Pro-Inflammatory Cytokines Enhanced In Vitro Cytotoxic Activity of Clostridioides difficile Toxin B in Enteric Glial Cells: The Achilles Heel of Clostridioides difficile Infection?

Bacterial infections are characterized by an inflammatory response, which is essential for infection containment but is also responsible for negative effects on the host. The pathogen itself may have evolved molecular mechanisms to antagonize the antimicrobial effects of an inflammatory response and to enhance its pathogenicity using inflammatory response mediators, such as cytokines. Clostridioides difficile (C. difficile) infection (CDI) causes gastrointestinal diseases with markedly increasing global incidence and mortality rates. The main C. difficile virulence factors, toxin A and B (TcdA/TcdB), cause cytopathic/cytotoxic effects and inflammation. We previously demonstrated that TcdB induces enteric glial cell (EGC) apoptosis, which is enhanced by the pro-inflammatory cytokine tumor necrosis factor alpha plus interferon gamma (CKs). However, it is unknown whether CKs-enhanced TcdB cytotoxicity (apoptosis/necrosis) is affected by the timing of the appearance of the CKs. Thus, we simulated in vitro, in our experimental model with TcdB and EGCs, three main situations of possible interactions between TcdB and the timing of CK stimulation: before TcdB infection, concomitantly with infection, or at different times after infection and persisting over time. In these experimental conditions, which all represent situations of possible interactions between C. difficile and the timing of CK stimulation, we evaluated apoptosis, necrosis, and cell cycle phases. The CKs, in all of these conditions, enhanced TcdB cytotoxicity, which from apoptosis became necrosis when CK stimulation persisted over time, and was most relevant after 48 h of TcdB:EGCs interaction. Particularly, the enhancement of apoptosis by CKs was dependent on the TcdB dose and in a less relevant manner on the CK stimulation time, while the enhancement of necrosis occurred always independently of the TcdB dose and CK stimulation time. However, since in all conditions stimulation with CKs strongly enhanced the TcdB cytotoxicity, it always had a negative impact on C. difficile pathogenicity. This study might have important implications for the treatment of CDI.

Role of the Alteration in Calcium Homeostasis in Cell Death Induced by Clostridioides difficile Toxin A and Toxin B.

Clostridioides difficile (C. difficile), responsible for 15-25% of gastrointestinal infections, causes health problems mainly due to the toxic activity of toxins A and B (Tcds). These are responsible for its clinical manifestations, including diarrhea, pseudomembranous colitis, toxic megacolon and death, with a mortality of 5-30% in primary infection, that increase following relapses. Studies on Tcd-induced cell death have highlighted a key role of caspases, calpains, and cathepsins, with involvement of mitochondria and reactive oxygen species (ROS) in a complex signaling pathway network. The complex response in the execution of various types of cell death (apoptosis, necrosis, pyroptosis and pyknosis) depends on the amount of Tcd, cell types, and Tcd receptors involved, and could have as initial/precocious event the alterations in calcium homeostasis. The entities, peculiarities and cell types involved in these alterations will decide the signaling pathways activated and cell death type. Calcium homeostasis alterations can be caused by calcium influx through calcium channel activation, transient intracellular calcium oscillations, and leakage of calcium from intracellular stores. These increases in cytoplasmic calcium have important effects on all calcium-regulated molecules, which may play a direct role in several cell death types and/or activate other cell death effectors, such as caspases, calpains, ROS and proapoptotic Bcl-2 family members. Furthermore, some support for the possible role of the calcium homeostasis alteration in Tcd-induced cell death originates from the similarity with cytotoxic effects that cause pore-forming toxins, based mainly on calcium influx through plasma membrane pores.

Cardiolipin-mediated temporal response to hydroquinone toxicity in human retinal pigmented epithelial cell line.

