José A. Ferragut

Global Foodomics strategy to investigate the health benefits of dietary constituents.

A global methodology, called Foodomics, which allows carrying out a comprehensive evaluation of the health benefits of food ingredients is presented in this work. The new methodology is based on the combination of several analytical platforms and data processing for Transcriptomics, Proteomics and Metabolomics studies, allowing the determination of changes induced by food ingredients at molecular level. Both, the whole methodological development and its potential are presented through the investigation of a case study following a hypothesis-free strategy. Namely, the chemopreventive effect of polyphenols from rosemary was examined on the total gene, protein and metabolite expression in human HT29 colon cancer cells. Conclusions on the bioactivity of polyphenols against colon cancer cells based on the results from each single platform (Transcriptomics, Proteomics or Metabolomics) are compared with the conclusions based on the integration of the whole results from the three platforms, corroborating the interest of using a global integrative strategy as Foodomics. To our knowledge, although many papers and reviews have been published on this topic, this is the first time that Transcriptomics, Proteomics and Metabolomics platforms are put together to study the health benefits from dietary ingredients against colon cancer cells at gene, protein and metabolite level. Advantages, drawbacks and current challenges of this global analytical strategy are discussed in this work. The results from our study provide new insights on the biological mechanisms involved in the cancer risk reduction properties of dietary constituents.

CE/LC-MS multiplatform for broad metabolomic analysis of dietary polyphenols effect on colon cancer cells proliferation

In this study, an analytical multiplatform is presented to carry out a broad metabolomic study on the anti‐proliferative effect of dietary polyphenols on human colon cancer cells. CE, RP/UPLC, and HILIC/UPLC all coupled to TOF MS were combined to achieve a global metabolomic examination of the effect of dietary polyphenols on HT29 colon cancer cells. By the use of a nontargeted metabolomic approach, metabolites showing significant different expression after the polyphenols treatment were identified in colon cancer cells. It was demonstrated that this multianalytical platform provided extensive metabolic information and coverage due to its complementary nature. Differences observed in metabolic profiles from CE‐TOF MS, RP/UPLC‐TOF MS, and HILIC/UPLC‐TOF MS can be mainly assigned to their different separation mechanisms without discarding the influence of the different tools used for data processing. Changes in glutathione metabolism with an enhanced reduced glutathione/oxidized glutathione (GSH/GSSG) ratio were detected in polyphenols‐treated cells. Moreover, significant alterations in polyamines content with important implications in cancer proliferation were observed after the treatment with polyphenols. These results from metabolomics can explain the chemopreventive effect of the tested dietary polyphenols on colon cancer and may be of importance for future prevention and/or treatment of this disease.

Histone deacetylase inhibitors induced caspase-independent apoptosis in human pancreatic adenocarcinoma cell lines.

The antitumor activity of the histone deacetylase inhibitors was tested in three well-characterized pancreatic adenocarcinoma cell lines, IMIM-PC-1, IMIM-PC-2, and RWP-1. These cell lines have been previously characterized in terms of their origin, the status of relevant molecular markers for this kind of tumor, resistance to other antineoplastic drugs, and expression of differentiation markers. In this study, we report that histone deacetylase inhibitors induce apoptosis in pancreatic cancer cell lines, independently of their intrinsic resistance to conventional antineoplastic agents. The histone deacetylase inhibitor–induced apoptosis is due to a serine protease–dependent and caspase-independent mechanism. Initially, histone deacetylase inhibitors increase Bax protein levels without affecting Bcl-2 levels. Consequently, the apoptosis-inducing factor (AIF) and Omi/HtrA2 are released from the mitochondria, with the subsequent induction of the apoptotic program. These phenomena require AIF relocalization into the nuclei to induce DNA fragmentation and a serine protease activity of Omi/HtrA2. These data, together with previous results from other cellular models bearing the multidrug resistance phenotype, suggest a possible role of the histone deacetylase inhibitors as antineoplastic agents for the treatment of human pancreatic adenocarcinoma.

Effect of dietary polyphenols on K562 leukemia cells: A Foodomics approach

In this work, a global Foodomics strategy has been applied to study the antiproliferative effect of dietary polyphenols from rosemary on two human leukemia lines, one showing a drug‐sensitive phenotype (K562), and another exhibiting a drug‐resistant phenotype (K562/R). To this aim, whole‐transcriptome microarray together with an MS‐based nontargeted analytical approach (via CE‐TOF MS and UPLC‐TOF MS) have been employed to carry out transcriptomics and metabolomics analyses, respectively. Functional enrichment analysis was done using ingenuity pathway analysis (IPA) software as a previous step for a reliable interpretation of transcriptomic and metabolomic profiles. Rosemary polyphenols altered the expression of approximately 1% of the genes covered by the whole transcriptome microarray in both leukemia cell lines. Overall, differences in the transcriptional induction of a number of genes encoding phase II detoxifying and antioxidant genes, as well as differences in the metabolic profiles observed in the two leukemia cell lines suggest that rosemary polyphenols may exert a differential chemopreventive effect in leukemia cells with different phenotypes. IPA predictions on transcription factor analysis highlighted inhibition of Myc transcription factor function by rosemary polyphenols, which may explain the observed antiproliferative effect of rosemary extract in the leukemia cells. Metabolomics analysis suggested that rosemary polyphenols affected differently the intracellular levels of some metabolites in two leukemia cell sublines. Integration of data obtained from transcriptomics and metabolomics platforms was attempted by overlaying datasets on canonical (defined) metabolic pathways using IPA software. This strategy enabled the identification of several differentially expressed genes in the metabolic pathways modulated by rosemary polyphenols providing more evidences on the effect of these compounds.

Is metabolomics reachable? Different purification strategies of human colon cancer cells provide different CE-MS metabolite profiles

In this work, four different metabolite purification approaches are investigated prior to metabolomics of human HT29 colon cancer cells. Namely, methanol deproteinization, ultrafiltration and two SPE methods using C18 and polymer‐based cartridges were studied. The extracts were characterized via a metabolomic approach based on the application of CE TOF MS (CE‐MS). CE‐MS analysis time was less than 20 min per sample and allowed the simultaneous and reproducible analysis of more than 80 metabolites in a single run with a minimum consumption of sample and reagents. Metabolome analysis revealed in some cases important differences among the studied metabolite purification procedures. No significant differences were observed in the metabolite profile using C18 and polymer‐based cartridges, or between ultrafiltration and methanol deproteinization. However, important differences were observed in the metabolomic profiles obtained from SPE and methanol deproteinization samples. These results demonstrate the crucial role of the metabolite purification strategy in metabolomics since it can bias (and in some cases mislead) the conclusions achieved by the metabolomic study.

Post-transcriptional Regulation of P-Glycoprotein Expression in Cancer Cell Lines

The present study of inhibitors shows that the histone deacetylase–induced increase in P-glycoprotein (Pgp) mRNA (MDR1 mRNA) does not parallel either an increase in Pgp protein or an increase in Pgp activity in several colon carcinoma cell lines. Furthermore, studying the polysome profile distribution, we show a translational control of Pgp in these cell lines. In addition, we show that the MDR1 mRNA produced in these cell lines is shorter in its 5′ end that the MDR1 mRNA produced in the MCF-7/Adr (human breast carcinoma) and K562/Adr (human erythroleukemia) cell lines, both of them expressing Pgp. The different size of the MDR1 mRNA is due to the use of alternative promoters. Our data suggest that the translational blockade of MDR1 mRNA in the colon carcinoma cell lines and in wild-type K562 cells could be overcome by alterations in the 5′ end of the MDR1 mRNA in the resistant variant of these cell lines, as in the case of the K562/Adr cell line. This is, to our knowledge, the first report demonstrating that the presence of an additional 5′ untranslated fragment in the MDR1 mRNA improves the translational efficiency of this mRNA. (Mol Cancer Res 2007;5(6):641–53)

Small tyrosine kinase inhibitors interrupt EGFR signaling by interacting with erbB3 and erbB4 in glioblastoma cell lines.

Signaling through the epidermal growth factor receptor (EGFR) is relevant in glioblastoma. We have determined the effects of the EGFR inhibitor AG1478 in glioblastoma cell lines and found that U87 and LN-229 cells were very sensitive to this drug, since their proliferation diminished and underwent a marked G(1) arrest. T98 cells were a little more refractory to growth inhibition and A172 cells did not undergo a G(1) arrest. This G(1) arrest was associated with up-regulation of p27(kip1), whose protein turnover was stabilized. EGFR autophosphorylation was blocked with AG1478 to the same extent in all the cell lines. Other small-molecule EGFR tyrosine kinase inhibitors employed in the clinic, such as gefitinib, erlotinib and lapatinib, were able to abrogate proliferation of glioblastoma cell lines, which underwent a G(1) arrest. However, the EGFR monoclonal antibody, cetuximab had no effect on cell proliferation and consistently, had no effect on cell cycle either. Similarly, cetuximab did not inhibit proliferation of U87 ΔEGFR cells or primary glioblastoma cell cultures, whereas small-molecule EGFR inhibitors did. Activity of downstream signaling molecules of EGFR such as Akt and especially ERK1/2 was interrupted with EGFR tyrosine kinase inhibitors, whereas cetuximab treatment could not sustain this blockade over time. Small-molecule EGFR inhibitors were able to prevent phosphorylation of erbB3 and erbB4, whereas cetuximab only hindered EGFR phosphorylation, suggesting that EGFR tyrosine kinase inhibitors may mediate their anti-proliferative effects through other erbB family members. We can conclude that small-molecule EGFR inhibitors may be a therapeutic approach for the treatment of glioblastoma patients.

Comprehensive foodomics study on the mechanisms operating at various molecular levels in cancer cells in response to individual rosemary polyphenols.

In this work, the contribution of carnosic acid (CA) and carnosol (CS), two major compounds present in rosemary, against colon cancer HT-29 cells proliferation is investigated using a comprehensive Foodomics approach. The Foodomics study reveals that CA induces transcriptional activation of genes that encode detoxifying enzymes and altered the expression of genes linked to transport and biosynthesis of terpenoids in the colon cancer cell line. Functional analysis highlighted the activation of the ROS metabolism and alteration of several genes involved in pathways describing oxidative degradation of relevant endogenous metabolites, providing new evidence about the transcriptional change induced by CA in HT-29 cells. Metabolomics analysis showed that the treatment with CA affected the intracellular levels of glutathione. Elevated levels of GSH provided additional evidence to transcriptomic results regarding chemopreventive response of cells to CA treatment. Moreover, the Foodomics approach was useful to establish the links between decreased levels of N-acetylputrescine and its degradation pathway at the gene level. The findings from this work and the predictions based on microarray data will help explore novel metabolic processes and potential signaling pathways to further elucidate the effect of CA in colon cancer cells.

Study of the Anticancer Properties of Tin(IV) Carboxylate Complexes on a Panel of Human Tumor Cell Lines

A group of organotin(IV) complexes were prepared: [SnCy3(DMNI)] (1), [SnCy3(BZDO)] (2), [SnCy3(DMFU)] (3), and [SnPh2(BZDO)2] (4), for which DMNIH=2,6‐dimethoxynicotinic acid, BZDOH=1,4‐benzodioxane‐6‐carboxylic acid, and DMFUH=2,5‐dimethyl‐3‐furoic acid. The cytotoxic activities of compounds 1–4 were tested against pancreatic carcinoma (PANC‐1), erythroleukemia (K562), and two glioblastoma multiform (U87 and LN‐229) human cell lines; they show very high antiproliferative activity, with IC50 values in the 150–700 nM range after incubation for 72 h. Distribution of cellular DNA upon treatment with 1–4 revealed that whereas compounds 1–3 induce apoptosis in most of the cell lines, compound 4 does not affect cell viability in any cell line tested, indicating a possible difference in cytotoxic mechanism. Studies with the daunomycin‐resistant K562/R cell line expressing P‐glycoprotein (Pgp) showed that compounds 1–4 are not substrates of this protein efflux pump, indicating that these compounds do not induce acquisition of multidrug resistance, which is associated with the overexpression of Pgp.

Multidrug resistance-associated protein (MRP1) gene is strongly expressed in gastric carcinomas. Analysis by immunohistochemistry and real-time quantitative RT-PCR

Aims : To assess MRP1 protein and MRP1 mRNA levels in gastric carcinomas and in non‐neoplastic mucosa remote from the tumours. MRP1 gene expression may play a role in the complex pattern of chemoresistance present in gastric carcinomas.