Prognostic implications of markers of the metabolic phenotype in human cutaneous melanoma.

BACKGROUND: Reprogramming of energy metabolism to enhanced aerobic glycolysis has been defined as a hallmark of cancer. OBJECTIVES: To investigate the role of the mitochondrial proteins, ß-subunit of the H+ -ATP synthase (ß-F1-ATPase), and heat-shock protein 60 (HSP60), and the glycolytic markers, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and pyruvate kinase M2 (PKM2), as well as the bioenergetic cellular (BEC) index, in melanoma progression. MATERIALS AND METHODS: The expression of energy metabolism proteins was assessed on a set of different melanoma cells representing the natural biological history of the disease: primary cultures of melanocytes, radial (WM35) and vertical (WM278) growth phases, and poorly (C81-61-PA) and highly (C8161-HA) aggressive melanoma cells. Cohorts of 63 melanocytic naevi, 55 primary melanomas and 35 metastases were used; and 113 primary melanoma and 33 metastases were used for validation. RESULTS: The BEC index was significantly reduced in melanoma cells and correlated with their aggressive characteristics. Overexpression of HSP60, GAPDH and PKM2 was detected in melanoma human samples compared with naevi, showing a gradient of increased expression from radial growth phase to metastatic melanoma. The BEC index was also significantly reduced in melanoma samples and correlated with worse overall and disease-free survival; the multivariate Cox analysis showed that the BEC index (hazard ratio 0·64; 95% confidence interval 0·4-1·2) is an independent predictor for overall survival. CONCLUSIONS: A profound alteration in the mitochondrial and glycolytic proteins and in the BEC index occurs in the progression of melanoma, which correlates with worse outcome, supporting that the alteration of the metabolic phenotype is crucial in melanoma transformation.

Neuropeptide Y expression in primary cutaneous melanoma.

BACKGROUND: Neuropeptide Y (NPY) is involved in the carcinogenesis of different tumours, especially neural crest-derived tumours. OBJECTIVE: The aim of our study is to investigate the expression of NPY on melanoma and its relation with prognostic histological parameters and survival. METHODS: This is a retrospective observational study of two independent series, with a total of 79 primary melanomas, diagnosed in two independent University Hospitals in Spain, from January 2000 to December 2004. RESULTS: We found a significant higher expression of NPY on superficial spreading melanoma and lentigo maligna (40%) (P = 0.030). Thinner tumours were associated with higher NPY expression (Clark level, P = 0.003; Breslow level, P = 0.012). Melanomas with low NPY expression were associated with intense cell proliferation (Ki-67, P = 0.034), high density of peritumoral mast cell infiltrates (P = 0.033) and low E-cadherin expression (P = 0.031). Melanomas with high NPY expression exhibited significant differences in terms of relapse time (median: 114 vs. 68 months, P = 0.008) and overall survival (114 vs. 74 months, P = 0.004). CONCLUSION: High expression of NPY was associated with better prognostic histological parameters, low peritumoral mast cells density, presence of adhesion proteins and better outcome.

Resistance of Nonmelanoma Skin Cancer to Nonsurgical Treatments. Part II: Photodynamic Therapy, Vismodegib, Cetuximab, Intralesional Methotrexate, and Radiotherapy. / Resistencias al tratamiento no quirúrgico en cáncer cutáneo no melanoma. Parte II: terapia fotodinámica, vismodegib, cetuximab, metotrexato intralesional y radioterapia.

A wide range of treatments is now available for nonmelanoma skin cancer, including 5-fluorouracil, ingenol mebutate, imiquimod, diclofenac, photodynamic therapy, methotrexate, cetuximab, vismodegib, and radiotherapy. All are associated with high clinical and histologic response rates. However, some tumors do not respond due to resistance, which may be primary or acquired. Study of the resistance processes is a broad area of research that aims to increase our understanding of the nature of each tumor and the biologic features that make it resistant, as well as to facilitate the design of new therapies directed against these tumors. In this second article, having covered the topical treatments of nonmelanoma skin cancer, we review resistance to other nonsurgical treatments, such as monoclonal antibodies against basal and squamous cell carcinomas, intralesional chemotherapy, photodynamic therapy, and radiotherapy.

