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.

Decurarization After Thoracic Anesthesia using sugammadex compared to neostigmine (DATA trial): a multicenter randomized double-blinded controlled trial.

BACKGROUND: Thoracic surgery is a high-risk surgery especially for the risk of postoperative pulmonary complications. Postoperative residual paralysis has been shown to be a risk factor for pulmonary complications. Nevertheless, there are few data in the literature concerning the use of neuromuscular blocking agent antagonists in patients undergoing lung surgery. METHODS: Seventy patients were randomized in three Italian centers to receive sugammadex or neostigmine at the end of thoracic surgery according to the depth of the residual neuromuscular block. The primary outcome was the time from reversal administration to a train-of-four ratio (TOFR) of 0.9. Secondary outcomes were the time to TOFR of 1.0, to extubation, to postanesthesia unit (PACU) discharge, postoperative complications until 30 days after surgery, and length of hospital stay. RESULTS: Median time to recovery to a TOFR of 0.9 was significantly shorter in the sugammadex group compared to the neostigmine one (88 vs. 278 s - P < 0.001). The percentage of patients who recovered to a TOFR of 0.9 within 5 min from reversal administration was 94.4% and 58.8% in the sugammadex and neostigmine groups, respectively (P < 0.001). The time to extubation, but not the PACU stay time, was significantly shorter in the sugammadex group. No differences were found between the study groups as regards postoperative complications and length of hospital stay. The superiority of sugammadex in shortening the recovery time was confirmed for both deep/moderate and shallow/minimal neuromuscular block. CONCLUSIONS: Among patients undergoing thoracic surgery, sugammadex ensures a faster recovery from the neuromuscular block and earlier extubation compared to neostigmine.

Potential role of the hydroxyl carboxylic acid receptor type 2 (HCAR2) in microglia pathophysiology: A possible cross-talk with C-X-C chemokine receptor 1 (CXCR1).

Following insults or injury, microglia cells are activated contributing to the cytotoxic response or by promoting an immune-mediated damage resolution. Microglia cells express HCA2R, a hydroxy carboxylic acid (HCA) receptor, which has been shown to mediate neuroprotective and anti-inflammatory effects. In this study we found that HCAR2 expression levels were increased in cultured rat microglia cells after Lipopolysaccharide (LPS) exposure. In a similar fashion, the treatment with MK 1903, a potent full agonist of HCAR2, increased the receptor protein levels. Moreover, HCAR2 stimulation prevented i) cells viability ii) morphological activation iii) pro/anti-inflammatory mediators production in LPS-treated cells. Likewise, HCAR2 stimulation reduced the proinflammatory mediators mRNA expression induced by neuronal chemokine fractalkine (FKN), a neuronal derived chemokine activating its unique receptor, chemokine receptor 1 (CX3CR1) on microglia surface. Interestingly, electrophysiological recordings in vivo revealed that MK1903 was able to prevent the increase of the nociceptive neurons (NS) firing activity mediated by the spinal FKN application in healthy rats. Collectively, our data demonstrate that HCAR2 is functionally expressed in microglia, by showing its capability to shift microglia toward an anti-inflammatory phenotype. Moreover, we indicated the contribute of HCAR2 in the FKN signaling and suggested a possible HCAR2/CX3CR1 functional interaction. This study paves the way for further investigations aimed at understanding the role HCAR2 as potential target in neuroinflammation-based CNS disorders. This article is part of the Special Issue on "The receptor-receptor interaction as a new target for therapy".

Novel insights in the clinical management of hyperimmune patients before and after transplantation.

Despite improvements in anti-Human Leucocyte Antigens antibody detection, identification, and characterization offer a better in peri-operative management techniques, antibodies remain a serious cause of morbidity and mortality for patients both before and after organ transplantation. Hyperimmune patients are disadvantaged by having to wait longer to receive an organ from a suitably matched donor. They could benefit from desensitization protocols in both pre- and post-transplantation period. Clinical studies are underway to highlight which best desensitization strategies could be assure the best outcome in both heart and kidney transplantation. Although most clinical evidence about desensitization strategies by using anti-CD20 monoclonal antibodies, proteasome inhibitors, anti-CD38 monoclonal antibodies, interleukin-6 blockade, cysteine protease and complement inhibitors, comes from kidney transplantation studies, many of the debated novel concepts can be easily applied to desensitization also in heart transplantation. Here, we discuss the candidates and recipients' management by using most common standard of care and novel therapeutics, desensitization endpoints, and strategies for future studies.

