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In this study, we screened a chemical library to find potent anticancer compounds that are less cytotoxic to non-cancerous cells. This study revealed that pyrazole PTA-1 is a potent anticancer compound. Additionally, we sought to elucidate its mechanism of action (MOA) in triple-negative breast cancer cells. Cytotoxicity was analyzed with the differential nuclear staining assay (DNS). Additional secondary assays were performed to determine the MOA of the compound. The potential MOA of PTA-1 was assessed using whole RNA sequencing, Connectivity Map (CMap) analysis, in silico docking, confocal microscopy, and biochemical assays. PTA-1 is cytotoxic at a low micromolar range in 17 human cancer cell lines, demonstrating less cytotoxicity to non-cancerous human cells, indicating a favorable selective cytotoxicity index (SCI) for the killing of cancer cells. PTA-1 induced phosphatidylserine externalization, caspase-3/7 activation, and DNA fragmentation in triple-negative breast MDA-MB-231 cells, indicating that it induces apoptosis. Additionally, PTA-1 arrests cells in the S and G2/M phases. Furthermore, gene expression analysis revealed that PTA-1 altered the expression of 730 genes at 24 h (198 upregulated and 532 downregulated). A comparison of these gene signatures with those within CMap indicated a profile similar to that of tubulin inhibitors. Subsequent studies revealed that PTA-1 disrupts microtubule organization and inhibits tubulin polymerization. Our results suggest that PTA-1 is a potent drug with cytotoxicity to various cancer cells, induces apoptosis and cell cycle arrest, and inhibits tubulin polymerization, indicating that PTA-1 is an attractive drug for future clinical cancer treatment.
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Antineoplásicos , Apoptosis , Puntos de Control del Ciclo Celular , Pirazoles , Neoplasias de la Mama Triple Negativas , Tubulina (Proteína) , Humanos , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Apoptosis/efectos de los fármacos , Pirazoles/farmacología , Pirazoles/química , Tubulina (Proteína)/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Antineoplásicos/farmacología , Polimerizacion/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Moduladores de Tubulina/farmacologíaRESUMEN
The cellular enzyme poly (ADP-ribose) polymerase-1 (PARP-1) regulates multiple processes that are potentially implicated in HIV-1 infection. However, the role of PARP-1 in HIV-1 infection remains controversial, with reports indicating or excluding that PARP-1 influence early steps of the HIV-1 life cycle. Most of these studies have been conducted with Vesicular Stomatitis virus Glycoprotein G (VSV-G)-pseudotyped, single-round infection HIV-1; limiting our understanding of the role of PARP-1 in HIV-1 replication. Therefore, we evaluated the effect of PARP-1 deficiency or inhibition in HIV-1 replication in human CD4+ T cells. Our data showed that PARP-1 knockout increased viral replication in SUP-T1 cells. Similarly, a PARP-1 inhibitor that targets PARP-1 DNA-binding activity enhanced HIV-1 replication. In contrast, inhibitors affecting the catalytic activity of the enzyme were inactive. In correspondence with the pharmacological studies, mutagenesis analysis indicated that the DNA-binding domain was required for the PARP-1 anti-HIV-1 activity, but the poly-ADP-ribosylation activity was dispensable. Our results also demonstrated that PARP-1 acts at the production phase of the viral life cycle since HIV-1 produced in cells lacking PARP-1 was more infectious than control viruses. The effect of PARP-1 on HIV-1 infectivity required Env, as PARP-1 deficiency or inhibition did not modify the infectivity of Env-deleted, VSV-G-pseudotyped HIV-1. Furthermore, virion-associated Env was more abundant in sucrose cushion-purified virions produced in cells lacking the enzyme. However, PARP-1 did not affect Env expression or processing in the producer cells. In summary, our data indicate that PARP-1 antagonism enhances HIV-1 infectivity and increases levels of virion-associated Env. Importance: Different cellular processes counteract viral replication. A better understanding of these interfering mechanisms will enhance our ability to control viral infections. We have discovered a novel, antagonist effect of the cellular enzyme poly (ADP-ribose) polymerase-1 (PARP-1) in HIV-1 replication. Our data indicate that PARP-1 deficiency or inhibition augment HIV-1 infectivity in human CD4+ T cells, the main HIV-1 target cell in vivo . Analysis of the mechanism of action suggested that PARP-1 antagonism increases in the virus the amounts of the viral protein mediating viral entry to the target cells. These findings identify for the first time PARP-1 as a host factor that regulates HIV-1 infectivity, and could be relevant to better understand HIV-1 transmission and to facilitate vaccine development.
