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
A higher incidence of coexisting lymphoproliferative disease and Kaposi's sarcoma (KS) has been reported. However, the association of Hodgkin's disease (HD) and KS is a rare condition, and only 41 cases have been found upon a literature review of the last 33 years. In this study the case of a 70 year-old male patient with consecutive appearance of Hodgkin's disease (HD) and Kaposi's sarcoma (KS) without evidence of HIV infection or other immunodeficiency is presented. The data suggest, that the association of KS with HD may be due to common pathogenic mechanisms, rather than a direct causal relationship, except the requirement of an impaired immune system for the development of KS. Recently development of HD and KS has been associated with EBV and HHV-8, respectively. Neither immunosuppression, nor EBV or HHV-8 infection alone result in development of HD or KS. There is no clear time relationship between the development of KS and HD when the two occurred together in the same patient. That finding, coupled with the rarity of the association, suggest, that the association is more likely coincidental.