Cryptomphalus aspersa mollusc eggs extract promotes migration and prevents cutaneous ageing in keratinocytes and dermal fibroblasts in vitro.

BACKGROUND: The search of substances that minimize cutaneous ageing has increased in the last few years. Previous studies have described the regenerative properties of the secretion of the mollusc Cryptomphalus aspersa (C. aspersa) when applied topically. OBJECTIVE: We evaluate the in vitro effects of a new product derived from the eggs of C. aspersa, IFC-CAF, on cell proliferation, migration, distribution of cytoskeletal proteins, production of extracellular components as well as its ability to prevent cutaneous ageing because of intrinsic or extrinsic factors (exposure to UVB) by determination of ageing markers. METHODS: We have used the human keratinocyte cell line (HaCaT cells), primary dermal fibroblasts (HDF) and senescent dermal fibroblasts (SHDF). The effects of the compound on cell proliferation and on the cell cycle were determined by the MTT colorimetric assay, estimation of total protein and/or trypan blue test and by flow cytometry, respectively. We also studied cell migration using the wound-healing migration assay, whereas ELISA assays, Western Blot and immunofluorescence microscopy were carried out to test the expression of proteins related to cytoskeleton, extracellular matrix and with ageing. RESULTS: We have found that IFC-CAF does not promote proliferation but induces migration of HaCaT, HDF and SHDF in a time- and dose-dependent manner; a better organization of cytoskeletal proteins (F-actin and vimentin) and promotes the production of extracellular components (fibronectin, collagen 1 and MMPs) and the adhesion to cell-substrate vinculin protein. IFC-CAF also prevents cutaneous ageing. The treatment decreases the expression of the ageing-related markers b-Gal, p53 and p16INK4 in SDDF cells, and improves cell survival after UVB irradiation and nuclear repair in HaCaT cells. CONCLUSION: IFC-CAF has regenerative properties and protects against ageing factors being, therefore, a potential therapeutic agent for treating or preventing skin ageing.

New experimental models of skin homeostasis and diseases.

Homeostasis, whose regulation at the molecular level is still poorly understood, is intimately related to the functions of epidermal stem cells. Five research groups have been brought together to work on new in vitro and in vivo skin models through the SkinModel-CM program, under the auspices of the Spanish Autonomous Community of Madrid. This project aims to analyze the functions of DNA methyltransferase 1, endoglin, and podoplanin in epidermal stem cell activity, homeostasis, and skin cancer. These new models include 3-dimensional organotypic cultures, immunodeficient skin-humanized mice, and genetically modified mice. Another aim of the program is to use skin-humanized mice to model dermatoses such as Gorlin syndrome and xeroderma pigmentosum in order to optimize new protocols for photodynamic therapy.

Comparative response to PDT with methyl-aminolevulinate and temoporfin in cutaneous and oral squamous cell carcinoma cells.

Cutaneous and Head and Neck squamous cell carcinoma (CSCC, HNSCC) are among the most prevalent cancers. Both types of cancer can be treated with photodynamic therapy (PDT) by using the photosensitizer Temoporfin in HNSCC and the prodrug methyl-aminolevulinate (MAL) in CSCC. However, PDT is not always effective. Therefore, it is mandatory to correctly approach the therapy according to the characteristics of the tumour cells. For this reason, we have used cell lines of CSCC (A431 and SCC13) and HNSCC (HN5 and SCC9). The results obtained indicated that the better response to MAL-PDT was related to its localization in the plasma membrane (A431 and HN5 cells). However, with Temoporfin all cell lines showed lysosome localization, even the most sensitive ones (HN5). The expression of mesenchymal markers and migratory capacity was greater in HNSCC lines compared to CSCC, but no correlation with PDT response was observed. The translocation to the nucleus of ß-catenin and GSK3ß and the activation of NF-κß is related to the poor response to PDT in the HNSCC lines. Therefore, we propose that intracellular localization of GSK3ß could be a good marker of response to PDT in HNSCC. Although the molecular mechanism of response to PDT needs further elucidation, this work shows that the most MAL-resistant line of CSCC is more sensitive to Temoporfin.

A secretion of the mollusc Cryptomphalus aspersa promotes proliferation, migration and survival of keratinocytes and dermal fibroblasts in vitro.

Regenerative properties of skin decrease with age, and thus, the search for substances that minimize cutaneous ageing has increased in the last few years. The secretion of the mollusc Cryptomphalus Aspersa (SCA) is a natural product that bears regenerative properties when applied topically. The purpose of this work is to study the in vitro effects of SCA on cell proliferation and migration, as well as on cell-cell (E-cadherin and ß-catenin) and cell-substrate (vinculin and ß1-integrin) adhesion proteins expression, using a human keratinocyte cell line (HaCaT cells) and primary dermal fibroblasts (HF). We tested the effects of SCA on cell proliferation using a colorimetric assay. In addition, SCA-induced changes on cell migration were studied by wound-healing assays. Besides, Western blot and immunofluorescence microscopy were carried out to test the expression of different cell adhesion proteins. We found that SCA promotes proliferation and migration of HaCaT cells in a time- and dose-dependent manner. Moreover, treatment with SCA increases the migratory behaviour and the expression of adhesion molecules in both HaCaT and HF. Finally, SCA also improves cell survival and promotes phosphorylation of FAK and nuclear localization of ß-catenin. These results shed light on the molecular mechanisms underlying the regenerative properties of SCA, based on its promoting effect on skin cell migration, proliferation and survival. Moreover, these results support future clinical uses of SCA in the regeneration of wounded tissues.

Photodynamic therapy reduces the histological features of actinic damage and the expression of early oncogenic markers.

BACKGROUND: Photodynamic therapy (PDT) has been shown to be effective in treating nonmelanoma skin cancer (NMSC), especially actinic keratosis (AK). Moreover, there is sufficient evidence of its effectiveness in preventing the appearance of premalignant and malignant lesions in organ transplant recipients. OBJECTIVES: To describe the molecular and genetic changes underlying this preventive effect. METHODS: Twenty-two patients with AK were treated with methyl aminolaevulinate and red light. Biopsies were performed before and 6 weeks after the treatment. Conventional histopathology and immunohistochemistry were carried out. RESULTS: Not only was a reduction in the dysplasia and elastosis observed, but also a decreased expression of Ki-67 and p53. The abnormal findings did not disappear completely in all cases. The expression of cyclin D1 remained stable. CONCLUSIONS: These findings show that PDT has the potential to reduce the histological signs of photoageing. Moreover, the reduction of Ki-67, a marker of proliferation and of p53, a marker of early skin carcinogenesis, indicates a reversal of the carcinogenic process. On the other hand, the fact that one treatment does not clear dysplasia and expression of p53 completely, and the persistence of cyclin D1, indicate that one single treatment, despite showing good clinical results, is not sufficient to clear completely the signs of chronic actinic damage, and thus the risk of NMSC.

Cytotoxic effects of Argentinean plant extracts on tumour and normal cell lines.

In the search for possible new anti-cancer agents, we investigated the effects of 75 aqueous and methanol extracts from 41 Argentinean plant species. The effect in cell growth was evaluated in the LM2 mammary adenocarcinoma cells. In a second stage, the highly active selected extracts were assayed in 3 other tumour cell lines: melanoma B16, bladder MB49 and lung A549; and 3 normal cell lines: mammary Hb4a and keratinocytes PAM212 and HaCat. Eight methanol extracts were found to be highly cytotoxic: Collaea argentina leaf, Iochroma australe leaf, Ipomoea bonariensis flower, Jacaranda mimosifolia flower, Solanum amygdalifolium flower, Solanum chacoense leaf, Solanum sisymbriifolium flower and Solanum verbascifolium flower. However, extract inhibition on cell growth was highly dependent on cell type. In general, except for the highly resistant cell lines, the inhibitory concentrations 50% were in the range of 10-150 µg/ml The eight extracts highly inhibited cell growth in a concentration-dependent manner, and in general the methanol extracts were always more active than the aqueous. Murine cells appear to be more sensitive than human cells to the cytotoxic action of the plant extracts. The human melanoma B16 line was the most resistant to four of the extracts. In terms of selectivity, S. verbascifolium was the species which showed most selectivity for tumour cells. Overall, this is one of the first studies focusing on southern South American native plants and their biological effects. Since some species of 5 genera analyzed have been reported to possess different degrees of alkaloid content, we examined microtubule structures after extract treatments. The eight extracts induced destabilization, condensation and aggregation of microtubules in LM2 cells, although no depolarization, typical of Vinca alkaloids damage was observed. In a near future, antitumour activity of purified fractions of the extracts administered at non-toxic doses will be assayed in transplantable murine tumour models.

