CAPE induces apoptosis in tumor pancreatic cells after inhibition of autophagy

PAPADEMETRIO DANIELA; SIMUNOVICH TANIA; CAVALIERE VICTORIA; FADER CLAUDIO; COLOMBO MARÍA INES; BLANCO GUILLERMO; HAJOS SILVIA; ALVAREZ ELIDA

Colorado Convention Center, Denver, CO. USA

Congreso; AACR 100th Annual Meeting 2009; 2009

Pancreas tumors are highly resistant to the action of chemotherapeutic drugs. The lack of apoptosis could be related to the instauration of an autophagic process. The aim of this study was to verify the presence of autophagy in a tumor pancreatic cell line, MIAPaCa-2, and to determine if the blockage of this process sensitized the cells to death by apoptosis by inhibition of NF-kB pathway. For that purpose, we analyzed the ability of the NF-kB inhibitor CAPE (Caffeic acid phenylehtyl ester) to induce apoptosis. This drug was able to induce a 59.0% of apoptosis after 6 days of treatment, whereas in basal conditions, the apoptosis measured was only of 10.3%, using the Annexin V-FITC/PI assay (p<0.001). In order to address if the MIAPaCa-2 cell line presented autophagy, cells were transfected with the autophagic protein RFP-LC3 and stained with monodansylcadaverine. In both cases, the results indicate that these cells presented autophagic features in basal conditions, which were inhibited when cells were pre-incubated with 4 mM of the autophagy inhibitor, 3-metyladenine (3-MA). We also analyzed the ratio beclin1/Bcl-2 after 24h post-treatment with CAPE by western blot. This value increased from 1.5 to 3 folds in those cells treated with the NF-kB inhibitor compared to basal conditions. Finally, with the purpose of studying if autophagy plays a role in the mechanisms by which the MIAPaCa-2 cells are resistant to die by apoptosis, we studied if cells pre-treated with 4 mM of 3-MA were sensitized to the action of CAPE. We observed that in only 3 days, CAPE induced a 48.0% of apoptosis in cells in which the autophagy process was inhibited, vs. a 14.0% and 1.0% in cells treated only with CAPE or 3-MA, respectively (p<0.001). Our data shows that long periods of time are required to induce apoptosis in the pancreas tumor cells MIAPaCa-2. We propose that autophagy is participating in the mechanisms that maintain these cells alive delaying the outcome of apoptotic death. We suggest that combined therapies with inhibitors of autophagy and inductors of apoptosis, such as CAPE, could be useful in the treatment of pancreas tumors.