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Carbon dots (CDs) are carbon nano materials (CNMs) that find use across several biological applications because of their water solubility, biocompatible nature, eco-friendliness, and ease of synthesis. Additionally, their physiochemical properties can be chemically tuned for further optimization towards specific applications. Here, we investigate the efficacy of C70-derived Graphene Acid Quantum Dots (GAQDs) in mitigating the transformation of soluble, monomeric Hen Egg-White Lysozyme (HEWL) to mature fibrils during its amyloidogenic trajectory. Our findings reveal that GAQDs exhibit dose-dependent inhibition of HEWL fibril formation (up to 70 % at 5 mg/mL) without affecting mitochondrial membrane potential or inducing apoptosis at the same density. Furthermore, GAQDs scavenged reactive oxygen species (ROS); achieving a 50 % reduction in ROS levels at a mere 100 µg/mL when exposed to a standard free radical generator. GAQDs were not only found to be biocompatible with a human neuroblastoma-derived SHSY-5Y cell line but also rescued the cells from rotenone-induced apoptosis. The GAQD-tolerance of SHSY-5Y cells coupled with their ability to restitute cells from rotenone-dependent apoptosis, when taken in conjunction with the biocompatibility data, indicate that GAQDs possess neuroprotective potential. The data position this class of CNMs as promising candidates for resolving aberrant cellular outputs that associate with the advent and progress of multifactorial neurodegenerative disorders including Parkinson's (PD) and Alzheimer's diseases (AD) wherein environmental causes are implicated (95 % etiology). The data suggest that GAQDs are a multifunctional carbon-based sustainable nano-platform at the intersection of nanotechnology and neuroprotection for advancing green chemistry-derived, sustainable healthcare solutions.
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Apoptosis , Grafito , Muramidasa , Puntos Cuánticos , Especies Reactivas de Oxígeno , Puntos Cuánticos/química , Humanos , Grafito/química , Grafito/farmacología , Especies Reactivas de Oxígeno/metabolismo , Muramidasa/química , Muramidasa/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/metabolismo , Animales , Tamaño de la Partícula , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/química , Carbono/química , Propiedades de Superficie , Potencial de la Membrana Mitocondrial/efectos de los fármacosRESUMEN
In an effort to identify novel anti-cancer agents, we employed a well-established High Throughput Screening (HTS) assay to assess the cytotoxic effect of compounds within the ChemBridge DIVERSet Library on a lymphoma cell line. This screen revealed a novel thiophene, F8 (methyl 5-[(dimethylamino)carbonyl]-4-methyl-2-[(3-phenyl-2-propynoyl) amino]-3-thiophenecarboxylate), that displays anti-cancer activity on lymphoma, leukemia, and other cancer cell lines. Thiophenes and thiophene derivatives have emerged as an important class of heterocyclic compounds that have displayed favorable drug characteristics. They have been previously reported to exhibit a broad spectrum of properties and varied uses in the field of medicine. In addition, they have proven to be effective drugs in various disease scenarios. They contain anti-inflammatory, anti-anxiety, anti-psychotic, anti-microbial, anti-fungal, estrogen receptor modulating, anti-mitotic, kinase inhibiting and anti-cancer activities, rendering compounds with a thiophene a subject of significant interest in the scientific community. Compound F8 consistently induced cell death at a low micromolar range on a small panel of cancer cell lines after a 48 h period. Further investigation revealed that F8 induced phosphatidylserine externalization, reactive oxygen species generation, mitochondrial depolarization, kinase inhibition, and induces apoptosis. These findings demonstrate that F8 has promising anti-cancer activity.
