Anticancer properties of phospholipase A2 fromDaboia siamensis venom on human skin melanoma cells

Phospholipase A2 (PLA2) is a major component of theDaboia siamensis venom, which is able to hydrolyse the membrane of various cells. For this reason, the activity of PLA2was investigated regarding its pharmaceutical properties. This study was conducted to explore the pharmacological properties of a PLA2from Daboia siamensis (dssPLA2) venom on human skin melanoma cell line (SK-MEL-28). Methods dssPLA2 was isolated by ion exchange and gel filtration columns. Various concentrations of dssPLA2were investigated for cytotoxic activity and inhibition of migration on SK-MEL-28 cells. Cell death analysis, mRNA expression levels of Notch I-III and BRAF V600E genes were also determined. Results dssPLA2 exhibited cytotoxicity on SK-MEL-28 for 24 and 72 h as compared with untreated cells. However, it had no toxic effects on CCD-1064sk cells under the same conditions. dssPLA2 (0.25 and 0.5 g/mL) induced 17.16 and 30.60 % of apoptosis, while activated 6.53 and 7.05 % of necrotic cells. dssPLA2 at 0.25, 0.5, 1 and 2 g/mL could inhibit migration on SK-MEL-28 cells for 24 h by 31.06, 41.66, 50 and 68.75 %, respectively. The action of dssPLA2 significantly reduced the levels of Notch I and BRAF V600E genes expression on SK-MEL-28 cells compared with untreated cells at 72 h. Conclusions This study indicates that dssPLA2 had potential effects of apoptosis, necrosis, cytotoxicity and inhibition of migration on SK-MEL-28 cells. dssPLA2 could possibly be a selective agent that targets cancer cells without affecting normal cells.

Desmoplastic melanoma associated with an intraepidermal lentiginous lesion: case report and literature review / Melanoma desmoplásico associado a lesão lentiginosa intraepidérmica, com evolução de 10 anos: relato de caso e revisão bibliográfica

Desmoplastic melanoma tends to present as firm, amelanotic papules. Microscopically, it reveals a proliferation of fusiform cells in the dermis and variable collagen deposition, as well as intraepidermal melanocytic proliferation of lentiginous type in most cases. Biopsy in a 61-year-old white male patient, who had received a diagnosis of lentigo maligna on his face 10 years before, revealed a proliferation of dermal pigmented spindle cells and collagen deposition, reaching the deep reticular dermis, with a lentiginous component. Immunohistochemistry with S-100, Melan-A and WT1 showed positivity, but it was weak with HMB45. Desmoplastic melanoma associated with lentigo maligna was diagnosed. Several authors discuss whether desmoplastic melanoma represents a progression from the lentiginous component or arises "de novo". Desmoplastic melanoma represents a minority of cases of primary cutaneous melanoma (less than 4%). Identification of lentigo maligna indicates that desmoplastic melanoma should be carefully investigated.

Os melanomas desmoplásicos apresentam-se como pápulas amelanóticas firmes; à microscopia exibem proliferação de células fusiformes na derme e variável deposição de colágeno, além de proliferação melanocítica lentiginosa, intraepidérmica, na maioria dos casos. Realizada biópsia de pele de paciente masculino, 61 anos, branco, com diagnóstico de lentigo maligno na face, há 10 anos. O exame histopatológico revela proliferação dérmica de células fusiformes pigmentadas e deposição de colágeno, invadindo até a profundidade da derme reticular, associado a componente lentiginoso; presença de positividade imuno-histoquímica com S-100, Melan-A e WT1, e marcação fraca com HMB45. Diagnóstico de melanoma desmoplásico, associado a lentigo maligno. Existe divergência quanto à origem do melanoma desmoplásico, a partir do componente lentiginoso ou "de novo", na ausência de lentigo associado. O melanoma desmoplásico representa uma minoria dos casos de melanoma cutâneo primário (menos de 4%). A presença de lentigo maligno pode servir de sinal de alerta para possível relação com melanoma desmoplásico.

