RESUMO
TGF-ß1 plays a crucial role in the pathogenesis of vascular fibrotic diseases. Chalcones are reportedly cancer chemo-preventive food components that are rich in fruits and vegetables. In this study, flavokawain A (FKA, 2-30 µM), a naturally occurring chalcone in kava extracts, was evaluated for its anti-fibrotic and antioxidant properties in TGF-ß1-stimulated vascular smooth muscle (A7r5) cells, as well as its underlying molecular mechanism of action. Immunofluorescence data showed down-regulated F-actin expression with FKA treatment in TGF-ß1-stimulated A7r5 cells. Western blotting demonstrated that FKA treatment suppressed the expression of α-SMA and fibronectin proteins under TGF-ß1 stimulation. Findings from wound-healing and invasion experiments showed that FKA inhibits TGF-ß1-mediated migration and invasion. Western blotting demonstrated that treatment with FKA down-regulated MMP-9 and MMP-2 and up-regulated TIMP-1 expression. Further evidence showed that FKA decreased TGF-ß1-mediated phosphorylation and the transcriptional activity of Smad3. TGF-ß1-induced excessive ROS production was remarkably reversed by FKA treatment in A7r5 cells, and inhibition by FKA or N-acetylcysteine (NAC) substantially diminished TGF-ß1-induced p-Smad3 activation and wound-healing migration. Interestingly, FKA-mediated antioxidant properties were associated with increased nuclear translocation of Nrf2 and elevated antioxidant response element (ARE) luciferase activity. Activation of Nrf2/ARE signaling was accompanied by the induction of HO-1, NQO-1 and γ-GCLC genes in FKA-treated A7r5 cells. Notably, silencing of Nrf2 (siRNA transfection) significantly diminished the FKA-mediated antioxidant effects, indicating that FKA may inhibit TGF-ß1-induced fibrosis through suppressing ROS generation in A7r5 cells. Our results suggested that anti-fibrotic and antioxidant activities of the chalcone flavokawain A may contribute to the development of food-based chemo-preventive drugs for fibrotic diseases.
Assuntos
Antioxidantes/farmacologia , Chalcona/análogos & derivados , Miócitos de Músculo Liso/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/genética , Espécies Reativas de Oxigênio/antagonistas & inibidores , Proteína Smad3/genética , Actinas/genética , Actinas/metabolismo , Animais , Elementos de Resposta Antioxidante/efeitos dos fármacos , Aorta/citologia , Aorta/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Chalcona/farmacologia , Fibronectinas/genética , Fibronectinas/metabolismo , Fibrose/prevenção & controle , Regulação da Expressão Gênica , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Modelos Biológicos , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Proteína Smad3/antagonistas & inibidores , Proteína Smad3/metabolismo , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
We reported in our previously executed studies that the fermented culture broth of Antrodia salmonea (AS), a mushroom used in Taiwanese folk medicine induced reactive oxygen species (ROS)-mediated apoptosis in human ovarian carcinoma cells. In this study, we studied the anticancer efficacies of AS (0-240 µg/ml) by examining the key molecular events implicated in cell death associated with autophagy in SKOV-3 and A2780 human ovarian carcinoma cells and clarified the fundamental molecular mechanisms. Treatment of ovarian carcinoma cells with AS-induced autophagic cell death mediated by increased microtubule-associated protein LC3-II, GFP-LC3 puncta, and acidic vesicular organelle (AVO) formation. These events are linked with the activation of p62/SQSTM1, the inhibition of ATG4B, the expression of ATG7, and the dysregulation of Beclin-1/Bcl-2 (i.e., B-cell lymphoma 2). N-acetylcysteine inhibited AS-induced ROS generation, which in turn constricted AS-induced LC3 conversion, AVO formation, and ATG4B inhibition, indicating ROS-mediated autophagy cell death. In addition, the 3-methyladenine (3-MA) or chloroquine (CQ)-induced autophagy inhibition decreased AS-induced apoptosis. Additionally, apoptosis inhibition by Z-VAD-FMK, a pan-caspase inhibitor, substantially suppressed AS-induced autophagy. Furthermore, AS-inhibited HER-2/ neu and PI3K/AKT signaling pathways which were reversed by autophagy inhibitors 3-MA and CQ. Thus, A. salmonea is a potential chemopreventive agent that is capable of activating ROS-mediated autophagic cell death in ovarian carcinoma cells.
Assuntos
Antineoplásicos/farmacologia , Antrodia , Morte Celular Autofágica/efeitos dos fármacos , Carcinoma Epitelial do Ovário/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Antineoplásicos/isolamento & purificação , Antrodia/química , Apoptose/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/metabolismo , Carcinoma Epitelial do Ovário/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/metabolismo , Transdução de SinaisRESUMO
Antrodia salmonea is well known in Taiwan as a traditional Chinese medicinal fungus and has demonstrated antioxidant, anti-inflammatory, and anticancer effects. However, the anticancer activity of A. salmonea against human ovarian cancer is still elusive. Therefore, we investigated the antiovarian tumor activity of a fermented culture broth of A. salmonea and exhibits its underlying molecular mechanism. A. salmonea shows a significant effect on cell viability in human ovarian carcinoma (SKOV-3 or A2780) cell lines with an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Increased terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells and annexin V-propidium iodide stained cells indicate that A. salmonea induces late apoptosis in SKOV-3 cells. Notably, treatment with A. salmonea induced the following events: Apoptosis; caspase-3, -8, -9 and poly(ADP-ribose) polymerase activation; first apoptosis signal (Fas) and Fas ligand activation; Bid cleavage; and Bax2-B-cell lymphoma 2 dysregulation. The results show that A. salmonea-induced apoptosis was mediated by both mitochondrial and death receptor pathways. An increase in intracellular reactive oxygen species (ROS) was also observed in A. salmonea-treated cells, whereas the antioxidant N-acetylcysteine (NAC) prevented A. salmonea-induced cell death and DNA fragmentation, indicating that A. salmonea-induced apoptosis was mediated by ROS generation. Interestingly, A. salmonea-induced apoptosis is associated with the suppression of human epidermal growth factor receptor-2 (HER-2/neu) and phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) expression in HER-2/neu overexpressing SKOV-3 cells. NAC significantly prevented A. salmonea-induced HER-2/neu depletion and PI3K/AKT inactivation, indicating that A. salmonea-triggered apoptosis is mediated by ROS-inhibited HER-2/neu signaling cascades. To our knowledge, this is the first report describing the anticancer activity of this potentially beneficial mushroom against human ovarian carcinoma.