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Antineoplásicos , Linfoma , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Línea Celular Tumoral , Tiofenos/farmacología , Proliferación Celular , Apoptosis , Antineoplásicos/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Ensayos de Selección de Medicamentos Antitumorales , Relación Estructura-Actividad , Estructura MolecularRESUMEN
A novel series of pyrazole-oxindole conjugates were prepared and characterized as potential cytotoxic agents by FT-IR, NMR and HR-MS. The cytotoxic activity of these compounds was tested in the Jurkat acute T cell leukemia, CEM acute lymphoblastic leukemia, MCF10â A mammary epithelial and MDA-MB 231 triple negative breast cancer cell lines. Among the tested conjugates, 5-methyl-3-((3-(1-phenyl)-3-(p-tolyl)-1H-pyrazol-4-yl)methylene)indolin-2-one 6h emerged as the most cytotoxic with a CC50 of 4.36+/-0.2â µM against Jurkat cells. The mechanism of cell death induced by 6h was investigated through the Annexin V-FITC assay via flow cytometry. Reactive oxygen species (ROS) accumulation, mitochondrial health and the cell cycle progression were also evaluated in cells exposed to 6h. Results demonstrated that 6h induces apoptosis in a dose-response manner, without generating ROS and/or altering mitochondrial health. In addition, 6h disrupted the cell cycle distribution causing an increase in DNA fragmentation (Sub G0-G1), and an arrest in the G0-G1 phase. Taken together, the 6h compound revealed a strong potential as an antineoplastic agent evidenced by its cytotoxicity in leukemia cells, the activation of apoptosis and restriction of the cell cycle progression.
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Antineoplásicos , Neoplasias , Humanos , Especies Reactivas de Oxígeno/metabolismo , Oxindoles/farmacología , Espectroscopía Infrarroja por Transformada de Fourier , Línea Celular Tumoral , Apoptosis , Antineoplásicos/química , Pirazoles/farmacologíaRESUMEN
In recent years, the thienopyrazole moiety has emerged as a pharmacologically active scaffold with antitumoral and kinase inhibitory activity. In this study, high-throughput screening of 2000 small molecules obtained from the ChemBridge DIVERset library revealed a unique thieno[2,3-c]pyrazole derivative (Tpz-1) with potent and selective cytotoxic effects on cancer cells. Compound Tpz-1 consistently induced cell death at low micromolar concentrations (0.19 µM to 2.99 µM) against a panel of 17 human cancer cell lines after 24 h, 48 h, or 72 h of exposure. Furthermore, an in vitro investigation of Tpz-1's mechanism of action revealed that Tpz-1 interfered with cell cycle progression, reduced phosphorylation of p38, CREB, Akt, and STAT3 kinases, induced hyperphosphorylation of Fgr, Hck, and ERK 1/2 kinases, and disrupted microtubules and mitotic spindle formation. These findings support the continued exploration of Tpz-1 and other thieno[2,3-c]pyrazole-based compounds as potential small-molecule anticancer agents.
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In this study, we identified a novel pyrazole-based derivative (P3C) that displayed potent cytotoxicity against 27 human cancer cell lines derived from different tissue origins with 50% cytotoxic concentrations (CC50) in the low micromolar and nanomolar range, particularly in two triple-negative breast cancer (TNBC) cell lines (from 0.25 to 0.49 µM). In vitro assays revealed that P3C induces reactive oxygen species (ROS) accumulation leading to mitochondrial depolarization and caspase-3/7 and -8 activation, suggesting the participation of both the intrinsic and extrinsic apoptotic pathways. P3C caused microtubule disruption, phosphatidylserine externalization, PARP cleavage, DNA fragmentation, and cell cycle arrest on TNBC cells. In addition, P3C triggered dephosphorylation of CREB, p38, ERK, STAT3, and Fyn, and hyperphosphorylation of JNK and NF-kB in TNBC cells, indicating the inactivation of both p38MAPK/STAT3 and ERK1/2/CREB signaling pathways. In support of our in vitro assays, transcriptome analyses of two distinct TNBC cell lines (MDA-MB-231 and MDA-MB-468 cells) treated with P3C revealed 28 genes similarly affected by the treatment implicated in apoptosis, oxidative stress, protein kinase modulation, and microtubule stability.
