RESUMO
The upregulation of the HER2 oncogene is associated with a variety of human cancers and is associated with poor prognosis. Baicalein is reported to have anti-tumor activity, but the molecular mechanism of this effect in HER2-positive cancer cells has not been studied. In this study, our data showed that baicalein can inhibit the proliferation and transformation potential of ovarian cancer cells overexpressing HER2. Baicalein treatment caused a dose-dependent inhibition of HER2 gene expression at the transcriptional level. Baicalein acted on ovarian cancer cells overexpressing HER2 to downregulate the PI3K/Akt signaling pathway downstream of HER2 and inhibit the expression or activity of downstream targets, such as VEGF and cyclin D1 and MMP2. Oral administration of baicalein supplemented with a pharmaceutical excipient significantly inhibited the growth of HER2-overexpressing ovarian SKOV-3 cancer xenografts in mice. These results suggest that downregulation of HER2 gene expression by baicalein at the transcriptional level contributes to inhibit the in vitro and in vivo proliferation and HER2-mediated malignant transformation of HER2-overexpressing ovarian cancer cells.
Assuntos
Neoplasias Ovarianas , Proteínas Proto-Oncogênicas c-akt , Humanos , Camundongos , Animais , Feminino , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Linhagem Celular Tumoral , Expressão Gênica , Proliferação de CélulasRESUMO
Ovarian cancer is a lethal gynecological cancer because drug resistance often results in treatment failure. The CHK2, a tumor suppressor, is considered to be an important molecular target in ovarian cancer due to its role in DNA repair. Dysfunctional CHK2 impairs DNA damage-induced checkpoints, reduces apoptosis, and confers resistance to chemotherapeutic drugs and radiation therapy in ovarian cancer cells. This provides a basis for finding new effective agents targeting CHK2 upregulation or activation to treat or prevent the progression of advanced ovarian cancer. Here, the results show that baicalein (5,6,7-trihydroxyflavone) treatment inhibits the growth of highly invasive ovarian cancer cells, and that baicalein-induced growth inhibition is mediated by the cell cycle arrest in the G2/M phase. Baicalein-induced G2/M phase arrest is associated with an increased reactive oxygen species (ROS) production, DNA damage, and CHK2 upregulation and activation. Thus, baicalein modulates the expression of DNA damage response proteins and G2/M phase regulatory molecules. Blockade of CHK2 activation by CHK2 inhibitors protects cells from baicalein-mediated G2/M cell cycle arrest. All the results suggest that baicalein has another novel growth inhibitory effect on highly invasive ovarian cancer cells, which is partly related to G2/M cell cycle arrest through the ROS-mediated DNA breakage damage and CHK2 activation. Collectively, our findings provide a molecular basis for the potential of baicalein as an adjuvant therapeutic agent in the treatment of metastatic ovarian cancer.
Assuntos
Células M , Neoplasias Ovarianas , Humanos , Feminino , Espécies Reativas de Oxigênio/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Linhagem Celular Tumoral , Pontos de Checagem do Ciclo Celular , Dano ao DNA , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Mitose , Apoptose , Ciclo CelularRESUMO
Hericium erinaceus (HE) is a common edible mushroom consumed in several Asian countries and considered to be a medicinal mushroom with neuroprotective effects. Erinacine A (EA) is a bioactive compound in Hericium erinaceus mycelium (HEM) that has been shown to have a neuroprotective effect against neurodegenerative diseases, e.g., Parkinson's disease (PD). Although the etiology of PD is still unclear, neuroinflammation may play an important role in causing dopaminergic neuron loss, which is a pathological hallmark of PD. However, glial cell activation has a close relationship with neuroinflammation. Thus, this study aimed to investigate the anti-neuroinflammatory and neuroprotective effects of EA on lipopolysaccharide (LPS)-induced glial cell activation and neural damage in vitro and in vivo. For the in vitro experiments, glial cells, BV-2 microglial cells and CTX TNA2 astrocytes were pretreated with EA and then stimulated with LPS and/or IFN-γ. The expression of proinflammatory factors in the cells and culture medium was analyzed. In addition, differentiated neuro-2a (N2a) cells were pretreated with EA or HEM and then stimulated with LPS-treated BV-2 conditioned medium (CM). The cell viability and the amount of tyrosine hydroxylase (TH) and mitogen-activated protein kinases (MAPKs) were analyzed. In vivo, rats were given EA or HEM by oral gavage prior to injection of LPS into the substantia nigra (SN). Motor coordination of the rats and the expression of proinflammatory mediators in the midbrain were analyzed. EA pretreatment prevented LPS-induced iNOS expression and NO production in BV-2 cells and TNF-α expression in CTX TNA2 cells. In addition, both EA and HEM pretreatment significantly increased cell viability and TH expression and suppressed the phosphorylation of JNK and NF- κB in differentiated N2a cells treated with CM. In vivo, both EA and HEM significantly improved motor dysfunction in the rotarod test and the amphetamine-induced rotation test and reduced the expression of TNF-α, IL-1ß and iNOS in the midbrain of rats intranigrally injected with LPS. The results demonstrate that EA ameliorates LPS-induced neuroinflammation and has neuroprotective properties.
