63ª Reunião Anual da SBPC

C. Ciências Biológicas - 3. Bioquímica - 6. Bioquímica

BENZALDEHYDE LIMONENE THIOSEMICARBAZONE PROMOTES ACTIVATION OF CASPASES IN HUMAN MELANOMA CELLS

Débora Cristina Silva dos Passos 1 Elisângela Ribeiro 1 Cecília Maria Alves de Oliveira 2 Lídia andreu Guillo 1

1. Depto de Biologia Celular e Molecular, UFG, Goiânia/GO 2. Drª. Depto de Biologia Celular e Molecular, UFG, Goiânia/GO 3. Profª. Drª. Depto de Química, UFG, Goiânia/GO 4. Profª. Drª./Orientadora - Depto de Biologia Celular e Molecular, UFG, Goiânia/GO

INTRODUÇÃO:

Melanoma is a type of cancer that arises from melanocytes and is notoriously resistant to radiation and chemotherapy. Apoptosis is a complex cellular event that converges to an activation of the cascade of proteases called caspases, culminating in cell death. Multiple complementary techniques are necessary to ascertain the occurrence of apoptotic mechanisms. Benzaldehyde limonene thiosemicarbazone (BLT) has previously demonstrated to inhibit the in vitro proliferation of human melanoma cells. In an attempt to assess the mechanism of action of BLT we have verified by flow cytometry and caspase assays, if the antiproliferative effect of BLT is related to induction of apoptosis in human melanoma cells.

METODOLOGIA:

Human melanoma SK-MEL-37 cells were treated with BLT, harvested and resuspended in ice cold 70% ethanol. For cell cycle analysis, ethanol-fixed cells were resuspended in 100 µL of PBS containing 10U/mL ribonuclease and 20 µg/mL propidium iodide (PI). Early and late apoptotic events were ascertained by incubation of fixed cells with annexin V-FITC and PI and further analyzed by flow cytometry. For colorimetric caspase assay, exponentially growing cells were treated with BLT (100 µM) and incubated for a period 8 and 15 hours. They were then harvested and resuspended in cell lysis buffer provided by the kit (Apotarget - Caspase Colorimetric Protease Assay Caspases-2, -3, -6, -8, -9, Invitrogen). Samples were then mixed with reaction buffer and the following caspase substrates were added VDVAD-pNA (for Caspase-2), DEVD-pNA (for Caspase-3), VEID-pNA (for Caspase-6), IETD-pNA (for Caspase-8), and LEHD-pNA (for Caspase-9). Samples were incubated at 37°C for 15h and absorbance was taken in a microplate reader at 405nm. Before and after treatments, morphology was observed and photographed in a NIKON E6000 Eclipse fluorescence microscope.

RESULTADOS:

The typical morphological changes of apoptotic process could be accomplished by visualization by microscopy. We observed the collapse of the plasma membrane following by the detachment of cells after 48 h of treatment. Flow cytometry analysis demonstrated that late apoptotic features were detected in 28,07% of cells after 48h of treatment. While the percentage of cells in G0/G1 phase decreased from 73 ± 2,9% to 51 ± 0,8% , cells in SG2/M phase increased from 15 ± 2,2% to 22 ± 0,8%, indicating that the growth inhibitory effect of BLT might be due to arrest of cells at SG2/M phase. In addition, we have observed the following activities after treatment with BLT for a period of 8h caspase2: 0,95 µM/µg protein; caspase-3: 15,61 µM/µg protein; caspase-6: 5,47 µM/µg protein; caspase-8: 3,78 µM/µg protein and caspase-9: 0,98 µM/µg protein. After a period of 15 h, we have observed: caspase-2: 0,34 µM/µg protein; caspase-3: 0,45 µM/µg protein, caspase-6: 0,86 µM/µg protein, caspase-8: 0,35 µM/µg protein and caspase-9: 0,18 µM/µg protein. According to these data, caspase 3 and 6 are involved in cell death observed after treatment with BLT.

CONCLUSÃO:

The results suggest that BLT induces apoptosis in human melanoma cells by caspase 3 and 6-dependent pathway and could be an attractive candidate for future preclinical in vivo studies.

Palavras-chave: Benzaldehyde limonene thiosemicarbazone, melanoma cells, apoptosis.