Homeobox D10 gene, a candidate tumor suppressor, is downregulated through promoter hypermethylation and associated with gastric carcinogenesis.

Homeobox D10 (HoxD10 ) gene plays a critical role in cell differentiation and morphogenesis during development. However, the function of HoxD10 in tumor progression remains largely unknown. We demonstrate that the expression of HoxD10 is commonly downregulated in gastric cancer tissues (n = 33) and cell lines (n = 8) relative to normal stomach tissues. Functionally, reexpression of HoxD10 results in significant inhibition of cell survival, induction of cell apoptosis, and impairment of cell migration and invasion. Moreover, ectopic expression of HoxD10 suppresses gastric tumor growth in a mouse xenograft model. To identify target candidates of HoxD10, we performed cDNA microarray and showed that HoxD10 regulates multiple downstream genes including IGFBP3. Reintroduction of HoxD10 transcriptionally upregulates IGFBP3, activates caspase 3 and caspase 8, and subsequently induces cell apoptosis. Methylation specific PCR revealed that HoxD10 promoter DNA was hypermethylated in gastric cancer cell lines. Additionally, 5-aza demethylation treatment could transiently reactivate the expression of HoxD10 in gastric cancer cells. HoxD10 promoter methylation frequently was detected in gastric cancer tissues obtained from endoscopic biopsies (85.7%, 24/28) and surgically resected samples (82.6%, 57/69). Intestinal metaplasia tissues showed a 60% methylation rate (18/30), but no detectable methylation in normal stomach tissues (0%, 0/10). Taken together, our results suggest that HoxD10 functions as a candidate tumor suppressor in gastric cancer, which is inactivated through promoter hypermethylation.

MicroRNA-122a functions as a novel tumor suppressor downstream of adenomatous polyposis coli in gastrointestinal cancers.

Aberrant regulation of APC/beta-catenin signaling pathway is common in the pathogenesis of colorectal and other cancers. Targets regulated by APC/beta-catenin signaling pathway play crucial roles in cancer development. In the current study, we aimed to illustrate the influence of APC/beta-catenin signaling pathway on expression of microRNAs, one new group of players important to carcinogenesis. Restoration of APC function in colorectal cancer cells led to the deregulation of several cancer-related microRNAs, such as miR-122a which was recognized as the liver-specific microRNA. MiR-122a was down-regulated in gastrointestinal cancer cell lines as well as primary carcinoma tissues. Inhibition of miR-122a could reverse wild-type APC-induced growth inhibition of gastrointestinal cancer cells while miR-122a mimic inhibited cell growth. In summary, we identified some cancer-related microRNAs regulated by APC/beta-catenin signaling pathway. The down-regulation of miR-122a mediated by aberrant APC/beta-catenin signaling is important to the pathogenesis of gastrointestinal cancers.

ZIC1 is downregulated through promoter hypermethylation in gastric cancer.

As one of major epigenetic changes responsible for tumor suppressor gene inactivation in the development of cancer, promoter hypermethylation was proposed as a marker to define novel tumor suppressor genes. In the current study we identified ZIC1 (Zic family member 1, odd-paired Drosophila homolog) as a novel tumor suppressor gene silenced through promoter hypermethylation in gastric cancer, the second leading cause of cancer death worldwide. In all of gastric cancer cells lines examined, ZIC1 expression was downregulated and such downregulation was accompanied with the hypermethylation of ZIC1 promoter. Demethylation treatment with 5-aza-2'-deoxycytidine (Aza) reversed ZIC1 downregulation, highlighting the importance of promoter methylation to ZIC1 downregulation in gastric cancer cells. Notably, ZIC1 expression was significantly downregulated in primary gastric carcinoma tissues in comparison with non-tumor adjacent gastric tissues (p<0.01). Accordingly, promoter methylation of ZIC1 was frequently detected in primary gastric carcinoma tissues (94.6%, 35/37) but not normal gastric tissues, indicating that promoter hypermethylation mediated ZIC1 downregulation may play an important role in gastric carcinogenesis. Indeed, ectopic expression of ZIC1 led to the growth inhibition of gastric cancer cells through the induction of S-phase cell cycle arrest (p<0.01). Our results revealed ZIC1 as a novel candidate tumor suppressor gene downregulated through promoter hypermethylation in gastric cancer.

Promoter hypermethylation mediates downregulation of thiamine receptor SLC19A3 in gastric cancer.

