F. A. Fitzpatrick

Evaluation of a 7-Day Continuous Intravenous Infusion of Decitabine: Inhibition of Promoter-Specific and Global Genomic DNA Methylation

PURPOSEnThe nucleoside analog 5-aza-2-deoxycytidine (5-aza-CdR, decitabine) is a potent inhibitor of DNA methylation in vitro. Cellular treatment with this agent induces the re-expression of methylation-silenced genes. It remains unclear to what extent this compound inhibits DNA methylation in vivo. A clinical study was designed to examine the molecular effects and toxicity of a continuous 1-week intravenous infusion of decitabine in solid tumor patients.nnnMETHODSnTen patients with refractory solid tumors were included in this study. Decitabine was administered at 2 mg/m(2)/d [DOSAGE ERROR CORRECTED] via continuous infusion for 168 hours. Quantitative polymerase chain reaction and high performance liquid chromatography were utilized to measure promoter-specific and global DNA methylation in peripheral-blood cells before and after treatment.nnnRESULTSnTransient grade III/IV neutropenia (two patients) and grade II thrombocytopenia (one patient) was observed at the lowest planned dose step (2 mg/m2/d for 7 days). Nonhematologic toxicities were not observed. Quantitative polymerase chain reaction demonstrated significant MAGE-1 promoter hypomethylation by 14 days after the start of treatment in all 13 treatment cycles examined. Significant genomic DNA hypomethylation was also seen by day 14 in 11 of 13 treatment cycles analyzed. Genomic DNA methylation reverted to baseline levels by 28 to 35 days after the start of treatment, demonstrating that inhibition of DNA methylation by decitabine is transient.nnnCONCLUSIONnA 168-hour continuous infusion of decitabine is well tolerated and results in the inhibition of promoter-specific and genomic DNA methylation in vivo. This treatment schedule is suitable for evaluation of decitabine in combination with agents whose activity may be enhanced by the reversal of DNA methylation-mediated gene silencing.

Reactive Lipid Species from Cyclooxygenase-2 Inactivate Tumor Suppressor LKB1/STK11 CYCLOPENTENONE PROSTAGLANDINS AND 4-HYDROXY-2-NONENAL COVALENTLY MODIFY AND INHIBIT THE AMP-KINASE KINASE THAT MODULATES CELLULAR ENERGY HOMEOSTASIS AND PROTEIN TRANSLATION

LKB1, a unique serine/threonine kinase tumor suppressor, modulates anabolic and catabolic homeostasis, cell proliferation, and organ polarity. Chemically reactive lipids, e.g. cyclopentenone prostaglandins, formed a covalent adduct with LKB1 in MCF-7 and RKO cells. Site-directed mutagenesis implicated Cys210 in the LKB1 activation loop as the residue modified. Notably, ERK, JNK, and AKT serine/threonine kinases with leucine or methionine, instead of cysteine, in their activation loop did not form a covalent lipid adduct. 4-Hydroxy-2-nonenal, 4-oxo-2-nonenal, and cyclopentenone prostaglandin A and J, which all contain α,β-unsaturated carbonyls, inhibited the AMP-kinase kinase activity of cellular LKB1. In turn, this attenuated signals throughout the LKB1 → AMP kinase pathway and disrupted its restraint of ribosomal S6 kinases. The electrophilic β-carbon in these lipids appears to be critical for inhibition because unreactive lipids, e.g. PGB1, PGE2, PGF2α, and TxB2, did not inhibit LKB1 activity (p > 0.05). Ectopic expression of cyclooxygenase-2 and endogenous biosynthesis of eicosanoids also inhibited LKB1 activity in MCF-7 cells. Our results suggested a molecular mechanism whereby chronic inflammation or oxidative stress may confer risk for hypertrophic or neoplastic diseases. Moreover, chemical inactivation of LKB1 may interfere with its physiological antagonism of signals from growth factors, insulin, and oncogenes.

Alkylation of the Tumor Suppressor PTEN Activates Akt and β-Catenin Signaling: A Mechanism Linking Inflammation and Oxidative Stress with Cancer

PTEN, a phosphoinositide-3-phosphatase, serves dual roles as a tumor suppressor and regulator of cellular anabolic/catabolic metabolism. Adaptation of a redox-sensitive cysteinyl thiol in PTEN for signal transduction by hydrogen peroxide may have superimposed a vulnerability to other mediators of oxidative stress and inflammation, especially reactive carbonyl species, which are commonly occurring by-products of arachidonic acid peroxidation. Using MCF7 and HEK-293 cells, we report that several reactive aldehydes and ketones, e.g. electrophilic α,β-enals (acrolein, 4-hydroxy-2-nonenal) and α,β-enones (prostaglandin A2, Δ12-prostaglandin J2 and 15-deoxy-Δ-12,14-prostaglandin J2) covalently modify and inactivate cellular PTEN, with ensuing activation of PKB/Akt kinase; phosphorylation of Akt substrates; increased cell proliferation; and increased nuclear β-catenin signaling. Alkylation of PTEN by α,β-enals/enones and interference with its restraint of cellular PKB/Akt signaling may accentuate hyperplastic and neoplastic disorders associated with chronic inflammation, oxidative stress, or aging.

Colon tumor mutations and epigenetic changes associated with genetic polymorphism: insight into disease pathways.

