Flavopiridol increases sensitization to gemcitabine in human gastrointestinal cancer cell lines and correlates with down-regulation of ribonucleotide reductase M2 subunit

Abstract

As a single agent, gemcitabine (2',2'-difluorodeoxycytidine) has shown minimal activity against gastrointestinal malignancies with only a modest improvement in survival in patients with pancreatic cancer. Recently, gemcitabine resistance has been associated with the up-regulation of mRNA and protein levels of the ribonucleotide reductase M2 subunit (RR-M2), a rate-limiting enzyme in DNA synthesis that is cell cycle regulated. In this study we show that flavopiridol, a cyclin-dependent kinase inhibitor, enhances the induction of apoptosis by gemcitabine in human pancreatic, gastric, and colon cancer cell lines. As determined by quantitative fluorescence microscopy, flavopiridol enhanced gemcitabine-induced apoptosis 10-15-fold in all of the cell lines tested in a sequence-dependent manner. This was confirmed by poly(ADP-ribose) polymerase cleavage and mitochondrial cytochrome c release. Colony formation assays confirmed the apoptotic rates, showing complete suppression of colony formation only after exposure to sequential treatment of G(24)-->F(24). This is associated with suppression of the RR-M2 protein. This appears to be related to down-regulation of E2F-1, a transcription factor that regulates RR-M2 transcription and hypophosphorylation of pRb. The proteasome inhibitor PS-341 could restore the protein levels of E2F-1 in G(24)-->F(24) treatment indicating that E2F-1 down-regulation is attributable to its increased degradation via ubiquitin-proteasome pathway. This also resulted in restoration of RR-M2 mRNA and protein. These results indicate that flavopiridol in gemcitabine-treated cells inhibits parts of the machinery necessary for the transcription induction of RR-M2. Thus, combining flavopiridol with gemcitabine may provide an important and novel new means of enhancing the efficacy of gemcitabine in the treatment of gastrointestinal cancers.