The luminescent Chalcones. Its potential use as a luminescent and antitumoral agents / Chalconas luminiscentes. Su uso potencial como agentes luminiscentes y antitumorales

Background: Hepatocellular carcinoma (HCC) is one of the most diagnosed cancers worldwide. Chemoprevention of HCC can be achieved using natural or synthetic compounds that reverse, suppress, detect, or prevent cancer progression. Objectives: In this study, both the antiproliferative effects and luminescent properties of 2'-hydroxychalcones were evaluated. Methods: Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay, spectroscopy assays, and density functional theory (DFT) calculations were used to determine the luminescent properties of 2 ́-hydroxychalcones. Results: Cytotoxic effects of 2 ́-hydroxychalcones were observed over the HepG2 and EA.hy926 cells. Since the chalcone moiety could be used as a fluorescent probe, these compounds may be helpful in cancer diagnosis and tumor localization. They may enable tumor observation and regression through the fluorescence during treatment; therefore, the compounds are a potential candidate as novel anticancer agents acting on human hepatomas. Conclusions: This report describes the chalcones' use as a specific luminescent biomarker in tumor cells. We also report the cellular uptake of 2'-hydroxychalcones, their cellular distribution, and the mechanisms that may be responsible for their cytotoxic effects

ANTECEDENTES: El carcinoma hepatocelular (CHC) es uno de los cánceres más diagnosticados en todo el mundo. La quimio prevención del CHC se puede lograr utilizando compuestos naturales o sintéticos que reviertan, supriman, detecten o prevengan la progresión del cáncer. OBJETIVOS: En este estudio, se investigó tanto los efectos antiproliferativos como las propiedades luminiscentes de las 2'-hidroxicalconas. MÉTODOS: La viabilidad celular se evaluó usando el ensayo colorimétrico (MTT), los ensayos de espectroscopia y los cálculos DFT se usaron para determinar las propiedades luminiscentes de las 2 ́-hidroxichalconas. RESULTADOS: Se observaron efectos citotóxicos sobre las líneas celulares del tipo HepG2 y EA.hy926. Dado que la estructura de la 2 ́-hidroxichalcona puede ser usada como sonda fluorescente, estos compuestos pueden ser útiles en el diagnóstico del cáncer y la localización del tumor, ya que pueden permitir la observación a través de la fluorescencia y la regresión del tumor durante el tratamiento, por lo que son candidatas potenciales como nuevos agentes anticancerígenos que podrían actuar sobre hepatomas humanos. CONCLUSIONES: Este trabajo describe el uso de las 2 ́-hidroxichalconas como un biomarcador luminiscente específico para células tumorales. También informamos la captación celular de 2>-hidroxicalconas, su distribución celular y los mecanismos que pueden ser responsables de sus efectos citotóxicos

Low-dose cisplatin exposure and SNAIL, Vimentin, E-cadherin expression in HEPG2 cell line / Exposición a dosis bajas de cisplatino y expresión de SNAIL, Vimentina, E-cadherina en línea celular HEPG2

Cisplatin, the first platinum compound approved for cancer treatment, is widely used in the treatment of various cancers including hepatocellular carcinoma (HCC). HCC incidence rates rise globally. Epithelial mesenchymal transition (EMT) is implicated in cancer invasion and metastasis, which are associated with increased mortality. Cisplatin dose might influence cancer invasion and metastatic behavior of the cells. The aim of the study was to investigate the effect of low-dose cisplatin treatment on EMT- related changes in HepG2 cells. Following treatment with 4 µM cisplatin, HepG2 cells were evaluated morphologically. Gene expression of E-cadherin, Vimentin, Snail1 was assessed by quantitative PCR. Immunofluorescence analyses of NA-K ATPase were performed. Although the low-dose cisplatin treated cells exhibited a more stretched morphology, no statistical difference was detected in gene expression of E-cadherin, Vimentin, Snail1 and immunofluorescence of NA-K ATPase. Findings on low-dose cisplatin effects in HepG2 might contribute to the knowledge of antineoplastic inefficacy by further understanding the molecular mechanisms of drug action.

El cisplatino, el primer compuesto de platino aprobado para el tratamiento del cáncer, es ampliamente utilizado en el tratamiento de varios tipos de cáncer, incluido el carcinoma hepatocelular (CHC). Las tasas de incidencia de CHC aumentan a nivel mundial. La transición mesenquimal epitelial (EMT) está implicada en la invasión del cáncer y la metástasis, que se asocian con un aumento de la mortalidad. La dosis de cisplatino podría influir en la invasión del cáncer y el comportamiento metastásico de las células. El objetivo del estudio fue investigar el efecto del tratamiento con dosis bajas de cisplatino en los cambios relacionados con la EMT en las células HepG2. Tras el tratamiento con cisplatino de 4 µM, se evaluaron morfológicamente las células HepG2. La expresión génica de E-cadherina, vimentina, caracol1 se evaluó mediante PCR cuantitativa. Se realizaron análisis de inmunofluorescencia de NA-K ATPasa . Aunque las células tratadas con cisplatino en dosis bajas exhibieron una morfología más estirada, no se detectaron diferencias estadísticas en la expresión génica de E-cadherina, vimentina, Snail1 e inmunofluorescencia de NA-K ATPasa. Los hallazgos sobre los efectos del cisplatino en dosis bajas en HepG2 podrían contribuir al conocimiento de la ineficacia antineoplásica al comprender mejor los mecanismos moleculares de la acción del fármaco.