Hydroquinone, a potent toxic agent of cigarette smoke, damages retinal pigmented epithelial cells by triggering oxidative stress and mitochondrial dysfunction, two events causally related to the development and progression of retinal diseases. The inner mitochondrial membrane is enriched in cardiolipin, a phospholipid susceptible of oxidative modifications which determine cell-fate decision. Using ARPE-19 cell line as a model of retinal pigmented epithelium, we analyzed the potential involvement of cardiolipin in hydroquinone toxicity. Hydroquinone exposure caused an early concentration-dependent increase in mitochondrial reactive oxygen species, decrease in mitochondrial membrane potential, and rise in the rate of oxygen consumption not accompanied by changes in ATP levels. Despite mitochondrial impairment, cell viability was preserved. Hydroquinone induced cardiolipin translocation to the outer mitochondrial membrane, and an increase in the colocalization of the autophagosome adapter protein LC3 with mitochondria, indicating the induction of protective mitophagy. A prolonged hydroquinone treatment induced pyroptotic cell death by cardiolipin-mediated caspase-1 and gasdermin-D activation. Cardiolipin-specific antioxidants counteracted hydroquinone effects pointing out that cardiolipin can act as a mitochondrial "eat-me signal" or as a pyroptotic cell death trigger. Our results indicate that cardiolipin may act as a timer for the mitophagy to pyroptosis switch and propose cardiolipin-targeting compounds as promising approaches for the treatment of oxidative stress-related retinal diseases.

Clostridioides difficile and colorectal cancer: a dangerous liaison.

Many colorectal diseases depend on complex interactions between several pathophysiological factors, including the intestinal microbiota. In recent years, the widespread use of antibiotics has been recognized as a main cause of intestinal dysbiosis and a favouring factor for Clostridioides difficile infection. The latter, in addition, causes infectious diarrhoea, pseudomembranous colitis, and toxic megacolon by means of its toxins (A and, especially, B), is characterized by frequent relapses; thus, its persistence in a host may be long-lasting. Based on recent experimental evidence, here we analyse the possibility that, similarly to other bacteria, Clostridioides difficile may be considered a potential carcinogen for colorectal cancer.

Clostridioides difficile Toxin B Induced Senescence: A New Pathologic Player for Colorectal Cancer?

Clostridioides difficile (C. difficile) is responsible for a high percentage of gastrointestinal infections and its pathological activity is due to toxins A and B. C. difficile infection (CDI) is increasing worldwide due to the unstoppable spread of C. difficile in the anthropized environment and the progressive human colonization. The ability of C. difficile toxin B to induce senescent cells and the direct correlation between CDI, irritable bowel syndrome (IBS), and inflammatory bowel diseases (IBD) could cause an accumulation of senescent cells with important functional consequences. Furthermore, these senescent cells characterized by long survival could push pre-neoplastic cells originating in the colon towards the complete neoplastic transformation in colorectal cancer (CRC) by the senescence-associated secretory phenotype (SASP). Pre-neoplastic cells could appear as a result of various pro-carcinogenic events, among which, are infections with bacteria that produce genotoxins that generate cells with high genetic instability. Therefore, subjects who develop IBS and/or IBD after CDI should be monitored, especially if they then have further CDI relapses, waiting for the availability of senolytic and anti-SASP therapies to resolve the pro-carcinogenic risk due to accumulation of senescent cells after CDI followed by IBS and/or IBD.

Cytotoxic synergism of Clostridioides difficile toxin B with proinflammatory cytokines in subjects with inflammatory bowel diseases.

