Giuseppina Granato

Markers of mitochondrial dysfunction during the diclofenac-induced apoptosis in melanoma cell lines

Melanoma is an aggressive cutaneous cancer, whose incidence is growing in recent years, especially in the younger population. The favorable therapy for this neoplasm consists in its early surgical excision; otherwise, in case of late diagnosis, melanoma becomes very refractory to any conventional therapy. Nevertheless, the acute inflammatory response occurring after excision of the primary melanoma can affect the activation and/or regulation of melanoma invasion and metastasis. Nonsteroidal anti-inflammatory drugs (NSAIDs), widely employed in clinical therapy as cyclooxygenase inhibitors, also display a cytotoxic effect on some cancer cell lines; therefore, their possible usage in combination with conventional chemo- and radio-therapies of tumors is being considered. In particular, diclofenac, one of the most common NSAIDs, displays its anti-proliferative effect in many tumor lines, through an alteration of the cellular redox state. In this study, the possible anti-neoplastic potential of diclofenac on the human melanoma cell lines A2058 and SAN was investigated, and a comparison was made with the results obtained from the nonmalignant fibroblast cell line BJ-5ta. Either in A2058 or SAN, the diclofenac treatment caused typical apoptotic morphological changes, as well as an increase of the number of sub-diploid nuclei; conversely, the same treatment on BJ-5ta had only a marginal effect. The observed decrease of Bcl-2/Bax ratio and a parallel increase of caspase-3 activity confirmed the pro-apoptotic role exerted by diclofenac in melanoma cells; furthermore, the drug provoked an increase of the ROS levels, a decrease of mitochondrial superoxide dismutase (SOD2), the cytosolic translocation of both SOD2 and cytochrome c, and an increase of caspase-9 activity. Finally, the cytotoxic effect of diclofenac was amplified, in melanoma cells, by the silencing of SOD2. These data improve the knowledge on the effects of diclofenac and suggest that new anti-neoplastic treatments should be based on the central role of mitochondrion in cancer development; under this concern, the possible involvement of SOD2 as a novel target could be considered.

Cancer: An Oxidative Crosstalk between Solid Tumor Cells and Cancer Associated Fibroblasts

Redox balance is associated with the regulation of several cell signalling pathways and functions. In fact, under physiological conditions, cells maintain a balance between oxidant and antioxidant systems, and reactive oxygen species (ROS) can act as second messengers to regulate cell proliferation, cell death, and other physiological processes. Cancer tissues usually contain higher levels of ROS than normal tissues, and this ROS overproduction is associated with tumor development. Neoplastic tissues are very heterogeneous systems, composed of tumor cells and microenvironment that has a critical role in tumor progression. Cancer associated fibroblasts (CAFs) represent the main cell type of tumor microenvironment, and they contribute to tumor growth by undergoing an irreversible activation process. It is known that ROS can be transferred from cancer cells to fibroblasts. In particular, ROS affect the behaviour of CAFs by promoting the conversion of fibroblasts to myofibroblasts that support tumor progression and dissemination. Furthermore, the wrecking of redox homeostasis in cancer cells and tumor microenvironment induces a metabolic reprogramming in tumor cells and cancer associated fibroblasts, giving advantage to cancer growth. This review describes the role of ROS in tumor growth, by focusing on CAFs activation and metabolic interactions between cancer cells and stromal fibroblasts.

