Trace elements in toenails in a population living near a modern municipal solid waste incinerator in Modena (Italy).

BACKGROUND AND OBJECTIVES: A cross-sectional biomonitoring study was performed in Modena (Italy) to assess trace element levels in toenails in a population living near a municipal solid waste incinerator (SWI), and investigate potential differences in their concentrations according to SWI emission exposure and other environmental and behavioral factors. METHODS: During the winter 2013/14 eligible subjects, aged 18-69 yrs, living within 4 km from SWI, were randomly selected from the population register. Toxic and essential element concentrations (As, Cd, Cr, Cu, Mn, Ni, Pb, Se, Zn) were analyzed in 489 toenail samples. Individual exposure to SWI emissions was estimated by using, as a tracer, fall-out maps of emitted particulate matter. Information on anthropometric parameters, lifestyles, diet, and road traffic, residential and work exposures were collected by questionnaires and objective measurements. Multivariate logistic regression analyses were carried out, separately for females and males. RESULTS: Excluding As, toxic elements were found, usually at low levels, in many samples, while essential elements, especially Cu and Zn, showed higher levels. Overall, no clear relationships between element levels and SWI exposure were observed, whereas associations with other environmental and lifestyle factors were found, including local food consumption, smoking and occupation. CONCLUSIONS: The low pollutant concentrations measured in SWI emissions could explain the absence of clear patterns in toenail levels across SWI exposure levels. The associations observed with other factors suggest that, at least in this specific population, other environmental exposures and personal behaviors could act as more important predictors of trace element uptake.

The Hydroxytyrosol Induces the Death for Apoptosis of Human Melanoma Cells.

Melanoma is the most aggressive form of skin cancer and one of the most treatment-refractory malignancies. In metastatic melanoma cell lines, we analysed the anti-proliferative and pro-apoptotic potentials of a phenolic component of olive oil, the hydroxytyrosol. In particular, through MTS assay, DeadEnd™ Colorimetric TUNEL assay, Annexin V binding and PI uptake, western blot experiment, intracellular reactive oxygen species (ROS) analysis, and the cell colony assay, we showed that the hydroxytyrosol treatment remarkably reduces the cell viability inducing the death for apoptosis of melanoma cells. Moreover, we showed that the hydroxytyrosol treatment of melanoma cells leads to a significant increase of p53 and γH2AX expression, a significant decrease of AKT expression and the inhibition of cell colony formation ability. Finally, we propose that the increased amount of intracellular reactive oxygen species (ROS) that may be related to the regulation of the pathways involved in the activation of apoptosis and in the inhibition of melanoma growth could be the strategy used by hydroxytyrosol to exert its functions in melanoma. Therefore, for its role in melanoma growth inhibition, the hydroxytyrosol treatment could deeply interfere with melanoma progression as a promising therapeutic option for the treatment of this highly invasive tumour.

Mitochondrial transplantation ameliorates acute limb ischemia.

OBJECTIVE: Acute limb ischemia (ALI), the most challenging form of ischemia-reperfusion injury (IRI) in skeletal muscle tissue, leads to decreased skeletal muscle viability and limb function. Mitochondrial injury has been shown to play a key role in skeletal muscle IRI. In previous studies, we have demonstrated that mitochondrial transplantation (MT) is an efficacious therapeutic strategy to replace or to augment mitochondria damaged by IRI, allowing enhanced muscle viability and function in cardiac tissue. In this study, we investigated the efficacy of MT in a murine ALI model. METHODS: C57BL/6J mice (male, 10-12 weeks) were used in a model of ALI. Ischemia was induced by applying a tourniquet on the left hindlimb. After 2 hours of ischemia, the tourniquet was released, and reperfusion of the hindlimb was re-established; either vehicle alone (n = 15) or vehicle containing mitochondria (n = 33) was injected directly into all the muscles of the hindlimb. Mitochondria were delivered at concentrations of 1 × 106 to 1 × 109 per gram wet weight to each muscle, and the animals were allowed to recover. Sham mice received no ischemia or injections but were anesthetized for 2 hours and allowed to recover. After 24 hours of recovery, limb function was assessed by DigiGait (Mouse Specifics Inc, Boston, Mass), and animals were euthanized; the gastrocnemius, soleus, and vastus medialis muscles were collected for analysis. RESULTS: After 24 hours of hindlimb reperfusion, infarct size (percentage of total mass) and apoptosis were significantly decreased (P < .001, each) in the gastrocnemius, soleus, and vastus medialis muscles in MT mice compared with vehicle mice for all mitochondrial concentrations (1 × 106 to 1 × 109 per gram wet weight). DigiGait analysis at 24 hours of reperfusion showed that percentage shared stance time was significantly increased (P < .001) and stance factor was significantly decreased (P = .001) in vehicle compared with MT and sham mice. No significant differences in percentage shared stance time (P = .160) or stance factor (P = .545) were observed between MT and sham mice. CONCLUSIONS: MT ameliorates skeletal muscle injury and enhances hindlimb function in the murine model of ALI.

