 µg/mL were found to significantly increase caspase-3 activity after 6, 12 and 24 h (P<0.01). However, only 100 µg/mL GSPE was found to significantly increase caspase-9 activity (P<0.001 at 6 and 12 h; P<0.05 at 24 h).ConclusionsGSPE inhibits the proliferation of HL-60/ADR cells by the induction of apoptosis in a dose-dependent manner via the Bax/Bcl-2 caspase-3/9 signaling pathway.

Axolotl Ambystoma mexicanum extract induces cell cycle arrest and differentiation in human acute myeloid leukemia HL-60 cells.

Acute myeloid leukemia is the most common form of acute leukemia in adults, constituting about 80% of cases. Although remarkable progress has been made in the therapeutic scenario for patients with acute myeloid leukemia, research and development of new and effective anticancer agents to improve patient outcome and minimize toxicity is needed. In this study, the antitumor activity of axolotl (AXO) Ambystoma mexicanum crude extract was assessed in vitro on the human acute myeloid leukemia HL-60 cell line. The anticancer activity was evaluated in terms of ability to influence proliferative activity, cell viability, cell cycle arrest, and differentiation. Moreover, gene expression analysis was performed to evaluate the genes involved in the regulation of these processes. The AXO crude extract exhibited antiproliferative but not cytotoxic activities on HL-60 cells, with cell cycle arrest in the G0/G1 phase. Furthermore, the AXO-treated HL-60 cells showed an increase in both the percentage of nitroblue tetrazolium positive cells and the expression of CD11b, whereas the proportion of CD14-positive cells did not change, suggesting that extract is able to induce differentiation toward the granulocytic lineage. Finally, the treatment with AXO extract caused upregulation of CEBPA, CEBPB, CEBPE, SPI1, CDKN1A, and CDKN2C, and downregulation of c-MYC. Our data clearly show the potential anticancer activity of Ambystoma mexicanum on HL-60 cells and suggest that it could help develop promising therapeutic agents for the treatment of acute myeloid leukemia.

Anticancer effects of an extract from a local planarian species on human acute myeloid leukemia HL-60 cells in vitro

Current anti-cancer drugs can cause many undesirable side effects to patients. Thus, there is a constant need to develop alternative therapeutic drugs. Bioactive compounds derived from natural products including animals, plants and microorganisms are being actively studied as sources for anticancer treatments. Freshwater planarians are important models for stem cell research and regeneration. However, to date, no studies on the biological activities of planaria extracts on cancer have been published. The aim of this study was to examine the potential antitumoral activity of the extract from planaria species-Malta (PSM) on human acute myeloid leukemia (AML) HL-60 cells. Antiproliferative activity was studied in terms of proliferation, apoptosis and differentiation. The expression of genes involved in the regulation of these important cellular processes was also analyzed using real-time PCR. PSM extract exhibited a selective cytotoxic effect on HL-60 cells when compared to normal lymphocytes. Furthermore, cell cycle analysis and Annexin V/PI assay showed that the extract induced apoptosis in HL-60 cells. The PSM extract induced myeloid differentiation with HL-60 cells showing a decreased nucleo/cytoplasmic ratio, an increase in nitroblue tetrazolium-positive cells, and CD11b- and CD14-positive cells. Finally, we also found that the PSM extract increased the expression of CEBPA, CEBPB, CEBPE, SPI1, BAX, CDKN1A and CDKN2C; whereas it reduced the expression of c-MYC and BCL2.This is the first study to reveal the antiproliferative, cytotoxic, and differentiation potential of PSM on HL-60 cells and suggests that it may have considerable potential for development as a novel natural product-based anticancer agent against AML.

GSK3β suppression inhibits MCL1 protein synthesis in human acute myeloid leukemia cells.