Cytotoxic effect of hydroalcoholic extract of Cota tinctoria (L) J. Gay on AGS and Hep-G2 cancer cell lines / Efecto citotóxico del extracto hidroalcohólico de Cota tinctoria (L) J. Gay sobre líneas celulares de cáncer AGS y Hep-G2

Cota tinctoria is a medicinal plant which has been used for management of cancer in folk medicine of various regions. The aim of present study is to investigate cytotoxic activity of different concentrations of hydroalcoholic extract of C. tinctoria flowers on gastric (AGS) and liver (Hep-G2) cancer cell lines as well as Human Natural GUM fibroblast (HUGU) cells. Cell mortality rates were examined after 24, 48 and 72 h incubations using the MTT assay. IC50of extract on AGS cells after 24, 48 and 72h was 1.46, 1.29 and 1.14 µg/mL respectively. The extract demonstrated IC50 of 5.15, 3.92 and 2.89 µg/mL on Hep-G2 cells after 24, 48 and 72 h respectively. No cytotoxic effect was detected on HUGU (Human Natural GUM fibroblast) cells. C. tinctoria seems to have a promising potential to be considered as a source for anticancer drug discovery. However, more experimental and clinical studies are required.

Cota tinctoria es una planta medicinal que se ha utilizado para el tratamiento del cáncer en la medicina popular de varias regiones. El objetivo del presente estudio es investigar la actividad citotóxica de diferentes concentraciones de extracto hidroalcohólico de flores de C. tinctoria en líneas celulares de cáncer gástrico (AGS) e hígado (Hep-G2), así como en células de fibroblasto GUM humano natural (HUGU). Se examinaron las tasas de mortalidad celular después de incubaciones de 24, 48 y 72 h utilizando el ensayo MTT. La CI50 del extracto en células AGS después de 24, 48 y 72 h fue de 1,46; 1,29 y 1,14 µg respectivamente. El extracto demostró una CI50 de 5,15, 3,92 y 2,89 µg/mL en células Hep-G2 después de 24, 48 y 72 h, respectivamente. No se detectó ningún efecto citotóxico en las células HUGU (fibroblasto GUM humano natural). C. tinctoria parece tener un potencial prometedor para ser considerada como una fuente de descubrimiento de fármacos contra el cáncer. Sin embargo, se requieren más estudios experimentales y clínicos.

Anti-proliferative effects of paroxetine alone or in combination with sorafenib in HepG2 cells

Abstract Hepatocellular carcinoma (HCC) is a common cause of cancer-related death. Sorafenib is the first approved drug for the treatment of advanced HCC. Depression is frequent in cancer patients. Moreover, sorafenib might exert depression as an adverse drug reaction and paroxetine, a selective serotonin reuptake inhibitor, is a recommended pharmacotherapy. This study aimed to investigate the potential synergistic effects of paroxetine and sorafenib on HepG2 cell proliferation and death. Paroxetine and sorafenib were administered to HepG2 cells as single-agents or in combination. Cell viability was determined with XTT cell viability assay. Cellular apoptosis and DNA content were assessed by flow cytometry. The expression of anti-apoptotic Bcl-2 was examined by immunofluorescence confocal microscopy. A lower dose of sorafenib was found to be required to inhibit cell proliferation when in combination with paroxetine. Similarly, the coadministration enhanced cellular apoptosis and resulted in cell cycle arrest. Confocal imaging revealed a remarkably lower cell density and increased expression of Bcl-2 following combined treatment of paroxetine with sorafenib. To our knowledge, this is the first study demonstrating the synergistic effect of paroxetine and sorafenib in HCC and might provide a potentially promising therapeutic strategy.

Xuezhikang alleviates lipid accumulation via AMPK activation in hepatocellular steatosis model

Abstract Xuezhikang (XZK) is an extract of Chinese red yeast rice. It has multiple protective effects in cardiovascular systems. However, the underlying mechanism by which XZK affects free fatty acid (FFA)-induced lipogenesis in hepatocellular steatosis model is still unknown. Herein, we investigated this mechanism in HepG2 cells. The HepG2 cells were treated with palmitate acid (PA) to induce lipogenesis. Then the PA-induced HepG2 cells were subsequently treated with XZK. After 24 h of treatment, we determined the intracellular triglyceride (TG) contents and average areas of lipid droplets. To study the involvement of AMPK signaling pathway, we pre-treated the PA-induced HepG2 cells with Compound C, an AMPK inhibitor, before XZK treatment. Expressions of p-AMPK and AMPK were determined by Western blot. The results showed that XZK decreased TG content and lipid accumulation in hepatocellular steatosis model. Compound C abolished the effects of XZK. These results demonstrated for the first time that XZK protects hepatocytes against lipid accumulation induced by free fatty acids. Its effects may be mediated by the activation of AMPK pathway.

