Antitumoral Activity of the Universal Methyl Donor S-Adenosylmethionine in Glioblastoma Cells.

Glioblastoma (GBM), the most frequent and lethal brain cancer in adults, is characterized by short survival times and high mortality rates. Due to the resistance of GBM cells to conventional therapeutic treatments, scientific interest is focusing on the search for alternative and efficient adjuvant treatments. S-Adenosylmethionine (AdoMet), the well-studied physiological methyl donor, has emerged as a promising anticancer compound and a modulator of multiple cancer-related signaling pathways. We report here for the first time that AdoMet selectively inhibited the viability and proliferation of U87MG, U343MG, and U251MG GBM cells. In these cell lines, AdoMet induced S and G2/M cell cycle arrest and apoptosis and downregulated the expression and activation of proteins involved in homologous recombination DNA repair, including RAD51, BRCA1, and Chk1. Furthermore, AdoMet was able to maintain DNA in a damaged state, as indicated by the increased γH2AX/H2AX ratio. AdoMet promoted mitotic catastrophe through inhibiting Aurora B kinase expression, phosphorylation, and localization causing GBM cells to undergo mitotic catastrophe-induced death. Finally, AdoMet inhibited DNA repair and induced cell cycle arrest, apoptosis, and mitotic catastrophe in patient-derived GBM cells. In light of these results, AdoMet could be considered a potential adjuvant in GBM therapy.

S-Adenosylmethionine Inhibits Colorectal Cancer Cell Migration through Mirna-Mediated Targeting of Notch Signaling Pathway.

Metastasis is a leading cause of mortality and poor prognosis in colorectal cancer (CRC). Thus, the identification of new compounds targeting cell migration represents a major clinical challenge. Recent findings evidenced a central role for dysregulated Notch in CRC and a correlation between Notch overexpression and tumor metastasis. MicroRNAs (miRNAs) have been reported to cross-talk with Notch for its regulation. Therefore, restoring underexpressed miRNAs targeting Notch could represent an encouraging therapeutic approach against CRC. In this context, S-adenosyl-L-methionine (AdoMet), the universal biological methyl donor, being able to modulate the expression of oncogenic miRNAs could act as a potential antimetastatic agent. Here, we showed that AdoMet upregulated the onco-suppressor miRNAs-34a/-34c/-449a and inhibited HCT-116 and Caco-2 CRC cell migration. This effect was associated with reduced expression of migration-/EMT-related protein markers. We also found that, in colorectal and triple-negative breast cancer cells, AdoMet inhibited the expression of Notch gene, which, by luciferase assay, resulted the direct target of miRNAs-34a/-34c/-449a. Gain- and loss-of-function experiments with miRNAs mimics and inhibitors demonstrated that AdoMet exerted its inhibitory effects by upregulating miRNAs-34a/-34c/-449a. Overall, these data highlighted AdoMet as a novel Notch inhibitor and suggested that the antimetastatic effects of AdoMet involve the miRNA-mediated targeting of Notch signaling pathway.

Polyphenol Extract from "Greco" Grape Canes: Characterization, Antioxidant Capacity, and Antitumor Effects on Cal-33 and JHU-SCC-011 Head and Neck Squamous Cell Carcinoma.

In the current study, we determined the antioxidant properties of "Greco" grape cane extracts, a typical cultivar of southern Italy. We also explored the anticancer activity of the polyphenol-rich fraction of the extract on head and neck squamous carcinoma cells (HNSCC) and investigated the underlying mechanism. Aqueous extracts were prepared at different pHs and extraction times and the total phenolic and reducing sugar contents were estimated. Radical Scavenging Activity (RSA), Ferric Reducing Antioxidant Power (FRAP), and Total Antioxidant Capacity (TAC) of the extracts were measured. A polyphenol-rich fraction, accounting for 6.7% by weight and characterized mainly by procyanidins and stilbenoids, was prepared from the extract obtained at pH 7 for 60 min. We demonstrated that the extract exerted a cytotoxic effect on HNSCC cell lines by inducing cell cycle arrest via cyclin downregulation and p21 upregulation, and by triggering apoptosis through caspase cascade activation, PARP-1 cleavage, and an increase in the Bax/Bcl-2 ratio. We furnished evidence that the polyphenol-rich fraction played the major role in the anticancer activity of the extract. These outcomes highlighted grape canes from the "Greco" cultivar as a valuable source of polyphenols that may represent good candidates for the design of innovative adjuvant therapies in the treatment of HNSCC.