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Pirazoles/toxicidad , Transducción de Señal , Neoplasias de la Mama Triple Negativas/patología , Caspasas/metabolismo , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Activación Enzimática/efectos de los fármacos , Exocitosis/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Invasividad Neoplásica , Proteínas de Neoplasias/metabolismo , Fosfatidilserinas/metabolismo , Fosforilación/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Pirazoles/química , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Tubulina (Proteína)/metabolismoRESUMEN
BACKGROUND: Breast cancer (BC) continues to have the second highest mortality amongst women in the United States after lung cancer. For 2021, the American Cancer Association predicted 281,550 new invasive breast cancer cases besides 49,290 new cases of non-invasive breast cancer and 43,600 deaths from the metastatic disease. A treatment modality is radiation therapy, which is given for local control as well as palliation of patient symptoms. The initial step of new drug development is in-vitro cell studies, which help describe new drug properties and toxicities. However, these models are not optimal, and better ones have yet to be determined. This study uses bioprinting technology to elucidate the sensitivity of tumor cells to the combination of palbociclib (PD) and letrozole (Let) treatment. We hypothesize that this technology could serve as a model to predict treatment outcomes more efficiently. METHODS: The breast cancer cell lines MCF7 and MDA-MB-231 as well as the normal breast epithelial cell line, MCF-10A, were treated with PD-Let with and without radiotherapy (RT), and cell viability was compared in pairwise fashion for thermally inkjet bioprinted (TIB) and manually seeded (MS) cells. RESULTS: In absence of radiation, the TIB MCF7 cells have 2.5 times higher viability than manually seeded (MS) cells when treated with 100 µM palbociclib and 10 µM letrozole, a 36% higher viability when treated with 50 µM palbociclib and 10 µM letrozole, and an 8% higher viability when treated with 10 µM palbociclib and 10 µM letrozole. With 10 Gy of radiation, TIB cells had a 45% higher survival rate than MS cells at the lowest palbociclib concentration and a 29% higher survival rate at the intermediate palbociclib concentration. Without radiation treatment, at a concentration of 10 µM PD-Let, TIB MDA-MB-231 cells show a 8% higher viability than MS cells when treated with 10 µM PD and 10 µM Let; at higher drug concentrations, the differences disappeared, but some 1.7% of the TIB MDA-MB-231 cells survived exposure to 150 µM of PD + 10 µM letrozole vs. none of the MS cells. These cells are more radiation sensitive than the other cell lines tested and less sensitive to the combo drug treatments. We observed an 18% higher survival of TIB MCF-10A cells without radiation treatment when exposed to 10 µM PD + 10 µM Let but no difference in cell survival between the two groups when radiation was applied. Independent of growth conditions, TIB cells did not show more resistance to radiation treatment than MS cells, but a higher resistance to the combo treatment was observed, which was most pronounced in the MCF-7 cell line. CONCLUSION: Based on these results, we suggest that TIB used in in-vitro models could be a feasible strategy to develop and/or test new anticancer drugs.