Clostridioides difficile (C. difficile) is progressively colonizing humans and animals living with humans. During this process, hypervirulent strains and mutated toxin A and B of C. difficile (TcdA and TcdB) are originating and developing. While in healthy subjects colonization by C. difficile becomes a risk after the use of antibiotics that alter the microbiome, other categories of people are more susceptible to infection and at risk of relapse, such as those with inflammatory bowel disease (IBD). Recent in vitro studies suggest that this increased susceptibility could be due to the strong cytotoxic synergism between TcdB and proinflammatory cytokines the tumor necrosis factor-alpha and interferon-gamma (CKs). Therefore, in subjects with IBD the presence of an inflammatory state in the colon could be the driver that increases the susceptibility to C. difficile infection and its progression and relapses. TcdB is internalized in the cell via three receptors: chondroitin sulphate proteoglycan 4; poliovirus receptor-like 3; and Wnt receptor frizzled family. Chondroitin sulphate proteoglycan 4 and Wnt receptor frizzled family are involved in cell death by apoptosis or necrosis depending on the concentration of TcdB and cell types, while poliovirus receptor-like 3 induces only necrosis. It is possible that cytokines could also induce a greater expression of receptors for TcdB that are more involved in necrosis than in apoptosis. Therefore, in subjects with IBD there are the conditions: (1) For greater susceptibility to C. difficile infection, such as the inflammatory state, and abnormalities of the microbiome and of the immune system; (2) for the enhancement of the cytotoxic activity of TcdB +Cks; and (3) for a greater expression of TcdB receptors stimulated by cytokines that induce cell death by necrosis rather than apoptosis. The only therapeutic approach currently possible in IBD patients is monitoring of C. difficile colonization for interventions aimed at reducing tumor necrosis factor-alpha and interferon-gamma levels when the infection begins. The future perspective is to generate bacteriophages against C. difficile for targeted therapy.

The Polyunsaturated Fatty Acid EPA, but Not DHA, Enhances Neurotrophic Factor Expression through Epigenetic Mechanisms and Protects against Parkinsonian Neuronal Cell Death.

ω-3 Polyunsaturated fatty acids (PUFAs) have been found to exert many actions, including neuroprotective effects. In this regard, the exact molecular mechanisms are not well understood. Parkinson's disease (PD) is the second most common age-related neurodegenerative disease. Emerging evidence supports the hypothesis that PD is the result of complex interactions between genetic abnormalities, environmental toxins, mitochondrial dysfunction, and other cellular processes, such as DNA methylation. In this context, BDNF (brain-derived neurotrophic factor) and GDNF (glial cell line-derived neurotrophic factor) have a pivotal role because they are both involved in neuron differentiation, survival, and synaptogenesis. In this study, we aimed to elucidate the potential role of two PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and their effects on BDNF and GDNF expression in the SH-SY5Y cell line. Cell viability was determined using the MTT assay, and flow cytometry analysis was used to verify the level of apoptosis. Transmission electron microscopy was performed to observe the cell ultrastructure and mitochondria morphology. BDNF and GDNF protein levels and mRNA were assayed by Western blotting and RT-PCR, respectively. Finally, methylated and hydroxymethylated DNA immunoprecipitation were performed in the BDNF and GDNF promoter regions. EPA, but not DHA, is able (i) to reduce the neurotoxic effect of neurotoxin 6-hydroxydopamine (6-OHDA) in vitro, (ii) to re-establish mitochondrial function, and (iii) to increase BNDF and GDNF expression via epigenetic mechanisms.

Clostridioides difficile toxin B alone and with pro-inflammatory cytokines induces apoptosis in enteric glial cells by activating three different signalling pathways mediated by caspases, calpains and cathepsin B.

