In vitro antitumor properties of a triazolyl aminoacyl(peptidyl) penicillin in murine melanoma cells

BELLIZZI YANINA; DELPICCOLO CM; MATA E.G.; CORNIER P.G.; REY O; BLANK VC; BOGGIÁN D.B; ROGUIN, L.P.

Mar del Plata

Congreso; LXI Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica, LXIV Reunión Anual de la Sociedad Argentina de Inmunología, XLVIII Reunión Científica Anual de la Sociedad Argentina de Farmacología Experimental; 2016

SAIC, SAI, SAFE, NANOMEDAR,AACYTAL

The triazolyl aminoacyl(peptidyl) penicillins (TAP) are novel hybrids compounds having in their structure a penicillanic core linked to a peptide portion via a triazole group. In a previous study, we showed that the derivative containing the dipeptide Leu-Phe (TAP6) exerts an antiproliferative potency around 30 times higher in HeLa (human cervix adenocarcinoma) and B16-F0 (murine melanoma) cells with respect to non-malignant cells. In this work, after exploring a wider panel of human and murine tumor cell lines, we showed that IC50 values obtained in human cells varied between 3-12 µM, while IC50 values ≥ 20 µM were determined in various murine cells except B16-F0, that showed an IC50= 3,5±0,3 µM. B16-F0 cells were then selected to investigate the mechanism of antitumor action of TAP6. By flow cytometry, we showed that TAP6 slowed down the cell cycle at the S phase and increase the percentage of early (10±3%) and late (45±4%) apoptotic cells. A significant enhancement (1,5-3 fold) of the expression levels of some hallmark proteins of the endoplasmic reticulum (ER) stress response was detected by Western blot after 6h of incubation. In addition, we found a reduction of the expression levels of Bcl-XL (0.40±0.05) and Bcl-2 (0.7±0.2), and an increase in the amount of the pro-apoptotic Bax protein (1.8±0.5). A time-dependent increment in the activity of caspase-3, -8 and -9 was also observed. The loss of the mitochondrial inner transmembrane potential was determined by flow cytometry, being the percentage of cells with reduced DiOC6(3) incorporation 30% after 3h of TAP6 exposure. Taken together, our results indicated that TAP6 is a potent and selective antitumor agent in different tumor cell lines. TAP6 activates an ER stress response that contributes together with the mitochondrial pathway to the induction of an apoptotic response. Even though we showed an increment of caspase-8 activity, the involvement of the death receptor pathway remained to be studied.