N-Acetylcysteine protects epithelial cells against the oxidative imbalance due to Clostridium difficile toxins

AbstractToxins A and B from the anaerobic bacterium Clostridium difficile are the causative agents of the antibiotic-associated pseudomembraneous colitis. At the subcellular level, they inhibit the Rho family GTPases, thus causing alterations of the actin cytoskeleton. The cytoskeletal integrity is also controlled by the redox state of cells. Therefore, we have evaluated whether an oxidative imbalance could be involved in the toxin-induced cytopathic effects. Our results indicate that both toxins induce oxidative stress with a significant depletion of protein SH-groups. These responses and the cytoskeleton-dependent cell retraction and rounding are significantly counteracted by N-acetylcysteine but not by α-tocopherol. Our study provides the first evidence that the thiol supplier N-acetylcysteine impairs the cellular intoxication by acting on the cytoskeleton integrity. This also suggests a possible beneficial role for this drug during therapeutic intervention

Modulating the metabolism by trimetazidine enhances myoblast differentiation and promotes myogenesis in cachectic tumor-bearing c26 mice.

Trimetazidine (TMZ) is a metabolic reprogramming agent able to partially inhibit mitochondrial free fatty acid β-oxidation while enhancing glucose oxidation. Here we have found that the metabolic shift driven by TMZ enhances the myogenic potential of skeletal muscle progenitor cells leading to MyoD, Myogenin, Desmin and the slow isoforms of troponin C and I over-expression. Moreover, similarly to exercise, TMZ stimulates the phosphorylation of the AMP-activated protein kinase (AMPK) and up-regulates the peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α), both of which are known to enhance the mitochondrial biogenesis necessary for myoblast differentiation. TMZ also induces autophagy which is required during myoblast differentiation and promotes myoblast alignment which allows cell fusion and myofiber formation. Finally, we found that intraperitoneally administered TMZ (5mg/kg) is able to stimulate myogenesis in vivo both in a mice model of cancer cachexia (C26 mice) and upon cardiotoxin damage. Collectively, our work demonstrates that TMZ enhances myoblast differentiation and promotes myogenesis, which might contribute recovering stem cell blunted regenerative capacity and counteracting muscle wasting, thanks to the formation of new myofibers; TMZ is already in use in humans as an anti-anginal drug and its repositioning might impact significantly on aging and regeneration-impaired disorders, including cancer cachexia, as well as have implications in regenerative medicine

Functional estrogen receptors of red blood cells. Do they influence intracellular signaling?

Background/Aims: Estrogen could play a key role in the mechanisms underlying sex-related disparity in the incidence of thrombotic events. We investigated whether estrogen receptors (ERs) were expressed in human red blood cells (RBCs), and if they affected cell signaling of erythrocyte constitutive isoform of endothelial NO-synthase (eNOS) and nitric oxide (NO) release. Methods: RBCs from 29 non-smoker volunteers (15 males and 14 females) aged between 20 and 40 years were analyzed by cytometry and western blot. In particular, content and distribution of ER-α and ER-β, tyrosine kinases and eNOS phosphorylation and NO release were analyzed. Results: We demonstrated that: i) both ER-α and ER-β were expressed by RBCs; ii) they were both functionally active; and iii) ERs distribution and function were different in males and females. In particular, ERs modulated eNOS phosphorylation and NO release in RBCs from both sexes, but they induced the phosphorylation of specific tyrosine residues of kinases linked to eNOS activation and NO release in the RBCs from females only. Conclusion: Collectively, these data suggest that ERs could play a critical role in RBC intracellular signaling. The possible implication of this signaling in sex-linked risk disparity in human cardiovascular diseases, e.g. in thrombotic events, may not be ruled out

Cannibalism of Live Lymphocytes by Human Metastatic but Not Primary Melanoma Cells

The phenomenon of cell cannibalism, which generally refers to the engulfment of cells within other cells, was described in malignant tumors, but its biological significance is still largely unknown. In the present study, we investigated the occurrence, the in vivo relevance, and the underlying mechanisms of cannibalism in human melanoma. As first evidence, we observed that tumor cannibalism was clearly detectable in vivo in metastatic lesions of melanoma and often involved T cells, which could be found in a degraded state within tumor cells. Then, in vitro experiments confirmed that cannibalism of T cells was a property of metastatic melanoma cells but not of primary melanoma cells. In particular, morphologic analyses, including time-lapse cinematography and electron microscopy, revealed a sequence of events, in which metastatic melanoma cells were able to engulf and digest live autologous melanoma-specific CD8+ T cells. Importantly, this cannibalistic activity significantly increased metastatic melanoma cell survival, particularly under starvation condition, supporting the evidence that tumor cells may use the eating of live lymphocytes as a way to ‘‘feed’’ in condition of low nutrient supply. The mechanism underlying cannibalism involved a complex framework, including lysosomal protease cathepsin B activity, caveolae formation, and ezrin cytoskeleton integrity and function. In conclusion, our study shows that human metastatic melanoma cells may eat live T cells, which are instead programmed to kill them, suggesting a novel mechanism of tumor immune escape. Moreover, our data suggest that cannibalism may represent a sort of ‘‘feeding’’ activity aimed at sustaining survival and progression of malignant tumor cells in an unfavorable microenvironment. (Cancer Res 2006; 66(7): 3629-38

What Is the Real Impact of Estrogen Receptor Status on the Prognosis and Treatment of HER2-Positive Early Breast Cancer?