Assuntos
Diterpenos/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Mediadores da Inflamação/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Fármacos Neuroprotetores/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Lipopolissacarídeos/imunologia , Microglia/imunologia , Doenças Neuroinflamatórias/etiologia , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/patologia , Doença de Parkinson/etiologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , RatosRESUMO
The overexpression of EGFR and/or ErbB2 occurs frequently in ovarian cancers and is associated with poor prognosis. The purpose of this study was to examine the anticancer effects and molecular mechanisms of berberine on human ovarian cancer cells with different levels of EGFR and/or ErbB2. We found that berberine reduced the motility and invasiveness of ovarian cancer cells. Berberine depleted both EGFR and ErbB2 in ovarian cancer cells. Furthermore, berberine suppressed the activation of the EGFR and ErbB2 downstream targets cyclin D1, MMPs, and VEGF by down-regulating the EGFR-ErbB2/PI3K/Akt signaling pathway. The berberine-mediated inhibition of MMP-2 and MMP-9 activity could be rescued by co-treatment with EGF. Finally, we demonstrated that berberine induced ErbB2 depletion through ubiquitin-mediated proteasome degradation. In conclusion, the suppressive effects of berberine on the ovarian cancer cells that differ in the expression of EGFR and ErbB2 may be mediated by the dual depletion of EGFR and/or ErbB2.
Assuntos
Berberina , Neoplasias Ovarianas , Berberina/farmacologia , Linhagem Celular Tumoral , Regulação para Baixo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Receptor ErbB-2/genéticaRESUMO
Ganoderma is one of the common medicinal mushrooms in traditional Chinese medicine. Previous researches have unveiled the multifaceted biological activity of Ganoderma extract. Ganoderma tsugae has been investigated the potential on curing prostate, colon, lung, epidermoid, breast and ovarian cancers, but not including endometrial cancer. Endometrial cancer is a gynecological malignant tumor with serious drug resistance problem in clinical cancer treatment. This study aimed to demonstrate the first study of Ganoderma on treating endometrial cancer. The Ganoderma tsugae ethanol extract (GTEE) could suppress the proliferation of endometrial cancer cells HEC-1-A, KLE, and AN3 CA. GTEE also induced G1/S phase arrest and mitochondria-mediated apoptosis in endometrial cancer cells. Furthermore, the Akt signaling pathway could be suppressed by GTEE. Therefore, our results suggest for the first time that GTEE has the potential to be an adjuvant therapeutic agent in the treatment of endometrial cancer.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Neoplasias do Endométrio/tratamento farmacológico , Ganoderma , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Medicamentos de Ervas Chinesas/uso terapêutico , Feminino , Humanos , Medicina Tradicional Chinesa , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: The prevention of age-related neurodegenerative disorders is an important issue in an aging society. Microglia-mediated neuroinflammation resulting in dopaminergic neuron loss may lead to the pathogenesis of Parkinson's disease (PD). Lipopolysaccharide (LPS), an endotoxin, induces neuroinflammatory microglial activation, contributing to dopaminergic neuron damage. Diosgenin is a phytosteroid sapogenin with a wide spectrum of pharmacological activities, e.g., anti-inflammatory activity. However, the preventive effect of diosgenin on neuroinflammation is not clear. Thus, in this study, we further investigated the neuroprotective effect of diosgenin on LPS-induced neural damage in vitro and in vivo. METHODS: For in vitro experiments, primary mesencephalic neuron-glia cultures and primary microglia cultures isolated from Sprague-Dawley rats were used. Cells were pretreated with diosgenin and then stimulated with LPS. The expression of proinflammatory cytokines or tyrosine hydroxylase (TH) in the cells was analyzed. In vivo, rats were fed a diet containing 0.1% (w/w) diosgenin for 4 weeks before being administered a unilateral substantia nigra (SN) injection of LPS. Four weeks after the LPS injection, the rats were assessed for lesion severity using the amphetamine-induced rotation test and TH immunohistochemistry. RESULTS: Diosgenin pretreatment prevented LPS-induced neurite shortening in TH-positive neurons in mesencephalic neuron-glia cultures. In addition, pretreatment of primary microglia with diosgenin significantly reduced tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) expression. Moreover, diosgenin pretreatment significantly suppressed LPS-induced extracellular signal-regulated kinase (ERK) activation. In vivo, the intranigral injection of LPS in rats fed a diosgenin-containing diet significantly improved motor dysfunction and reduced TH expression in SN. CONCLUSION: These results support the effectiveness of diosgenin in protecting dopaminergic neurons from LPS-induced neuroinflammation.