Morphological and biochemical effects of carnosic acid on human hepatocellular carcinoma HepG2 cells / Efectos Morfológicos y Bioquímicos del ácido Carnósico en Células de Carcinoma Hepatocelular Humano HepG2

Background/aim: Autophagic cell death and apoptosis of tumor cells has become one of the main objectives in cancer treatment, whereas tumor cell lines are mainly used in studies for providing important data for the evaluation of potential anti cancer substances. In this study, our objective was to evaluate morphological and biochemical changes including rate of apoptosis and Alpha Fetoprotein (AFP) levels at different concentrations of Carnosic Acid (CA) on Human Hepatocellular Carcinoma HepG2 Cells.Materials and methods: Human Hepatocellular Carcinoma (7th passage HepG2 cells) Cell lines were cultured on 11 µM D263M schott glass coverslips placed in 12-well plates and were treated with DMSO, 1, 2.5, 5 and 10 µM concentrations of CA for 24, 48 and 72 hours. Morphological and biochemical data were recorded daily including apoptosis rates demonstrated by Caspase 3, Annexin V expressions under inverted light and Immunofluorescence microscopy, then data were analyzed for statistical significance. AFP, albumin and total protein levels were analyzed spectrophotometricaly for biochemical evaluation.Results: Our results showed that CA significantly inhibited HepG2 cell proliferation in a dose and time dependant manner and significantly caused the formation of autophagic vacuoles starting from 5µM and reaching significance at 10 µM concentrations. Significant decrease was observed in AFP when 48 and 72 hours expressions were examined, with the lowest level reached at 72 hours in the 10 µM CA group. Additionally, increase in albumin levels reached significance only in the 48 h group whereas non-significant increases were also observed in 24 h and 72 h groups.Conclusion: Our current study demonstrates significant increase in apoptosis rates by Carnosic Acid mainly at 10µM concentrations, supporting its anticancer effect on HepG2 cells. These findings are also supported by changes in biochemical analyses of Albumin and AFP levels at 10 µM concentrations.

Antecedentes / objetivos: La muerte celular autofágica y la apoptosis de células tumorales se ha convertido en uno de los principales objetivos en el tratamiento del cáncer, mientras que las líneas celulares tumorales se utilizan principalmente en estudios para proporcionar datos importantes para la evaluación de posibles sustancias anticancerígenas. En este estudio, nuestro objetivo fue evaluar los cambios morfológicos y bioquímicos, incluida la tasa de apoptosis y los niveles de alfa fetoproteína (AFP) a diferentes concentraciones de ácido carnósico (CA) en células de carcinoma hepatocelular humano HepG2.Materiales y métodos: Carcinoma hepatocelular humano (HepG2).Las líneas celulares se cultivaron en cubreobjetos de vidrio Schott D263M de 11 µM colocados en placas de 12 pocillos y se trataron con DMSO, concentraciones de CA 1, 2,5, 5 y 10 µM durante 24, 48 y 72 horas. Los datos morfológicos y bioquímicos se registraron diariamente, incluidas las tasas de apoptosis demostradas por Caspasa 3, las expresiones de Anexina V bajo luz invertida y microscopía de inmunofluorescencia, luego se analizaron los datos para determinar la significación estadística. Los niveles de AFP, albúmina y proteínas totales se analizaron espectrofotométricamente para evaluación bioquímica.Resultados: Nuestros resultados mostraron que CA inhibió significativamente la proliferación de células HepG2 de una manera dependiente de la dosis y el tiempo y causó significativamente la formación de vacuolas autofágicas comenzando desde 5 µM y alcanzando significancia a concentraciones de 10 µM. Se observó una disminución significativa en la AFP cuando se examinaron las expresiones de 48 y 72 horas, alcanzando el nivel más bajo a las 72 horas en el grupo de CA 10 µM. Además, el aumento en los niveles de albúmina alcanzó significación solo en el grupo de 48 h, mientras que también se observaron aumentos no significativos en los grupos de 24 hy 72 h.Conclusión: Nuestro estudio demuestra un aumento significativo en las tasas de apoptosis por el ácido carnósico principalmente a concentraciones de 10 µM, lo que respalda su efecto anticancerígeno en las células HepG2. Estos hallazgos también están respaldados por cambios en los análisis bioquímicos de los niveles de albúmina y AFP a concentraciones de 10 µM.

Efecto del extracto hidroalcohólico de la planta Lampaya medicinalis Phil. (Verbenaceae) sobre la esteatosis inducida in vitro en hepatocitos humanos / Effect of the hydroalcoholic extract of the plant Lampaya medicinalis Phil. (Verbenaceae) on in vitro-induced steatosis in human hepatocytes

