Joshua Ka-Shun Ko

Involvement of neutrophils and free radicals in the potentiating effects of passive cigarette smoking on inflammatory bowel disease in rats.

BACKGROUND & AIMS Cigarette smoking is associated with inflammatory bowel diseases (IBDs), particularly Crohns disease, in humans. The aim of this study was to examine whether passive cigarette smoking aggravates experimental IBD in rats and to clarify the underlying mechanisms. METHODS Rats were exposed to cigarette smoke (CS) for 1 hour once daily for 4 days before induction of IBD by 2,4, 6-trinitrobenzene sulfonic acid (TNBS)-ethanol enema and were then killed at 2, 6, or 24 hours later. RESULTS Preexposure to CS significantly potentiated colonic damage induced by TNBS. TNBS-ethanol enema caused a pronounced increase in colonic myeloperoxidase activity, leukotriene B(4) level, and also inducible nitric oxide synthase activity, its protein, and messenger RNA expression. These parameters were all significantly increased further by exposure to CS. In contrast, increased colonic superoxide dismutase activity after TNBS-ethanol enema was attenuated by CS exposure. The potentiating effects of CS exposure on TNBS-induced IBD were significantly alleviated after pretreatment with cyclosporin A (an immunosuppressant), N (G)-nitro-L-arginine methylester (a nitric oxide synthase inhibitor), and dimethyl sulfoxide (a hydroxyl radical scavenger). CONCLUSIONS The results show that promotion of neutrophil infiltration and free radical production contributed significantly to the potentiating effect of passive cigarette smoking on experimental IBD.

Novel herbal flavonoids promote apoptosis but differentially induce cell cycle arrest in human colon cancer cell

SummaryFormononetin is a novel herbal isoflavonoid isolated from Astragalus membranaceus, a medicinal plant that possesses antitumorigenic property. We attempted to compare the anticarcinogenic mechanism of formononetin with that of the known proapoptotic flavonoid isoliquiritigenin (ISL) in human cancer cells. We first evaluated the effects of formononetin and ISL on HCT 116 colon cancer cell viability. Immunofluorescence staining was then performed to observe the morphological changes of cancer cells undergoing apoptosis, which had been substantiated using Annexin V-FITC/propidium iodide staining. Western immunoblotting and flow cytometry were also employed to study parameters associated with apoptosis and cell proliferation. Our data show that formononetin and ISL both inhibited the growth of colon cancer cells and promoted apoptosis. These processes were accompanied by caspase activation and downregulation of the antiapoptotic proteins Bcl-2 and Bcl-xL. Besides, the novel proapoptotic protein NSAID-activated gene (NAG-1) and its upstream regulator were overexpressed in drug-treated cells. Nevertheless, only ISL was found to induce a G2 arrest. These findings exemplify that both formononetin and ISL could cause growth inhibition and facilitate apoptosis in colon cancer cells, while only ISL is capable of inducing phase-specific cell cycle arrest. This suggests that the anticarcinogenic activities of different herbal flavonoids may involve both common and differential mechanisms of action, which could be developed as potential anticancer drugs.

A novel anticancer effect of Astragalus saponins: transcriptional activation of NSAID-activated gene.

Astragalus membranaceus has been used to ameliorate the side effects of antineoplastic drugs because of its immunomodulating nature. We had recently demonstrated that total Astragalus saponins (AST) possess anticarcinogenic and proapoptotic properties in human colon cancer cells and tumor xenograft. In this study, we identified NSAID‐activated gene (NAG‐1) as a potential molecular target of AST. The growth‐inhibitory and proapoptotic effects of AST were assessed in a panel of human cancer cell lines. Hoechst 33342 nuclear staining, Annexin V‐FITC/propidium iodide staining, Western immunoblotting, real‐time PCR, luciferase reporter assay and electrophoretic mobility shift assay were conducted to determine the association of NAG‐1 and related transcription factors with AST during its regulation of apoptotic activities. Moreover, the combined proapoptotic and NAG‐1 promoting activities of AST and/or inhibitors of the PI3K‐Akt pathway were also examined. AST caused overexpression of NAG‐1, leading to PARP cleavage and apoptosis. The induction of NAG‐1 promoter activity by the drug was associated with increased gene expression, in addition to prior increase in Egr‐1 expression and DNA binding activity. AST‐induced NAG‐1 activation was intensified when PI3K inhibitor LY294002 or Akt inhibitor was co‐treated and reversed by NAG‐1 siRNA transfection. Nevertheless, the extent of NAG‐1 induction could not be altered by the ERK inhibitor PD98059. Our results indicate that NAG‐1 is a potential molecular target of AST in its antitumorigenic and proapoptotic actions, which would have additive effects when used along with PI3K‐Akt inhibitors. The information obtained could facilitate future development of a novel target‐specific chemotherapeutic agent with known molecular pathway.