Previous studies have shown that glycogen synthase kinase 3β (GSK3β) suppression is a potential strategy for human acute myeloid leukemia (AML) therapy. However, the cytotoxic mechanism associated with GSK3β suppression remains unresolved. Thus, the underlying mechanism of N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418)-elicited GSK3β suppression in the induction of AML U937 and HL-60 cell death was investigated in this study. Our study revealed that AR-A014418-induced MCL1 downregulation remarkably elicited apoptosis of U937 cells. Furthermore, the AR-A014418 treatment increased p38 MAPK phosphorylation and decreased the phosphorylated Akt and ERK levels. Activation of p38 MAPK subsequently evoked autophagic degradation of 4EBP1, while Akt inactivation suppressed mTOR-mediated 4EBP1 phosphorylation. Furthermore, AR-A014418-elicited ERK inactivation inhibited Mnk1-mediated eIF4E phosphorylation, which inhibited MCL1 mRNA translation in U937 cells. In contrast to GSK3α, GSK3β downregulation recapitulated the effect of AR-A014418 in U937 cells. Transfection of constitutively active GSK3β or cotransfection of constitutively activated MEK1 and Akt suppressed AR-A014418-induced MCL1 downregulation. Moreover, AR-A014418 sensitized U937 cells to ABT-263 (BCL2/BCL2L1 inhibitor) cytotoxicity owing to MCL1 suppression. Collectively, these results indicate that AR-A014418-induced GSK3β suppression inhibits ERK-Mnk1-eIF4E axis-modulated de novo MCL1 protein synthesis and thereby results in U937 cell apoptosis. Our findings also indicate a similar pathway underlying AR-A014418-induced death in human AML HL-60 cells.

The pan-Bcl-2 inhibitor obatoclax promotes differentiation and apoptosis of acute myeloid leukemia cells

SummaryOne of the key features of acute myeloid leukemia (AML) is the arrest of differentiation at the early progenitor stage of myelopoiesis. Therefore, the identification of new agents that could overcome this differentiation block and force leukemic cells to enter the apoptotic pathway is essential for the development of new treatment strategies in AML. Regarding this, herein we report the pro-differentiation activity of the pan-Bcl-2 inhibitor, obatoclax. Obatoclax promoted differentiation of human AML HL-60 cells and triggered their apoptosis in a dose- and time-dependent manner. Importantly, obatoclax-induced apoptosis was associated with leukemic cell differentiation. Moreover, decreased expression of Bcl-2 protein was observed in obatoclax-treated HL-60 cells. Furthermore, differentiation of these cells was accompanied by the loss of their proliferative capacity, as shown by G0/G1 cell cycle arrest. Taken together, these findings indicate that the anti-AML effects of obatoclax involve not only the induction of apoptosis but also differentiation of leukemic cells. Therefore, obatoclax represents a promising treatment for AML that warrants further exploration.

Inhibition of UBE2L6 attenuates ISGylation and impedes ATRA-induced differentiation of leukemic cells.

Ubiquitin/ISG15‐conjugating enzyme E2L6 (UBE2L6) is a critical enzyme in ISGylation, a post‐translational protein modification that conjugates the ubiquitin‐like modifier, interferon‐stimulated gene 15 (ISG15), to target substrates. Previous gene expression studies in acute promyelocytic leukemia (APL) cells showed that all‐trans‐retinoic acid (ATRA) altered the expression of many genes, including UBE2L6 (200‐fold) and other members of the ISGylation pathway. Through gene expression analyses in a cohort of 98 acute myeloid leukemia (AML) patient samples and in primary neutrophils from healthy donors, we found that UBE2L6 gene expression is reduced in primary AML cells compared with normal mature granulocytes. To assess whether UBE2L6 expression is important for leukemic cell differentiation—two cell line models were employed: the human APL cell line NB4 and its ATRA‐resistant NB4R counterpart, as well as the ATRA‐sensitive human AML HL60 cells along with their ATRA‐resistant subclone—HL60R. ATRA strongly induced UBE2L6 in NB4 APL cells and in ATRA‐sensitive HL60 AML cells, but not in the ATRA‐resistant NB4R and HL60R cells. Furthermore, short hairpin (sh)RNA‐mediated UBE2L6 depletion in NB4 cells impeded ATRA‐mediated differentiation, suggesting a functional role for UBE2L6 in leukemic cell differentiation. In addition, ATRA induced ISG15 gene expression in NB4 APL cells, leading to increased levels of both free ISG15 protein and ISG15 conjugates. UBE2L6 depletion attenuated ATRA‐induced ISG15 conjugation. Knockdown of ISG15 in NB4 APL cells inhibited ISGylation and also attenuated ATRA‐induced differentiation. In summary, we demonstrate the functional importance of UBE2L6 in ATRA‐induced neutrophil differentiation of APL cells and propose that this may be mediated by its catalytic role in ISGylation.

