Gema Alcarraz-Vizán

Modulation of pentose phosphate pathway during cell cycle progression in human colon adenocarcinoma cell line HT29

Cell cycle regulation is dependent on multiple cellular and molecular events. Cell proliferation requires metabolic sources for the duplication of DNA and cell size. However, nucleotide reservoirs are not sufficient to support cell duplication and, therefore, biosynthetic pathways should be upregulated during cell cycle. Here, we reveal that glucose‐6‐phosphate dehydrogenase (G6PDH) and transketolase (TKT), the 2 key enzymes of oxidative and nonoxidative branches of the pentose phosphate pathway (PPP), respectively, which is necessary for nucleotide synthesis, are enhanced during cell cycle progression of the human colon cancer cell line HT29. These enhanced enzyme activities coincide with an increased ratio of pentose monophosphate to hexose monophosphate pool during late G1 and S phase, suggesting a potential role for pentose phosphates in proliferating signaling. Isotopomeric analysis distribution of nucleotide ribose synthesized from 1,2‐13C2‐glucose confirms the activation of the PPP during late G1 and S phase and reveals specific upregulation of the oxidative branch. Our data sustain the idea of a critical oxidative and nonoxidative balance in cancer cells, which is consistent with a late G1 metabolic check point. The distinctive modulation of these enzymes during cell cycle progression may represent a new strategy to inhibit proliferation in anticancer treatments.

Characterization of the metabolic changes underlying growth factor angiogenic activation: identification of new potential therapeutic targets.

Angiogenesis is a fundamental process to normal and abnormal tissue growth and repair, which consists of recruiting endothelial cells toward an angiogenic stimulus. The cells subsequently proliferate and differentiate to form endothelial tubes and capillary-like structures. Little is known about the metabolic adaptation of endothelial cells through such a transformation. We studied the metabolic changes of endothelial cell activation by growth factors using human umbilical vein endothelial cells (HUVECs), [1,2-(13)C(2)]-glucose and mass isotopomer distribution analysis. The metabolism of [1,2-(13)C(2)]-glucose by HUVEC allows us to trace many of the main glucose metabolic pathways, including glycogen synthesis, the pentose cycle and the glycolytic pathways. So we established that these pathways were crucial to endothelial cell proliferation under vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) stimulation. A specific VEGF receptor-2 inhibitor demonstrated the importance of glycogen metabolism and pentose cycle pathway. Furthermore, we showed that glycogen was depleted in a low glucose medium, but conserved under hypoxic conditions. Finally, we demonstrated that direct inhibition of key enzymes to glycogen metabolism and pentose phosphate pathways reduced HUVEC viability and migration. In this regard, inhibitors of these pathways have been shown to be effective antitumoral agents. To sum up, our data suggest that the inhibition of metabolic pathways offers a novel and powerful therapeutic approach, which simultaneously inhibits tumor cell proliferation and tumor-induced angiogenesis.

A Lyophilized Red Grape Pomace Containing Proanthocyanidin-Rich Dietary Fiber Induces Genetic and Metabolic Alterations in Colon Mucosa of Female C57BL/6J Mice

Diet plays a decisive role in promoting or preventing colon cancer. However, the specific effects of some nutrients remain unclear. The capacity of fruit and vegetables to prevent cancer has been associated with their fiber and antioxidant composition. We investigated whether consumption of a lyophilized red grape pomace containing proanthocyanidin-rich dietary fiber (grape antioxidant dietary fiber, GADF) by female C57BL/6J mice would affect the serum metabolic profile or colon mucosa gene expression using NMR techniques and DNA microarray, respectively. The mice were randomly assigned to 2 groups that for 2 wk consumed a standard rodent diet and were gavaged with 100 mg/kg body weight GADF suspended in water or an equivalent volume of plain tap water (10 mL/kg body weight). The amount of fiber supplemented was calculated to equal the current recommended daily levels of fiber consumption for humans. The inclusion of dietary GADF induced alterations in the expression of tumor suppressor genes and proto-oncogenes as well as the modulation of genes from pathways, including lipid biosynthesis, energy metabolism, cell cycle, and apoptosis. Overexpression of enzymes pertaining to the xenobiotic detoxifying system and endogenous antioxidant cell defenses was also observed. In summary, the genetic and metabolic profiles induced by GADF were consistent with the preventive effects of fiber and polyphenols. On the basis of these observations, we propose that GADF may contribute to reducing the risk of colon cancer.

Chaperones ameliorate beta cell dysfunction associated with human islet amyloid polypeptide overexpression.