Delayed Transplantation of Autologous Mitochondria for Cardioprotection in a Porcine Model.

BACKGROUND: We have previously demonstrated the efficacy of mitochondrial transplantation (MT) for the treatment of ischemia-reperfusion injury (IRI). We now investigate the efficacy of delayed MT by intracoronary administration in a model of regional IRI as a strategy for cardioprotection. METHODS: Female Yorkshire pigs (40-50 kg; n = 16) underwent 30 minutes of ischemia by snaring of the left anterior descending artery, and the hearts were then reperfused for 120 minutes. At that point, vehicle only or autologous mitochondria (1 × 109 in 5 mL of vehicle) were delivered as a bolus to the left coronary ostium, followed by a further 120-minute reperfusion. RESULTS: Echocardiographic analysis demonstrated that hearts receiving delayed MT after regional IRI had enhanced ejection fraction (P = .019), fractional shortening (P = .022), and fractional area change (P = .011) at 240 minutes of reperfusion compared with the untreated pigs. At the end of reperfusion there was a difference between the groups in measures of global left ventricular (LV) function such as LV end-diastolic pressure (P = .015) and rate of rise of LV pressure (P = .021). No significant differences were found between the groups in the area at risk (P = .48). Infarct size (% area at risk) was significantly decreased in hearts receiving MT compared with hearts receiving vehicle only (P < .001). CONCLUSIONS: Delayed MT by intracoronary injection appreciably decreases myocardial infarct size, increasing regional and global myocardial function. These results suggest that this can be a viable treatment modality in IRI, thus reducing long-term morbidity and mortality in cardiac surgical patients.

Preischemic autologous mitochondrial transplantation by intracoronary injection for myocardial protection.

OBJECTIVE: To investigate preischemic intracoronary autologous mitochondrial transplantation (MT) as a therapeutic strategy for prophylactic myocardial protection in a porcine model of regional ischemia-reperfusion injury (IRI). METHODS: The left coronary artery was cannulated in Yorkshire pigs (n = 26). Mitochondria (1 × 109) or buffer (vehicle [Veh]) were delivered as a single bolus (MTS) or serially (10 injections over 60 minutes; MTSS). At 15 minutes after injection, the heart was subjected to temporary regional ischemia (RI) by snaring the left anterior descending artery. After 30 minutes of RI, the snare was released, and the heart was reperfused for 120 minutes. RESULTS: Coronary blood flow (CBF) and myocardial function were increased temporarily during the pre-RI period. Following 30 minutes of RI, MTS and MTSS hearts had significantly increased CBF that persisted throughout reperfusion (Veh vs MTS and MTSS; P = .04). MTS and MTSS showed a significantly enhanced ejection fraction (Veh vs MTS, P < .001; Veh vs MTSS, P = .04) and developed pressure (Veh vs MTS, P < .001; Veh vs MTSS, P = .03). Regional function, assessed through segmental shortening (Veh vs MTS, P = .03; Veh vs MTSS, P < .001), fractional shortening (Veh vs MTS, P < .001; Veh vs MTSS, P = .04), and strain analysis (Veh vs MTS, P = .002; Veh vs MTSS, P = .003), was also significantly improved. Although there was no difference in the area at risk between treatment groups, infarct size was significantly reduced in both MT groups (Veh vs MTS and MTSS, P < .001). CONCLUSIONS: Preischemic MT by single or serial intracoronary injections provides prophylactic myocardial protection from IRI, significantly decreasing infarct size and enhancing global and regional function.