Variation in genes associated with serum levels of proteins may be useful for examining specific disease pathways. Using data from a large study of colon cancer, we examine genetic variants in insulin, inflammation, estrogen, metabolizing enzymes, and energy homeostasis genes to explore associations with microsatellite instability (MSI), CpG Island methylator phenotype (CIMP), mutations of p53 in exons 5 through 8, and mutations in codons 12 and 13 of Ki-ras. Insulin-related genes were associated with CIMP-positive and MSI tumors, with the strongest associations among aspirin users. The Fok1 vitamin D receptor (VDR) polymorphism was associated with CIMP-positive/Ki-ras-mutated tumors; the Poly A and CDX2 VDR polymorphisms were associated only with Ki-ras-mutated tumors. NAT2 was associated with CIMP-positive/Ki-ras-mutated tumors but not with MSI tumors. The TCF7L2 rs7903146 polymorphism was associated with p53 mutated tumors. Most associations varied by recent aspirin/NSAID use: IL6 rs1800796 and rs1800795 polymorphisms were associated inversely with tumor mutations in the presence of aspirin/NSAIDs; POMC significantly reduced risk of Ki-ras-mutated tumors when aspirin/NSAIDs were not used; the TCF7L2 rs7903146 was associated with reduced risk of Ki-ras-mutated tumors in the presence of aspirin and increased risk in the absence of aspirin. These data, although exploratory, identify specific tumor subsets that may be associated with specific exposures/polymorphism combinations. The important modifying effects of aspirin/NSAIDs on associations with genetic polymorphisms reinforce the underlying role of inflammation in the etiology of colon cancer.

Fiberoptic Resonance Raman Spectroscopy to Measure Carotenoid Oxidative Breakdown in Live Tissues

Based on compelling epidemiologic and corroboratory in vitro studies, carotenoids are thought to have great potential as dietary prevention against cancer. Yet, carotenoid-based chemopreventive trials have found very contradictory results. Definitive conclusions from these trials are hampered by an inability to accurately and safely measure carotenoids in specific tissues at risk of cancer development. Raman spectroscopy has been proposed as an optical technology with which to analyze various molecules in live tissues. One major obstacle that impedes the clinical use of this powerful technology is the lack of a fiberoptic Raman probe suitable for endoscopic tissue evaluation. A single-fiber resonance Raman Spectroscope capable of noninvasive “optical biopsies” to measure carotenoid concentrations in live tissues has been developed. The accuracy of this Raman instrument was confirmed by comparison with more standard methods of spectrophotometry and high-pressure liquid chromatography using solubilized β-carotene (BC) and BC-loaded cells before use in a small patient cohort. This Raman instrument detected intact BC as well as BC oxidative breakdown as a decrement of its Raman signal in cells. Use of the Raman instrument in our small cohort study showed its feasibility for measuring human tissues and raised some potentially intriguing possibilities about BC tissue pharmacokinetics and oxidative biology. Based on these results, our newly developed single fiberoptic resonance Raman instrument may provide a very useful method of measuring carotenoids and their oxidative breakdown within live tissue during future carotenoid chemopreventive trials. This proof-of-concept study provides the foundation to justify future validation of our Raman prototype. Cancer Prev Res; 3(4); 529–38. ©2010 AACR.

Evaluating 2-chlorodeoxycytidine for its novel mechanism as a DNA methylation inhibitor

3125 Background: Many tumor suppressor genes are silenced by epigenetic alterations of CpG islands in the promoter regions. A clinical example of this is seen in colorectal cancer. Although the adenomatous polyposis coli gene is frequently somatically mutated in colon carcinoma, methylation of the adenomatous polyposis coli gene is seen in early colon adenomas. Thus, epigenetic silencing of tumor suppressor genes may play a role in the progression of colon polyps to carcinomas. Further, reactivation of critical tumor suppressor genes in carcinomas may decrease chemotherapy resistance. 5-aza-deoxycytidine is a DNA methyltransferase inhibitor. This drug has problems of DNA mutagenesis. We are evaluating 2-chlorodeoxycytidine for its inhibitory properties of s-adenosylhomocysteine hydrolase. Since s-adenosylmethionine is the only source of methyl donor for all mammalian methyltransferases, we can indirectly inhibit DNA methylation by depleting s-adenosylmethionine.nnnMETHODSnA growth inhibition experiment is done comparing HT-29 cells treated with 2-chlorodeoxycytidine and cells treated with vehicle. This time point is then used for future experiments. Genomic DNA is isolated and digested with a methylation sensitive restriction enzyme. Methylation sensitive endonuclease digested DNA will be amplified by real time polymerase chain reaction to assess for promoter methylation. We will next evaluate global methylation level of the genome using HPLC. We have also performed microarray on vehicle and treated samples. The effect of 2-chlorodeoxycytidine compared to 5-aza-deoxycytidine on gene expression is evaluated.nnnRESULTSn300 nM 2-chlorodeoxycytidine inhibits cell proliferation within 48 hours. Many known genes silenced by methylation, including genes in the interferon pathway, were upregulated by at least 2 fold after 48 hours of 2-chlorodeoxycytidine.nnnCONCLUSIONn2-chlorodeoxycytidine, a known adenosine deaminase inhibitor, also may indirectly inhibit DNA methylation, reactivating critical tumor suppressor genes in various malignancies. [Table: see text].