Human Promyelocytic Leukemia Cell Line Research Articles

A method for detecting protein expression in single cells using the C1™ Single-Cell Auto Prep System (TECH2P.874)

Abstract Recent improvements in microfluidics and biochemistry have enabled single-cell molecular analysis, providing new insight into the heterogeneity of cell populations. The C1 Single-Cell Auto Prep System is an automated platform that streamlines the isolation and processing of 96 individual, live cells for RNA and DNA analysis. Single-cell protein profiling is a direct complement to genomic analysis as it provides additional insights into key molecular mechanisms and system biology. To enable this, we adapted a highly multiplexed protein detection system (Proseek Multiplex Oncology, Olink Bioscience) for use on the C1 System. We analyzed 192 individual cells from two human promyelocytic leukemia cell lines (HL60 and K562) and compared these result against 16 FACS-sorted cells in tube format. These results indicated that four key proteins (i.e. CASP3, CSTB, CD69 and MPO) are differentially expressed between the two cell lines and vary from cell-to-cell. For example, the human protein MPO, produced by neutrophils for their microbicidal activity, was differentially expression between individual HL60 and K562 cells, suggesting varying levels of neutrophil activation within the cell populations. This work demonstrates highly-multiplexed protein detection at the single-cell level and shows promise to have sufficient sensitivity, reproducibility and scale to effectively interrogate a statistically significant numbers of individual cells.

Dystroglycan depletion inhibits the functions of differentiated HL-60 cells

Dystroglycan has recently been characterized in blood tissue cells, as part of the dystrophin glycoprotein complex but to date nothing is known of its role in the differentiation process of neutrophils. We have investigated the role of dystroglycan in the human promyelocytic leukemic cell line HL-60 differentiated to neutrophils. Depletion of dystroglycan by RNAi resulted in altered morphology and reduced properties of differentiated HL-60 cells, including chemotaxis, respiratory burst, phagocytic activities and expression of markers of differentiation. These findings strongly implicate dystroglycan as a key membrane adhesion protein involved in the differentiation process in HL-60 cells.

Spectroscopic Characterization,In VitroCytotoxicity, and Antioxidant Activity of Mixed Ligand Palladium(II) Chloride Complexes Bearing Nucleobases

Mixed-ligand palladium(II) chloride complexes bearing the nucleobases, adenine (Ad), cytosine (Cyt), and guanine (Gua), have been synthesized and characterized by UV-vis spectrophotometric methods, magnetic susceptibility, molar conductivity, elemental analysis, FTIR, and1H-NMR. The complexes were found to have the composition cis-[PdCl2(Gua)(Cyt)], cis-[PdCl2(Ad)(Cyt)], and cis-[PdCl2(Ad)(Gua)]. A four-coordinated square-planar geometry is proposed for these Pd(II) complexes based on magnetic evidence and electronic spectra. The complexes as well as the free nucleobase ligands were tested for theirin vitrocytotoxicity on human promyelocytic leukemia (HL60) and human histiocytic leukemia (U937) cell lines. cis-[PdCl2(Ad)(Gua)] showed IC50values of 11.29 ± 2.91 and 8.31 ± 1.44 μM against HL60 and U937, respectively, which was higher than that of the positive control (curcumin) against U937. The complexes also showed significant antioxidant activity when tested against 2,2-diphenyl-1-picrylhydrazylradical (DPPH).