INTRODUCCIÓN: La enfermedad del hígado graso no alcohólico (EHGNA) es la forma más común de enfermedad hepática. A nivel celular se caracteriza por la acumulación de triglicéridos (TG) en forma de gotas lipídicas (GL) dando lugar a esteatosis e inflamación. Entre los factores relevantes para la síntesis de TG se encuentran las enzimas DGAT1/2 que catalizan la etapa final de la síntesis de TG, y la proteína FABP4 que transporta lípidos intracelulares y se expresa en modelos de enfermedad hepática dependiente de obesidad. Por otra parte, TNF-α es una reconocida citoquina involucrada en el proceso inflamatorio en la EHGNA. La medicina popular del norte de Chile ha utilizado la planta Lampaya medicinalis Phil. (Verbenaceae) para el tratamiento de algunas enfermedades inflamatorias. OBJETIVO: Evaluar el efecto de un extracto hidroalcóholico de lampaya (EHL) sobre la esteatosis y expresión de marcadores de inflamación en hepatocitos tratados con ácidos grasos. Diseño experimental: Estudio in vitro en cultivos de la línea celular humana HepG2 tratadas con ácido oleico (AO) y ácido palmítico (AP). MÉTODOS: Se incubó hepatocitos HepG2 con AO/AP por 24 horas en presencia o no de EHL. Se evaluó la presencia de GL y el contenido de TG intracelulares por Oil Red O y Nile Red, respectivamente. La expresión de DGAT1/2, FABP4 y TNF-α fue evaluada por qPCR. RESULTADOS: Los hepatocitos tratados con AO/AP mostraron un aumento en las GL y TG, así como una mayor expresión de DGAT2 en comparación al control. El cotratamiento con EHL revirtió los efectos inducidos por AO/AP. CONCLUSIONES: EHL revierte el incremento en las GL, TG y en la expresión de DGAT2 inducido por AO/AP en células HepG2. Estos hallazgos sugieren un efecto hepatoprotector de la Lampaya contra la esteatosis, y apoyarían su uso complementario en el tratamiento de patologías con componente inflamatorio como la EHGNA.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease. At the cellular level, it is characterized by the accumulation of triglycerides (TG) in the form of lipid droplets (LD), which leads to steatosis and inflammation. Among relevant factors for TG synthesis are the enzymes DGAT1/2 catalyzing the final stage of TG synthesis, and the protein FABP4 which transports intracellular lipids and is expressed in cell models of obesity-dependent liver disease. Additionally, TNF-α is a cytokine involved in the inflammatory process associated to NAFDL. Lampaya medicinalis Phil. (Verbenaceae) is a plant used in folk medicine in northern Chile to treat some inflammatory diseases. OBJECTIVE: To evaluate the effect of the hydroalcoholic extract of lampaya (HEL) on steatosis and the expression of inflammatory markers in hepatocytes treated with fatty acids. Study design: In vitro study in cultures of the human HepG2 cell line treated with oleic acid (OA) and palmitic acid (PA). METHODS: HepG2 hepatocytes were incubated with OA/PA for 24 hours in the presence and absence of HEL. The formation of LD and the accumulation of intracellular TG were assessed by Oil Red O and Nile Red, respectively. The expression of DGAT1/2, FABP4 and TNF-α was assessed by qPCR. RESULTS: The treatment with OA/PA increased the levels of LD and TG as well as the expression of DGAT2 in HepG2 hepatocytes compared to control cells. HEL cotreatment counteracted OA/PA-induced effects. CONCLUSIONS: HEL prevents the increase in LD and TG levels and DGAT2 expression induced by OA/PA in HepG2 cells. These findings suggest that lampaya may have a protective effect against hepatic steatosis, which would support its complementary use in the treatment of pathologies associated with inflammation, such as NAFLD.

mRNAs biomarker related to the control of proliferation and cell death in HepG2/C3A spheroid and monolayer cultures treated with piperlongumine

Background: Cell culture (spheroid and 2D monolayer cultures) is an essential tool in drug discovery. Piperlongumine (PLN), a naturally occurring alkaloid present in the long pepper (Piper longum), has been implicated in the regulation of GSTP1 activity. In vitro treatment of cancer cells with PLN increases ROS (reactive oxygen species) levels and induces cell death, but its molecular mode of action has not been entirely elucidated. Methods: In this study, we correlated the antiproliferative effects (2D and 3D cultures) of PLN (CAS 20069­09-4, Sigma-Aldrich) with morphological and molecular analyses in HepG2/C3A cell line. We performed assays for cytotoxicity (MTT), comet assays for genotoxicity, induction of apoptosis, analysis of the cell cycle phase, and analysis of the membrane integrity by flow cytometry. Relative expression of mRNA of genes related to proliferation, apoptosis, cell cycle control, metabolism of xenobiotics, and reticulum endoplasmic stress. Results: PLN reduced the cell proliferation by the cell cycle arrest in G2/M. Changes in the mRNA expression for CDKN1A (4.9x) and CCNA2 (0.5x) of cell cycle control genes were observed. Cell death occurred due to apoptosis, which may have been induced by increased expression of proapoptotic mRNAs (BAK1, 3.1x; BBC3, 2.4x), and by an increase in 9 and 3/7 active caspases. PLN induced cellular injury by ROS generation and DNA damage. DNA damage induced MDM2 signaling (3.0x) associated with the appearance of the monastral spindle in mitosis. Genes associated with ROS degradation also showed increased mRNA expression (GSR, 2.0x; SOD1, 2.1x). PLN induce endoplasmic reticulum stress with the increase in the mRNA expression of ERN1 (4.5x) and HSPA14 (2.2x). The xenobiotic metabolism showed increased mRNA expression for CYP1A2 (2.2x) and CYP3A4 (3.4x). In addition to 2D culture, PLN treatment also inhibited the growth of 3D culture (spheroids). Conclusion: Thus, the findings of our study show that several gene expression biomarkers (mRNAs) and monastral spindle formation indicated the many pathways of damage induced by PLN treatment that contributes to its antiproliferative effects