Cephasinenoside A, a new cephalotane diterpenoid glucoside from Cephalotaxus sinensis

A new cephalotane diterpenoid glucoside, cephasinenoside A (1), was isolated from the twigs and leaves of Cephalotaxus sinensis (Rehd. et Wils.) Li. The structure of 1 was established on the basis of spectroscopic analyses and ECD calculations. Compound 1 represents the first cephalotane diterpenoid glycoside which inhibits the proliferation of human acute myeloid leukemia HL-60 cells with a GI50 value of 7.17 ± 1.03 μM.

Knockdown of diacylglycerol kinase zeta (DGKZ) induces apoptosis and G2/M phase arrest in human acute myeloid leukemia HL-60 cells through MAPK/survivin/caspase pathway.

Diacylglycerol kinase zeta (DGKZ) is associated with the pathogenesis of a variety of malignant diseases, but its biological function on acute myeloid leukemia (AML) has not been explored. The aim of this study was to analyze apoptosis induced by knockdown of DGKZ and its mechanism in human acute myeloid leukemia HL-60 cells. qRT-PCR was carried out to detect the expression of DGKZ in HL-60, THP-1, Jurkat, K562, and CD34 cell lines. Additionally the expression of DGKZ in AML cells obtained from patients were detected by qRT-PCR. Cell Counting Kit-8 (CCK-8) assay was used to determine the viability of HL-60 cells DGKZ knocked down. Apoptosis and cell cycle phase of HL-60 cells after DGKZ knockdown were evaluated by flow cytometry. Western blot analysis was performed to investigate expressions of the proteins related to apoptosis and cell cycle. Results showed that expression of DGKZ was significantly higher in HL-60 and AML cells obtained from patients than those of Jurkat, THP-1, K562 and human CD34 cell. Compared with the shCtrl group, DGKZ was markedly knocked down in HL-60 cells transfected with lentivirus encoding shRNA. DGKZ knockdown significantly inhibited the proliferation and induced cycle arrest at the G2/M phase in HL-60 cells. The expressions of MAPK, caspase-3, caspase-8, cytochrome C markedly increased and p-MAPK and survivin decreased in HL-60 cells after DGKZ knockdown. The results suggest that knockdown of DGKZ can induce apoptosis and G2/M phase arrest in human acute myeloid leukemia HL-60 cells through the MAPK/survivin/caspase pathway.

Synthesis of New N1Arylpiperazine Substituted Xanthine Derivatives and Evaluation of their Antioxidant and Cytotoxic Effects.