New porphyrin amino acid conjugates: synthesis and photodynamic effect in human epithelial cells.

The efficacy of new porphyrin amino acid conjugates as photosensitizers for photodynamic therapy (PDT) were assayed in vitro on tumoral (HeLa) and on non tumoral (HaCaT) human cell lines. The conjugates stable in liposomes are able to penetrate efficiently in the cytoplasm of cultured cancer and normal cells. No dark cytotoxicity is observed at the same concentration used for PDT cell treatment and during long incubation time (24h). The cell survival after the PDT treatment with visible light is dependent upon light exposure level and compound concentration. The tested compounds show higher photocytotoxicity in tumoral HeLa cells than in no tumoral HaCaT cells. The results suggest that these amino acid porphyrin conjugates are potential photosensitizers for PDT.

Expression of p53 and p16 in actinic keratosis, bowenoid actinic keratosis and Bowen's disease.

INTRODUCTION: Bowen's disease (BD) and bowenoid actinic keratosis (bAK) have traditionally been differentiated according to the presence or absence of dysplasia in the follicular epithelium. p16 has been suggested to be a useful tool to make the differential diagnosis between BD and AK and as a marker of bad prognosis. MATERIALS: Five biopsies of BD, five of AK and five of bAK where stained for p53 and p16. RESULTS: All lesions showed positive immunostaining of p53, affecting to the lower two thirds of the epidermis in BD and bAK, and only the basal layer in non-bAK. All the BD and bAK cases were positive for p16, showing a similar immunostaining pattern, whereas no staining was observed in non-bAK. DISCUSSION AND CONCLUSION: These findings suggest a common pathogenic mechanism for BD and bAK. bAK might have worse prognosis than AK. p16 might not be useful as a tool for differential diagnosis between AK and BD because bAK and BD show an extremely similar immunohistochemical pattern.

Influence of the Exposome on Skin Cancer. / La influencia del exposoma en el cáncer de piel.

Skin cancer is the most frequent type of cancer in humans. While exposure to solar radiation is the most widely known and relevant causal factor, the different degrees of individual risk have not been fully elucidated. Epidemiological studies show how the risk of skin cancer is affected by other types of radiation (eg, ionizing radiation), pesticides, particulate matter in air pollution, toxins (eg, arsenic) in water and some foods. Some living entities, such as polyomavirus and human papillomavirus, can also cause specific types of cancer. Lastly, lifestyle factors such as stress, sleep, and exercise may play a role, although only a few studies shed light on these factors. The abovementioned factors make up the exposome of skin cancer, that is, the set of environmental exposures that, together with the genome and microbiome, determine the onset of disease.

Porphycenes: facts and prospects in photodynamic therapy of cancer.