V Brazilian consensus guidelines for detection of anti-cell autoantibodies on hep-2 cells

Abstract Background: The V Brazilian Consensus for determination of autoantibodies against cellular constituents on HEp-2 cells, held in Brasilia (DF, Brazil) on August 27, 2016, discussed the harmonization between the Brazilian Consensus on ANA (BCA) guidelines and the International Consensus on ANA Patterns (ICAP) recommendations (www.anapatterns.org). Initial guidelines were formulated by the group of Brazilian experts with the purpose of guiding and enabling Brazilian clinical laboratories to adopt recommendations and to provide a common standard for national and international consensuses. Mainbody: Twenty Brazilian researchers and experts from universities and clinical laboratories representing the various geographical regions of the country participated in the meeting. Three main topics were discussed, namely the harmonization between the BCA guidelines and latest recommendations of the ICAP initiative, the adjustment of the terminology and report on HEp-2 patterns, and a reassessment of quality assurance parameters. For the three topics, our aim was to establish specific guidelines. All recommendations were based on consensus among participants. There was concrete progress in the adjustment of the BCA guidelines to match the ICAP guidelines. To a certain extent, this derives from the fact that ICAP recommendations were largely based on the algorithm and recommendations of the IV Brazilian ANA Consensus, as consistently recognized in the ICAP publications and presentations. However, although there is great overlap between the two Consensuses, there are some point divergences. These specific items were individually and extensively discussed, and it was acknowledged that in several points ICAP improved recommendations previously issued by the Brazilian ANA Consensus and these changes were readily implemented. Regarding some specific topics, the BCA panel of experts felt that the previously issued recommendations remained relevant and possibly will require further discussion with ICAP. The term anti-cell antibodies was adopted as the recommended designation, recognizing that the assay addresses antibodies against antigens in the nucleus and in other cell compartments. However, the acronym ANA HEp-2 was maintained due to historical and regulatory reasons. It was also signalized that the latest trend in ICAP is to adopt the term Indirect Immunofluorescent Assay on HEp-2 cell substrate (HEp-2 IIFA). In addition, the quality assurance strategies previously presented were ratified and emphasized. Conclusion: The V BCA edition was successful in establishing an overall harmonization with the ICAP recommendations for interpretation of the HEp-2 IIFA test, pinpointing the perspectives in filling the remaining gaps between both initiatives.

BjussuLAAO-II induces cytotoxicity and alters DNA methylation of cell-cycle genes in monocultured/co-cultured HepG2 cells

The use of animal venoms and their toxins as material sources for biotechnological applications has received much attention from the pharmaceutical industry. L-amino acid oxidases from snake venoms (SV-LAAOs) have demonstrated innumerous biological effects and pharmacological potential against different cancer types. Hepatocellular carcinoma has increased worldwide, and the aberrant DNA methylation of liver cells is a common mechanism to promote hepatic tumorigenesis. Moreover, tumor microenvironment plays a major role in neoplastic transformation. To elucidate the molecular mechanisms responsible for the cytotoxic effects of SV-LAAO in human cancer cells, this study aimed to evaluate the cytotoxicity and the alterations in DNA methylation profiler in the promoter regions of cell-cycle genes induced by BjussuLAAO-II, an LAAO from Bothrops jaracussu venom, in human hepatocellular carcinoma (HepG2) cells in monoculture and co-culture with endothelial (HUVEC) cells. Methods: BjussuLAAO-II concentrations were 0.25, 0.50, 1.00 and 5.00 μg/mL. Cell viability was assessed by MTT assay and DNA methylation of the promoter regions of 22 cell-cycle genes by EpiTect Methyl II PCR array. Results: BjussuLAAO-II decreased the cell viability of HepG2 cells in monoculture at all concentrations tested. In co-culture, 1.00 and 5.00 μg/mL induced cytotoxicity (p < 0.05). BjussuLAAO-II increased the methylation of CCND1 and decreased the methylation of CDKN1A in monoculture and GADD45A in both cell-culture models (p < 0.05). Conclusion: Data showed BjussuLAAO-II induced cytotoxicity and altered DNA methylation of the promoter regions of cell-cycle genes in HepG2 cells in monoculture and co-culture models. We suggested the analysis of DNA methylation profile of GADD45A as a potential biomarker of the cell cycle effects of BjussuLAAO-II in cancer cells. The tumor microenvironment should be considered to comprise part of biotechnological strategies during the development of snake-toxin-based novel drugs.(AU)

Antiplasmodial and antileishmanial activities of compounds from Piper tuberculatum Jacq fruits

Abstract INTRODUCTION This study assessed the activity of compounds from Piper tuberculatum against Plasmodium falciparum and Leishmania guyanensis. METHODS The effects of compounds from P. tuberculatum fruits on P. falciparum and L. guyanensis promastigote growth in vitro were determined. Hemolytic action and cytotoxicity in HepG2 and J774 cells were measured. RESULTS Three compounds showed strong antiplasmodial activity and one compound showed strong antileishmanial activity. Two compounds were non-toxic to HepG2 cells and all were toxic to J774 cells. The compounds showed no hemolytic activity. CONCLUSIONS The tested compounds from P. tuberculatum exhibited antiparasitic and cytotoxic effects.