Cancer is one of the leading causes of morbidity and mortality worldwide, with approximately 14 million new cases in 2012, with most of the clinically used drugs being ineffective. Methylxanthines have raised more interest in research on modifying their structure because of their diverse biological activity. In addition, the piperazine nucleus is one of the most important heterocycles exhibiting remarkable pharmacological activities. The structure of the obtained compounds was characterized and elucidated by IR, 1H and 13C NMR and LCMS spectral analysis. The purity of the substances was proven by corresponding melting points and elemental analysis. The antioxidant activity was evaluated by four common methods - DPPH, ABTS, FRAP and lipid peroxidation assay. The cytotoxic effects of the tested series were evaluated using the standard MTT-dye reduction assay on three tumour cell lines. A series of new xanthine derivatives comprising an arylpiperazine moiety at N1 were synthesized. The cytotoxicity against human T-cell leukemia cell SKW-3, human acute myeloid leukemia HL-60 and human Bcell precursor leukemia cell REH was evaluated. The relationship between the structure and citotoxicity of the compounds was investigated by quantitative structure-activity relationship (QSAR) analysis and the important structural parameters were drawn. The highest antioxidant activity was demonstrated by compound 6c. The highest cytotoxic effect was observed for compound 6f. It was found that cytotoxicity against SKW-3 depends on the electron density distribution in the structures. Branching of the molecular skeleton and introduction of heteroatoms like fluorine and sulfur in the structures also significantly improved the antiproliferative activity of the compounds.

Notopterol-induced apoptosis and differentiation in human acute myeloid leukemia HL-60 cells.

Purpose: This study aims to observe the effects of notopterol on the apoptosis and differentiation of HL-60 cells and to explore the underlying molecular mechanisms.Methods: Cell viability was assessed using sulforhodamine B assay. Cell proliferation was determined by the trypan blue dye exclusion test. Colony-forming units were assayed in methylcellulose. Apoptosis assays were carried out by annexin V-fluorescein isothiocyanate(FITC)/propidium iodide (PI) double staining, Hoechst 33342 staining, mitochondrial membrane potential, and Western blot. Wright–Giemsa staining, nitroblue tetrazolium (NBT) reduction assay, CD11b and CD14 and Western blot were detected for induction of differentiation. In addition, cell-cycle phase distribution was analyzed by flow cytometry and Western blot. The combination therapy of notopterol and all-trans retinoic acid (ATRA) on HL-60 cells was examined.Results: Notopterol obviously inhibited the growth of HL-60 cells with an IC50 value of 40.32 μM and remarkably reduced the number of colonies by 10, 20, and 40 µM. In addtion, notopterol induced the percentage of apoptotic HL-60 cells, reduced the mitochondrial membrane potential, decreased the protein expresstion of Bcl-2 and Mcl-1, and increased the expression of Bax, cleavage of caspase 9, caspase 3, and PARP. As for cell differentiation, notopterol clearly induced chromatin condensation; increased the nucleocytoplasmic ratio, nitroblue tetrazolium-positive cells, expression of CD14 and CD11b, and protein expression of c-Jun and Jun B, and decreased c-myc. Furthermore, notopterol induced the G0/G1 cell-cycle arrest as determined using flow cytometry, which may be related to the regulation of cell-cycle-related proteins p53, CDK2, CDK4, Cyclin D1, Cyclin E, and survivin. The combined use of notopterol and ATRA did not enhance the apoptotic effect as evidenced by cell viability test and Hoechst 33342. However, the combination of notopterol and ATRA enhanced the effect of inducing differentiation when compared with using either notopterol or ATRA alone, which can be evidenced by the increased nucleocytoplasmic ratio, NBT positive cells, and expression of CD14.Conclusion: This is the first time it has been demonstrated that notopterol could induce apoptosis, differentiation, and G0/G1 arrest in human AML HL-60 cells, suggesting that notopterol has potential therapeutic effects on AML. The combination application of notopterol (20 and 40 μM) and ATRA (2 μM) could augment differentiation of HL-60 cells.

Overexpression of MYCT1 Inhibits Proliferation and Induces Apoptosis in Human Acute Myeloid Leukemia HL-60 and KG-1a Cells in vitro and in vivo.