Assuntos
Diosgenina/farmacologia , Neurônios Dopaminérgicos/metabolismo , Lipopolissacarídeos/toxicidade , Microglia/metabolismo , Doenças Neurodegenerativas , Fármacos Neuroprotetores/farmacologia , Animais , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Neuritos/metabolismo , Doenças Neurodegenerativas/induzido quimicamente , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/prevenção & controle , Ratos , Ratos Sprague-DawleyRESUMO
Oral squamous cell carcinoma (OSCC) is the fifth common cause of cancer mortality in Taiwan with high incidence and recurrence and needs new therapeutic strategies. In this study, ursolic acid (UA), a triterpenoid, was examined the antitumor potency in OSCC cells. Our results showed that UA inhibited the proliferation of OSCC cells in a dose- and time-dependent manner in both Ca922 and SCC2095 oral cancer cells. UA induced caspase-dependent apoptosis accompanied with the modulation of various biological biomarkers including downregulating Akt/mTOR/NF-κB signaling, ERK, and p38. In addition, UA inhibited angiogenesis as evidenced by abrogation of migration/invasion and blocking MMP-2 secretion in Ca922 cells. Interestingly, UA induced autophagy in OSCC cells, as manifested by LC3B-II conversion and increased p62 expression and accumulation of autophagosomes. Inhibition by autophagy inhibitor enhanced UA-mediated apoptosis in Ca922 cells. The experiment provides a rationale for using triterpenoid in the treatment of OSCC.
Assuntos
Inibidores da Angiogênese/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Carcinoma de Células Escamosas/patologia , Neoplasias Bucais/patologia , Triterpenos/farmacologia , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Humanos , Neoplasias Bucais/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Ácido UrsólicoRESUMO
Resistance to the current therapies is the main clinical challenge in the treatment of lethal metastatic prostate cancer (mPCa). Developing novel therapeutic approaches with effective regimes and minimal side effects for this fatal disease remain a priority in prostate cancer study. In the present study, we demonstrated that a traditional Chinese medicine, quality-assured Ganoderma tsugae ethanol extract (GTEE), significantly suppressed cell growth and metastatic capability and caused cell cycle arrest through decreasing expression of cyclins in mPCa cells, PC-3 and DU145 cells. GTEE also induced caspase-dependent apoptosis in mPCa cells. We further showed the potent therapeutic efficacy of GTEE by inhibiting subcutaneous PC-3 tumor growth in a xenograft model. The in vitro and in vivo efficacies on mPCa cells were due to blockade of the PI3K/Akt and MAPK/ERK signaling pathways associated with cancer cell growth, survival and apoptosis. These preclinical data provide the molecular basis for a new potential therapeutic approach toward the treatment of lethal prostate cancer progression.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Ganoderma/química , Animais , Antineoplásicos Fitogênicos/química , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/química , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Medicina Tradicional Chinesa , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Alcohol dehydrogenase (ADH) is the principal enzyme responsible for the metabolism of ethanol. Human ADH constitutes a complex family of isozymes and allozymes with striking variation in kinetic properties and tissue distribution. The liver and the gastrointestinal tract are the major sites for first-pass metabolism (FPM). The quantitative contributions of ADH isozymes and ethnically distinct allozymes to cellular ethanol metabolism remain poorly understood. To address this issue, kinetic mechanism and the steady-state full-rate equations for recombinant human class I ADH1A, ADH1B (including allozymes ADH1B1, ADH1B2, and ADH1B3), ADH1C (including allozymes ADH1C1 and ADH1C2), class II ADH2, and class IV ADH4 were determined by initial velocity, product inhibition, and dead-end inhibition experiments in 0.1 M sodium phosphate at pH 7.5 and 25 °C. Models of the hepatic and gastrointestinal metabolisms of ethanol were constructed by linear combination of the numerical full-rate equations of the component isozymes and allozymes in target organs. The organ simulations indicate that in homozygous ADH1B*1/*1 livers, a representative genotype among ethnically distinct populations due to high prevalence of the allele, major contributors at 1 to 10 mM ethanol are ADH1B1 (45% to 24%) and the ADH1C allozymes (54% to 40%). The simulated activities at 1 to 50 mM ethanol for the gastrointestinal tract (total mucosae of ADH1C*1/*1-ADH4 stomach and the ADH1C*1/*1-ADH2 duodenum and jejunum) account for 0.68%-0.76% of that for the ADH1B*1/*1-ADH1C*1/*1 liver, suggesting gastrointestinal tract plays a relatively minor role in the human FPM of ethanol. Based on the flow-limited sinusoidal perfusion model, the simulated hepatic Kmapp, Vmaxapp, and Ci at a 95% clearance of ethanol for ADH1B*1/*1-ADH1C*1/*1 livers are compatible to that documented in hepatic vein catheterization and pharmacokinetic studies with humans that controlled for the genotypes. The model simulations suggest that slightly higher or similar ethanol elimination rates for ADH1B*2/*2 and ADH1B*3/*3 individuals compared with those for ADH1B*1/*1 individuals may result from higher hepatocellular acetaldehyde.
