Claudio Luparello

Cadmium and mitochondria

The heavy metal cadmium (Cd) a pollutant associated with several modern industrial processes, is absorbed in significant quantities from cigarette smoke, water, food and air contaminations. It is known to have numerous undesirable effects on health in both experimental animals and humans, targeting kidney, liver and vascular system. The molecular mechanism accounting for most of the biological effects of Cd are not well-understood and the toxicity targets are largely unidentified. The present review focuses on important recent advances about the effects of cadmium on mitochondria of mammalian cells. Mitochondria are the proverbial powerhouses of the cell, running the fundamental biochemical processes that produce energy from nutrients using oxygen. They are among the key intracellular targets for different stressors including Cd. This review provides new additional informations on the cellular and molecular aspects of the interaction between Cd and cells, emphasizing alterations of mitochondria as important events in Cd cytotoxicity, thus representing an important basis for understanding the mechanisms of cadmium effect on the cells.

Tissue inhibitor of metalloprotease (TIMP)-1 and proliferative behaviour of clonal breast cancer cells

In the present paper, we have examined whether human tissue inhibitor of metalloprotease‐1 (hTIMP‐1) is able to exert a growth factor‐like effect on two clonal cell lines (BC‐3A and BC‐61), isolated from a parental line of human breast carcinoma cells (8701‐BC), and endowed with different growth and invasive behaviour ‘in vitro’ and in nude mouse. The data obtained indicate that only the more tumorigenic clonal cell line (BC‐61) is responsive to hTIMP‐1 treatment by increasing its proliferative rate in a dose‐dependent manner. It was also found that BC‐61 cells selectively express a transmembrane protein of about 80u2009kDa able to bind hTIMP‐1 ‘in vitro’ and ‘in vivo’ with high affinity (Kd of 0.07 ± 0.004 u2009 nM), and that treatment of BC‐61 cells with a proliferation‐promoting concentration of hTIMP‐1 is able to stimulate tyrosine‐targeted phosphorylation. The cumulative results obtained strongly support the hypothesis that hTIMP‐1, ‘classically’ regarded as a collagenase inhibitor, may be a crucial element of the extracellular signalling network during breast cancer development by controlling cell growth phenotype in autocrine and paracrine manner, and that intratumoural heterogeneity for the biological response to TIMP‐1 may exist within the composite cell population of the primary tumour site.

Purinergic Receptors Influence the Differentiation of Human Mesenchymal Stem Cells

Adult stem cells, including adipose tissue-derived mesenchymal stem cells (MSCs) or ectomesenchymal dental follicle cells (DFCs), attract considerable attention for their potential to differentiate into lineages, which are of major interest in the field of Regenerative Medicine. Purinergic receptors exert a wide range of biological actions in many cell and tissue types through extracellular nucleotides. Little is known about P2 receptors in adult stem cells and changes in their expression levels during differentiation. All known P2 receptors have been investigated, and a variety of P2X and P2Y receptor subtypes were detected in MSCs. Studies investigating intracellular calcium levels on receptor stimulation demonstrated that the found P2 receptors are metabolically active. Interestingly, up- or downregulation of several P2 receptor subtypes at gene and protein level was observed during adipogenic and osteogenic differentiation, and the effect on differentiation was directly influenced by both the application of agonists/antagonists and apyrase-induced nucleotide cleavage. Here, we show for the first time that the combination of several P2 receptors plays a role in the differentiation of adult stem cells. The expression pattern of the P2 receptors, as well as their fate in differentiation, varies in stem cells of mesenchymal origin if compared with stem cells of ectomesenchymal origin. The subtypes P2X6, P2Y4, and P2Y14 seem to be pivotal regulators in MSC commitment, as they are regulated in both adipogenic and osteogenic differentiation of adipose tissue-derived stem cells and DFCs. These findings provide new insights into the differentiation processes and might reveal novel options to influence stem cell fate in future applications.

Parathyroid hormone-related peptide and 8701-BC breast cancer cell growth and invasion in vitro: evidence for growth-inhibiting and invasion-promoting effects

It has been previously reported that 8701-BC cells, derived from a primary carcinoma of the breast, constitutively express parathyroid hormone-related peptide (PTHrP) gene and that N-terminal PTHrP immunoreactivity can be found in cell medium. Here we have firstly measured immunoreactive PTHrP in 8701-BC cell medium using antibodies raised against midregion and C-terminal fragments, and also demonstrated the expression of PTH/PTHrP receptor by 8701-BC cells. Secondly, we have examined the role, if any, elicited by diverse PTHrP domains on 8701-BC cell proliferation, and invasive behaviour in vitro related to production of extracellular proteolytic enzymes. Our data show that PTHrP [1-34], and, to a minor extent, [67-86] and [107-139], are anti-mitogenic but invadogenic for 8701-BC cells, and suggest that diverse enzymatic activities may contribute to cell invasion in response to different PTHrP fragments. In light of the present data on a chemoattractive role for PTHrP in vitro, we hypothesize that this protein might intervene in local control of the invasive process in breast carcinoma.