Delineation of the protective action of zinc sulfate on ulcerative colitis in rats

The protective action of zinc compounds in Crohns disease-like inflammatory bowel disease in animals has been shown. A similar action of zinc sulfate on ulcerative colitis has not been defined. The present study aimed to delineate the protective action of zinc sulfate and the pathogenic mechanisms of 2,4-dinitrobenzene sulfonic acid (DNBS)-induced ulcerative colitis in rats. Zinc sulfate at different concentrations was given either orally (p.o.) or rectally (p.r.) to rats at 42, 48, 66 and 72 h following the induction of colonic inflammation by DNBS. Rats were killed 96 h after instillation of DNBS rectally to assess the severity of colonic damage, myeloperoxidase and xanthine oxidase activities. The involvement of mast cell degranulation and histamine release in the pathogenesis of DNBS-induced colitis was determined by using a mast cell stabilizer (ketotifen) and histamine receptor blockers (terfenadine and ranitidine). DNBS given rectally produced inflammation and ulceration in rats with a pathology resembling ulcerative colitis. Myeloperoxidase activity but not xanthine oxidase activity was sharply increased by this agent. Intrarectal administration of zinc solution and parenteral injection of histamine blockers significantly reduced tissue damage and myeloperoxidase but not xanthine oxidase activity. Ketotifen, a mast cell stabilizer, also significantly decreased mucosal injury and myeloperoxidase activity in the colon. In conclusion, mast cell degranulation followed by histamine release plays an important role in the pathogenesis of DNBS-induced ulcerative colitis. Zinc given rectally has a therapeutic effect against this colitis model, perhaps through the reduction of inflammation and inhibition of the above pathogenic mechanisms.

Inflammatory bowel disease: etiology, pathogenesis and current therapy.

Ulcerative colitis (UC) and Crohns disease (CD) constitute the two major groups of idiopathic disorders in inflammatory bowel disease (IBD). Environmental factors, genetic factors and immune responses have been considered as the major etiology of IBD. Despite the diversified pathogenesis of the disease, no guaranteed curative therapeutic regimen has been developed so far. This review summarizes the knowledge on the pathophysiology and current treatment approaches of IBD. Since IBD is caused by excessive and tissue- disruptive inflammatory reactions of the gut wall, down-regulation of the immune responses may allow the damaged mucosa to heal and reset the physiological functions of the gut back to normal. Current pharmacotherapy through modulation of neutrophil-derived factors, cytokines, adhesion molecules and reactive oxygen/nitrogen metabolites has been utterly described. Categories of treatment modalities include corticosteroids, aminosalicylates, immunomodulators, antibiotics, probiotics, and a series of unique novel agents. The use of anti-tumor necrosis factor monoclonal antibody (Infliximab), recombinant anti-inflammatory cytokines and related gene therapy has been covered. In addition, discussions on dietary supplementation and heparin treatment are also included. The anti-inflammatory and immunoregulatory potential of investigational agents such as nicotine and the filtered protective compounds from tobacco smoke, as well as active herbal medicinal compounds were tested in our previous experimental works, whereas promising findings have been presented here. With the discovery of novel target-oriented agents, more effective and relatively harmless approaches of IBD therapy could be established to achieve a curative outcome. Indeed, more experimental and clinical studies are needed to confirm the relevance of these therapies.

Astragalus saponins downregulate vascular endothelial growth factor under cobalt chloride-stimulated hypoxia in colon cancer cells

BackgroundOur ongoing research has revealed that total saponins extracted from the medicinal herb Radix Astragali (AST) exhibits significant growth-inhibitory and proapoptotic effects in human cancer cells. In the present study, the potential of AST in controlling angiogenesis was further investigated with elaboration of the underlying molecular mechanism in human colon cancer cell and tumor xenograft.ResultsAST decreased the protein level of VEGF and bFGF in HCT 116 colon cancer cells in a time- and dose-dependent manner. Among the Akt/mTOR signal transduction molecules being examined, AST caused PTEN upregulation, reduction in Akt phosphorylation and subsequent activation of mTOR. AST also suppressed the induction of HIF-1α and VEGF under CoCl2-mimicked hypoxia. These effects were intensified by combined treatment of AST with the mTOR inhibitor rapamycin. Despite this, our data also indicate that AST could attenuate cobalt chloride-evoked COX-2 activation, while such effect on COX-2 and its downstream target VEGF was intensified when indomethacin was concurrently treated. The anti-carcinogenic action of AST was further illustrated in HCT 116 xenografted athymic nude mice. AST significantly suppressed tumor growth and reduced serum VEGF level in vivo. In the tumor tissues excised from AST-treated animals, protein level of p-Akt, p-mTOR, VEGF, VEGFR1 and VEGFR2 was down-regulated. Immunohistochemistry has also revealed that AST effectively reduced the level of COX-2 in tumor sections when compared with that in untreated control.ConclusionTaken together, these findings suggest that AST exerts anti-carcinogenic activity in colon cancer cells through modulation of mTOR signaling and downregulation of COX-2, which together reduce VEGF level in tumor cells that could potentially suppress angiogenesis.

