Estudo das propriedades anticâncer In vitro e In vivo de triterpenos cucurbitanos (Cucurbitacinas) obtidos da planta Cayaponia racemosa COGN

Abstract

Cucurbitacins are a group of highly oxygenated tetracyclic triterpenoids. This study aimed to evaluate the anticancer properties of three cucurbitacins (2,3,16,20(R),25-pentahydroxy-cucurbita-5-ene-22-ona (1), 2,3,16,20(R),25-pentahydroxy-cucurbita-5,23(E)-diene-22-ona (deacetylpicracin, 2) and 2,3,16,20(R),25-pentahydroxy-cucurbita-5-ene-11,22-dione (cucurbitacin P, 3) on in vitro and in vivo models. First, the cytotoxic activity on human peripheral mononuclear blood cells (PMBC) by alamar blue method, cell viability by tripan blue exclusion method, morphological changes using hematoxylin/eosin (HE) staining, proliferative capability using BrdU incorporation method and the induction of apoptosis using acridine orange/ethidium bromide (AO/EB) staining were evaluated. None of cucurbitacins was cytotoxic to PMBC in alamar blue test (IC50>25μg/ml). The tripan blue exclusion test revealed that, except compound 3 (2.5μg/ml), all cucurbitacins (2.5 and 5.0μg/ml) reduced the number of viable HL-60 cells, and none of them changed the number of non-viable cells. The morphological analysis of cells treated with 3 indicated the presence of many apoptotic cells (cell retraction, DNA fragmentation and vacuoles) in both concentrations, confirming data obtained by AO/EB staining. Cells treated with 1 showed intense deposition of granules in the cytoplasm (eosinophilia), DNA fragmentation and irregularities in the plasma membrane. Compound 2 caused intense vacuolization, deposition of granules in the cytoplasm and plasma membrane disruption. Regarding the antiproliferative activity, compounds 1 and 2 were active. Compound 1 was the most active, showing BrdU incorporation inhibition of 17 (2.5μg/ml) and 22% (5.0µg/ml), followed by compound 2 (5.0μg/ml: 17%). The AO/EB staining showed drastic reduction in the number of viable cells with increasing number of apoptotic cells for all compounds, without increasing the number of necrotic cells. Then, flow cytometry analyses were performed. The evaluation of cell membrane integrity showed membrane disruption only on those cells treated with compound 1 (5.0μg/mL). The assessment of nuclear DNA content distribution revealed that compound 2 induced cell cycle arrest at S phase. Finally, mitochondrial membrane depolarization was observed in cells treated with compound 2 (5.0μg/ml). On Sarcoma 180 tumor model, compound 1 showed 52% and 62% of antitumoral activity, alone (25mg/Kg/day) or in association to the chemotherapeutic agent 5-FU (10 + 10 mg/kg/day), respectively. Moreover, alone or associated with 5-FU, 1 caused increasing on spleens weight. Histopathological analysis of liver, kidneys and spleen showed signs of slight toxicity and potentially reversible and discrete alterations. In conclusion, cucurbitacins tested showed antiproliferative activity on HL-60 tumor cell line associated with induction of apoptosis. Additionally, compound 1 had moderate in vivo anticancer activity in Sarcoma 180 tumor model.