gms | German Medical Science

Background: Pancreatic cancer is a tumor with high intrinsic chemotherapy resistance. This has been linked to the overexpression of anti-apoptotic bcl-2. Knockdown of bcl-2 by siRNA inhibits proliferation and induces apoptosis in pancreatic cancer cells in vitro and in vivo (Ocker et al., Gut 2005;54:1298-308). We now investigated if and to what extend the knockdown of bcl-2 sensitises pancreatic cancer cells to gemcitabine treatment in vitro and in vivo.

Methods: Knockdown of bcl-2 was achieved in YAP C pancreatic cancer cells by transfecting specific siRNA or shRNA constructs. After 72 h, different concentrations of Gemcitabine were added and apoptosis was determined by propidium iodide staining. Changes in protein and mRNA levels were determined by western blotting and quantitative RT-PCR. 5x106 cells were injected subcutaneously into male NMRI mice. Mice were treated intraperitoneally with low dose Gemcitabine (8 mg/kg every 4th day) and siRNA against bcl-2 or GFP. Tumor growth was determined daily and verified by immunohistochemistry, qRT-PCR and western blotting after 21 days.

Results: Gemcitabine inudeced apoptosis at 0.1 µM or higher but was ineffective at 0.01 µM or below. The pre-transfection of bcl-2-specific siRNA and the addition of 0.01 µM Gemcitabine lead to a time-dependent induction of apoptosis, reaching 50% after 72 h of Gemcitabine treatment. Apoptosis was not induced in combinations of mock or control siRNA transfections with 0.01 µM Gemcitabine. In vivo, 8 mg/kg every 4th day Gemcitabine alone or in combination with siRNA against GFP lead to a 50% reduction in tumour size after 21 days compared to saline controls. Combination of Gemcitabine with bcl-2-specific siRNA lead to a stable tumor size for 21 days, with a tumor size of 12.5% of untreated controls.

Conclusions: RNAi-mediated knockdown of bcl-2 sensitizes human pancreatic cancer cells to Gemcitabine-induced apoptosis in vitro and delays growth of YAP C xenografts in nude mice. This strategy might contribute to the treatment of pancreatic cancer.