Nella Maria Ardizzone

Upon oxidative stress, the antiapoptotic Hsp60/procaspase-3 complex persists in mucoepidermoid carcinoma cells.

Hsp60, a mitochondrial chaperonin highly conserved during evolution, has been found elevated in the cytosol of cancer cells, both in vivo and in vitro, but its role in determining apoptosis during oxidative stress (OS) has not yet been fully elucidated. The aim of the present work was to study the effects of OS on Hsp60 levels and its interactions with procaspase- 3 (p-C3) and p53 in tumor cells. NCI-H292 (mucoepidermoid carcinoma) cells were exposed to various concentrations of hydrogen peroxide (H2O2) for 24 hours. Cell viability was determined by Trypan blue and MTT assays. DNA damage was assessed by the Comet assay, and apoptosis was measured by the AnnexinV cytofluorimetric test. Exposure to increasing concentrations of H2O2 resulted in a reduction of cell viability, DNA damage, and early apoptotic phenomena. Hsp60, p-C3, p53, and p21 were assessed by Western blotting and immunocytochemistry before and after OS. Hsp60 and p-C3 were present before and after OS induction. Immunoprecipitation experiments showed an Hsp60/p-C3 complex before OS that persisted after it, while an Hsp60/p53 complex was not detected in either condition. The presence of wild type (wt) p53 was confirmed by RT-PCR, and p21 detection suggested p53 activation after OS. We postulate that, although OS may induce early apoptosis in NCI-H292 cells, Hsp60 exerts an anti-apoptotic effect in these cells and, by extension, it may do so in other cancer cells.

Cardiac Stem Cell Research: An Elephant in the Room?

Heart disease is the leading cause of death in the industrialized world, and stem cell therapy seems to be a promising treatment for injured cardiac tissue. To reach this goal, the scientific community needs to find a good source of stem cells that can be used to obtain new myocardium in a very period range of time. Since there are many ethical and technical problems with using embryonic stem cells as a source of cells with cardiogenic potential, many laboratories have attempted to isolate potential cardiac stem cells from several tissues. The best candidates seem to be cardiac “progenitor” and/or “stem” cells, which can be isolated from subendocardial biopsies from the same patient or from embryonic and/or fetal myocardium. Regardless of the technique used to isolate and characterize these cells, it appears that the different cells isolated from adult myocardium to date are all phenotypic variations of a unique cell type that expresses several markers, such as c‐Kit, CD34, MDR‐1, Sca‐1, CD45, nestin, or Isl‐1, in various combinations. Anat Rec, 292:449–454, 2009.

Expression of Heat Shock Proteins HSP10, HSP27, HSP60, HSP70, and HSP90 in Urothelial Carcinoma of Urinary Bladder

AIM: Heat shock proteins (HSPs) have essential roles in a number of pathophysiologic conditions including carcinogenesis and represent a group of novel molecular markers in cancer management. The aim of this study is to explore the expression status of HSPs in bladder urothelial carcinoma (BUC) patients. METHODS: The immunohistochemical staining of HSP10, HSP27, HSP60, HSP70, and HSP90 were done in 46 vesical BUC patients with different grades (G) and stages (T). Statistical analyses were performed to determine whether there was any correlation between tumoral HSP expression and both G and T of tumors. RESULTS: We found a significant correlation between high grading (G ≥ 2) and tumor tissues positive for HSP10 and HSP90 staining (P 1) and tumor tissues expressing HSP70 (P 1 tumors (P < 0.005). No relationship was found between tumoral T status and HSP10, HSP27 and HSP90 expression. Finally, we found a significant correlation between the high-graded (G ≥ 2) neoplasms and the percentage of tumoral cells positive for HSP10, HSP27, HSP70, or HSP90 (P < 0.005). CONCLUSION: This is the first report to show the presence of HSP10 in bladder cancer tissues, with its expression correlated to the tumoral grading. These data may also be valuable for developing new molecular anti-cancer therapies.

Effect of conjugated linoleic acid on testosterone levels in vitro and in vivo after an acute bout of resistance exercise

Abstract Macaluso, F, Morici, G, Catanese, P, Ardizzone, NM, Marino Gammazza, A, Bonsignore, G, Giudice, GL, Stampone, T, Barone, R, Farina, F, and Di Felice, V. Effect of conjugated linoleic acid on testosterone levels in vitro and in vivo after an acute bout of resistance exercise. J Strength Cond Res 26(6): 1667–1674, 2012—The purposes of the present study were to investigate the effect of conjugated linoleic acid (CLA) supplementation on testosterone levels in vitro on a cell line derived from Leydig cells (R2C) and in vivo in the blood of physically active subjects before and after a resistance exercise bout. In vitro R2C cells were treated with different CLA concentrations (0–30 &mgr;M) for 24 and 48 hours. After treatment, supernatant media were tested to determine testosterone secretion. The CLA increased the testosterone secretion only after 48 hours. In vivo, 10 resistance-trained male subjects, in a double-blind placebo-controlled and crossover study design were randomized for 3 weeks of either 6 g·d−1 CLA or placebo. Blood was drawn pre and post each resistance exercise bout to determine the total testosterone and sex hormone–binding globulin (SHBG) levels. No significant differences were observed for total testosterone or SHBG pre and post each resistance exercise bout; although after the resistance exercise bouts, total testosterone increased moderately (effect size = moderate), whereas after CLA supplementation, there was a large increase in total testosterone (effect size = large). CLA supplementation induced an increase in testosterone levels in Leydig cells in vitro after 48 hours but not in vivo before and after a resistance exercise bout. These findings suggest that CLA supplementation may promote testosterone synthesis through a molecular pathway that should be investigated in the future, although this effect did not have an anabolic relevance in our in vivo model.

