Doerthe Kuester

Oxidative stress in ulcerative colitis-associated carcinogenesis.

Oxidative stress is defined as an imbalance between generation of reactive oxygen species (ROS) and decreased antioxidant defense systems. Oxidative stress develops particularly in inflammatory reactions because the inflammatory cells, neutrophils, and macrophages produce large amounts of ROS. It has been known for a long time that oxidative stress in inflamed tissue can pave the way for malignant tumors, and that it is a major pathogenetic factor for the well-established correlation between inflammatory diseases and cancer. Oxidative stress has long been associated with the pathogenesis of chronic inflammatory bowel disease (IBD)-related colorectal cancer. This article provides an overview of the pathology of ROS and presents recent advances concerning the role of ROS in IBD-related colorectal carcinogenesis (Fig. 1).

Thymoquinone Triggers Inactivation of the Stress Response Pathway Sensor CHEK1 and Contributes to Apoptosis in Colorectal Cancer Cells

There are few reports describing the role of p53-dependent gene repression in apoptotic cell death. To identify such apoptosis-associated p53 target genes, we used the pro-oxidant plant-derived drug thymoquinone and compared p53+/+ and p53-/- colon cancer cells HCT116. The p53 wild-type (wt) status correlated with more pronounced DNA damage and higher apoptosis after thymoquinone treatment. A significant up-regulation of the survival gene CHEK1 was observed in p53-/- cells in response to thymoquinone due to the lack of transcriptional repression of p53. In p53-/- cells, transfection with p53-wt vector and CHEK1 small interfering RNA treatment decreased CHEK1 mRNA and protein levels and restored apoptosis to the levels of the p53+/+ cells. p53-/- cells transplanted to nude mice treated with thymoquinone up-regulated CHEK1 expression and did not undergo apoptosis unlike p53+/+ cells. Immunofluorescence analysis revealed that the apoptosis resistance in p53-/- cells after thymoquinone treatment might be conveyed by shuttling of CHEK1 into the nucleus. We confirmed the in vivo existence of this CHEK1/p53 link in human colorectal cancer, showing that tumors lacking p53 had higher levels of CHEK1, which was accompanied by poorer apoptosis. CHEK1 overexpression was correlated with advanced tumor stages (P = 0.03), proximal tumor localization (P = 0.02), and worse prognosis (1.9-fold risk, univariate Cox regression; Kaplan-Meier, P = 0.04). We suggest that the inhibition of the stress response sensor CHEK1 might contribute to the antineoplastic activity of specific DNA-damaging drugs.

Thymoquinone reduces mouse colon tumor cell invasion and inhibits tumor growth in murine colon cancer models

We have shown that thymoquinone (TQ) is a potent antitumor agent in human colorectal cancer cells. In this study, we evaluated TQs therapeutic potential in two different mice colon cancer models [ 1,2 ‐dimethyl hydrazine (DMH) and xenografts]. We also examined TQ effects on the growth of C26 mouse colorectal carcinoma spheroids and assessed tumor invasion in vitro. Mice were treated with saline, TQ, DMH, or combinations once per week for 30 weeks and the multiplicity, size and distribution of aberrant crypt foci (ACF) and tumors were determined at weeks 10, 20 and 30. TQ injected intraperitoneally (i.p.) significantly reduced the numbers and sizes of ACF at week 10; ACF numbers were reduced by 86%. Tumor multiplicity was reduced at week 20 from 17.8 in the DMH group to 4.2 in mice injected with TQ. This suppression was observed at week 30 and was long‐term; tumors did not re‐grow even when TQ injection was discontinued for 10 weeks. In a xenograft model of HCT116 colon cancer cells, TQ significantly (P < 0.05) delayed the growth of the tumor cells. Using a matrigel artificial basement membrane invasion assay, we demonstrated that sub‐cyto‐toxic doses of TQ (40μM) decreased C26 cell invasion by 50% and suppressed growth in three‐dimensional spheroids. Apoptotic signs seen morphologically were increased significantly in TQ‐treated spheroids. TUNEL staining of xenografts and immunostaining for caspase 3 cleavage in DMH tumors confirmed increased apoptosis in mouse tumors in response to TQ. These data should encourage further in vivo testing and support the potential use of TQ as a therapeutic agent in human colorectal cancer.