Assuntos
Antrodia/química , Carcinoma/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Receptor ErbB-2/genética , Acetilcisteína/farmacologia , Antioxidantes/metabolismo , Apoptose/genética , Carcinoma/genética , Carcinoma/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Medicina Tradicional Chinesa , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genéticaRESUMO
Chalcone flavokawain B (FKB) possesses a chemopreventive and anti-cancer activity. Doxorubicin is a chemotherapeutic DNA intercalating agent widely used in malignancy treatment. The present study investigated whether synergistic effects exist between the combination of FKB (1.25-5 µg/mL) and doxorubicin (0.5 µg/mL) on the apoptosis and autophagy in human gastric cancer (AGS) cells, and the possible in vitro and in vivo mechanisms. The MTT assay measured cell viability. Various apoptotic-, autophagy-associated protein expression was determined by the Western blot technique. FKB+doxorubicin synergy was estimated by the Chou-Talalay combination index (CI) method. In vivo studies were performed on BALB/c mice. Results showed that compared to FKB/doxorubicin treatments, low doses of FKB+doxorubicin suppressed AGS cell growth. FKB potentiated doxorubicin-induced DNA fragmentation, apoptotic cell death, and enhanced doxorubicin-mediated mitochondrial, death receptor pathways. FKB+doxorubicin activated increased LC3-II accumulation, p62/SQSTM1 expression, and AVO formation as compared to the FKB/doxorubicin alone treatments indicating autophagy in these cells. The death mechanism in FKB+doxorubicin-treated AGS cells is due to the activation of autophagy. FKB+doxorubicin-mediated dysregulated Bax/Bcl-2, Beclin-1/Bcl-2 ratios suggested apoptosis, autophagy induction in AGS cells. FKB+doxorubicin-induced LC3-II/AVOs downregulation was suppressed due to an apoptotic inhibitor Z-VAD-FMK. Whereas, 3-methyladenine/chloroquine weakened FKB+doxorubicin-induced apoptosis (decreased DNA fragmentation/caspase-3). Activation of ERK/JNK may be involved in FKB+doxorubicin-induced apoptosis and autophagy. FKB+doxorubicin-triggered ROS generation, but NAC attenuated FKB+doxorubicin-induced autophagic (LC3 accumulation) and apoptotic (caspase-3 activation and PARP cleavage) cell death. FKB+doxorubicin blocked gastric cancer cell xenografts in nude mice in vivo as compared to FKB/doxorubicin alone treatments. FKB and doxorubicin wielded synergistic anti-tumor effects in gastric cancer cells and is a promising therapeutic approach.
RESUMO
BACKGROUND: UVA irradiation-induced skin damage/photoaging is associated with redox imbalance and collagen degradation. OBJECTIVE: Dermato-protective efficacies of trans-cinnamic acid (t-CA), a naturally occurring aromatic compound have been investigated against UVA irradiation, and elucidated underlying molecular mechanism. METHODS: Human foreskin fibroblast-derived (Hs68) cells and nude mice were treated with t-CA prior to UVA exposure, and assayed the anti-photoaging effects of t-CA. RESULTS: We found t-CA (20-100⯵M) pretreatment substantially ameliorated UVA (3â¯J/cm2)-induced cytotoxicity, and inhibited intracellular ROS production in Hs68 cells. UVA-induced profound upregulation of metalloproteinase (MMP)-1/-3 and degradation of type I procollagen in dermal fibroblasts were remarkably reversed by t-CA, possibly through inhibition of AP-1 (c-Fos, but not c-Jun) translocation. The t-CA-mediated anti-photoaging properties are associated with increased nuclear translocation of Nrf2. Activation of Nrf2 signaling is accompanied with induction of HO-1 and γ-GCLC expressions in t-CA-treated fibroblasts. Furthermore t-CA-induced Nrf2 translocation is mediated through PKC, AMPK, CKII or ROS signaling cascades. This phenomenon was confirmed with respective pharmacological inhibitors, GF109203X, Compound C, CKII inhibitor or NAC, which blockade t-CA-induced Nrf2 activation. Silencing of Nrf2 signaling with siRNA showed no anti-photoaging effects of t-CA against UVA-induced ROS production, loss of HO-1 and type I collagen degradation in fibroblasts. In vivo evidence on nude mice revealed that t-CA pretreatment (20 or 100â¯mM/day) significantly suppressed MMP-1/-3 activation and maintained sufficient type I procollagen levels in biopsied skin tissue against UVA irradiation (3â¯J/cm2/day for 10-day). CONCLUSION: t-CA treatment diminished UVA-induced photoaging/collagen degradation, and protected structural integrity of the skin.