BackgroundThe Keap1-Nrf2 pathway has been reported to be impaired in several cancers. However, the status of Keap1-Nrf2 system in human colorectal cancer (CRC) has not been elucidated.MethodsWe used colorectal cancer (CRC) cell lines and surgical specimens to investigate the methylation status of the KEAP1 promoter region as well as expression of Nrf2 and its downstream antioxidative stress genes, NQO-1 and AKR1C1.ResultsDNA sequencing analysis indicated that all mutations detected were synonymous, with no amino acid substitutions. We showed by bisulfite genomic sequencing and methylation-specific PCR that eight of 10 CRC cell lines had hypermethylated CpG islands in the KEAP1 promoter region. HT29 cells with a hypermethylated KEAP1 promoter resulted in decreased mRNA and protein expression but unmethylated Colo320DM cells showed higher expression levels. In addition, treatment with the DNA methyltransferase inhibitor 5-Aza-dC combined with the histone deacetylase inhibitor trichostatin A (TSA) increased KEAP1 mRNA expression. These result suggested that methylation of the KEAP1 promoter regulates its mRNA level. Time course analysis with the Nrf2-antioxidant response element (ARE) pathway activator t-BHQ treatment showed a rapid response within 24 h. HT29 cells had higher basal expression levels of NQO-1 and AKR1C1 mRNA than Colo320DM cells. Aberrant promoter methylation of KEAP1 was detected in 53% of tumor tissues and 25% of normal mucosae from 40 surgical CRC specimens, indicating that cancerous tissue showed increased methylation of the KEAP1 promoter region, conferring a protective effect against cytotoxic anticancer drugs.ConclusionHypermethylation of the KEAP1 promoter region suppressed its mRNA expression and increased nuclear Nrf2 and downstream ARE gene expression in CRC cells and tissues.

Mesenchymal stromal cells (MSCs), also called mesenchymal stem cells, migrate and function as stromal cells in tumor tissues. The effects of MSCs on tumor growth are controversial. In this study, we showed that MSCs increase proliferation of tumor cells in vitro and promote tumor growth in vivo. We also further analyzed the mechanisms that underlie these effects. For use in in vitro and in vivo experiments, we established a bone marrow-derived mesenchymal stromal cell line from cells isolated in C57BL/6 mice. Effects of murine MSCs on tumor cell proliferation in vitro were analyzed in a coculture model with B16-LacZ cells. Both coculture with MSCs and treatment with MSC-conditioned media led to enhanced growth of B16-LacZ cells, although the magnitude of growth stimulation in cocultured cells was greater than that of cells treated with conditioned media. Co-injection of B16-LacZ cells and MSCs into syngeneic mice led to increased tumor size compared with injection of B16-LacZ cells alone. Identical experiments using Lewis lung carcinoma (LLC) cells instead of B16-LacZ cells yielded similar results. Consistent with a role for neovascularization in MSC-mediated tumor growth, tumor vessel area was greater in tumors resulting from co-injection of B16-LacZ cells or LLCs with MSCs than in tumors induced by injection of cancer cells alone. Co-injected MSCs directly supported the tumor vasculature by localizing close to vascular walls and by expressing an endothelial marker. Furthermore, secretion of leukemia inhibitory factor, macrophage colony-stimulating factor, macrophage inflammatory protein-2 and vascular endothelial growth factor was increased in cocultures of MSCs and B16-LacZ cells compared with B16-LacZ cells alone. Together, these results indicate that MSCs promote tumor growth both in vitro and in vivo and suggest that tumor promotion in vivo may be attributable in part to enhanced angiogenesis.

The 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, also called statins, are commonly used as lipid-lowering drugs that inhibit cholesterol biosynthesis. An anticancer effect, as a pleiotropic function of certain statins, has been hypothesized. In the present study, we investigated the effect of simvastatin, one of the natural statins, on cell proliferation, cell cycle, invasive activity, and molecular expressions associated with cell-extracellular matrix adhesion, signal transduction, and DNA synthesis in Tu167 and JMAR cells from head and neck squamous cell carcinoma. The addition of simvastatin resulted in a dose-dependent inhibition of cell growth and migration into the extracellular matrix. Considerable morphological changes occurred after treatment with simvastatin, demonstrating loss of cell adhesion and disruption of actin filaments in cytoplasm. The inhibitory effect of simvastatin on cell proliferation seemed to be associated with cell cycle arrest and increased expression of p21, p27, and activated caspase-3. The expression of β β β β1-integrin, a counter adhesion for the extracellular matrix, phosphorylated FAK, and phosphorylated ERK was decreased by treatment with simvastatin. The proapoptotic effect of simvastatin was inhibited by treatment with mevalonate. cDNA microarray assay demonstrated that molecular changes resulting from treatment with simvastatin included the up-regulation of cell cycle regulators and apoptosisinducing factors and the down-regulation of integrin-associated molecules and cell proliferation markers. Of down-regulated genes induced by simvastatin treatment, a significant depletion of thymidylate synthase was confirmed using western blot analysis. These results imply that simvastatin has the potential to be effective for the prevention of the growth and metastasis of cancer cells. S imvastatin, one of the 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, derived from fungal fermentation, is currently used widely as a safe and effective therapeutic agent in the treatment of hypercholesterolemia, contributing to the reduction in morbidity and mortality of atherosclerosis and coronary artery disease.(1) HMG-CoA reductase inhibitors are also commonly referred to as the statins. In addition to their original role in lowering serum cholesterol levels, statins exert antiproliferative and proapoptotic effects in cancer cells by causing cell cycle arrest at the G1-S phase.(2) An anticancer effect with in vitro simvastatin treatment has been reported in several human malignancies, including multiple myeloma, malignant lymphoma, small cell lung carcinoma, and nasopharyngeal undifferentiated carcinoma.(3-6) However, the efficacy and the molecular mechanism of simvastatin on tumor progression has yet to be clarified.Statins target mevalonate, one of the cholesterol precursors, which is catalyzed by HMG-CoA reductase. Overexpression of mevalonate has been reported to be associated with cell survival and proliferation of cancer cells.(7) Another mechanism that plays a ro...