Homo-AMPA in the periaqueductal grey modulates pain and rostral ventromedial medulla activity in diabetic neuropathic mice.

The 2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)-butyric acid, homo-AMPA, an analog of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and 2-aminoadipic acid, has shown no activity towards ionotropic and metabotropic glutamate 1, 2, 3, 4, 5, and 7 receptors (mGluR1-7), agonist activity at mGluR6 while the activity at mGluR8 was never investigated. The effect of homo-AMPA on pain control has been never investigated. In this study we evaluated the effect of intra-ventrolateral periaqueductal grey (VL PAG) microinjections of homo-AMPA on pain responses and the activity of pain-responding neurons of the rostral ventromedial medulla (RVM), the "pronociceptive" ON cells, and the "antinociceptive" OFF cells. The study was performed in control and diabetic neuropathic mice. Homo-AMPA decreased mechanical allodynia in diabetic neuropathic mice. Homo-AMPA increased also the latency to tail-flick, decreased the ongoing activity, the pain stimulus-evoked burst of firing, and the duration of the burst of the ON cells in both, control and neuropathic mice. Homo-AMPA also increased the ongoing activity, decreased and delayed the pause of the OFF cells in control mice. Unlike the retina, we did not find the transcript and protein for mGluR6 in the VL PAG. Alpha-methyl-serine-O-phosphate, a group III mGluRs antagonist, blocked the anti-allodynic effect of homo-AMPA. Considering the absence of both, mGluR6 in VL-PAG and homo-AMPA activity at mGluR4 and mGluR7 at the dose used, mGluR8 could be the target on which homo-AMPA produces the observed effects. The target of homo-AMPA capable of evoking analgesia at a very low dose and in conditions of diabetic neuropathy deserves further consideration.

PEA-OXA ameliorates allodynia, neuropsychiatric and adipose tissue remodeling induced by social isolation.

Chronic social isolation generates a persistent state of stress associated with obesity along with some neuro-endocrine disorders and central behavioral sequelae (eg anxiety, depression, aggression, and allodynia). In this study, we evaluated the effect of social isolation on body weight, depressive- and anxious-aggressive-like behavior, as well as on phenotypic changes of adipocytes from visceral adipose tissue of control (group-housed) or socially isolated (single-housed) male mice. The effect of treatment with pentadecyl-2-oxazoline (PEA-OXA), a natural alpha2 antagonist and histamine H3 protean partial agonist, on these alterations was also evaluated. Single or group-housed mice treated with vehicle or PEA-OXA underwent body weight, mechanical allodynia, anxious-, depressive- and aggressive-like behavior measurements. Proliferation rate, apoptosis, senescence, expression of fat lineage genes, lipid droplets and proinflammatory cytokines were measured on white adipose tissue adipocytes from group- or single-housed mice. Single housed mice developed weight gain, mechanical allodynia at the von Frey test, aggressiveness in the resident intruder test, depression- and anxiety-like behavior in the tail suspension and hole drop tests, respectively. Single housed mice receiving PEA-OXA showed a general resolution of both, physical-metabolic and behavioral alterations associated with social isolation. Furthermore, adipocytes from the adipose tissue of socially isolated mice showed an evident inflamed phenotype (i.e. a reduced rate of proliferation, apoptosis, senescence, and ROS hyper-production together with an increased expression of IL-1ß, IL-10, IL-17, and TNF-α and a decrease of IL-6). The treatment with PEA-OXA on adipocytes from single housed mice produced a protective/anti-inflammatory phenotype with an increased expression of brown adipose tissue biomarker. This study confirms that persistent stress caused by social isolation predisposes to obesity and neuropsychiatric disorders. PEA-OXA, through its multi-target activity on alpha2 adrenoceptor and histamine H3 receptors, which have recently aroused great interest in the neuropsychiatric field, reduces weight gain, systemic pro-inflammatory state, allodynia, and affective disorders associated with social isolation.