The cathepsin family and their role in colorectal cancer

Cathepsins are a class of globular proteases, initially described as intracellular peptide hydrolases, although several cathepsins also have extracellular functions. Cathepsins B, C, F, H, L, K, O, S, V, W, and X are cysteine proteases of the papain family, and represent the largest and best-known class of the cathepsins. Cathepsin G is a serine carboxypeptidases, and cathepsins D and E are aspartic proteases. Cathepsins are synthesized as inactive proenzymes and processed to become mature and active enzymes. Endogenous protein inhibitors, such as cystatins and some serpins, inhibit active enzymes. As primarily lysosomal proteases, cathepsins play important roles in proteolysis during physiological processes, as well as in several diseases. On the basis of their ability to degrade extracellular matrix proteins, cathepsins have been implicated to play a role in invasion and metastasis of colorectal cancer. In the present review, the role of cathepsins in the disease process of colorectal cancers and the correlation of cathepsin expression and activity with clinicopathological features is discussed. Furthermore, we give an overview of the recent developments of cathepsins in animal models and in in vitro experiments of colorectal disease, and provide information on inhibitors of cathepsins as possible therapeutics.

Naturally Occurring Regulatory T cells (CD4+, CD25high, FOXP3+) in the Antrum and Cardia are Associated with Higher H. pylori Colonization and Increased Gene Expression of TGF-β1

Background: Helicobacter pylori causes gastric inflammation. Despite the induction of H. pylori‐specific B‐ and T cells, the immune response is not sufficient to clear the infection. Regulatory T cells (Treg cells) suppress the activation and proliferation of antigen‐specific T cells and mediate immunologic tolerance. FOXP3 was shown to be expressed in a subset of Treg cells known as ‘naturally occurring Treg cells’. These cells have not been sufficiently studied in context to H. pylori‐induced inflammation in human gastric mucosa.

Proteinase-activated receptor-2 in the pathogenesis of gastroesophageal reflux disease.

OBJECTIVES:The proteinase-activated receptor-2 (PAR-2) is activated by serine proteases and has been demonstrated to induce proinflammatory and neuroinflammatory effects. It is considered to alter transepithelial resistance and mediates visceral hypersensitivity. This study aimed to evaluate the expression of PAR-2 in human esophageal mucosa of patients with gastroesophageal reflux disease (GERD) in relation to mucosal alterations.METHODS:The study included 123 patients with GERD stratified to erosive reflux disease (n=50), non-erosive reflux disease (n=46), and reflux-negative patients as controls (n=27). Endoscopic and histopathological characterization was performed according to the Los Angeles classification and modified Ismail-Beigi criteria, respectively. PAR-2 expression was analyzed by quantitative reverse transcription (RT)-PCR and immunohistochemistry. The gene expression levels of interleukin (IL)-8 were determined by quantitative RT-PCR and correlated to PAR-2 expression in each patient. Performing in vitro studies, esophageal squamous cell lines (KYSE 150, KYSE 450) were incubated, adjusted to different pH (7.0, 6.0, and 5.0), and exposed to bile acids and PAR-2-activation peptide (SLIGKV-NH2).RESULTS:PAR-2 gene expression was 7- to 10-fold upregulated (P<0.0001) in the mucosa of patients with GERD and correlated positively with IL-8 expression and with histomorphological alterations (dilated intercellular spaces, papillary elongation, basal cell hyperplasia (BCH); P<0.01). Immunohistochemistry showed an intense staining of PAR-2 throughout all epithelial layers in patients with GERD compared with controls (P=0.0005). In vitro studies revealed a 1.5- to 20-fold induction of PAR-2 gene expression in esophageal squamous cells by acidified medium (P<0.01), but not by additional bile acids. The activation of PAR-2 leads to expression and secretion of IL-8.CONCLUSIONS:This study provides evidence of the functional importance of PAR-2-mediated pathways in the pathogenesis of GERD and GERD-associated mucosal alterations and inflammatory changes.

Inflammation and Barrett's carcinogenesis.

Barretts esophagus (BE) is one of the most common premalignant lesions in which normal squamous epithelium of the esophagus is replaced by metaplastic columnar epithelium. Esophageal adenocarcinoma (EA) develops through progression from BE to low- and high-grade dysplasia (LGD/HGD) and to adenocarcinoma. It is widely accepted that inflammation can increase cancer risk, promoting tumor progression. Therefore, inflammation is regarded as the seventh hallmark of cancer. In recent years, the inflammation-cancer connection of Barretts carcinogenesis has been intensively studied, unraveling genetic abnormalities. Besides genetic alterations, inflammation is also epigenetically linked to loss of protein expression through transcriptional silencing via promoter methylation. Key mediators linking inflammation and Barretts carcinogenesis include reactive oxygen species (ROS), NFκB, inflammatory cytokines, prostaglandins, and specific microRNAs (miRNAs). Therefore, the decipherment of molecular pathways that contain these and novel inflammatory key mediators is of major importance for diagnosis, therapy, and prognosis. The detailed elucidation of the signaling molecules involved in Barretts carcinogenesis will be important for the development of pharmaceutical inhibitors. We herein give an overview of the current knowledge of the inflammation-mediated genetic and epigenetic alterations involved in Barretts carcinogenesis. We highlight the role of oxidative stress and deregulated DNA damage checkpoints besides the NFκB pathway.