In type 2 diabetes, beta-cell dysfunction is thought to be due to several causes, one being the formation of toxic protein aggregates called islet amyloid, formed by accumulations of misfolded human islet amyloid polypeptide (hIAPP). The process of hIAPP misfolding and aggregation is one of the factors that may activate the unfolded protein response (UPR), perturbing endoplasmic reticulum (ER) homeostasis. Molecular chaperones have been described to be important in regulating ER response to ER stress. In the present work, we evaluate the role of chaperones in a stressed cellular model of hIAPP overexpression. A rat pancreatic beta-cell line expressing hIAPP exposed to thapsigargin or treated with high glucose and palmitic acid, both of which are known ER stress inducers, showed an increase in ER stress genes when compared to INS1E cells expressing rat IAPP or INS1E control cells. Treatment with molecular chaperone glucose-regulated protein 78 kDa (GRP78, also known as BiP) or protein disulfite isomerase (PDI), and chemical chaperones taurine-conjugated ursodeoxycholic acid (TUDCA) or 4-phenylbutyrate (PBA), alleviated ER stress and increased insulin secretion in hIAPP-expressing cells. Our results suggest that the overexpression of hIAPP induces a stronger response of ER stress markers. Moreover, endogenous and chemical chaperones are able to ameliorate induced ER stress and increase insulin secretion, suggesting that improving chaperone capacity can play an important role in improving beta-cell function in type 2 diabetes.

Histone deacetylase inhibition results in a common metabolic profile associated with HT29 differentiation

Cell differentiation is an orderly process that begins with modifications in gene expression. This process is regulated by the acetylation state of histones. Removal of the acetyl groups of histones by specific enzymes (histone deacetylases, HDAC) usually downregulates expression of genes that can cause cells to differentiate, and pharmacological inhibitors of these enzymes have been shown to induce differentiation in several colon cancer cell lines. Butyrate at high (mM) concentration is both a precursor for acetyl-CoA and a known HDAC inhibitor that induces cell differentiation in colon cells. The dual role of butyrate raises the question whether its effects on HT29 cell differentiation are due to butyrate metabolism or to its HDAC inhibitor activity. To distinguish between these two possibilities, we used a tracer-based metabolomics approach to compare the metabolic changes induced by two different types of HDAC inhibitors (butyrate and the non-metabolic agent trichostatin A) and those induced by other acetyl-CoA precursors that do not inhibit HDAC (caprylic and capric acids). [1,2-13C2]-d-glucose was used as a tracer and its redistribution among metabolic intermediates was measured to estimate the contribution of glycolysis, the pentose phosphate pathway and the Krebs cycle to the metabolic profile of HT29 cells under the different treatments. The results demonstrate that both HDAC inhibitors (trichostatin A and butyrate) induce a common metabolic profile that is associated with histone deacetylase inhibition and differentiation of HT29 cells whereas the metabolic effects of acetyl-CoA precursors are different from those of butyrate. The experimental findings support the concept of crosstalk between metabolic and cell signalling events, and provide an experimental approach for the rational design of new combined therapies that exploit the potential synergism between metabolic adaptation and cell differentiation processes through modification of HDAC activity.

Islet amyloid polypeptide exerts a novel autocrine action in β-cell signaling and proliferation

The toxic effects of human islet amyloid polypeptide (IAPP) on pancreatic islets have been widely studied. However, much less attention has been paid to the physiologic actions of IAPP on pancreatic β cells, which secrete this peptide together with insulin upon glucose stimulation. Here, we aimed to explore the signaling pathways and mitogenic actions of IAPP on β cells. We show that IAPP activated Erk1/2 and v‐akt murine thymoma viral oncogene homolog 1 (Akt) at the picomolar range (10‐100 pM) in mouse pancreatic islets and MIN6 β cells cultured at low glucose concentrations. In contrast, IAPP decreased the induction of these pathways by high glucose levels. Consistently, IAPP induced a 1.7‐fold increase of β‐cell proliferation at low‐glucose conditions, whereas it reduced β‐cell proliferation at high glucose levels. Strikingly, the specific antagonist of the IAPP receptor AC187 (100 nM) decreased the activation of Erk1/2 and Akt and reduced β‐cell proliferation by 24% in glucose‐stimulated β cells, uncovering a key role of endogenously released IAPP in β‐cell responses to glucose. We conclude that exogenously added IAPP exerts a dual effect on β‐cell mitogenic signaling and proliferation, depending on the glucose concentration. Importantly, secreted IAPP contributes to the signaling and mitogenic response of β cells to glucose through an autocrine mechanism.— Visa, M., Alcarraz‐Vizán, G., Montane, J., Cadavez, L., Castaño, C., Villanueva‐Peñacarrillo, M. L., Servitja, J.‐M., Novials, A. Islet amyloid polypeptide exerts a novel autocrine action in β‐cell signaling and proliferation. FASEB J. 29, 2970‐2979 (2015). www.fasebj.org

Epicatechin gallate impairs colon cancer cell metabolic productivity.

Green tea and grape phenolics inhibit cancer growth and modulate cellular metabolism. Targeting the tumor metabolic profile is a novel therapeutic approach to inhibit cancer cell proliferation. Therefore, we treated human colon adenocarcinoma HT29 cells with the phenolic compound epicatechin gallate (ECG), one of the main catechins in green tea and the most important catechin in grape extracts, and evaluated its antiproliferation effects. ECG reduced tumor viability and induced apoptosis, necrosis, and S phase arrest in HT29 cells. Later, biochemical determinations combined with mass isotopomer distribution analysis using [1,2-(13)C2]-D-glucose as a tracer were used to characterize the metabolic network of HT29 cells in response to different concentrations of ECG. Glucose consumption was importantly decreased after ECG treatment. Moreover, metabolization of [1,2-(13)C2]-D-glucose indicated that the de novo synthesis of fatty acids and the pentose phosphate pathway were reduced in ECG-treated cells. Interestingly, ECG inhibited the activity of transketolase and glucose-6-phosphate dehydrogenase, the key enzymes of the pentose phosphate pathway. Our data point to ECG as a promising chemotherapeutic agent for the treatment of colon cancer.

