Autophagy prevents apoptotic death in pancreatic tumor.

DANIELA L. PAPADEMETRIO; VICTOEIA CAVALIERE; TANIA C. SIMUNOVICH; SUSANA N. COSTANTINO; TOMÁS LOMBARDO; MARÍA I. COLOMBO; GUILLERMO BLANCO; CLAUDIO M. FADER AND ELIDA ALVAREZ.

Simposio; Keystone Symposia on Molecular and Cellular Biology. Autophagy.; 2011

Infiltrating ductal adenocarcinoma of the pancreas (PDA) accounts for over 95% of all exocrine pancreatic malignancies. Infiltrating PDA is almost uniformly fatal, with a five-year survival of less than 5%. The median survival for patients with PDA is 4–6 months, in part because the disease usually only becomes clinically apparent at late stages, and because it resists all forms of conventional radiotherapy and chemotherapy that includes the administration of Gemcitabine (2’-2’-difluorodeoxycytidine). In several tumors, autophagy process constitutes a resistant mechanism to pro-apoptotic stimuli. The aim of this study was to verify if Gemcitabine was able to induce apoptotic death of human pancreatic tumor cells after autophagy inhibition, in vitro and in vivo in a xenograph model. For that, we analyzed the capability of Gemcitabine (0.01-1000µg/ml) to induce apoptosis alone or after pre-treatment with 3-metyladenine (3-MA) 10mM on MIAPaCa-2 and PANC-1 cell lines, using Annexin V-FITC/PI and TUNEL assay. In all cases Gemcitabine increased in a 30% the number of apoptotic cells when autophagy process is inhibited (p<0.001). The treatment of MIAPaCa-2 and PANC-1 cells with Gemcitabine 1000 µg/ml produced a (44.5±4.8)% and (23.7±4.4) of apoptotic cells, respectively. When cells were pretreated with 10 mM of 3-MA, the percentage reach to (70.1±8.3)% and (41.1±6.1)%. Then, we assayed if the pan-caspase inhibitor, z-VAD-fmk, was able to revert the pro-apoptotic effect of Gemcitabine alone or in combination with 3-MA. Z-VAD-fmk reduced the percentages of apoptotic cells in a 65% (p<0.001) on MIAPaCa-2 cells but does not have any significant effect on PANC-1 cells. In order to determine the apoptotic pathway involved, we analyzed the level expression of pro-caspase 3 and PARP cleavage, after 48 h and AIF, after 24 h of treatment. Gemcitabine induced, on MIAPaCa-2 cells, a 3-fold increase of pro-caspase 3 levels and the pretreatment with 3-MA increased in a 50% the cleavage of PARP and diminished the pro-caspase 3 levels in a 30%. No effect was observed on PARP cleavage and pro-caspase 3 levels on PANC-1, but 3-MA + Gemcitabine increased in a 4.2-fold the AIF ratio nuclear/cytoplasmic. We analyzed the effect of Gemcitabine alone or in combination with 3-MA on the tumor size of a xenograph model of MIAPaCa-2 cells and the morphology of tumor cells after treatment by electron microscopy. We observed that both Gemcitabine and 3-MA + Gemcitabine were able to inhibited tumor growth. When we analyzed tumor cell morphology, we saw that Gemcitabine exacerbated autophagy process meanwhile in combination with 3-MA induced apoptosis. Taken together, our results suggest that Gemcitabine exacerbates autophagy and if this mechanism is inhibited, induces apoptosis in vitro and in vivo, in a caspase-dependent manner on MIAPaCa-2 but caspase-independent way, on PANC-1 cells.   Acknowledgements This work was supported by grants from SECYT UBA B011 and CONICET PIP #0199