S-Adenosylmethionine Increases the Sensitivity of Human Colorectal Cancer Cells to 5-Fluorouracil by Inhibiting P-Glycoprotein Expression and NF-κB Activation.

Colorectal cancer (CRC) is the second deadliest cancer worldwide despite significant advances in both diagnosis and therapy. The high incidence of CRC and its poor prognosis, partially attributed to multi-drug resistance and antiapoptotic activity of cancer cells, arouse strong interest in the identification and development of new treatments. S-Adenosylmethionine (AdoMet), a natural compound and a nutritional supplement, is well known for its antiproliferative and proapoptotic effects as well as for its potential in overcoming drug resistance in many kinds of human tumors. Here, we report that AdoMet enhanced the antitumor activity of 5-Fluorouracil (5-FU) in HCT 116p53+/+ and in LoVo CRC cells through the inhibition of autophagy, induced by 5-FU as a cell defense mechanism to escape the drug cytotoxicity. Multiple drug resistance is mainly due to the overexpression of drug efflux pumps, such as P-glycoprotein (P-gp). We demonstrate here that AdoMet was able to revert the 5-FU-induced upregulation of P-gp expression and to decrease levels of acetylated NF-κB, the activated form of NF-κB, the major antiapoptotic factor involved in P-gp-related chemoresistance. Overall, our data show that AdoMet, was able to overcome 5-FU chemoresistance in CRC cells by targeting multiple pathways such as autophagy, P-gp expression, and NF-κB signaling activation and provided important implications for the development of new adjuvant therapies to improve CRC treatment and patient outcomes.

Mutual Correlation between Non-Coding RNA and S-Adenosylmethionine in Human Cancer: Roles and Therapeutic Opportunities.

Epigenetics includes modifications in DNA methylation, histone and chromatin structure, and expression of non-coding RNAs (ncRNAs), especially microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). Knowledge of the relationships between S-adenosylmethionine (AdoMet or SAM), the universal methyl donor for all epigenetic methylation reactions and miRNAs or lncRNAs in human cancer may provide helpful insights for the development of new end more effective anticancer therapeutic approaches. In recent literature, a complex network of mutual interconnections between AdoMet and miRNAs or lncRNAs has been reported and discussed. Indeed, ncRNAs expression may be regulated by epigenetic mechanisms such as DNA and RNA methylation and histone modifications. On the other hand, miRNAs or lncRNAs may influence the epigenetic apparatus by modulating the expression of its enzymatic components at the post-transcriptional level. Understanding epigenetic mechanisms, such as dysregulation of miRNAs/lncRNAs and DNA methylation, has become of central importance in modern research. This review summarizes the recent findings on the mechanisms by which AdoMet and miRNA/lncRNA exert their bioactivity, providing new insights to develop innovative and more efficient anticancer strategies based on the interactions between these epigenetic modulators.

Mi-RNA-888-5p Is Involved in S-Adenosylmethionine Antitumor Effects in Laryngeal Squamous Cancer Cells.

(1) Purpose: The methyl donor S-Adenosylmethionine (AdoMet) has been widely explored as a therapeutic compound, and its application-alone or in combination with other molecules-is emerging as a potential effective strategy for the treatment and chemoprevention of tumours. In this study, we investigated the antitumor activity of AdoMet in Laryngeal Squamous Cell Carcinoma (LSCC), exploring the underlying mechanisms. (2) Results: We demonstrated that AdoMet induced ROS generation and triggered autophagy with a consistent increase in LC3B-II autophagy-marker in JHU-SCC-011 and HNO210 LSCC cells. AdoMet induced ER-stress and activated UPR signaling through the upregulation of the spliced form of XBP1 and CHOP. To gain new insights into the molecular mechanisms underlying the antitumor activity of AdoMet, we evaluated the regulation of miRNA expression profile and we found a downregulation of miR-888-5p. We transfected LSCC cells with miR-888-5p inhibitor and exposed the cells to AdoMet for 48 and 72 h. The combination of AdoMet with miR-888-5p inhibitor synergistically induced both apoptosis and inhibited cell migration paralleled by the up-regulation of MYCBP and CDH1 genes and of their targets. (3) Conclusion: Overall, these data highlighted that epigenetic reprogramming of miRNAs by AdoMet play an important role in inhibiting apoptosis and migration in LSCC cell lines.

