Flavopiridol increases sensitization to gemcitabine in human gastrointestinal cancer cell lines and correlates with down-regulation of ribonucleotide reductase M2 subunit.
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.
Jung
C P
CP
Laboratory of New Drug Development and Gastrointestinal Oncology Research, Division of Solid Tumor Oncology, Department of Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA.
Motwani
M V
MV
Schwartz
G K
GK
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000187:drug effects / Q000378:metabolism; D019938:Cyclin D1 / Q000187:drug effects / Q000378:metabolism; D019927:Cyclin E / Q000187:drug effects / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000378:metabolism; D003574:Cytochrome c Group / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004357:Drug Synergism; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005419:Flavonoids / Q000494:pharmacology*; D005770:Gastrointestinal Neoplasms / Q000188:drug therapy / Q000201:enzymology / Q000473:pathology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009097:Multienzyme Complexes / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010880:Piperidines / Q000494:pharmacology*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D021122:Protein Subunits; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000187:drug effects / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism; D014157:Transcription Factors / Q000187:drug effects / Q000378:metabolism; D014316:Tritium; D014407:Tumor Cells, Cultured; D014410:Tumor Stem Cell Assay; D000093542:Gemcitabine
D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D019927:Cyclin E; D003574:Cytochrome c Group; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D004791:Enzyme Inhibitors; D005419:Flavonoids; D009097:Multienzyme Complexes; D010880:Piperidines; D021122:Protein Subunits; D012333:RNA, Messenger; D016160:Retinoblastoma Protein; D014157:Transcription Factors; D003841:Deoxycytidine; D014316:Tritium; D019938:Cyclin D1; C077990:alvocidib; D012264:Ribonucleotide Reductases; D011065:Poly(ADP-ribose) Polymerases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D013936:Thymidine; D000093542:Gemcitabine