The thymidylate synthase inhibitor 5-fluorouracil (5-FU) is used widely for chemotherapy of colorectal carcinoma. Recent studies showed that 5-FU affects polyamine metabolism in colon carcinoma cells. We therefore examined whether combinations of 5-FU with drugs that specifically target polyamine metabolism, i.e. N 1 ,N 11 -diethylnorspermine (DENSPM) or ␣-difluoromethylornithine (DFMO), have synergistic effects in killing HCT116 colon carcinoma cells with wild-type or absent p53. Our results showed that simultaneous 5-FU and DENSPM, a spermine analogue, synergistically increased transcript levels of the polyamine catabolism enzyme spermidine/spermine N 1 -acetyltransferase, depleted spermine and spermidine, increased acetylated spermidine, and produced synergistic tumor cell apoptosis in both p53 wild-type and p53-null variants. By contrast, simultaneous combination of 5-FU with DFMO, an inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase, depleted putrescine but did not produce synergistic cell killing. Some pre-treatment and post-treatment regimens of DENSPM and DFMO were antagonistic to 5-FU depending on cellular p53 status. Protein and transcriptome expression analysis showed that combined 5-FU and DENSPM treatment activated caspase 9, but not caspase 3, and significantly suppressed NADH dehydrogenases and cytochrome c oxidases, consistent with the observed increase in hydrogen peroxide, loss of mitochondrial membrane potential, and release of cytochrome c. Our findings demonstrate the importance of the polyamine pathway in 5-FU effects and suggest that the combination of 5-FU with DENSPM has potential for development as therapy for colorectal carcinoma.The fluoropyrimidine 5-fluorouracil (5-FU) 1 has been widely used a chemotherapy agent in the treatment of many types of solid tumors (1-3). To improve the efficacy of 5-FU, several other drugs such as leucovorin, irinotecan, or oxaliplatin have been used recently in combination with 5-FU in preclinical and clinical trials that have produced promising results (4 -6).5-FU achieves its therapeutic effect by several known mechanisms. It binds to and inhibits thymidylate synthase, which converts 2-deoxyuridine to thymidylate, thus interfering with DNA synthesis. 5-FU incorporates into RNA, leading to disruption of RNA processing (7-9). Our recent functional genomic studies have rediscovered that 5-FU also affects polyamine pathways to reduce the levels of polyamines in treated colon cancer cells (10), an observation that was originally made by Russell et al. in 1974 in a liver cancer xenograft mouse model (11). Thus, it appears that 5-FU targets two major cellular processes, i.e. polyamine metabolism as well as processing and synthesis of DNA and RNA. These two processes may have intimate inter-relationships because polyamines bind DNA and RNA and affect chromosomal structure, RNA stability, and translation (12).Spermidine, spermine, and their diamine precursor putrescine are natural polyamines in living organisms and play critical roles in pr...

BACKGROUND.Imatinib has demonstrated marked clinical efficacy against gastrointestinal stromal tumor (GIST). Microarray technology, real‐time polymerase chain reaction (PCR) validation, and fluorodeoxyglucose‐positron emission tomography (FDG‐PET) imaging were used to study the early molecular effects of imatinib antitumor activity in GIST.METHODS.After exposure of sensitive and resistant sarcoma cell lines to imatinib for 24 to 48 hours, the changes in gene expression were evaluated using a 1146 unique pathway array with Western blot validation. Real‐time PCR was used to confirm changes in gene expression in human GIST samples (preimatinib biopsy and postimatinib surgical specimen after 3–7 days of therapy). FDG‐PET was performed to correlate radiographic findings with the effects of imatinib on gene expression in GIST.RESULTS.In all, 55 genes demonstrated a ≥ 2‐fold change after imatinib treatment of the GIST882 cells. Among these genes there was up‐regulation of insulin‐like growth factor binding protein‐3 (IGFBP‐3), a protein that modulates proliferation and apoptosis. Western blot analysis confirmed the increase of IGFBP‐3 only in imatinib‐sensitive GIST882 cells. Up to a 7‐fold induction (49% mean increase; P = .08) of IGFBP‐3 mRNA was found in tumor samples from patients with low residual FDG uptake, whereas there was an up to 12‐fold reduction (−102% mean decrease; P = .03) in IGFBP‐3 in those patients with high residual FDG uptake after imatinib therapy.CONCLUSIONS.In the current study, imatinib appears to regulate numerous genes and specifically induces IGFBP‐3 in GIST cells and tumor samples. IGFBP‐3 levels also were found to be inversely correlated with residual FDG uptake in GIST patients early in imatinib therapy. These initial observations suggest that IGFBP‐3 is an important early marker of antitumor activity of imatinib in GIST. Cancer 2006. © 2006 American Cancer Society.

BackgroundImatinib mesylate has significantly improved survival and quality of life of patients with gastrointestinal stromal tumors (GISTs). However, the molecular mechanism through which imatinib exerts its anti-tumor effects is not clear. Previously, we found up-regulation of insulin-like growth factor binding protein-3 (IGFBP3) expression in imatinib-responsive GIST cells and tumor samples. Because IGFBP3 regulates cell proliferation and survival and mediates the anti-tumor effects of a number of anti-cancer agents through both IGF-dependent and IGF-independent mechanisms, we hypothesized that IGFBP3 mediates GIST cell response to imatinib. To test this hypothesis, we manipulated IGFBP3 levels in two imatinib-responsive GIST cell lines and observed cell viability after drug treatment.ResultsIn the GIST882 cell line, imatinib treatment induced endogenous IGFBP3 expression, and IGFBP3 down-modulation by neutralization or RNA interference resulted in partial resistance to imatinib. In contrast, IGFBP3 overexpression in GIST-T1, which had no detectable endogenous IGFBP3 expression after imatinib, had no effect on imatinib-induced loss of viability. Furthermore, both the loss of IGFBP3 in GIST882 cells and the overexpression of IGFBP3 in GIST-T1 cells was cytotoxic, demonstrating that IGFBP3 has opposing effects on GIST cell viability.ConclusionThis data demonstrates that IGFBP3 has dual, opposing roles in modulating GIST cell viability and response to imatinib in vitro. These preliminary findings suggest that there may be some clinical benefits to IGFBP3 therapy in GIST patients, but further studies are needed to better characterize the functions of IGFBP3 in GIST.

The clinical application of tyrosine kinase inhibitors for cancer treatment represents a therapeutic breakthrough. The rationale for development of these compounds rests on the observation that tyrosine kinases are central components of the cellular signaling apparatus and are deregulated in many human malignancies. Successful tyrosine kinase inhibitors target aberrant pathways that are critical for tumor cell viability. Herein, we will review the current state-of-the-art using imatinib mesylate (Gleevec TM , Glivec ® , CGP57148, formerly STI571; Novartis Pharmaceuticals) as it is used in the treatment of gastrointestinal stromal tumors (GISTs).GISTs, the most common mesenchymal tumors of the gastrointestinal tract, originate from the neoplastic transformation of the intestinal pacemaker cell, the interstitial cell of Cajal. GISTs commonly have mutations in the kit gene, resulting in a gain-of-function mutation and ligand-independent constitutive activation of the Kit receptor tyrosine kinase. The use of imatinib has greatly increased the therapeutic efficacy for this otherwise chemotherapy-resistant tumor. Resistance to the drug is the major cause of treatment failure, emphasizing the need for researchers to understand Kit signaling pathways so as to identify new therapeutic targets. This article will review the development, pharmacology, and clinical trials of imatinib in the setting of GIST. The potential role of imatinib in the postoperative and preoperative setting will be discussed.