Clostridioides difficile infection (CDI) causes nosocomial/antibiotic-associated gastrointestinal diseases with dramatically increasing global incidence and mortality rates. The main C. difficile virulence factors, toxins A and B (TcdA/TcdB), cause cytopathic/cytotoxic effects and inflammation. We demonstrated that TcdB induces caspase-dependent, mitochondria-independent enteric glial cell (EGC) apoptosis that is enhanced by the pro-inflammatory cytokines TNF-α and IFN-γ (CKs) by increasing caspase-3/7/9 and PARP activation. Because this cytotoxic synergism is important for CDI pathogenesis, we investigated the apoptotic pathways involved in TcdB- and TcdB + CK-induced apoptosis indepth. EGCs were pre-treated with the inhibitors BAF or Q-VD-OPh (pan-caspase), Z-DEVD-fmk (caspase-3/7), Z-IETD-fmk (caspase-8), PD150606 (calpains), and CA-074Me (cathepsin B) 1 h before TcdB exposure, while CKs were given 1.5 h after TcdB exposure, and assays were performed at 24 h. TcdB and TcdB + CKs induced apoptosis through three signalling pathways activated by calpains, caspases and cathepsins, which all are involved both in induction and execution apoptotic signalling under both conditions but to different degrees in TcdB and TcdB + CKs especially as regards to signal transduction mediated by these proteases towards downstream effects (apoptosis). Calpain activation by Ca2+ influx is the first pro-apoptotic event in TcdB- and TcdB + CK-induced EGC apoptosis and causes caspase-3, caspase-7 and PARP activation. PARP is also directly activated by calpains which are responsible of about 75% of apoptosis in TcdB and 62% in TcdB + CK which is both effector caspase-dependent and -independent. Initiator caspase-8 activation mediated by TcdB contributes to caspase-3/caspase-7 and PARP activation and is responsible of about 28% of apoptosis in both conditions. Caspase-3/caspase-7 activation is weakly responsible of apoptosis, indeed we found that it mediates 27% of apoptosis only in TcdB. Cathepsin B contributes to triggering pro-apoptotic signal and is responsible in both conditions of about 35% of apoptosis by a caspase-independent manner, and seems to regulate the caspase-3 and caspase-7 cleaved fragment levels, highlighting the complex interaction between these cysteine protease families activated during TcdB-induced apoptosis. Further a relevant difference between TcdB- and TcdB + CK-induced apoptosis is that TcdB-induced apoptosis increased slowly reaching at 72 h the value of 18.7%, while TcdB + CK-induced apoptosis increased strongly reaching at 72 h the value of 60.6%. Apoptotic signalling activation by TcdB + CKs is enriched by TNF-α-induced NF-κB signalling, inhibition of JNK activation and activation of AKT. In conclusion, the ability of C. difficile to activate three apoptotic pathways represents an important strategy to overcome resistance against its cytotoxic activity.

Invisible steps for a global endemy: molecular strategies adopted by Clostridioides difficile.

Clostridioides difficile infection (CDI) is on the rise worldwide and is associated with an increase in deaths and socio-health burden. C. difficile has become ubiquitous in anthropized environments because of the extreme resistance of its spores. Based on the epidemiological data and knowledge of molecular pathogenesis of C. difficile, it is possible to predict its progressive colonization of the human population for the following reasons: first, its global spread is unstoppable; second, the toxins (Tcds) produced by C. difficile, TcdA and TcdB, mainly cause cell death by apoptosis, but the surviving cells acquire a senescence state that favours persistence of C. difficile in the intestine; third, proinflammatory cytokines, tumour necrosis factor-α and interferon-γ, induced during CDI, enhance the cytotoxicity of Tcds and can increase the survival of senescent cells; fourth, Tcds block mobility and induce apoptosis in immune cells recruited at the infection site; and finally, after remission from primary infection or relapse, C. difficile causes functional abnormalities in the enteric glial cell (EGC) network that can result in irritable bowel syndrome, characterized by a latent inflammatory response that contributes to C. difficile survival and enhances the cytotoxic activity of low doses of TcdB, thus favouring further relapses. Since a 'global endemy' of C. difficile seems inevitable, it is necessary to develop an effective vaccine against Tcds for at-risk individuals, and to perform a prophylaxis/selective therapy with bacteriophages highly specific for C. difficile. We must be aware that CDI will become a global health problem in the forthcoming years, and we must be prepared to face this menace.

Proinflammatory Cytokines: Possible Accomplices for the Systemic Effects of Clostridioides difficile Toxin B.

Clostridioides difficile infection (CDI) has a serious impact on the healthcare system, and most of its pathogenic effects are mainly due to the activity of toxins A and B (TcdA and TcdB, respectively). The molecular mechanisms of their cytotoxic activity are well known, especially in the colon, where the infection occurs and normally remains localized. However, the mechanisms causing toxic effects on various systemic organs (extraintestinal manifestations) with frequent lethal outcomes in some patients affected by CDI are still poorly understood. Few studies are available that demonstrate low serum levels of Tcds in both experimental animal models and patients with CDI. Until now, it has remained unclear how low levels of circulating Tcds could lead to serious toxic effects. On the basis of our previous in vitro studies, in which the proinflammatory cytokines TNF-alpha and IFN-gamma strongly potentiated the toxic activity of low doses of TcdB, we hypothesize that the presence of both TcdB in the circulation and a systemic proinflammatory cytokine storm may be responsible for the selective severe effects of TcdB in some patients. This may occur in patients with severe CDI and systemic Tcds, in whom proinflammatory cytokines such as TNF-alpha and IFN-gamma reach a significant concentration in the circulation. This hypothesis could identify therapeutic interventions based on the reduction or neutralization of the indirect toxic action of these cytokines.