The photodynamic process induces cell damage and death by the combined effect of a photosensitizer (PS), visible light, and molecular oxygen, which generate singlet oxygen ((1)O(2)) and other reactive oxygen species that are responsible for cytotoxicity. The most important application of this process with increasing biomedical interest is the photodynamic therapy (PDT) of cancer. In addition to hematoporphyrin-based drugs, 2nd generation PSs with better photochemical properties are now studied using cell cultures, experimental tumors and clinical trials. Porphycene is a structural isomer of porphyrin and constitutes an interesting new class of PS. Porphycene derivatives show higher absorption than porphyrins in the red spectral region (lambda > 600 nm, epsilon > 50000 M-(1)cm(-1)) owing to the lower molecular symmetry. Photophysical and photobiological properties of porphycenes make them excellent candidates as PSs, showing fast uptake and diverse subcellular localizations (mainly membranous organelles). Several tetraalkylporphycenes and the tetraphenyl derivative (TPPo) induce photodamage and cell death in vitro. Photodynamic treatments of cultured tumor cells with TPPo and its palladium(II) complex induce cytoskeletal changes, mitotic blockage, and dose-dependent apoptotic or necrotic cell death. Some pharmacokinetic and phototherapeutic studies on experimental tumors after intravenous or topical application of lipophilic alkyl-substituted porphycene derivatives are known. Taking into account all these features, porphycene PSs should be very useful for PDT of cancer and other biomedical applications.

Meso-tetraphenylporphyrin: photosensitizing properties and cytotoxic effects on cultured tumor cells.

In this study we analyzed photosensitizing and photodamaging properties of the hydrophobic meso-tetraphenylporphyrin (TPP, incorporated into liposomes) on HeLa cells. Under the fluorescence microscope, red fluorescence by TPP was detected on the cell surface. TPP followed by violet-blue or red irradiation led to cell death, blebs and plasma membrane deformations appearing immediately after photodynamic treatment. Production of singlet oxygen by TPP was studied by analyzing tryptophan photodegradation, which increased in the presence of D2O and was abolished by NaN3. Present results suggest that the plasma membrane is the main cellular target for TPP, which could be a valuable photosensitizing drug in studies on photodynamic therapy of cancer.

Necrotic cell death induced by photodynamic treatment of human lung adenocarcinoma A-549 cells with palladium(II)-tetraphenylporphycene.

In this study we describe photodamaging and photokilling effects of palladium(II)-tetraphenylporphycene (PdTPPo) (previously incorporated into dipalmitoylphosphatidylcholine liposomes) on the human lung adenocarcinoma A-549 cell line. No dark cytotoxicity was found when the drug was applied at 10(-6) M or 5 x 10(-7) M for 1 or 18 h, respectively. After 1-h treatment with 10(-7) M or 5 x 10(-7) M PdTPPo followed by red light irradiation for variable times, dose-dependent lethal effects were observed in A-549 cells. Apoptosis was not found after the above photodynamic treatments or under even milder sublethal conditions. In contrast to HeLa cells subjected to PdTPPo photosensitization where either apoptosis or necrosis were induced, morphological analysis and electrophoretical DNA pattern of A-549 cells always revealed a clearly necrotic death mechanism. However, A-549 cells died by apoptosis after serum and L-glutamine deprivation, indicating that only the photodynamically induced apoptosis was inhibited. Immunofluorescent labeling revealed that microtubules and actin microfilaments were immediately and strongly damaged by photodynamic treatments with PdTPPo. No metaphase arrest and/or mitotic alterations were observed after phototreatments. Present results show that the cell type plays a fundamental role in relation to the apoptotic or necrotic response to photosensitization, and that cytoskeletal components are important targets implicated in cell death processes.

The pesticide malathion induces alterations in actin cytoskeleton and in cell adhesion of cultured breast carcinoma cells.

We have studied the effects of the organophosphorous pesticide malathion on cell viability, actin cytoskeleton, cell adhesion complex E-cadherin/beta-catenin, and Rho and Rac1 GTPases from the human mammary carcinoma cell line MCF-7. Malathion induced cell lethality, determined by the MTT assay, depending on the treatment conditions. Cells incubated with low concentrations of malathion, 16-32 microg/ml, showed high survival rates (>95%) at any evaluated time (1-5 days), whereas complete cell lethality was found using 512 microg/ml and 5 days of treatment. Deep morphological changes were induced with high doses of 64 and 128 microg/ml, and long incubation time (5 days); cells showed perinuclear vacuoles, rounding, shrinkage, and a gradual loss of adhesion. These changes were related to a decrease in the expression of the adhesion molecules, E-cadherin and beta-catenin, and to the distribution and reactivity of actin microfilaments to TRITC-phalloidin. Disruption of microfilaments, accompanied by the collapse of actin to perinuclear region, were characteristic of cells with loss of adhesion. At lower concentrations, some cells presented deformations on the plasma membrane as lamellipodia-like structures, which were particularly evident from 32 to 128 microg/ml. Conversely, we observed an increase in the expression of Rho and Rac1 GTPases, modulators of actin cytoskeleton and cell adhesion.

