Eunok Im

Toll-Like Receptor 5 Engagement Modulates Tumor Development and Growth in a Mouse Xenograft Model of Human Colon Cancer

BACKGROUND & AIMS Toll-like receptor (TLR)-dependent signaling was proposed as immunotherapeutic targets against invading pathogens and tumorigenesis. Here, we investigated whether TLR5-dependent signaling modulates colonic tumor development in mouse xenograft model of human colon cancer. METHODS The expression of myeloid differentiation factor 88 (MyD88) or TLR5 was stably knocked down in human colon cancer cells (DLD-1). Nude mice were subcutaneously implanted with MyD88-knocked down (KD), TLR5-KD, or control cells (n = 16) to examine the pathophysiology of tumor xenografts. Protein microarray assessed the differential expression of cytokines in these tumors. Leukocyte infiltration and tumor angiogenesis were assessed by immunohistochemistry with antibodies against neutrophil (Gr-1, 7/4) or macrophage-specific antigens (CD68, F4-80) and the vascular endothelial cell marker CD31, respectively. Tumor xenografts from DLD-1 cells were treated with flagellin (5.0 microg/kg, 1 injection/every 2 days for 3 weeks), and tumor regression and histopathology were examined. RESULTS Lack of MyD88 or TLR5 expression dramatically enhanced tumor growth and inhibited tumor necrosis in mouse xenografts of human colon cancer. In contrast, TLR5 activation by peritumoral flagellin treatment substantially increased tumor necrosis, leading to significant tumor regression. Tumors from MyD88-KD or TLR5-KD cells revealed the reduced production of neutrophil attracting chemokines (epithelial cell-derived neutrophil-activating peptide-78, macrophage-inflammatory protein alpha, and interleukin-8). Consequently, neutrophil infiltration was dramatically diminished in MyD88- or TLR5-KD xenografts, whereas tumor-associated macrophage infiltration or angiogenesis was not changed. CONCLUSIONS TLR5 engagement by flagellin mediates innate immunity and elicits potent antitumor activity, indicating that TLR5-dependent signaling could be a potential immunotherapeutic target to modulate colonic tumors.

Novel bile acid derivatives induce apoptosis via a p53-independent pathway in human breast carcinoma cells

We have compared the anti-proliferative effects of ursodeoxycholic acid (UDCA), chenodeoxycholic acid (CDCA) and their derivatives, HS-1183, HS-1199 and HS-1200, on MCF-7 (wild-type p53) and MDA-MB-231 (mutant p53) cells. While UDCA and CDCA exhibited no significant effect, their novel derivatives inhibited the proliferation of both cell lines in a concentration-dependent manner, concomitant with apoptotic nuclear changes and the increase of a sub-G1 population and DNA fragmentation. Furthermore, we also observed an increase in the ratio of pro-apoptotic protein Bax to anti-apoptotic protein Bcl-2 and cleavages of lamin B and poly(ADP-ribose) polymerase (PARP) in MCF-7 and MDA-MB-231 cells. Cell cycle related proteins, cyclin D1 and D3, as well as retinoblastoma protein (pRb) were down-regulated, while the level of cyclin-dependent kinase inhibitor p21(WAF1/CIP1) was increased in both cancer cells after treatment with novel bile acids. These findings suggest that these cytotoxic effects of novel bile acid derivatives on human breast carcinoma cells were mediated via apoptosis through a p53-independent pathway.

TRIF Mediates Toll-like Receptor 5-induced Signaling in Intestinal Epithelial Cells

