RESUMO
Curcumin has anticancer functions in various tumors. It has been shown to induce apoptosis through p53-dependent pathways. p73 gene is a member of the p53 family which encodes both a tumor suppressor (transactivation-competent p73 (TAp73)) and a putative oncogene (dominant-negative p73 (DNp73)); the former shares similarity with the tumor suppressor p53, and the latter behaves as dominant-negative proteins that interfere with the activity of TAp73. To understand the p73-dependent mechanisms that are engaged during curcumin-induced apoptosis, we established a p73 overexpression cell models using p53-deficient Hep3B cells (Hep3B(TAp73/DNp73)). Our results demonstrated that curcumin at concentrations of 40 and 80 µM induced DNA damage, increased TAp73/DNp73 ratio, and also led to apoptosis in the Hep3B(TAp73/DNp73) cells. The apoptotic cell death was concurrent with the loss of mitochondrial membrane potential; release of cytochrome c from mitochondria; and the cleavage of caspase 9, caspase 3, and poly(ADP-ribose) polymerase (PARP). These results demonstrated a p73-dependent mechanism for curcumin-induced apoptosis that involves the mitochondria-mediated pathway.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Curcumina/farmacologia , Proteína Tumoral p73/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Transdução de Sinais , Ativação TranscricionalRESUMO
AIM: To investigate the mechanism for bradykinin (BK) to stimulate intestinal secretomotor neurons and intestinal chloride secretion. METHODS: Muscle-stripped guinea pig ileal preparations were mounted in Ussing flux chambers for the recording of short-circuit current (Isc). Basal Isc and Isc stimulated by BK when preincubated with the BK receptors antagonist and other chemicals were recorded using the Ussing chamber system. Prostaglandin E2 (PGE2) production in the intestine was determined by enzyme immunologic assay (EIA). RESULTS: Application of BK or B2 receptor (B2R) agonist significantly increased the baseline Isc compared to the control. B2R antagonist, tetrodotoxin and scopolamine (blockade of muscarinic receptors) significantly suppressed the increase in Isc evoked by BK. The BK-evoked Isc was suppressed by cyclooxygenase (COX)-1 or COX-2 specific inhibitor as well as nonselective COX inhibitors. Preincubation of submucosa/mucosa preparations with BK for 10 min significantly increased PGE2 production and this was abolished by the COX-1 and COX-2 inhibitors. The BK-evoked Isc was suppressed by nonselective EP receptors and EP4 receptor antagonists, but selective EP1 receptor antagonist did not have a significant effect on the BK-evoked Isc. Inhibitors of PLC, PKC, calmodulin or CaMKII failed to suppress BK-induced PGE2 production. CONCLUSION: The results suggest that BK stimulates neurogenic chloride secretion in the guinea pig ileum by activating B2R, through COX increasing PGE2 production. The post-receptor transduction cascade includes activation of PLC, PKC, CaMK, IP3 and MAPK.
RESUMO
OBJECTIVE: To explore the effect of catalpol on choline acetyl-transferase and M receptor affinity in a PC12 cell model and a rat model induced by beta-amyloid 25-35 (Aß25-35). METHODS: In PC12 cells, catalpol (10µmol/l, 100µmol/) or saline was retained in the medium and Aß25-35 (final concentration 20µmol/l) was added. Choline acetyl-transferase (ChAT) expression was determined by immunocytochemistry, ChAT activity measured by radioenzymatic assay, and M receptor (muscarinic receptor) affinity determined by (3)H-QNB binding test. In Wistar rats, Aß25-35 was injected intracerebroventricularly to establish AD model. After injection of Aß25-35, the rats were injected catalpol at 5 and 10mg/kgd(-1) intraperitoneally for the next 7 days, and saline for the control rats. ChAT expression, ChAT activity and M receptor affinity were tested. Cells and rats all were divided into four groups: Group A (control), Group B (model), Group C (catalpol low dose), and Group D (catalpol high dose). RESULTS: Compared with control, both PC12 cell and rat AD models showed decreased expression and activity of ChAT (p<0.01), but M receptor affinity remained the same (p>0.05). Compared with model group, treatment of catalpol increased expression and activity of ChAT of PC12 cell and rat AD model induced by Aß25-35, p<0.05 or p<0.01 respectively. But there was no difference of M receptor affinity among the four groups (p>0.05). M receptor affinity remained the same as concentration of catalpol increased gradually in atropine competition experiments (p>0.05). CONCLUSIONS: Catalpol could regulate the cholinergic nerve system function from its effect on ChAT and may have beneficial effect for treatment of AD, but had no effect on M receptor affinity.
Assuntos
Córtex Cerebral/enzimologia , Córtex Cerebral/fisiopatologia , Colina O-Acetiltransferase/metabolismo , Colina/metabolismo , Glucosídeos Iridoides/farmacologia , Receptores Muscarínicos/metabolismo , Doença de Alzheimer/enzimologia , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Modelos Animais de Doenças , Masculino , Células PC12 , Ratos , Ratos WistarRESUMO
Aminopeptidase N (APN) is important in tumour processes. The present study detected the antitumour activity of the novel APN inhibitor DH12a, which is an indoline2,3dione derivative. In the present study, Bestatin, a clinical APN inhibitor was used as a positive control. The expression of APN in the ES-2 and 3AO cell lines were assessed using flow cytometry and the drug inhibition constants of DH12a (Ki=13.15 µM) and Bestatin (Ki=16.57 µM) were assessed using a double reciprocal method of competitive inhibition. The in vitro effects of DH12a on cell proliferation were assessed using a 3(4,5dimethylthiazol2yl)2,5diphenyl tetrazolium bromide assay on human cell lines of ES2 (IC50=43.8 µM), A549 (inhibition rate=41.5% at 160 µM DH12a), HL60 (inhibition rate=47.83% at 160 µM DH12a) and 3AO (IC50=70.2 µM). The inhibition rates were consistently higher than those of Bestatin. The effects of DH12a on cell migration (inhibition rates in ES2 cells and 3AO cells were 56.4 and 76.5%, respectively at 15 µM) and invasion (inhibition rates in ES2 cells and 3AO cells were 75.6 and 66.5%, respectively at 15 µM) were assessed using transwell plates. The in vivo effects of DH12a on tumour proliferation and lung tumour metastasis were determined using an H22 xenograft mice model, where DH12a was administered in combination with genotoxic 5fluorouracil. The antitumour activities of DH12a in vivo were also greater than those of Bestatin. In conclusion, the in vitro effects of DH12a on tumour proliferation, migration and invasion were consistent with the in vivo effects. In addition, DH12a exhibited greater antitumour properties compared with Bestatin.