Schedule-dependent molecular effects of the proteasome inhibitor bortezomib and gemcitabine in pancreatic cancer

Background. 26S proteasome inhibitors are a novel class of compounds entering clinical trials as a method to increase tumor sensitivity to standard chemotherapy. We determined the effect of alternate sequencing regimens of a proteasome inhibitor and gemcitabine on molecular and cellular responses in pancreatic cancer cells. Materials and methods. MIA-PaCa-2 human pancreatic cancer cells were treated with the proteasome inhibitor bortezomib either before, simultaneously or following exposure to gemcitabine. Expression of the cell cycle proteins p21^WAF1/CIP1 and p27^KIP1, and the anti-apoptotic protein BCL-2 were determined by Western blotting. Cell cycle changes and immediate or delayed induction of apoptosis were quantitated. Results. Gemcitabine followed by bortezomib induced the greatest induction of apoptosis and long-term inhibition of cell growth. Bortezomib treatment led to accumulation of p21^WAF1/CIP1 and p27^KIP1 and decreased BCL-2; gemcitabine decreased p27^KIP1, induced BCL-2 and had no effect on p21^WAF1/CIP1. When these agents were given in combination or sequence, intermediate changes in these proteins were observed, and the alterations did not correlate with immediate or delayed induction of apoptosis. Conclusions. Inhibition of the 26S proteasome following chemotherapy appears to be the most effective regimen, though changes in BCL-2, p21^WAF1/CIP1, p27^KIP1 do not necessarily correlate with the cellular effects when various sequences are examined. Therefore, these proteins may not be the most appropriate surrogate markers of efficacy of this regimen. These data provide the background for the development of the optimal regimen to be used in clinical trials.