EFEITO DA CAMPOMANESIA ADAMANTIUM (CAMBESS.) O. BERG EM MODELO DE MELANOMA: APLICAÇÃO DO IR-780 IN VITRO E IN VIVO

Abstract

Cancer is a group of malignant diseases with rapid and aggressive development, and melanoma is considered one of the most serious types due to high possibility of metastasis. Brazil has biodiversity available for study of bioactive for treatment of various diseases. Cerrado is a biome where fruit of Campomanesia adamantium (guavira) is found, popularly used as a medicine and described as having anti-proliferative potential on tumor cells, therefore, effect of this fruit on melanoma lung metastasis model was evaluated. Peel and pulp extracts were obtained by maceration in dichloromethane solvent and used to research antiproliferative potential in seven cell lines, two of which were non-tumorous, using SRB and MTT methods, obtaining data on 50% of growth inhibition, total growth inhibition and selectivity index using NIH-3T3 line. Furthermore, parameters were evaluated for using IR-780 tumor marker in In-Vivo Xtreme equipment to evaluate samples against B16-F10 lineage. Both extracts were active mainly against U251 lineage (glioblastoma), with peel extract being more active (GI50 = 4.89 ug/ml, IS = 51.3) than pulp extract (GI50 = 32.1 ug/ml ml, IS = 8.7). Peel extract showed, at the highest concentration, a cytocidal effect for most of tumor lines tested. In IR-780 absorption test by B16-F10 cells (murine melanoma), best visualization of cell viability was obtained using marker at concentration of 20 µM for a cell density of 2x105. Furthermore, simultaneous staining with nuclear dye Hoechst 33342 showed the accumulation of IR-780 in cell cytosol. In vivo tests, three tumor models (n=3) were performed by inoculating 5x105 cells in male mice of BALB/c lineage, one dorsal subcutaneous nodule and two methods of obtaining pulmonary metastasis, by intraperitoneal inoculation and by venous inoculation through caudal lateral vein. 200µL of IR-780 at 0.45 mg kg-1 intraperitoneally was used in last 24 hours of experiment to obtain fluorescence images, satisfactory results were obtained in metastasis model, by venous inoculation, and the use of marker in two other models was unfeasible. To determine activity of extracts in vivo, animals were inoculated with B16-F10 cells, through lateral caudal vein, on first day of experiment. On 15th day, they were distributed into five groups (n=6), where control group received no treatment, Olive group received treatment with 100µL of pure extra virgin olive oil, DOX group was treated with 200µL of doxorubicin 5 mg/kg, DEGPU was treated with pulp extract and DEGPE group was treated with peel extract, both diluted in extra virgin olive oil at 92.5 mg/kg/100µL intraperitoneally, all groups received a single dose of treatment. In last 24h of study, on 29th day, groups also received IR-780. Animals were weighed on different days, without showing a statistically significant difference between groups. Feed consumption was evaluated in last three days of experiment, where a significant difference was found only between Control group and Olive group. Regarding metastasis, statistical difference (p<0.05) was between groups treated with extracts and Control group, indicating a great reduction in lung metastasis, especially in DEGPU group, confirmed by macroscopic and histological analyzes of lungs. Images obtained in ex vivo analysis indicated that fluorescence intensity of IR-780 was inversely proportional to lung involvement, suggesting the feasibility of using this marker for this type of analysis. Therefore, it is concluded that C. adamantium extracts limited tumor growth and metastasis implantation and that IR-780 can be used to determine cell viability and tumor lung involvement.