Evaluation of cytotoxic effects of 7-dehydrocholesterol on melanoma cells

Ultraviolet radiation is the main cause of skin cancers, and melanoma is the most serious form of tumor. There is no therapy for advanced-stage melanoma and its metastasis because of their high resistance to various anticancer therapies. Human skin is an important metabolic organ in which occurs photoinduced synthesis of vitamin D3 from 7-dehydrocholesterol (7-DHC). 7-DHC, the precursor of cholesterol biosynthesis, is highly reactive and easily modifiable to produce 7-DHC-derived compounds. The intracellular levels of 7-DHC or its derivatives can have deleterious effects on cellular functionality and viability. In this study we evaluated the effects on melanoma cell lines of 7-DHC as such and for this aim we used much care to minimize 7-DHC modifications. We found that from 12 to 72 h of treatment 82-86% of 7-DHC entered the cells, and the levels of 7-DHC-derived compounds were not significant. Simultaneously, reactive oxygen species production was significantly increased already after 2h. After 24 h and up to 72 h, 7-DHC-treated melanoma cells showed a reduction in cell growth and viability. The cytotoxic effect of 7-DHC was associated with an increase in Bax levels, decrease in Bcl-2/Bax ratio, reduction of mitochondrial membrane potential, increase in apoptosis-inducing factor levels, unchanged caspase-3 activity, and absence of cleavage of PARP-1. These findings could explain the mechanism through which 7-DHC exerts its cytotoxic effects. This is the first report in which the biological effects found in melanoma cells are mainly attributable to 7-DHC as such.

Ligand-based chemoinformatic discovery of a novel small molecule inhibitor targeting CDC25 dual specificity phosphatases and displaying in vitro efficacy against melanoma cells

CDC25 phosphatases are important regulators of the cell cycle and represent promising targets for anticancer drug discovery. We recently identified NSC 119915 as a new quinonoid CDC25 inhibitor with potent anticancer activity. In order to discover more active analogs of NSC 119915, we performed a range of ligand-based chemoinformatic methods against the full ZINC drug-like subset and the NCI lead-like set. Nine compounds (3, 5–9, 21, 24, and 25) were identified with Ki values for CDC25A, -B and -C ranging from 0.01 to 4.4 μM. One of these analogs, 7, showed a high antiproliferative effect on human melanoma cell lines, A2058 and SAN. Compound 7 arrested melanoma cells in G2/M, causing a reduction of the protein levels of CDC25A and, more consistently, of CDC25C. Furthermore, an intrinsic apoptotic pathway was induced, which was mediated by ROS, because it was reverted in the presence of antioxidant N-acetyl-cysteine (NAC). Finally, 7 decreased the protein levels of phosphorylated Akt and increased those of p53, thus contributing to the regulation of chemosensitivity through the control of downstream Akt pathways in melanoma cells. Taken together, our data emphasize that CDC25 could be considered as a possible oncotarget in melanoma cells and that compound 7 is a small molecule CDC25 inhibitor that merits to be further evaluated as a chemotherapeutic agent for melanoma, likely in combination with other therapeutic compounds.

Involvement of breast cancer associated fibroblasts in tumor development, therapy resistance and evaluation of potential therapeutic strategies

Breast cancer is the most common cancer in women, which incidence has increased in recent years. It is constituted by very heterogeneous tissue characterized by an abnormal microenvironment regulating tumor progression and providing evasion from cancer therapies. Breast cancer-associated fibroblasts (BCAFs) are the main cell type of breast cancer microenvironment and can represent up to 80% of the tumor mass. In particular, BCAFs induce cancer initiation, proliferation, invasion and metastasis by undergoing an activation process associated with the secretion of growth factors, cytokines, and paracrine interactions. Therapy resistance is the main cause of poor therapeutic results or even failure in breast cancer patients. Despite recent advances in breast cancer management, there is a need for new prognostic markers and novel agents for targeting key signalling pathways to either improve the efficacy of the current therapies, or reduce toxicity. In this view, BCAFs represent markers useful to clinical diagnosis, therapy, and prognosis of breast cancer. This review focuses on the role of BCAFs in cancer, and describes the processes of endocrine/chemotherapy resistance linked to BCAFs action. Moreover, it points to molecules and pathways regulating therapy resistance induced by BCAFs. Finally, potential therapeutic strategies targeting BCAFs and offering new tools in breast cancer therapy are highlighted.

