P53 tumor suppressor is required for efficient execution of the death program following treatment with a cytotoxic limonoid obtained from Melia azedarach

MARIANA BELÉN JORAY; FLORENCIA VILLAFAÑEZ; MARÍA LAURA GONZÁLEZ; MARÍA INÉS CRESPO; JERÓNIMO LAIOLO; SARA MARÍA PALACIOS; JOSÉ LUIS BOCCO; GASTÓN SORIA; MARÍA CECILIA CARPINELLA

Lisboa

Encuentro; COST ACTION CM1407 2nd Trainning School. Synthesis, isolation and structural elucidation of bioactive compounds.; 2017

The fact that cancer still has high mortality rates encourages researchers to find novel candidate compounds for therapies. The genus Melia is well-known as a rich and valuable source of bioactive tetranortriterpenoids known as limonoids. In previous research conducted by our group, the fractionation of a kernel extract obtained from Melia azedarach L. led to the isolation of meliartenin, an antifeedant limonoid, which exists as a mixture with its tautomeric isomer 12-hydroxyamoorastatin.In this study, the antitumor activity of the interchangeable isomeric mixture (1) was evaluated over a panel of human tumor-derived cell lines, resulting in a cytotoxic effect that was highly selective of the human colorectal carcinoma cell line HCT116 over the rest of the assayed cell lines. Mechanistic analysis revealed that treatment with 1 induced a time-dependent increase in the number of cells transiting the S-phase of the cell cycle, with a similar activity from 0.4 uM to that obtained with the positive control hydroxyurea (HU) at 48 and 72 h. Subsequent detection with an anti-BrdU antibody confirmed a sustained anti-proliferative effect on HCT116 cells which is remarkable considering that compounds shown to induce arrest at the S-phase are quite rare. In addition to slowing down the cellular progression through S-phase, flow cytometry analysis after SYTOX Red stain showed that 1 induced cell death in a time-dependent manner, reaching percentages of death higher than 53% from 0.2 μM at 72 h. Furthermore, Annexin V/SYTOX Red double staining demonstrated that this compound was able to induce apoptosis in HCT116 cells with values higher than 70% at 72 h. Taken together our results suggest that this active principle triggers apoptosis due to a sustained blockage of S-phase progression. It was also found that 1 was not only capable of killing HCT116 but also strongly impaired its clonogenic potential at a concentration as low as 0.04 uM. This is a very important finding, since drugs that suppress clonogenicity hold potential to treat a variety of tumors by inhibiting their ability to expand and colonize. In the light of our findings and considering the key role of p53 in the integration of signals from diverse pathways that regulate the cell cycle and apoptosis we evaluated whether the biological effects mediated by 1 were related to the induction and activity of p53 and its downstream target p21. The increased expression profile of these proteins was similar to treatment with HU. It was found that p53-dependent apoptosis was responsible for the cytotoxicity observed in HCT116 after treatment with 1. The fact that p53, but not p21, was linked to the cytotoxic effect mediated by this compound strongly suggests that only the pro-apoptotic functions of p53 are critical for this effect. Taken together, the results described in this work position compound 1 as a strong antitumoral agent, with great potential for targeting p53+ tumors. The present investigation is the first effort toward a comprehensive understanding of the mechanisms underlying the anticancer activity of compound 1. This information is of great value for positioning 1 as a promising antitumor agent that may be used to improve anticancer therapy.