Midregion Parathyroid Hormone‐Related Protein Inhibits Growth and Invasion In Vitro and Tumorigenesis In Vivo of Human Breast Cancer Cells

Parathyroid hormone‐related protein (PTHrP) is critical for normal mammary development and is overexpressed by breast cancers. PTHrP is a peptide hormone that undergoes extensive post‐translational processing, and PTHrP(38–94)‐amide is one of the mature secretory forms of the peptide. In this study, we explored the effect of PTHrP(38–94)‐amide in a panel of six breast cancer cell lines “in vitro” and in MDA‐MB231 cells “in vivo” specifically examining cell viability, proliferation, invasiveness, and growth in nude mice. PTHrP(38–94)‐amide markedly inhibited proliferation and also caused striking toxicity and accelerated cell death in breast cancer cells. In addition, direct injection of PTHrP(38–94)‐amide into MDA‐MB231 breast cancer cells passaged in immunodeficient mice produced a marked reduction in tumor growth. These studies (i) indicate breast cancer cells are one of the few tissues in which specific effects of midregion PTHrP have been established to date, (ii) support a role for midregion secretory forms of PTHrP in modulating not only normal but also pathological mammary growth and differentiation, (iii) add further evidence for the existence of a specific midregion PTHrP receptor, and (iv) provide a novel molecule for modeling of small molecule analogues that may have anti‐breast cancer effects.

Type V collagen regulates the expression of apoptotic and stress response genes by breast cancer cells.

Type V collagen is a “minor” component of normal human breast stroma, which is subjected to over‐deposition in cases of ductal infiltrating carcinoma (DIC). We reported that, if used as a culture substrate for the DIC cell line 8701‐BC, it exhibited poorly‐adhesive properties and restrained the proliferative and motile behavior of the cell subpopulation able to attach onto it. Moreover, this collagen species was able to trigger DNA fragmentation and impair survival of 8701‐BC cells. In this study, we have extended our investigation with the aim to obtain further evidence that the death induced by type V collagen was of the apoptotic type by (i) microscopic detection and quantitation of Apoptag‐labeled cells, (ii) analysis of the expression levels of selected genes coding for apoptosis‐linked factors, caspases, and stress‐response proteins by conventional and semi‐quantitative multiplex PCR, and (iii) evaluation of the extent of caspase activation by chromogenic assay. We report here that type V collagen is able to determine an increase in the percentage of Apoptag‐positive cells, to up‐regulate Bcl‐xS, Bad, Dap kinase, hsf‐1, mthsp75, caspase‐1, ‐5, ‐8, ‐9, and ‐14, whilst down‐regulating Bcl‐2, Bcl‐xβ, and hsp60. Treatment of cell lysates with chromogenic tetrapeptide substrates specific for caspase‐1, ‐5, ‐8, and ‐9 demonstrated a marked increase of enzymatic activity in the presence of type V collagen. Our data validate 8701‐BC cell line as a suitable “in vitro” model for further and more detailed studies on the molecular mechanisms of the death response induced by type V collagen on primary DIC cells.

Cytotoxic effects of Jay Amin hydroxamic acid (JAHA), a ferrocene-based class I histone deacetylase inhibitor, on triple-negative MDA-MB231 breast cancer cells.

The histone deacetylase inhibitors (HDACis) are a class of chemically heterogeneous anticancer agents of which suberoylanilide hydroxamic acid (SAHA) is a prototypical member. SAHA derivatives may be obtained by three-dimensional manipulation of SAHA aryl cap, such as the incorporation of a ferrocene unit like that present in Jay Amin hydroxamic acid (JAHA) and homo-JAHA [ Spencer , et al. ( 2011 ) ACS Med. Chem. Lett. 2 , 358 - 362 ]. These metal-based SAHA analogues have been tested for their cytotoxic activity toward triple-negative MDA-MB231 breast cancer cells. The results obtained indicate that of the two compounds tested, only JAHA was prominently active on breast cancer cells with an IC(50) of 8.45 μM at 72 h of treatment. Biological assays showed that exposure of MDA-MB231 cells to the HDACi resulted in cell cycle perturbation with an alteration of S phase entry and a delay at G(2)/M transition and in an early reactive oxygen species production followed by mitochondrial membrane potential (MMP) dissipation and autophagy inhibition. No annexin binding was observed after short- (5 h) and longer (24 and 48 h) term incubation with JAHA, thereby excluding the promotion of apoptosis by the HDACi. Although caution must be exercised in extrapolation of in vitro results to the in vivo situation for which research on animals and human trials are needed, nevertheless JAHA treatment possesses the potential for its development as an agent for prevention and/or therapy of aggressive breast carcinoma, thus prompting us to get more insight into the molecular basis of its antibreast cancer activity.