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BACKGROUND: Breast cancer is the most frequently diagnosed cancer in women worldwide. Pyronaridine (PND), an antimalarial drug, was shown to exert anticancer activity on seventeen different human cancer cells, seven from female breast tissue. Additionally, PND induced apoptosis via mitochondrial depolarization, alteration of cell cycle progression, and DNA intercalation. However, the molecular target of PND in cells was not elucidated. OBJECTIVE: Here, we have further investigated PND's mode of action by using transcriptome analysis. Preclinical studies were also performed to determine whether PND could affect tumor progression in a human breast cancer xenograft in mice. Moreover, we assessed the combined efficacy of PND with well-known anticancer drugs. METHODS: Transcriptome analyses of PND-treated cancer cells were performed. Topoisomerase II activity was evaluated by an in vitro assay. In addition, daily oral administration of PND was given to mice with human breast cancer xenografts. The differential nuclear staining assay measured in-vitro cell toxicity. RESULTS: The transcriptome signatures suggested that PND might act as a topoisomerase II inhibitor. Thus, topoisomerase inhibition assays were performed, providing evidence that PND is a bona fide topoisomerase II inhibitor. Also, in-vivo studies suggest that PND hinders tumor progression. Besides, combination studies of PND with anticancer drugs cisplatin and gemcitabine revealed higher cytotoxicity against cancer cells than individual drug administration. CONCLUSION: The findings provide evidence that PND is a topoisomerase II inhibitor and can hinder cancer progression in an animal model, further demonstrating PND's favorable characteristics as a repurposed anticancer drug.
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Communicated by Ramaswamy H. Sarma.
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Simulación de Dinámica Molecular , Simulación del Acoplamiento MolecularRESUMEN
Lactoferrin has gained extensive attention due to its ample biological properties. In this study, recombinant human lactoferrin carrying humanized glycosylation (rhLf-h-glycan) expressed in the yeast Pichia pastoris SuperMan5, which is genetically glycoengineered to efficiently produce functional humanized glycoproteins inclosing (Man)5(GlcNAc)2 Asn-linked glycans, was analyzed, inspecting its potential toxicity against cancer cells. The live-cell differential nuclear staining assay was used to quantify the rhLf-h-glycan cytotoxicity, which was examined in four human cell lines: acute lymphoblastic leukemia (ALL) CCRF-CEM, T-cell lymphoblastic lymphoma SUP-T1, cervical adenocarcinoma HeLa, and as control, non-cancerous Hs27 cells. The defined CC50 values of rhLf-h-glycan in CCRF-CEM, SUP-T1, HeLa, and Hs27 cells were 144.45 ± 4.44, 548.47 ± 64.41, 350 ± 14.82, and 3359.07 ± 164 µg/mL, respectively. The rhLf-h-glycan exhibited a favorable selective cytotoxicity index (SCI), preferentially killing cancer cells: 23.25 for CCRF-CEM, 9.59 for HeLa, and 6.12 for SUP-T1, as compared with Hs27 cells. Also, rhLf-h-glycan showed significant antiproliferative activity (P < 0.0001) at 24, 48, and 72 h of incubation on CCRF-CEM cells. Additionally, it was observed via fluorescent staining and confocal microscopy that rhLf-h-glycan elicited apoptosis-associated morphological changes, such as blebbing, nuclear fragmentation, chromatin condensation, and apoptotic bodies in ALL cells. Furthermore, rhLf-h-glycan-treated HeLa cells revealed shrinkage of the microfilament structures, generating a speckled/punctuated pattern and also caused PARP-1 cleavage, a hallmark of apoptosis. Moreover, in ALL cells, rhLf-h-glycan altered cell cycle progression inducing the G2/M phase arrest, and caused apoptotic DNA fragmentation. Overall, our findings revealed that rhLf-h-glycan has potential as an anticancer agent and therefore deserves further in vivo evaluation.
