Quantitative patterns of Hsps in tubular adenoma compared with normal and tumor tissues reveal the value of Hsp10 and Hsp60 in early diagnosis of large bowel cancer

Large bowel carcinogenesis involves accumulation of genetic alterations leading to transformation of normal mucosa into dysplasia and, lastly, adenocarcinoma. It is pertinent to elucidate the molecular changes occurring in the pre-neoplastic lesions to facilitate early diagnosis and treatment. Heat shock proteins (Hsps), many of which are molecular chaperones, are implicated in carcinogenesis, and their variations with tumor progression encourage their study as biomarkers. There are many reports on Hsps and cancer but none to our knowledge on their systematic quantification in pre-neoplastic lesions of the large bowel. We performed immunohistochemical determinations of Hsp10, Hsp60, Hsp70, and Hsp90 in biopsies of large bowel tubular adenomas with moderate grade of dysplasia and compared to normal mucosa and adenocarcinoma with a moderate grade of differentiation (G2). A significant elevation of Hsp10 and Hsp60 only, i.e., in the absence of elevation of Hsp70 or Hsp90, in both epithelium and lamina propria was found in tubular adenoma by comparison with normal mucosa. In contrast, adenocarcinoma was characterized by the highest levels of Hsp10 and Hsp60 in epithelium and lamina propria, accompanied by the highest levels of Hsp70 only in epithelium and of Hsp90 only in lamina propria, by comparison with normal and tubular adenoma counterparts. Hsp10 and Hsp60 are promising biomarkers for early diagnosis of tubular adenoma and for its differentiation from more advanced malignant lesions. Hsp10 and Hsp60 may be implicated in carcinogenesis from its very early steps and, thus, are potentially convenient targets for therapy

Modulation of hippocampal neurogenesis by Nano-Pulsed Laser Therapy

Neurogenesis is a physiological process through which new neurons are generated and it occurs throughout life, subverting the old dogma stating to the inability of the adult brain to replace neurons. Specifically, neurogenesis take place thanks to the presence of neuronal stem cells (NSCs) located in the subventricular zone of the lateral ventricle and in the subgranular zone of the dentate gyrus of the hippocampus. Data present in the literature demonstrate that neurogenesis in the hippocampus decreases during aging and it is impaired in neurodegenerative diseases, such as Alzheimer’s disease, and after traumatic brain injury (TBI). Traumatic Brain Injury (TBI) is a chronic disease that occurs after a head trauma and the results of which are permanent. One of the main area affected by TBI is the hippocampus a brain region that plays a pivotal role in learning and memory. For this reason there is an increased interest in TBI research, particularly as it relates to finding a possible cure, which currently does not exist. The goal of the research I conducted was to stimulate NSCs to proliferate and differentiate so as to restore the neurogenesis process in the brain. Specifically, I tested the effect of a highly innovative non-invasive device that combines the benefits of both near infrared laser light (808nm) and ultrasound waves, optoacoustically generated with each short (10ns) high-energy (15mJ) laser pulse within the tissue. Dr. Micci’s laboratory demonstrated the beneficial effect of NPLT in a rat model of brain trauma. However, the mechanism by which NPLT stimulates NSCs is not well understood. At the completion of this project I will have gained valuable knowledge of the effects of NPLT on neurogenesis and provided a unique therapeutic strategy based on a self-repair via stimulation of NSCs that are already present in the brain

DUX4 Role in Normal Physiology and in FSHD Muscular Dystrophy

In the last decade, the sequence-specific transcription factor double homeobox 4 (DUX4) has gone from being an obscure entity to being a key factor in important physiological and pathological processes. We now know that expression of DUX4 is highly regulated and restricted to the early steps of embryonic development, where DUX4 is involved in transcriptional activation of the zygotic genome. While DUX4 is epigenetically silenced in most somatic tissues of healthy humans, its aberrant reactivation is associated with several diseases, including cancer, viral infection and facioscapulohumeral muscular dystrophy (FSHD). DUX4 is also translocated, giving rise to chimeric oncogenic proteins at the basis of sarcoma and leukemia forms. Hence, understanding how DUX4 is regulated and performs its activity could provide relevant information, not only to further our knowledge of human embryonic development regulation, but also to develop therapeutic approaches for the diseases associated with DUX4. Here, we summarize current knowledge on the cellular and molecular processes regulated by DUX4 with a special emphasis on FSHD muscular dystrophy

The histone deacetylase inhibitor SAHA induces HSP60 nitration and its extracellular release by exosomal vesicles in human lung-derived carcinoma cells

