Human Promyelocytic Leukemia Cells Research Articles

Effect of methoxy stilbenes\u2014analogs of resveratrol\u2014on the viability and induction of cell cycle arrest and apoptosis in human myeloid leukemia cells

The present study aimed to evaluate the cytotoxicity and its mechanism of five synthetic methoxy stilbenes, namely 3,4,4ʹ-trimethoxy, 3,4,2ʹ-trimethoxy, 3,4,2ʹ,4ʹ-tetramethoxy, 3,4,2ʹ,6ʹ-tetramethoxy, and 3,4,2ʹ,4ʹ,6ʹ-pentamethoxy-trans-stilbenes (MS), in comparison with resveratrol (RSV). Human promyelocytic (HL-60) and monocytic leukemia (THP-1) cells were treated with the tested compounds for 24 h, and cytotoxicity, cell cycle distribution, and apoptosis were evaluated. Significant differences were found in the susceptibility of these cell lines to all stilbenes, including RSV. The THP-1 cells were more resistant to cytotoxic activity of these compounds than HL-60 cells. Among the tested stilbenes, 3,4,4ʹ-tri-MS and 3,4,2ʹ,4ʹ-tetra-MS exhibited higher cytotoxicity toward both cell lines than RSV and the other methoxy stilbenes. This activity might be related to cell cycle arrest at the G2/M phase and induction of apoptosis. In this regard, 3,4,4ʹ-tri-MS and 3,4,2ʹ,4ʹ-tetra-MS at highest concentrations increased the p53 protein level particularly in HL-60 cells. Moreover, treatment with these derivatives increased the ratio of the proapoptotic Bax protein to the antiapoptotic Bcl-xl protein, suggesting the induction of apoptosis through the intrinsic mitochondrial pathway in both cell lines. Further studies are required to fully elucidate the mechanism of these activities.

Overexpression of ATG5 Gene Makes Granulocyte-Like HL-60 Susceptible to Release Reactive Oxygen Species

Neutrophils represent the first line of defense against pathogens using various strategies, such as phagocytosis, production of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) formation. Recently, an autophagy-independent role of autophagy related (ATG) gene 5 in immune cells, including neutrophils, was emphasized. Our aim was to investigate the role of ATG5 protein in neutrophils’ antimicrobial functions, proliferation and apoptosis. To this end, we used genetically modified human promyelocytic leukemia (HL-60) cells overexpressing ATG5, differentiated toward granulocyte-like cells with all-trans retinoic acid (ATRA) and dimethylformamide. The level of differentiation, phagocytosis, proliferation and apoptosis were determined by flow cytometry. ROS production and NETs release was assessed by fluorometry and fluorescent microscopy. ATG5 gene expression was evaluated by real-time PCR, whereas the protein level of ATG5 and LC3-II was determined by Western blot. We did not observe the induction of autophagy in differentiated HL-60 cells overexpressing ATG5. The increased expression of ATG5 affects the differentiation of HL-60 cells with ATRA, ROS production and phagocytosis. However, we did not detect changes in NETs release. Moreover, ATG5 protects differentiated HL-60 cells from apoptosis but does not cause changes in proliferation rate.

Detecting Basal Myeloperoxidase Activity in Living Systems with a Near-Infrared Emissive "Turn-On" Probe.

Detecting myeloperoxidase (MPO) activity in living organisms is important because MPO contributes to the pathogenesis of many diseases such as rheumatoid arthritis and other inflammatory diseases, artherosclerosis, neurodegenerative disease, and some cancers. However, rapid and effective methods for the detection of basal MPO activity in living systems have not yet been reported. Herein, we report a near-infrared (NIR) emissive "turn-on" probe FD-301 that can specifically bind to MPO and accurately measure MPO activity in living cells and in vivo via a rapid response to initial hypochlorous acid (HOCl), produced by MPO. Notably, FD-301 could detect the basal level of MPO activity in human promyelocytic leukemia cells (HL-60) and could discriminate between MPO high-expression and low-expression cells. Furthermore, FD-301 was successfully applied to in vivo imaging of MPO in MPO-dependent diseases, such as arthritis and inflammatory bowel disease.