Assuntos
Álcool Desidrogenase/metabolismo , Etanol/metabolismo , Trato Gastrointestinal/metabolismo , Fígado/metabolismo , Álcool Desidrogenase/genética , Trato Gastrointestinal/enzimologia , Genótipo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Fígado/enzimologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Ipomoea batatas has long been used in folk medicine for the treatment of hyperglycemia or as a food additive for the prevention of type 2 diabetes. However, neither the plant extract nor its active components have been evaluated systematically. In this work four crude extracts, including n-hexane- (IBH), 95% MeOH- (IBM), n-BuOH- (IBB), and H2O-soluble (IBW) fractions, were prepared by fractionation of a methanolic extract of purple I. batatas leaves. Twenty-four pure compounds 1-24 were then isolated by various chromatographic techniques and their structures identified from NMR and MS data. Glucose uptake assays in differentiated 3T3-L1 adipocytes and rat primary hepatocytes, as well as western blot analysis, were carried out to evaluate the antidiabetic activity of this species. The IBH crude fraction, with methyl decanoate (22) as a major and active compound, showed the greatest effect on glucose uptake, most likely via activation of Glut4 and regulation of the PI3K/AKT pathway. Quercetin 3-O-ß-d-sophoroside (1), quercetin (3), benzyl ß-d-glucoside (10), 4-hydroxy-3-methoxybenzaldehyde (12), and methyl decanoate (22) could be important components contributing to the antidiabetic effects. We conclude that purple I. batatas leaves have potential as an antidiabetic plant source and the active constituents 1, 3, 10, 12, and 22 are promising lead candidates for future investigation.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Glucose/metabolismo , Ipomoea batatas/química , Extratos Vegetais/farmacologia , Células 3T3-L1/efeitos dos fármacos , Animais , Metabolismo dos Carboidratos/efeitos dos fármacos , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Extratos Vegetais/química , Folhas de Planta/química , RatosRESUMO
Overexpression of the amyloid precursor protein (APP) and the hyperphosphorylation of the tau protein are vital in the understanding of the cause of Alzheimer's disease (AD). As a consequence, regulation of the expression of both APP and tau proteins is one important approach in combating AD. The APP and tau proteins can be targeted at the levels of transcription, translation and protein structural integrity. This paper reports the utilization of a bi-cistronic vector containing either APP or tau internal ribosome entry site (IRES) elements flanked by ß-galactosidase gene (cap-dependent) and secreted alkaline phosphatase (SEAP) (cap-independent) to discern the mechanism of action of memantine, an N-methyl-D-aspartate (NMDA) receptor antagonist. Results indicate that memantine could reduce the activity of both the APP and tau IRES at a concentration of ~10 µM (monitored by SEAP activity) without interfering with the cap-dependent translation as monitored by the ß-galactosidase assay. Western blot analysis of the tau protein in neuroblastoma (N2A) and rat hippocampal cells confirmed the halting of the expression of the tau proteins. We also employed this approach to identify a preparation named NB34, extracts of Boussingaultia baselloides (madeira-vine) fermented with Lactobacillus spp., which can function similarly to memantine in both IRES of APP and Tau. The water maze test demonstrated that NB34 could improve the spatial memory of a high fat diet induced neurodegeneration in apolipoprotein E-knockout (ApoE-/-) mice. These results revealed that the bi-cistronic vector provided a simple, and effective platform in screening and establishing the mechanistic action of potential compounds for the treatment and management of AD.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Precursor de Proteína beta-Amiloide/genética , Fármacos do Sistema Nervoso Central/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Sítios Internos de Entrada Ribossomal , Proteínas tau/genética , Doença de Alzheimer/etiologia , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Linhagem Celular Tumoral , Dieta Hiperlipídica/efeitos adversos , Avaliação Pré-Clínica de Medicamentos , Expressão Gênica/efeitos dos fármacos , Masculino , Memantina/farmacologia , Camundongos Knockout , Terapia de Alvo Molecular , Especificidade de Órgãos , Aprendizagem Espacial/efeitos dos fármacos , Proteínas tau/metabolismoRESUMO