S-Adenosyl-l-Methionine Overcomes uL3-Mediated Drug Resistance in p53 Deleted Colon Cancer Cells.

PURPOSE: In order to study novel therapeutic approaches taking advantage of natural compounds showing anticancer and anti-proliferative effects, we focused our interest on S-adenosyl-l-methionine, a naturally occurring sulfur-containing nucleoside synthesized from adenosine triphosphate and methionine by methionine adenosyltransferase, and its potential in overcoming drug resistance in colon cancer cells devoid of p53. RESULTS: In the present study, we demonstrated that S-adenosyl-l-methionine overcomes uL3-mediated drug resistance in p53 deleted colon cancer cells. In particular, we demonstrated that S-adenosyl-l-methionine causes cell cycle arrest at the S phase; inhibits autophagy; augments reactive oxygen species; and induces apoptosis in these cancer cells. CONCLUSIONS: Results reported in this paper led us to propose S-adenosyl-l-methionine as a potential promising agent for cancer therapy by examining p53 and uL3 profiles in tumors to yield a better clinical outcomes.

Therapeutic Potential of the Natural Compound S-Adenosylmethionine as a Chemoprotective Synergistic Agent in Breast, and Head and Neck Cancer Treatment: Current Status of Research.

The present review summarizes the most recent studies focusing on the synergistic antitumor effect of the physiological methyl donor S-adenosylmethionine (AdoMet) in association with the main drugs used against breast cancer and head and neck squamous cell carcinoma (HNSCC), two highly aggressive and metastatic malignancies. In these two tumors the chemotherapy approach is recommended as the first choice despite the numerous side effects and recurrence of metastasis, so better tolerated treatments are needed to overcome this problem. In this regard, combination therapy with natural compounds, such as AdoMet, a molecule with pleiotropic effects on multiple cellular processes, is emerging as a suitable strategy to achieve synergistic anticancer efficacy. In this context, the analysis of studies conducted in the literature highlighted AdoMet as one of the most effective and promising chemosensitizing agents to be taken into consideration for inclusion in emerging antitumor therapeutic modalities such as nanotechnologies.

Annurca apple polyphenol extract promotes mesenchymal-to-epithelial transition and inhibits migration in triple-negative breast cancer cells through ROS/JNK signaling.

Aberrant activation of epithelial-to-mesenchymal transition has been shown to correlate with triple-negative breast cancer (TNBC) progression and metastasis. Thus, the induction of the reverse process might offer promising opportunities to restrain TNBC metastatic spreading and related mortality. Recently, the Annurca apple polyphenol extract (APE) has been highlighted as a multi-faceted agent that selectively kills TNBC cells by ROS generation and sustained JNK activation. Here, by qualitatively and quantitatively monitoring the real-time movements of live cells we provided the first evidence that APE inhibited the migration of MDA-MB-231 and MDA-MB-468 TNBC cells and downregulated metalloproteinase-2 and metalloproteinase-9. In MDA-MB-231 cells APE decreased SMAD-2/3 and p-SMAD-2/3 levels, increased E-cadherin/N-cadherin protein ratio, induced the switch from N-cadherin to E-cadherin expression and greatly reduced vimentin levels. Confocal and scanning electron microscopy imaging of APE-treated MDA-MB-231 cells evidenced a significant cytoskeletal vimentin and filamentous actin reorganization and revealed considerable changes in cell morphology highlighting an evident transition from the mesenchymal to epithelial phenotype with decreased migratory features. Notably, all these events were reverted by N-acetyl-L-cysteine and JNK inhibitor SP600125 furnishing evidence that APE exerted its effects through the activation of ROS/JNK signaling. The overall data highlighted APE as a potential preventing agent for TNBC metastasis.

