RESUMO
Ginkgo biloba (EGb761) is a widely used botanical drug. Several reports indicate that EGb761 confers preventive as well as anti-tumorigenic properties in a variety of tumors, including hepatocellular carcinoma (HCC). We here evaluate functional effects and molecular alterations induced by EGb761 in hepatoma cells and non-malignant hepatocytes. Hepatoma cell lines, primary human HCC cells and immortalized human hepatocytes (IH) were exposed to various concentrations (0-1000 µg/ml) of EGb761. Apoptosis and proliferation were evaluated after 72h of EGb761 exposure. Response to oxidative stress, tumorigenic properties and molecular changes were further investigated. While anti-oxidant effects were detected in all cell lines, EGb761 promoted anti-proliferative and pro-apoptotic effects mainly in hepatoma cells. Consistently, EGb761 treatment caused a significant reduction in colony and sphere forming ability in hepatoma cells and no mentionable changes in IH. Transcriptomic changes involved oxidative stress response as well as key oncogenic pathways resembling Nrf2- and mTOR signaling pathway. Taken together, EGb761 induces differential effects in non-transformed and cancer cells. While treatment confers protective effects in non-malignant cells, EGb761 significantly impairs tumorigenic properties in cancer cells by affecting key oncogenic pathways. Results provide the rational for clinical testing of EGb761 in preventive and therapeutic strategies in human liver diseases.
Assuntos
Carcinogênese/efeitos dos fármacos , Ginkgo biloba/química , Extratos Vegetais/farmacologia , Transcriptoma/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Ginkgo biloba/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/química , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
Organ fibrosis is characterized by a chronic wound-healing response, with excess deposition of extracellular matrix components. Here, collagen type I represents the most abundant scar component and a primary target for antifibrotic therapies. Liver fibrosis can progress to cirrhosis and primary liver cancer, which are the major causes of liver related morbidity and mortality. However, a (pro-)collagen type I specific therapy remains difficult and its therapeutic abrogation may incur unwanted side effects. We therefore designed tetracycline-regulated procollagen alpha1(I) short hairpin (sh)RNA expressing mice that permit a highly efficient inducible knockdown of the procollagen alpha1(I) gene in activated (myo-)fibroblasts, to study the effect of induced procollagen type I deficiency. Transgenic mice were generated using recombinase-mediated integration in embryonic stem cells or zinc-finger nuclease-aided genomic targeting combined with miR30-shRNA technology. Liver fibrosis was induced in transgenic mice by carbon tetrachloride, either without or with doxycycline supplementation. Doxycycline treated mice showed an 80-90% suppression of procollagen alpha1(I) transcription and a 40-50% reduction in hepatic collagen accumulation. Procollagen alpha1(I) knockdown also downregulated procollagens type III, IV and VI and other fibrosis related parameters. Moreover, this was associated with an attenuation of chronic inflammation, suggesting that collagen type I serves not only as major scar component, but also as modulator of other collagens and promoter of chronic inflammation.
Assuntos
Colágeno Tipo I/genética , Proteínas da Matriz Extracelular/genética , Cirrose Hepática/prevenção & controle , Pró-Colágeno/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Cirrose Hepática/genética , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/farmacologiaRESUMO
Although allergen-specific immunotherapy is a clinically effective therapy for IgE-mediated allergic diseases, the risk of IgE-mediated adverse effects still exists. For this reason, chemically modified allergoids have been introduced, which may destroy IgE-binding sites while T-cell activation should be retained. The aim of the study was to analyse the differences between intact allergens and differently modified/aggregated allergoids concerning their internalization as well as T-cell and basophil activation. For this purpose human monocyte-derived immature dendritic cells (DC) were incubated with Phleum pratense or Betula verrucosa pollen extract or with the corresponding allergoids, modified with formaldehyde or glutaraldehyde. After an additional maturation process, the antigen-loaded mature DC were co-cultured with autologous CD4(+) T cells. Allergenicity was tested by leukotriene release from basophils. In addition, the uptake of intact allergens and allergoids by immature DC was analysed. The proliferation of, as well as the interleukin-4 (IL-4), IL-10, IL-13 and interferon-γ production by, CD4(+) T cells which had been stimulated with glutaraldehyde allergoid-treated DC was reduced compared with CD4(+) T cells stimulated with intact allergen-treated or formaldehyde allergoid-treated DC. In line with this, glutaraldehyde-modified allergoids were more aggregated and were internalized more slowly. Furthermore, only the allergoids modified with glutaraldehyde induced a decreased leukotriene release by activated basophils. These findings suggest that IgE-reactive epitopes were destroyed more efficiently by modification with glutaraldehyde than with formaldehyde under the conditions chosen for these investigations. Glutaraldehyde-modified allergoids also displayed lower T-cell stimulatory capacity, which is mainly the result of greater modification/aggregation and diminished uptake by DC.