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Inflammatory bowel disease (IBD) patients have an increased risk of developing colitis-associated colon cancer (CAC); however, the basis for inflammation-induced genetic damage requisite for neoplasia is unclear. Several studies have shown that IBD patients have signs of increased oxidative damage, which could be a result of genetic and environmental factors such as an excess in oxidant molecules released during chronic inflammation, mitochondrial dysfunction, a failure in antioxidant capacity, or oxidant promoting diets. It has been suggested that chronic oxidative environment in the intestine leads to the DNA lesions that precipitate colon carcinogenesis in IBD patients. Indeed, several preclinical and clinical studies show that different endogenous and exogenous antioxidant molecules are effective at reducing oxidation in the intestine. However, most clinical studies have focused on the short-term effects of antioxidants in IBD patients but not in CAC. This review article examines the role of oxidative DNA damage as a possible precipitating event in CAC in the context of chronic intestinal inflammation and the potential role of exogenous antioxidants to prevent these cancers.

Inflammatory bowel disease patients have a greatly increased risk of developing colitis-associated colon cancer (CAC); however, the basis for inflammation-induced genetic damage requisite for neoplasia is unclear. Using three models of CAC, we find that sustained inflammation triggers 8-oxoguanine DNA lesions. Strikingly, antioxidants or iNOS inhibitors reduce 8-oxoguanine and polyps in CAC models. Because the mismatch repair (MMR) system repairs 8-oxoguanine and is frequently defective in colorectal cancer (CRC), we test whether 8-oxoguanine mediates oncogenesis in a Lynch syndrome (MMR-deficient) model. We show that microbiota generates an accumulation of 8-oxoguanine lesions in MMR-deficient colons. Accordingly, we find that 8-oxoguanine is elevated in neoplastic tissue of Lynch syndrome patients compared to matched untransformed tissue or non-Lynch syndrome neoplastic tissue. While antioxidants reduce 8-oxoguanine, they do not reduce CRC in Lynch syndrome models. Hence, microbe-induced oxidative/nitrosative DNA damage play causative roles in inflammatory CRC models, but not in Lynch syndrome models., It is unclear how microbial-induced inflammation promotes neoplastic transformation in colitis-associated cancer (CAC). Here, the authors use models of CAC to show that inflammation induces 8-oxoguanine lesions in DNA, and that antioxidants can reduce these DNA lesions as well as CAC.

Background Human polo-like kinase 1 (PLK1), a highly conserved serine/threonine kinase is a key player in several essential cell-cycle events. PLK1 is considered an oncogene and its overexpression often correlates with poor prognosis of cancers, including colorectal cancer (CRC). However, regulation of PLK1 expression in colorectal cells was never studied earlier and it is currently unknown if PLK1 regulates differentiation and apoptosis of CRC. Methods PLK1 expression was analyzed by real-time PCR and western blotting. Transcriptional regulation was studied by reporter assay, gene knock-down, EMSA and ChIP. Results PLK1 expression was down-regulated during butyrate-induced differentiation of HT-29 and other CRC cells. Also, PLK1 down-regulation mediated the role of butyrate in CRC differentiation and apoptosis. We report here a novel transcriptional regulation of PLK1 by butyrate. Transcription factors CCAAT/enhancer-binding protein α (C/EBPα) and Oct-1 share an overlapping binding site over the PLK1 promoter. Elevated levels of C/EBPα by butyrate treatment of CRC cells competed out the activator protein Oct-1 from binding to the PLK1 promoter and sequestered it. Binding of C/EBPα was associated with increased deacetylation near the transcription start site (TSS) of the PLK1 promoter, which abrogated transcription through reduced recruitment of RNA polymerase II. We also found a synergistic role between the synthetic PLK1-inhibitor SBE13 and butyrate on the apoptosis of CRC cells. Conclusion This study offered a novel p53-independent regulation of PLK1 during CRC differentiation and apoptosis. General significance Down-regulation of PLK1 is one of the mechanisms underlying the anti-cancer role of dietary fibre-derived butyrate in CRC.

In a recent issue of Science, Wilson et al. (2019) provide direct evidence that the bacterial-produced colibactin alkylates DNA in vivo, resulting in DNA adducts, which mediates its genotoxic effect. This work reinforces the role of colibactin-producing bacteria in colon cancer pathogenesis.

