Gemcitabine induces apoptosis death after autophagy inhibition in pancreatic tumor

PAPADEMETRIO D; CAVALIERE V; FADER C; LOMBARDO T; SIMUNOVICH T; COSTANTINO S; COLOMBO MI; HAJOS SE; BLANCO G; ALVAREZ E

Vancouver, Canadá

Simposio; Keystone Symposia; 2010

Keystone

Pancreatic cancer is an aggressive disease whose incidence has risen in the last two decades, being the fourth cause of cancer death in the Western world. Currently, gemcitabine (2’-2’-difluorodeoxycytidine) represents the standard chemotherapy in all stages of pancreatic adenocarcinoma. However, survival benefit and clinical impact remains modest due to a high degree of intrinsic and acquired resistance. In several tumors, the lack of apoptosis could be related to the instauration of an autophagy process. The aim of this study was to verify if the blockage of autophagy process sensitized the cells to death by apoptosis after treatment with Gemcitabine. For that, we analyzed the capability of Gemcitabine (10-1000mg/ml) to induce apoptosis alone or after pre-treatment with 3-metyladenine (3-MA) 10mM on MIAPaCa-2 cell line, using the Annexin V-FITC/PI assay. In all cases Gemcitabine increased in a 30% the number of Annexin + cells when the autophagy process was inhibited (52.6%) vs. Gemcitabine alone (22.0%) (p<0.001). Similar results were obtained by TUNEL assay. Then, we analyzed de variation of the levels of several proteins by western blot. The treatment with Gemcitabine in 3-MA pretreated cells decreased the levels of both Beclin-1 and Bcl-XL in a 50 %, meanwhile the cleavage of PARP increased in a 40%, indicating an increment in the caspase 3 activity. By contrast, Gemcitabine alone increased in a 60% the beclin-1 levels and diminised the cleavage of PARP in a 30% (p<0.01). Bcl-XL remained invariant respect to basal condition.