L-securinine induces apoptosis in the human promyelocytic leukemia cell line HL-60 and influences the expression of genes involved in the PI3K/AKT/mTOR signaling pathway

The Securinega alkaloids are a class of natural products isolated from plants of the Euphorbiaceae family. L-securinine induces apoptosis in the human promyelocytic leukemia cell line HL-60 indicating its potential as an efficient natural antitumor drug with low toxicity. The aim of the present study was to investigate the apoptotic effects of L-securinine on HL-60 cells and to explore its potential underlying molecular mechanism(s) as an antitumor agent. HL-60 cells were cultured with L-securinine. The proliferation and changes in cell morphology were evaluated by cell counting Kit-8 (CCK-8) assay and electron microscopy, respectively. Induction of apoptosis and cell cycle progression were investigated by flow cytometry. The PI3K/AKT/mTOR pathway gene expression was measured by quantitative PCR (qPCR). L-securinine decreased the viability of HL-60 cells in a dose- and time-dependent manner, with IC50 values at 24, 48 and 72 h post-treatment of 47.88, 23.85 and 18.87 µmol/l, respectively. Numerous apoptotic bodies were observed in the HL-60 cells treated with 25 µmol/l L-securinine for 48 h. L-securinine at 12.5, 25 and 50 µmol/l increased the rate of apoptosis in HL-60 cells, and G1/S phase progression was retarded. Furthermore, L-securinine induced downregulation of PI3K, AKT and mTOR gene expression and upregulation of PTEN gene expression in a dose-dependent manner. In conclusion, L-securinine induces apoptosis and inhibition of cell cycle progression in HL-60 cells by modulation of the PI3K/AKT/mTOR pathway gene expression. These observations indicate the potential of L-securinine as an antitumor agent.

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.

EGF Receptor-Dependent Mechanism May be Involved in the Tamm–Horsfall Glycoprotein-Enhanced PMN Phagocytosis via Activating Rho Family and MAPK Signaling Pathway

Our previous studies showed that urinary Tamm–Horsfall glycoprotein (THP) potently enhanced polymorphonuclear neutrophil (PMN) phagocytosis. However, the domain structure(s), signaling pathway and the intracellular events responsible for THP-enhanced PMN phagocytosis remain to be elucidated. THP was purified from normal human urine. The human promyelocytic leukemia cell line HL-60 was induced to differentiate into PMNs by all-trans retinoid acid. Pretreatment with different MAPK and PI3K inhibitors was used to delineate signaling pathways in THP-enhanced PMN phagocytosis. Phosphorylation of molecules responsible for PMN phagocytosis induced by bacterial lipopolysaccharide (LPS), THP, or human recombinant epidermal growth factor (EGF) was evaluated by western blot. A p38 MAPK inhibitor, SB203580, effectively inhibited both spontaneous and LPS- and THP-induced PMN phagocytosis. Both THP and LPS enhanced the expression of the Rho family proteins Cdc42 and Rac that may lead to F-actin re-arrangement. Further studies suggested that THP and EGF enhance PMN and differentiated HL-60 cell phagocytosis in a similar pattern. Furthermore, the EGF receptor inhibitor GW2974 significantly suppressed THP- and EGF-enhanced PMN phagocytosis and p38 and ERK1/2 phosphorylation in differentiated HL-60 cells. We conclude that EGF receptor-dependent signaling may be involved in THP-enhanced PMN phagocytosis by activating Rho family and MAP kinase.

Microfluidic device capable of medium recirculation for non-adherent cell culture.

We present a microfluidic device designed for maintenance and culture of non-adherent mammalian cells, which enables both recirculation and refreshing of medium, as well as easy harvesting of cells from the device. We demonstrate fabrication of a novel microfluidic device utilizing Braille perfusion for peristaltic fluid flow to enable switching between recirculation and refresh flow modes. Utilizing fluid flow simulations and the human promyelocytic leukemia cell line, HL-60, non-adherent cells, we demonstrate the utility of this RECIR-REFRESH device. With computer simulations, we profiled fluid flow and concentration gradients of autocrine factors and found that the geometry of the cell culture well plays a key role in cell entrapping and retaining autocrine and soluble factors. We subjected HL-60 cells, in the device, to a treatment regimen of 1.25% dimethylsulfoxide, every other day, to provoke differentiation and measured subsequent expression of CD11b on day 2 and day 4 and tumor necrosis factor-alpha (TNF-α) on day 4. Our findings display perfusion sensitive CD11b expression, but not TNF-α build-up, by day 4 of culture, with a 1:1 ratio of recirculation to refresh flow yielding the greatest increase in CD11b levels. RECIR-REFRESH facilitates programmable levels of cell differentiation in a HL-60 non-adherent cell population and can be expanded to other types of non-adherent cells such as hematopoietic stem cells.