Inflammatory bowel disease (IBD) is a group of inflammatory disorders of the intestine caused by dysregulated T-cell mediated immune response against commensal microflora. Probiotics are reported as therapeutically effective against IBD. However, variable efficacy of the live probiotic strains, difference in survival and persistence in the gut between the strains and the lack of insight into the mechanisms of probiotic action limit optimal therapeutic efficacy. Our aims were to evaluate the lactobacillus strains isolated from the North Indian population for the generation of regulatory cells and cytokines in the intestine, to study their effects on pro-inflammatory mediators in the mouse model of inflammatory bowel disease and to explore the underlying mechanisms of their actions. Among the selected lactobacillus strains, Lactobacillus casei Lbs2 (MTCC5953) significantly suppressed lipopolysaccharide-induced pro-inflammatory cytokine (TNF-alpha, IL-6) secretion. Both live and heat-killed Lbs2 polarized Th0 cells to T-regulatory (Treg) cells in vitro, increased the frequency of FoxP3(+) Treg cells in the mesenteric lymph nodes (MLNs) and alleviated macroscopic and histopathological features of colitis in probiotic-fed mice. Moreover, the levels of IL-12, TNF-alpha and IL-17A were suppressed, while IL-10 and TGF-beta levels were augmented in the colonic tissues of Lbs2-treated mice. The induced Treg (iTreg) cells secreted IL-10 and TGF-beta and exerted suppressive effects on the proliferation of effector T-cells. Adoptive transfer of iTreg cells ameliorated the disease manifestations of murine colitis and suppressed the levels of TNF-alpha and IL-17A. Finally, Lbs2 effects were mediated by Toll-like receptor 2 (TLR2) activation on the dendritic cells. This study identified live and heat-killed Lbs2 as putative therapeutic candidates against IBD and highlighted their Toll-like receptor 2-dependent immunomodulatory and regulatory function.

Spleen tyrosine kinase (Syk), a non-receptor tyrosine kinase, regulates tumor progression, either negatively or positively, depending on the tissue lineage. Information about the role of Syk in colorectal cancers (CRC) is limited, and conflicting reports have been published. We studied Syk expression and its role in differentiation and apoptosis of the colonocytes. Here, we reported for the first time that expression of two transcript variants of Syk is suppressed in colonocytes during butyrate-induced differentiation, which mediates apoptosis of HT-29 cells. Despite being a known HDAC inhibitor, butyrate deacetylates histone3/4 around the transcription start site (TSS) of Syk. Histone deacetylation precludes the binding of RNA Polymerase II to the promoter and inhibits transcription. Since butyrate is a colonic metabolite derived from undigested fibers, our study offers a plausible explanation of the underlying mechanisms of the protective role of butyrate as well as the dietary fibers against CRC through the regulation of Syk. We also report that combined use of butyrate and highly specific Syk inhibitor BAY61-3606 does not enhance differentiation and apoptosis of colonocytes. Instead, BAY completely abolishes butyrate-induced differentiation and apoptosis in a Syk- and ERK1/2-dependent manner. While butyrate dephosphorylates ERK1/2 in HT-29 cells, BAY re-phosphorylates it, leading to its activation. This study describes a novel mechanism of butyrate action in CRC and explores the role of Syk in butyrate-induced differentiation and apoptosis. In addition, our study highlights those commercial small molecule inhibitors, although attractive drug candidates should be used with concern because of their frequent off-target effects. J. Cell. Biochem. 118: 191-203, 2017. © 2016 Wiley Periodicals, Inc.

Toll-like receptor 5 (TLR5) expression in the intestinal epithelial cells (IECs) is critical to maintain health, as underscored by multiple intestinal and extra-intestinal diseases in mice genetically engineered for IEC-specific TLR5 knockout. A gradient of expression exists in the colonic epithelial cells from the cecum to the distal colon. Intriguingly, an identical gradient for the dietary metabolite, butyrate also exists in the luminal contents. However, both being critical for intestinal homeostasis and immune response, no studies examined the role of butyrate in the regulation of TLR5 expression. We showed that butyrate transcriptionally upregulates TLR5 in the IECs and augments flagellin-induced immune responses. Both basal and butyrate-induced transcription is regulated by differential binding of Sp-family transcription factors to the GC-box sequences over the TLR5 promoter. Butyrate activates two different protein kinase C isoforms to dephosphorylate/acetylate Sp1 by serine/threonine phosphatases and phosphorylate Sp3 by ERK-MAPK, respectively. This resulted in Sp1 displacement from the promoter and binding of Sp3 to it, leading to p300 recruitment and histone acetylation, activating transcription. This is the first study addressing the mechanisms of physiological TLR5 expression in the intestine. Additionally, a novel insight is gained into Sp1/Sp3-mediated gene regulation that may apply to other genes.