Folic acid modified lipid-bilayer coated mesoporous silica nanoparticles co-loading paclitaxel and tanshinone IIA for the treatment of acute promyelocytic leukemia

In this work, paclitaxel (Ptx) combined with tanshinone IIA (TanIIA) was found to show synergistic effect on inducing apoptosis of human acute promyelocytic leukemia (APL) cell line NB4, and the anti-tumor effect was strongest when its molar ratio was 1:1. To enhance the efficacy and reduce side effects, an active targeting drug delivery system with mesoporous silica nanoparticles (MSNs) coated with folic acid (FA) modified PEGylated lipid-bilayer (LB) membrane (FA-LB-MSNs) was established for co-loading drugs. The drug loadings of Ptx and TanIIA in FA-LB-MSNs were 5.5% and 1.8%, respectively. Compared with the uncoated MSNs, the FA-LB-MSNs showed a sustained drug release, and Ptx and TanIIA released synchronously from the carriers. By means of biological adhesion between FA and its receptors, the uptake of FA-LB-MSNs by NB4 cells was significantly higher than that of uncoated preparations, and Ptx combined with TanIIA had strong synergistic effect to enhance the apoptosis and differentiation of NB4 cells. The results of pharmacodynamics in vivo showed that the FA-LB-MSNs targeted tumor in nude mice more effectively than the compared formulations without FA modification. The Ptx and TanIIA-loaded FA-LB-MSNs group showed significantly better effects on inducing apoptosis and inhibiting tumor growth than the reference groups, which agreed with the results of anti-tumor experiments in vitro. Furthermore, no toxicity was observed to the heart, liver, spleen, lung and kidney of the tumor-bearing animals, indicating good biocompatibility of the prepared novel nanocarriers. This study confirmed the synergistic therapeutic effect of Ptx and TanIIA on APL, and the superior of FA-LB-MSNs as co-loaded nanocarriers for active targeted therapy of tumors.

A new steroidal glycoside from the fruits of Solanum myriacanthum

A new cholestane-type steroidal glycoside, solamyriaside A (1), was isolated from the fruits of Solanum myriacanthum Dunal (Solanaceae), along with two known steroidal glycosides, namely, solaviaside A (2) and aculeatiside A (3), and three known steroidal alkaloid glycosides, namely, solamargine (4), khasianine (5) and solasonine (6), which were isolated for the first time from this plant. Based on spectroscopic data as well as chemical evidence, 1 was determined to be 3-O-α-L-rhamnopyranosyl-(1→2)-O-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranosyl-22R,25R-cholest-5-ene-3β,16α,22,26-tetraol 26-O-β-D-glucopyranoside. The cytotoxic activity of 1–6 against HL-60 human promyelocytic leukaemia cells was examined. Compounds 4–6 showed cytotoxic activity. Among them, 4 exhibited the strongest activity with an IC50 value of 4.64 ± 0.17 μM, similar to the activity of cisplatin, a positive control.

Antibacterial and in vitro Anticancer Study of Methanol Extracts of Clitoria ternatea Leaves

Clitoria ternatea is often known as butterfly pea plant by the natives and is one of the major plants used in Ayurveda and traditional medicine worldwide. It is also used in food and cosmetic industries for various purposes. In this study, methanol leaf extract of Clitoria ternatea was studied for its antibacterial and in vitro anticancer study against human promyelocytic leukemia cells (HL60). Secondary metabolites like alkaloids, phlobatannin, triterpenoids, flavonoids, lipids, steroids, terpenoids, tannins and glycosides are identified in the extract by phytochemical screening. Leaf extract showed the presence of ethers and carboxylic acid groups (GCMS analysis). The leaf extract showed resistance against Salmonella typhi. Thus, from this study it can be concluded Clitoria ternatea leaves methanol extract has bioactive compounds showing anticancer activity against HL60 cells.

An acylhydroquinone derivative produces OXPHOS uncoupling and sensitization to BH3 mimetic ABT-199 (Venetoclax) in human promyelocytic leukemia cells.

