3-nitrotyrosine and Skin-Neoplasms

Topics: Aldehydes; Animals; Carcinogenesis; DNA Damage; Female; Humans; Melanoma; Metformin; Mice; Oxidative Stress; Radiation Injuries, Experimental; Radiodermatitis; Skin; Skin Neoplasms; Tumor Suppressor Protein p53; Tyrosine; Ultraviolet Rays

Increased expression and prognostic significance of major redox regulator nuclear factor erythroid-2-related factor (Nrf2) is recognized in many cancers. Our aim was to investigate the role of oxidative stress markers in melanoma.. We characterized the immunohistochemical expression of Nrf2, kelch-like ECH-associated protein 1 (Keap1), BRAF(V600E), 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nitrotyrosine in 36 nevi, 14 lentigo maligna and 71 malignant melanomas. We measured Nrf2 expression in melanoma cell lines and conducted cytotoxicity assays combining BRAF/NRAS ablation and H2O2treatment.. Nuclear Nrf2 expression in melanoma correlated with deeper Breslow (p<0.0005), invasive phenotype (Clark III-V) (p=0.011), nodular growth (p=0.001) and worse melanoma-specific survival (p=0.008). Absence of 8-OHdG in the endothelium was a greater significant predictor of poor prognosis (p=0.024) than ulceration (p=0.17) and had a similar impact on prognosis as Breslow (p=0.024). A decrease of Nrf2 followed the BRAF/NRAS inhibition, but combination of inhibitor with H2O2did not increase cytotoxicity.. Nrf2 and 8-OHdG influence prognosis in melanoma.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aged; Cell Line, Tumor; Deoxyguanosine; Female; Humans; Intracellular Signaling Peptides and Proteins; Kaplan-Meier Estimate; Kelch-Like ECH-Associated Protein 1; Male; Melanoma; NF-E2-Related Factor 2; Oxidative Stress; Proto-Oncogene Proteins B-raf; Signal Transduction; Skin Neoplasms; Tyrosine

This study highlights the systemic oxidative changes in patients submitted to primary cutaneous melanoma removal. Cutaneous melanoma is highly aggressive and its incidence is increasing worldwide. We evaluated systemic oxidative stress (OS) and 3-nitrotyrosine (3-NT) expression in melanoma tissue in relation to the Breslow thickness in patients under surveillance. Forty-three patients with cutaneous melanoma and 50 healthy volunteers were recruited. Patients were divided into two groups according to the tumor's Breslow thickness: T1/T2 (<2 mm) and T3/T4 (≥2 mm). Systemic OS and inflammatory mediators were evaluated in plasma, and the 3-NT expression was analyzed via immunohistochemistry. Compared with the controls, the patients had lower blood levels of reduced glutathione, higher malondialdehyde and thiol levels, and a higher total radical-trapping antioxidant parameter to uric acid ratio. The C-reactive protein and γ-glutamyl transpeptidase were increased only in the T3/T4 group. High levels of 3-NT were present only in T3/T4 patients. Our data suggested that a correlation exists between the Breslow thickness and a systemic pro-oxidant status, and that oxidative changes induced by the melanoma remain in the microenvironment post-surgery, demonstrating a role for oxygen species in melanoma.

Topics: Adult; Aged; Female; Humans; Male; Melanoma; Melanoma, Cutaneous Malignant; Middle Aged; Oxidative Stress; Skin Neoplasms; Tumor Microenvironment; Tyrosine; Young Adult

Merkel cell carcinoma (MCC) is an aggressive, polyomavirus-linked skin cancer. Although CD8 lymphocyte infiltration into the tumor is strongly correlated with improved survival, these cells are absent or sparse in most MCCs. We investigated whether specific mechanisms of T-cell migration may be commonly disrupted in MCC tumors with poor CD8 lymphocyte infiltration. Intratumoral vascular E-selectin, critical for T-cell entry into skin, was downregulated in the majority (52%) of MCCs (n=56), and its loss was associated with poor intratumoral CD8 lymphocyte infiltration (P<0.05; n=45). Importantly, survival was improved in MCC patients whose tumors had higher vascular E-selectin expression (P<0.05). Local nitric oxide (NO) production is one mechanism of E-selectin downregulation and it can be tracked by quantifying nitrotyrosine, a stable biomarker of NO-induced reactive nitrogen species (RNS). Indeed, increasing levels of nitrotyrosine within MCC tumors were associated with low E-selectin expression (P<0.05; n=45) and decreased CD8 lymphocyte infiltration (P<0.05, n=45). These data suggest that one mechanism of immune evasion in MCC may be restriction of T-cell entry into the tumor. Existing therapeutic agents that modulate E-selectin expression and/or RNS generation may restore T-cell entry and could potentially synergize with other immune-stimulating therapies.