Cytotoxic Properties of Lyophilized Beers in a Malignant Cell Line

Moderate beer consumption can be considered as part of a healthy diet since it may protect against heart disease, cancer and osteoporosis. The protective effects of beer reside in its polyphenol content whose chemical composition appears extremely complex. In the present study, five commercial beers with different polyphenol content (ranging between 690 - 2400 μM equivalents of quercetin) were employed to investigate their cytotoxic effect in vitro on HL-60 cells derived from a human promyelocytic leukemia cell line. A significant reduction in cell viability was measured after 48 hours treatment. Lyophilized beers with higher polyphenol content showed the highest cytotoxicity compared to those with lower concentrations. However, when the assay was performed applying equal amounts of total polyphenols from different lyophilized beers, the sample possessing lowest amount of polyphenols (690 μM equivalents of quercetin) resulted the most effective in reducing cell viability. These data suggest that the biological activities of polyphenols present in beer are not simply dependent upon their total concentration, but qualitative profile and relative concentrations are even more important in determining their antiproliferative effects on cancer cells.

Differences in the Toxicities of Trichothecene Mycotoxins, Deoxynivalenol and Nivalenol, in Cultured Cells

To illustrate the differences in toxicities between deoxynivalenol (DON) and nivalenol (NIV), cell proliferation, cytokine secretion, and the involvement of heat shock protein 90 (Hsp90) were investigated. Both toxins retarded the proliferation of all four cell lines tested. NIV was more potent than DON in human promyelocytic leukemia cell line HL60, human lymphoblastic leukemia cell line MOLT-4, and rat aortic myoblast cell line A-10. In contrast, both toxins exhibited almost equivalent potencies in human hepatoblastoma cell line HepG2. If both toxins exert their toxicities through the same mechanism, one should be more potent than the other, regardless of cell types. While exposure to DON significantly induced the secretion of anti-hematopoietic cytokines macrophage inflammatory protein-1α (MIP-1α/ CCL3) and MIP-1β/CCL4, treatment with NIV decreased the secretion of these cytokines in HL60 cells, indicating that the toxicity mechanisms of these toxins differ. An Hsp90-specific inhibitor radici col canceled the effect of DON on these cytokine secretions, indicating that Hsp90 plays a crucial role in these DON-induced cytokine secretions in HL60 cells. Conversely, the results of treatment with NIV and radicicol indicate that radicicol does not mitigate the effect of NIV. When viewing the above results collectively, although these toxins share highly similar chemical structures, there are evident differences in their toxicities.

Evaluation of in Vitro Antioxidative, Cytotoxic and Apoptotic Activities of Rheum ribes Ethyl Acetate Extracts

Rheum species are medicinally important plants due to the presence of anthracene derivatives. This study was designed to determine the antioxidative, cytotoxic and apoptotic properties of Rheum ribes shoot and root ethyl acetate extracts using human promyelocytic leukemia (HL-60) cell line as a model system. R. ribes shoot and root dry powder samples were prepared and extracted with ethyl acetate. The extracts were revealed to be a potential scavenger of DPPH radicals (IC50 value of 206.28 μg/ml for shoot and 10.92 μg/ml for root) and the chemical composition of the extracts was quantified by colorimetric determination of total phenol (GAE) and flavonoid (CAE) contents. HL–60 cells were cultured in the presence of various concentrations of extracts up to 72 h. R. ribes inhibited the survival of HL-60 cells in a concentration- and time-dependent manner, shown by XTT assay. R. ribes caused HL-60 cells apoptosis via formation of phosphatidylserine externalization, as evidenced by flow cytometry. Exposure of HL-60 cells to higher concentrations of extracts for 72 h resulted in a shift of 87% of the cell population from normal to the early/late apoptotic stage. These findings suggest that Rheum ribes ethyl acetate root extracts exhibits potential antioxidant and cytotoxic properties against HL-60 cells better than shoot extracts and exert their toxicity via induction of apoptosis.