Since cancer cells have different mitochondrial bioenergetic requirements than non-cancerous cells, therapeutic inhibition of its mitochondrial functionality continues to be an important target for anticancer drug discovery. In this study, a series of acylhydroquinones with different acyl-chain length, and their chlorinated derivatives, in the aromatic ring, synthesized by Fries rearrangement under microwave irradiation, were evaluated for their anticancer activity in two leukemia cell lines. Findings from the primary and secondary screening of the 18 acylhydroquinones, tested at 5µM on acute promyelocytic leukemia HL-60 and acute lymphoblastic leukemia CEM cells lines, identified an acylchlorohydroquinone (12) with a highly selective anti-proliferative effect toward HL-60 cells. This compound induced S-phase arrest in the cell cycle progression of HL-60 cells with insignificant toxicity on leukemic CEM cells and non-cancerous Hs27 cells. In HL-60 leukemic cells, 12 triggered increased mitochondrial NADH oxidation, increased respiration in presence of oligomycin (state 4o), mitochondrial depolarization, and ROS production, suggesting an uncoupling of OXPHOS. This provoked a metabolic adaptation dependent on AMPK/ACC/autophagy axis, having the mitochondrial β-oxidation a pro-survival role since the combination of 12 and etomoxir, a carnitine palmitoyl-transferase (CPT) inhibitor promoted extensive HL-60 cell death. Finally, 12-induced metabolic stress sensitized to HL-60 cells to cell death by the FDA-approved anti-leukemic drug ABT-199, a BH3 mimetic. Therefore, our results suggest that acylchlorohydroquinone is a promising scaffold in anti-promyelocytic leukemia drug research.

Synthesis of 6-unsubstituted 2-oxo, 2-thioxo, and 2-amino-3,4-dihydropyrimidines and their antiproliferative effect on HL-60 cells

A general and efficient synthetic method for 6-unsubstituted 3,4-dihydropyrimidin-2(1H)-thiones 6 and -ones 7 has been developed. In three-component reactions, the reactivity of reagents serving as the C5–C6 fragment of the dihydropyrimidine ring was compared. The reaction of thiourea or urea 12, aldehydes 13, and ethyl 3-dimethylaminoacrylate 9 in the presence of a catalytic amount of AlCl3 by heating smoothly proceeds to give 6 and 7 in high yields. A synthetic novelty of our protocol is as follows: 1) Lewis acid-mediated reaction, 2) good to high yields, 3) broad scope as for aldehydes, and ureas. Hitherto unavailable 6-unsubstituted 2-aminodihydropyridimidine 8 has been obtained from the 2-thioxo derivative 6 by a stepwise method involving a substitution reaction with the amine at the 2-position. These 6-unsubstituted compounds 6, 7, and some 6-methyl derivatives 16 were assessed for their antiproliferative effect on the human promyelocytic leukemia cell line, HL-60. The 4-propyl-6-methyl derivative 16b showed relatively strong activity with the IC50 value of 952 nM.

Steroidal glycosides from the bulbs of Lilium speciosum

Steroidal glycosides (1-21), including eight new spirostanol glycosides (1-8) and a new cholestane glycoside (9), were isolated from the bulbs of Lilium speciosum. The structures of the new compounds were determined based on spectroscopic data analysis and hydrolytic cleavage reactions. The isolated compounds and their aglycones (1a, 8a, and 9a) were evaluated for their cytotoxicity against HL-60 human acute promyelocytic leukemia cells and A549 human lung carcinoma cells. Compounds 12 and 21 exhibited moderate cytotoxic activity toward HL-60 and A549 cells, with IC50 values of 6.9 μM and 5.1 μM against HL-60 cells, and 7.6 μM and 4.4 μM against A549 cells, respectively. Compound 20 only showed moderate cytotoxicity toward A549 cells, with an IC50 value of 6.7 μM.