Effects of S­adenosyl­L­methionine on the invasion and migration of head and neck squamous cancer cells and analysis of the underlying mechanisms.

S­Adenosyl­L­methionine (AdoMet) is the principal methyl donor in transmethylation reactions fundamental to sustaining epigenetic modifications. Over the past decade, AdoMet has been extensively investigated for its anti­proliferative, pro­apoptotic and anti­metastatic roles in several types of human cancer. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide, and is an aggressive type of cancer that is associated with a high recurrence rate, metastasis and poor treatment outcomes. The present study demonstrates, for the first time, to the best of our knowledge, that AdoMet induces cell cycle arrest and inhibits the migratory and invasive ability of two different HNSCC cell lines, oral Cal­33 and laryngeal JHU­SCC­011 cells. In both cell lines, AdoMet attenuated cell cycle progression, decreased the protein level of several cyclins and downregulated the expression of p21 cell cycle inhibitor. Moreover, AdoMet was able to inhibit Cal­33 and JHU­SCC­011 cell migration in a dose­dependent manner after 24 and 48 h, respectively, and also induced a significant reduction in the cell invasive ability, as demonstrated by Matrigel invasion assay monitored by the xCELLigence RTCA system. Western blot analysis of several migration and invasion markers confirmed the inhibitory effects exerted by AdoMet on these processes and highlighted AKT, ß­catenin and small mothers against decapentaplegic (SMAD) as the main signaling pathways modulated by AdoMet. The present study also demonstrated that the combination of AdoMet and cisplatin synergistically inhibited HNSCC cell migration. Taken together, these findings demonstrate that the physiological compound, AdoMet, affects the motility and extracellular matrix invasive capability in HNSCC. Thus, AdoMet may prove to be a good candidate for future drug development against metastatic cancer.

Structures of catalytic cycle intermediates of the Pyrococcus furiosus methionine adenosyltransferase demonstrate negative cooperativity in the archaeal orthologues.

Methionine adenosyltransferases catalyse the biosynthesis of S-adenosylmethionine, the primary methyl group donor in biochemical reactions, through the condensation of methionine and ATP. Here, we report the structural analysis of the Pyrococcus furiosus methionine adenosyltransferase (PfMAT) captured in the unliganded, substrate- and product-bound states. The conformational changes taking place during the enzymatic catalytic cycle are allosterically propagated by amino acid residues conserved in the archaeal orthologues to induce an asymmetric dimer structure. The distinct occupancy of the active sites within a PfMAT dimer is consistent with a half-site reactivity that is mediated by a product-induced negative cooperativity. The structures of intermediate states of PfMAT reported here suggest a distinct molecular mechanism for S-adenosylmethionine synthesis in Archaea, likely consequence of the evolutionary pressure to achieve protein stability under extreme conditions.

S-Adenosylmethionine Inhibits Cell Growth and Migration of Triple Negative Breast Cancer Cells through Upregulating MiRNA-34c and MiRNA-449a.

Triple-negative breast cancer (TNBC) is one of the most common malignancies worldwide and shows maximum invasiveness and a high risk of metastasis. Recently, many natural compounds have been highlighted as a valuable source of new and less toxic drugs to enhance breast cancer therapy. Among them, S-adenosyl-L-methionine (AdoMet) has emerged as a promising anti-cancer agent. MicroRNA (miRNA or miR)-based gene therapy provides an interesting antitumor approach to integrated cancer therapy. In this study, we evaluated AdoMet-induced modulation of miRNA-34c and miRNA-449a expression in MDA-MB-231 and MDA-MB-468 TNBC cells. We demonstrated that AdoMet upregulates miR-34c and miR-449a expression in both cell lines. We found that the combination of AdoMet with miR-34c or miR-449a mimic strongly potentiated the pro-apoptotic effect of the sulfonium compound by a caspase-dependent mechanism. For the first time, by video time-lapse microscopy, we showed that AdoMet inhibited the in vitro migration of MDA-MB-231 and MDA-MB-468 cells and that the combination with miR-34c or miR-449a mimic strengthened the effect of the sulfonium compound through the modulation of ß-catenin and Small Mother Against Decapentaplegic (SMAD) signaling pathways. Our results furnished the first evidence that AdoMet exerts its antitumor effects in TNBC cells through upregulating the expression of miR-34c and miR-449a.

