Resposta celular associada à expressão de galectina-3 em linhagens de melanoma expostas a irradiação / Cellular response associated to galectin-3 expression in exposed irradiation melanoma cells

O câncer de pele é um dos mais frequentes entre humanos, sendo o melanoma o tipo menos comum, mas com grande importância devido à agressividade que ele apresenta. Um dos principais agentes etiológicos deste tipo de tumor é a radiação ultravioleta proveniente da luz solar. A fração de radiação ultravioleta B (UVB) gera dano no DNA e induz alterações nas células da pele após a exposição prolongada e sem proteção. A resposta à luz UVB em melanócitos e melanomas é diferente, mostrando a importância do perfil celular. O efeito genotóxico da luz UVB pode alterar a expressão de moléculas como galectina-3 e MAPKs, desencadeando respostas UVB-dependentes. Galectina-3 é uma lectina que reconhece beta-galactosídeos e está envolvida na regulação de diversos processos celulares que modificam a viabilidade celular e a proliferação. Esta molécula é ubiquamente expressa apresentando um comportamento específico dependendo da sua localização subcelular. No presente trabalho mostramos que a distribuição de galectina-3 em melanoma e melanócitos é ampla, encontrando-se tanto no núcleo como no citoplasma, podendo ser modificada após irradiação UVB ou ainda secretada para o meio extracelular. Além disso, observamos que a luz UVB ativa a via de MAPKs, proteínas quinases ativadas por mitógenos envolvidas no crescimento, sobrevivência, diferenciação e resposta a estresse, em melanócitos e em melanomas poucos minutos após a exposição à UVB. Uma maior atividade de p38 e de ERK é evidenciada em melanomas, enquanto que em melanócitos a via de p38 é a mais ativa, corroborando a noção de que a resposta celular à luz UVB difere entre melanócitos e melanoma. As moléculas p38 e JNK são proteínas quinases ativada pelo estresse (SAPK). A via de JNK não é tão responsiva em alguns melanomas, mas ativação desta molécula parece estar envolvida com a sobrevivência celular e a translocação mitocondrial após UVB...

Skin cancer is the most common cancer among humans, melanoma being the least common type but very important due to its aggressive behavior. A major etiologic agent of this type of tumor is ultraviolet radiation from the sunlight. The ultraviolet B rays (UVB) cause DNA damage and induce alterations over the skin cells after prolonged exposition without protection. The UVB response in melanocytes and melanoma cells is different. This shows the importance of the cellular profile. The genotoxic effect of UVB light can alter the expression of molecules such as galectine-3 and MAPKs and also triggers multiple responses UVB-dependent. Galectin-3 is a lectin that recognizes beta-galactosides. It is involved in the regulation of many cellular processes that modify cellular viability and proliferation and presents specific behavior depending on its subcellular localization. In the present study we showed that galectine-3 distribution in melanoma cells and melanocytes is large, lying both in the nucleus and in the cytoplasm. After UVB irradiation this distribution could be modified or even galactine-3 secreted itself into the extracellular space. Moreover, we observed that UVB light activates the mitogen-activated protein kinase pathway (MAPK) involved in growth, survival, differentiation and stress-response in melanocytes and in melanoma cells just a few minutes after exposure. An increased activity of p38 and ERK was observed in melanomas, while in melanocytes just p38 pathway was highly active, supporting the notion that the cellular response to UVB light differs between melanocytes and melanoma cells. The molecules p38 and JNK are stress-activated protein kinases (SAPK). The JNK pathway is not responsive in some melanoma cells, but the activation of this molecule appears to be involved in cell survival and mitochondrial translocation after being exposed to UVB. Inhibition of JNK leads to increased cell death in irradiated and non-irradiated melanocytic lineage...

A comparative study of UV-induced cell signalling pathways in human keratinocyte-derived cell lines.

Ultraviolet (UV) radiation can activate the p38 mitogen-activated protein kinase (MAPK), Jun N-terminal kinase (JNK) and nuclear factor-κB (NFκB) pathways in skin cells. HaCaT cells are widely used as a primary keratinocyte substitute to study these pathways. However, like most squamous cell carcinomas (SCCs), it contains a dysfunctional p53. It is unclear if HaCaT cells activate these signalling pathways similarly to SCC cells (Colo16) or to primary human epidermal keratinocytes (HEK). In this study, the UV activation (UVA, UVB, UVA+B, UVB+A) of p38 MAPK, JNK and NFκB pathways, and TNFα secretion by HEK, HaCaT and Colo16 cells were investigated. The signalling pathway activation was UV-type and dose-dependent with UVB+A radiation inducing a high p38 and JNK activation. HaCaT cells exhibited 2- to 4-fold higher activity of the p38 (771% at 60 min) and JNK (794% at 30 min) pathways following UVB+A radiation than did HEK cells (p38: 367% at 15 min and JNK: 184% at 30 min). While both HaCaT and Colo16 cells did not activate the NFκB pathway, Colo16 cells had a lower p38 and higher JNK activity than HaCaT cells. Irradiated HaCaT cells produced less TNFα (UVB: 3.5 pg/ml), while HEK cells produced the most (UVB: 1,296 pg/ml). When co-exposed to IL1α, irradiated HaCaT had the greatest fold of TNFα release (UVB: 16.2-fold, UVA+B: 8.9-fold and UVB+A: 6.1-fold). The pattern of activation and TNFα secretion of HaCaT cells mirrored that of Colo16 cells. It is likely that the presence of molecular alterations in HaCaT cells may be responsible for its different responses to that seen for HEK cells. The results of this study suggest caution in using HaCaT cells as a substitute for normal keratinocytes in investigating UV-induced cells signalling pathways.

