Claudia Campanella

Hsp60 is actively secreted by human tumor cells

Background Hsp60, a Group I mitochondrial chaperonin, is classically considered an intracellular chaperone with residence in the mitochondria; nonetheless, in the last few years it has been found extracellularly as well as in the cell membrane. Important questions remain pertaining to extracellular Hsp60 such as how generalized is its occurrence outside cells, what are its extracellular functions and the translocation mechanisms that transport the chaperone outside of the cell. These questions are particularly relevant for cancer biology since it is believed that extracellular chaperones, like Hsp70, may play an active role in tumor growth and dissemination. Methodology/Principal Findings Since cancer cells may undergo necrosis and apoptosis, it could be possible that extracellular Hsps are chiefly the result of cell destruction but not the product of an active, physiological process. In this work, we studied three tumor cells lines and found that they all release Hsp60 into the culture media by an active mechanism independently of cell death. Biochemical analyses of one of the cell lines revealed that Hsp60 secretion was significantly reduced, by inhibitors of exosomes and lipid rafts. Conclusions/Significance Our data suggest that Hsp60 release is the result of an active secretion mechanism and, since extracellular release of the chaperone was demonstrated in all tumor cell lines investigated, our observations most likely reflect a general physiological phenomenon, occurring in many tumors.

The odyssey of Hsp60 from tumor cells to other destinations includes plasma membrane-associated stages and Golgi and exosomal protein-trafficking modalities.

Background In a previous work we showed for the first time that human tumor cells secrete Hsp60 via exosomes, which are considered immunologically active microvesicles involved in tumor progression. This finding raised questions concerning the route followed by Hsp60 to reach the exosomes, its location in them, and whether Hsp60 can be secreted also via other mechanisms, e.g., by the Golgi. We addressed these issues in the work presented here. Principal Findings We found that Hsp60 localizes in the tumor cell plasma membrane, is associated with lipid rafts, and ends up in the exosomal membrane. We also found evidence that Hsp60 localizes in the Golgi apparatus and its secretion is prevented by an inhibitor of this organelle. Conclusions/Significance We propose a multistage process for the translocation of Hsp60 from the inside to the outside of the cell that includes a combination of protein traffic pathways and, ultimately, presence of the chaperonin in the circulating blood. The new information presented should help in designing future strategies for research and for developing diagnostic-monitoring means useful in clinical oncology.

Human Hsp10 and Early Pregnancy Factor (EPF) and their relationship and involvement in cancer and immunity: Current knowledge and perspectives

This article is about Hsp10 and its intracellular and extracellular forms focusing on the relationship of the latter with Early Pregnancy Factor and on their roles in cancer and immunity. Cellular physiology and survival are finely regulated and depend on the correct functioning of the entire set of proteins. Misfolded or unfolded proteins can cause deleterious effects and even cell death. The chaperonins Hsp10 and Hsp60 act together inside the mitochondria to assist protein folding. Recent studies demonstrated that these proteins have other roles inside and outside the cell, either together or independently of each other. For example, Hsp10 was found increased in the cytosol of different tumors (although in other tumors it was found decreased). Moreover, Hsp10 localizes extracellularly during pregnancy and is often indicated as Early Pregnancy Factor (EPF), which is released during the first stages of gestation and is involved in the establishment of pregnancy. Various reports show that extracellular Hsp10 and EPF modulate certain aspects of the immune response with anti-inflammatory effects in patients with autoimmune conditions improving clinically after treatment with recombinant Hsp10. Moreover, Hsp10 and EPF are involved in embryonic development, acting as a growth factor, and in cell proliferation/differentiation mechanisms. Therefore, it becomes evident that Hsp10 is not only a co-chaperonin, but an active player in its own right in various cellular functions. In this article, we present an overview of various aspects of Hsp10 and EPF as they participate in physiological and pathological processes such as the antitumor response and autoimmune diseases.

Hsp60 chaperonopathies and chaperonotherapy: targets and agents

Introduction: Hsp60 (Cpn60) assembles into a tetradecamer that interacts with the co-chaperonin Hsp10 (Cpn10) to assist client polypeptides to fold, but it also has other roles, including participation in pathogenic mechanisms. Area covered: Hsp60 chaperonopathies are pathological conditions, inherited or acquired, in which the chaperone plays a determinant etiologic-pathogenic role. These diseases justify selection of Hsp60 as a target for developing agents that interfere with its pathogenic effects. We provide information on how to proceed. Expert opinion: The information available encourages the development of ways to improve Hsp60 activity (positive chaperonotherapy) when deficient or to block it (negative chaperonotherapy) when pathogenic. Many questions are still unanswered and obstacles are obvious. More information is needed to establish when and why autologous Hsp60 becomes a pathogenic autoantigen, or induces cytokine formation and inflammation, or favors carcinogenesis. Clarification of these points will take considerable time. However, analysis of the Hsp60 molecule and a search for active compounds aimed at structural sites that will affect its functioning should continue without interruption. No doubt that some of these compounds will offer therapeutic hopes and will also be instrumental for dissecting structure–function relationships at the biochemical and biological (using animal models and cultured cells) levels.

