Alessandro Arcucci

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.

Effect of DNOC, Ferbam and Imidan exposure on mouse sperm morphology

DNOC, Ferbam and Imidan were tested in (C3H X C57BL/6) F1 mice to assess their potential testicular toxicity. Chemicals were administered i.p. and per os at different doses for 5 consecutive days. After 35 days the testicular was toxicity was evaluated by measuring the testicular weights, the sperm counts and the percentage of abnormal sperm. DNOC and Imidan failed to induce teratospermia in mice treated by both routes of administration. Conversely Ferbam induced a statistically significant increase in teratospermia only following per os administration to mice at a dose of 1000 mg/kg b.w./day. These data indicate that per os administration of Ferbam succeeded in producing active metabolites able to interfere with the differentiation process of spermatogenic cells.

Erratum - Analysis of extracellular superoxide dismutase and Akt in ascending aortic aneurysm with tricuspid or bicuspid aortic valve

This correct the article published on European Journal of Histochemistry 2014;58:200-206 doi: 10.4081/ejh.2014.2383.

In vitro cultured progenitors and precursors of cardiac cell lineages from human normal and post-ischemic hearts

The demonstration of the presence of dividing primitive cells in damaged hearts has sparked increased interest about myocardium regenerative processes. We examined the rate and the differentiation of in vitro cultured resident cardiac primitive cells obtained from pathological and normal human hearts in order to evaluate the activation of progenitors and precursors of cardiac cell lineages in post-ischemic human hearts. The precursors and progenitors of cardiomyocyte, smooth muscle and endothelial lineage were identified by immunocytochemistry and the expression of characteristic markers was studied by western blot and RT-PCR. The amount of proteins characteristic for cardiac cells (alpha-SA and MHC, VEGFR-2 and FVIII, SMA for the precursors of cardiomyocytes, endothelial and smooth muscle cells, respectively) inclines toward an increase in both alpha-SA and MHC. The increased levels of FVIII and VEGFR2 are statistically significant, suggesting an important re-activation of neoangiogenesis. At the same time, the augmented expression of mRNA for Nkx 2.5, the trascriptional factor for cardiomyocyte differentiation, confirms the persistence of differentiative processes in terminally injured hearts. Our study would appear to confirm the activation of human heart regeneration potential in pathological conditions and the ability of its primitive cells to maintain their proliferative capability in vitro. The cardiac cell isolation method we used could be useful in the future for studying modifications to the microenvironment that positively influence cardiac primitive cell differentiation or inhibit, or retard, the pathological remodeling and functional degradation of the heart.

Protein engineering on enzymes of the peptide elongation cycle in Sulfolobus solfataricus.

The present article is a review of the work done on the elongation factors EF-1 alpha, EF-2 and EF-1 beta isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. The molecular, physical and biochemical properties of the intact, truncated, mutant or chimeric forms are described and compared.

Altered expression of integrins in RSV-transformed chick epiphyseal chondrocytes

Chondrocytes have been shown to express both in vivo and in vitro a number of integrins of the beta1-, beta3- and beta5-subfamilies (Biorheology 37 (2000) 109). Normal and v-Src-transformed chick epiphyseal chondrocytes (CEC) display different adhesion properties. While normal CEC with time in culture tends to increase their adhesion to the substrate by organizing focal adhesions and actin stress fibers, v-Src-transformed chondrocytes display a refractile morphology and disorganization of actin cytoskeleton. We wondered whether the reduced adhesion and spreading of v-Src-transformed chondrocytes could be ascribed to changes in integrin expression and/or function. Integrin expression by normal CEC is studied and compared to v-Src-transformed chick chondrocytes, using monoclonal and polyclonal antibodies to integrins alpha- and beta-chains. We show the presence of alpha1-, alpha3-, alphav-, alpha6-, beta1- and beta3-chains on CEC, with very low levels of alpha2- and alpha5-chains. Alphav chain associates with multiple beta subunits in normal and transformed chondrocytes. With the exception of alpha1- and alpha2-chains, the levels of the integrin chains analyzed are higher in transformed chondrocytes as compared with normal chondrocytes. In spite of the increased levels of integrin expression, transformed chondrocytes exhibit loss of focal adhesion and actin stress fibers and low adhesion activity on several extracellular matrix constituents. These observations raise the possibility that, in addition to its effects on global pattern of integrin expression, v-Src can influence integrin function in chondrocytes.

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.