The mechanisms of proapoptotic effects of rottlerin in pancreatic cancer cells involve interaction with Bcl-2 protein

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Background: Pancreatic cancer (PaCa) cells are resistant to apoptosis, which is one reason for their poor responsiveness to chemo- and radiotherapy. Several plant-derived polyphenols were shown to stimulate apoptosis in PaCa cells, although the underlying mechanisms remain unknown. In this study we determine the effects of polyphenolic phytochemical rottlerin on pancreatic cancer cells survival, both in vitro and in the orthotopic model of pancreatic cancer. We showed that rottlerin directly interacts with pro-survival effects of Bcl-2 and Bcl-xL proteins in PaCa cells resulting in mitochondrial dysfunctional apoptosis. Methods: Apoptosis in MIA PaCa-2 and PANC-1 cells was assessed by measuring DNA fragmentation and AnV/PI staining. Cytochrome c release and mitochondrial membrane potential (ΔΨm) were measured in both intact and digitonin-permeabilized cells using Western blot and ΔΨm-sensitive TPP⁺ electrode, respectively. Caspase-3 activity and processing were measured with a fluorogenic assay and Western blot, respectively. Bcl-xL-Bad interaction in cells was measured by co-immunoprecipitation, and complex formation between recombinant Bcl-xL and Bad using fluorescence polarization. Results: Both rottlerin (0.5-5μM) and Bcl-xL inhibitor BH3I-2’ (10-100μM) dose-dependently stimulated apoptosis in PaCa cells. In particular, rottlerin (5μM) and BH3I-2’ (25μM) increased apoptosis in PaCa cells as much as three fold. Both rottlerin and BH3I-2’ markedly stimulated cytochrome c release, mitochondrial depolarization and caspase-3 activation in both cell lines. Both agents stimulated cytochrome c release and mitochondrial depolarization if added to isolated mitochondria, suggesting the direct interaction with mitochondria in PaCa cells. Furthermore rottlerin and BH3I-2’ both decreased association between Bcl-xL and Bad suggesting that both act though inhibiting anti-apoptotic effects of Bcl-2 and Bcl-xL. Injections of 0.5 mg/kg rottlerin for 4 weeks decreased tumor size and increased apoptosis in the orthotopic mouse model of pancreatic cancer; rottlerin was not toxic for the animals. Conclusions: Rottlerin sensitizes PaCa cells to apoptosis both in vitro and in vivo, likely, through inhibition of anti-apoptotic Bcl-2 proteins. Rottlerin is a promising agent for pancreatic cancer treatment.