Mitochondrial transplantation enhances murine lung viability and recovery after ischemia-reperfusion injury.

The most common cause of acute lung injury is ischemia-reperfusion injury (IRI), during which mitochondrial damage occurs. We have previously demonstrated that mitochondrial transplantation is an efficacious therapy to replace or augment mitochondria damaged by IRI, allowing for enhanced muscle viability and function in cardiac tissue. Here, we investigate the efficacy of mitochondrial transplantation in a murine lung IRI model using male C57BL/6J mice. Transient ischemia was induced by applying a microvascular clamp on the left hilum for 2 h. Upon reperfusion mice received either vehicle or vehicle-containing mitochondria either by vascular delivery (Mito V) through the pulmonary artery or by aerosol delivery (Mito Neb) via the trachea (nebulization). Sham control mice underwent thoracotomy without hilar clamping and were ventilated for 2 h before returning to the cage. After 24 h recovery, lung mechanics were assessed and lungs were collected for analysis. Our results demonstrated that at 24 h of reperfusion, dynamic compliance and inspiratory capacity were significantly increased and resistance, tissue damping, elastance, and peak inspiratory pressure (Mito V only) were significantly decreased (P < 0.05) in Mito groups as compared with their respective vehicle groups. Neutrophil infiltration, interstitial edema, and apoptosis were significantly decreased (P < 0.05) in Mito groups as compared with vehicles. No significant differences in cytokines and chemokines between groups were shown. All lung mechanics results in Mito groups except peak inspiratory pressure in Mito Neb showed no significant differences (P > 0.05) as compared with Sham. These results conclude that mitochondrial transplantation by vascular delivery or nebulization improves lung mechanics and decreases lung tissue injury.

Dibutyltin(IV) and Tributyltin(IV) Derivatives of meso-Tetra(4-sulfonatophenyl)porphine Inhibit the Growth and the Migration of Human Melanoma Cells.

Melanoma is the most aggressive and deadly form of skin cancer, which is largely due to its propensity to metastasize. Therefore, with the aim to inhibit the growth and the metastatic dissemination of melanoma cells and to provide a novel treatment option, we studied the effects of the melanoma treatment with two organotin(IV) complexes of the meso-tetra(4-sulfonato-phenyl)porphine, namely (Bu2Sn)2TPPS and (Bu3Sn)4TPPS. In particular, we showed that nanomolar concentrations of (Bu2Sn)2TPPS and (Bu3Sn)4TPPS are sufficient to inhibit melanoma cell growth, to increase the expression of the full-length poly (ADP-ribose) polymerase (PARP-1), to induce the cell cycle arrest respectively at G2/M and G0/G1 through the inhibition of the Cyclin D1 expression and to inhibit cell colony formation. Nanomolar concentrations of (Bu2Sn)2TPPS and (Bu3Sn)4TPPS are also sufficient to inhibit the melanoma cell migration and the expression of some adhesion receptors. Moreover, we report that (Bu2Sn)2TPPS and (Bu3Sn)4TPPS act downstream of BRAF, mainly bypassing its functions, but targeting the STAT3 signalling protein. Finally, these results suggest that (Bu2Sn)2TPPS and (Bu3Sn)4TPPS may be effective therapeutic strategies for their role in the inhibition of melanoma growth and migration.

Alloreactivity and allorecognition of syngeneic and allogeneic mitochondria.

Previously, we have demonstrated that the transplantation of autologous mitochondria is cardioprotective. No immune or autoimmune response was detectable following the single injection of autologous mitochondria. To expand the therapeutic potential and safety of mitochondrial transplantation, we now investigate the immune response to single and serial injections of syngeneic and allogeneic mitochondria delivered by intraperitoneal injection. Our results demonstrate that there is no direct or indirect, acute or chronic alloreactivity, allorecognition or damage-associated molecular pattern molecules (DAMPs) reaction to single or serial injections of either syngeneic or allogeneic mitochondria.

A Novel Biological Strategy for Myocardial Protection by Intracoronary Delivery of Mitochondria: Safety and Efficacy.