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.

Abstract C160: New class of DNA-alkylating agents with a suitable tolerability profile created for use in antibody-drug conjugates (ADCs).

Abstract The recent FDA approvals of brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla) has spurred tremendous interest in ADCs for the treatment of cancer. Most clinical-stage ADCs, including the two approved ones, utilize a tubulin-acting small molecule as the cytotoxic payload. There is strong interest in preparing DNA-acting payload agents because some cancers are more responsive to DNA-acting agents than to tubulin-acting ones. The key challenge has been developing a DNA-acting payload agent with the necessary tolerability profile as well as potency. We have developed a new class of DNA-acting agents for use in ADCs, IGNs, which demonstrate the desired profile. Initially, our IGNs were indolino-benzodiazepine dimers that were highly potent in vitro (IC50 ∼10−12 M) by virtue of their ability to both alkylate and cross-link DNA. However, a resultant IGN-ADC did not have the desired in vivo tolerability, a known problem with highly potent DNA-acting agents. This IGN-ADC was found to be intolerable in mice (4.4 mg/kg, weekly x 3), resulting in delayed toxicity and mortality. To address this, we tested modifying the saturation level of the di-imine cross-linking groups by generating a mono-imine compound that could still alkylate DNA, but no longer act as a cross-linker. This new, mono-imine IGN, 2m (alkylator only), was highly potent, but with at least 2-fold better tolerability (9.1 mg/kg, weekly x 3) and, most importantly, not associated with delayed toxicity. To assess its potency in an ADC format, 2m and the lead IGN di-imine compound, 2d (crosslinker/alkylator) were conjugated to the folate receptor-α (FRα)-binding antibody, anti-FRα, and the CD33-binding antibody, anti-CD33, via amide bonds. In spite of their different imine saturation levels, anti-FRα-2d and anti-FRα-2m were both highly potent against the FRα-expressing KB cell line (IC50 values of 15 pM and 20 pM, respectively). The addition of excess unconjugated antibody abolished the cytotoxic effect (>3 nM), thus demonstrating that the activity of the conjugate is antigen specific. Similarly, anti-CD33-2d and anti-CD33-2m displayed comparable potency against a panel of CD33-expressing cell lines. This included the human promyelocytic leukemia cell line, NB4, where these two IGN-ADCs had IC50 values in the picomolar range despite the low level of antigen expression on the target cells (∼10,000 molecules/cell). In vivo, the anti-FRα-2m conjugate was found to be effective against KB tumors in mouse xenograft models at doses that were well below the MTD. The mechanism of action, potency, tolerability, and lack of delayed toxicity make IGNs a promising new class of cytotoxic small molecules for use in ADCs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C160. Citation Format: Michael Miller, Nathan Fishkin, Wei Li, Emily Reid, Katie Archer, Erin Maloney, Yelena Kovtun, Gregory Jones, Megan Ellis, Rajeeva Singh, Kathleen Whiteman, Jan Pinkas, Ravi Chari. New class of DNA-alkylating agents with a suitable tolerability profile created for use in antibody-drug conjugates (ADCs). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C160.