BACKGROUND: Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are principal enzymes responsible for metabolism of ethanol (EtOH). Functional polymorphisms of ADH1B, ADH1C, and ALDH2 genes occur among racial populations. This study aimed to systematically determine the functional expressions and cellular localization of ADH and ALDH family members in human small bowel. METHODS: One hundred and seventeen surgical specimens of duodenal mucosae, 34 jejunal mucosal specimens, and 14 paired specimens of stomach, duodenum, and jejunum from same individuals were investigated. The isozyme/allozyme expression patterns of ADH and ALDH were identified by isoelectric focusing, and the ADH/ALDH activities were assayed spectrophotometrically. The protein contents of ADH/ALDH isozymes were determined by immunoblotting using the corresponding purified class-specific antibodies, and the cellular localizations were detected by immunohistochemistry and histochemistry. RESULTS: The activities of ADH1C*1/*1 allelotype were significantly higher than those of the ADH1C*1/*2 allelotype in duodenum (p < 0.001) and in jejunum (p < 0.05); and the activity of ADH2-expressing phenotype was significantly higher than that of the ADH2-missing phenotype in duodenum (p < 0.05). The activities of ALDH2-inactive phenotype were not significantly different from those of the ALDH2-active phenotype in duodenum and jejunum. Stomach exhibited significantly lower ADH activity (p < 0.05), and duodenum displayed significantly lower ALDH activity (p < 0.001) comparing the paired gastric, duodenal, and jejunal mucosae of same individuals. Gender and age did not significantly influence the ADH and ALDH activities in duodenum. The isozyme protein contents in duodenum and jejunum were in the following decreasing order: ALDH1A1, ADH1/ALDH2, ADH3, ADH2, and ALDH3A1. Villous epithelial cells, cryptic Paneth cells, and Brunner's gland ductal cells revealed a greater immunostaining intensity with ADH1, ALDH1A1, and ALDH2. CONCLUSIONS: ADH and ALDH isozymes are differentially expressed in the various cell types of duodenum and jejunum. The results suggest that proximal small intestine can substantively contribute to first-pass metabolism of EtOH under certain conditions and that cytotoxic acetaldehyde and EtOH perturbation of retinol metabolism might play an etiological role in the pathogenesis of small bowel.
Assuntos
Álcool Desidrogenase/biossíntese , Aldeído Desidrogenase/biossíntese , Etanol/metabolismo , Intestino Delgado/enzimologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Álcool Desidrogenase/metabolismo , Álcool Desidrogenase/fisiologia , Aldeído Desidrogenase/metabolismo , Aldeído Desidrogenase/fisiologia , Duodeno/enzimologia , Duodeno/metabolismo , Feminino , Mucosa Gástrica/metabolismo , Humanos , Mucosa Intestinal/enzimologia , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Isoenzimas/biossíntese , Isoenzimas/metabolismo , Isoenzimas/fisiologia , Jejuno/enzimologia , Jejuno/metabolismo , Masculino , Pessoa de Meia-Idade , Fenótipo , Estômago/enzimologiaRESUMO
BACKGROUND: The extract of freshwater clams has been used to protect the body against liver diseases in traditional folk medicine. This study aims at investigating the effects of freshwater clam extract on activated hepatic stellate cells (aHSCs), which are critical contributors to liver fibrosis. METHODS: The aHSCs used in this study were derived from hepatic stellate cells that were isolated and purified from the livers of male Wistar rats and then transformed into the activated phenotype by culturing on uncoated plastic dishes. Freshwater clam extract (CE) was collected after the outflow from the live freshwater clams in a water bath at 100°C for 60 min. The effects of CE on aHSCs were analyzed by MTT assay, flow cytometry, Oil Red O (ORO) staining, western blot, and real-time RT-PCR. RESULTS: The results indicated that CE suppressed the proliferation of aHSCs through G0/G1 cell cycle arrest by downregulating cyclin D1 and upregulating p27. The expression levels of a-SMA, collagen I, TGF-ß, and TNF-α were inhibited in the CE-treated aHSCs. In addition, the CE treatment increased the lipid contents in aHSCs by promoting PPARγ expression. Furthermore, CE modulated the expression of ECM-related genes, i.e., by upregulating MMP-9 and downregulating TIMP-II. CONCLUSIONS: These data revealed that CE could induce the deactivation of aHSCs. We therefore suggest that CE has potential as an adjuvant therapeutic agent against hepatic fibrosis.
