u-0126 and caffeic-acid-phenethyl-ester

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a survival rate of 4-6 months from diagnosis. PDAC is the fourth leading cause of cancer-related death in the Western world, with a mortality rate of 10 cases per 100,000 population. Chemotherapy constitutes only a palliative strategy, with limited effects on life expectancy.. To investigate the biological response of PDAC to mitogen-activated protein kinase (MAPK) and NF-kappaB (NF-kB) inhibitors and the role of autophagy in the modulation of these signaling pathways in order to address the challenge of developing improved medical protocols for patients with PDAC.. Two ATCC cell lines, MIAPaCa-2 and PANC-1, were used as PDAC models. Cells were exposed to inhibitors of MAPK or NF-kB survival pathways alone or after autophagy inhibition. Several aspects were analyzed, as follows: cell proliferation, by [(3)H]TdR incorporation; cell death, by TUNEL assay, regulation of autophagy by LC3-II expression level and modulation of pro-and anti-apoptotic proteins by Western blot.. We demonstrated that the inhibition of the MAPK and NF-kB survival pathways with U0126 and caffeic acid phenethyl ester (CAPE), respectively, produced strong inhibition of pancreatic tumor cell growth without inducing apoptotic death. Interestingly, U0126 and CAPE induced apoptosis after autophagy inhibition in a caspase-dependent manner in MIA PaCa-2 cells and in a caspase-independent manner in PANC-1 cells.. Here we present evidence that allows us to consider a combined therapy regimen comprising an autophagy inhibitor and a MAPK or NF-kB pathway inhibitor as a possible treatment strategy for pancreatic cancer.

Topics: Antineoplastic Agents; Apoptosis; Autophagy; Butadienes; Caffeic Acids; Carcinoma, Pancreatic Ductal; Cell Growth Processes; Cell Line, Tumor; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; NF-kappa B; Nitriles; Pancreatic Neoplasms; Phenylethyl Alcohol; Protein Kinase Inhibitors; Survival Rate

To investigate apoptosis of osteoarthritic (OA) chondrocytes stimulated with different inhibitors targeting tumor necrosis factor-alpha (TNFα) pathway, we isolated first passage chondrocytes from OA patients and then treated them with the inhibitors in combination with TNFα, and then collected the stimulated chondrocytes for Western blotting. Chondrocytes from OA patients expressed cleaved caspase-3 and PARP, suggesting apoptotic background. We here, validated that 10 ng/ml of TNFα couldn't induce more chondrocytes apoptosis. PI3K inhibitor LY294002 or NF-κB inhibitor CAPE, but not mTOR inhibitor rapamycin and MEK1/2 inhibitor U0126 in combination with TNFα could facilitate apoptosis. CAPE-induced more apoptosis could be explained by c-FLIP downregulation more than cIAP1 upregulation. And, we showed the first time that PI3K-NF-κB pathway, but not mTOR pathway could prevent chondrocytes apoptosis induced by a pro-apoptotic factor TNFα and call for attention while trying to inhibit NF-κB as a therapeutic target.

Topics: Aged; Apoptosis; Butadienes; Caffeic Acids; Caspase 3; Cells, Cultured; Chondrocytes; Chromones; Female; Humans; Middle Aged; Morpholines; NF-kappa B; Nitriles; Osteoarthritis; Phenylethyl Alcohol; Phosphoinositide-3 Kinase Inhibitors; Poly(ADP-ribose) Polymerases; Sirolimus; TOR Serine-Threonine Kinases; Tumor Necrosis Factor-alpha

Chitinases are produced in significant quantities by hosts defending against infections with chitin-containing organisms. However, little is known about the immune response of exogenous chitinase in human epithelial cells. IL-8 has been suggested to have a role in the pathogenesis of the allergenic inflammation of bronchial asthma. We examined whether Streptomyces griseus (S. griseus) chitinase-induced IL-8 on airway epithelium and identified the involvement of intracellular signalling pathways. H292 cells were treated with S. griseus chitinase with different concentrations and times. The IL-8 levels were determined by specific human IL-8 enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction. Using a series of pharmacological inhibitors, we examined the upstream signalling pathway responsible for IL-8 expression in response to S. griseus chitinase. Cells exposed to S. griseus chitinase showed higher level of IL-8 protein production and mRNA expression. Cells stimulated by S. griseus chitinase resulted in the activation of protein kinase C (PKC), extracellular signal-regulated kinase (ERK) and nuclear factor kappa-B (NF-kB) pathways. Inhibitors of Ca(2+)-dependent PKC (Ro-31-8220, calphostin C and Go6976) significantly abolished chitinase-induced expression of IL-8. However, Ca(2+)-independent PKC inhibitor (rottlerin) did not inhibit IL-8 expression. Through ERK inhibitor (U0126) and NF-kB inhibitor (caffeine acid phenethyl ester) treatment, it was proven that ERK and NF-kB regulated chitinase-induced IL-8 expression. We concluded that S. griseus chitinase-induced IL-8 expression was regulated by the activation of Ca(2+/-)-dependent PKC, ERK and NF-kB in human airway epithelial cells.

Topics: Blotting, Western; Butadienes; Caffeic Acids; Carbazoles; Cell Line, Tumor; Chitinases; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Humans; Indoles; Interleukin-8; Naphthalenes; NF-kappa B; Nitriles; Phenylethyl Alcohol; Protein Kinase C; Protein Kinase Inhibitors; Respiratory Mucosa; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction