Alberto Valdés, Virginia García-Cañas *, Carolina Simó, Clara Ibáñez, Vicente Micol, Jose A. Ferragut, Alejandro Cifuentes - PDF

Alberto Valdés, Virginia García-Cañas *, Carolina Simó, Clara Ibáñez, Vicente Micol, Jose A. Ferragut, Alejandro Cifuentes Laboratory of Foodomics, Institute of Food Science Research (CIAL), CSIC, Nicolas

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Alberto Valdés, Virginia García-Cañas *, Carolina Simó, Clara Ibáñez, Vicente Micol, Jose A. Ferragut, Alejandro Cifuentes Laboratory of Foodomics, Institute of Food Science Research (CIAL), CSIC, Nicolas Cabrera 9, Campus de Cantoblanco, Madrid, Spain. Institute of Molecular and Cellular Biology, Miguel Hernandez University. Avda. Universidad s/n, Elche, Alicante, Spain. ABSTRACT: In previous works, rosemary extracts (REs) obtained in our laboratory using supercritical fluid extraction exhibited inhibitory effect on proliferation of several cancer cell lines. However, the observed antiproliferative activity was not correlated to any compound present in the extract. In order to address this question, in this work the contribution of carnosic acid (CA) and carnosol (CS), two major compounds present in the RE, against colon cancer HT-29 cells proliferation is investigated using a comprehensive Foodomics approach. Although CA and CS exhibit additive antiproliferative effect when they are combined in solution at a molar ratio of 6.9:1, the results reveal that CA contributes more significantly than CS to the activity of RE against colon cancer cells proliferation. The Foodomics study reveals that CA induces transcriptional activation of genes that encode detoxifying enzymes and altered the expression of genes linked to other relevant functions such as 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 evidences 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 evidences 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 exploring novel metabolic processes and potential signaling pathways to further elucidate de effect of CA in colon cancer cells. Rosmarinus officinalis L., commonly known as rosemary, is a Labiatae species from Mediterranean origin. Phenolic diterpenes as carnosic acid (CA) and carnosol (CS) are abundant in rosemary leaves, representing approximately 5% of the dry weight of rosemary leaves, and accounting for 90% of the antioxidant activity of rosemary extracts. 1 For many years, the potent antioxidant activity of rosemary extracts has attracted great interest for the preservation of food material, and recently the European Union (EU) has approved the use of rosemary extracts as additives for food preservation. 2 In addition to their strong antioxidant properties, rosemary extracts have been also of interest for their anti-inflammatory and anticancer activities. 3 A great body of evidence from in vitro and in vivo studies indicates that rosemary extracts exert chemoprotective effects through different mechanisms. 4,5 Such chemoprotective activity has also conferred upon rosemary extracts cancer preventive properties. 6 Thus, the antiproliferative effect of rosemary extracts have been studied on leukemia, 7 ovarian cancer, 8 liver cancer 9 and colon cancer 10 cells. The anticancer activities of the major rosemary diterpenes, CA and CS, have been also object of several studies In addition to the antiproliferative and antiinvasive activities of CA, in vitro and in vivo neuroprotection is one of the most widely studied biological activities that have been attributed to this particular diterpene. 16,17 In general, rosemary extracts, as well as CA and CS, show differential antiproliferative activity in several human tumor cell lines. 18 One of the mechanisms underlying the antiproliferative activity of these compounds is linked to their ability for altering cell cycle progression. However, although both diterpenes are structurally related, they affect different phases of cell cycle. Furthermore, these compounds appear to exert a different effect on cell cycle phase distribution depending on the concentration assayed and the cell type. 10,15 Apart from the possible alteration of cell cycle regulatory genes, other specific mechanisms associated to the antiproliferative activity of rosemary compounds cannot be dismissed since they seem to be differentially effective against cells with specific mutational patterns. 7 However, comprehensive studies centered on the investigation of the mechanisms operating at various molecular levels in cancer cells in response to individual rosemary polyphenols are still lacking. In this regard, a global methodology such as Foodomics, based on the combination of several omics platforms and data processing, is well suited to perform comprehensive evaluations of the health benefits of food ingredients In previous work, a polyphenol-enriched rosemary extract (RE) obtained in our laboratory using supercritical fluid extraction exhibited inhibitory effect on proliferation of several cancer cell models. 7,10,24,25 Our findings suggested that the RE altered some cell cycle regulatory genes as well as various signaling pathways in colon cancer cells. However, the observed antiproliferative activity has not been correlated to any compound present in the extract yet. In order to address this question, the contribution of two major compounds, CA and CS, present in the RE, to the activity of the extract on HT-29 cell proliferation is investigated in this work. To achieve this, we have conducted proliferation inhibition assays and studied potential synergistic, additive or antagonistic effects of both diterpenes. Then, we focused our experiments on the effects of CA on cell cycle distribution analyses in combination with a Foodomics evaluation based on genome-wide transcriptomics and metabolomics analysis to investigate the cellular and molecular changes operating in HT-29 cells in response to CA treatments. Functional enrichment analysis was applied as a previous step for a reliable data interpretation obtained from transcriptomics and metabolomics, and for cross-platform data integration. CA and CS were purchased as pure standards (Sigma-Aldrich, St. Louis, MO, USA). The rosemary extract (RE) was obtained from dried rosemary leaves using supercritical CO 2 and 7% ethanol at 150 bar as reported by Herrero et al. 26 Chemical characterization of the RE indicated that two main diterpenes, CA and CS, were found at high concentrations in the RE, namely, and 37.1 µg/mg extract, respectively. Dry extract and standards samples were dissolved in DMSO (Sigma-Aldrich) at the appropriate concentrations and stored as aliquots at -80 ºC until use. Colon adenocarcinoma HT-29 cells obtained from ATCC (American Type Culture Collection, LGC Promochem, UK) were grown in McCoy s 5A supplemented with 10% heat-inactivated fetal calf serum, 50 U/mL penicillin G, and 50 U/mL streptomycin, at 37 ºC in humidified atmosphere and 5% CO 2. When cells reached ~50% confluence, they were trypsinized, neutralized with culture medium, plated in different culture plates and allowed to adhere overnight at 37 ºC. In order to study the effect of rosemary extracts on the proliferation of HT-29 line, cells were seeded onto 96-well culture plates at 10,000 cells/cm 2, permitted to adhere overnight at 37 ºC, and exposed to different treatments with different concentrations of CA, CS or RE for h depending on the experiment. After incubation with the bioactive compounds for the indicated time in each case, cell proliferation was estimated by the MTT assay as follows: 0.5 mg/ml of MTT reagent (Sigma-Aldrich) was added and incubated for 3 h at 37 ºC in humidified 5% CO 2 /air atmosphere. After the incubation, the media were aspirated and 100 µl of DMSO was added to each well to dissolve the formazan (the metabolic product of MTT). Then, the absorbance at 570 nm was measured in a microplate reader (Multiskan FC Microplate Photometer, Thermo Fisher Scientific, Vantaa, Finland). Results are provided as the mean ± 95% confidence interval of at least three independent experiments, each performed in triplicate. Cell viability at the beginning of the treatment (time zero) was used to calculate the percent of growth (PG) and the following parameters related to cell proliferation: GI50 (50% growth inhibition), TGI (total growth inhibition), and LC50 (50% cell death). These parameters were calculated according to the NIH definitions. 27 Combination assays were performed by treating HT-29 cells with appropriate concentrations of CA, CS and mixtures with the same molar ration of both. Cell proliferation inhibition was determined using MTT assay, as previously described. In the assessment of synergism, antagonism and additive effects, the combination index (CI) method was used according to Chou- Talalay equation. 28 The calculations were performed using CompuSyn software from Biosoft (Cambridge, UK), which takes into account both the potency (Dm or IC50) and shape of the dose-effect curve. CI 1 indicates synergism; CI = 1 indicates an additive effect; and CI 1 indicates antagonism. Results are shown as the mean ± standard error of the mean of three independent experiments, each performed in triplicate. Cells were seeded onto tissue culture dishes at 10,000 cells/cm 2, permitted to adhere overnight at 37 ºC, and incubated with 12.5 µg/ml CA or 30 µg/ml RE in complete culture medium up to 72 h. Then, cell cycle distribution was measured using flow cytometry at different time points. Briefly, for cell cycle distribution of DNA content, control and treated cells were trypsinized, washed with PBS, and fixed with 70 % cold ethanol at -20 ºC for at least 24 h. Then, fixed cells were resuspended in 0.5 ml PI/RNase Staining Buffer (BD Pharmingen, San Jose, CA, USA), incubated for 15 min in the dark, and analyzed on a Gallios flow cytometer equipped with a 0.75 W argon laser set at 488 nm (Beckman Coulter, Miami Lakes, FL, USA). Events were gated for peak width and area to exclude subcellular debris and aggregates. A total of 10,000 events were recorded for each sample and a frequency histogram of peak area was generated and analysed using Cylchred (V ) software (University of Wales College of Medicine, Cardiff, UK). Results are provided as the mean of the percentage of treated minus control samples ± SEM (standard error of the mean) of at least three independent experiments, each performed in triplicate. The results were analyzed using the analysis of variance (ANOVA) with Tukey post hoc test and differences were considered significant at p 0.05. A Foodomics strategy, involving transcriptomics and metabolomics profiling, was applied to study molecular mechanisms operating in HT-29 cells after CA treatment. Comparative transcriptomic analysis was performed on HT-29 cells incubated with 9.9 µg/ml CA, and their respective untreated controls. Microarray analyses and data processing were performed with triplicate samples for each experimental condition as described previously using Human Gene 1.0ST chips (Affymetrix). 10 After quality assessment using Expression ConsoleTM (Affymetrix), CEL files were processed using the Robust Multi-Array (RMA) normalization in the BioConductor package affy for R ( 29 Significance analysis was performed using the BioConductor package limma. 30 Moderated t statistics was used for significance analysis of each probe set and each contrast, and then false discovery rate was estimated using Benjamini and Hochberg s method for multiple testing correction. 31 To identify the statistically most significant changes in gene expression, microarray data were subjected to gene filtering based on the statistical significance (false discovery ratio, FDR, applied on moderated t statistics, adjusted p value 0.05). Reverse transcription quantitative PCR (RTqPCR) was used to confirm relative changes in mrna levels of selected genes from microarray datasets. Further details are available in the Supporting Information (Supporting methods). In this work, computational tools were used for functional enrichment and pathway analysis as a previous step for a reliable data interpretation obtained from microarray analysis. The bioinformatic tool Ingenuity Pathway Analysis (IPA, Ingenuity Systems, USA) was used in order to interpret the gene expression data in the context of biological processes and pathways. To this aim, the Core Analysis function included in IPA was applied to analyze the lists of differentially expressed genes (DEGs) identified in microarray analysis. In each analysis, expression parameters cut-offs were set at 0.4 as M-value cutoff that corresponds to expression ratios (fold change) 1.3 for up-regulated and 0.8 for down-regulated genes. From the gene set, up- and down-regulated identifiers were defined as value parameters for the analysis. Based on the list of identifiers, IPA performs functional enrichment analysis in order to identify the biological processes and functions overrepresented in a given list of genes. The p-value, calculated with the Fischer's exact test, reflected the likelihood that the association between a set of genes in our dataset and a related biological function is significant (p-value 0.05). The biological functions that were expected to be increased or decreased according to the gene expression changes in our dataset were identified using the IPA regulation z-score algorithm. A positive or negative z-score value indicates that a function is predicted to be increased or decreased in treated relative to untreated cells. Metabolic profiles from HT-29 cells were determined using two complementary analytical platforms (CE-TOF MS and HILIC/UHPLC-TOF MS) in order to widen the metabolic coverage. CE-TOF MS was carried out using a P/ACE 5500 CE system (Beckman Instruments, Fullerton, CA, USA) connected to a TOF MS instrument (microtof model) from Bruker Daltonics (Bremen, Germany) by an orthogonal electrospray ionization (ESI) interface G1607A from Agilent Technologies (Palo Alto, CA, USA). Metabolites were separated in a fused silica capillary (90 cm, 50 μm i.d.) filled with 3M formic acid as electrolyte applying a voltage of +27 kv at 25ºC. Isopropanol-water (1:1, v/v) was delivered as the sheath liquid at 0.24 ml/min. ESI-TOF MS was performed in the positive ion mode, and the capillary voltage was set at 4 kv. The flow rate of heated dry nitrogen gas at 200 C was maintained at 0.4 bar. Other conditions regarding external and internal calibration of the TOF-MS are given as Supporting Information. UHPLC-TOF MS was carried out using an Agilent 1290 system connected to a quadrupole-time-of-flight (Q/TOF) 6540 (Agilent Technologies) operating in the positive ion mode. Hydrophilic interaction liquid chromatography (HILIC) was performed on a ZORBAX HILIC Plus HT (2.1 mm 50 mm, 1.8 m) column maintained at 30 ºC. Elution was performed using phase A (water with 5 mm ammonium formate at ph 5.8) and phase B (acetonitrile with 0.1% (v/v) formic acid). Detailed elution gradient is given as Supporting Information. TOF-MS operation parameters were the following: capillary voltage, 4000 V; nebulizer pressure, 40 psi; drying gas flow rate, 10 L/min; gas temperature, 200 C; skimmer voltage, 45 V; fragmentor voltage was 125 V; m/z mass scan. Conditions regarding internal mass calibration and external calibration of the Q/TOF mass spectrometer are given as Supporting Information. Metabolomic data signals obtained from CE-MS and UHPLC-MS were exported to the MS exchange format mzxml using Trapper (available at trapper). CE-MS data was then processed with MZmine program (version 2.7.2). 32 Parameters applied for mass detection, peak deconvolution and sample alignment are described elsewhere. 33 LC-MS data was processed using XCMS package written in the platform-independent programming language R. 34 The resulting sample files were then converted to peakml format to execute the mzmatch.r package. 35 Parameters for LC-MS analysis have been already summarized. 36 Finally peaks showing high variability within the same group (i.e.control and treated cells) were removed (with a value of median/average 1.5) in both CE-MS and LC-MS data sets. The resulting output.csv data tables of high quality timealigned detected peaks with their corresponding migration/retention time, m/z and peak area obtained for each sample were merged into one file to perform statistical analysis using STATISTICA program (v.7, Statsoft, Tulsa, OK, USA, Significant molecular features were detected applying an ANOVA with p-value set at Both CE- MS and LC-MS data processing and statistical analysis were carried out using a PC Intel Core i7-2600k Processor (8M Cache, up to 3.80 GHz and 64 bits) operating under Microsoft Windows 7. The metabolites showing significant differences (p 0.05) were subjected to tentative identification process by matching the experimental accurate m/z values and those contained in different free available databases, namely, Human Metabolome Database, 37 METLIN, 38 and KEGG, 39 with a mass accuracy window of 10 ppm. When available, metabolite standards from Sigma-Aldrich were used to confirm identifications. In previous work, a polyphenol-enriched rosemary extract (RE) obtained in our laboratory using supercritical fluid extraction exhibited inhibitory effect on proliferation of cancer cell models. 7,10,24,25 Chemical characterization of the extract indicated the presence of CA and CS at concentrations of and 37.1 µg/mg extract, respectively, as well as other minor polyphenols (caffeic acid and p-coumaric acid). However, the observed antiproliferative activity of the extract has not been yet correlated to any compound present in the mixture. In order to address this point, the effect of the two major diterpenes (CA and CS) found in the extract, for which pure standards are commercially available, was investigated on HT-29 cells. To determine the antiproliferative effect of CA and CS in relation to the RE, HT-29 cells were incubated with increasing concentrations of RE, CA and CS (from 0 to 33.2 μg/ml) for 24 h (Figure 1A), 48 h (Figure 1B) and 72 h (Figure 1C) and cell proliferation was analyzed by the MTT assay. As it can be observed in Figure 1, after treatment with increasing concentrations of CA, dose-dependent reduction of cell proliferation was observed starting from 8.3 µg/ml at 48 and 72 h (Figure 1B and 1C). Moreover, a considerable reduction (~ 40%) of cell proliferation is observed between 24 and 72 h after treatment with 12.5 μg/ml CA. The treatment with CS also resulted in a reduction of cell proliferation in a dose- and timedependent manner similar to that observed for CA (Figure 1). In order to deep in the mechanisms that can explain the antiproliferative activity of these diterpenes, the response parameters, growth inhibition (GI50), total growth inhibition (TGI) as an indicator for cytostaticity, and lethal concentration (LC50) indicative for the cytotoxic level of effect were also determined for 24, 48 and 72 h incubation times (Supporting Information, Table S1). Although the inhibitory activities of both diterpenes were similar, our results indicate that CA exerted a slightly stronger effect than CS on cell proliferation provided by the lower values obtained for the parameters at the different incubation times. Moreover, the maximum inhibitory effect of CA was achieved at 48 h while CS showed its strongest inhibitory activity after 72 h. According to the literature, both diterpenes have demonstrated different in vitro cytotoxic and cytostatic effects depending on the cell type, concentration, and time of exposition. 10,1
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