RESUMO
Pyruvate kinase M2 (PKM2) is a key enzyme responsible for the final step of glycolysis. It is still unclear whether PKM2 is involved in reactive oxygen species (ROS)-mediated cytotoxicity in gastrointestinal cancer, and what mechanisms are involved. One duodenal (AZ521) and two gastric (NUGC and SCM-1) cancer cell lines were treated with an indole-3-carbinol derivative OSU-A9, which caused cytotoxicity in acute myeloid leukemia through ROS generation. OSU-A9 caused a dose- and time-dependent cytotoxicity and induced apoptosis in duodenal and gastric cancer cells through ROS generation. Pretreatment with ROS scavengers rescued cancer cells from apoptosis and concomitant poly (ADP-ribose) polymerase cleavage, implying a key role of ROS in OSU-A9-induced cell death. Moreover, OSU-A9-induced ROS generation decreased protein levels of pTyr105-PKM2, and this effect was rescued by pretreatment with ROS scavengers. Interestingly, pTyr105-PKM2 protein levels decreased in the cell nucleus rather than in the cytoplasm. PKM2 overexpression partially rescued the survival of duodenal and gastric cancer cells treated with OSU-A9. Furthermore, the anticancer activity of OSU-A9 extended in vivo, as OSU-A9 administered by oral gavage suppressed the growth of AZ521 xenograft tumors in nude mice without obvious toxicity. In conclusion, OSU-A9 inhibited duodenal and gastric cancer cell proliferation through ROS generation and caused a subsequent decrease in nuclear pTyr105-PKM2 protein. These findings provide evidence for the non-canonical activity of PKM2 in cancer cell survival. Furthermore, they highlight the potential role of PKM2 as a future therapeutic target for duodenal and gastric cancer.
Assuntos
Neoplasias Duodenais/enzimologia , Indóis/farmacologia , Nitrobenzenos/farmacologia , Piruvato Quinase/antagonistas & inibidores , Piruvato Quinase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Gástricas/enzimologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Neoplasias Duodenais/tratamento farmacológico , Humanos , Indóis/uso terapêutico , Masculino , Metanol/análogos & derivados , Camundongos , Camundongos Nus , Nitrobenzenos/uso terapêutico , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Neoplasias Gástricas/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
BACKGROUND: Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are major enzymes responsible for metabolism of ethanol. Genetic polymorphisms of ADH1B, ADH1C, and ALDH2 occur among racial populations. The metabolic effect and metabolites contribute to pathogenesis of pancreatic injury. The goal of this study was to determine the functional expressions and cellular localization of ADH and ALDH families in human pancreas. METHODS: Fifty five surgical specimens of normal pancreas as well as 15 samples each for chronic pancreatitis and pancreatic cancer from archival formalin-fixed paraffin-embedded tissue specimens were investigated. Class-specific antibodies were prepared by affinity chromatographies from rabbit antisera raised against recombinant human ADH1C1, ADH4, ADH5, ADH7, ALDH1A1, ALDH2, and ALDH3A1. The isozyme expression patterns of ADH/ALDH were identified by isoelectric focusing, and the activities were assayed spectrophotometrically. The protein contents of ADH/ALDH isozymes were determined by immunoblotting, and the cellular localizations were detected by immunohistochemistry and histochemistry. RESULTS: At 33 mM ethanol, pH 7.5, the activities were significantly different between allelic phenotypes of ADH1B. The activity of ALDH2-inactive phenotypes was slightly lower than ALDH2-active phenotypes at 200 microM acetaldehyde. The protein contents were in the following decreasing order: ALDH1A1, ALDH2, ADH1, and ADH5. ADH1B was detected in the acinar cells and ADH1C in the ductular, islet, and stellate cells. The expression of ADH1C appeared to be increased in the activated pancreatic stellate cells in chronic pancreatitis and pancreatic cancer. CONCLUSIONS: Alcohol dehydrogenase and ALDH family members are differentially expressed in the various cell types of pancreas. ADH1C may play an important role in modulation of activation of pancreatic stellate cells.