As an important way to inactivate tumor suppressor genes (TSGs) during cancer development, promoter hypermethylation can be used to define novel TSGs and identify biomarkers for cancer diagnosis. SLC19A3 (solute carrier family 19, member 3) was found to be such a biomarker. SLC19A3 expression was downregulated in gastric cancer cell lines (71%, 5/7) and restored after pharmacological demethylation. Notably, hypermethylation of SLC19A3 promoter was detected in gastric cancer cell lines (57%, 4/7), primary gastric carcinoma tissues (51%, 52/101) and precancerous lesion (intestinal metaplasia) tissues (32%, 8/25). Exogenous SLC19A3 expression caused growth inhibition of gastric cancer cells. In summary, SLC19A3 was epigenetically downregulated in gastric cancer. Methylation of SLC19A3 promoter could be a novel biomarker for early gastric cancer development.

Cathelicidin stimulates colonic mucus synthesis by up-regulating MUC1 and MUC2 expression through a mitogen-activated protein kinase pathway.

Mucus forms the physical barrier along the gastrointestinal tract. It plays an important role to prevent mucosal damage and inflammation. Our animal study showed that antibacterial peptide 'cathelicidin' increased mucus thickness and prevented inflammation in the colon. In the current study, we examined the direct effect and mechanisms by which the peptide increased mucus synthesis in a human colonic cell line (HT-29). Human cathelicidin (LL-37) dose-dependently (10-40 microg/ml) and significantly stimulated mucus synthesis by increasing the D-[6-(3)H] glucosamine incorporation in the cells. Real-time PCR data showed that addition of LL-37 induced more than 50% increase in MUC1 and MUC2 mRNA levels. Treatment with MUC1 and MUC2 siRNAs normalized the stimulatory action of LL-37 on mucus synthesis. LL-37 also activated the phosphorylation of mitogen-activated protein (MAP) kinase in the cells. A specific inhibitor of the MAP kinase pathway, U0126, completely blocked the increase of MUC1 and MUC2 expression as well as mucus synthesis by LL-37. Taken together, LL-37 can directly stimulate mucus synthesis through activation of MUC1 and MUC2 expression and MAP kinase pathway in human colonic cells.

Probiotic Lactobacillus rhamnosus GG enhances gastric ulcer healing in rats.

Probiotics are widely used as functional foods which have been advocated for the maintenance of gastrointestinal microflora equilibrium and treatment of gastrointestinal disorders. However, studying the role of probiotics in peptic ulcer disease is limited. The aim of the present study is to investigate the effect of a probiotic strain Lactobacillus rhamnosus GG on gastric ulcer and to elucidate the mechanisms involved. Gastric kissing ulcers were induced in rats by acetic acid (60% v/v). L. rhamnosus GG was given intragastrically at 10(8) cfu/day or 10(9) cfu/day for three consecutive days after ulcer induction. L. rhamnosus GG successfully colonized in the gastric mucosa especially at the ulcer margin. It also significantly and dose-dependently reduced gastric ulcer area. Cell apoptosis to cell proliferation ratio was strongly decreased and accompanied by significant up-regulation of ornithine decarboxylase (ODC) and B-cell lymphoma 2 (Bcl-2) protein expression at the ulcer margin. Angiogenesis was also significantly stimulated together with the induction of vascular endothelial growth factor (VEGF) expression. Furthermore, L. rhamnosus GG up-regulated the phosphorylation level of epidermal growth factor receptor (EGF receptor) without altering the total EGF receptor expression. These findings suggested that L. rhamnosus GG enhanced gastric ulcer healing via the attenuation of cell apoptosis to cell proliferation ratio and increase in angiogenesis. Regulators of these processes such as ODC, Bcl-2, VEGF and EGF receptor are likely to be involved in the healing action of L. rhamnosus GG for gastric ulcer.

A new role for cathelicidin in ulcerative colitis in mice.

Cathelicidin, an antimicrobial peptide of the innate immune system, modulates microbial growth, wound healing, and inflammation. However, its association with inflammatory bowel diseases (IBDs) is unknown. Our objective was to determine whether cathelicidin would exert a modulatory effect on the progression of IBD and, if so, investigate the mechanism of action through which this effect occurred. We evaluated the potential for a synthetic cathelicidin, the mouse cathelin-related antimicrobial peptide (mCRAMP), to prevent the initiation and promote the healing of lesions from inflammatory colitis that was experimentally induced in mice with dextran sulfate sodium (DSS). During the experiment, mCRAMP was given: (i) as a parallel treatment starting together with 3% DSS feeding, and (ii) as a posttreatment starting 7 days after 3% DSS feeding. The body weight, fecal microflora populations, clinical symptoms, and histologic findings of colonic tissues were measured. Relative gene expression of mucins (MUC1, MUC2, MUC3, and MUC4) in colonic tissues was determined by real-time polymerase chain reaction. Intrarectal administration of mCRAMP ameliorated DSS-induced colitis with negligible effects on mucosal healing. The peptide also significantly reduced the increased number of fecal microflora in colitis animals. It reversed the decline of colonic mucus thickness during colitis through upregulation of the expression of mucin genes. Treatment with mCRAMP also prevented colitis development by suppressing the induction of apoptosis by DSS. The current study demonstrates for the first time that intrarectal administration of cathelicidin may be a novel therapeutic option for IBDs.

