Christoph-Thomas Germer

Toll-like receptor (TLR) 7 and TLR8 expression on CD133+ cells in colorectal cancer points to a specific role for inflammation-induced TLRs in tumourigenesis and tumour progression

Toll-like receptor (TLR) stimulation results in activation of NF-κB, a key modulator in driving inflammation to cancer and mitogen-activated protein kinases that have been shown to recruit mitotic and cyclooxygenase-2 (COX-2) induced pathways in carcinogenesis. Here we asked whether different TLR, COX-2 and stem cell marker expression profiles in colorectal cancer (CRC) provide further evidence for this hypothesis from a clinical perspective. We analysed gene and protein expression of TLR7-TLR10, COX-2 and CD133 as a marker for colon-initiating cells in CRC patients (n=65). Gene analysis demonstrated significantly upregulated TLR7-TLR10 and COX-2 expression in CRC tumour tissues. Analysis of isolated tumour cells from primary tumours showed co-expression of TLR7 and TLR8 with CD133 and gave evidence for a subpopulation of colon cancer-initiating cells. In multivariate analyses TLR8 expression was found to be an independent prognostic factor. Persistent TLR-specific activation of NF-κB in CRC and particularly in tumour-initiating cells may thus sustain further tumour growth and progression through perpetuated signalling known from inflammatory and tissue repair mechanisms with consecutive self-renewal in pluripotent tumour cells. Activation through self-ligands or viral RNA fragments may putatively maintain this inflammatory process, suggesting a key role in cancer progression.

Expression of Foxp3 in Colorectal Cancer but Not in Treg Cells Correlates with Disease Progression in Patients with Colorectal Cancer

Background Regulatory T cells (Treg) expressing the transcription factor forkhead-box protein P3 (Foxp3) have been identified to counteract anti-tumor immune responses during tumor progression. Besides, Foxp3 presentation by cancer cells itself may also allow them to evade from effector T-cell responses, resulting in a survival benefit of the tumor. For colorectal cancer (CRC) the clinical relevance of Foxp3 has not been evaluated in detail. Therefore the aim of this study was to study its impact in colorectal cancer (CRC). Methods and Findings Gene and protein analysis of tumor tissues from patients with CRC was performed to quantify the expression of Foxp3 in tumor infiltrating Treg and colon cancer cells. The results were correlated with clinicopathological parameters and patients overall survival. Serial morphological analysis demonstrated Foxp3 to be expressed in cancer cells. High Foxp3 expression of the cancer cells was associated with poor prognosis compared to patients with low Foxp3 expression. In contrast, low and high Foxp3 level in tumor infiltrating Treg cells demonstrated no significant differences in overall patient survival. Conclusions Our findings strongly suggest that Foxp3 expression mediated by cancer cells rather than by Treg cells contribute to disease progression.

Differential role of Rho GTPases in intestinal epithelial barrier regulation in vitro.

Maintenance of intestinal epithelial barrier functions is crucial to prevent systemic contamination by microbes that penetrate from the gut lumen. GTPases of the Rho‐family such as RhoA, Rac1, and Cdc42 are known to be critically involved in the regulation of intestinal epithelial barrier functions. However, it is still unclear whether inactivation or activation of these GTPases exerts barrier protection or not. We tested the effects of Rho GTPase activities on intestinal epithelial barrier functions by using the bacterial toxins cytotoxic necrotizing factor 1 (CNF‐1), toxin B, C3 transferase (C3 TF), and lethal toxin (LT) in an in vitro model of the intestinal epithelial barrier. Incubation of cell monolayers with CNF‐1 for 3 h induced exclusive activation of RhoA whereas Rac1 and Cdc42 activities were unchanged. As revealed by FITC‐dextran flux and measurements of transepithelial electrical resistance (TER) intestinal epithelial permeability was significantly increased under these conditions. Inhibition of Rho kinase via Y27632 blocked barrier destabilization of CNF‐1 after 3 h. In contrast, after 24 h of incubation with CNF‐1 only Rac1 and Cdc42 but not RhoA were activated which resulted in intestinal epithelial barrier stabilization. Toxin B to inactivate RhoA, Rac1, and Cdc42 as well as Rac1 inhibitor LT increased intestinal epithelial permeability. Similar effects were observed after inhibition of RhoA/Rho kinase signaling by C3 TF or Y27632. Taken together, these data demonstrate that both activation and inactivation of RhoA signaling increased paracellular permeability whereas activation of Rac1 and Cdc42 correlated with stabilized barrier functions. J. Cell. Physiol. 226: 1196–1203, 2011.