The Optimization of HL60-IL6 Assay and its Application in the Pyrogen Detection of Monoclonal Antibody

Background: The HL60-IL6 assay has been initially established, but the process of the assay and calculation was not simplified. And there are no reports on whether it can be applied to detect pyrogen contamination in the monoclonal antibody. Objective: The study aimed to improve the HL60/IL-6 assay and detect the pyrogens in the monoclonal antibody drug by HL60-IL6 assay. Methods: The human promyelocytic leukemia cell line (HL-60) was incubated with pyrogen standard solution, such as lipopolysaccharide (LPS), zymosan and lipoteichoic acid (LTA),or monoclonal antibody sample solution for 48 hours, and then cytokines interleukin-6 (IL-6),secreted from HL-60, were measured by ELISA. The study further described the standard curves on OD (Optical Density) value of IL-6 responding to pyrogen stimulation, and determined the content of pyrogen in the monoclonal antibody production after validation. In addition, the sensitivity of HL60 to three pyrogens was evaluated to establish one standard curve to determine endotoxin and non-endotoxin level. Then, the credibility of standard curves was evaluated. After improvement of the assay, 9 monoclonal antibody batches were assayed for pyrogens in parallel with the Rabbit Pyrogen Test (RPT) and HL60/IL-6 assay. Results: It was achieved that the standard curve between OD value of IL-6 and pyrogen concentration was established. Then, it was found that the sensitivity of HL60 responding to LPS was the weakest, as a result of which, only LPS standard curve needs to be described in each test for detection of pyrogens. Besides, to evaluate the credibility of standard curve, the parameters of the standard curve were restricted and the resulting interpretation was also specified. 3 Bevacizumab batches failed the RPT, which also showed pyrogenic contamination by the HL60/IL-6 assay. Conclusion: HL60-IL6 assay was improved and can be applied to pyrogen detection of monoclonal antibody.

Triterpenes from Poria cocos are revealed as potential retinoid X receptor selective agonists based on cell and in silico evidence.

Poria cocos is an edible and medicinal fungus that is widely used in Traditional Chinese Medicines as well as in modern applications. Retinoid X receptor (RXR) occupies a central place in nuclear receptor signaling, and a pharmacological RXR-dependent pathway is involved in myeloid cell function. Here, structural information for 82 triterpenes from P.cocos and 17 known RXR agonists was collected in a compound library and retrieved for a molecular docking study. Three triterpenes, 16α-hydroxytrametenolic acid (HTA), pachymic acid (PA), and polyporenic acid C (PPAC), were identified as novel RXR-specific agonists based on luciferase reporter assays and in silico evidence. Treatment with HTA, PA, and PPAC significantly induced differentiation of the human promyelocytic leukemia cell line HL-60 with EC50 values of 21.0±0.52, 6.7±0.37, and 9.4±0.65μM, respectively. These effects were partly blocked by the RXR antagonist UVI3003, suggesting that an RXR-dependent pathway may play an important role in their anti-acute promyelocytic leukemia (APL) effects. Taken together, triterpenes from P.cocos are revealed as naturally occurring RXR selective agonists with the potential for anti-cancer activity. These results suggest a novel approach to the treatment or prevention of APL.

Novel Steroidal Glycosides from the Whole Plants of Helleborus foetidus.

Phytochemical analysis of the whole Helleborus foetidus plants identified 28 steroidal glycosides (1-28), including 20 novel spirostanol glycosides (1-20) and a novel furostanol glycoside (21). The structures of the newly identified compounds were elucidated by two-dimensional NMR spectroscopy and hydrolytic cleavage. Compounds 12, 13, and 15 were determined to be spirostanol trisdesmosides bearing sugar moieties at the C-1, -21, and -24 hydroxy groups of the aglycone unit. The isolated compounds were subsequently evaluated for cytotoxic activity against HL-60 human promyelocytic leukemia cells and A549 human lung carcinoma cells. In particular, 7 showed cytotoxic activity against the HL-60 and A549 cells, with IC50 values of 5.9 and 6.6 µM, respectively, whereas 19 was selectively cytotoxic to A549 cells with an IC50 value of 5.5 µM.

