Inhibition of extracellular matrix production and remodeling by doxycycline in smooth muscle cells.

Alterations in the extracellular matrix (ECM) production and remodeling of smooth muscle cells (SMCs) have been implicated in processes related to the differentiation in atherosclerosis. Due to the anti-atherosclerotic properties of the tetracyclines, we aimed to investigate whether cholesterol supplementation changes the effect of doxycycline over the ECM proteins synthesis and whether isoprenylated proteins and Rho A protein activation are affected. SMC primary culture isolated from chicks exposed to atherogenic factors in vivo (a cholesterol-rich diet, SMC-Ch), comparing it with control cultures isolated after a standard diet (SMC-C). After treatment with 20 nM doxycycline, [H3]-proline and [H3]-mevalonate incorporation were used to measure the synthesis of collagen and isoprenylated proteins, respectively. Real-time PCR was assessed to determine col1a2, col2a1, col3a1, fibronectin, and mmp2 gene expression and the pull-down technique was applied to determine the Rho A activation state. A higher synthesis of collagens and isoprenylated proteins in SMC-Ch than in SMC-C was determined showing that doxycycline inhibits ECM production and remodeling in both SMC types of cultures. Moreover, preliminary results about the effect of doxycycline on protein isoprenylation and Rho A protein activation led us to discuss the possibility that membrane G-protein activation pathways could mediate the molecular mechanism.

Interplay Between Gemcitabine and Erlotinib Over Pancreatic Adenocarcinoma Cells.

OBJECTIVES: Pancreatic ductal adenocarcinoma remains as a chemoresistant disease with the poorest prognosis. Gemcitabine has been the standard treatment during the last decade. Erlotinib, a tyrosine kinase inhibitor, in combination with gemcitabine produces a small increase in survival. However, these results remain insufficient. The aim of this study was to investigate the molecular interplay in vitro between them regarding their effects over cytotoxicity, proliferation, apoptosis, and invasion. METHODS: Using the human pancreatic cancer cell lines Panc-1 and BxPC-3 in vitro, the effects of gemcitabine and erlotinib therapy on growth, proliferation, and invasion were tested by cytotoxicity, cell cycle, and Annexin V-Fluorescein Isothiocyanate analysis, reverse transcription polymerase chain reaction, protein expression, and Chip assays. RESULTS: Therapy decreased cell proliferation causing G0/G1 phase cell cycle arrest with induction of apoptosis in the Panc-1 cell line. This blockade was associated with increased p27 expression. Besides, treatments enhanced the nuclear factor-κB (NF-κB) pathway and the binding of NF-κB to the promoters of genes related to the proliferation and the evasion of apoptosis. CONCLUSIONS: Our data suggest that, although gemcitabine and erlotinib exert antiproliferative effects over pancreatic cancer cell lines, the gemcitabine-induced activation of NF-κB expression and its DNA-binding activities are important drawbacks of this treatment against pancreatic cancer.

Prognosis Relevance of Serum Cytokines in Pancreatic Cancer.

The overall survival of patients with pancreatic ductal adenocarcinoma is extremely low. Although gemcitabine is the standard used chemotherapy for this disease, clinical outcomes do not reflect significant improvements, not even when combined with adjuvant treatments. There is an urgent need for prognosis markers to be found. The aim of this study was to analyze the potential value of serum cytokines to find a profile that can predict the clinical outcome in patients with pancreatic cancer and to establish a practical prognosis index that significantly predicts patients' outcomes. We have conducted an extensive analysis of serum prognosis biomarkers using an antibody array comprising 507 human cytokines. Overall survival was estimated using the Kaplan-Meier method. Univariate and multivariate Cox's proportional hazard models were used to analyze prognosis factors. To determine the extent that survival could be predicted based on this index, we used the leave-one-out cross-validation model. The multivariate model showed a better performance and it could represent a novel panel of serum cytokines that correlates to poor prognosis in pancreatic cancer. B7-1/CD80, EG-VEGF/PK1, IL-29, NRG1-beta1/HRG1-beta1, and PD-ECGF expressions portend a poor prognosis for patients with pancreatic cancer and these cytokines could represent novel therapeutic targets for this disease.

The potential role of the glycoprotein osteoactivin/glycoprotein nonmetastatic melanoma protein B in pancreatic cancer.