Mitochondrial dysfunction is the determinant insult of ischemia-reperfusion injury. Autologous mitochondrial transplantation involves supplying one's healthy mitochondria to the ischemic region harboring damaged mitochondria. The authors used in vivo swine to show that mitochondrial transplantation in the heart by intracoronary delivery is safe, with specific distribution to the heart, and results in significant increase in coronary blood flow, which requires intact mitochondrial viability, adenosine triphosphate production, and, in part, the activation of vascular KIR channels. Intracoronary mitochondrial delivery after temporary regional ischemia significantly improved myocardial function, perfusion, and infarct size. The authors concluded that intracoronary delivery of mitochondria is safe and efficacious therapy for myocardial ischemia-reperfusion injury.

Mitochondrial transplantation prolongs cold ischemia time in murine heart transplantation.

BACKGROUND: Cold ischemia time (CIT) causes ischemia‒reperfusion injury to the mitochondria and detrimentally effects myocardial function and tissue viability. Mitochondrial transplantation replaces damaged mitochondria and enhances myocardial function and tissue viability. Herein we investigated the efficacy of mitochondrial transplantation in enhancing graft function and viability after prolonged CIT. METHODS: Heterotopic heart transplantation was performed in C57BL/6J mice. Upon heart harvesting from C57BL/6J donors, 0.5 ml of either mitochondria (1 × 108 in respiration buffer; mitochondria group) or respiration buffer (vehicle group) was delivered antegrade to the coronary arteries via injection to the coronary ostium. The hearts were excised and preserved for 29 ± 0.3 hours in cold saline (4°C). The hearts were then heterotopically transplanted. A second injection of either mitochondria (1 × 108) or respiration buffer (vehicle) was delivered antegrade to the coronary arteries 5 minutes after transplantation. Grafts were analyzed for 24 hours. Beating score, graft function, and tissue injury were measured. RESULTS: Beating score, calculated ejection fraction, and shortening fraction were significantly enhanced (p < 0.05), whereas necrosis and neutrophil infiltration were significantly decreased (p < 0.05) in the mitochondria group as compared with the vehicle group at 24 hours of reperfusion. Transmission electron microscopy showed the presence of contraction bands in vehicle but not in mitochondria grafts. CONCLUSIONS: Mitochondrial transplantation prolongs CIT to 29 hours in the murine heart transplantation model, significantly enhances graft function, and decreases graft tissue injury. Mitochondrial transplantation may provide a means to reduce graft failure and improve transplantation outcomes after prolonged CIT.

Human biomonitoring of polycyclic aromatic hydrocarbonsand metals in the general population residing near the municipal solid waste incinerator of Modena, Italy.

BACKGROUND AND OBJECTIVES: A cross-sectional biomonitoring study was carried out to investigate exposure to incinerator emission in relation to the body burden of selected biomarkers in the population living around the plant. METHODS: Approximately 500 people, aged 18-69 yrs, living within 4 km from the incinerator were randomly selected form the population register. Exposure was measured through fall-out maps of particulate matter (PM), used as tracer for incinerator emissions. Ten metabolized polycyclic aromatic hydrocarbons (PAHs), from naphthalene to chrysene, 1-hydroxypyrene and twelve metals (Cd, Cr, Cu, Hg, Ni, Pb, Ni, Zn, V, Tl, As, Sn) were measured in spot urine samples. Confounders, such as diet, smoking, traffic, occupation and personal characteristics were assessed by questionnaires and objective measurements, and included into multivariate linear regression models. RESULTS: Metal concentrations in urine were in line with or higher than Italian reference limits, besides Cr and V with more than twofold concentrations. Metal levels did not show clear association to exposure categories. Most abundant PAHs were naphthalene (median 26.2 ng/L) and phenanthrene (7.4 ng/L). All PAHs, but benz[a]anthracene and 1-hydroxypyrene, were found in more than 52% of samples, and included in regression models. Significant associations between urinary PAHs and exposure were found, strong for fluorene, and weaker for naphthalene, fluoranthene and pyrene. Results were confirmed by sensitivity analyses. Correlation with variables reported in literature were observed. CONCLUSIONS: The study indicates that the emissions were very low and highlights that specific urinary PAHs provided useful information about the internal dose arising from incinerator emission.

