Chemoprevention, especially through the use of naturally occurring phytochemicals capable of impeding the process of one or more steps of carcinogenesis process, is a promising approach for cancer management. Despite promising results in preclinical settings, its applicability to humans has met with limited success largely due to inefficient systemic delivery and bioavailability of promising chemopreventive agents. Here, we introduce the concept of nanochemoprevention, which uses nanotechnology for enhancing the outcome of chemoprevention. We encapsulated green tea polyphenol epigallocatechin-3-gallate (EGCG) in polylactic acid-polyethylene glycol nanoparticles and observed that encapsulated EGCG retains its biological effectiveness with over 10-fold dose advantage for exerting its proapoptotic and angiogenesis inhibitory effects, critically important determinants of chemopreventive effects of EGCG in both in vitro and in vivo systems. Thus, this study could serve as a basis for the use of nanoparticle-mediated delivery to enhance bioavailability and limit any unwanted toxicity of chemopreventive agents, such as EGCG.

Helicobacter pylori infects the human stomach by escaping the host immune response. One mechanism of bacterial survival and mucosal damage is induction of macrophage apoptosis, which we have reported to be dependent on polyamine synthesis by arginase and ornithine decarboxylase. During metabolic back-conversion, polyamines are oxidized and release H(2)O(2), which can cause apoptosis by mitochondrial membrane depolarization. We hypothesized that this mechanism is induced by H. pylori in macrophages. Polyamine oxidation can occur by acetylation of spermine or spermidine by spermidine/spermine N(1)-acetyltransferase prior to back-conversion by acetylpolyamine oxidase, but recently direct conversion of spermine to spermidine by the human polyamine oxidase h1, also called spermine oxidase, has been demonstrated. H. pylori induced expression and activity of the mouse homologue of this enzyme (polyamine oxidase 1 (PAO1)) by 6 h in parallel with ornithine decarboxylase, consistent with the onset of apoptosis, while spermidine/spermine N(1)-acetyltransferase activity was delayed until 18 h when late stage apoptosis had already peaked. Inhibition of PAO1 by MDL 72527 or by PAO1 small interfering RNA significantly attenuated H. pylori-induced apoptosis. Inhibition of PAO1 also significantly reduced H(2)O(2) generation, mitochondrial membrane depolarization, cytochrome c release, and caspase-3 activation. Overexpression of PAO1 by transient transfection induced macrophage apoptosis. The importance of H(2)O(2) was confirmed by inhibition of apoptosis with catalase. These studies demonstrate a new mechanism for pathogen-induced oxidative stress in macrophages in which activation of PAO1 leads to H(2)O(2) release and apoptosis by a mitochondrial-dependent cell death pathway, contributing to deficiencies in host defense in diseases such as H. pylori infection.

Macrophage activation is a critical step in host responses during bacterial infections. Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine metabolism, has been well studied in epithelial cells and is known to have essential roles in many different cellular functions. However, its role in regulating macrophage function during bacterial infections is not well characterized. We demonstrate that macrophage-derived ODC is a critical regulator of M1 macrophage activation during both Helicobacter pylori and Citrobacter rodentium infection. Myeloid-specific Odc deletion significantly increased gastric and colonic inflammation, respectively, and enhanced M1 activation. Add-back of putrescine, the product of ODC, reversed the increased macrophage activation, indicating that ODC and putrescine are regulators of macrophage function. Odc-deficient macrophages had increased histone 3, lysine 4 (H3K4) monomethylation, and H3K9 acetylation, accompanied by decreased H3K9 di/trimethylation both in vivo and ex vivo in primary macrophages. These alterations in chromatin structure directly resulted in up-regulated gene transcription, especially M1 gene expression. Thus, ODC in macrophages tempers antimicrobial, M1 macrophage responses during bacterial infections through histone modifications and altered euchromatin formation, leading to the persistence and pathogenesis of these organisms. macrophage polarization | polyamines | histone modifications | ornithine decarboxylase | Helicobacter pylori

BACKGROUND & AIMS Helicobacter pylori-induced gastric carcinogenesis has been linked to the microbial oncoprotein CagA. Spermine oxidase (SMO) metabolizes the polyamine spermine into spermidine and generates H2O2 that causes apoptosis and DNA damage. We determined if pathogenic effects of CagA are attributable to SMO. METHODS Levels of SMO, apoptosis, and DNA damage (8-oxoguanosine) were measured in gastric epithelial cell lines infected with cagA+ or cagA− H. pylori strains, or transfected with a CagA expression plasmid, in the absence or presence of SMO small interfering RNA, or an SMO inhibitor. The role of CagA in induction of SMO and DNA damage was assessed in H. pylori-infected gastritis tissues from humans, gerbils, and both wild-type and hypergastrinemic INS-GAS mice, using immunohistochemistry and flow cytometry. RESULTS cagA+ strains or ectopic expression of CagA, but not cagA− strains, led to increased levels of SMO, apoptosis, and DNA damage in gastric epithelial cells, and knockdown or inhibition of SMO blocked apoptosis and DNA damage. There was increased SMO expression, apoptosis, and DNA damage in gastric tissues from humans infected with cagA+, but not cagA− strains. In gerbils and mice, DNA damage was CagA-dependent and present in cells that expressed SMO. Gastric epithelial cells with DNA damage that were negative for markers of apoptosis accounted for 42–69% of cells in gerbils and INS-GAS mice with dysplasia and carcinoma. CONCLUSIONS By inducing SMO, H. pylori CagA generates cells with oxidative DNA damage, and a subpopulation of these cells are resistant to apoptosis and thus at high risk for malignant transformation.