Sonia Perales

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.

Effect of Oxysterol-Induced Apoptosis of Vascular Smooth Muscle Cells on Experimental Hypercholesterolemia

Smooth muscle cells (SMCs) undergo changes related to proliferation and apoptosis in the physiological remodeling of vessels and in diseases such as atherosclerosis and restenosis. Recent studies also have demonstrated the vascular cell proliferation and programmed cell death contribute to changes in vascular architecture in normal development and in disease. The present study was designed to investigate the apoptotic pathways induced by 25-hydroxycholesterol in SMCs cultures, using an in vivo/in vitro cell model in which SMCs were isolated and culture from chicken exposed to an atherogenic cholesterol-rich diet (SMC-Ch) and/or an antiatherogenic fish oil-rich diet (SMC-Ch-FO). Cells were exposed in vitro to 25-hydroxycholesterol to study levels of apoptosis and apoptotic proteins Bcl-2, Bcl-XL and Bax and the expression of bcl-2 and bcl-xL, genes. The quantitative real-time reverse transcriptase-polymerase chain reaction and the Immunoblotting western blot analysis showed that 25-hydroxycholesterol produces apoptosis in SMCs, mediated by a high increase in Bax protein and Bax gene expression. These changes were more marked in SMC-Ch than in SMC-Ch-FO, indicating that dietary cholesterol produces changes in SMCs that make them more susceptible to 25-hydroxycholesterol-mediated apoptosis. Our results suggest that the replacement of a cholesterol-rich diet with a fish oil-rich diet produces some reversal of cholesterol-induced changes in the apoptotic pathways induced by 25-hydroxycholesterol in SMCs cultures, making SMCs more resistant to apoptosis.

Influence of cholesterol and fish oil dietary intake on nitric oxide-induced apoptosis in vascular smooth muscle cells

Apoptosis of vascular smooth muscle cells (SMC) is critically involved in the progression of atherosclerosis. We previously reported that dietary cholesterol intake induces changes in SMC at molecular and gene expression levels. The objectives of the present study were to investigate the differential response to nitric oxide of vascular SMC obtained from chicks after cholesterol and fish oil dietary intake and to examine effects on the main pro-apoptotic and anti-apoptotic genes. Dietary cholesterol intake reduced the Bcl-2/Bax (anti-apoptotic/pro-apoptotic) protein ratio in SMC, making them more susceptible to apoptosis. When cholesterol was withdrawn and replaced with a fish oil-enriched diet, the Bcl-xl/Bax protein ratio significantly increased, reversing the changes induced by cholesterol. The decrease in c-myc gene expression after apoptotic stimuli and the increase in Bcl-xl/Bax ratio indicate that fish oil has a protective role against apoptosis in SMC. Nitroprussiate-like nitric oxide donors exerted an intensive action on vascular SMC cultures. However, SMC-C (isolated from animals fed with control diet) and SMC-Ch (isolated from animals fed with cholesterol-enriched diet) responded differently to nitric oxide, especially in their bcl-2 and bcl-xl gene expression. SMC isolated from animals fed with cholesterol-enriched and then fish oil-enriched diet (SMC-Ch-FO cultures) showed an intermediate apoptosis level (Bcl-2/Bax ratio) between SMC-C and SMC-Ch, induction of c-myc expression and elevated p53 expression. These findings indicate that fish oil protects SMC against apoptosis.

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.

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.

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-&kgr;B (NF-&kgr;B) pathway and the binding of NF-&kgr;B 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-&kgr;B expression and its DNA-binding activities are important drawbacks of this treatment against pancreatic cancer.

Effect of HMG-CoA Reductase Inhibition on Vascular Smooth Muscle Cells Extracellular Matrix Production: Role of RhoA

Cholesterol-lowering effects apart, statins can improve the endothelial function, stabilize the atherosclerotic plaques, decrease the oxidative stress and inflammation and inhibit the thrombogenic response by means of the inhibition of isoprenoids, which serve as lipid attachments for intracellular signaling molecules. We aimed to evaluate whether the effect of statins on RhoA activity mediate extracellular matrix production, particularly affecting collagen type I, in smooth muscle cells (SMCs). Our results showed that lovastatin decreased collagen expression in primary cultured chicken SMCs as determined by incorporation of [H3]-proline, RT-PCR and immunocytochemistry. This fall was parallel to that found in Rho A activity. Similar results were found when GGTI-298, a RhoA inhibitor, was added to the culture medium. Mevalonate or geranylgeranyl pyrophosphate reverted these effects. In order to elucidate the role of Rho A in these events we transfected the cell line A10 (rat SMCs) with constitutively active (G14V) or dominant negative RhoA (T19N) constructs. The last ones showed similar results regarding collagen production that those stated above in lovastatin treated primary SMC cultures. Constitutively active RhoA transfected cells showed the opposite effects. Next we performed a promoter activity assay to exclude post-transcriptional mechanisms implicated in these studies. We found a similar pattern in col1a2 promoter activity to that found in collagen expression. Our results have demonstrated that statins regulate the activation of RhoA through its isoprenylation, which is crucial for the regulation of extracellular matrix synthesis in SMCs.

Fish oil supplementation reverses the effect of cholesterol on apoptotic gene expression in smooth muscle cells.

BackgroundNutritional control of gene regulation guides the transformation of smooth muscle cells (SMC) into foam cells in atherosclerosis. Oxidative stress has been reported in areas of lipid accumulation, activating proliferation genes. Suppression of oxidative stress by antioxidant administration reduces this activation and the progression of lesions. We hypothesized that fish oil consumption may protect against atherosclerotic vascular disease. The study objective was to determine the effects of dietary cholesterol and fish-oil intake on the apoptotic pathways induced by 25-hydroxycholesterol (25-HC) in SMC cultures.MethodsAn in vivo/in vitro cell model was used, culturing SMC isolated from chicks exposed to an atherogenic cholesterol-rich diet with 5% of cholesterol (SMC-Ch) alone or followed by an anti-atherogenic fish oil-rich diet with 10% of menhaden oil (SMC-Ch-FO) and from chicks on standard diet (SMC-C). Cells were exposed to 25-HC, studying apoptosis levels by flow cytometry (Annexin V) and expressions of caspase-3, c-myc, and p53 genes by quantitative real-time reverse transcriptase-polymerase chain reaction. Results: Exposure to 25-HC produced apoptosis in all three SMC cultures, which was mediated by increases in caspase-3, c-myc, and p53 gene expression. Changes were more marked in SMC-Ch than in SMC-C, indicating that dietary cholesterol makes SMC more susceptible to 25-HC-mediated apoptosis. Expression of p53 gene was elevated in SMC-Ch-FO. This supports the proposition that endogenous levels of p53 protect SMC against apoptosis and possibly against the development of atherosclerosis. Fish oil attenuated the increase in c-myc levels observed in SMC-C and SMC-Ch, possibly through its influence on the expression of antioxidant genes.ConclusionReplacement of a cholesterol-rich diet with a fish oil-rich diet produces some reversal of the cholesterol-induced changes, increasing the resistance of SMC to apoptosis.