Clostridioides difficile Infection in Patients with Inflammatory Bowel Disease May be Favoured by the Effects of Proinflammatory Cytokines on the Enteroglial Network.

Clostridioides difficile infection is widespread throughout countries and represents an important cause of nosocomial diarrhoea, with relatively high morbidity. This infection often occurs in patients with inflammatory bowel diseases and may complicate their clinical picture. Here, we propose, on the basis of evidence from basic science studies, that in patients affected by inflammatory bowel diseases, this infection might be facilitated by a derangement of the enteric glial cell (EGC) network caused by the effects of proinflammatory cytokines, such as tumour necrosis factor alpha and interferon gamma, which enhance the cytotoxic effects of C. difficile toxin B on EGCs. This hypothesis, if confirmed, could open the door to alternative treatment approaches to fight C. difficile infection.

The efficacy of the anticancer 3-bromopyruvate is potentiated by antimycin and menadione by unbalancing mitochondrial ROS production and disposal in U118 glioblastoma cells.

Metabolic reprogramming of tumour cells sustains cancer progression. Similar to other cancer cells, glioblastoma cells exhibit an increased glycolytic flow, which encourages the use of antiglycolytics as an effective complementary therapy. We used the antiglycolytic 3-bromopyruvate (3BP) as a metabolic modifier to treat U118 glioblastoma cells and investigated the toxic effects and the conditions to increase drug effectiveness at the lowest concentration. Cellular vitality was not affected by 3BP concentrations lower than 40 µM, although p-Akt dephosphorylation, p53 degradation, and ATP reduction occurred already at 30 µM 3BP. ROS generated in mitochondria were enhanced at 30 µM 3BP, possibly by unbalancing their generation and their disposal because of glutathione peroxidase inhibition. ROS triggered JNK and ERK phosphorylation, and cyt c release outside mitochondria, not accompanied by caspases-9 and -3 activation, probably due to 3BP-dependent alkylation of cysteine residues at caspase-9 catalytic site. To explore the possibility of sensitizing cells to 3BP treatment, we exploited 3BP effects on mitochondria by using 30 µM 3BP in association with antimycin A or menadione concentrations that in themselves exhibit poor toxicity. 3BP effect on cyt c release and cell vitality loss was potentiated due the greater oxidative stress induced by antimycin or menadione association with 3BP, supporting a preeminent role of mitochondrial ROS in 3BP toxicity. Indeed, the scavenger of mitochondrial superoxide MitoTEMPO counteracted 3BP-induced cyt c release and weakened the potentiating effect of 3BP/antimycin association. In conclusion, the biochemical mechanisms leading U118 glioblastoma cells to viability loss following 3BP treatment rely on mitochondrial ROS-dependent pathways. Their potentiation at low 3BP concentrations is consistent with the goal to minimize the toxic effect of the drug towards non-cancer cells.

The cytotoxic synergy between Clostridioides difficile toxin B and proinflammatory cytokines: an unholy alliance favoring the onset of Clostridioides difficile infection and relapses.

Clostridioides difficile infection (CDI) represents an important health problem worldwide, with significant morbidity and mortality. This infection has also high recurrence rates, whose pathophysiological grounds are still poorly understood. Based on our experiments in vitro with Clostridioides difficile toxin B and existing experimental and clinical evidence, we propose that primary CDI and relapses might be favored by a mechanism that involves the enhancement of the toxicity of toxin B by proinflammatory cytokines, tumor necrosis factor alpha, and interferon gamma on the enteric glial cells and their network in an environment characterized by a strong dysmicrobism.