Serological assessment of gastric mucosal atrophy in gastric cancer

BackgroundNon-invasive tools for gastric cancer screening and diagnosis are lacking. Serological testing with the detection of pepsinogen 1 (PG1), pepsinogen 2 (PG2) and gastrin 17 (G17) offers the possibility to detect preneoplastic gastric mucosal conditions. Aim of this study was to assess the performance of these serological tests in the presence of gastric neoplasia.MethodsHistological and serological samples of 118 patients with gastric cancer have been assessed for tumor specific characteristics (Laurén type, localisation), degree of mucosal abnormalities (intestinal metaplasia, atrophy) and serological parameters (PG1, PG2, PG1/2-ratio, G17, H. pylori IgG, CagA status). Association of the general factors to the different serological values have been statistically analyzed.ResultsPatients with intestinal type gastric cancer had lower PG1 levels and a lower PG1/2-ratio compared to those with diffuse type cancer (p = 0.003). The serum levels of PG2 itself and G17 were not significantly altered. H. pylori infection in general had no influence on the levels of PG1, PG2 and G17 in the serum of gastric cancer patients. There was a trend towards lower PG1 levels in case of positive CagA-status (p = 0.058). The degree of both intestinal metaplasia and atrophy correlated inversely with serum levels for PG1 and the PG1/2-ratio (p < 0.01). Laurén-specific analysis revealed that this is only true for intestinal type tumors. Univariate ANOVA revealed atrophy and CagA-status as the only independent factors for low PG1 and a low PG1/2-ratio.ConclusionsGlandular atrophy and a positive CagA status are determinant factors for decreased pepsinogen 1 levels in the serum of patients with gastric cancer. The serological assessment of gastric atrophy by analysis of serum pepsinogen is only adequate for patients with intestinal type cancer.

Silencing of MGMT expression by promoter hypermethylation in the metaplasia–dysplasia–carcinoma sequence of Barrett’s esophagus

To determine the relevance of MGMT in Barretts carcinogenesis, we analyzed promotor hypermethylation and expression of MGMT in Barretts adenocarcinomas and its paired precursor lesions from 133 patients using a methylation-specific PCR, real-time RT-PCR and immunohistochemistry. Hypermethylation was detected in 78.9% of esophageal adenocarcinomas, in 100% of Barretts intraepithelial neoplasia, in 88.9% of Barretts metaplasia, but only in 21.4% of normal esophageal mucosa samples (P<0.001) and correlated significantly with downregulation of MGMT transcripts (P=0.048) and protein expression (P=0.02). Decrease of protein expression was significantly correlated with progressed stage of disease, lymph node invasion and tumor size. We conclude, that aberrant promoter methylation of MGMT is a frequent and early event during tumorigenesis of Barretts esophagus. High prevalence of MGMT hypermethylation may represent a candidate marker for improved diagnosis and targeted therapy in Barretts adenocarcinoma.

P16 alterations increase the metastatic potential of endometrial carcinoma.

OBJECTIVE The aim of this study was to investigate the role of p16 in tumorigenesis of endometrial carcinoma (EC). METHODS Expression of p16 protein was analyzed using immunohistochemistry. The methylation status of p16 promoter region was determined by methylation-specific PCR. Deletion analysis of the p16 gene was performed by PCR-analyses. RESULTS Aberrant protein expression of p16 was observed in 18 of 46 (39.2%) ECs and correlated significantly with p16 alterations, including gene deletions in 26 of 46 (56.5%) ECs and promoter hypermethylation in 8 of 46 (17.4%) ECs (p<0.001). A significant increase in the frequency of p16 alterations from early stage (I-II) to advanced stage (III-IV) ECs was observed (p=0.002). There was no significant correlation between p16 protein expression and the clinico-pathological features of EC. The development of metastases correlated significantly with the frequency of p16 alterations: p16 alterations were detected in 14 of 15 (93.3%) PTs with metastases and in only 18 of 31 (58.1%) PTs without metastases (p=0.018). The genetic comparison of 15 primary ECs and their paired metastases revealed that in most of the cases the deleted region of p16 gene remains the same or becomes larger during the progression from primary tumor to its corresponding metastases. CONCLUSION Our results suggest that p16 alterations and particularly p16 gene deletions in ECs are associated with increased incidence of metastases.