Annurca apple polyphenol extract selectively kills MDA-MB-231 cells through ROS generation, sustained JNK activation and cell growth and survival inhibition.

Polyphenols represent the most studied class of nutraceuticals that can be therapeutics for a large spectrum of diseases, including cancer. In this study, we investigated for the first time the antitumor activities of polyphenol extract from Annurca apple (APE) in MDA-MB-231 triple negative breast cancer cells, and we explored the underlying mechanisms. APE selectively inhibited MDA-MB-231 cell viability and caused G2/M phase arrest associated with p27 and phospho-cdc25C upregulation and with p21 downregulation. APE promoted reactive oxygen species (ROS) generation in MDA-MB-231 cells while it acted as antioxidant in non-tumorigenic MCF10A cells. We demonstrated that ROS generation represented the primary step of APE antitumor activity as pretreatment with antioxidant N-acetylcysteine (NAC) prevented APE-induced G2/M phase arrest, apoptosis, and autophagy. APE downregulated Dusp-1 and induced a significant increase in JNK/c-Jun phosphorylation that were both prevented by NAC. Moreover, downregulation of JNK by its specific inhibitor SP600125 significantly diminished the anticancer activity of APE indicating that ROS generation and sustained JNK activation represented the main underlying mechanism of APE-induced cell death. APE also inhibited AKT activation and downregulated several oncoproteins, such as NF-kB, c-myc, and ß-catenin. In light of these results, APE may be an attractive candidate for drug development against triple negative breast cancer.

AdoMet triggers apoptosis in head and neck squamous cancer by inducing ER stress and potentiates cell sensitivity to cisplatin.

S-Adenosyl-l-methionine (AdoMet) is a naturally and widely occurring sulfonium compound that plays a primary role in cell metabolism and acts as the principal methyl donor in many methylation reactions. AdoMet also exhibits antiproliferative and proapoptotic activities in different cancer cells. However, the molecular mechanisms underlying the effects exerted by AdoMet have only been partially studied. In the current study, we evaluated the antiproliferative effect of AdoMet on Cal-33 oral and JHU-SCC-011 laryngeal squamous cancer cells to define the underlying mechanisms. We demonstrated that AdoMet induced apoptosis in Cal-33 and JHU-SCC-011 cells, involving a caspase-dependent mechanism paralleled by an increased Bax/Bcl-2 ratio. Moreover, we showed, for the first time, that AdoMet induced ER-stress in Cal-33 cells and activated the unfolded protein response, which can be responsible for apoptosis induction through the activation of CHOP and JNK. In addition, AdoMet-induced ER-stress was followed by autophagy with a consistent increase in the levels of the autophagic marker LC3B-II, which was indeed potentiated by the autophago-lysosome inhibitor chloroquine. As both escape from apoptosis and decreased activation of JNK are mechanisms of resistance to cisplatin (cDPP), an agent usually used in cancer therapy, we have evaluated the effects of AdoMet in combination with cDPP on Cal-33 cells. Our data showed that the combined treatment resulted in a strong synergism in inhibiting cell proliferation and in enhancing apoptosis via intrinsic mechanism. These results demonstrate that AdoMet has ER-stress-mediated antiproliferative activity and synergizes with cDDP on cell growth inhibition, thus providing the basis for its use in new anticancer strategies.

S-Adenosylmethionine regulates apoptosis and autophagy in MCF-7 breast cancer cells through the modulation of specific microRNAs.