Phosphodiesterase-4 inhibition as a therapeutic approach to treat capillary leakage in systemic inflammation

•  A specific therapy to treat capillary leakage in systemic inflammation and sepsis is not available at present. •  Recent studies demonstrated that reduced cAMP levels in endothelial cells contribute to inflammation‐induced breakdown of the endothelial barrier. •  The present study demonstrates that systemically applied phosphodiesterase‐4 inhibitors to increase endothelial cAMP are effective to prevent and to treat capillary leakage followed by improved microcirculation in a rodent model of systemic inflammation. •  These data suggest a highly clinically relevant and applicable approach to stabilize capillary leakage in sepsis and systemic inflammation.

Pathogenesis of colonic diverticular disease

PurposeThis paper aims to review the current evidence regarding pathogenesis of colonic diverticular disease and its complications, which are a major health problem in the Western world.MethodsBased on selective Medline searches, relevant literature was indentified regarding pathogenesis of (1) diverticulosis/formation of diverticula, (2) diverticulitis/inflammation of diverticula, (3) complicated diverticulitis/perforation, and (4) diverticular bleeding.ResultsPathogenesis of colonic diverticula is regarded as a multifactorial process, involving dietary factors (Western low-fiber diet), structural changes of the colonic wall (altered musculature, collagen, elastin, etc.) and functional changes (motility disorder, increased intraluminal pressure). Genetic changes are also discussed and aging is also a key factor. Pathogenesis of inflammation (diverticulosis) is regarded as a result of “microperforations” at the fundus of the diverticulum, and not an “abscessed diverticulum” due to an impacted fecolith. Histamine and its receptors do also seem to play a role, corresponding with the promising prophylactic approach with probiotics. Pathogenesis of complicated diverticulitis is characterized by perforation, which is the cardinal feature. Furthermore, an intensive inflammatory infiltrate with macrophages is found in surgical specimens, even after antibiotic pretreatment. Steroid intake and immunosuppression are risk factors and only recently a glucocorticoid-induced tumor necrosis factor-receptor has been suggested to resemble the molecular link. Diverticular bleeding is a distinct disease process—which does usually take place without diverticulitis—and is due to eccentric rupture of the vas rectum.ConclusionsThe pathophysiology of diverticular disease is multifactorial. Some of the current evidence has important implications for clinical practice, e.g., the suggested role of steroid intake and immunosuppression for complicated diverticulitis.

Natural resistance to ascorbic acid induced oxidative stress is mainly mediated by catalase activity in human cancer cells and catalase-silencing sensitizes to oxidative stress

BackgroundAscorbic acid demonstrates a cytotoxic effect by generating hydrogen peroxide, a reactive oxygen species (ROS) involved in oxidative cell stress. A panel of eleven human cancer cell lines, glioblastoma and carcinoma, were exposed to serial dilutions of ascorbic acid (5-100 mmol/L). The purpose of this study was to analyse the impact of catalase, an important hydrogen peroxide-detoxifying enzyme, on the resistance of cancer cells to ascorbic acid mediated oxidative stress.MethodsEffective concentration (EC50) values, which indicate the concentration of ascorbic acid that reduced the number of viable cells by 50%, were detected with the crystal violet assay. The level of intracellular catalase protein and enzyme activity was determined. Expression of catalase was silenced by catalase-specific short hairpin RNA (sh-RNA) in BT-20 breast carcinoma cells. Oxidative cell stress induced apoptosis was measured by a caspase luminescent assay.ResultsThe tested human cancer cell lines demonstrated obvious differences in their resistance to ascorbic acid mediated oxidative cell stress. Forty-five percent of the cell lines had an EC50 > 20 mmol/L and fifty-five percent had an EC50 < 20 mmol/L. With an EC50 of 2.6–5.5 mmol/L, glioblastoma cells were the most susceptible cancer cell lines analysed in this study. A correlation between catalase activity and the susceptibility to ascorbic acid was observed. To study the possible protective role of catalase on the resistance of cancer cells to oxidative cell stress, the expression of catalase in the breast carcinoma cell line BT-20, which cells were highly resistant to the exposure to ascorbic acid (EC50: 94,9 mmol/L), was silenced with specific sh-RNA. The effect was that catalase-silenced BT-20 cells (BT-20 KD-CAT) became more susceptible to high concentrations of ascorbic acid (50 and 100 mmol/L).ConclusionsFifty-five percent of the human cancer cell lines tested were unable to protect themselves against oxidative stress mediated by ascorbic acid induced hydrogen peroxide production. The antioxidative enzyme catalase is important to protect cancer cells against cytotoxic hydrogen peroxide. Silenced catalase expression increased the susceptibility of the formerly resistant cancer cell line BT-20 to oxidative stress.