The putative BH3 mimetic S1 sensitizes leukemia to ABT-737 by increasing reactive oxygen species, inducing endoplasmic reticulum stress, and upregulating the BH3-only protein NOXA

S1 is a putative BH3 mimetic proposed to inhibit BCL2 and MCL1 based on cell-free assays. However, we previously demonstrated that it failed to inhibit BCL2 or induce apoptosis in chronic lymphocytic leukemia (CLL) cells, which are dependent on BCL2 for survival. In contrast, we show here that S1 rapidly increases reactive oxygen species, initiates endoplasmic reticulum stress, and upregulates the BH3-only protein NOXA. The BCL2 inhibitors, ABT-737, ABT-263, and ABT-199, have demonstrated pro-apoptotic efficacy in cell lines, while ABT-263 and ABT-199 have demonstrated efficacy in early clinical trials. Resistance to these inhibitors arises from the upregulation of anti-apoptotic factors, such as MCL1, BFL1, and BCLXL. This resistance can be induced by co-culturing CLL cells on a stromal cell line that mimics the microenvironment found in patients. Since NOXA can inhibit MCL1, BFL1, and BCLXL, we hypothesized that S1 may overcome resistance to ABT-737. Here we demonstrate that S1 induces NOXA-dependent sensitization to ABT-737 in a human promyelocytic leukemia cell line (NB4). Furthermore, S1 sensitized CLL cells to ABT-737 ex vivo, and overcame resistance to ABT-737 induced by co-culturing CLL cells with stroma.

Design, synthesis and antitumor activity of a novel series of PAC-1 analogues

A novel series of first procaspase activating compound(PAC-1) analogues was designed, synthesized and evaluated for antitumor activity towards two cell lines[human promyelocytic leukemia cell line(HL60) and human embryonic lung fibroblast cell line(HLF)] by the MTT[3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazo-liumromide]_method in vitro. The structures of all the compounds were confirmed by 1H NMR, MS and elemental analysis. Among the compounds synthesized,(E)-2-[(3-{[4-(tert-butyl)benzyl](methyl)amino}propyl)(methyl)amino]-N′-[4-(diethylamino)-2-hydroxybenzylidene]acetohydrazide(compound 6n) exhibits a good anti-proliferative activity to the majority of tumor cells tested, and selectively cleaves cancer cells. Thus, compound 6n was identified as promising lead compound for further structural modification.

Vinyl Sulfone-Based Peptidomimetics as Anti-Trypanosomal Agents: Design, Synthesis, Biological and Computational Evaluation

A series of vinyl sulfone-containing peptidomimetics were rationally designed, synthesized, and evaluated against Trypanosoma brucei brucei . These electrophilic compounds are likely to exert their antitrypanosomal activity via inhibition of trypanosomal cysteine proteases, TbCatB and rhodesain, through alkylation of a key cysteine residue within the protease active site. The series was designed to present complementary groups to naturally recognized peptide substrates while probing tolerance to a range of substitutions at the P1, P1', and P2 positions. The most potent compound, 29 (EC50 = 70 nM, T. b. brucei whole cell assay), displayed minimal toxicity (>785 times selectivity) when assayed for cytotoxicity against the human promyelocytic leukemia (HL-60) cell line. Cells treated with compound 29, as with K777 (2), exhibited an increase in both the number of multinucleated cells and cells with swollen flagellar pockets. Computational analysis revealed a strong correlation between the hypothetical binding mode in TbCatB/rhodesain and trypanocidal activity in vitro.

P-Toluenesulfonic acid-catalyzed solvent-free synthesis and biological evaluation of new 1-(4′,6′-dimethylpyrimidin-2′-yl)-5-amino-4H-3-arylpyrazole derivatives