OBJECTIVES: Pancreatic ductal adenocarcinoma is still one of the deadliest solid cancers so the finding of new therapeutic approaches and novel targets are of utmost importance. Glycoprotein nonmetastatic melanoma protein B (GPNMB), initially termed glycoprotein nonmetastatic gene B and also named osteoactivin (OA), is a type 1 transmembrane protein that has been recently found to play a role in cancer cell proliferation, angiogenesis, and invasion. Due to its potential responsibility in cancer aggressiveness, the main objective of this work was to assess the role of GPNMB/OA in human pancreatic cancer. METHODS: Using the human pancreatic cancer cell line Panc-1 in vitro, the effects of GPNMB on growth, proliferation, and invasion were tested by BrdU uptake, cell cycle and Annexin V-FITC analysis, RT-PCR, protein expression, and invasion chamber assays. RESULTS: Our results showed that GPNMB/OA protein expression prevents cells from apoptosis-enhancing proliferation and represents a novel modulator of the invasion and metastasis in pancreatic cancer cells. CONCLUSIONS: Due to its main membrane localization in cancer cells and its role in the aggressiveness of pancreatic cancer, GPNMB/OA could represent a novel targeted therapy for pancreatic cancer being attractive for antibody-based therapies.

Serum cytokine profile in patients with pancreatic cancer.

OBJECTIVE: Pancreatic ductal adenocarcinoma is a deadly disease because of late diagnosis and chemoresistance. We aimed to find a panel of serum cytokines representing diagnostic and predictive biomarkers for pancreatic cancer. METHODS: A cytokine antibody array was performed to simultaneously identify 507 cytokines in sera of patients with pancreatic cancer and healthy controls. The nonparametric Mann-Whitney U test was used to pairwise compare the controls, the pretreated patients, and the posttreated patients. Fold changes greater than or equal to 1.5 or less than or equal to 1/1.5 were considered significant. Receiver operating characteristic curves were used to assess the performance of the model. A leave-one-out cross-validation was used for estimating prediction error. RESULTS: Comparing the sera of pretreated patients against the control samples, the cytokines fibroblast growth factor 10 (FGF-10/keratinocyte growth factor-2 (KGF-2), chemokine (C-X-C motif) ligand 11 interferon inducible T cell alpha chemokine (I-TAC)/chemokine [C-X-C motif] ligand 11 (CXCL11), oncostatin M (OSM), osteoactivin/glycoprotein nonmetastatic melanoma protein B, and stem cell factor (SCF) were found significantly overexpressed. Besides, the cytokines CD30 ligand/tumor necrosis factor superfamily, member 8 (TNFSF8), chordin-like 2, FGF-10/KGF-2, growth/differentiation factor 15, I-TAC/CXCL11, OSM, and SCF were differentially expressed in response to treatment. CONCLUSIONS: We propose a role for FGF-10/KGF-2, I-TAC/CXCL11, OSM, osteoactivin/glycoprotein nonmetastatic melanoma protein B, and SCF as novel diagnostic biomarkers. CD30 ligand/TNFSF8, chordin-like 2, FGF-10/KGF-2, growth/differentiation factor 15, I-TAC/CXCL11, OSM, and SCF might represent as predictive biomarkers for gemcitabine and erlotinib response of patients with pancreatic cancer.

Cyclic fluctuations of 3-hydroxy-3-methylglutaryl-CoA reductase in aortic smooth muscle cell cultures.

The cyclic fluctuations of HMG-CoA reductase activity and mRNA are reportedly related to feeding the cells in culture or to variations in food consumption by the animals over a 24-h cycle. In this work, we demonstrate cyclic increments in HMG-CoA reductase activity in smooth muscle cells (SMC) not associated with the culture feeding. Since reductase activity also shows a marked rise preceding the S phase, one of the major goals of the present work was to evaluate this dual role of reductase activity and mRNA fluctuations related to the cell cycle and to food intake in the SMC-C/SMC-Ch cultures derived from control-fed (SMC-C) and cholesterol-fed (SMC-Ch) chicks. The period and amplitude oscillations in HMG-CoA reductase activity varied depending on culture conditions: lipoprotein-deficient serum vs. FBS, young vs. senescent cells, or confluent vs. nonconfluent cultures. The HMG-CoA reductase mRNA concentration showed a marked rise after feeding not correlated to the fluctuation activity, suggesting posttranscriptional modulation. Reductase activity and mRNA were down-regulated in SMC-Ch. Since the nutritional culture conditions were the same in both cell lines, these findings indicate that consumption of a high-cholesterol diet by the animals prior to the establishment of the SMC cultures induced changes in the HMG-CoA reductase gene expression in-aortic SMC.