Topics: Aged; Antigens, Differentiation, T-Lymphocyte; Blood Vessels; Carcinoma, Merkel Cell; CD8-Positive T-Lymphocytes; E-Selectin; Female; Humans; Immunotherapy, Adoptive; Male; Membrane Glycoproteins; Middle Aged; Skin; Skin Neoplasms; Tyrosine

Exposure to ultraviolet radiation (UVR) can lead to a range of deleterious responses in the skin. An important form of damage is the DNA photolesion cyclobutane pyrimidine dimer (CPD). CPDs can be highly mutagenic if not repaired prior to cell division and can lead to UV-induced immunosuppression, making them potentially carcinogenic. UVR exposure also produces vitamin D, a prehormone. Different shapes of the steroid hormone 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] can produce biological responses through binding either to its cognate nuclear receptor (VDR) to regulate gene transcription or to the VDR associated with plasma membrane caveolae to produce, via signal transduction, nongenomic physiologic responses. Here, we show that both 1,25(OH)₂D₃ and 1α,25(OH)₂-lumisterol (JN), a conformationally restricted analogue that can generate only nongenomic responses, are effective inhibitors of UV damage in an immunocompetent mouse (Skh:hr1) model susceptible to UV-induced tumors. Both 1,25(OH)₂D₃ and JN significantly reduced UVR-induced CPD, apoptotic sunburn cells, and immunosuppression. Furthermore, these compounds inhibited skin tumor development, both papillomas and squamous cell carcinomas, in these mice. The observed reduction of these UV-induced effects by 1,25(OH)₂D₃ and JN suggests a role for these compounds in prevention against skin carcinogenesis. To the best of our knowledge, this is the first comprehensive report of an in vivo long-term biological response generated by chronic dosing with a nongenomic-selective vitamin D steroid.

Topics: Animals; Anticarcinogenic Agents; Apoptosis; Calcitriol; Carcinoma, Squamous Cell; Cell Line; Humans; Immunosuppressive Agents; Mice; Receptors, Calcitriol; Signal Transduction; Skin Neoplasms; Tumor Suppressor Protein p53; Tyrosine; Ultraviolet Rays

The in vitro reactivity of capsanthin (1) and fucoxanthin (2) with peroxynitrite was investigated, and the reaction products produced by scavenging with peroxynitrite were analyzed. (14'Z)-Nitrocapsanthin (3) and 12-nitrocapsanthin (4) were isolated from the products of the reaction of capsanthin with peroxynitrite. Similarly, (14Z)-15-nitrofucoxanthin (5), (11Z)-11-nitrofucoxanthin (6), and (14Z,9'Z)-15-nitrofucoxanthin (7) were obtained from the reaction of peroxynitrite reaction with fucoxanthin. Capsanthin and fucoxanthin inhibited the nitration of tyrosine by peroxynitrite. Furthermore, nitrocapsanthins (3 and 4) and nitrofucoxanthins (5 and 6) exhibited an inhibitory effect on Epstein-Barr virus early antigen activation in Raji cells and an antiproliferative effect on human pancreatic carcinoma. Moreover, nitrocapsanthins (3 and 4) inhibited carcinogensis of mouse skin tumors initiated by 7,12-dimethylbenz[a]anthracene (DMBN).

Topics: Animals; Anticarcinogenic Agents; Burkitt Lymphoma; Cell Line, Tumor; Female; Free Radical Scavengers; Humans; Mice; Mice, Inbred ICR; Pancreatic Neoplasms; Papilloma; Peroxynitrous Acid; Skin Neoplasms; Tyrosine; Xanthophylls

UV light-based damage to skin cells can cause photoaging and skin cancer. A major cause of UV light-induced damage to skin is increased free radicals, such as superoxides. Increased superoxides can cause oxidative and nitrative damage to cell components. Thus, agents that counteract these damages may have therapeutic value. Herein, we show that angiopoietin-1 (ang1) prevented and blocked H(2)O(2)-induced increases in superoxides in human spontaneously immortalized keratinocyte line, HaCaT, and primary melanocytes (HeMn). Ang1 prevented H(2)O(2)-induced increases in damage to DNA (8-hydroxy-2'-deoxyguanosine) and proteins (nitrotyrosinylation). Ang1 promoted skin cell metabolism/viability, adhesion, and akt and MAPK(p42/44) activations. Using multi-gene transcriptional profiling, we found that skin cells express integrin subunits {(beta(1), beta(4-6), beta(8), alpha(v), alpha(2), alpha(3), alpha(6) (HaCaT)), (beta(1), beta(3), beta(5), beta(8), alpha(v), alpha(3) (HeMn))} and lack tie2 receptor mRNA. Integrin antibodies (alpha(v), beta(1)) disrupted skin cell adhesion to ang1 and ang1-induced decreases in superoxides. Our findings show that ang1 blocks free radical damage to skin cells and may be clinically useful to prevent and/or reduce photoaging and skin cancer.