L-Ascorbic Acid and α-Tocopherol Synergistically Triggers Apoptosis Inducing Antileukemic Effects of Arsenic Trioxide via Oxidative Stress in Human Acute Promyelocytic Leukemia Cells.

Chemosensitization is an effective strategy to overcome the drawbacks of arsenic trioxide (As2O3) treatment, which may be possible through the use of dietary supplements in combination. The present investigation evaluates the synergistic mechanism of action of vitamins, such as L-ascorbic acid (L-AA) and α-tocopherol (α-TOC) in As2O3 chemotherapy using human leukemia (HL-60) cells. In vitro assays on the cytotoxicity of As2O3 and vitamins and cellular apoptotic evidences were done; a proteomic investigation with mass spectrometry was also performed. The combination of L-AA and α-TOC potentiates As2O3 cytotoxicity in HL-60 cells, substantiated by depletion in antioxidant status, mitochondrial transmembrane potential, and inhibition of nuclear factor erythroid 2-related factor 2 and B-cell lymphoma 2 transcription factors. Mass spectrometry results showed decreased expression of proteins regulating cell cycle and translation in cells treated with As2O3, L-AA, and α-TOC when compared with As2O3-treated sample. In addition, this combination treatment identified numerous proteins associated with apoptosis and cell stress. HL-60 cells became more prone to As2O3 on exposure to L-AA and α-TOC, indicating that this combination may be a promising approach to increase the outcome of As2O3 chemotherapy.

Seleninic Acid Potassium Salts as Water-Soluble Biocatalysts with Enhanced Bioavailability.

Organoselenium compounds are well-known glutathione peroxidase (GPx) mimetics that possess antioxidants/prooxidant properties and are able to modulate the concentration of reactive oxygen species (ROS), preventing oxidative stress in normal cells or inducing ROS formation in cancer cells leading to apoptosis. The purpose of this study was the synthesis of potent GPx mimics with antioxidant and anticancer activity along with improved bioavailability, as a result of good solubility in protic solvents. As a result of our research, glutathione peroxidase (GPx) mimetics in the form of water-soluble benzeneseleninic acid salts were obtained. The procedure was based on the synthesis of 2-(N-alkylcarboxyamido)benzeneselenenic acids, through the oxidation of benzisoselenazol-3(2H)-ones or analogous arenediselenides with an amido group, which were further converted to corresponding potassium salts by the treatment with potassium tert-butanolate. All derivatives were tested as potential antioxidants and anticancer agents. The areneseleninic acid salts were significantly better peroxide scavengers than analogous acids and the well-known organoselenium antioxidant ebselen. The highest activity was observed for the 2-(N-ethylcarboxyamido)benzeneselenenic acid potassium salt. The strongest cytotoxic effect against breast cancer (MCF-7) and human promyelocytic leukemia (HL-60) cell lines was found for 2-(N-cyclohexylcarboxyamido)benzeneselenenic acid potassium salt and the 2-(N-ethylcarboxyamido)benzeneselenenic acid, respectively. The structure–activity correlations, including the differences in reactivity of benzeneseleninic acids and corresponding salts were evaluated.

A Balance between Turning and Persistent Motion is Critical for Fast and Efficient 3-Dimensional Neutrophil Migration

While much research has been dedicated to identifying the cascade of specific biochemical processes involved in the recruitment of neutrophils, much less is known about the mechanical events driving their directed migration. In particular, it is still unclear how neutrophils generate the necessary traction forces to migrate across three-dimensional (3-D) extravascular spaces and the effects of matrix porosity on turning events and persistence during this process. In this study, we examine the effects of extracellular matrix properties on the mechanics of 3-D neutrophil motility in collagen gels. We embedded neutrophil-like differentiated human promyelocytic leukemia (dHL-60) cells in collagen matrices of different concentrations containing fluorescent micro-beads. To induce directed migration, we introduced the chemokine formyl-Methionyl-Leucyl-Phenylalanine (fMLP) to dHL-60 cells in a custom build device. We then used brightfield microscopy to track chemotaxing cells and both confocal and fluorescent microscopy techniques to image the movement of the embedded micro-beads and fluorescently labeled cells. We find that turning events, mediated by the Arp 2/3 complex, are crucial for fast 3-D neutrophil migration. Additionally, the Arp 2/3 complex and myosin II play an increasingly important role in 3-D migration as matrices become denser. Finally, we find that cellular force patterns vary in response to matrices with various degrees of pore size heterogeneities and cell's intrinsic abilities to engage in path-finding and contractility. The results from our study show that neutrophils migrating in 3-D environments employ distinct mechanical mechanisms that depend on the local structure of their mechanical environments. Our work has implications for understanding how neutrophils interact physically with their surrounding environment what conditions may be necessary for fast 3-D neutrophil migration.