Efecto de la activación del receptor sensor de calcio sobre la expresión de genes lipogénicos en células HepG2 / Effect of calcium sensing receptor activation on lipogenic gene expression in HepG2 cells

Introduction: The Calcium Sensing Receptor (CaSR) is expressed in human fat cells, and its stimulation may be associated with adipose tissue dysfunction. The multisystemic character of obesity and the search of deepening the scope of the activation of CaSR in this disorder allows us to study the response of this protein in tissues that differ from adipose. Objective: To evaluate the effect of CaSR activation on the expression of lipogenic genes in a model of excess glucose and fatty acids in HepG2 human liver cells. Materials and methods: The effect of the calcimimetic cinacalcet (allosteric agonist of CaSR) on the content of triglycerides (fluorimetry) in a model of glucose supply and on the expression of lipogenic genes (qPCR) in hyperglycemia and hyperlipidemia conditions in the Liver cell line HepG2. Results: Cinacalcet, glucose (25 mM) and oleic acid (0.6 mM) did not affect cell viability. Activation of CaSR in the presence of glucose failed to increase the intracellular triglyceride content at 72 hours. Under these conditions, no response was observed for the factors coding for lipogenic genes (SREBP1c and FAS) at 24 hours of stimulation with cinacalcet in the liver cells. In the case of the over supply of fatty acids, the HepG2 cells did not show a variation in the gene expression of the DGAT enzymes after exposure to cinacalcet. Conclusion:Under conditions of glucose exposure, cinacalcet did not show a response in the triglyceride content, nor in the expression of genes related to hepatic lipogenesis. Therefore, stimulation of CaSR would not be associated with hepatic steatosis in HepG2 cells exposed to glucose.

Inducible T-cell co-stimulators regulate the proliferation and invasion of human hepatocellular carcinoma HepG2 cells

Abstract Background This study determined the regulatory effects of inducible T-cell co-stimulators (ICOS) in human hepatocellular carcinoma HepG2 cells using a RNA interference (RNAi) technique. Methods A RNAi technique was used to knockdown the expression of ICOS. ICOS expression after knockdown was detected as mRNA and protein levels by RT-PCR and Western blot, respectively. A MTT colorimetric assay was used to detect cell proliferation, and the Transwell assay was used to detect cell invasion. Western blot was carried out to detect the level of Bcl-2, AKT, and PI3K protein expression in different groups. Results The proliferation of HepG2 cells were significantly decreased after ICOS siRNA transfection (EG group). Similarly, the results of the Transwell experiment showed that invasion of HepG2 cells in the EG group was clearly reduced compared to the negative control (NC) and blank control groups (CON). Western blot analysis showed that knockdown of ICOS expression reduced the levels of Bcl-2 and AKT, and also significantly up-regulated the level of PI3K phosphorylation (P < 0.01). Conclusion Down-regulating ICOS expression in HepG2 cells suppressed cell proliferation and invasion. The underlying mechanism may be related to the expression of the downstream factor, PI3K/AKT.

Low density lipoprotein modified silica nanoparticles loaded with docetaxel and thalidomide for effective chemotherapy of liver cancer

In the present study, we successfully developed a docetaxel (DTX) and thalidomide (TDD) co-delivery system based on low density lipoprotein (LDL) modified silica nanoparticles (LDL/SLN/DTX/TDD). By employing the tumor homing property of LDL and the drug-loading capability of silica nanoparticles, the prepared LDL/SLN/DTX/TDD was expected to locate and specifically deliver the loaded drugs (DTX and TDD) to achieve effective chemotherapy of liver cancer. In vitro analysis revealed that nano-sized LDL/SLN/DTX/TDD with decent drug loading capabilities was able to increase the delivery efficiency by targeting the low density lipoprotein receptors, which were overexpressed on HepG2 human hepatocellular liver carcinoma cell line, which exerted better cytotoxicity than unmodified silica nanoparticles and free drugs. In vivo imaging and anti-cancer assays also confirmed the preferable tumor-homing and synergetic anti-cancer effects of LDL/SLN/DTX/TDD.

The hepatic ectonucleotide pyrophosphatase/phosphodiesterase 1 gene mRNA abundance is reduced by insulin and induced by dexamethasone

Hormones regulate hepatic gene expressions to maintain metabolic homeostasis. Ectonucleotide pyrophosphatase/phosphodiesterase 1 has been thought to interfere with insulin signaling. To determine its potential role in the regulation of metabolism, we analyzed its gene (Enpp1) expression in the liver of rats experiencing fasting and refeeding cycles, and in primary rat hepatocytes and human hepatoma HepG2 cells treated with insulin and dexamethasone using northern blot and real-time PCR techniques. Hepatic Enpp1 expression was induced by fasting and reduced by refeeding in the rat liver. In primary rat hepatocytes and HepG2 hepatoma cells, insulin reduced Enpp1 mRNA abundance, whereas dexamethasone induced it. Dexamethasone disrupted the insulin-reduced Enpp1 expression in primary hepatocytes. This is in contrast to the responses of the expression of the cytosolic form of phosphoenolpyruvate carboxykinase gene to the same hormones, where insulin reduced it significantly in the process. In addition, the dexamethasone-induced Enpp1 gene expression was attenuated in the presence of 8-Br-cAMP. In conclusion, we demonstrated for the first time that hepatic Enpp1 is regulated in the cycle of fasting and refeeding, a process that might be attributed to insulin-reduced Enpp1 expression. This insulin-reduced Enpp1 expression might play a role in the development of complications in diabetic patients.