Purpose Alterations in Smad4 signaling and its loss causes genomic instability and head and neck squamous cell carcinoma (HNSCC), suggesting that agents which target both Smad4-dependent and -independent pathways could control HNSCC. Experimental Design Resveratrol efficacy was evaluated against HNSCC cells, FaDu, Cal27, Det562, and Cal27-Smad4 for viability, DNA damage, cell cycle progression, and apoptosis, as well as DNA damage, γH2AX expression and foci formation (γH2AX and Brca1). Resveratrol efficacy was also examined in nude mice for FaDu xenograft growth. Xenografts were analyzed for γH2AX and cleaved caspase-3. Results Resveratrol (5-50 μM) suppressed viability and induced DNA damage in FaDu and Cal27 cells, but not in NHEK and HFF, showing its selectivity towards HNSCC cells; however, Det562 cells were resistant to resveratrol even at 100 μM. Cal27 cells stably transfected with Smad4 showed similar resveratrol effects as parental Cal27 indicating that a lack of resveratrol effect in Det562 cells was independent of Smad4 status in these cells. Furthermore, resveratrol caused S phase arrest and apoptotic death of FaDu and Cal27 cells together with induction of Brca1 and γH2AX foci. Resveratrol (50 mg/kg bw) treatment also inhibited FaDu tumor growth in nude mice, and γH2AX and cleaved caspase-3 were strongly increased in xenografts from resveratrol-treated mice compared to controls. Conclusion Our findings for the first time showed anti-proliferative, DNA damaging and apoptotic effects of resveratrol in HNSCC cells independent of Smad4 status, both in vitro and in vivo, suggesting that more studies are needed to establish its potential usefulness against HNSCC.

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AimsOmeprazole is mainly metabolized by the polymorphic cytochrome P450 (CYP) 2C19. The inhibitory effect of fluvoxamine, an inhibitor of CYP2C19 as well as CYP1A2, on the metabolism of omeprazole was compared between different genotypes for CYP2C19. MethodsEighteen volunteers, of whom six were homozygous extensive metabolizers (EMs), six were heterozygous EMs and six were poor metabolizers (PMs) for CYP2C19, participated in the study. A randomized double-blind, placebo-controlled crossover study was performed. All subjects received two six-day courses of either daily 50 mg fluvoxamine or placebo in a randomized fashion with a single oral 40 mg dose of omeprazole on day six in both cases. Plasma concentrations of omeprazole and its metabolites, 5-hydroxyomeprazole, omeprazole sulphone, and fluvoxamine were monitored up to 8 h after the dosing. ResultsDuring placebo administration, geometric means of peak concentration ( C max ), under the plasma concentration-time curve from 0 to 8 h (AUC(0,8 h)) and elimination half-life ( t 1/2 ) of omeprazole were 900 ng ml -1 , 1481 ng ml -1 h, and 0.6 h in homozygous EMs, 1648 ng ml -1 , 4225 ng ml -1 h, and 1.1 h in heterozygous EMs, and 2991 ng ml -1 , 11537 ng ml -1 h, and 2.8 h in PMs, respectively. Fluvoxamine treatment increased C max of omeprazole by 3.7-fold (95%CI, 2.4, 5.0-fold, P < 0.01) and 2.0-fold (1.4, 2.6-fold, P < 0.01), AUC(0,8 h) by 6.0-fold (3.3, 8.7-fold, P < 0.001) and 2.4-fold (1.7, 3.2-fold, P < 0.01), AUC(0, • ) by 6.2-fold (3.0, 9.3-fold, P < 0.01) and 2.5-fold (1.6, 3.4-fold, P < 0.001) and prolonged t 1 / 2 by 2.6-fold (1.9, 3.4-fold, P < 0.001) and 1.4-fold (1.02, 1.7-fold, P < 0.05), respectively. However, no pharmacokinetic parameters were changed in PMs. The AUC(0,8 h) ratios of 5-hydroxyomeprazole to omeprazole were decreased with fluvoxamine in homozygous EMs ( P < 0.05) and heterozygous EMs ( P < 0.01). ConclusionsEven a low dose of fluvoxamine increased omeprazole exposure in EMs, but did not increase omeprazole exposure in PMs after a single oral dose of omeprazole. These findings confirm a potent inhibitory effect of fluvoxamine on CYP2C19 activity. The bioavailability of omeprazole might, to some extent, be increased through inhibition of P-glycoprotein during fluvoxamine treatment.N. Yasui-Furukori et al. 48857 :4 Br J Clin Pharmacol

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The elderly EMs showed wide variance in the in vivo CYP2C19 activity and were phenotypically closer to the elderly PMs than the young EMs were to the young PMs. Some of the elderly homozygous EMs, as well as heterozygous EMs, have a metabolic activity similar to PMs, and the CYP2C19 genotype may therefore not be as useful as phenotyping in the elderly.