Caffeic acid and chlorogenic acid cytotoxicity, genotoxicity and impact on global DNA methylation in human leukemic cell lines.

Dietary phenolic compounds such as caffeic and chlorogenic acid exert an antiproliferative effect and modulate the gene-specific DNA methylation status in human breast tumor cells, but it remains unclear whether they interfere with global DNA methylation in human leukemia cells. We examined whether caffeic and chlorogenic acid (1-250 µM) exert antitumor action in human promyelocytic leukemia cells (HL-60) and human acute T-cell leukemia cells (Jurkat). Caffeic and chlorogenic acid did not reduce cell viability in the two cell lines, as assessed using the neutral red uptake and MTT assays. These phenolic acids (1-100 μM) neither induced DNA damage (comet assay) nor increased the micronuclei frequency (micronucleus assay) in HL-60 and Jurkat cells, indicating that they were not genotoxic or mutagenic. Analysis of global DNA methylation levels using a 5-mC DNA ELISA kit revealed that chlorogenic acid at a non-cytotoxic concentration (100 μM) induced global DNA hypomethylation in Jurkat cells, but not in HL-60 cells, suggesting that it exerts a cell-specific effect. Caffeic acid did not change global DNA methylation. As other phenolic compounds, chlorogenic acid probably modulates DNA methylation by targeting DNA methyltransferases. The hypomethylating action of chlorogenic acid can be beneficial against hematological malignances whose pathogenic processes involve impairment of DNA methylation.

Saponins from the rhizomes of Chamaecostus subsessilis and their cytotoxic activity against HL60 human promyelocytic leukemia cells

Context: Species of the Costaceae family have been traditionally used for the treatment of infections, tumors and inflammatory diseases. Chamaecostus subsessilis (Nees & Mart.) C. D. Specht & D.W. Stev. (Costaceae) is a native medicinal plant with distribution in the Cerrado forest ecosystem of Central Brazil. In our previous work, the antitumor potential of the chloroform fraction (CHCl3 Fr) from rhizomes ethanol extract (REEX) of C. subsessilis was determined against a series of tumor cell lines (HL60, Jurkat, MDA-MB231, MCF-7, HTC-116 and THP-1) using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) colorimetric assay. Aims: To isolate and identify the cytotoxic compounds present in CHCl3 Fr from REEX of C. subsessilis. Methods: The bio-guided chromatographic fractionation of the CHCl3 Fr was performed by High-Performance Liquid Chromatography (HPLC) and preparative Thin Layer Chromatography (prep-TLC). The cytotoxicity of the isolated compounds was evaluated against human promyelocytic leukemia cells (HL60) and human embryonic renal non-tumor cell lineage (HEK-293) by MTT assay. Results: Dioscin and gracillin were identified as active compounds from CHCl3 Fr from REEX of C. subsessilis. They showed IC50 values of 5.3 µM and 14.0 µM, respectively, against HL60 cells, but they did not demonstrate selectivity compared to HEK-293 cells. Conclusions: These results corroborate with the popular use of species of the Costaceae family as a source of antitumor agents. To the best of our knowledge, this is the first time that these saponins are isolated from this species.

An Opsonophagocytic Killing Assay for the Evaluation of Group A Streptococcus Vaccine Antisera.

