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.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Antineoplastic Agents / pharmacology*
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Apoptosis / drug effects
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Blotting, Western
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Cell Cycle Proteins / drug effects
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Cell Cycle Proteins / metabolism
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Cyclin D1 / drug effects
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Cyclin D1 / metabolism
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Cyclin E / drug effects
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Cyclin E / metabolism
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Cysteine Endopeptidases / drug effects
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Cysteine Endopeptidases / metabolism
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Cytochrome c Group / drug effects
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Cytochrome c Group / metabolism
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DNA-Binding Proteins*
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Deoxycytidine / analogs & derivatives
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Deoxycytidine / pharmacology*
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Dose-Response Relationship, Drug
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Down-Regulation
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Drug Synergism
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E2F Transcription Factors
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E2F1 Transcription Factor
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Enzyme Inhibitors / pharmacology*
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Flavonoids / pharmacology*
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Gastrointestinal Neoplasms / drug therapy
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Gastrointestinal Neoplasms / enzymology
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Gastrointestinal Neoplasms / pathology*
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Gemcitabine
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Gene Expression Regulation, Enzymologic / drug effects
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Humans
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Mitochondria / drug effects
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Mitochondria / metabolism
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Multienzyme Complexes / drug effects
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Multienzyme Complexes / metabolism
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Phosphorylation / drug effects
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Piperidines / pharmacology*
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Poly(ADP-ribose) Polymerases / metabolism
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Proteasome Endopeptidase Complex
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Protein Subunits
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RNA, Messenger / drug effects
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RNA, Messenger / genetics
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RNA, Messenger / metabolism
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Retinoblastoma Protein / drug effects
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Retinoblastoma Protein / metabolism
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Ribonucleotide Reductases / antagonists & inhibitors*
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Ribonucleotide Reductases / genetics
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Ribonucleotide Reductases / metabolism
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Thymidine / metabolism
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Transcription Factors / drug effects
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Transcription Factors / metabolism
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Tritium
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Tumor Cells, Cultured
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Tumor Stem Cell Assay
Substances
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Antineoplastic Agents
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Cell Cycle Proteins
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Cyclin E
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Cytochrome c Group
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DNA-Binding Proteins
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E2F Transcription Factors
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E2F1 Transcription Factor
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E2F1 protein, human
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Enzyme Inhibitors
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Flavonoids
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Multienzyme Complexes
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Piperidines
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Protein Subunits
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RNA, Messenger
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Retinoblastoma Protein
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Transcription Factors
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Deoxycytidine
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Tritium
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Cyclin D1
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alvocidib
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Ribonucleotide Reductases
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Poly(ADP-ribose) Polymerases
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Cysteine Endopeptidases
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Proteasome Endopeptidase Complex
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Thymidine
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Gemcitabine