Differential diagnosis in primary and metastatic cutaneous melanoma by FT-Raman spectroscopy / Diagnóstico diferencial no melanoma primário e metastático por espectroscopia FT-Raman

PURPOSE: To qualify the FT-Raman spectral data of primary and metastatic cutaneous melanoma in order to obtain a differential diagnosis. METHODS: Ten normal human skin samples without any clinical or histopathological alterations, ten cutaneous melanoma fragments, and nine lymph node metastasis samples were used; 105, 140 and 126 spectra were obtained respectively. Each sample was divided into 2 or 3 fragments of approximately 2 mm³ and positioned in the Raman spectrometer sample holder in order to obtain the spectra; a monochrome laser light Nd:YAG at 1064 nm was used to excite the inelastic effect. RESULTS: To differentiate the three histopathological groups according to their characteristics extracted from the spectra, data discriminative analysis was undertaken. Phenylalanine, DNA, and Amide-I spectral variables stood out in the differentiation of the three groups. The percentages of correctly classified groups based on Phenylalanine, DNA, and Amide-I spectral features was 93.1 percent. CONCLUSION: FT-Raman spectroscopy is capable of differentiating melanoma from its metastasis, as well as from normal skin.

OBJETIVO: Qualificar os dados espectrais FT-Raman do melanoma cutâneo primário e metastático e assim realizar o diagnóstico diferencial. MÉTODOS: Foram utilizadas amostras de 10 fragmentos de pele sem alterações clínicas ou histopatológicas, 10 de melanomas cutâneos e 9 de metástases linfonodais; 105, 140 and 126 espectros foram obtidos respectivamente. Cada amostra foi dividida em 2 ou 3 frações de 2 mm³ e posicionada no porta amostras do espectrômetro Raman para obtenção dos espectros, por meio da excitação do espalhamento inelástico pelo laser de Nd:YAG em 1064 nm incididos na amostra. RESULTADOS: Para diferenciar os três grupos formados de acordo com as características fornecidas pelos espectros, realizamos a análise discriminante dos dados. As variáveis espectrais Fenilalanina, DNA e Amida-I se destacaram na capacidade de diferenciação dos três grupos histológicos. A porcentagem de classificação correta utilizando estes critérios foi de 93,1 por cento; o que mostra a eficiência da análise realizada. CONCLUSÃO: A espectroscopia FT-Raman é capaz de diferenciar o melanoma de sua metástase, assim como da pele normal.

Procoagulant properties of human MV3 melanoma cells

A correlation between cancer and prothrombotic states has long been described. More recently, a number of studies have focused on the procoagulant mechanisms exhibited by tumor cells. In the present study, we dissected the molecular mechanisms responsible for the procoagulant activity of MV3, a highly aggressive human melanoma cell line. It was observed that tumor cells strongly accelerate plasma coagulation as a result of: i) expression of the blood clotting initiator protein, a tissue factor, as shown by flow cytometry and functional assays (factor Xa formation in the presence of cells and factor VIIa), and ii) direct activation of prothrombin to thrombin by cells, as evidenced by hydrolysis of the synthetic substrate, S-2238, and the natural substrate, fibrinogen. This ability was highly potentiated by the addition of exogenous factor Va, which functions as a co-factor for the enzyme factor Xa. In contrast, prothrombin activation was not observed when cells were previously incubated with DEGR-factor Xa, an inactive derivative of the enzyme. Moreover, a monoclonal antibody against bovine factor Xa reduced the prothrombin-converting activity of tumor cells. In conclusion, the data strongly suggest that MV3 cells recruit factor Xa from the culture medium, triggering an uncommon procoagulant mechanism.