Group A Streptococcus (GAS) is a major cause of global mortality, yet there are no licensed GAS vaccines. Vaccine progress has been hampered, in part, by a lack of standardized assays able to quantify antibody function in test antisera. The most widely used assay was developed over 50years ago by Rebecca Lancefield and relies on human whole blood as a source of complement and neutrophils. Recently, an opsonophagocytic killing (OPK) assay has been developed for GAS by adapting the OPK methods utilized in Streptococcus pneumoniae vaccine testing. This assay uses dimethylformamide (DMF)-differentiated human promyelocytic leukemia cells (HL-60 cells) as a source of neutrophils and baby rabbit complement, thus removing the major sources of variation in the Lancefield assays. This protocol outlines methods for performing a GAS OPK assay including titering test sera to generate an opsonic index. This in vitro assay could aid in selecting vaccine candidates by demonstrating whether candidate-induced antibodies lead to complement deposition and opsonophagocytic killing.

Toxicity and Antitumor Activity of a Thiophene-Acridine Hybrid.

The antitumor effects of thiophene and acridine compounds have been described; however, the clinical usefulness of these compounds is limited due to the risk of high toxicity and drug resistance. The strategy of molecular hybridization presents the opportunity to develop new drugs which may display better target affinity and less serious side effects. Herein, 2-((6-Chloro-2-methoxy-acridin-9-yl)amino)-5,6,7,8-tetrahydro-4H-cyclohepta[b]-thiophene-3-carbonitrile (ACS03), a hybrid thiophene–acridine compound with antileishmanial activity, was tested for toxicity and antitumor activity. The toxicity was evaluated in vitro (on HaCat and peripheral blood mononuclear cells) and in vivo (zebrafish embryos and acute toxicity in mice). Antitumor activity was also assessed in vitro in HCT-116 (human colon carcinoma cell line), K562 (chronic myeloid leukemic cell line), HL-60 (human promyelocytic leukemia cell line), HeLa (human cervical cancer cell line), and MCF-7 (breast cancer cell line) and in vivo (Ehrlich ascites carcinoma model). ACS03 exhibited selectivity toward HCT-116 cells (Half maximal inhibitory concentration, IC50 = 23.11 ± 1.03 µM). In zebrafish embryos, ACS03 induced an increase in lactate dehydrogenase, glutathione S-transferase, and acetylcholinesterase activities. The LD50 (lethal dose 50%) value in mice was estimated to be higher than 5000 mg/kg (intraperitoneally). In vivo, ACS03 (12.5 mg/kg) induced a significant reduction in tumor volume and cell viability. In vivo antitumor activity was associated with the nitric oxide cytotoxic effect. In conclusion, significant antitumor activity and weak toxicity were recorded for this hybrid compound, characterizing it as a potential anticancer compound.

Differential proteolytic activation of the Bacillus thuringiensis Cry41Aa parasporin modulates its anticancer effect.

Bacillus thuringiensis (Bt) is a gram positive spore forming bacterium which produces intracellular protein crystals toxic to a wide variety of insect larvae and is the most commonly used biological pesticide worldwide. More recently, Bt crystal proteins known as parasporins have been discovered, that have no known insecticidal activity but target some human cancer cells exhibiting strong cytocidal activities with different toxicity spectra and varied activity levels. Parasporin-3, also called Cry41Aa, has only been shown to exhibit cytocidal activity towards HL-60 (Human promyelocytic leukemia cells) and HepG2 (Human liver cancer cells) cell lines after being proteolytically cleaved. In order to understand this activation mechanism various mutations were made in the N-terminal region of the protein and the toxicity against both HepG2 and HL-60 cell lines was evaluated. Our results indicate that only N-terminal cleavage is required for activation and that N-terminally deleted mutants show some toxicity without the need for proteolytic activation. Furthermore, we have shown that the level of toxicity towards the two cell lines depends on the protease used to activate the toxin. Proteinase K-activated toxin was significantly more toxic towards HepG2 and HL-60 than trypsin-activated toxin. N-terminal sequencing of activated toxins showed that this difference in toxicity is associated with a difference of just two amino acids (serine and alanine at positions 59 and 60, respectively) which we hypothesize occlude a binding motif.