BACKGROUND: To get insight into the molecular mechanisms underlying the anti-tumor activity of S-adenosyl-l-methionine (AdoMet), we analyzed AdoMet-induced modulation of microRNAs (miRNAs) expression profile in MCF-7 breast cell line and its correlation with cancer-related biological pathways. METHODS: MiRNA expression profiling was performed using a TaqMan MiRNA Array, following 500 µM AdoMet-treatment. The results were confirmed by Quantitative real-time PCR analysis. MCF-7 were transfected with miR-34a, miR-34c and miR-486-5p, mimics and inhibitors in presence or not of 500 µM AdoMet for 72 h. Apoptosis and autophagy were analyzed by flow cytometry and the modulation of the main antiproliferative signaling pathways were evaluated by Western blotting. The potential mRNA targets for each miRNA were identified by the TargetScan miRNA target prediction software. RESULTS: Twenty-eight microRNAs resulted differentially expressed in AdoMet-treated MCF-7 cells compared to control cells. Among them, miRNA-34a and miRNA-34c were up-regulated while miRNA-486-5p was down-regulated. Moreover, we confirmed the ability of AdoMet to regulate these miRNAs in MDA-MB 231 breast cancer cell line. We demonstrate that, in MCF7 cells, the combination of either miR-34a or miR-34c mimic with AdoMet greatly potentiated the pro-apoptotic effect of AdoMet, by a caspase-dependent mechanism and activates p53 acetylation by inhibiting SIRT1 and HDAC1 expression. We also showed that miR-486-5p inhibitor induces autophagy and enhances AdoMet-induced autophagic process by increasing PTEN expression and by inhibiting AKT signaling. CONCLUSIONS: Our findings provide the first evidence that AdoMet can regulate miRNA expression in MCF-7 increasing our knowledge on the molecular basis of the antitumor effect of the sulfonium compound and suggest the use of AdoMet as an attractive miRNA-mediated chemopreventive and therapeutic strategy in breast cancer.

S-Adenosylmethionine-mediated apoptosis is potentiated by autophagy inhibition induced by chloroquine in human breast cancer cells.

The naturally occurring sulfonium compound S-adenosyl-L-methionine (AdoMet) is an ubiquitous sulfur-nucleoside that represents the main methyl donor in numerous methylation reactions. In recent years, it has been shown that AdoMet possesses antiproliferative properties in various cancer cells, but the molecular mechanisms at the basis of the effect induced by AdoMet have been only in part investigated. In the present study, we found that AdoMet strongly inhibited the proliferation of breast cancer cells MCF-7 by inducing both autophagy and apoptosis. AdoMet consistently enhanced the levels of the autophagy markers beclin-1 and LC3B-II, and caused a significant increase of pro-apoptotic Bax/Bcl-2 ratio paralleled by poly (ADP ribose) polymerase (PARP) and caspase 9, and 6 cleavage. Notably, AdoMet, already at low doses, raised the percentage of cells in G2 /M phase of cell cycle by down-regulating the expression of cell cycle-regulatory proteins cyclin B and cyclin E with a remarkable increase of p53, p27, and p21. We also evaluated the combination of AdoMet and the autophagy inhibitor chloroquine (CLC) showing that autophagy block is synergistic in inducing both growth inhibition and apoptosis. These effects were paralleled by a strong inhibition of the activity of AKT and of the downstream effector mTOR and by an increased cleavage of caspase-6 and PARP. These data suggest, for the first time, that autophagy can act as an escape mechanism from the apoptotic activity of AdoMet, and that AdoMet could be used in combination with CLC or its analogs in the treatment of breast cancer.

Pro-oxidant and pro-apoptotic activity of polyphenol extract from Annurca apple and its underlying mechanisms in human breast cancer cells.