Acetamidine-Based iNOS Inhibitors as Molecular Tools to Counteract Inflammation in BV2 Microglial Cells.

Neurodegenerative diseases are associated with increased levels of nitric oxide (NO) mainly produced by microglial cells through inducible nitric oxide synthase (iNOS) whose expression is induced by inflammatory stimuli. NO can both exert cytotoxic functions and induce a metabolic switch by inhibiting oxidative phosphorylation and upregulating glycolytic flux. Here, we investigated whether two newly synthesized acetamidine based iNOS inhibitors, namely CM292 and CM544, could inhibit lipopolysaccharide (LPS)-induced BV2 microglial cell activation, focusing on both inflammatory and metabolic profiles. We found that CM292 and CM544, without affecting iNOS protein expression, reduced NO production and reverted LPS-induced inflammatory and cytotoxic response. Furthermore, in the presence of the inflammatory stimulus, both the inhibitors increased the expression of glycolytic enzymes. In particular, CM292 significantly reduced nuclear accumulation of pyruvate kinase M2, increased mitochondrial membrane potential and oxygen consumption rate, and augmented the expression of pyruvate dehydrogenase, pointing to a metabolic switch toward oxidative phosphorylation. These data confirm the role played by NO in the connection between cell bioenergetics profile and inflammation, and suggest the potential usefulness of iNOS inhibitors in redirecting microglia from detrimental to pro-regenerative phenotype.

Crosstalk between Long-Term Sublethal Oxidative Stress and Detrimental Inflammation as Potential Drivers for Age-Related Retinal Degeneration.

Age-related retinal degenerations, including age-related macular degeneration (AMD), are caused by the loss of retinal pigmented epithelial (RPE) cells and photoreceptors. The pathogenesis of AMD, deeply linked to the aging process, also involves oxidative stress and inflammatory responses. However, the molecular mechanisms contributing to the shift from healthy aging to AMD are still poorly understood. Since RPE cells in the retina are chronically exposed to a pro-oxidant microenvironment throughout life, we simulated in vivo conditions by growing ARPE-19 cells in the presence of 10 µM H2O2 for several passages. This long-term oxidative insult induced senescence in ARPE-19 cells without affecting cell proliferation. Global proteomic analysis revealed a dysregulated expression in proteins involved in antioxidant response, mitochondrial homeostasis, and extracellular matrix organization. The analyses of mitochondrial functionality showed increased mitochondrial biogenesis and ATP generation and improved response to oxidative stress. The latter, however, was linked to nuclear factor-κB (NF-κB) rather than nuclear factor erythroid 2-related factor 2 (Nrf2) activation. NF-κB hyperactivation also resulted in increased pro-inflammatory cytokines expression and inflammasome activation. Moreover, in response to additional pro-inflammatory insults, senescent ARPE-19 cells underwent an exaggerated inflammatory reaction. Our results indicate senescence as an important link between chronic oxidative insult and detrimental chronic inflammation, with possible future repercussions for therapeutic interventions.

Guanylin, Uroguanylin and Guanylate Cyclase-C Are Expressed in the Gastrointestinal Tract of Horses.

Guanylate cyclase-C (GC-C) is a multifunctional receptor encoded by the GUCY2C gene, representing an attractive target for therapy in several gastrointestinal diseases in humans. Little is known about this system in horses. We investigated for the first time the gene expression of guanylin, uroguanylin and GC-C receptors in different horse's gastrointestinal tracts. Tissue samples from stomach, duodenum, jejunum, ileum, head and body of cecum, left and right dorsal colon, left and right ventral colon, pelvic flexure, transverse colon, descending colon and rectum were collected from adult horses within 1 h post mortem. For each sample, total RNA was extracted from 100 mg of ground tissue, and qRT-PCR performed on GUCA2a, GUCA2b and GUCY2 transcripts on a CFX96 Touch instrument. Data analysis was carried out with Bio-Rad CFX Manager software, and genes of interest normalized relative to the abundance of the two reference genes (SDHA, HPRT). Additionally, the protein expression levels of GC-C receptor were analyzed through western blotting. A common pattern of expression throughout the gastrointestinal lumen for all three investigated transcripts was found. The expression of GUCA2a, GUCA2b and GUCY2 genes was higher in jejunum, ileum, descending colon and rectum. The levels of expression of GC-C protein confirmed these data. The findings of this study might open new scenarios for the therapeutic approach to enteric diseases of horse using selective agonists of GC-C.