HSP60 undergoes changes in quantity and distribution in some types of tumors suggesting a participation of the chaperonin in the mechanism of transformation and cancer progression. Suberoylanilide hydroxamic acid (SAHA), a member of a family of histone deacetylase inhibitors (HDACi), has anti-cancer potential but its interaction, if any, with HSP60 has not been elucidated. We investigated the effects of SAHA in a human lung-derived carcinoma cell line (H292). We analysed cell viability and cycle; oxidative stress markers; mitochondrial integrity; HSP60 protein and mRNA levels; and HSP60 post-translational modifications, and its secretion. We found that SAHA is cytotoxic for H292 cells, interrupting the cycle at the G2/M phase, which is followed by death; cytotoxicity is associated with oxidative stress, mitochondrial damage, and diminution of intracellular levels of HSP60; HSP60 undergoes a post-translational modification and becomes nitrated; and nitrated HSP60 is exported via exosomes. We propose that SAHA causes ROS overproduction and mitochondrial dysfunction, which leads to HSP60 nitration and release into the intercellular space and circulation to interact with the immune system. These successive steps might constitute the mechanism of the anti-tumor action of SAHA and provide a basis to design supplementary therapeutic strategies targeting HSP60, which would be more efficacious than the compound alone

Curcumin Affects HSP60 Folding Activity and Levels in Neuroblastoma Cells

The fundamental challenge in fighting cancer is the development of protective agents able to interfere with the classical pathways of malignant transformation, such as extracellular matrix remodeling, epithelial–mesenchymal transition and, alteration of protein homeostasis. In the tumors of the brain, proteotoxic stress represents one of the main triggering agents for cell transformation. Curcumin is a natural compound with anti-inflammatory and anti-cancer properties with promising potential for the development of therapeutic drugs for the treatment of cancer as well as neurodegenerative diseases. Among the mediators of cancer development, HSP60 is a key factor for the maintenance of protein homeostasis and cell survival. High HSP60 levels were correlated, in particular, with cancer development and progression, and for this reason, we investigated the ability of curcumin to affect HSP60 expression, localization, and post-translational modifications using a neuroblastoma cell line. We have also looked at the ability of curcumin to interfere with the HSP60/HSP10 folding machinery. The cells were treated with 6, 12.5, and 25 µM of curcumin for 24 h, and the flow cytometry analysis showed that the compound induced apoptosis in a dose-dependent manner with a higher percentage of apoptotic cells at 25 µM. This dose of curcumin-induced a decrease in HSP60 protein levels and an upregulation of HSP60 mRNA expression. Moreover, 25 µM of curcumin reduced HSP60 ubiquitination and nitration, and the chaperonin levels were higher in the culture media compared with the untreated cells. Furthermore, curcumin at the same dose was able to favor HSP60 folding activity. The reduction of HSP60 levels, together with the increase in its folding activity and the secretion in the media led to the supposition that curcumin might interfere with cancer progression with a protective mechanism involving the chaperonin

Post-translational modifications of hsp60 and its extracellular release via exosomes are induced by the histone deacetylase inhibitor (HDACi) SAHA in the mucoepidermoid tumor H292 cells.

The chaperonin Hsp60 has multiple functions, among which that of supporting the growth of some type of tumours (1). HDACi (histone-deacetylase inhibitors) are drugs that regulate gene expression via modulation of epigenetic mechanisms, and induce tumor-cell death (2). Here, we show that in the tumor cells H292 the HDACi SAHA decreases the intracellular level of Hps60 and promotes its extracellular trafficking by exosomal vesicles. SAHA caused a time- and dose-dependent decrease in cell viability with a G/2M cell-cycle arrest at 24 h and cell death at 48 h. These effects were accompanied by production of reactive oxygen species and mitochondrial membrane-potential dissipation. The marked decrease in Hsp60 level in SAHA-treated cells was not related to proteasomal degradation since it was not affected by the addition of the proteasome inhibitor MG132. Moreover, the analysis of post-translational modifications of Hsp60 revealed that SAHA treatment induced a modest reduction in the ubiquitination of the protein, with no effect on its acetylation state, but did cause a marked increase in tyrosine-nitrated Hsp60. This effect was related to oxidative stress since it was prevented by the anti-oxidant N-acetylcysteine. Most importantly, we showed for the first time that SAHA markedly increases extracellular Hsp60 export via exosomes, which might explain the concomitant decrease of the intracellular chaperonin. Our results suggest that SAHA modifies Hsp60 by nitration and stimulates its extracellular export via exosomes. Since Hsp60-bearing exosomes have been implicated in effective anti-tumour responses, and since elevated intracellular levels of Hsp60 have been related to the arrest of tumour-cell death, our data offer clues to explore what might be as yet uncharacterized mechanisms by which SAHA works as antitumor drug. 1. Rappa et al. (2012) HSP-molecular chaperones in cancer biogenesis and tumour therapy: an overview. Anticancer Res. 32, 5139-5150. 2. Lauricella et al. (2012) SAHA/TRAIL combination induces detachment and anoikis of MDA-MB231 and MCF-7 breast cancer cells. Biochimie 94, 287-299