The HLA-DR mediated signalling increases the migration and invasion of melanoma cells, the expression and lipid raft recruitment of adhesion receptors, PD-L1 and signal transduction proteins.

The constitutive expression of Major Histocompatibility Complex (MHC) class II molecules is restricted to professional Antigen-Presenting Cells (APCs), nevertheless almost 50% of melanomas express constitutively the MHC class II molecules. Therefore, in two MHC class II constitutive expressing melanoma cell lines we studied the signalling mediated by the HLA-DR molecules in the aim to understand the consequence of class II mediated signalling on metastatic dissemination of melanoma. In particular, we reported that the HLA-DR mediated signalling play a new role in melanoma progression, increasing the migration and invasion of melanoma cells. Furthermore, we showed that the HLA-DR mediated signalling increases the expression and the lipid raft localisation of class II molecules, PD-L1 receptor, Integrin and CAM adhesion receptors, FAK, AKT and STAT3 signalling proteins. We also showed that the HLA-DR mediated signalling increases the activation of FAK, AKT, ERK, PKC and STAT3 signalling proteins and the expression of ILK, PAX, BRAF, ERK and PKC. Indeed, the results showed suggest that the HLA-DR mediated signalling provides a platform useful to frustrate an effective anti-tumour response and to increase melanoma migration and metastatic dissemination of this cancer.

High-dose Ionizing Radiation Regulates Gene Expression Changes in the MCF7 Breast Cancer Cell Line.

BACKGROUND: Intraoperative electron radiation therapy (IOERT) is a therapeutic technique which administers a single high dose of ionizing radiation immediately after surgical tumor removal. IOERT induces a strong stress response: both tumor and normal cells activating pro- and antiproliferative cell signaling pathways. Following treatment, several genes and factors are differently modulated, producing an imbalance in cell fate decision. However, the contribution of these genes and pathways in conferring different cell radiosensitivity and radioresistance needs to be further investigated, in particular after high-dose treatments. Despite the documented and great impact of IOERT in breast cancer care, and the trend for dose escalation, very limited data are available regarding gene-expression profiles and cell networks activated by IOERT or high-dose treatment. The aim of the study was to analyze the main pathways activated following high radiation doses in order to select for potential new biomarkers of radiosensitivity or radioresistance, as well as to identify therapeutic targets useful in cancer care. MATERIALS AND METHODS: We performed gene-expression profiling of the MCF7 human breast carcinoma cell line after treatment with 9- and 23-Gy doses (conventionally used during IOERT boost and exclusive treatments, respectively) by cDNA microarrays. Real-Time Quantitative Reverse Transcription PCR (qRT-PCR), immunofluorescence and immunoblot experiments were performed to validate candidate IOERT biomarkers. We also conducted clonogenic tests and cellular senescence assays to monitor for radiation-induced effects. RESULTS: The analyses highlighted a transcriptome dependent on the dose delivered and a number of specific key genes that may be proposed as new markers of radiosensitivity. Cell and molecular traits observed in MCF7 cells revealed a typical senescent phenotype associated with cell proliferation arrest after treatments with 9- and 23-Gy doses. CONCLUSION: In this study, we report genes and cellular networks activated following high-dose IOERT. The selected validated genes were used to design two descriptive models for each dose delivered. We believe that this study could contribute to the understanding over the complex mechanisms which regulate cell radiosensitivity and radioresistance in order to improve personalized radiotherapeutic treatment.

Focusing on genera to improve species identification: revised systematics of the ciliate Spirostomum.

Although many papers dealing with the description of new ciliate taxa are published each year, species taxonomy and identification in most groups of the phylum Ciliophora remain confused. This is largely due to a scarcity of surveys on the systematics of immediately higher levels (genera and families) providing data for old and new species together. Spirostomum is a common and distinctive inhabitant of fresh- and brackish water environments, including artificial and eutrophic ones, and is a good model for applied ecology and symbiosis research. Despite this, only 3 of the numerous species are commonly cited, and no studies have yet confirmed their monophyly, with the consequence that reproducibility of the results may be flawed. In this paper we present morphological and molecular data for 30 Spirostomum populations representing 6 different morphospecies, some of which were collected in previously unreported countries. We performed a detailed revision of Spirostomum systematics combining literature surveys, new data on hundreds of organisms and statistical and phylogenetic analyses; our results provide insights on the evolution, ecology and distribution of known morphospecies and a novel one: Spirostomum subtilis sp. n. We also offer tools for quick species identification.

