Molecular mechanism(s) of actinomycin-D induced sensitization of pancreatic cancer cells to CD95 mediated apoptosis

The death receptor CD95 transduces apoptotic death signaling in many cell types. However, in pancreatic tumor cells CD95 mediated apoptotic machinery is blocked by unknown protein(s). We and others have recently demonstrated that actinomycin-D (ActD) treatment induces sensitization of pancreatic cancer cells as well as other cell types to CD95 mediated apoptosis. In addition, NF-κB/Akt system has been implicated in the processes of CD95 mediated apoptosis, however the precise mechanism by which ActD sensitizes pancreatic tumor cells to CD95 mediated apoptosis is still unknown. In the present study, we demonstrated that HPAC and PANC1 pancreatic cancer cells constitutively express high levels of NF-κB and phosphorylated form of Akt. ActD at a dose that is known to sensitize pancreatic cancer cells to CD95 mediated apoptosis abrogated the DNA binding activity of NF-κB but did not affect expression of phosphorylated Akt. Co-treatment of pancreatic cancer cells with ActD and agonist anti-CD95 antibodies showed no effect on the abrogation on the DNA binding activity of NF-κB, but decreased the expression of phosphorylated Akt. Moreover, treatment with PI3-kinase inhibitor LY294002 did not show any sensitization of pancreatic cancer cells to CD95 mediated apoptosis. Our data suggest that ActD sensitizes pancreatic cancer cells to CD95 mediated apoptosis through the abrogation of DNA binding activity of NF-κB, rather than PI3-kinase/Akt system. Over-expression of phosphorylated Akt in pancreatic cancer cells is controlled by effective apoptotic signaling from CD95 receptors, but do not protect tumor cells from CD95 induced apoptosis. Thus, our results indicate that modulation of NF-κB activity rather then Akt may provide a useful tool to sensitize pancreatic cancer cells to CD95 mediated apoptosis.