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Citoesqueleto de Actina/efectos de los fármacos , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Lactoferrina/farmacología , Línea Celular Tumoral , Células HeLa , Humanos , Proteínas Recombinantes , SaccharomycetalesRESUMEN
Ni-Cu nanoparticles have been synthesized by reducing Ni and Cu from metal precursors using a sol-gel route followed by annealing at 300 °C for 1, 2, 3, 6, 8, and 10 h for controlled self-regulating magnetic hyperthermia applications. Particle morphology and crystal structure revealed spherical nanoparticles with a cubic structure and an average size of 50, 60, 53, 87, and 87 nm for as-made and annealed samples at 300 °C for 1, 3, 6, and 10 h, respectively. Moreover, hysteresis loops indicated ferromagnetic behavior with saturation magnetization (Ms) ranging from 13-20 emu/g at 300 K. Additionally, Zero-filed cooled and field cooled (ZFC-FC) curves revealed that each sample contains superparamagnetic nanoparticles with a blocking temperature (TB) of 196-260 K. Their potential use for magnetic hyperthermia was tested under the therapeutic limits of an alternating magnetic field. The samples exhibited a heating rate ranging from 0.1 to 1.7 °C/min and a significant dissipated heating power measured as a specific absorption rate (SAR) of 6-80 W/g. The heating curves saturated after reaching the Curie temperature (Tc), ranging from 30-61 °C within the therapeutic temperature limit. An in vitro cytotoxicity test of these Ni-Cu samples in biological tissues was performed via exposing human breast cancer MDA-MB231 cells to a gradient of concentrations of the sample with 53 nm particles (annealed at 300 °C for 3 h) and reviewing their cytotoxic effects. For low concentrations, this sample showed no toxic effects to the cells, revealing its biocompatibility to be used in the future for in vitro/in vivo magnetic hyperthermia treatment of cancer.
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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 5 µM 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 ß-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.
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Antineoplásicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Hidroquinonas/química , Fosforilación Oxidativa/efectos de los fármacos , Sulfonamidas/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Humanos , Hidroquinonas/farmacología , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Bioprinting technology merges engineering and biological fields and together, they possess a great translational potential, which can tremendously impact the future of regenerative medicine and drug discovery. However, the molecular effects elicited by thermal inkjet bioprinting in breast cancer cells remains elusive. Previous studies have suggested that bioprinting can be used to model tissues for drug discovery and pharmacology. We report viability, apoptosis, phosphorylation, and RNA sequence analysis of bioprinted MCF7 breast cancer cells at separate timepoints post-bioprinting. An Annexin A5-FITC apoptosis stain was used in combination with flow cytometry at 2 and 24 h post-bioprinting. Antibody arrays using a Human phospho-MAPK array kit was performed 24 h post-bioprinting. RNA sequence analysis was conducted in samples collected at 2, 7, and 24 h post-bioprinting. The post-bioprinting cell viability averages were 77 and 76% at 24 h and 48 h, with 31 and 64% apoptotic cells at 2 and 24 h after bioprinting. A total of 21 kinases were phosphorylated in the bioprinted cells and 9 were phosphorylated in the manually seeded controls. The RNA seq analysis in the bioprinted cells identified a total of 12,235 genes, of which 9.7% were significantly differentially expressed. Using a ±2-fold change as the cutoff, 266 upregulated and 206 downregulated genes were observed in the bioprinted cells, with the following 5 genes uniquely expressed NRN1L, LUCAT1, IL6, CCL26, and LOC401585. This suggests that thermal inkjet bioprinting is stimulating large scale gene alterations that could potentially be utilized for drug discovery. Moreover, bioprinting activates key pathways implicated in drug resistance, cell motility, proliferation, survival, and differentiation.
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Combustion-derived nanomaterials are noxious ultrafine (<100 nm) aerosol by-products of human activity. They pose threats to pulmonary health due to their small size, allowing them to penetrate alveoli causing detrimental responses downstream. Information regarding the cellular activity that connects nanocarbon particle exposure to poor pulmonary health remains lacking. We hypothesized that low-dose and long-term administrations of carbonaceous nanoparticles contribute to lung irritation by adversely affecting respiratory cells that function as the first line of defense. Responses to ultrafine black carbon (UBC), a key component of airborne pollutants, by human lung A549, murine lung LA4 epithelial cells, human peripheral-blood monocytes THP1, and murine macrophages RAW264.7 were investigated. The cells were first plated on day zero and were fed fresh UBC suspended in culture media on days one, four, and seven. The exposure regimen included three different concentrations of UBC. On day ten, all cells were harvested, washed, and assayed. The impact on cellular viability revealed that UBC was only moderately cytotoxic, while metabolic activity was significantly diminished in a dose-dependent manner. Additionally, beta-galactosidase proportionally increased with UBC concentration compared to untreated cells, indicating that cellular senescence was promoted across all cell types. The implemented regimen caused minimal toxicity yet demonstrated different cellular modifications across the cell lines of both species, inducing changes to enzyme vitality and cellular fitness. The data suggested that compounding nanosized black carbon exposure could negatively impair overall pulmonary health by distinctively modifying intracellular behavior.