HER2+ early breast cancer is a heterogeneous disease, comprising all the intrinsic breast cancer subtypes. The only biomarker available nowadays for anti-HER2 treatment selection is HER2 status itself, but estrogen receptor (ER) status is emerging as a robust predictive marker within HER2+ disease. In this Perspective, we discuss the biological and clinical differences between patients with HER2+/ER-positive (ER+) disease versus those with HER2+/ ER-negative (ER-neg) tumors, namely, short-term and long-term (>5 years after diagnosis) prognosis, response to neoadjuvant treatment and benefit from adjuvant anti-HER2–targeted therapies. We also address other possible biomarkers to be used for patient selection in future clinical trials, such as gene signatures, PAM50 subtypes, tumor-infiltrating lymphocytes, PIK3CA mutations, and changes in Ki67 score during treatment and discuss their limitations. Finally, we suggest new clinical trial designs that can have an impact on clinical practice, aiming to test treatment deescalation separately for patients with HER2+/ER+ and HER2+/ER-neg tumors. We also propose an integrated classification of HER2+ disease, comprising DNA, RNA, protein expression, and microenvironment characteristics, in order to identify those tumors that are truly “HER2-addicted” and may benefit the most from anti-HER2 treatment

Role of GD3-CLIPR-59 Association in Lymphoblastoid T Cell Apoptosis Triggered by CD95/Fas

We previously found that a directional movement of the raft component GD3 towards mitochondria, by its association with microtubules, was mandatory to late apoptogenic events triggered by CD95/Fas. Since CLIPR-59, CLIP-170-related protein, has recently been identified as a microtubule binding protein associated with lipid rafts, we analyzed the role of GD3-CLIPR-59 association in lymphoblastoid T cell apoptosis triggered by CD95/Fas. To test whether CLIPR-59 could play a role at the raft-microtubule junction, we performed a series of experiments by using immunoelectron microscopy, static or flow cytometry and biochemical analyses. We first assessed the presence of CLIPR-59 molecule in lymphoblastoid T cells (CEM). Then, we demonstrated that GD3-microtubule interaction occurs via CLIPR-59 and takes place at early time points after CD95/Fas ligation, preceding the association GD3-tubulin. GD3-CLIPR-59 association was demonstrated by fluorescence resonance energy transfer (FRET) analysis. The key role of CLIPR-59 in this dynamic process was clarified by the observation that silencing CLIPR-59 by siRNA affected the kinetics of GD3-tubulin association, spreading of GD3 towards mitochondria and apoptosis execution. We find that CLIPR-59 may act as a typical chaperone, allowing a prompt interaction between tubulin and the raft component GD3 during cell apoptosis triggered by CD95/Fas. On the basis of the suggested role of lipid rafts in conveying pro-apoptotic signals these results disclose new perspectives in the understanding of the mechanisms by which raft-mediated pro-apoptotic signals can directionally reach their target, i.e. the mitochondria, and trigger apoptosis execution

Clinical outcomes after palbociclib with or without endocrine therapy in postmenopausal women with hormone receptor positive and HER2-negative metastatic breast cancer enrolled in the TREnd trial

Currently, there is limited data regarding the effectiveness of standard subsequent line therapies such as endocrine therapy, chemotherapy, or targeted agents after progression on CDK4/6 inhibitor-based regimens. This paper describes time-to-treatment failure beyond progression on palbociclib or palbociclib+endocrine therapy in patients enrolled in the phase II, multicenter TREnd trial. Our results indicate that there is limited benefit from post-palbociclib treatment, regardless of the type of therapy received. A small population of long responders were identified who demonstrated ongoing benefit from a subsequent line of endocrine therapy after progression to palbociclib-based regimens. A translational research program is ongoing on this population of outliers

A serum metabolomics classifier derived from elderly patients with metastatic colorectal cancer predicts relapse in the adjuvant setting

Adjuvant treatment for patients with early stage colorectal cancer (eCRC) is currently based on suboptimal risk stratification, especially for elderly patients. Metabolomics may improve the identification of patients with residual micrometastases after surgery. In this retrospective study, we hypothesized that metabolomic fingerprinting could improve risk stratification in patients with eCRC. Serum samples obtained after surgery from 94 elderly patients with eCRC (65 relapse free and 29 relapsed, after 5-years median follow up), and from 75 elderly patients with metastatic colorectal cancer (mCRC) obtained before a new line of chemotherapy, were retrospectively analyzed via proton nuclear magnetic resonance spectroscopy. The prognostic role of metabolomics in patients with eCRC was assessed using Kaplan–Meier curves. PCA-CA-kNN could discriminate the metabolomic fingerprint of patients with relapse-free eCRC and mCRC (70.0% accuracy using NOESY spectra). This model was used to classify the samples of patients with relapsed eCRC: 69% of eCRC patients with relapse were predicted as metastatic. The metabolomic classification was strongly associated with prognosis (p-value 0.0005, HR 3.64), independently of tumor stage. In conclusion, metabolomics could be an innovative tool to refine risk stratification in elderly patients with eCRC. Based on these results, a prospective trial aimed at improving risk stratification by metabolomic fingerprinting (LIBIMET) is ongoing