Assuntos
Álcool Desidrogenase/metabolismo , Aldeído Desidrogenase/metabolismo , Depressores do Sistema Nervoso Central/metabolismo , Etanol/metabolismo , Pâncreas/enzimologia , Neoplasias Pancreáticas/enzimologia , Pancreatite/enzimologia , Adulto , Idoso , Álcool Desidrogenase/genética , Aldeído Desidrogenase/genética , Aldeído-Desidrogenase Mitocondrial , Feminino , Regulação da Expressão Gênica , Genótipo , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/enzimologia , Células Estreladas do Fígado/patologia , Humanos , Isoenzimas/metabolismo , Masculino , Pessoa de Meia-Idade , Pâncreas/citologia , Pâncreas/patologia , Neoplasias Pancreáticas/patologia , Pancreatite/patologia , FenótipoRESUMO
Human alcohol dehydrogenase (ADH) constitutes a complex family with diversified functions. Rabbit antihuman class I, II, III, and IV ADH antisera were prepared and used as probes to compare cross-reactivity with the isozymes across classes by semiquantitative Western blotting and quantitative enzyme-linked immunosorbent assay (ELISA). The interclass cross-reactivities with the noncognate isozymes by ELISA, generally approximately 0-35%, appeared considerably lower than those of the intraclass cross-reactivities except with the class IV isozyme. The anti-ADH1B1, ADH1C1, and ADH3 antisera, but not the anti-ADH2, exhibited approximately 80% cross-reactivity with ADH4. The intraclass cross-reactivities among class I isozymes ADH1A, ADH1B1, and ADH1C1 with anti-ADH1B1 or anti-ADH1C1 antisera were approximately 90%. Immunohistochemistry detecting with class-specific antibodies for ADH1-4 isolated from the corresponding antisera demonstrated that ADH4 was the predominant isoform expressed in the basal and suprabasal layer of human esophagus mucosa, whereas it was virtually devoid in the adjacent squamous cell carcinoma. Thus, the setup is more valuable for scanning ADH expression at protein level in different tissues and under different conditions, and maybe not as a tool for classification.
Assuntos
Álcool Desidrogenase/classificação , Adulto , Álcool Desidrogenase/imunologia , Aldeído Oxirredutases/imunologia , Animais , Anticorpos , Western Blotting , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Neoplasias Esofágicas/enzimologia , Esôfago/enzimologia , Humanos , Imuno-Histoquímica , Isoenzimas/imunologia , Masculino , CoelhosRESUMO
Alcohol dehydrogenase (ADH) is the principal enzyme responsible for metabolism of ethanol. Human ADH constitutes a complex isozyme family with striking variations in kinetic function and tissue distribution. Liver and gastrointestinal tract are the major sites for first-pass metabolism (FPM). Their relative contributions to alcohol FPM and degrees of the inhibitions by aspirin and its metabolite salicylate, acetaminophen and cimetidine remain controversial. To address this issue, mathematical organ modeling of ethanol-oxidizing activities in target tissues and that of the ethanol-drug interactions were constructed by linear combination of the corresponding numerical rate equations of tissue constituent ADH isozymes with the documented isozyme protein contents, kinetic parameters for ethanol oxidation and the drug inhibitions of ADH isozymes/allozymes that were determined in 0.1 M sodium phosphate at pH 7.5 and 25 °C containing 0.5 mM NAD(+). The organ simulations reveal that the ADH activities in mucosae of the stomach, duodenum and jejunum with ADH1C*1/*1 genotype are less than 1%, respectively, that of the ADH1B*1/*1-ADH1C*1/*1 liver at 1-200 mM ethanol, indicating that liver is major site of the FPM. The apparent hepatic KM and Vmax for ethanol oxidation are simulated to be 0.093 ± 0.019 mM and 4.0 ± 0.1 mmol/min, respectively. At 95% clearance in liver, the logarithmic average sinusoidal ethanol concentration is determined to be 0.80 mM in accordance with the flow-limited gradient perfusion model. The organ simulations indicate that higher therapeutic acetaminophen (0.5 mM) inhibits 16% of ADH1B*1/*1 hepatic ADH activity at 2-20 mM ethanol and that therapeutic salicylate (1.5 mM) inhibits 30-31% of the ADH1B*2/*2 activity, suggesting potential significant inhibitions of ethanol FPM in these allelotypes. The result provides systematic evaluations and predictions by computer simulation on potential ethanol FPM in target tissues and hepatic ethanol-drug interactions in the context of tissue ADH isozymes.