Diagnóstico precoz de metástasis de melanoma mediante biopsia de linfonodo centinela: informe preliminar / Early diagnosis of melanoma metastasis by sentinel lymph node biopsy

Se presenta un estudio de linfonodo centinela y la experiencia inicial en el diagnóstico de micro metástasis linfática en melanoma maligno de piel, en un período de dos años. Se identificó en el pre operatorio el sitio del linfonodo centinela mediante albúmina marcada con Tc99 y, en el intra operatorio, con tinción vital con azul de isosulfán para seleccionar a los pacientes con micro metástasis que se pueden beneficiar con tratamiento quirúrgico linfático, descartar la cirugía ganglionar en los pacientes con linfonodo centinela negativo y reducir la morbilidad asociada al tratamiento quirúrgico. El criterio de inclusión incluyó pacientes con tumores Clark III o superior. Se incluyeron 6 pacientes; 2 Clark III, 3 Clark IV y un paciente Clark V. El diagnóstico de micro metástasis se realizó mediante tinción hematoxilina-eosina y estudio histoquímico de antígenos Melan A, S 100 y HMB 45. En dos pacientes Clark III y uno Clark IV, el estudio con H-E e inmunohistoquímica no identificó micro metástasis. Un paciente Clark IV no evidenció metástasis en el primer estudio con H-E; sin embargo, la inmunohistoquímica mostró micro metástasis con los tres marcadores tumorales. La revisión de las placas H-E confirmó el hallazgo. Un paciente con micro etapa IV y uno con micro etapa V mostraron metástasis a la tinción H_E y se confirmó la presencia de anticuerpos para los marcadores Melan A, S 100 y HMB 45. En este estudio el uso de Melan A, S 100 y HMB 45 mejoró la sensibilidad del diagnóstico histológico de metástasis linfáticas en un 20 por ciento y redujo a la mitad la indicación de disección ganglionar regional.

Effect of 3 plant extracts on b16-bl6 melanoma cell growth and metastasis in C57bl/6 mice

En el presente estudio, se investigaron los posibles efectos de extractos acuosos de tres plantas amazónicas, Uncaria tomentosa (UT), Petiveria alliacea (PA) y Phyllantus niruri (PN), sobre el crecimiento y la metástasis de células de melanoma B16/BL6 en ratones C57BL/6, y sobre los niveles séricos del factor de necrosis tumoral alfa (TNF-iÀ), la interleucina-6 (IL-6) y el componente amiloide P (SAP). Ninguno de los tres extractos inhibió el crecimiento de las células B16/BL6, ni de las líneas celulares tumorales humanas, HT 29 y Caco-2, in vitro. Los niveles séricos de IL-6 y SAP, y la respuesta de TNF-¡À sérico a LPS resultaron elevados en los animales inoculados i.v. con las células de melanoma. Sólo el UT, inyectado i.p., redujo significativamente la metástasis a pulmón de un inóculo i.v. de células B16/BL6, y produjo un retardo en el crecimiento de un tumor primario. Además, sólo el extracto de UT produjo disminución de la respuesta sérica de las dos citocinas proinflamatorias, TNF-¡À e IL-6. UT amerita futuros estudios como potencial agente anticáncer.

Several useful anticancer drugs have been derived from plants. The growth and metastasis of tumours in the mousemay be used to study plant extracts with therapeutic potential. In the present study we investigated the possible effects of aqueous extracts of three Amazonian plants (Uncaria tomentosa [UT], Petiveria alliacea [PA] and Phyllantus niruri [PN]) on the growth and metastasis of B16/BL6 melanoma cells in the C57BL/6 mouse, and on the serum levels of tumor necrosis factor alpha (TNF-a), interleukin-6 (IL-6) and serum amyloid P component (SAP). None of the three extracts inhibited the growth of the B16/BL6 cells in vitro, or of two other tumor cell lines, HT-29 and Caco-2. Serum levels of IL-6 and SAP, and the TNF-¦Á serum response to LPS rose in the animals inoculated i.v. with melanoma cells. Only UT, when injected i.p. every other day for 14 days, was able to significantly reduce lung metastases after an i.v. inoculation of melanoma cells or to delay the appearance and growth of solid tumors after s.c. inoculation. In addition, only UT was able to reduce the serum response of the two proinflammatory cytokines, TNF-a and IL-6. No effect on SAP levels was observed. UT merits further study as an anticancer agent.