AT-101, a small molecule inhibitor of anti-apoptotic Bcl-2 family members, activates the SAPK/JNK pathway and enhances radiation-induced apoptosis.

BACKGROUND: Gossypol, a naturally occurring polyphenolic compound has been identified as a small molecule inhibitor of anti-apoptotic Bcl-2 family proteins. It induces apoptosis in a wide range of tumor cell lines and enhances chemotherapy- and radiation-induced cytotoxicity both in vitro and in vivo. Bcl-2 and related proteins are important inhibitors of apoptosis and frequently overexpressed in human tumors. Increased levels of these proteins confer radio- and chemoresistance and may be associated with poor prognosis. Consequently, inhibition of the anti-apoptotic functions of Bcl-2 family members represents a promising strategy to overcome resistance to anticancer therapies. METHODS: We tested the effect of (-)-gossypol, also denominated as AT-101, radiation and the combination of both on apoptosis induction in human leukemic cells, Jurkat T and U937. Because activation of the SAPK/JNK pathway is important for apoptosis induction by many different stress stimuli, and Bcl-X(L) is known to inhibit activation of SAPK/JNK, we also investigated the role of this signaling cascade in AT-101-induced apoptosis using a pharmacologic and genetic approach. RESULTS: AT-101 induced apoptosis in a time- and dose-dependent fashion, with ED50 values of 1.9 and 2.4 microM in Jurkat T and U937 cells, respectively. Isobolographic analysis revealed a synergistic interaction between AT-101 and radiation, which also appeared to be sequence-dependent. Like radiation, AT-101 activated SAPK/JNK which was blocked by the kinase inhibitor SP600125. In cells overexpressing a dominant-negative mutant of c-Jun, AT-101-induced apoptosis was significantly reduced. CONCLUSION: Our data show that AT-101 strongly enhances radiation-induced apoptosis in human leukemic cells and indicate a requirement for the SAPK/JNK pathway in AT-101-induced apoptosis. This type of apoptosis modulation may overcome treatment resistance and lead to the development of new effective combination therapies.

Inhibition of UVA-induced c-Jun N-terminal kinase activity results in caspase-dependent apoptosis in human keratinocytes.

Inhibition of c-Jun N-terminal kinase (JNK) with the pharmacologic inhibitor SP600125 in UVA-irradiated HaCaT cells and human primary keratinocytes resulted in dramatic phenotypic changes indicative of cell death. These phenotypic changes correlated with caspase 8, 9 and 3 activations as well as cleavage of the caspase substrate polyADP-ribose polymerase (PARP). Morphologic analysis and analysis of sub-G0 DNA content confirmed apoptotic cell death in these keratinocytes after combination treatment. Addition of the general caspase inhibitor zVAD-fmk to combination-treated HaCaT cells was able to completely block caspase activation, PARP cleavage, the increase in sub-G0 DNA content and the classic morphologic features of apoptosis, indicating that this combination treatment resulted in caspase-dependent apoptotic cell death. zVAD-fmk treatment of primary keratinocytes was able to completely inhibit caspase activation and PARP cleavage, reduce morphologic apoptosis at lower concentrations of SP600125 and decrease the sub-G(0) DNA content detected after UVA + SP600125 treatment. However, cell death and a significant amount of debris was still detected after caspase inhibitor treatment, particularly with 125 nM SP600125. At subconfluent conditions and low passage, primary keratinocytes were more sensitive to UVA irradiation alone than HaCaT cells. In conclusion, we have observed that inhibition of UVA-induced JNK activity with the pharmacologic inhibitor SP600125 resulted in caspase-dependent apoptotic cell death in both the immortalized keratinocyte cell line HaCaT and primary keratinocytes. However, the increased sensitivity of primary keratinocytes to experimental stress may have also resulted in direct cellular injury and caspase-independent cell death.

Ultraviolet irradiation suppresses T cell activation via blocking TCR-mediated ERK and NF-kappa B signaling pathways.