MYC target 1 (MYCT1), a direct target gene of c-Myc, is a novel candidate tumor suppressor gene first cloned from laryngeal squamous cell carcinoma. The downregulation of MYCT1 has been reported to be associated with carcinogenesis. However, the role of MYCT1 in the development and progress of acute myeloid leukemia (AML) remains unknown and requires further investigation. In this study, we first found that the expression level of MYCT1 was significantly lower in the bone marrow (BM) derived from AML patients than that from healthy individuals. The low expression of MYCT1 in AML BM may be due to the hypermethylation in its promoter. MYCT1 expression was strongly associated with French–American–British classifications of AML. The low expression level of MYCT1 was more often observed in patients of M1, M5 and M6 types. In vitro, lentiviral particles carrying the complete CDS of MYCT1 gene were used to mediate the forced overexpression of MYCT1 in two AML cell lines, HL-60 and KG-1a. MYCT1 overexpression significantly inhibited cell proliferation, arrested cell cycle at G0/G1 phase, and downregulated the expression of cyclins D and E. Moreover, MYCT1 overexpression triggered apoptosis in AML cells, which was accompanied by enhanced cleavage of caspase-3 and -9, upregulated expression of B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), and downregulated Bcl-2. Finally, in BALB/c nude mice bearing xenograft tumors generated by HL-60 and KG-1a cells, we noted that the intratumoral injection of MYCT1 lentivirus repressed tumor growth and led to massive apoptosis. In summary, our results reveal that MYCT1’s promoter is hypermethylated and its expression is downregulated in the BM of AML patients. MYCT1 plays a tumor-suppressive role, and it may serve as a promising target for the genetic therapeutic strategy in treating AML.

Effects of Arsenic Trioxide on Cdc20 and Mad2 in Acute Myeloid Leukemia HL-60 Cell Line

To investigate the effects of arsenic trioxide (As2O3) on Cdc20 and Mad2 in process of AML HL-60 cell proliferation. The proliferation of HL-60 cells was detected by CCK-8 method at different concentrations of arsenic trioxide for 24, 48 and 72 hours. The cell morphological changes were observed by inverted microscopy. The expressions of Mad2 and Cdc20 mRNA and protein in HL-60 cells treated with As2O3 for 48 h were detected by real-time PCR and Western blot respectively. Arsenic trioxide significantly inhibited the HL-60 cell proliferation and displayed a good time-dose correlation. RT-PCR and Western blot showed that the expression of Mad2 was up-regulated and the expression of Cdc20 was down-regulated in HL-60 cells treated with arsenic trioxide of different concentration (4,8,10 µmol/L). Arsenic trioxide can inhibit the human acute myeloid leukemia HL-60 cell proliferation, and its mechanism may be related with up-regulation of Mad2 expression and down-regulation of Cdc20 expression.

Determination of the Antiproliferative Activity of New Theobromine Derivatives and Evaluation of Their In Vitro Hepatotoxic Effects.

A new series of N-substituted 1-benzyltheobromine-8-thioacetamides were designed and synthesized. Their anti-proliferative activity against human chronic myelocytic leukemia cell K562, human T-cell leukemia cell SKW-3 and human acute myeloid leukemia HL-60 was evaluated. For the tested compounds a concentrationdependent cytotoxic activity was observed, with 7g outlined as the most active compound within the series. The targed compounds were obtained in yields of 56 to 85% and their structures were elucidated by FTIR, (1)H NMR, (13)C NMR and microanalyses. The compounds purity was proven by elemental analysis and spectral data. In general, the compounds showed low hepatotoxicity on sub-cellular and cellular level. On isolated rat microsomes only 7d showed toxic effect while theobromine, 1-benzyl-theobromine-thioacetic acid (BTTA) and the other new theobromine derivatives were devoid of toxicity. In isolated rat hepatocytes, when compared to theobromine and BTTA, 7f showed lower cytotoxic effects, and 7d exerted higher cytotoxicity. The results indicate 7g as a promising structure for the design of future compounds with low hepatotoxicity and good antiproliferative activity.