Generation and analysis of spheroids from human primary skin myofibroblasts: an experimental system to study myofibroblasts deactivation

Myofibroblasts are activated fibroblasts involved in tissue repair and cancer. They are characterized by de novo expression of α-smooth muscle actin (α-SMA), immunoregulatory phenotype and paracrine interaction with normal and tumorigenic cells leading to cell proliferation. At the end of wound-healing myofibroblasts undergo apoptotic cell death, whereas in vitro-activated fibroblasts are also subjected to a programmed necrosis-like cell death, termed nemosis, associated with cyclooxygenase-2 (COX-2) expression induction and inflammatory response. Furthermore, myofibroblasts form clusters during wound healing, fibrotic states and tumorigenesis. In this study, we generated and analysed clusters such as spheroids from human primary cutaneous myofibroblasts, which represent a part of stromal microenvironment better than established cell lines. Therefore, we evaluated apoptotic or necrotic cell death, inflammation and activation markers during myofibroblasts clustering. The spheroids formation did not trigger apoptosis, necrotic cell death and COX-2 protein induction. The significant decrease of α-SMA in protein extracts of spheroids, the cytostatic effect exerted by spheroids conditioned medium on both normal and cancer cell lines and the absence of proliferation marker Ki-67 after 72 h of three-dimensional culture indicated that myofibroblasts have undergone a deactivation process within spheroids. The cells of spheroids reverted to adhesion growth preserved their proliferation capability and can re-acquire a myofibroblastic phenotype. Moreover, the spontaneous formation of clusters on plastic and glass substrates suggests that aggregates formation could be a physiological feature of cutaneous myofibroblasts. This study represents an experimental model to analyse myofibroblasts deactivation and suggests that fibroblast clusters could be a cell reservoir regulating tissues turnover.

Analysis of SOD3 and Akt in ascending aortic aneurysm

Ascending aortic aneurysm (AsAA) is divided into three different forms: syndromic, familial non-syndromic, and degenerative. Bicuspid aortic valve (BAV), occuring in 2% of the population, is the most frequent cardiac congenital abnormality, associated to AsAA. All the different forms of AsAAs are a consequence of cystic medial necrosis (CMN), characterized by apoptotic loss of smooth muscle cells (SMCs), fragmentation of elastic and collagen fibers and increased accumulation of mucoid material. The extracellular superoxide dismutase (SOD3) is a Cu/Zn enzyme, affecting redox state and homeostasis of extracellular matrix (ECM) (1). Moreover, the outsidein signalling from ECM modulates intracellular pathways regulating many cellular functions. The multifunctional Akt pathway affects survival and cellular proliferation and has important effects on the cardiovascular function. In this study we examined the relevance of SOD3 and Akt in AsAA pathogenesis. To this aim, the SOD3 and Akt protein levels were evaluated in normal ascending aortic tissues (n=6) and in tissues from AsAAs associated both to tricuspid aortic valve (TAV) (n=6) and BAV (n=6); moreover, we measured SOD3 activity in sera from healthy donors and patients with AsAA. Our data showed a reduction of SOD3 and phospho-Akt (pAkt) protein levels in AsAAs from BAV patients compared to normal donors; on the other hand, no differences emerged in SOD3 activity. Furthermore, immunohistochemical analysis performed on normal and pathological ascending aortic tissues showed a SOD3 immunostaining in both extracellular space and tunica media cells from normal ascending aortic tissues; conversely, no SOD3 immunostaining was detected in AsAAs tissues from both TAV and BAV patients. Our data show that SOD3 and pAkt could be associated to AsAA pathogenesis and suggest a link between ECM homeostasis and Akt survival pathway.

Metabolic Reprogramming of Cancer Associated Fibroblasts: The Slavery of Stromal Fibroblasts

Cancer associated fibroblasts (CAFs) are the main stromal cell type of solid tumour microenvironment and undergo an activation process associated with secretion of growth factors, cytokines, and paracrine interactions. One of the important features of solid tumours is the metabolic reprogramming that leads to changes of bioenergetics and biosynthesis in both tumour cells and CAFs. In particular, CAFs follow the evolution of tumour disease and acquire a catabolic phenotype: in tumour tissues, cancer cells and tumour microenvironment form a network where the crosstalk between cancer cells and CAFs is associated with cell metabolic reprogramming that contributes to CAFs activation, cancer growth, and progression and evasion from cancer therapies. In this regard, the study of CAFs metabolic reprogramming could contribute to better understand their activation process, the interaction between stroma, and cancer cells and could offer innovative tools for the development of new therapeutic strategies able to eradicate the protumorigenic activity of CAFs. Therefore, this review focuses on CAFs metabolic reprogramming associated with both differentiation process and cancer and stromal cells crosstalk. Finally, therapeutic responses and potential anticancer strategies targeting CAFs metabolic reprogramming are reviewed.