Protein disulfide isomerase ameliorates β-cell dysfunction in pancreatic islets overexpressing human islet amyloid polypeptide

Human islet amyloid polypeptide (hIAPP) is the major component of amyloid deposits in islets of type 2 diabetic patients. hIAPP misfolding and aggregation is one of the factors that may lead to β-cell dysfunction and death. Endogenous chaperones are described to be important for the folding and functioning of proteins. Here, we examine the effect of the endoplasmic reticulum chaperone protein disulfide isomerase (PDI) on β-cell dysfunction. Among other chaperones, PDI was found to interact with hIAPP in human islet lysates. Furthermore, intrinsically recovered PDI levels were able to restore the effect of high glucose- and palmitate-induced β-cell dysfunction by increasing 3.9-fold the glucose-stimulated insulin secretion levels and restoring insulin content up to basal control values. Additionally, PDI transduction decreased induced apoptosis by glucolipotoxic conditions. This approach could reveal a new therapeutic target and aid in the development of strategies to improve β-cell dysfunction in type 2 diabetic patients.

Inhibition of BACE2 counteracts hIAPP-induced insulin secretory defects in pancreatic β-cells

BACE2 (β‐site APP‐cleaving enzyme 2) is a protease localized in the brain, where it appears to play a role in the development of Alzheimer disease (AD). It is also found in the pancreas, although its biologic function is not fully known. Amyloidogenic diseases, including AD and type 2 diabetes mellitus (T2D), share the accumulation of abnormally folded and insoluble proteins that interfere with cell function. Islet amyloid polypeptide (IAPP) deposits are a key pathogenic feature of T2D. Within this context, we found by global gene expression profiling that BACE2 was up‐regulated in the rat pancreatic β‐cell line INS1E stably transfected with human IAPP gene (hIAPP‐INS1E). Glucose‐stimulated insulin secretion (GSIS) in hIAPP‐INS1E cells was 30% lower than in INS1E cells. Additionally, INS1E cells transfected with a transient overexpression of BACE2 showed a 60% decrease in proliferation, a 3‐fold increase in reactive oxygen species production, and a 25% reduction in GSIS compared to control cells. Remarkably, silencing of endogenous BACE2 in hIAPP‐INS1E cells resulted in a significant improvement in GSIS (3‐fold increase vs. untransfected cells), revealing the significant role of BACE2 expression in β‐cell function. Thus, BACE2 inhibition may be useful to recover insulin secretion in hIAPP‐INS1E defective cells and may be proposed as a therapeutic target for T2D.‐ Montane, J., Servitja, J.‐M., Novials, A. Inhibition of BACE2 counteracts hIAPP‐induced insulin secretory defects in pancreatic β‐cells. FASEB J. 29, 95–104 (2015). www.fasebj.org

Validation of NCM460 cell model as control in antitumor strategies targeting colon adenocarcinoma metabolic reprogramming: trichostatin A as a case study.

BACKGROUND Cancer cells have extremely active metabolism, which supports high proliferation rates. Metabolic profiles of human colon cancer cells have been extensively studied, but comparison with non-tumour counterparts has been neglected. METHODS Here we compared the metabolic flux redistribution in human colon adenocarcinoma cells (HT29) and the human colon healthy cell line NCM460 in order to identify the main pathways involved in metabolic reprogramming. Moreover, we explore if induction of differentiation in HT29 by trichostatin A (TSA) reverts the metabolic reprogramming to that of NCM460. Cells were incubated with [1,2-(13)C2]-d-glucose as a tracer, and Mass Isotopomer Distribution Analysis was applied to characterize the changes in the metabolic flux distribution profile of the central carbon metabolism. RESULTS We demonstrate that glycolytic rate and pentose phosphate synthesis are 25% lower in NCM460 with respect to HT29 cells. In contrast, Krebs cycle activity in the former was twice that recorded in the latter. Moreover, we show that TSA-induced HT29 cell differentiation reverts the metabolic phenotype to that of healthy NCM460 cells whereas TSA does not affect the metabolism of NCM460 cells. CONCLUSIONS We conclude that pentose phosphate pathway, glycolysis, and Krebs cycle are key players of colon adenocarcinoma cellular metabolic remodeling and that NCM460 is an appropriate model to evaluate the results of new therapeutic strategies aiming to selectively target metabolic reprogramming. GENERAL SIGNIFICANCE Our findings suggest that strategies to counteract robust metabolic adaptation in cancer cells might open up new avenues to design multiple hit and targeted therapies.