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Breast cancer is the most frequently diagnosed cancer among women worldwide. Here, recombinant human lactoferrin (rhLf) expressed in Pichia pastoris was tested for its potential cytotoxic activity on a panel of six human breast cancer cell lines. The rhLf cytotoxic effect was determined via a live-cell HTS imaging assay. Also, confocal microscopy and flow cytometry protocols were employed to investigate the rhLf mode of action. The rhLf revealed an effective CC50 of 91.4 and 109.46 µg/ml on non-metastatic and metastatic MDA-MB-231 cells, with favorable selective cytotoxicity index values, 11.68 and 13.99, respectively. Moreover, rhLf displayed satisfactory SCI values on four additional cell lines, MDA-MB-468, HCC70, MCF-7 and T-47D (1.55-3.34). Also, rhLf provoked plasma membrane blebbing, chromatin condensation and cell shrinkage in MDA-MB-231 cells, being all three apoptosis-related morphological changes. Also, rhLf was able to shrink the microfilaments, forming a punctuated cytoplasmic pattern in both the MDA-MB-231 and Hs-27 cells, as visualized in confocal photomicrographs. Moreover, performing flow cytometric analysis, rhLf provoked significant phosphatidylserine externalization, cell cycle arrest in the S phase and apoptosis-induced DNA fragmentation in MDA-MB-231 cells. Hence, rhLf possesses selective cytotoxicity on breast cancer cells. Also, rhLf caused apoptosis-associated morphologic changes, disruption of F-actin cytoskeleton organization, phosphatidylserine externalization, DNA fragmentation, and arrest of the cell cycle progression on triple-negative breast cancer MDA-MB-231 cells. Overall results suggest that rhLf is using the apoptosis pathway as its mechanism to inflict cell death. Findings warranty further evaluation of rhLf as a potential anti-breast cancer drug option.
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Citoesqueleto de Actina/efectos de los fármacos , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Lactoferrina/farmacología , Neoplasias de la Mama Triple Negativas/patología , Antineoplásicos/aislamiento & purificación , Antineoplásicos/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Lactoferrina/genética , Lactoferrina/aislamiento & purificación , Lactoferrina/metabolismo , Fosfatidilserinas/metabolismo , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
In the last 15 years, pyridazinone derivatives have acquired extensive attention due to their widespread biological activities and pharmacological applications. Pyridazinones are well known for their anti-microbial, anti-viral, anti-inflammatory, anti-cancer, and cardiovascular activities, among others. In this study, we evaluated the anti-cancer activity of a new pyridazinone derivative and propose it as a potential anti-neoplastic agent in acute promyelocytic leukemia cells. Pyr-1 cytotoxicity was assessed on several human cancer and two non-cancerous cell lines by the DNS assay. Pyr-1 demonstrated potent cytotoxicity against 22 human cancer cell lines, exhibiting the most favorable selective cytotoxicity on leukemia (CEM and HL-60), breast (MDA-MB-231 and MDA-MB-468), and lung (A-549) cancer cell lines, when compared with non-cancerous breast epithelial MCF-10A cells. Analyses of apoptosis/necrosis pathways, reactive oxygen species (ROS) production, mitochondria health, caspase-3 activation, and cell cycle profile were performed via flow cytometry. Both hmox-1 RNA and protein expression levels were evaluated by quantitative real-time PCR and Western blotting assays, respectively. Pyr-1 induced apoptosis in acute promyelocytic leukemia cells as confirmed by phosphatidylserine externalization, mitochondrial depolarization, caspase-3 activation, DNA fragmentation, and disrupted cell cycle progression. Additionally, it was determined that Pyr-1 generates oxidative and proteotoxic stress by provoking the accumulation of ROS, resulting in the overexpression of the stress-related hmox-1 mRNA transcripts and protein and a marked increase in poly-ubiquitinated proteins. Our data demonstrate that Pyr-1 induces cell death via the intrinsic apoptosis pathway by accumulating ROS and by impairing proteasome activity.