Enhancement of gastric mucosal integrity by Lactobacillus rhamnosus GG.

The gastric mucosa is frequently exposed to different exogenous and endogenous ulcerative agents. Alcoholism is one of the risk factors for the development of mucosal damage in the stomach. This study aimed to assess if a probiotic strain Lactobacillus rhamnosus GG (LGG) is capable of protecting the gastric mucosa from acute damage induced by intragastric administration of ethanol. Pre-treatment of rats with LGG at 10(9) cfu/ml twice daily for three consecutive days markedly reduced ethanol-induced mucosal lesion area by 45%. LGG pre-treatment also significantly increased the basal mucosal prostaglandin E(2) (PGE(2)) level. In addition, LGG attenuated the suppressive actions of ethanol on mucus-secreting layer and transmucosal resistance and reduced cellular apoptosis in the gastric mucosa. It is suggested that the protective action of LGG on ethanol-induced gastric mucosal lesions is likely attributed to the up-regulation of PGE(2), which could stimulate the mucus secretion and increase the transmucosal resistance in the gastric mucosa. All these would protect mucosal cells from apoptosis in the stomach.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone from cigarette smoke stimulates colon cancer growth via beta-adrenoceptors.

Cigarette smoking is a risk factor for colorectal cancer. It is suggested that 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine, mediates the carcinogenic action of cigarette smoking by promoting cancer growth. In the present study, the proliferative response of a cultured colon cancer cell line HT-29 to NNK was determined. It was found that NNK dose-dependently stimulated HT-29 cell proliferation. In this regard, the stimulatory action of NNK was abolished by atenolol and ICI 118,551, a beta1- and beta2-selective antagonist, respectively. In addition, cell growth was stimulated by the nonselective adrenergic agonist, noradrenaline, and more effectively by the beta-selective agonist, isoproterenol. The second message cyclic AMP level for beta-adrenoceptor activation was elevated by isoproterenol and NNK treatment. These agents also up-regulated cyclooxygenase-2 expression, cytosolic phospholipase A2 expression, and prostaglandin E2 release. Beta2-adrenoceptor blockade with ICI 118,551, in contrast, significantly decreased cyclooxygenase-2 expression, cytosolic phospholipase A2 expression and prostaglandin E2 release induced by NNK and isoproterenol. To conclude, it is proposed that NNK stimulates HT-29 cell proliferation through beta-adrenoceptors, preferentially beta2 receptors. Activation of the beta-adrenoceptors, and the consequent cyclic AMP elevation coupled with the downstream arachidonic acid pathway, is perhaps an important mechanistic cascade in the promotion of colon cancer growth. These findings partly elucidate the carcinogenic actions of cigarette smoke and shed new light on the novel modulatory role of beta-adrenoceptors in the development of colon cancer.

Nicotine induces cyclooxygenase-2 and vascular endothelial growth factor receptor-2 in association with tumor-associated invasion and angiogenesis in gastric cancer.

Blockade of angiogenesis is a promising strategy to suppress tumor growth, invasion, and metastasis. Vascular endothelial growth factor (VEGF), which binds to tyrosine kinase receptors [VEGF receptors (VEGFR) 1 and 2], is the mediator of angiogenesis and mitogen for endothelial cells. Cyclooxygenase-2 (COX-2) plays an important role in the promoting action of nicotine on gastric cancer growth. However, the action of nicotine and the relationship between COX-2 and VEGF/VEGFR system in tumorigenesis remain undefined. In this study, the effects of nicotine in tumor angiogenesis, invasiveness, and metastasis were studied with sponge implantation and Matrigel membrane models. Nicotine (200 microg/mL) stimulated gastric cancer cell proliferation, which was blocked by SC-236 (a highly selective COX-2 inhibitor) and CBO-P11 (a VEGFR inhibitor). This was associated with decreased VEGF levels as well as VEGFR-2 but not VEGFR-1 expression. Topical injection of nicotine enhanced tumor-associated vascularization, with a concomitant increase in VEGF levels in sponge implants. Again, application of SC-236 (2 mg/kg) and CBO-P11 (0.4 mg/kg) partially attenuated vascularization by approximately 30%. Furthermore, nicotine enhanced tumor cell invasion through the Matrigel membrane by 4-fold and promoted migration of human umbilical vein endothelial cells in a cocultured system with gastric cancer cells. The activity of matrix metalloproteinases 2 and 9 and protein expressions of plasminogen activators (urokinase-type plasminogen activator and its receptor), which are the indicators of invasion and migration processes, were increased by nicotine but blocked by COX-2 and VEGFR inhibitors. Taken together, our results reveal that the promoting action of nicotine on angiogenesis, tumor invasion, and metastasis is COX-2/VEGF/VEGFR dependent.

