Emily Kai Yee Lam

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 μg/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 ∼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.

4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone from Cigarette Smoke Stimulates Colon Cancer Growth via β-Adrenoceptors

The influence of germ line BRCA2 unclassified variants (UCV), including missense mutations and in-frame deletions and insertions on BRCA2 function and on cancer risk, has not been defined although these mutations account for 43% of all identified BRCA2 sequence alterations. To investigate the effects of UCVs on BRCA2 function, we compared mutant and wild-type forms of BRCA2 using assays of cellular survival and viability, homologous recombination repair, and genome instability. We confirm that the effects of known deleterious mutations can be distinguished from neutral polymorphisms and wild-type BRCA2 in these assays, and we characterize the influence of a series of UCVs on BRCA2 function. We also describe how the results from the assays can be combined with data from analysis of cosegregation of the UCVs with cancer, co-occurrence of the UCVs with other deleterious mutations, and interspecies sequence variation in a comprehensive framework in an effort to better distinguish between disease predisposing and neutral UCVs. This combined approach represents a useful means of addressing the functional significance and cancer relevance of UCVs in BRCA2.

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.

1070 Differential Expression of MicroRNAs in Plasma of Colorectal Cancer Patients: A Potential Marker for Colorectal Cancer Screening

Objective: MicroRNAs (miRNAs) have been shown to anticipate great cancer diagnostic potential. We investigated whether plasma miRNAs could discriminate patients with and without colorectal cancer (CRC). Methods: This study was divided into three phases: (i) Marker discovery using real-time PCR-based miRNA profiling on plasma, corresponding cancerous and adjacent non-cancerous colonic tissues of 5 CRC patients, along with plasma from 5 healthy individuals as controls. (ii) Marker selection and validation by real-time quantitative RT-PCR on a small set of plasma. (iii) Independent validation on a large set of plasma from 90 CRC patients, 20 gastric cancer patients, 20 patients with inflammatory bowel disease (IBD) and 50 healthy controls. Results: Of the panel of 95 miRNAs analyzed, 5 miRNAs were up-regulated both in plasma and tissue samples. All the 5 miRNAs were validated on the plasma of 25 CRC patients and 20 healthy controls. Both miR-17-3p and miR-92 were significantly elevated in CRC patients (p Conclusion: MiR-92 is significantly elevated in plasma of CRC patients and can be a potential noninvasive molecular marker for CRC screening.

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.

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 α (TGFα), 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 TGFα-dependent transactivation of EGFR and its related signaling pathway to induce proliferation of gastric epithelial cells.

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 µg/ml) and significantly stimulated mucus synthesis by increasing the D‐[6‐3H] 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. J. Cell. Biochem. 104: 251–258, 2008.

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 β-adrenoceptors. The stimulatory action of adrenaline on colon cancer growth was blocked by atenolol and ICI 118,551, a β(1)- and β(2)-selective antagonist, respectively. This signified the role of β-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 β(1)- and β(2)-adrenoceptors by a COX-2 dependent pathway.

Involvement of Kv1.1 and Nav1.5 in proliferation of gastric epithelial cells

In the present study, patch clamp experiments demonstrated the expression of multiple ionic currents, including a Ba2+‐sensitive inward rectifier K+ current (IKir), a 4‐aminopyridine‐ (4‐AP) sensitive delayed rectifier K+ current (IKDR), and a nifedipine‐sensitive, tetrodotoxin‐resistant inward Na+ current (INa.TTXR) in the non‐transformed rat gastric epithelial cell line RGM‐1. RT‐PCR revealed molecular identities of mRNAs for the functional ionic currents, including Kir1.2 for IKir, Kv1.1, Kv1.6, and Kv2.1 for IKDR, and Nav1.5 for INa.TTXR. Pharmacologic blockade of Kv and Nav, but not Kir, suppressed RGM‐1 cell proliferation. To further elucidate which subtypes of the ion channels were involved in cell proliferation, RNA interference was employed to knockdown specific gene expression. Downregulation of Kv1.1 or Nav1.5 by RNA interference suppressed RGM‐1 cell proliferation. To conclude, our study is the first to delineate the expression of ion channels and their functions as growth modulators in gastric epithelial cells. J. Cell. Physiol. 207: 437–444, 2006.

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 [3H]‐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)‐α stimulated while interleukin (IL)‐1β suppressed RGM‐1 cell proliferation, suggesting that IL‐1β but not TNF‐α may play a part in the mediation of the antiproliferative effect of macrophage‐conditioned medium on gastric epithelial cells. In contrast, IL‐1β suppressed while TNF‐α 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.