Topics: Angiopoietin-1; Cell Adhesion; Cell Line, Transformed; Cell Survival; DNA Damage; Gene Expression Profiling; Humans; Hydrogen Peroxide; Integrins; Keratinocytes; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Oxidants; Oxidative Stress; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Skin; Skin Aging; Skin Neoplasms; Tyrosine

The ability of macrophages to produce reactive nitrogen species, particularly nitric oxide (NO) is correlated with an enhanced microbicidal or tumoricidal activity during pathogenic or tumoral invasion, respectively. NO reacts in water with oxygen and its reactive intermediates to yield, among others, nitrite and nitrate, which are relatively, stable anions. In this study, we show the varying concentrations of nitrite and nitrate present in different body fluids during AK-5 tumor growth and regression in Wistar rats. We have followed the tumor progression profile and the corresponding levels of nitrite and nitrate present in three major body compartments: the tumor mass; the serum which is the intermediary site; and the peritoneal compartment which is the priming ground for the macrophages. We are thus able to show that the status of the tumor has a direct correlation with macrophage activation and motility to different sites in the body. We also demonstrate after in vitro coculture, that the levels of nitrite and nitrate secreted by the macrophages correlate with their cytocidal capacity.

Topics: Animals; Ascites; Ascitic Fluid; Blotting, Western; Cells, Cultured; Coculture Techniques; Histiocytoma, Benign Fibrous; Macrophage Activation; Macrophages; Neoplasm Transplantation; Neoplasms, Experimental; Nitrates; Nitrites; Proteins; Rats; Rats, Wistar; Skin Neoplasms; Tyrosine

Spontaneous regression of AK-5 tumor in syngeneic hosts reported earlier involves the interplay of Th1-type cytokines and cell-mediated immunity. Upon subcutaneous transplantation of AK-5 cells, there was accumulation of immune cells in the peritoneum, of which macrophages were the predominant type and were found to be in a hyperactive state. They released macrophage-derived tumoricidal mediators like NO, O2(-), and ONOO(-) which exhibited potent cytotoxic activity against AK-5 cells in vitro. Interestingly, there was a dramatic disappearance of these hyperactive cells from the peritoneal cavity which correlated well with the onset of tumor regression at the subcutaneous site. Direct labeling of these cells in the peritoneum by the tracking dye PKH26 showed their migration to the tumor site. Similarly, frozen tumor sections when scanned under confocal microscope clearly exhibited fluorescent macrophages embedded into the tumor. Immunohistochemical sections of these intratumoral macrophages showed nitrotyrosine residues, indicating their contribution in the free-radical-mediated AK-5 cell death, thereby leading to successful tumor remission. These observations suggest a directional migration of the hyperactivated peritoneal population to the tumor site. We have also confirmed the influx of macrophages and other immune cells into the peritoneum after sc transplantation of Meth A tumor cells in Balb/c mice. Our studies suggest a role for the peritoneal compartment in imparting appropriate stimulus to the immune cells prior to their participation in the antitumor immune response. These studies suggest a novel route of macrophage trafficking via the peritoneum.

Topics: Animals; Apoptosis; Ascitic Fluid; Cell Communication; Cell Movement; Cytokines; Cytotoxicity, Immunologic; Free Radicals; Macrophages; Neoplasm Regression, Spontaneous; Neoplasms; Peritoneum; Rats; Rats, Wistar; Skin Neoplasms; Tumor Cells, Cultured; Tyrosine

Nitric oxide (NO) is a small molecule produced during the conversion of L-arginine to L-citrulline by NO synthase (NOS). Several isoforms of NOS exist, of which the Ca2+-independent, inducible NOS (iNOS or NOS2) is most prominently expressed by macrophages. iNOS activity and increased levels of iNOS have been found in various tumours and tumour cell lines but not in normal tissues; however, the precise role of NO in tumour progression has yet to be elucidated. We studied the expression of iNOS in paraffin sections of 41 benign naevi and 52 primary malignant melanomas (MM) of the skin, as well as in 13 metastatic MM. In addition, nitrotyrosine, indicative of NO production and formation of peroxynitrite, was studied in frozen sections of 13 naevi and 30 MM. Virtually all naevi expressed iNOS, but very few expressed nitrotyrosine, indicating either that iNOS in naevi is functionally inactive, or that naevus cells lack reactive oxygen radicals and thus do not form peroxynitrite. Normal melanocytes in adjacent uninvolved skin were unreactive for both markers. In MM, iNOS was most frequently expressed in the 'pure' and 'invasive' radial growth phase (RGP), whereas expression in the vertical growth phase (VGP) and metastatic phase occurred only in 76% of cases; moreover, in these latest phases of tumour progression, iNOS staining was weak and focal. We conclude that iNOS is expressed de novo in most benign pigment cell lesions. In MM (iNOS-generated) NO appears to play an important part in the early steps of invasion (i.e. the 'invasive' RGP), where it may stimulate neo-angiogenesis and may be a prerequisite for further tumour progression; this view is also supported by the finding of iNOS in the associated blood vessels in the papillary dermis. Finally, our data suggest that (iNOS-generated) NO plays a less significant part in the VGP and in metastatic melanoma.