Gentamicin Targets Acid Sphingomyelinase in Cancer: The Case of the Human Gastric Cancer NCI-N87 Cells.

Emerging literature implicates acid sphingomyelinase in tumor sensitivity/resistance to anticancer treatments. Gentamicin is a drug commonly used as an antimicrobial but its serendipity effects have been shown. Even though many evidences on the role of gentamicin in cancer have been reported, its mechanism of action is poorly understood. Here, we explored acid sphingomyelinase as a possible new target of gentamicin in cancer. Since gastric cancer is one of the most common cancers and represents the second cause of death in the world, we performed the study in NCI-N87 gastric cancer cell line. The effect of the drug resulted in the inhibition of cell proliferation, including a reduction of cell number and viability, in the decrease of MIB-1 proliferative index as well as in the upregulation of cyclin-dependent kinase inhibitor 1A and 1B (CDKN1A and CDKN1B), and growth arrest and DNA-damage 45A (GADD45A) genes. The cytotoxicity was apoptotic as shown by FACS analysis. Additionally, gentamicin reduced HER2 protein, indicating a minor tumor aggressiveness. To further define the involvement of sphingomyelin metabolism in the response to the drug, gene and protein expression of acid and neutral sphingomeylinase was analyzed in comparison with phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and vitamin D receptor (VDR), molecules involved in cancer. Gentamicin induced a downregulation of PTEN, VDR, and neutral sphingomyelinase and a strong upregulation of acid sphingomyelinase. Of note, we identified the same upregulation of acid sphingomyelinase upon gentamicin treatment in other cancer cells and not in normal cells. These findings provide new insights into acid sphingomyelinase as therapeutic target, reinforcing studies on the potential role of gentamicin in anticancer therapy.

Clostridium difficile toxin B induces senescence in enteric glial cells: A potential new mechanism of Clostridium difficile pathogenesis.

Clostridium difficile infection (CDI) causes nosocomial/antibiotic-associated diarrhea and pseudomembranous colitis, with dramatic incidence/mortality worldwide. C. difficile virulence factors are toxin A and toxin B (TcdB) which cause cytopathic/cytotoxic effects and inflammation. Until now studies were focused on molecular effects of C. difficile toxins (Tcds) on different cells while unexplored aspect is the status/fate of cells that survived their cytotoxicity. Recently we demonstrated that enteric glial cells (EGCs) are susceptible to TcdB cytotoxicity, but several EGCs survived and were irreversibly cell-cycle arrested and metabolically active, suggesting that EGCs could became senescent. This is important because allowed us to evaluate the not explored status/fate of cells surviving Tcds cytotoxicity, and particularly if TcdB induces senescence in EGCs. Rat-transformed EGCs were treated with 10 ng/ml TcdB for 6 h-48 h, or for 48 h, followed by incubation for additional 4 or 11 days in absence of TcdB (6 or 13 total days). Senescence markers/effectors were examined by specific assays. TcdB induces senescence in EGCs, as demonstrated by the senescence markers: irreversible cell-cycle arrest, senescence-associated-ß­galactosidase positivity, flat morphology, early and persistent DNA damage (ATM and H2AX phosphorylation), p27 overexpression, pRB hypophosphorylation, c­Myc, cyclin B1, cdc2 and phosphorylated-cdc2 downregulation, Sirtuin­2 and Sirtuin­3 overexpression. TcdB-induced EGC senescence is dependent by JNK and AKT activation but independent by ROS, p16 and p53/p21 pathways. In conclusion, TcdB induces senescence in EGCs. The extrapolation of these results to CDI leads to hypothesize that EGCs that survived TcdB, once they have acquired a senescence state, could cause irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), and tumors due to persistent inflammation, transfer of senescence status and stimulation of pre-neoplastic cells.