Antiproliferative effect of urolithin A, the ellagic acid-derived colonic metabolite, on hepatocellular carcinoma HepG2. 2. 15 cells by targeting Lin28a/let-7a axis

An abnormality in the Lin28/let-7a axis is relevant to the progression of hepatitis B virus (HBV)-positive hepatocellular carcinoma (HCC), which could be a novel therapeutic target for this malignant tumor. The present study aimed to investigate the antiproliferative and anti-invasive effects of urolithin A in a stable full-length HBV gene integrated cell line HepG2.2.15 using CCK-8 and transwell assays. The RNA and protein expressions of targets were assessed by quantitative PCR and western blot, respectively. Results revealed that urolithin A induced cytotoxicity in HepG2.2.15 cells, which was accompanied by the cleavage of caspase-3 protein and down-regulation of Bcl-2/Bax ratio. Moreover, urolithin A suppressed the protein expressions of Sp-1, Lin28a, and Zcchc11, and elevated the expression of microRNA let-7a. Importantly, urolithin A also regulated the Lin28a/let-7a axis in transient HBx-transfected HCC HepG2 cells. Furthermore, urolithin A decelerated the HepG2.2.15 cell invasion, which was involved in suppressing the let-7a downstream factors HMGA2 and K-ras. These findings indicated that urolithin A exerted the antiproliferative effect by regulating the Lin28a/let-7a axis and may be a potential supplement for HBV-infected HCC therapy.

Efecto mutagénico y genotóxico, y expresión de los genes Rad51C , Xiap , P53 y Nrf2 inducidos por extractos antipalúdicos de plantas recolectadas en el Vaupés medio, Colombia / Mutagenicity, genotoxicity and gene expression of Rad51C, Xiap, P53 and Nrf2 induced by antimalarial extracts of plants collected from the middle Vaupés region, Colombia

Resumen Introducción. Dada la resistencia de Plasmodium a los medicamentos antipalúdicos, es necesario encontrar nuevas alternativas terapéuticas para su tratamiento y control. Con base en el saber indígena colombiano, se recopilaron extractos de plantas del Vaupés medio con potencial efecto antipalúdico. Objetivo. Evaluar el efecto mutagénico y genotóxico, y la expresión de los genes Rad51C, Xiap, P53 yNrf2, inducidos por cuatro extractos etanólicos con actividad anti-Plasmodium(R001, T002, T015 y T028). Materiales y métodos. Se evaluó el potencial mutagénico de cuatro extractos etanólicos con efecto antiplasmódico utilizando el test de Ames y el efecto genotóxico, con un ensayo del cometa; asimismo, se analizó la expresión de los genes Rad51C, Xiap, P53 y Nrf2 en células HepG2. Resultados. Los extractos no fueron mutágenos en la cepa TA98 de Salmonella typhimurium en presencia y ausencia de actividad metabólica de la fracción S9. En la cepa TA100, los extractos R001, T015 y T028 se comportaron como mutágenos débiles en presencia de S9, con índices mutagénicos de 1,58; 1,38; 1,53 y 1,61, respectivamente; T015 tuvo el mismo comportamiento en ausencia de S9, con un índice mutagénico de 1,36. En el ensayo del cometa, todos los extractos provocaron daño de categorías 1 o 2, con colas de cometas entre 36,7 y 51,48 µm de longitud; sin embargo, el índice dedaño genético sugirió que los tratamientos afectaron la mayoría de las células. En los genes en estudio, los extractos R001 y T028 indujeron una sobreexpresiónde 1,84 a 3,99 frente a las células sin tratar de los genes Xiap y P53. Conclusiones. Los resultados evidenciaron que el extracto T002 fue el más seguro, ya que presentó actividad anti-Plasmodium, no fue citotóxico en las células HepG2, no fue mutágeno, causó daño de categoría 1 en el ADN y no modificó la expresión de los genes evaluados.