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Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Piridazinas/farmacología , Antineoplásicos/síntesis química , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Caspasas/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Fragmentación del ADN , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Necrosis/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Proteínas Ubiquitinadas , Proteína X Asociada a bcl-2/metabolismoRESUMEN
[This corrects the article DOI: 10.1039/C8RA08334G.].
RESUMEN
BACKGROUND: This study presents a prediction of putative miRNA within several Human Papillomavirus (HPV) types by using bioinformatics tools and a strategy based on sequence and structure alignment. Currently, little is known about HPV miRNAs. METHODS: Computational methods have been widely applied in the identification of novel miRNAs when analyzing genome sequences. Here, ten whole-genome sequences from HPV-6, -11, -16, -18, -31, -33, -35, -45, -52, and -58 were analyzed. Software based on local contiguous structure-sequence features and support vector machine (SVM), as well as additional bioinformatics tools, were utilized for identification and classification of real and pseudo microRNA precursors. RESULTS: An initial analysis predicted 200 putative pre-miRNAs for all the ten HPV genome variants. To derive a smaller set of pre-miRNAs candidates, stringent validation criteria was conducted by applying <â10 ΔG value (Gibbs Free Energy). Thus, only pre-miRNAs with total scores above the cut-off points of 90% were considered as putative pre-miRNAs. As a result of this strategy, 19 pre-miRNAs were selected (hpv-pre-miRNAs). These novel pre-miRNAs were located in different clusters within HPV genomes and some of them were positioned at splice regions. Additionally, the 19 identified pre-miRNAs sequences varied between HPV genotypes. Interestingly, the newly identified miRNAs, 297, 27b, 500, 501-5, and 509-3-5p, were closely implicated in carcinogenesis participating in cellular longevity, cell cycle, metastasis, apoptosis evasion, tissue invasion and cellular growth pathways. CONCLUSIONS: The novel putative miRNAs candidates could be promising biomarkers of HPV infection and furthermore, could be targeted for potential therapeutic interventions in HPV-induced malignancies.
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Biología Computacional/métodos , Genoma Viral , MicroARNs/análisis , Papillomaviridae/genética , Alineación de Secuencia/métodos , Homología de Secuencia de Ácido Nucleico , Secuencia de Bases , ADN Viral/análisis , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Interacciones Huésped-Patógeno/genética , Humanos , MicroARNs/genética , Papillomaviridae/patogenicidad , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/virología , Análisis de Secuencia de ADN/métodosRESUMEN
The potent antimalarial drug pyronaridine (PND) was tested for its potential as an anticancer drug. After exposing cancerous (17) and non-cancerous (2) cells to PND for 72 hr, PND was found to exhibit consistent and potent cytotoxic activity at low micromolar (µM) concentrations that ranged from 1.6 µM to 9.4 µM. Moreover, PND exerted a significant selective cytotoxicity index (SCI) on five out of seven breast cancer cell lines tested, with favorable values of 2.5 to 4.4, as compared with the non-cancerous breast MCF-10A cell line. By using the same comparison, PND exhibited a significant SCI on three out of four leukemia/lymphoma cell lines with promising values of 3.3 to 3.5. One breast cancer and one leukemia cell line were tested further in order to determine the likely mode of action of PND. PND was found to consistently elicit phosphatidylserine externalization, mitochondrial depolarization, and DNA fragmentation, in both the triple negative MDA-MB-231 breast cancer and HL-60 leukemia cell lines. In addition, PND treatment altered cell cycle progression in both cancer cells. Subsequent DNA mobility-shift assays, UV-Visible spectroscopic titrations, and circular dichroism (CD) experiments revealed that PND intercalates with DNA. The findings presented in this study indicates that PND induces apoptosis and interfered with cell cycle progression of cancer cell lines and these results indicate that this drug has the potential as a repurposed drug for cancer therapy.