Toll-like receptors (TLRs) associate with adaptor molecules (MyD88, Mal/TIRAP, TRAM, and TRIF) to mediate signaling of host-microbial interaction. For instance, TLR4 utilizes the combination of both Mal/TIRAP-MyD88 (MyD88-dependent pathway) and TRAM-TRIF (MyD88-independent pathway). However, TLR5, the specific receptor for flagellin, is known to utilize only MyD88 to elicit inflammatory responses, and an involvement of other adaptor molecules has not been suggested in TLR5-dependent signaling. Here, we found that TRIF is involved in mediating TLR5-induced nuclear factor κB (NFκB) and mitogen-activated protein kinases (MAPKs), specifically JNK1/2 and ERK1/2, activation in intestinal epithelial cells. TLR5 activation by flagellin permits the physical interaction between TLR5 and TRIF in human colonic epithelial cells (NCM460), whereas TLR5 does not interact with TRAM upon flagellin stimulation. Both primary intestinal epithelial cells from TRIF-KO mice and TRIF-silenced NCM460 cells significantly reduced flagellin-induced NFκB (p105 and p65), JNK1/2, and ERK1/2 activation compared with control cells. However, p38 activation by flagellin was preserved in these TRIF-deficient cells. TRIF-KO intestinal epithelial cells exhibited substantially reduced inflammatory cytokine (keratinocyte-derived cytokine, macrophage inflammatory protein 3α, and IL-6) expression upon flagellin, whereas control cells from TRIF-WT mice showed robust cytokine expression by flagellin. Compare with TRIF-WT mice, TRIF-KO mice were resistant to in vivo intestinal inflammatory responses: flagellin-mediated exacerbation of colonic inflammation and dextran sulfate sodium-induced experimental colitis. We conclude that in addition to MyD88, TRIF mediates TLR5-dependent responses and, thereby regulates inflammatory responses elicited by flagellin/TLR5 engagement. Our findings suggest an important role of TRIF in regulating host-microbial communication via TLR5 in the gut epithelium.

The anticancer effect of probiotic Bacillus polyfermenticus on human colon cancer cells is mediated through ErbB2 and ErbB3 inhibition

A wealth of data implicates that ErbB receptors have essential roles in tumor development. Probiotic bacteria are known to exert an anticancer activity in animal studies. Bacillus polyfermenticus (B.P.), a probiotic bacterium, has been clinically used for a variety of gastrointestinal disorders in East Asia. Here, we investigated the effect of B.P. on the growth of tumors and its putative mechanism of actions. Conditioned medium of B.P. cultures (B.P. CM) inhibited the growth of human colon cancer cells including HT‐29, DLD‐1 and Caco‐2 cells. Moreover, B.P. CM suppressed colony formation of HT‐29 cells cultured on soft agar and reduced carcinogen‐induced colony formation of normal colonocytes. Furthermore, data from the mouse xenograft model of human colon cancer cells showed reduced tumor size in B.P. CM‐injected mice when compared to E. coli conditioned medium‐injected mice. Exposure of B.P. CM to HT‐29 cells for 24 hr, 48 hr and 2 weeks reduced ErbB2 and ErbB3 protein expression as well as mRNA levels. Moreover, cyclin D1 expression that is required for ErbB‐dependent cell transformation was decreased by B.P. CM. Furthermore, transcription factor E2F‐1 that regulates cyclin D1 expression was also decreased by B.P. CM. These results show that B.P. inhibits tumor growth and its anticancer activity occurs, at least in part, through suppressing ErbB2 and ErbB3. Taken together, our study suggests that this probiotic may be clinically used as a prophylactic treatment to prevent colon cancer development.

Activation of Corticotropin-Releasing Factor Receptor 2 Mediates the Colonic Motor Coping Response to Acute Stress in Rodents