Numerous mechanisms are believed to contribute to the role of dietary fiber-derived butyrate in the protection against the development of colorectal cancers (CRCs). To identify the most crucial butyrate-regulated genes, we exploited whole genome microarray of HT-29 cells differentiated in vitro by butyrate treatment. Butyrate differentiates HT-29 cells by relaxing the perturbation, caused by mutations of Adenomatous polyposis coli (APC) and TP53 genes, the most frequent mutations observed in CRC. We constructed protein-protein interaction network (PPIN) with the differentially expressed genes after butyrate treatment and extracted the hub genes from the PPIN, which also participated in the APC-TP53 network. The idea behind this approach was that the expression of these hub genes also regulated cell differentiation, and subsequently CRC prognosis by evading the APC-TP53 mutational effect. We used mRNA expression profile of these critical hub genes from seven large CRC cohorts. Logistic Regression showed strong evidence for association of these common hubs with CRC recurrence. In this study, we exploited PPIN to reduce the dimension of microarray biologically and identified five prognostic markers for the CRC recurrence, which were validated across different datasets. Moreover, these five biomarkers we identified increase the predictive value of the TNM staging for CRC recurrence.

Caveolin-1(CAV1) is a tyrosine-phosphorylated scaffold protein of caveolae with multiple interacting partners. It functions both as an oncogene and a tumour suppressor depending upon the cellular contexts. In the early stage of colorectal cancers (CRC), CAV1 suppresses tumour progression, while over-expression of CAV1 reduced the tumourigenicity of colon carcinoma cells. In contrast, elevated level of CAV1 was reported in stage III CRC. To address this ambiguity, we studied the functional role and the regulation of CAV1 expression during colonocyte differentiation and apoptosis. Here, we reported for the first time that CAV1 expression was increased during colonocyte differentiation and mediated butyrate-induced differentiation and apoptosis of HT29 cells. CAV1 expression was silenced by promoter hypermethylation in HT-29 cells and reactivated by prolonged histone hyperacetylation of the promoter upon treatment of the cells with butyrate. However, the methylation status was unaltered by butyrate. We for the first time showed that HDAC inhibitor-mediated transactivation of CAV1 was regulated by methylation density of the promoter. Our study further explains the underlying mechanisms of the anti-cancer property of butyrate in CRC.

Cathelicidin antimicrobial peptide is a key component of the host innate immune system. It is constitutively expressed by the intestinal epithelial cells, but induced at further higher levels by different host-derived and microbial stimuli, including the ligands for Toll-like receptors (TLRs). While the underlying mechanisms of cathelicidin expression remain incompletely understood, altered expression may be associated with gastro-intestinal infections and inflammatory diseases. We demonstrate here that viral double-stranded RNA and its synthetic analog poly(I:C) are potent and tissue-specific inducers of cathelicidin mRNA and protein expression in the mouse as well as human intestinal epithelial cells. Reporter assays showed that poly(I:C) transcriptionally regulates murine cathelicidin-related antimicrobial peptide (mCRAMP) by recruiting Sp1 transcription factor to the GC-box cis-regulatory element at -71bp of the mCRAMP putative promoter. Sp1 recruitment to the endogenous mCRAMP promoter was confirmed by chromatin immunoprecipitation (ChIP) assays. Immunoblotting, qPCR, ChIP and siRNA-mediated gene knockdown studies revealed that the activation of phosphatidylinositol 3-kinase/protein kinase Cζ pathways in poly(I:C)-stimulated cells underlies Sp1 phosphorylation and recruitment to the mCRAMP promoter, leading to enhanced transcription. We further showed that intra-rectal poly(I:C) administration in mice reduces intestinal bacterial load and mucosal inflammation following Shigella flexneri 2a infection by inducing mCRAMP expression in the colonic epithelial cells. This study reports novel regulatory mechanisms of cathelicidin expression that may be targeted to treat gastro-intestinal infections.

Background Chemokines play key roles in immune homeostasis and inflammatory response. Considering the role of Ccl20 and Toll-like receptor 9 (TLR9) in gut homeostasis and inflammatory bowel disease (IBD), regulation of Ccl20 by bacterial DNA, the TLR9 ligand, merits in-depth studies. Methods We analyzed Ccl20 expression in various epithelial cell (EC) lines by q-PCR and ELISA. In-vivo expression was investigated in isolated murine colonocytes by immunoblotting. Transcriptional regulation of Ccl20 was studied by reporter assays, gene knock-down, electrophoretic mobility shift assay and chromatin immunoprecipitation. Activation of upstream kinases was checked by immunoblotting. Results We showed low levels of Ccl20 expression in mouse colonic ECs, but marked induction by in vivo treatment with bacterial DNA. This corroborated with persistent Ccl20 induction in different EC lines. We found involvement of MAP-kinases during the early hours after stimulation, and a novel AP-1site (− 252 bp) regulated the expression in colonic ECs. More importantly, mutually exclusive transcriptional regulation by AP-1 (cjun/cfos) and non-canonical NF-κB (RelB/p52) downstream of MEK-ERK and NIK-IKK-α-NF-κB2 (p100) phosphorylation, respectively was responsible for persistent Ccl20 expression in the colonic cells, while canonical NF-κB isoforms played no role. Conclusions Persistent Ccl20 induction by TLR9 in colonic ECs involves early and delayed activation of two independent signaling pathways. This is the first report of non-canonical NF-κB activation and Ccl20 expression in the colonic ECs by TLR9. General significance Our study will help to better understand immune regulation by Ccl20 in the intestine and may be exploited for future development of novel therapeutics against IBD.