HSP90 and eNOS partially co‐localize and change cellular localization in relation to different ECM components in 2D and 3D cultures of adult rat cardiomyocytes

Background information. Cultivation techniques promoting three‐dimensional organization of mammalian cells are of increasing interest, since they confer key functionalities of the native ECM (extracellular matrix) with a power for regenerative medicine applications. Since ECM compliance influences a number of cell functions, Matrigel‐based gels have become attractive tools, because of the ease with which their mechanical properties can be controlled. In the present study, we took advantage of the chemical and mechanical tunability of commonly used cell culture substrates, and co‐cultures to evaluate, on both two‐ and three‐dimensional cultivated adult rat cardiomyocytes, the impact of ECM chemistry and mechanics on the cellular localization of two interacting signalling proteins: HSP90 (heat‐shock protein of 90 kDa) and eNOS (endothelial nitric oxide synthase).

Atrial natriuretic peptide and CD34 overexpression in human idiopathic dilated cardiomyopathies.

Idiopathic dilated cardiomyopathy (IDCM) is a primary myocardial disease of unknown cause characterized by ventricular chamber enlargement with impaired contractile function. In familial forms of IDCM, mutations of genes coding for cytoskeletal proteins related to force transmission, such as dystrophin, cardiac actin, desmin, and δ‐sarcoglycan, have been identified. Here, we report the data of a retrospective investigation carried out to evaluate the expression of atrial natriuretic peptide (ANP), CD34, troponin T and nestin in the myocardium of patients affected with IDCM. Formalin‐fixed and paraffin‐embedded consecutive tissue sections from the ventricular wall of 10 human normal hearts (NH) following forensic autopsy and 22 IDCM (living explanted hearts) were studied using primary monoclonal antibodies against ANP, CD34, troponin T and nestin by immunohistochemistry. Myocardial fibers were counted independently by three pathologists. Statistics included analysis of variance, log‐rank test for Kaplan‐Meier analysis, and kappa assessment for intra‐ and inter‐observer variability. ANP and CD34 were significantly overexpressed in IDCM compared to NH (p<0.05). Conversely, troponin T and nestin expression levels did not show significant variation. Inter‐observer kappa statistics showed a value of 0.87 and intra‐observer kappa statistics a value of 0.98. Evaluation of the marker distribution in the myocardium of patients with IDCM CD34 expression curve was similar to that of troponin T (p<0.0001), although two groups could be identified. Patients with a difference of more than 20 myocardial fibers in expression of CD34 and troponin T had a somewhat less favorable survival although the difference was not significant. The analysis of cells positive for troponin T resulted in a similar number of cardiac fibers between NH and IDCM. This is in agreement with cardiac enlargement present in IDCM, which is due to ventricular dilatation rather than increased number of myocytes. Moreover, the expression of nestin, a marker of activation of myocardial precursors, did not change either, and this may confirm that there are no hyperplastic phenomena in the IDCM pathogenesis. The increase in ANP‐positive cells in IDCM could be a consequence of neurohormonal activation due to a decline in the impaired myocyte contractility. Furthermore, since it was already shown that ANP could be important in the control of vascular remodeling, we postulated that the increase in CD34‐positive cells might be functionally correlated with the increase in ANP production. Differential expression of CD34 and troponin T might be used in future studies to evaluate their prognostic value.

Human Recombinant Vasostatin‐1 May Interfere with Cell–Extracellular Matrix Interactions

Abstract:  Vasostatin‐1 (VS‐1), the N‐terminal fragment derived from the cleavage of chromogranin A (CgA), has been shown to exert several biological activities on several tissues and organs. Recently, it has been reported that human recombinant VS‐1 (STA‐CGA1‐78) may alter myocardial contractility in eel, frog, and rat hearts. In this article we have explored if STA‐CGA1‐78 can induce intracellular cascades interacting both with adhesion molecules and/or extracellular matrix (ECM), components, that is, involvement of the heat shock protein 90 (HSP90) and the endothelial NOS (eNOS), known to be implicated in signal transduction mechanisms affecting myocardial contractility. We used 3D cultured adult rat cardiomyocytes cultivated over fibronectin or fibroblasts or embedded in matrigel or collagen type I. Aurion‐conjugated VS‐1 (Au‐STA‐CGA1‐78) has been used to identify possible sites of interaction of this molecule with the cell membrane. We found that in our 3D culture, cell–ECM interactions played a crucial role in the cellular localization of HSP90 as well as in the expression of eNOS. VS‐1 appeared to modulate cell–ECM interactions, thereby remarkably leading to a different cellular localization of HSP90. Moreover, Au‐STA‐CGA1‐78 was never detected inside the cell nor overlapping the plasma membrane, but nearby the outer side of the cardiomyocyte plasmalemma, at a particular distance, typical of integrins. On the whole, these data suggest that VS‐1 does not have a classic receptor on the membrane but that integrins may represent a nonconventional VS‐1 receptor modulating eNOS signaling pathway.