BACKGROUND & AIMS Corticotropin-releasing factor receptor-1 (CRF(1)) mediates the stress-induced colonic motor activity. Less is known about the role of CRF(2) in the colonic response to stress. METHODS We studied colonic contractile activity in rats and CRF(2)-/-, CRF-overexpressing, and wild-type mice using still manometry; we analyzed defecation induced by acute partial-restraint stress (PRS), and/or intraperitoneal injection of CRF ligands. In rats, we monitored activation of the colonic longitudinal muscle myenteric plexus (LMMP) neurons and localization of CRF(1) and CRF(2) using immunohistochemical and immunoblot analyses. We measured phosphorylation of extracellular signal-regulated kinase 1/2 by CRF ligands in primary cultures of LMMP neurons (PC-LMMPn) and cyclic adenosine monophosphate (cAMP) production in human embryonic kidney-293 cells transfected with CRF(1) and/or CRF(2). RESULTS In rats, a selective agonist of CRF(2) (urocortin 2) reduced CRF-induced defecation (>50%), colonic contractile activity, and Fos expression in the colonic LMMP. A selective antagonist of CRF(2) (astressin(2)-B) increased these responses. Urocortin 2 reduced PRS-induced colonic contractile activity in wild-type and CRF-overexpressing mice, whereas disruption of CRF(2) increased PRS-induced colonic contractile activity and CRF-induced defecation. CRF(2) colocalized with CRF(1) and neuronal nitric oxide synthase in the rat colon, LMMP, and PC-LMMPn. CRF-induced phosphorylation of extracellular signal-regulated kinase in PC-LMMPn; this was inhibited or increased by a selective antagonist of CRF(1) (NBI35965) or astressin(2)-B, respectively. The half maximal effective concentration, EC(50), for the CRF-induced cAMP response was 8.6 nmol/L in human embryonic kidney-293 cells that express only CRF(1); this response was suppressed 10-fold in cells that express CRF(1) and CRF(2). CONCLUSIONS In colon tissues of rodents, CRF(2) activation inhibits CRF(1) signaling in myenteric neurons and the stress-induced colonic motor responses. Disruption of CRF(2) function impairs colonic coping responses to stress.

Apoptosis and modulation of cell cycle control by synthetic derivatives of ursodeoxycholic acid and chenodeoxycholic acid in human prostate cancer cells

The effects of synthetic derivatives of ursodeoxycholic acid (UDCA), HS-1183, and chenodeoxycholic acid (CDCA), HS-1199 and HS-1200, on the proliferation of human prostate carcinoma PC-3 cells were investigated. Whereas CDCA and UDCA had no effects on the growth of cells in a concentration range we have tested, HS-1199 and HS-1200 completely inhibited the cell proliferation, and HS-1183 showed a weak inhibitory activity. This proliferation-inhibitory effect of the synthetic bile acid derivatives was due to the induction of apoptosis, which was confirmed by observing DNA fragmentation, chromatin condensation and cleavage of PARP. Flow cytometric analysis also revealed that the synthetic bile acid derivatives arrested the cell cycle progression at the G1 phase, which effects were associated with inhibition of phosphorylation of pRB and enhanced binding of pRB and E2F-1. They also suppressed Cdk2 and cyclin E-dependent kinase activities without changes of their expressions. Furthermore, the synthetic bile acids increased the levels of Cdk inhibitor, p21WAF1/CIP1, expression and activated the reporter construct of p21WAF1/CIP1 promoter in p53-independent manner, and p21WAF1/CIP1 proteins induced by the synthetic bile acid derivatives were associated with Cdk2 and proliferating cell nuclear antigen. These distinctive features suggest that it is possible to create the new drugs useful for cancer therapy from the synthetic bile acid derivatives as lead compounds.

Baicalein, an active component of Scutellaria baicalensis Georgi, induces apoptosis in human colon cancer cells and prevents AOM/DSS-induced colon cancer in mice.

Flavonoids have been demonstrated to provide health benefits in humans. Baicalein (5,6,7-trihydroxyflavone) is a phenolic flavonoid compound derived mainly from the root of Scutellaria baicalensis Georgi, a medicinal plant traditionally used in oriental medicine. Baicalein is widely used in Korean and Chinese herbal medicines as anti-inflammatory and anticancer therapy. However, the molecular mechanisms of its activity remain poorly understood and warrant further investigation. This study was performed to investigate the anticancer effect of baicalein on HCT116 human colon cancer cells and the tumor preventing capacity of baicalein on colitis-associated cancer in mice. In in vivo experiments, we induced colon tumors in mice by azoxymethane (AOM) and dextran sulfate sodium (DSS) and evaluated the effects of baicalein on tumor growth. Baicalein treatment on HCT116 cells resulted in a concentration-dependent inhibition of cell growth and induction of apoptotic cell death. The induction of apoptosis was determined by morphological changes and cleavage of poly(ADP-ribose) polymerase. Baicalein also suppressed the activation of NF-κB through PPARγ activation. These results indicate that the anti-inflammatory effects of baicalein may be mediated through PPARγ activation. Finally, administration with baicalein significantly decreased the incidence of tumor formation with inflammation. Our findings suggest that baicalein is one of the candidates for the prevention of inflammation-associated colon carcinogenesis.

