RESUMO
CONTEXT: Fitzroya cupressoides (Molina) I. M. Johnst. and Austrocedrus chilensis (D. Don) Pic.Serm. & Bizzarri are two Chilean Cupressaceae that are naturally resistant to biodegradation. Secondary metabolites from these species display a variety of biological activities. OBJECTIVE: To evaluate the antiproliferative activity of two lignans, a diterpene and a flavonol isolated from A. chilensis and F. cupressoides, to elucidate their cytological effects on P3X murine myeloma cells. MATERIALS AND METHODS: The antiproliferative activity of yatein, isotaxiresinol, ferruginol, and isorhamnetin was evaluated in vitro using the MTT assay. The effect of yatein at the cellular level, due to its high antiproliferative activity was evaluated. P3X cells treated for 24 h with 12.5 and 25 µg/mL of yatein were also examined at the cytological level using immunofluorescence and scanning and transmission electron microscopy. RESULTS: Yatein, a lignan isolated from A. chilensis, potentially inhibited P3X murine myeloma cell proliferation, resulting in approximately 75% cell death in response to a 25 µg/mL treatment with the lignan. P3X cells lost membrane integrity at the nuclear and cytoplasmic levels, including organelles, in response to yatein treatment (12.5 µg/mL), and we observed changes in the cytoplasmic organization and distribution of microtubules. The other compounds tested had low activity. DISCUSSION AND CONCLUSIONS: Yatein is a lignan precursor of podophyllotoxin, a key agent in anticancer drugs. Due to its structural similarities to podophyllotoxin, yatein could have similar cytoplasmic target(s), such as the microtubular apparatus. These findings suggest that yatein may be of potential pharmacological interest and warrants further investigation in human cell lines.
Assuntos
4-Butirolactona/análogos & derivados , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Cupressaceae , Dioxóis/farmacologia , 4-Butirolactona/isolamento & purificação , 4-Butirolactona/farmacologia , 4-Butirolactona/uso terapêutico , Animais , Antineoplásicos/isolamento & purificação , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Dioxóis/isolamento & purificação , Dioxóis/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB CRESUMO
High frequencies of stem-like memory T cells in infusion products correlate with superior patient outcomes across multiple T cell therapy trials. Herein, we analyzed a published CRISPR activation screening to identify transcriptional regulators that could be harnessed to augment stem-like behavior in CD8+ T cells. Using IFN-γ production as a proxy for CD8+ T cell terminal differentiation, LMO4 emerged among the top hits inhibiting the development of effectors cells. Consistently, we found that Lmo4 was downregulated upon CD8+ T cell activation but maintained under culture conditions facilitating the formation of stem-like T cells. By employing a synthetic biology approach to ectopically express LMO4 in antitumor CD8+ T cells, we enabled selective expansion and enhanced persistence of transduced cells, while limiting their terminal differentiation and senescence. LMO4 overexpression promoted transcriptional programs regulating stemness, increasing the numbers of stem-like CD8+ memory T cells and enhancing their polyfunctionality and recall capacity. When tested in syngeneic and xenograft tumor models, LMO4 overexpression boosted CD8+ T cell antitumor immunity, resulting in enhanced tumor regression. Rather than directly modulating gene transcription, LMO4 bound to JAK1 and potentiated STAT3 signaling in response to IL-21, inducing the expression of target genes (Tcf7, Socs3, Junb, and Zfp36) crucial for memory responses. CRISPR/Cas9-deletion of Stat3 nullified the enhanced memory signature conferred by LMO4, thereby abrogating the therapeutic benefit of LMO4 overexpression. These results establish LMO4 overexpression as an effective strategy to boost CD8+ T cell stemness, providing a new synthetic biology tool to bolster the efficacy of T cell-based immunotherapies.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Linfócitos T CD8-Positivos , Proteínas com Domínio LIM , Fator de Transcrição STAT3 , Transdução de Sinais , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/imunologia , Linfócitos T CD8-Positivos/imunologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT3/metabolismo , Camundongos , Animais , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Humanos , Transdução de Sinais/imunologia , Transdução de Sinais/genética , Interleucinas/genética , Interleucinas/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologiaRESUMO
In this in vitro study, for the first time was evaluated the antioxidant and anti-inflammatory effect of an Oleuropein-enriched extract (OleE) on bovine mammary epithelial cell line (BME-UV1). OleE was obtained from olives leaves and characterized by HPLC and NMR analysis. Cell viability test indicated that OleE at concentrations of 7.8 up to 250 µg/mL did not exert cytotoxic effect. At concentration of 31.2 up to 250 µg/mL, a dose dependent reduction of ROS production induced by hydrogen peroxide was observed. In addition, OleE at 62.5, 125 and 250 µg/mL showed a dose-dependent reduction in gene expression of TNF, IL1B, and IL10 after exposure to LPS. The downregulation of ROS production and cytokines expression in BME-UV1 by OleE confirmed the antioxidant and anti-inflammatory properties. In vivo experiments will be necessary for future applications of OleE as natural feed supplement in dairy cattle to reduce incidence of oxidative stress in peripartal period.
Assuntos
Antioxidantes , Olea , Bovinos , Animais , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Linhagem Celular , Espécies Reativas de Oxigênio/metabolismo , Células Epiteliais , Anti-Inflamatórios/farmacologia , Extratos Vegetais/farmacologia , Extratos Vegetais/metabolismoRESUMO
Pomegranate peel extracts prepared in our laboratories from a waste of juice fruit processing were tested on bovine peripheral blood mononuclear cells to evaluate the effects on viability, oxidative stress and proliferation. The (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay pointed out that the extracts were not cytotoxic at the tested concentrations (0.1, 1.0, and 10 µg/mL). A moderate protective effect against Reactive Oxygen Species production induced by hydrogen peroxide or lipopolysaccharide and a significant anti-proliferative activity against proliferation induced by concanavalin A were observed on cell lines treated with the extracts at 10 µg/mL. Based on these results, pomegranate peel extracts seem promising as feed supplement for dairy cattle, in particular around calving, when the animals are subjected to an increase of the metabolic activity, responsible for oxidative stress and diseases. However, in vivo studies are needed to investigate the stability of the extracts across the bovine gastrointestinal tract barrier.
Assuntos
Leucócitos Mononucleares/citologia , Extratos Vegetais/farmacologia , Punica granatum/química , Animais , Antioxidantes/farmacologia , Bovinos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Concanavalina A/farmacologia , Frutas/química , Peróxido de Hidrogênio/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/químicaRESUMO
Pomegranate peel extracts (PPE) were tested for the first time on BME-UV1, a valid cellular model to study the bovine mammary epithelial metabolism, to evaluate the effects on the oxidative stress and inflammatory status. Based on the statistical analysis of MTT data, PPE at 0.1, 1.0 and 10 µg/mL resulted not cytotoxic after 24 h, 48 h and 7 days of treatment. At the same concentrations, PPE induced a reduction of ROS production elicited by the addition of hydrogen peroxide or lipopolysaccharide evidencing an antioxidant effect confirmed also by a decrease of malondialdehyde. At 10 µg/mL, PPE reduced pro-inflammatory cytokines expressions showing an anti-inflammatory effect on BME-UV1 treated with lipopolysaccharide. Although in vivo experiments are necessary, the results of this study are promising for future applications of PPE as feed supplement for dairy cattle, in particular around calving, when the animals are more subject to oxidative stress