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.

Ribosome Biogenesis and Cancer: Overview on Ribosomal Proteins.

Cytosolic ribosomes (cytoribosomes) are macromolecular ribonucleoprotein complexes that are assembled from ribosomal RNA and ribosomal proteins, which are essential for protein biosynthesis. Mitochondrial ribosomes (mitoribosomes) perform translation of the proteins essential for the oxidative phosphorylation system. The biogenesis of cytoribosomes and mitoribosomes includes ribosomal RNA processing, modification and binding to ribosomal proteins and is assisted by numerous biogenesis factors. This is a major energy-consuming process in the cell and, therefore, is highly coordinated and sensitive to several cellular stressors. In mitochondria, the regulation of mitoribosome biogenesis is essential for cellular respiration, a process linked to cell growth and proliferation. This review briefly overviews the key stages of cytosolic and mitochondrial ribosome biogenesis; summarizes the main steps of ribosome biogenesis alterations occurring during tumorigenesis, highlighting the changes in the expression level of cytosolic ribosomal proteins (CRPs) and mitochondrial ribosomal proteins (MRPs) in different types of tumors; focuses on the currently available information regarding the extra-ribosomal functions of CRPs and MRPs correlated to cancer; and discusses the role of CRPs and MRPs as biomarkers and/or molecular targets in cancer treatment.

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.

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.

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.

Role of Autophagy in Cancer Cell Response to Nucleolar and Endoplasmic Reticulum Stress.

Eukaryotic cells are exposed to many internal and external stimuli that affect their fate. In particular, the exposure to some of these stimuli induces stress triggering a variety of stress responses aimed to re-establish cellular homeostasis. It is now established that the deregulation of stress response pathways plays a central role in cancer initiation and progression, allowing the adaptation of cells to an altered state in the new environment. Autophagy is a tightly regulated pathway which exerts "housekeeping" role in physiological processes. Recently, a growing amount of evidence highlighted the crucial role of autophagy in the regulation of integrated stress responses, including nucleolar and endoplasmic reticulum. In this review, we attempt to afford an overview of the complex role of nucleolar and endoplasmic reticulum stress-response mechanisms in the regulation of autophagy in cancer and cancer treatment.

Arachidyl amido cholanoic acid improves liver glucose and lipid homeostasis in nonalcoholic steatohepatitis via AMPK and mTOR regulation.

BACKGROUND: Arachidyl amido cholanoic acid (Aramchol) is a potent downregulator of hepatic stearoyl-CoA desaturase 1 (SCD1) protein expression that reduces liver triglycerides and fibrosis in animal models of steatohepatitis. In a phase IIb clinical trial in patients with nonalcoholic steatohepatitis (NASH), 52 wk of treatment with Aramchol reduced blood levels of glycated hemoglobin A1c, an indicator of glycemic control. AIM: To assess lipid and glucose metabolism in mouse hepatocytes and in a NASH mouse model [induced with a 0.1% methionine and choline deficient diet (0.1MCD)] after treatment with Aramchol. METHODS: Isolated primary mouse hepatocytes were incubated with 20 µmol/L Aramchol or vehicle for 48 h. Subsequently, analyses were performed including Western blot, proteomics by mass spectrometry, and fluxomic analysis with 13C-uniformly labeled glucose. For the in vivo part of the study, male C57BL/6J mice were randomly fed a control or 0.1MCD for 4 wk and received 1 or 5 mg/kg/d Aramchol or vehicle by intragastric gavage for the last 2 wk. Liver metabolomics were assessed using ultra-high-performance liquid chromatography-time of flight-MS for the determination of glucose metabolism-related metabolites. RESULTS: Combination of proteomics and Western blot analyses showed increased AMPK activity while the activity of nutrient sensor mTORC1 was decreased by Aramchol in hepatocytes. This translated into changes in the content of their downstream targets including proteins involved in fatty acid (FA) synthesis and oxidation [P-ACCα/ß(S79), SCD1, CPT1A/B, HADHA, and HADHB], oxidative phosphorylation (NDUFA9, NDUFB11, NDUFS1, NDUFV1, ETFDH, and UQCRC2), tricarboxylic acid (TCA) cycle (MDH2, SUCLA2, and SUCLG2), and ribosome (P-p70S6K[T389] and P-S6[S235/S236]). Flux experiments with 13C-uniformely labeled glucose showed that TCA cycle cataplerosis was reduced by Aramchol in hepatocytes, as indicated by the increase in the number of rounds that malate remained in the TCA cycle. Finally, liver metabolomic analysis showed that glucose homeostasis was improved by Aramchol in 0.1MCD fed mice in a dose-dependent manner, showing normalization of glucose, G6P, F6P, UDP-glucose, and Rbl5P/Xyl5P. CONCLUSION: Aramchol exerts its effect on glucose and lipid metabolism in NASH through activation of AMPK and inhibition of mTORC1, which in turn activate FA ß-oxidation and oxidative phosphorylation.