Assuntos
Álcool Desidrogenase/metabolismo , Etanol/metabolismo , Mucosa Gástrica/metabolismo , Intestino Delgado/metabolismo , Fígado/metabolismo , Modelos Biológicos , Preparações Farmacêuticas/metabolismo , Acetaminofen/farmacologia , Aspirina/farmacologia , Cimetidina/farmacologia , Genótipo , Humanos , Intestino Delgado/efeitos dos fármacos , Isoenzimas/metabolismo , Cinética , Fígado/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Ácido Salicílico/farmacologia , Estômago/efeitos dos fármacos , Especificidade por SubstratoRESUMO
BACKGROUND AND PURPOSE: Klebsiella pneumoniae liver abscess with metastatic complications is an emerging infectious disease in Taiwan. The present study aimed to identify virulence genes involved in the pathogenicity of K. pneumoniae. METHODS: The closely related Escherichia coli genome array was employed to study the expression of the putative genome of K. pneumoniae. Total mRNA expression levels of a K. pneumoniae strain (designated National Taiwan University Hospital [NTUH]-K2044), isolated from a patient with liver abscess, and another strain (designated NTUH-K9), from a patient with sepsis only, were compared on the E. coli array. RNA blot was used to reconfirm mRNA expression in NTUH-K9, K2044 and in 9 other sepsis strains and 9 other liver abscess strains. RESULTS: One of the genes which was found to be highly expressed in NTUH-K2044, designated aldA, was selected for further study. The aldA gene codes for the enzyme aldehyde dehydrogenase (aldehyde:NAD[P](+) oxidoreductase; ALDH). Kinetic properties of ALDH isolated from the 2 strains, designated K2044 ALDH and K9 ALDH respectively, were characterized. The isolated recombinant K2044 ALDH and K9 ALDH, both with subunit molecular weight 55 kDa, exhibited similar substrate specificity and coenzyme preference with glycolaldehyde (V(max)/K(m) = 27 and 17 U/mg/mM, respectively) and glyceraldehyde (maximum velocity [V(max)]/ Michaelis constant [K(m)] = 42 and 30 U/mg/mM, respectively) being the much better substrates and NAD(+) being the preferred coenzyme (K(m) = 0.28 and 0.23 mM, respectively). Unlike K9 ALDH, K2044 ALDH displayed inhibition at high concentrations of glycolaldehyde (substrate inhibition constant [K(i)] = 7.4 mM) and glyceraldehyde (K(i) = 2.6 mM). CONCLUSION: The expression of the aldA gene is higher in K. pneumoniae strains from patients with liver abscess. The aldA gene encodes functional ALDH and can use glycolaldehyde and glyceraldehydes as substrates.
Assuntos
Aldeído Desidrogenase/genética , Clonagem Molecular , Klebsiella pneumoniae/genética , Sequência de Aminoácidos , Astemizol , Sequência de Bases , Escherichia coli/genética , Humanos , Infecções por Klebsiella , Klebsiella pneumoniae/enzimologia , Abscesso Hepático/microbiologia , Dados de Sequência Molecular , Especificidade por Substrato , Virulência/genéticaRESUMO
Background. Liver fibrosis is a significant liver disease in Asian countries. Sedum mexicanum Britt. (SM) has been claimed to have antihepatitis efficacy. In traditional folk medicine, a solution of boiling water-extracted SM (SME) is consumed to prevent and treat hepatitis. However, its efficacy has not yet been verified. The purpose of this study was to investigate the in vitro effect of SME on hepatoprotection. Methods. Hepatic stellate cells (HSCs) and hepatocytes (HCs) were isolated from the livers of the rats by enzymatic digestion and density gradient centrifugation. Results. Treating the HCs and aHSCs with SME caused a dose-dependent decrease in the viability of aHSCs but not that of HCs. In addition, treatment with SME resulted in apoptosis of aHSCs, as determined by DAPI analysis and flow cytometry. SME also increased the amount of cleaved caspase-3, cleaved caspase-9, and cleaved poly ADP-ribose polymerase (PARP) in aHSCs. Furthermore, SME treatment induced a dose-dependent reduction in Bcl-2 expression and increased the expression of Bax in aHSCs. Conclusions. SME did not cause cytotoxicity in HCs, but it induced apoptosis in aHSCs through the mitochondria-dependent caspase-3 pathway. Therefore, SME may possess therapeutic potential for liver fibrosis.
RESUMO
Background. Purple sweet potato leaves (PSPL) are widely grown and are considered a healthy vegetable in Taiwan. PSPL contain a high content of flavonoids, and the boiling water-extracted PSPL (PSPLE) is believed to prevent metabolic syndrome. However, its efficacy has not yet been verified. Therefore, we investigated the effect of PSPLE on adipocytes. Methods. The differentiated 3T3-L1 cells used in this study were derived from preadipocytes that were differentiated into adipocytes using an adipogenic agent (insulin, dexamethasone, and 3-isobutyl-1-methylxanthine); approximately 90% of the cells were differentiated using this method. Results. Treating the differentiated 3T3-L1 cells with PSPLE caused a dose-dependent decrease in the number of adipocytes rather than preadipocytes. In addition, treatment with PSPLE resulted in apoptosis of the differentiated 3T3-L1 cells as determined by DAPI analysis and flow cytometry. PSPLE also increased the expression of cleaved caspase-3 and poly ADP-ribose polymerase (PARP). Furthermore, PSPLE induced downregulation of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) gene expression in the differentiated 3T3-L1 cells. Conclusions. These results suggest that PSPLE not only induced apoptosis but also downregulated inflammation-associated genes in the differentiated 3T3-L1 cells.