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.
Subject(s)
Antineoplastic Agents/pharmacology , DNA-Binding Proteins , Deoxycytidine/pharmacology , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Gastrointestinal Neoplasms/pathology , Piperidines/pharmacology , Ribonucleotide Reductases/antagonists & inhibitors , Apoptosis/drug effects , Blotting, Western , Cell Cycle Proteins/drug effects , Cell Cycle Proteins/metabolism , 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/metabolism , Deoxycytidine/analogs & derivatives , Dose-Response Relationship, Drug , Down-Regulation , Drug Synergism , E2F Transcription Factors , E2F1 Transcription Factor , Gastrointestinal Neoplasms/drug therapy , Gastrointestinal Neoplasms/enzymology , Gene Expression Regulation, Enzymologic/drug effects , Humans , Mitochondria/drug effects , Mitochondria/metabolism , Multienzyme Complexes/drug effects , Multienzyme Complexes/metabolism , Phosphorylation/drug effects , Poly(ADP-ribose) Polymerases/metabolism , Proteasome Endopeptidase Complex , Protein Subunits , RNA, Messenger/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Retinoblastoma Protein/drug effects , Retinoblastoma Protein/metabolism , Ribonucleotide Reductases/genetics , Ribonucleotide Reductases/metabolism , Thymidine/metabolism , Transcription Factors/drug effects , Transcription Factors/metabolism , Tritium , Tumor Cells, Cultured , Tumor Stem Cell Assay , GemcitabineABSTRACT
Because of serious side effects, the indications for intralesional steroid injection of adnexal hemangiomas are unclear. Of 23 children with such lesions who were examined over a period of 9 years, 9 had no evidence of amblyopia and needed no intervention. Five required steroids intralesionally and/or systemically because of threatened occlusion of the pupillary axis. The remaining 9 were considered at risk of anisometropic amblyopia because of induced astigmatism: 5 received injections and 4 were treated with glasses and/or patching alone. The visual, refractive, and cosmetic results of the injected and conservatively managed anisometropes were similar. We recommend that steroid injection be reserved for patients with threatened occlusion of the visual axis and for those with severe astigmatism or amblyopia refractory to conservative management.