ZIC1 is downregulated through promoter hypermethylation, and functions as a tumor suppressor gene in colorectal cancer.

The transcription factor, Zinc finger of the cerebellum (ZIC1), plays a crucial role in vertebrate development. Recently, ZIC1 has also been found to participate in the progression of human cancers, including medulloblastomas, endometrial cancers, and mesenchymal neoplasms. However, the function of ZIC1 in colon cancer progression has not been defined. In this study, we demonstrate ZIC1 to be silenced or significantly downregulated in colon cancer cell lines. These effects were reversed by demethylation treatment with 5-aza-2'-deoxycytidine (Aza). ZIC1 expression is also significantly downregulated in primary colorectal cancer tissues relative to adjacent non-tumor tissues (p = 0.0001). Furthermore, methylation of ZIC1 gene promoter is frequently detected in primary tumor tissues (85%, 34/40), but not in adjacent non-tumor tissues. Ectopic expression of ZIC1 suppresses cell proliferation and induces apoptosis, which is associated with MAPK and PI(3)K/Akt pathways, as well as the Bcl-xl/Bad/Caspase3 cascade. To identify target candidates of ZIC1, we employed cDNA microarray and found that 337 genes are downregulated and 95 genes upregulated by ectopic expression of ZIC1, which were verified by 10 selected gene expressions by qRT-PCR. Taken together, our results suggest that ZIC1 may potentially function as a tumor suppressor gene, which is downregulated through promoter hypermethylation in colorectal cancers.

Effects of adrenaline in human colon adenocarcinoma HT-29 cells.

AIMS: Stress has been implicated in the development of cancers. Adrenaline levels are increased in response to stress. The effects of adrenaline on colon cancer are largely unknown. The aims of the study are to determine the effects of adrenaline in human colon adenocarcinoma HT-29 cells and the possible underlying mechanisms involved. MAIN METHODS: The effect of adrenaline on HT-29 cell proliferation was determined by [(3)H] thymidine incorporation assay. Expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) were detected by Western blot. Matrix metalloproteinase-9 (MMP-9) activity and prostaglandin E(2) (PGE(2)) release were determined by zymography and enzyme immunoassay, respectively. KEY FINDINGS: Adrenaline stimulated HT-29 cell proliferation. This was accompanied by the enhanced expression of COX-2 and VEGF in HT-29 cells. Adrenaline also upregulated MMP-9 activity and PGE(2) release. Adrenaline stimulated HT-29 cell proliferation which was reversed by COX-2 inhibitor sc-236. COX-2 inhibitor also reverted the action of adrenaline on VEGF expression and MMP-9 activity. Further study was performed to determine the involvement of ß-adrenoceptors. The stimulatory action of adrenaline on colon cancer growth was blocked by atenolol and ICI 118,551, a ß(1)- and ß(2)-selective antagonist, respectively. This signified the role of ß-adrenoceptors in this process. In addition, both antagonists also abrogated the stimulating actions of adrenaline on COX-2, VEGF expression, MMP-9 activity and PGE(2) release in HT-29 cells. SIGNIFICANCE: These results suggest that adrenaline stimulates cell proliferation of HT-29 cells via both ß(1)- and ß(2)-adrenoceptors by a COX-2 dependent pathway.

Shift of homeostasis from parenchymal regeneration to fibroblast proliferation induced by lipopolysaccharide-activated macrophages in gastric mucosal healing in vitro.