Abstracts Introduction: Due to Plasmodium resistance to antimalarial drugs, it is important to find new therapeutic alternatives for malaria treatment and control. Based on the knowledge of Colombian indigenous communities, we collected extracts of plants with potential antimalarial effects from the middle Vaupés region. Objective: To evaluate the mutagenic and genotoxic effects, as well as the gene expression of Rad51C, Xiap, P53 and Nrf2 induced by four ethanolic extracts with antimalarial activity (R001, T002, T015 and T028). Materials and methods: We evaluated four ethanolic extracts with antimalarial activity using the Ames test to assess mutagenicity, and the comet assay on HepG2 cells to determine the genotoxicicity. We also evaluated the expression of Rad51C, Xiap, P53 and Nrf2 from HepG2 cells stimulated with the four extracts. Results: None of the four extracts was mutagenic in Salmonella typhimurium TA98 strain in the presence and absence of S9 metabolic activity. Extracts R001, T015 and T028 were weakly mutagenic on the TA100 strain in the presence of S9, with mutagenic indexes (MI) of 1.58, 1.53 and 1.61, respectively. The T015 strain showed the same behavior without S9 with an MI of 1.36. The results of the comet assay showed that the four extracts produced category 1 or 2 damage, with comets between 36.7 and 51.48 µm in length. However, the genetic damage index suggested that most of the cells were affected by the treatments. Regarding gene expression, extracts R001 and T028 induced an overexpression of genes Xiap and P53 with an 1.84 to 3.99 fold-change compared with untreated cells. Conclusions: These results revealed that the T002 extract was the safest as it had antimalarial activity and was not cytotoxic on HepG2 cells. Moreover, it was not mutagenic and it only produced category 1 damage on the DNA. Also, the extract did not induce a change in the expression of the tested genes.

Subversion of immunoproteasome subunit expression in dengue virus serotype 2-infected HepG2 cells

Abstract: INTRODUCTION: Infection with all serotypes of dengue virus (DV) results in augmented antigen presentation by MHC class I molecules. However, the upregulation of immunoproteasome subunits only results from infection with two serotypes. This study aims to elucidate changes in the expression of immunoproteasome subunits resulting from infection with DV, particularly DV serotype 2 (DV2). METHODS: HepG2 cells were grown in various culture milieu. Total cellular RNA and proteins were extracted and quantified. RESULTS: Results demonstrated sequestration of immunoproteasome subunits LMP2 and LMP7 in DV2-infected cells. CONCLUSIONS: This study provides insights into the mechanisms underlying immune evasion by DV.

Evaluation of the antiplasmodial and leishmanicidal potential of Myrciaria dubia (Myrtaceae) extract

Abstract INTRODUCTION: Malaria and leishmaniasis are prevalent in tropical regions, which have environmental characteristics that are highly favorable to protozoa and vectors of these diseases; the transmission of these infections in sub-tropical regions, although recognized, represents only a small fraction of cases. Plants are constantly being used in the search for and acquisition of new drugs, and many compounds derived from them have been used to combat various diseases. In this study, we evaluated the action of the dichloromethanolic extract of Myrciaria dubia leaves against the protozoa Plasmodium falciparum, Leishmania amazonensis, Leishmania braziliensis, and Leishmania chagasi through bioassays. METHODS The extract from M. dubia was tested for its anti-P. falciparum activity in an anti-histidine-rich protein II immunosorbent assay. The antileishmanial assays were performed using the resazurin method, while cytotoxicity against human hepatoma (HepG2) strain was determined using the colorimetric MTT [3-(4, 5-dimethyl-2- thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide] method. RESULTS The M. dubia extract presented a half-maximal inhibitory concentration equal to 2.35 (1.05)μg/mL for P. falciparum, 190.73 (6.41) μg/mL for L. amazonensis, and greater than equal to 200µg/mL for L. chagasi and L. braziliensis strains. The cytotoxic concentration for 50% of the cells was above 500μg/mL for HepG2, indicating no toxicity and greater selectivity against parasites. CONCLUSIONS The results obtained indicate the presence of antiplasmodial and leishmanicidal bioactive compounds in the dichloromethanolic extracts of M. dubia leaves, and point towards future studies to elucidate the mechanism of action for each physiological effect.

miR-205 promotes proliferation and invasion of laryngeal squamous cell carcinoma by suppressing CDK2AP1 expression

BACKGROUND: The aberrant expression of microRNAs (miRNAs) has been found in various types of cancer. miR-205 was reported to be upregulated in laryngeal squamous cell carcinoma (LSCC) tissues, however, the mechanisms by which miR-205 functions as a regulator of LSCC are largely unknown. RESULTS: In this study, Real-time qPCR and Western blot assay showed that expression of miR-205 was upregulated and expression of cyclin-dependent kinase 2-associated protein 1 (CDK2AP1) was downregulated in LSCC tissues. The expression levels of miR-205 were negatively related to those of CDK2AP1 in LSCC tissues and cell lines. Moreover, we found that miR-205 was the upstream regulator of CDK2AP1 and could suppress the CDK2AP1 expression in LSCC cells. 3-(4,5-dimethylthiazal-2-yl)-2,5-diphenyl-tetrazolium bromide assays and transwell invasion assay were performed to test the proliferation and invasion of LSCC cells. Gelatin zymography was used to detect the activity of MMP2 and MMP9. CDK2AP1, c-Myc and CyclinD1 expression in cells was assessed with Western blotting. We found that miR-205 was the upstream regulator of CDK2AP1 and could suppress the expression of CDK2AP1 in LSCC cells. In addition, miR-205 significantly induced cell proliferation and invasion by suppressing CDK2AP1 expression. Consistent with miR-205 inhibitors, overexpressed CDK2AP1 suppressed the activity of MMP2 and MMP9 and c-Myc and CyclinD1 expression in LSCC cells. CONCLUSION: These findings help us to better elucidate the molecular mechanisms of LSCC progression and provide a new theoretical basis to further investigate miR-205 as a potential biomarker and a promising approach for LSCC treatment.