Topics: Biomarkers, Tumor; Disease Progression; Enzyme Induction; Humans; Isoenzymes; Macrophages; Melanoma; Neoplasm Metastasis; Nevus; Nitric Oxide Synthase; Skin Neoplasms; Tyrosine

The present studies examined the temporal sequence of inducible nitric oxide synthase (iNOS) gene expression and the cellular sources of iNOS protein and of 3-nitrotyrosine, as a marker of production of nitric oxide-derived reactive nitrogen intermediates during murine multi-stage carcinogenesis. Levels of iNOS mRNA in dorsal skin isolated from acetone-treated female Sencar mice were 2.5-fold higher than iNOS gene expression detected in cutaneous tissue isolated from Sencar mice at 1, 3, 6, 10, 16 and 22 weeks after exposure to a single topical application of 25 nmol 7,12-dimethylbenz[a]anthracene (DMBA) followed by repetitive applications of 2 microgram 17-O-tetradecanoylphorbol-13-acetate (TPA). Papillomas isolated at 16 and 22 weeks of a tumor promotion protocol also had low levels of iNOS mRNA. The diminished levels of iNOS mRNA inversely correlated with the extent of TPA-induced epidermal hyperplasia. In acetone-treated mouse skin, iNOS immunospecific antibody binding was localized to the stratum corneum and suprabasal keratinocytes. In contrast, iNOS protein was present in lower amounts and was localized to the upper-most suprabasal keratinocytes in cutaneous tissue isolated at 22 weeks following a single exposure to either 25 nmol DMBA or acetone and repetitive applications of 2 microgram TPA. At all time points examined over the 22 week time period of papilloma growth, infiltrating neutrophils within the dermis bound significant levels of anti-iNOS antibodies. The production of iNOS by neutrophils within the dermis correlated with the formation of protein nitrotyrosination within the dermal tissue, as detected by 3-nitrotyrosine-specific antibodies. The present studies indicate that NOS and reactive nitrogen intermediates, including peroxynitrite, are produced specifically by dermal neutrophils during the tumor promotion process at time points that correspond to simultaneous production of reactive oxygen intermediates. Conversely, iNOS is simultaneously down-regulated in hyperplastic epidermis and in papillomas.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Acetone; Animals; Carcinogens; DNA Primers; Enzyme Induction; Female; Keratinocytes; Mice; Mice, Inbred Strains; Models, Biological; Nitric Oxide Synthase; Polymerase Chain Reaction; RNA, Messenger; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tyrosine

8-Hydroxy-2'-deoxyguanosine (8-OHdG) is a mutation-prone (G:C to T:A transversion) DNA base-modified product generated by reactive oxygen species or photodynamic action. G:C to T:A transversions are observed in the p53 and ras genes of UVB-induced skin cancers of mice and in squamous and basal cell carcinomas of human skin exposed to sunlight. In the current study, 8-OHdG formation was evaluated in the epidermis of hairless mice after repeated exposure to UVB, and possible mechanisms involved were studied. Exposure of hairless mice to either 3.4 [2 minimal erythema dose (MED)] or 16.8 (10 MED) kJ/m2 of UVB three times a week for 2 wk induced a 2.5- or 6.1-fold increase, respectively, in the levels of 8-OHdG in DNA, compared to the unexposed controls. An immunohistochemical method using a monoclonal antibody specific for 8-OHdG showed stronger and more extensive staining in the nuclei of UV-irradiated epidermal cells than in those of nonirradiated cells. Western blots probed with antibodies against 4-hydroxy-2-nonenal-modified proteins confirmed the involvement of reactive oxygen species in the epidermal damage induced by chronic UVB exposure. 3-Nitro-L-tyrosine was detected in western blots in a concentration-dependent manner, suggesting that peroxynitrite derived from the reaction of nitric oxide and superoxide, both of which were probably released from inflammatory cells, was involved in modifying the DNA bases. Therefore, the formation of 8-OHdG after UVB exposure appears to be regulated by at least three pathways: photodynamic action, lipid peroxidation, and inflammation and may play a role in sunlight-induced skin carcinogenesis.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Deoxyguanosine; Male; Mice; Mice, Hairless; Pyrimidine Dimers; Skin; Skin Neoplasms; Tyrosine; Ultraviolet Rays