Spheroids from human primary skin myofibroblasts as experimental system for myofibroblast deactivation studies

Myofibroblasts are activated fibroblasts, involved in tissue repair and cancer, characterized by de novo expression of alpha smooth muscle actin (α-SMA), increased secretion of growth factors and immunoregulatory phenotype [1]. At the end of wound healing myofibroblasts undergo apoptotic cell death, whereas in vitro they are also subjected to a programmed necrosis-like cell death, termed nemosis, associated with cyclooxygenase-2 (COX-2) expression induction and inflammatory response [1,2]. Moreover, fibroblasts form clusters during wound healing, fibrotic states and tumorigenesis. In this investigation, we produced and analysed clusters such as spheroids from human primary cutaneous myofibroblasts to evaluate apoptotic or necrotic cell death, inflammation and activation markers during myofibroblasts clustering. The spheroids formation does not induce apoptosis, necrotic cell death and COX-2 protein induction. The significant decrease of α-SMA in protein extracts of spheroids, the anti- migratory effect of spheroid-conditioned medium on normal cell lines and the absence of proliferation marker Ki-67 after 72 h of three-dimensional culture indicated that myofibroblasts undergo a deactivation process within spheroids. The cells of spheroids, reverted to adhesion growth, preserve their proliferation capability and are able to reacquire a myofibroblastic phenotype. Furthermore, the spontaneous formation of clusters and spheroids on plastic and glass substrates suggests that aggregates formation could be a physiological feature of cutaneous myofibroblasts. This study represents an experimental model to analyse myofibroblasts deactivation and indicates that fibroblast clusters could be a cell reservoir regulating tissue turnover.

Positional memory of fibroblasts may affect efficiency of iPSC reprogramming

Induced Pluripotent Stem cells (iPSC) are pluripotent stem cells reprogrammed from adult somatic cells. Although iPSC hold great potential for applications in regenerative medicine, technical problems, mostly related to the low efficiency of reprogramming, are yet to be solved. Since the most used cells for iPSC reprogramming are skin fibroblasts (FB), and since FB preserve positional memory, we hypothesize that the anatomic origin of FB might influence iPSC reprogramming.We isolated FB from skin of five different sites (neck, arm, thigh, breast, abdomen) of 13 patients undergoing plastic surgery or from heart wall or ascending aorta wall of the explanted heart of 3 patients receiving heart transplantation. FB from different anatomic sites and control FB from neonatal foreskin, were cultured for one week to evaluate morphology, proliferation rate and proneness to apoptosis. Additionally, expression of vimentin, cadherin, smooth muscle actin and Factor VIII was investigated to exclude the presence of other cell types. Transcriptome analysis including genes involved in stemness maintenance, embryogenesis, cell growth, activation and development, was performed by real-time PCR. Despite the similar morphology of FB from different sites, and immunopositivity for vimentin, along with the absence of other cell type markers, FB isolated from abdomen and heart had 1.5-fold higher doubling time, while FB from heart, abdomen and breast were less susceptible to apoptosis. Intriguingly, Real-Time PCR revealed that in abdomen, breast, neck, arm and heart FB genes involved in cell growth, development, proliferation, and migration, as TM4SF1, GPC4, CSPG2, DDIT4, ID1 were up-regulated, while genes regulating embryogenesis and tissue morphogenesis, like VCAN, FN1, HOXA5, CD49a were up-regulated in FB isolated from abdomen, arm and heart. However, all FBs had transcripts of markers of Mesenchymal Stem Cells (MSC), as CD105 and CD90. Our results provide evidence that human adult FB from different sites have different genetic program. Therefore, FB may respond to reprogram technology in different manner, thus affecting reprogramming efficiency. While offering novel perspective of the reprogramming technology, our study also demonstrates that abdomen and breast FB share cardiac genetic signature of cardiac FB while expressing markers of MSC and they might represent the ideal cell for cardiac reprogramming.