Photodynamic damage to HeLa cell microtubules induced by thiazine dyes.

The aim of this study was to analyze possible alterations of the microtubule cytoskeleton of cultured cells subjected to photodynamic treatments with the thiazine dyes methylene blue or toluidine blue. Indirect immunofluorescence labeling of alpha-tubulin was performed in HeLa cells after 1 or 18 h of incubation with thiazines followed by red-light irradiation for 15 min [leading to surviving fractions (SF) of about 65% (SF65) or 1% (SF1), respectively]. Untreated control cells showed the normal distribution of interphase microtubules, whereas considerable or severe disorganization of the microtubule network was observed after SF65 or SF1 photodynamic treatments, respectively. A great amount of blebs showing homogeneous fluorescence was also found on the cell surface after SF1 treatments. Possible mechanisms responsible for the photodamage to microtubules induced by thiazine dyes are briefly discussed.

Photokilling mechanisms induced by zinc(II)-phthalocyanine on cultured tumor cells.

The photosensitizing effects of liposomal zinc(II)-phthalocyanine (ZnPc) on HeLa cells, with emphasis on morphological changes and mechanisms for cell death, have been studied. No dark toxicity for ZnPc alone was found. Incubation for 1 h with ZnPc followed by red light irradiation induced a variable decrease in the surviving of cells, which was related to both drug concentration and irradiation time. A lethal photodynamic effect (100% of the cells are killed: LD100) was induced by 5 x 10-6 M ZnPc and 5-min irradiation, whereas a sublethal effect (60% of the cells are killed: LD60) was detected with 10 7 M ZnPc and 3 min of red light. Toluidine blue and Hoechst 33258 staining showed characteristic alterations of cell morphology. Numerous bubbles on the plasma membrane were found immediately after an LD100 treatment, and a necrotic morphology appeared 24 h later. On the contrary, severe cell shrinkage with nuclear fragmentation. characteristic of apoptosis. was observed 8 and 24 h after LD60 treatments. In this case, propidium iodide-acridine orange labeling and the TUNEL assay confirmed the occurrence of apoptosis. The highest amount of apoptotic cells appeared 24 h after LD60 treatments, particularly in detached cells, as revealed by cell counting and DNA electrophoresis. Both apoptotic and necrotic mechanisms for cell death occur in HeLa cells in dependence on the experimental protocol of ZnPc photodynamic treatments.

Photodamage induced by Zinc(II)-phthalocyanine to microtubules, actin, alpha-actinin and keratin of HeLa cells.

We have studied the photosensitizing effects of zinc(II)-phthalocyanine (ZnPc) on the cytoskeleton of HeLa cells using sublethal (10(-7) M, followed by 1 or 3 min of red light to induce 20%, LD20, or 60%, LD60, cell death, respectively) or lethal (5 x 10(-6) M and 15 min of irradiation, LD100) experimental conditions. The immunofluorescent analysis of the cytoskeleton showed a variable photodamage to microtubules (MT), actin microfilaments (AF) and intermediate filaments of keratin (KF), as well as on alpha-actinin, which was dependent on treatment conditions. Both sublethal treatments induced deep alterations on interphase and mitotic MT. The mitotic index increased with time with the maximum at 18 h (12%) or 24 h (14%) after LD20 or LD60, respectively. The alterations on AF and alpha-actinin were much more severe than those observed on KF at any evaluated time. With the exception of the KF, which remained partially organized, the MT and AF network was severely damaged by the lethal treatment. Western blot analysis for alpha-tubulin, G-actin and alpha-actinin from soluble and insoluble fractions confirmed the results observed by immunofluorescence, thus indicating that these cytoskeletal components are involved in cell damage and death by ZnPc photosensitization.