Supramolecular assemblies based on complexes of nonionic amphiphilic cyclodextrins and a meso-tetra(4-sulfonatophenyl)porphine tributyltin(IV) derivative: potential nanotherapeutics against melanoma.

Amphiphilic cyclodextrin (ACyD) provides water-soluble and adaptable nanovectors by modulating the balance between the hydrophobic and hydrophilic chains at both CyD sides. This work aimed to design nanoassemblies based on nonionic and hydrophilic ACyD (SC6OH) for the delivery of a poor-water-soluble organotin(IV)-porphyrin derivative [(Bu3Sn)4TPPS] to melanoma cancer cells. To characterize the porphyrin derivatives under simulated physiological conditions, a speciation was performed using complementary techniques. In aqueous solution (≤ 20 µM), (Bu3Sn)4TPPS primarily exists as a monomer (2 in Figure 1), as suggested by the low static anisotropy (ρ ≈ 0.02) with a negligible formation of porphyrin supramolecular aggregates. MALDI-TOF spectra indicate the presence of moieties (i.e., [(Bu3Sn)3TPPS](-)) that are derivatives of the monomeric species. Spectrofluorimetry coupled with potentiometric measurements primarily assesses the presence of the hydrolytic [(Bu3Sn)4TPPS (OH)4](4-) species under physiological conditions. Nanoassemblies of (Bu3Sn)4TPPS/SC6OH were prepared by dispersion of organic films in PBS at pH 7.4 and were investigated using a combination of spectroscopic and morphological techniques. The UV-vis and emission fluorescence spectra of the (Bu3Sn)4TPPS/SC6OH reveal shifts in the peculiar bands of the organotin(IV)-porphyrin derivative due to its interaction with the ACyD supramolecular assemblies in aqueous solution. The mean size was within the range of 100-120 nm. The ξ-potential was negative (-16 mV) for the (Bu3Sn)4TPPS/SC6OH nanoassemblies, with an entrapment efficiency of approximately 67%. The intracellular delivery, cytotoxicity, nuclear morphology and cell growth kinetics were evaluated via fluorescence microscopy on A375 human melanoma cells. The delivery of (Bu3Sn)4TPPS by ACyD with respect to free (Bu3Sn)4TPPS increases the internalization efficiency and cytotoxicity to induce apoptotic cell death and, at lower concentrations, changes the cellular morphology and prevents cell proliferation.

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.

Effects of human leukocyte antigen (HLA)-DR engagement on melanoma cells.

Melanoma cells often display constitutive expression of the major histocompatibility complex (MHC) class II molecules which is associated with a higher metastatic dissemination. The MHC class II molecules during T cell/ professional antigen-presenting cell (APC) interactions are localized, as signalling receptors, into membrane lipid rafts which are thought to be sites of signalling complex assembly. Therefore, with the aim of understanding the molecular mechanisms used by melanoma cells to frustrate an effective anti-tumour response we stimulated a MHC class II constitutive expressing melanoma cell line with a specific antibody that mimics the interaction of T-cell receptor (TCR) with class II molecules. In stimulated melanoma cells we showed through Western blotting and immunofluorescence experiments the recruitment of HLA-DR molecules in lipid raft compartments as well as the c-Jun NH2-terminal kinase activations as a consequence of the class II engagement. Furthermore, we showed that SDS-stable HLA-DR-peptide complexes are recruited in lipid rafts of stimulated melanoma cells. Therefore, in light of the results reported, our hypothesis is that the redistribution of class II molecules into lipid raft microdomains of stimulated melanoma cells as well as the associated activation of signalling pathways, could be useful for melanoma cells to frustrate an effective anti-tumour response.

Apoptosis and cell growth arrest in A375 human melanoma cells by diorganotin(IV) and triorganotin(IV) complexes of [meso-Tetra(4-sulfonatophenyl)porphine] manganese(III)chloride.