Exosome levels in human body fluids: A tumor marker by themselves?

&NA; Despite considerable research efforts, the finding of reliable tumor biomarkers remains challenging and unresolved. In recent years a novel diagnostic biomedical tool with high potential has been identified in extracellular nanovesicles or exosomes. They are released by the majority of the cells and contain detailed molecular information on the cell of origin including tumor hallmarks. Exosomes can be isolated from easy accessible body fluids, and most importantly, they can provide several biomarkers, with different levels of specificity. Recent clinical evidence shows that the levels of exosomes released into body fluids may themselves represent a predictive/diagnostic of tumors, discriminating cancer patients from healthy subjects. The aim of this review is to highlight these latest challenging findings to provide novel and groundbreaking ideas for successful tumor early diagnosis and follow‐up. Graphical Abstract Exosomes as diagnostic tools. Starting from the patient (center of the figure), exosomes measurement in plasma can be useful for early diagnosis and follow‐up of virtually every type of cancer (left side). Exosomes obtained from primary tumor cell cultures may be used as valuable controls in terms of expression of tumor biomarkers (right side). Figure. No caption available.

Convergent Sets of Data from In Vivo and In Vitro Methods Point to an Active Role of Hsp60 in Chronic Obstructive Pulmonary Disease Pathogenesis

Background It is increasingly clear that some heat shock proteins (Hsps) play a role in inflammation. Here, we report results showing participation of Hsp60 in the pathogenesis of chronic obstructive pulmonary diseases (COPD), as indicated by data from both in vivo and in vitro analyses. Methods and Results Bronchial biopsies from patients with stable COPD, smoker controls with normal lung function, and non-smoker controls were studied. We quantified by immunohistochemistry levels of Hsp10, Hsp27, Hsp40, Hsp60, Hsp70, Hsp90, and HSF-1, along with levels of inflammatory markers. Hsp10, Hsp40, and Hsp60 were increased during progression of disease. We found also a positive correlation between the number of neutrophils and Hsp60 levels. Double-immunostaining showed that Hsp60-positive neutrophils were significantly increased in COPD patients. We then investigated in vitro the effect on Hsp60 expression in bronchial epithelial cells (16HBE) caused by oxidative stress, a hallmark of COPD mucosa, which we induced with H2O2. This stressor determined increased levels of Hsp60 through a gene up-regulation mechanism involving NFkB-p65. Release of Hsp60 in the extracellular medium by the bronchial epithelial cells was also increased after H2O2 treatment in the absence of cell death. Conclusions This is the first report clearly pointing to participation of Hsps, particularly Hsp60, in COPD pathogenesis. Hsp60 induction by NFkB-p65 and its release by epithelial cells after oxidative stress can have a role in maintaining inflammation, e.g., by stimulating neutrophils activity. The data open new scenarios that might help in designing efficacious anti-inflammatory therapies centered on Hsp60 and applicable to COPD.

A comparative analysis of the products of GROEL-1 gene from Chlamydia trachomatis serovar D and the HSP60 var1 transcript from Homo sapiens suggests a possible autoimmune response.

Chlamydia trachomatis serovar D produces large quantities of HSP60‐1 during infections, which accumulate inside the host cell inducing autoimmunity. We compare the aminoacid sequences of the human HSP60 with the bacterial counterpart to better elucidate how CTHSP60 may simulate HSP60 from human origin during infection and may induce an autoimmune response. As a result of the comparison we suggest several possible epitopes of the CTHSP60, which may induce autoimmunity.

Hsp60, a novel target for antitumor therapy: Structure-function features and prospective drugs design

Heat shock protein 60 kDa (Hsp60) is a chaperone classically believed to be involved in assisting the correct folding of other mitochondrial proteins. Hsp60 also plays a role in cytoprotection against cell stressors, displaying for example, antiapoptotic potential. Despite the plethora of studies devoted to the mechanism of Hsp60s function, especially in prokaryotes, fundamental issues still remain unexplored, including the definition of its role in cancer. Key questions still unanswered pertain to the differences in structure-function features that might exist between the well-studied prokaryotic GroEL and the largely unexplored eukaryotic Hsp60 proteins. In this article we discuss these differences in sequence, structure, and roles of Hsp60, focusing on the human ortholog with the view of devising compounds to block its ability to favour tumor-cell growth and survival. Compounds currently known to directly or indirectly affect Hsp60 functions, such as protein folding, HIF-1α accumulation, or Hsp60-induced cell proliferation, are discussed along with strategies that might prove effective for developing Hsp60-targeting drugs for anticancer therapy.

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.

Heat Shock Protein 60 Levels in Tissue and Circulating Exosomes in Human Large Bowel Cancer Before and After Ablative Surgery

Heat shock protein 60 (Hsp60) is a chaperonin involved in tumorigenesis, but its participation in tumor development and progression is not well understood and its value as a tumor biomarker has not been fully elucidated. In the current study, the authors presented evidence supporting the theory that Hsp60 has potential as a biomarker as well as a therapeutic target in patients with large bowel cancer.