UV irradiation is carcinogenic and immunosuppressive. Previous studies indicate that UV-mediated alteration of APCs and induction of suppressor T cells play a critical role in UV-induced immune suppression. In this study, we show that UV irradiation can directly (independently of APCs and suppressor T cells) inhibit T cell activation by blocking TCR-mediated phosphorylation of ERK and IkappaB via overactivation of the p38 and JNK pathways. These events lead to the down-modulation of c-Jun, c-Fos, Egr-1, and NF-kappaB transcription factors and thereby inhibit production of cytokines, e.g., IL-2, IL-4, IFN-gamma, and TNF-alpha, upon TCR stimulation. We also show that UV irradiation can suppress preactivated T cells, indicating that UV irradiation does not only impair T cell function in response to T cell activation, but can also have systemic effects that influence ongoing immune responses. Thus, our data provide an additional mechanism by which UV irradiation directly suppresses immune responses.

Xanthophylls and alpha-tocopherol decrease UVB-induced lipid peroxidation and stress signaling in human lens epithelial cells.

Epidemiological studies suggest that consumption of vegetables rich in the xanthophylls lutein (LUT) and zeaxanthin (ZEA) reduces the risk for developing age-related cataract, a leading cause of vision loss. Although LUT and ZEA are the only dietary carotenoids present in the lens, direct evidence for their photoprotective effect in this organ is not available. The present study examined the effects of xanthophylls and alpha-tocopherol (alpha-TC) on lipid peroxidation and the mitogen-activated stress signaling pathways in human lens epithelial (HLE) cells following ultraviolet B light (UVB) irradiation. When presented with LUT, ZEA, astaxanthin (AST), and alpha-TC as methyl-beta-cyclodextrin complexes, HLE cells accumulated the lipophiles in a concentration- and time-dependent manner with uptake of LUT exceeding that of ZEA and AST. Pretreatment of cultures with either 2 micromol/L xanthophyll or 10 micromol/L alpha-TC for 4 h before exposure to 300 J/m(2) UVB radiation decreased lipid peroxidation by 47-57% compared with UVB-treated control HLE cells. Pretreatment with the xanthophylls and alpha-TC also inhibited UVB-induced activation of c-JUN NH(2)-terminal kinase (JNK) and p38 by 50-60 and 25-32%, respectively. There was substantial inhibition of UVB-induced JNK and p38 activation for cells containing <0.20 and approximately 0.30 nmol xanthophylls/mg, respectively, whereas >2.3 nmol alpha-TC/mg protein was required to significantly decrease UVB-induced stress signaling. These data suggest that xanthophylls are more potent than alpha-TC for protecting human lens epithelial cells against UVB insult.

Apoptotic and necrotic mechanisms of stress-induced human lens epithelial cell death.

Exposure to ultraviolet radiation (UVR) and reactive oxygen species (ROS) can damage the human lens and contribute to cataract formation. Recent evidence suggests that apoptosis in lens epithelial cells (LEC) is an initiating event in noncongenital cataract formation in humans and animals. The present study examines the cellular and molecular mechanisms by which environmental (ultraviolet B [UVB]) and chemical (hydrogen peroxide [H(2)O(2)], t-butyl hydroperoxide [TBHP]) stress induces cell death in an SV-40 immortalized human lens epithelial (HLE) cell line. Treatment of HLE cells with UVB, H(2)O(2), and TBHP significantly decreased cell density with LD50 values of 350 J/m(2), 500 muM, and 200 muM, respectively. Cellular morphology, DNA fragmentation, and annexin/propidium iodide staining consistent with apoptosis was observed only in UVB-treated cells, whereas lactate dehydrogenase (LDH) release was significantly higher in H(2)0(2)- and TBHP-treated cells. In addition, activation of apoptotic stress-signaling proteins, including c-Jun NH2-terminal kinase (JNK), caspase-3, and DNA fragmentation factor 45 (DFF45) was observed only in UVB-treated cells. Inhibition of JNK activity increased UVB-induced cell death, suggesting that this pathway may serve a prosurvival role in HLE cells. These findings suggest UVB predominantly induces apoptosis in HLE cells, whereas H(2)O(2) and TBHP induce necrosis.

Activation of c-Jun N-terminal kinase is required for ultraviolet B-induced apoptosis of murine peritoneal macrophages in vitro.

The mechanisms of ultraviolet B (UVB)-induced apoptosis and the role of c-Jun N-terminal kinase (JNK) mitogen activated protein kinase (MAPK) in murine peritoneal macrophages, the terminally differentiated non-dividing cells were investigated. Exposure of macrophages to UVB 100 mJ/cm2 induced rapid apoptosis concurrent with activation of JNK and mitochondrial cytochrome c release leading to procaspase-3 activation. Late into the UVB-induced apoptosis, a caspase-mediated cleavage of Bid was observed. Caspase inhibitors N-Benzylocarbonyl-Val-Asp-fluoromethyl ketone and N-Acetyl-Asp-Glu-Val-Asp-aldehyde inhibited the UVB-induced apoptosis without preventing the release of cytochrome c and JNK activation. The inhibition of JNK MAPK prevented UVB-induced apoptosis, concomitant with inhibition in cytochrome c release and procaspase-3 activation. However, it had no effect on procaspase-8 activation. These results indicate that activation of JNK MAPK upstream of caspases might play an important role in the apoptotic process of macrophages exposed to UVB irradiation.