and inflammatory diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Glândulas Mamárias Animais/citologia , Extratos Vegetais/farmacologia , Punica granatum/química , Animais , Bovinos , Linhagem Celular , Citocinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Feminino , Frutas/química , Peróxido de Hidrogênio/farmacologia , Lipopolissacarídeos/farmacologia , Malondialdeído/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Lavandin essential oil (LEO), a natural sterile hybrid obtained by crossbreeding L. angustifolia × L. latifolia, is mainly composed by active components belonging to the family of terpenes endowed with relevant anti-proliferative activity, which can be enhanced by proper application of nanotechnology. In particular, this study reports the chemical characterization and the screening of the anti-proliferative activity on different human cell lines of pure and nano-formulated lavandin essential oil (EO). LEO and its formulation (NanoLEO) were analyzed by HS/GC-MS (Headspace/Gas Chromatography-Mass Spectrometry) to describe and compare their chemical volatile composition. The most abundant compounds were linalool and 1,8-cineole (LEO: 28.6%; 27.4%) (NanoLEO: 60.4%; 12.6%) followed by α-pinene (LEO: 9.6%; NanoLEO: 4.5%), camphor (LEO: 6.5%; NanoLEO: 7.0%) and linalyl acetate (LEO: 6.5%; NanoLEO: 3.6%). The cytotoxic effects of LEO and NanoLEO were investigated on human neuroblastoma cells (SHSY5Y), human breast adenocarcinoma cells (MCF-7), human lymphoblastic leukemia cells (CCRF CEM), human colorectal adenocarcinoma cells (Caco-2) and one normal breast epithelial cell (MCF10A) by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)-assay. Caco-2, MCF7 and MCF10A normal cells resulted more resistant to the treatment with LEO, while CCRF-CEM and SHSY5Y cells were more sensitive. The antiproliferative effect of LEO resulted amplified when the essential oil was supplied as nanoformulation, mainly in Caco-2 cells. Scanning and transmission electron microscopy investigations were carried out on Caco-2 cells to outline at ultrastructural level possible affections induced by LEO and NanoLEO treatments.
RESUMO
The aim of this study was to determine the anti-inflammatory potential of pomegranate peel extracts (PPE) prepared from waste material of pomegranate juice production both in vitro on Caco-2 cells and ex vivo using porcine colonic tissue explants. Caco-2 cells were stimulated in vitro by TNF and colonic tissue explants were stimulated ex vivo with lipopolysaccharide (LPS). Both tissues were co-treated with PPE at 0, 1.0, 2.5, 5.0, 10 and 25 µg/mL. The secretion of CXCL8 in the supernatant of both experiments was determined by enzyme linked immunosorbent assay (ELISA) and the relative expression of inflammatory cytokines were evaluated in the colonic tissue by quantitative polymerase chain reaction (QPCR). The 2.5 to 25 µg/mL of PPE suppressed CXCL8 (p < 0.001) in the Caco-2 cells, whereas CXCL8 production was suppressed by only 5 and 25 µg/mL (p < 0.01) of PPE in the colonic explants. The 5 µg/mL of PPE also suppressed the expression of IL1A (p < 0.05), IL6 (p < 0.01) and CXCL8 (p < 0.05) in LPS challenged colonic tissues compared to controls. In conclusion, the 5 µg/mL of PPE consistently elicits strong anti-inflammatory activity. These results support the potential of bioactive compounds from the waste peel of pomegranate in terms of their anti-inflammatory activity in cells and tissues of the gastrointestinal tract.
Assuntos
Anti-Inflamatórios/farmacologia , Frutas/química , Lythraceae , Extratos Vegetais/farmacologia , Animais , Células CACO-2/efeitos dos fármacos , Colo/efeitos dos fármacos , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Interleucina-8/metabolismo , Lipopolissacarídeos , Reação em Cadeia da Polimerase , SuínosRESUMO
Ethanolic extracts of Salvia officinalis leaves demonstrated antibacterial activity against Bacillus cereus. Fractionation of the extracts led to the isolation of the most active antibacterial compound, which, from spectroscopic and LC-MS evidence, was proved to be the diterpene, methyl carnosate.