Wound healing in the gastrointestinal tract is an orderly process involving orchestrated responses of various cell types. Lipopolysaccharides (LPS) are major components of the outer membrane of Gram-negative bacteria, which are known to impair gastric ulcer healing in animals. The influence of LPS on intercellular communication in wound healing, however, is unknown. We examined the effects of LPS-induced macrophage activation on the proliferative response in cultured rat gastric epithelial cells (RGM-1) and fibroblasts JHU-25. Rat peritoneal resident macrophages were activated with increasing doses of LPS. The supernatant from the activated macrophage preparation, designated as macrophage-conditioned medium, was then used to treat RGM-1 or JHU-25 cells. Cell proliferation and migration were determined by [(3)H]-thymidine incorporation and a monolayer wound-healing assay, respectively. Macrophage-conditioned medium significantly suppressed RGM-1 cell proliferation but had no effect on cell migration. The same medium, however, increased JHU-25 cell proliferation. LPS treatment alone suppressed JHU-25 cell proliferation while it had no effect on RGM-1 cell proliferation, indicating that the differential effects of the macrophage-conditioned medium on cell proliferation were elicited by the factors derived from macrophages. In this regard, tumor necrosis factor (TNF)-alpha stimulated while interleukin (IL)-1beta suppressed RGM-1 cell proliferation, suggesting that IL-1beta but not TNF-alpha may play a part in the mediation of the antiproliferative effect of macrophage-conditioned medium on gastric epithelial cells. In contrast, IL-1beta suppressed while TNF-alpha had no effect on JHU-25 cell proliferation. Collectively, LPS-activated macrophages delay gastric mucosal regeneration but promote fibroblast proliferation in vitro. Such changes may partly elucidate the detrimental effect of bacterial infection on tissue repair in the stomach.

The cationic host defense peptide rCRAMP promotes gastric ulcer healing in rats.

Cathelicidin, a cationic host defense peptide, has been shown to promote cutaneous wound repair and reaches high levels in the gastric mucosa during infection and inflammation. Therefore, we investigated whether this peptide contributes to gastric ulcer healing in rats. Ulcer induction increased the expression of rat cathelicidin rCRAMP in the gastric mucosa. Further increase in expression of rCRAMP by local injection of rCRAMP-encoding plasmid promoted ulcer healing by enhancing cell proliferation and angiogenesis. rCRAMP directly stimulated proliferation of cultured rat gastric epithelial cells (RGM-1), which was abolished by inhibitors of matrix metalloproteinase (MMP), epidermal growth factor receptors (EGFR) tyrosine kinase, or mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase. rCRAMP also increased EGFR and ERK1/2 phosphorylation via an MMP-dependent mechanism. Knockdown of transforming growth factor alpha (TGFalpha), which is a ligand of EGFR, by small interfering RNA completely nullified the mitogenic signals evoked by rCRAMP in RGM-1 cells. These findings suggest that rCRAMP exhibits prohealing activity in stomachs through TGFalpha-dependent transactivation of EGFR and its related signaling pathway to induce proliferation of gastric epithelial cells.

Reduced adiposity in bitter melon (Momordica charantia)-fed rats is associated with increased lipid oxidative enzyme activities and uncoupling protein expression.

To further explore the antiobesity effect of freeze-dried bitter melon (BM) juice, activities of mitochondrial lipid oxidative enzymes as well as the expression of uncoupling proteins and their transcription coactivator peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1alpha) were determined in diet-induced obese (DIO) rats. Rats were fed high-fat (HF) diets to induce obesity, and the effect of BM was assessed at doses of 0.75, 1.0, or 1.25% (wt:wt). In a dose-response experiment, BM-supplemented rats had lower energy efficiency (g weight gained/kJ consumed), visceral fat mass, serum glucose, and insulin resistance index, but higher plasma norepinephrine than unsupplemented rats (P < 0.05). Hepatic and skeletal muscle triglyceride concentrations were lower in supplemented HF diet-fed rats than in unsupplemented HF diet-fed rats (P < 0.05). An HF diet supplemented with BM elevated activities of hepatic and muscle mitochondrial carnitine palmitoyl transferase-I (CPT-I) and acyl-CoA dehydrogenase (AD) (P < 0.05). In another experiment, BM (1.0 g/100 g) lowered visceral fat mass but increased serum adiponectin concentration in HF diet-fed rats (P < 0.05). In the final study, rats were fed the HF diet with 0, 1.0 or 1.25% BM. Both groups of BM-supplemented rats had higher uncoupling protein 1 in brown adipose tissue (P < 0.05) and uncoupling protein 3 in red gastrocnemius muscle (P < 0.05), measured by Western blotting and RT-PCR, than the controls. The expression of the transcription coactivator PGC-1alpha in both tissues was also significantly elevated in the BM-supplemented rats (P < 0.05). The present results suggest that decreased adiposity in BM-supplemented rats may result from lower metabolic efficiency, a consequence of increased lipid oxidation and mitochondrial uncoupling.