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.

Protective role of cyclooxygenase inhibitors in the adverse action of passive cigarette smoking on the initiation of experimental colitis in rats

Clinical and experimental findings had indicated that cigarette smoke exposure, and cyclooxygenase-2, are strongly associated with inflammatory bowel disease. The present study aimed to evaluate the role of cyclooxygenase-2 in the pathogenesis of experimental inflammatory bowel disease as well as in the adverse action of cigarette-smoke exposure. Rats were pretreated with different cyclooxygenase-2 inhibitors (indomethacin, nimesulide, or SC-236 (4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide)) along with cigarette-smoke exposure before 2,4,6-trinitrobenzenesulfonic acid-enema. Results indicated that pretreatment with cyclooxygenase-2 inhibitors not only protected against 2,4,6-trinitrobenzenesulfonic acid-induced inflammatory bowel disease, but also attenuated the potentiating effect of cigarette-smoke exposure on colonic damage. Furthermore, the colonic cyclooxygenase-2 protein and mRNA expression was markedly induced by 2,4,6-trinitrobenzenesulfonic acid-enema, and it was potentiated further by cigarette-smoke exposure, while the cyclooxygenase-1 expression was not changed. The present study suggests that the highly induced cyclooxygenase-2 expression not only plays a pathogenic role in 2,4,6-trinitrobenzenesulfonic acid-induced inflammatory bowel disease, but also contributes to the adverse action of cigarette-smoke exposure on this disorder.

Astragalus membranaceus: A Review of its Protection Against Inflammation and Gastrointestinal Cancers

Astragalus membranaceus is a major medicinal herb commonly used in many herbal formulations in the practice of traditional Chinese medicine (TCM) to treat a wide variety of diseases and body disorders. Among its diversified clinical applications, the potential use of this herb and its chemical constituents in treatments of inflammatory diseases and cancers has been actively investigated in recent years. Astragalus-based treatments have demonstrated significant amelioration of the toxicity induced by other concurrently administered orthodox drugs (e.g., immunosuppressants and cancer chemotherapeutics). The major components of Astragalus membranaceus are polysaccharides, flavonoids, and saponins. Contemporary use of Astragalus membranaceus mainly focuses on its immunomodulating, anti-oxidant, and anti-inflammatory, as well as anticancer effects. In this paper, we summarize the properties of Astragalus membranaceus and its major constituents in the biological system based on experimental and clinical studies. The antitumorigenic mechanisms of a novel Astragalus saponins extract called AST in treating various gastrointestinal cancers are highlighted. We discuss in detail how the Astragalus herb and AST influence the immune system, modulate various cancer signaling pathways, and interact with specific transcription molecules during protection against gastrointestinal inflammation and cancers. This information could help clinicians and scientists develop novel target-specific and effective therapeutic agents that are deprived of major systemic side effects, so as to establish a better treatment regimen in the battle against inflammatory diseases and cancers of the gut.

Beneficial intervention of experimental colitis by passive cigarette smoking through the modulation of cytokines in rats.

Abstract Background Epidemiologic observations have indicated that cigarette smoking decreases the risk of ulcerative colitis, but the modes of action remain anonymous. The present study aimed to investigate the beneficial effects of passive cigarette smoking using an animal colitis model. We hypothesized that the underlying mechanisms may involve immunoregulation of cytokines. Methods Experimental colitis was induced in rats by enema administration of 2,4-dinitrobenzene sulfonic acid (DNBS). Passive cigarette smoking by rats was performed for 1 hour once daily, from 3 days before DNBS enema until they were sacrificed on day 8. Other groups of DNBS-treated rats received therapeutic treatment of cyclosporin A or pentoxifylline, a tumor necrosis factor (TNF)-α inhibitor. Macroscopic and histologic damage were graded, and the colonic levels of different cytokines and the levels/activities of parameters related to neutrophil activation were also measured. Results DNBS-induced colonic damage was improved in passive-cigarette-smoking rats. This was accompanied by attenuation of the elevated colonic myeloperoxidase and inducible nitric oxide synthase activities and leukotriene B4 level. Likewise, the augmentation in colonic levels of TNF-α, interleukin (IL)-1β, and IL-6 in colitis rats was also alleviated by passive cigarette smoking. In contrast, the deprivation of colonic IL-10 during colitis was preserved in cigarette-smoking rats. These effects were similarly accomplished by pentoxifylline and, to some degree, by cyclosporin A. Conclusions The results support the idea that the beneficial effects of passive cigarette smoking in experimental colitis involved immunoregulation of cytokines in colonic tissues.