Palmitate lipotoxicity in enteric glial cells: Lipid remodeling and mitochondrial ROS are responsible for cyt c release outside mitochondria.

Enteric glial cells (EGCs) are components of the enteric nervous system, an organized structure that controls gut functions. EGCs may be vulnerable to different agents, such as bacterial infections that could alter the intestinal epithelial barrier, allowing bacterial toxins and/or other agents possessing intrinsic toxic effect to access cells. Palmitate, known to exhibit lipotoxicity, is released in the gut during the digestion process. In this study, we investigated the lipotoxic effect of palmitate in cultured EGCs, with particular emphasis on palmitate-dependent intracellular lipid remodeling. Palmitate but not linoleate altered mitochondrial and endoplasmic reticulum lipid composition. In particular, the levels of phosphatidic acid, key precursor of phospholipid synthesis, increased, whereas those of mitochondrial cardiolipin (CL) decreased; in parallel, phospholipid remodeling was induced. CL remodeling (chains shortening and saturation) together with palmitate-triggered mitochondrial burst, caused cytochrome c (cyt c) detachment from its CL anchor and accumulation in the intermembrane space as soluble pool. Palmitate decreased mitochondrial membrane potential and ATP levels, without mPTP opening. Mitochondrial ROS permeation into the cytosol and palmitate-induced ER stress activated JNK and p38, culminating in Bim and Bax overexpression, factors known to increase the outer mitochondrial membrane permeability. Overall, in EGCs palmitate produced weakening of cyt c-CL interactions and favoured the egress of the soluble cyt c pool outside mitochondria to trigger caspase-3-dependent viability loss. Elucidating the mechanisms of palmitate lipotoxicity in EGCs may be relevant in gut pathological conditions occurring in vivo such as those following an insult that may damage the intestinal epithelial barrier.

Nicotine induces apoptosis in human osteoblasts via a novel mechanism driven by H2O2 and entailing Glyoxalase 1-dependent MG-H1 accumulation leading to TG2-mediated NF-kB desensitization: Implication for smokers-related osteoporosis.

Nicotine contained in cigarette smoke contributes to the onset of several diseases, including osteoporosis, whose emerging pathogenic mechanism is associated with osteoblasts apoptosis. Scanty information is available on the molecular mechanisms of nicotine on osteoblasts apoptosis and, consequently, on an important aspect of the pathogenesis of smokers-related osteoporosis. Glyoxalase 1 (Glo1) is the detoxification enzyme of methylglyoxal (MG), a major precursor of advanced glycation end products (AGEs), potent pro-apoptotic agents. Hydroimidazolone (MG-H1) is the major AGE derived from the spontaneous MG adduction of arginine residues. The aim of this study was to investigate whether, and by means of which mechanism, the antiglycation defence Glo1 was involved in the apoptosis induced by 0.1 and 1µM nicotine in human primary osteoblasts chronically exposed for 11 and 21 days. By using gene overexpression/silencing and scavenging/inhibitory agents, we demonstrated that nicotine induces a significant intracellular accumulation of hydrogen peroxide (H2O2) that, by inhibiting Glo1, drives MG-H1 accumulation/release. MG-H1, in turn, triggers H2O2 overproduction via receptor for AGEs (RAGE) and, in parallel, an apoptotic mitochondrial pathway by inducing Transglutaminase 2 (TG2) downregulation-dependent NF-kB desensitization. Measurements of H2O2, Glo1 and MG-H1 circulating levels in smokers compared with non-smokers or in smokers with osteoporosis compared with those without this bone-related disease supported the results obtained in vitro. Our findings newly pose the antiglycation enzymatic defense Glo1 and MG-H1 among the molecular events involved in nicotine-induced reactive oxygen species-mediated osteoblasts apoptosis, a crucial event in smoker-related osteoporosis, and suggest novel exposure markers in health surveillance programmes related to smokers-associated osteoporosis.