Flavopiridol Increases Sensitization to Gemcitabine in Human Gastrointestinal Cancer Cell Lines and Correlates with Down-Regulation of Ribonucleotide Reductase M2 Subunit

• Published in: Clinical cancer research 2001-08, Vol.7 (8), p.2527-2536
• Main Authors: JUNG, Christoph P, MOTWANI, Monica V, SCHWARTZ, Gary K
• Format: Article
• Language: English
• Subjects: Antineoplastic agents; Antineoplastic Agents - pharmacology; Apoptosis - drug effects; Biological and medical sciences; Blotting, Western; Cell Cycle Proteins - drug effects; Cell Cycle Proteins - metabolism; Chemotherapy; Cyclin D1 - drug effects; Cyclin D1 - metabolism; Cyclin E - drug effects; Cyclin E - metabolism; Cysteine Endopeptidases - drug effects; Cysteine Endopeptidases - metabolism; Cytochrome c Group - drug effects; Cytochrome c Group - secretion; Deoxycytidine - analogs & derivatives; Deoxycytidine - pharmacology; DNA-Binding Proteins; Dose-Response Relationship, Drug; Down-Regulation; Drug Synergism; E2F Transcription Factors; E2F1 Transcription Factor; Enzyme Inhibitors - pharmacology; Flavonoids - pharmacology; Gastrointestinal Neoplasms - drug therapy; Gastrointestinal Neoplasms - enzymology; Gastrointestinal Neoplasms - pathology; Gene Expression Regulation, Enzymologic - drug effects; Humans; Medical sciences; Mitochondria - drug effects; Mitochondria - metabolism; Multienzyme Complexes - drug effects; Multienzyme Complexes - metabolism; Pharmacology. Drug treatments; Phosphorylation - drug effects; Piperidines - pharmacology; Poly(ADP-ribose) Polymerases - metabolism; Proteasome Endopeptidase Complex; Protein Subunits; Retinoblastoma Protein - drug effects; Retinoblastoma Protein - metabolism; Ribonucleotide Reductases - antagonists & inhibitors; Ribonucleotide Reductases - genetics; Ribonucleotide Reductases - metabolism; RNA, Messenger - drug effects; RNA, Messenger - genetics; RNA, Messenger - metabolism; Thymidine - metabolism; Transcription Factors - drug effects; Transcription Factors - metabolism; Tritium; Tumor Cells, Cultured; Tumor Stem Cell Assay
• ISSN: 1078-0432; 1557-3265

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.