A solvent-free quick synthesis of 1-(4′,6′-dimethylpyrimidin-2′-yl)-5-amino-4H-3-arylpyrazoles (3) was accomplished by grinding 2-hydrazino-4,6-dimethylpyrimidine (1) and β-ketonitriles (2) in the presence of p-Toluenesulfonic acid as a catalyst. Subsequently, 5-aminopyrazoles (3) were converted to their corresponding N-acetamide (4) and N-trifluoroacetamide (5) derivatives by treating (3) with acetic anhydride/acetic acid and trifluoroacetic anhydride/trifluoroacetic acid, respectively. The newly synthesized compounds were fully characterized using IR, NMR (1H, 13C, and 19F), mass spectral studies, and elemental analyses. All of the fifteen compounds were screened for their in vitro antibacterial activity against two Gram-positive and two Gram-negative pathogenic bacteria such as Bacillus pumilus, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, respectively. Additionally, nine of these compounds were screened for their cytotoxicity against the human Caucasian promyelocytic leukemia (HL-60) cell line using the alamarBlue® assay. Preliminary results reveal that compounds 3a, 3b, 3d, 5a, and 5d are showing moderate-to-significant cytotoxic and antibacterial activity.

6-C-Methyl Flavonoids Isolated from Pinus densata Inhibit the Proliferation and Promote the Apoptosis of the HL-60 Human Promyelocytic Leukaemia Cell Line

Three structurally related 6-C-methyl flavonoids isolated from Pinus densata, including 5,4'-dihydroxy-3,7,8-trimethoxy-6-C-methylflavone (PD1), 5,7,4'-trihydroxy-3,8-dimethoxy-6-C-methylflavone (PD2), and 5,7,4'-trihydroxy-3-methoxy-6-C-methylflavone (PD3), were tested for their ability to inhibit the proliferation and promote the apoptosis of the HL-60 human leukaemia cell line. Cytotoxicity assays in the HL-60 human cancer cell line demonstrated that 5,4'-dihydroxy-3,7,8-trimethoxy-6-C-methylflavone exhibited the most potent cytotoxicity of the three structurally related 6-C-methyl flavonoids. 5,4'-Dihydroxy-3,7,8-trimethoxy-6-C-methylflavone inhibited the proliferation of HL-60 cells in a dose-dependent manner with an IC₅₀ of 7.91 µM (48 h treatment). Furthermore, 5,4'-dihydroxy-3,7,8-trimethoxy-6-C-methylflavone-induced apoptosis was associated with mitochondrial membrane disruption and cytochome c release. Flow cytometry analyses revealed an increase in the hypodiploid population in 5,4'-dihydroxy-3,7,8-trimethoxy-6-C-methylflavone-treated HL-60 cells. Treatment with a concentration of 5,4'-dihydroxy-3,7,8-trimethoxy-6-C-methylflavone that induced apoptosis activated caspase-3 but did not activate caspase-1. A caspase-3 inhibitor (Ac-DEVD-CHO), but not a caspase-1 inhibitor (Ac-YVAD-CHO), reversed the cytotoxic effects of 5,4'-dihydroxy-3,7,8-trimethoxy-6-C-methylflavone in HL-60 cells. These data demonstrated that 5,4'-dihydroxy-3,7,8-trimethoxy-6-C-methylflavone effectively induced the apoptosis of HL-60 cells and exhibited significant anticancer activity via the mitochondrial caspase-3-dependent apoptosis pathway.

Fluorogenic sialic acid glycosides for quantification of sialidase activity upon unnatural substrates

Herein we report the synthesis of N-acetyl neuraminic acid derivatives as 4-methylumbelliferyl glycosides and their use in fluorometrically quantifying human and bacterial sialidase activity and substrate specificities. We found that sialidases in the human promyelocytic leukemic cell line HL60 were able to cleave sialic acid substrates with fluorinated C-5 modifications, in some cases to a greater degree than the natural N-acetyl functionality. Human sialidases isoforms were also able to cleave unnatural substrates with bulky and hydrophobic C-5 modifications. In contrast, we found that a bacterial sialidase isolated from Clostridium perfringens to be less tolerant of sialic acid derivatization at this position, with virtually no cleavage of these glycosides observed. From our results, we conclude that human sialidase activity is a significant factor in sialic acid metabolic glycoengineering efforts utilizing unnatural sialic acid derivatives. Our fluorogenic probes have enabled further understanding of the activities and substrate specificities of human sialidases in a cellular context.