In previous studies we have demonstrated that two derivatives of meso-Tetra(4-sulfonatophenyl)porphine (TPPS), (Bu2Sn)2TPPS and (Bu3Sn)4TPPS, cause apoptotic death of A375 melanoma cells and, at lower concentrations, arrest of cell proliferation. In the present study, we examined if the manganese metal inside the porphyrin cavity could improve the efficacy of this class of compounds. Thus, [meso-Tetra(4-sulfonatophenyl)porphine]Mn(III)Cl (=MnTPPS) derivatives, namely (Me2Sn)2MnTPPS, (Bu2Sn)2MnTPPS, (Me3Sn)4MnTPPS and (Bu3Sn)4MnTPPS, were tested on the A375 human melanoma cell line. A cytotoxicity assay showed that (Bu2Sn)2MnTPPS and (Bu3Sn)4MnTPPS were highly cytotoxic by inducing apoptosis in melanoma cells, as shown by DNA fragmentation analysis and by apoptotic nuclei fluorescence, and when used at lower concentrations, they affected only cellular proliferation. An arrest of cell proliferation was also observed with (Me3Sn)4MnTPPS, but at the highest concentrations used. Moreover, the lower concentration of (Bu3Sn)4MnTPPS induced a change in cell morphology, from a polygonal to an elongated and spindle-shaped phenotype, likewise to its cognate (Bu3Sn)4TPPS, previously tested. Western blotting analysis showed indeed that both tributyltin compounds, i.e. (Bu3Sn)4MnTPPS and (Bu3Sn)4TPPS, lowered levels of the major proteins involved in tumorigenesis: ß-catenin, c-myc and snail. We also demonstrated that all compounds entered the cells and localized in the nuclei. In conclusion, our results show that, in spite of the Mn(III) metal introduction, the butyl derivatives always have a higher efficacy than methyl derivatives, and the tributyltin compounds in particular have an interesting effect in vitro on A375 cell proliferation.

Effects of two organotin(IV)(sulfonatophenyl)porphinates on MAPKs and on the growth of A375 human melanoma cells.

Previously we showed apoptotic induction in A375 human melanoma cells using two complexes of the meso-tetra(4-sulfonatophenyl)porphinate (TPPS), (Bu2Sn)2TPPS and (Bu3Sn)4TPPS. To understand how these compounds activate apoptosis in melanoma cells we studied MAPKs and the (Bu2Sn)2TPPS and (Bu3Sn)4TPPS cellular uptake. Western blotting experiments showed activated protein kinases ERK 1/2, JNK and p38 in 10 microM (Bu2Sn)2TPPS- and 1 microM (Bu3Sn)4TPPS-treated melanoma cells, which suggests that the three MAP kinases are involved in the apoptotic death of A375-treated cells. By taking advantage of the porphyrin fluorescence, we found a fast concentration of (Bu2Sn)2TPPS and (Bu3Sn)4TPPS in the nucleus and in the nucleoli compared to TPPS. A significantly reduced growth of A375 human melanoma cells was also observed after only 48 h treatment by using 500 nM of (Bu2Sn)2TPPS or 80 nM of (Bu3Sn)4TPPS. A strong slowdown of cell growth and loss of cell-cell interactions were visible by in vitro wound repair assay.

Diorganotin(IV) and triorganotin(IV) complexes of meso-tetra(4-sulfonatophenyl)porphine induce apoptosis in A375 human melanoma cells.

The cytotoxic effect of several diorganotin(IV) and triorganotin(IV)-meso-tetra(4-sulfonatophenyl)porphine derivatives was tested and only the (Bu(2)Sn)(2)TPPS and the (Bu(3)Sn)(4)TPPS showed cytotoxicity on A375 human melanoma cells. To examine the pathway of (Bu(2)Sn)(2)TPPS or (Bu(3)Sn)(4)TPPS induced A375 cell death, DNA fragmentation analysis, Annexin V binding and PI uptake as well as caspases activation analysis by Western blot were carried out. A375 cells treated exhibited several typical characteristics of apoptosis. Both the (Bu(2)Sn)(2)TPPS and the (Bu(3)Sn)(4)TPPS compounds activate caspase-8 and caspase-9 leading to caspase-3 activation. Thus, we propose that these two porphirin derivatives lead to the apoptosis of human melanoma cells via both death receptor-mediated and mitochondrial apoptotic pathways.