Cadmium as a transcriptional modulator in human cells

Cadmium (Cd) is an underground mineral widely used in the steel industry, in plastics, and as a component of batteries. It is an industrial and environmental pollutant released as an air contaminant from fertilizers and, more prominently, in the form of wastewater. Food, drinking water, and, mainly, inhalation of smoke from cigarettes are sources of daily exposure of humans to the heavy metal. Although Cd has no known useful function for humans as well as other organisms, it appears to evoke in cells a number of responses that involve not only death signaling but also protective reactions against the toxicity. This finding prompted a number of experimental studies aimed to elucidate the cellular and molecular aspects of Cd-dependent regulation of gene expression and signal transduction pathways in different model system. Here, the authors briefly review the role of Cd as a transcriptional regulator in diverse cytotypes of human origin, focusing in particular on its effects on two classes of genes, i.e., stress-response genes such as metallothioneins (MTs), heme oxygenase, and heat shock proteins (hsps), and apoptosis-related genes, but giving also an overview of many other examples of genes involved in cell metabolism and both intracellular and extracellular signalization whose expression levels are controlled by Cd.

PTHrP [67-86] regulates the expression of stress proteins in breast cancer cells inducing modifications in urokinase-plasminogen activator and MMP-1 expression

It was previously reported that a midregion domain of parathyroid hormone-related protein (PTHrP), that is, [67-86]-amide, is able to restrain growth and promote matrigel penetration by the 8701-BC cell line, derived from a biopsy fragment of a primary ductal infiltrating carcinoma of the human breast, and that cell invasion in vitro is drastically impaired by inactivation of urokinase-plasminogen activator (uPa). In this study we started a more detailed investigation of the possible effects on gene expression arising from the interaction between PTHrP [67-86]-amide and 8701-BC breast cancer cells by a combination of conventional-, differential display-and semi-quantitative multiplex-polymerase chain reaction (PCR) assays. We present here the first evidence that the upregulation of some stress-related genes, most noticeably heat shock factor binding protein-1 (hsbp1) and heat shock protein 90 (hsp-90), is involved in the acquisition of an in vitro more invasive phenotype by cells treated with midregion PTHrP. This is conceivably accomplished by sequestering and inactivating heat shock factor-1 (hsf1) which is able to recognize Ets transcription-factor-binding sites present in some gene promoters, such as those of uPa and matrix metalloprotease-1 (MMP-1). In fact, our data show that incubation of PTHrP [67-86]-amide-treated cells with either antisense hsbp1-oligonucleotide or geldanamycin, an hsp90-inactivating antibiotic, results in downregulation of uPa and upregulation of MMP-1, and in a prominent inhibition of cell invasion in matrigel-containing Transwell chambers. Alternatively, incubation of untreated 8701-BC cells with quercetin, a flavonoid known to decrease the amount of free hsf1, is found to induce upregulation of uPa and downregulation of MMP-1, and an increase of matrigel invasion by cells, thus providing further supporting data of the involvement of hsf unavailability on the modulation of uPa and MMP-1 expression and on cell invasive behaviour. These studies confirm a previous postulate that over-secretion of uPa, rather than of other extracellular proteases, is a primary condition for the increase of invasive activity triggered by PTHrP [67-86]-amide in vitro, and support a role for midregion forms of PTHrP in potentially affecting pathological mammary growth and differentiation. They also identify two new key protagonists in the complex scenario of breast tumor cell invasiveness in vitro, that is, hsbp1 and hsp90, which deserve further and more extensive studies as potential and attractive molecular targets for anti-breast cancer treatments.

Adhesion, growth and cytoskeletal characteristics of 8701-BC breast carcinoma cells cultured in the presence of type V collagen☆

Type V collagen is one of the minor components of the extracellular matrix (ECM) whose content is increased in cases of ductal infiltrating carcinomas of the breast. In order to clarify its biological role, we have investigated the effect of this molecule, both as substrate and as soluble factor, on the behaviour of a breast carcinoma cell line (8701-BC) grown in vitro. Cell-collagen adhesion was monitored for 24 h from plating in the absence or presence of serum. The influence of type V collagen on cell growth was followed during 9 days of culture, and the actin-vinculin arrangement was studied by simultaneous fluorescent immuno-staining. The results indicate that type V collagen is not a permissive substrate for neoplastic cell proliferation and dissemination in vitro.