Spleen tyrosine kinase (Syk), a non-receptor tyrosine kinase, regulates tumor progression, either negatively or positively, depending on the tissue lineage. Information about the role of Syk in colorectal cancers (CRC) is limited, and conflicting reports have been published. We studied Syk expression and its role in differentiation and apoptosis of the colonocytes. Here, we reported for the first time that expression of two transcript variants of Syk is suppressed in colonocytes during butyrate-induced differentiation, which mediates apoptosis of HT-29 cells. Despite being a known HDAC inhibitor, butyrate deacetylates histone3/4 around the transcription start site (TSS) of Syk. Histone deacetylation precludes the binding of RNA Polymerase II to the promoter and inhibits transcription. Since butyrate is a colonic metabolite derived from undigested fibers, our study offers a plausible explanation of the underlying mechanisms of the protective role of butyrate as well as the dietary fibers against CRC through the regulation of Syk. We also report that combined use of butyrate and highly specific Syk inhibitor BAY61-3606 does not enhance differentiation and apoptosis of colonocytes. Instead, BAY completely abolishes butyrate-induced differentiation and apoptosis in a Syk- and ERK1/2-dependent manner. While butyrate dephosphorylates ERK1/2 in HT-29 cells, BAY re-phosphorylates it, leading to its activation. This study describes a novel mechanism of butyrate action in CRC and explores the role of Syk in butyrate-induced differentiation and apoptosis. In addition, our study highlights those commercial small molecule inhibitors, although attractive drug candidates should be used with concern because of their frequent off-target effects. J. Cell. Biochem. 118: 191-203, 2017. © 2016 Wiley Periodicals, Inc.

We report here a 3.2-Mb draft assembled genome of Lactobacillus casei Lbs2. The bacterium shows probiotic and immunomodulatory activities. The genome assembly and annotation will help to identify molecules and pathways responsible for interaction between the host immune system and the microbe.

PGE2, the major product of cyclooxygenases implicated in carcinogenesis, is significantly upregulated in cervical cancer. PGE2 via prostanoid receptor EP4 stimulates proliferation and motility while inhibiting apoptosis and immune surveillance. It promotes angiogenesis by stimulating the production of pro-angiogenic factors. The present study demonstrates GW627368X, a highly selective competitive EP4 antagonist, which hinders cervical cancer progression by inhibiting EP4/epithelial growth factor receptor (EGFR) interactive signaling. GW627368X reduced protein kinase A (PKA) phosphorylation which in turn leads to decreased cAMP response element-binding protein (CREB) activation. Decreased PKA phosphorylation also directly enhanced Bax activity and in part reduced glycogen synthase kinase 3 (GSK3)β phosphorylation. Owing to the interactive signaling between EP4 and EGFR, GW627368X lowered EGFR phosphorylation in turn reducing Akt, mitogen-activated protein kinase (MAPK) and GSK3β activity significantly. Sublethal dose of GW627368X was found to reduce the nuclear translocation of β-catenin in a time dependent manner along with time-dependent decrease in cytoplasmic as well as whole-cell β-catenin. Decreased CREB and β-catenin transcriptional activity restricts the aberrant transcription of key genes like EP4, cyclooxygenase (COX)-2, vascular endothelial growth factor and c-myc, which ultimately control cell survival, proliferation and angiogenesis. Reduced activity of EGFR resulted in enhanced expression of 15-hydroxyprostaglandin dehydrogenase increasing PGE2 degradation thereby blocking a positive feedback loop. In xenograft model, dose-dependent decrease in cancer proliferation was observed characterized by reduction in tumor mass and volume and a marked decrease in Ki67 expression. A diminished CD31 specific staining signified decreased tumor angiogenesis. Reduced expression of pAkt, pMAPK, pEGFR and COX-2 validated in vitro results. GW627368X therefore effectively inhibits tumor survival, motility, proliferation and angiogenesis by blocking EP4/EGFR interactive signaling. EP4 is a potent therapeutic target in cervical cancer and can be explored in combination with conventional therapies to attain superior outcomes and to overcome complications associated with organ toxicities, therapeutic resistance and disease relapse.