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.

Comparison Between Swan-Ganz Catheter and Minimally Invasive Hemodynamic Monitoring During Liver Transplantation: Report of a Monocentric Case Series.

INTRODUCTION: The aim of the present investigation was to retrospectively evaluate the utilization of Swan-Ganz catheter during orthotopic liver transplantation as opposed to FloTrac/Vigileo in selected cases, comparing a number of clinical outcomes across postoperative hospitalization. MATERIALS AND METHODS: Before 2015 all recipients received pulmonary artery catheter (Swan-Ganz group, n = 109). After 2015 Swan-Ganz was used only if coronary artery disease or high-grade portal hypertension or Child-Pugh C were present; the remaining recipients were assigned to FloTrac/Vigileo monitoring (Mini group, n =100). A number of clinical outcomes were considered. RESULTS: Donor's Risk Index was similar between groups (median value 1.7, P = .27). Anthropometric characteristics of the recipients were similar in the 2 groups. There were no significant differences in the proportion of patients with Child-Pugh C (P = .873), coronary artery disease (P = .18), and grade of portal hypertension (P = .733). The Model for End-Stage Liver Disease score was slightly higher in the Mini group: (9 [7-11] vs 9 [8-12], Swan-Ganz vs Mini, respectively, P < .035). Swan-Ganz utilization decreased over time (92% vs 26%, Swan-Ganz vs Mini, P < .001). Upon admission to the intensive care unit, patients of the Mini group presented a higher SAPS II score with similar values of Sequential Organ Failure Assessment score. Days on mechanical ventilation were similar between groups. The incidence of graft failure was similar between groups (2% vs 5%, Swan-Ganz and Mini group respectively, P = .376). Recipients' hospital length of stay was similar (13 days [11-19] vs 14 [11-20], P < .083). CONCLUSIONS: Our data suggest that the intraoperative utilization of FloTrac/Vigileo for oncologic patients with low grade end stage liver disease is reasonably safe.

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.

Evolution shapes the responsiveness of the D-box enhancer element to light and reactive oxygen species in vertebrates.

The circadian clock is a highly conserved cell-autonomous mechanism that directs daily rhythms in most aspects of biology. Daily entrainment by environmental signals, notably light, is essential for its function. However, our understanding of the mechanisms and the evolution of photic entrainment remains incomplete. Fish represent attractive models for exploring how light regulates the circadian clock due to the direct light sensitivity of their peripheral clocks. Central to this property is the light induced expression of clock genes that is mediated by D-box enhancer elements. Here, using zebrafish cells, we reveal that the light responsive D-box enhancer serves as a nuclear target for reactive oxygen species (ROS). We demonstrate that exposure to short wavelengths of visible light triggers increases in ROS levels via NADPH oxidase activity. Elevated ROS activates the JNK and p38 MAP kinases and in turn, induces clock gene expression via the D-box. In blind cavefish and mammals, where peripheral clocks are no longer entrained by direct illumination, ROS levels are still increased upon light exposure. However, in these species ROS no longer induces D-box driven clock gene transcription. Thus, during evolution, alterations in ROS-responsive signal transduction pathways underlie fundamental changes in peripheral clock photoentrainment.