Corticotropin-Releasing Hormone Family of Peptides Regulates Intestinal Angiogenesis

BACKGROUND & AIMS The corticotrophin-releasing hormone (CRH) family of peptides modulates intestinal inflammation and the CRH receptor 2 (CRHR2) suppresses postnatal angiogenesis in mice. We investigated the functions of CRHR1 and CRHR2 signaling during intestinal inflammation and angiogenesis. METHODS The activities of CRHR1 and CRHR2 were disrupted by genetic deletion in mice or with selective antagonists. A combination of in vivo, ex vivo, and in vitro measures of angiogenesis were used to determine their activity. CRHR1(-/-) mice and CRHR2(-/-) mice with dextran sodium sulfate-induced colitis were analyzed in comparison with wild-type littermates (controls). RESULTS Colitis was significantly reduced in mice in which CRHR1 activity was disrupted by genetic deletion or with an antagonist, determined by analyses of survival rate, weight loss, histological scores, and cytokine production. Inflammation was exacerbated in mice in which CRHR2 activity was inhibited by genetic deletion or with an antagonist, compared with controls. The inflamed intestines of CRHR1(-/-) mice had reduced microvascular density and expression of vascular endothelial growth factor (VEGF)-A, whereas the intestines of CRHR2(-/-) mice had increased angiogenesis and VEGF-A levels. An antagonist of VEGFR2 activity alleviated colitis in CRHR2(-/-) mice. Ex vivo aortic vessel outgrowth was reduced when CRHR1 was deficient but increased when CRHR2 was deficient. The CRHR1 preferred agonist CRH stimulated tube formation, proliferation, and migration of cultured intestinal microvascular endothelial cells by phosphorylating Akt, whereas the specific CRHR2 agonist Urocortin III had opposite effects. CONCLUSION CRHR1 promotes intestinal inflammation, as well as endogenous and inflammatory angiogenesis whereas CRHR2 inhibits these activities.

Src Family Kinases Promote Vessel Stability by Antagonizing the Rho/ROCK Pathway

Src family kinases (SFKs) are one of the signaling enzymes that contribute to angiogenesis, but their precise input to the various phases of the angiogenic program has not been defined. Using an in vitro model system, we discovered that SFKs promoted the formation of tubes and prevented their regression. They suppressed regression by activating the ERK pathway that antagonized the Rho/ROCK pathway, which was essential for regression. These studies reveal that SFKs contribute to several phases of the angiogenic program and identify the downstream effectors by which SFKs stabilize tubes.

Elevated lipopolysaccharide in the colon evokes intestinal inflammation, aggravated in immune modulator-impaired mice

Frequency of gram-negative bacteria is markedly enhanced in inflamed gut, leading to augmented LPS in the intestine. Although LPS in the intestine is considered harmless and, rather, provides protective effects against epithelial injury, it has been suggested that LPS causes intestinal inflammation, such as necrotizing enterocolitis. Therefore, direct effects of LPS in the intestine remain to be studied. In this study, we examine the effect of LPS in the colon of mice instilled with LPS by rectal enema. We found that augmented LPS on the luminal side of the colon elicited inflammation in the small intestine remotely, not in the colon; this inflammation was characterized by body weight loss, increased fluid secretion, enhanced inflammatory cytokine production, and epithelial damage. In contrast to the inflamed small intestine induced by colonic LPS, the colonic epithelium did not exhibit histological tissue damage or inflammatory lesions, although intracolonic LPS treatment elicited inflammatory cytokine gene expression in the colon tissues. Moreover, we found that intracolonic LPS treatment substantially decreased the frequency of immune-suppressive regulatory T cells (CD4(+)/CD25(+) and CD4(+)/Foxp3(+)). We were intrigued to find that LPS-promoted intestinal inflammation is exacerbated in immune modulator-impaired IL-10(-/-) and Rag-1(-/-) mice. In conclusion, our results provide evidence that elevated LPS in the colon is able to cause intestinal inflammation and, therefore, suggest a physiological explanation for the importance of maintaining the balance between gram-negative and gram-positive bacteria in the intestine to maintain homeostasis in the gut.