Among nutraceuticals, polyphenols represent the most intriguing and studied class of compounds that can be therapeutics for a large spectrum of the most common diseases, including cancer. Although polyphenols are well known as potent antioxidants, a pro-oxidant effect has been associated with a pro-apoptotic function of these compounds in various types of tumor cells. Annurca apple, a southern Italian variety, is characterized by an extremely high content of polyphenols and displays a stronger antioxidant activity compared with other varieties. In the present study we explored the antiproliferative effect of Annurca apple polyphenol extract (APE) in human breast cancer MCF-7 cells and we investigated the underlying mechanisms. Results showed that at 500 µM catechin equivalent (EqC) APE acts as a pro-oxidant increasing thiobarbituric acid-reactive species cell content of approximately 6-fold more than the untreated cells. We found that APE strongly inhibits the proliferation of MCF-7 cells by inducing G2/M cell cycle arrest and apoptosis. Immunoblot analysis demonstrated that APE treatment increases the levels of p53 and p21, downregulates the expression of the cell cycle regulatory protein cyclin D1, and inhibits ERK1/2 phosphorylation. Moreover, APE treatment caused a marked increase of pro-apoptotic Bax/Bcl-2 ratio paralleled by caspase-9, -6, -7, and poly(ADP ribose) polymerase cleavage. Altogether our data indicate that APE, at elevated concentrations, acts as a potent pro-oxidant and antiproliferative agent able to downregulate ERK1/2 pathway leading to cell cycle inhibition and apoptosis and provides a rationale for its potential use in the development of novel therapeutics towards breast cancer.

Protective Effect of Tyrosol and S-Adenosylmethionine against Ethanol-Induced Oxidative Stress of Hepg2 Cells Involves Sirtuin 1, P53 and Erk1/2 Signaling.

Oxidative stress plays a major role in ethanol-induced liver damage, and agents with antioxidant properties are promising as therapeutic opportunities in alcoholic liver disease. In the present work, we investigated the effect of S-adenosylmethionine (AdoMet), Tyrosol (Tyr), and their combination on HepG2 cells exposed to ethanol exploring the potential molecular mechanisms. We exposed HepG2 cells to 1 M ethanol for 4 and 48 h; thereafter, we recorded a decreased cell viability, increase of intracellular reactive oxygen species (ROS) and lipid accumulation, and the release into culture medium of markers of liver disease such as triacylglycerol, cholesterol, transaminases, albumin, ferritin, and homocysteine. On the other hand, AdoMet and Tyrosol were able to attenuate or antagonize these adverse changes induced by acute exposure to ethanol. The protective effects were paralleled by increased Sirtuin 1 protein expression and nuclear translocation and increased ERK1/2 phosphorylation that were both responsible for the protection of cells from apoptosis. Moreover, AdoMet increased p53 and p21 expression, while Tyrosol reduced p21 expression and enhanced the expression of uncleaved caspase 3 and 9, suggesting that its protective effect may be related to the inhibition of the apoptotic machinery. Altogether, our data show that AdoMet and Tyrosol exert beneficial effects in ethanol-induced oxidative stress in HepG2 cells and provide a rationale for their potential use in combination in the prevention of ethanol-induced liver damage.

Efficient Fludarabine-Activating PNP From Archaea as a Guidance for Redesign the Active Site of E. Coli PNP.

The combination of the gene of purine nucleoside phosphorylase (PNP) from Escherichia coli and fludarabine represents one of the most promising systems in the gene therapy of solid tumors. The use of fludarabine in gene therapy is limited by the lack of an enzyme that is able to efficiently activate this prodrug which, consequently, has to be administered in high doses that cause serious side effects. In an attempt to identify enzymes with a better catalytic efficiency than E. coli PNP towards fludarabine to be used as a guidance on how to improve the activity of the bacterial enzyme, we have selected 5'-deoxy-5'-methylthioadenosine phosphorylase (SsMTAP) and 5'-deoxy-5'-methylthioadenosine phosphorylase II (SsMTAPII), two PNPs isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. Substrate specificity and catalytic efficiency of SsMTAP and SsMTAPII for fludarabine were analyzed by kinetic studies and compared with E. coli PNP. SsMTAP and SsMTAPII share with E. coli PNP a comparable low affinity for the arabinonucleoside but are better catalysts of fludarabine cleavage with k(cat)/K(m) values that are 12.8-fold and 6-fold higher, respectively, than those reported for the bacterial enzyme. A computational analysis of the interactions of fludarabine in the active sites of E. coli PNP, SsMTAP, and SsMTAPII allowed to identify the crucial residues involved in the binding with this substrate, and provided structural information to improve the catalytic efficiency of E. coli PNP by enzyme redesign.