Soluble VE-cadherin is involved in endothelial barrier breakdown in systemic inflammation and sepsis

AIMS Microvascular endothelial barrier breakdown in sepsis precedes organ failure and death in patients. We tested the hypothesis that the formation of endothelium-derived soluble vascular endothelial (VE)-cadherin fragments (sVE-cadherin) is involved in inflammation-induced endothelial barrier disruption. METHODS AND RESULTS Incubation of human dermal microvascular endothelial cells (HDMEC) with tumour necrosis factor-α (TNF-α) and bacterial lipopolysaccharide (LPS) led to endothelial barrier disruption which correlated with significantly increased sVE-cadherin at a size of ∼90 kDa in cell culture supernatants. Inhibition of the VE-cadherin-cleaving disintegrin and metalloproteinase ADAM10 using GI254023X attenuated inflammation-induced formation of sVE-cadherin and endothelial barrier disruption, suggesting ADAM10-mediated shedding as a mechanism underlying sVE-cadherin release. Formation of VE-cadherin fragments at 90 and 110 kDa was observed when recombinant VE-cadherin (rVE-cadherin) was digested with recombinant ADAM10. Mass spectrometry of the VE-cadherin fragments showed that they originated from cleavage of the extracelluar domain and thereby several cleavage sites of ADAM10 were identified. Atomic force microscopy measurements demonstrated that cell culture supernatants containing sVE-cadherin and application of rVE-cadherin blocked VE-cadherin binding. Accordingly rVE-cadherin dose-dependently led to loss of endothelial barrier functions in HDMEC monolayers. Finally, in patients suffering from severe sepsis or septic shock with clinical signs of a microvascular leackage, serum levels of sVE-cadherin were significantly increased. CONCLUSION Taken together, formation of sVE-cadherin is associated and contributes to inflammation-induced breakdown of endothelial barrier functions by inhibition of VE-cadherin binding. The underlying mechanism of VE-cadherin cleavage involves ADAM10 and appears to be of clinical relevance since sVE-cadherin was augmented in patients with severe sepsis.

CIP2A Influences Survival in Colon Cancer and Is Critical for Maintaining Myc Expression

The cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncogenic factor that stabilises the c-Myc protein. CIP2A is overexpressed in several tumours, and expression levels are an independent marker for long-term outcome. To determine whether CIP2A expression is elevated in colon cancer and whether it might serve as a prognostic marker for survival, we analysed CIP2A mRNA expression by real-time PCR in 104 colon cancer samples. CIP2A mRNA was overexpressed in colon cancer samples and CIP2A expression levels correlated significantly with tumour stage. We found that CIP2A serves as an independent prognostic marker for disease-free and overall survival. Further, we investigated CIP2A-dependent effects on levels of c-Myc, Akt and on cell proliferation in three colon cancer cell lines by silencing CIP2A using small interfering (si) and short hairpin (sh) RNAs. Depletion of CIP2A substantially inhibited growth of colon cell lines and reduced c-Myc levels without affecting expression or function of the upstream regulatory kinase, Akt. Expression of CIP2A was found to be dependent on MAPK activity, linking elevated c-Myc expression to deregulated signal transduction in colon cancer.

Hyperthermic Intraperitoneal Chemotherapy in Patients with Peritoneal Carcinomatosis: Role of Heat Shock Proteins and Dissecting Effects of Hyperthermia

BackgroundIn patients with isolated peritoneal carcinomatosis (PC) of gastrointestinal cancer, hyperthermic intraperitoneal chemotherapy (HIPEC) represents a promising treatment option integrated into multimodal concepts. Heat shock proteins (HSP) seem to play a major role in cellular stress during HIPEC therapy. We analyzed differentially hyperthermic conditions and HSPs responsible for cell stress–mediated repair mechanisms in tumor tissues from patients who underwent HIPEC therapy and in an in vitro hyperthermic model.MethodsTumor tissues from our patient cohort with isolated PC were selected for further analysis when representative material was available before and after HIPEC therapy. To further dissect the role of HSPs under conditions of hyperthermia, gene and protein expression was additionally determined, together with cellular apoptosis and proliferation in human HT-29 colon cancer cells.ResultsDifferently up-regulated HSP70/72 and HSP90 gene and protein expression was found in all investigated patient tumors. In vitro studies confirmed observations from clinical tumor analysis as underlying HSP-mediated cell stress mechanisms. Moreover, results from proliferation and apoptosis assays combined with differentiated HSP expression analysis demonstrated the relevance of preselecting specific target temperatures to achieve optimal toxic effects on remaining tumor cells in vivo.ConclusionsTherapeutic approaches like HIPEC to achieve antiproliferative and apoptosis-inducing cellular effects in patients with PC are negatively influenced by highly conserved HSP mechanisms in tumor cells. This study shows for the first time that specific hyperthermic conditions are necessary to be established to achieve optimal toxic effects on tumor cells during HIPEC therapy, a finding that opens potentially new therapeutic strategies.

What happens in stapled transanal rectum resection

INTRODUCTION: Stapled transanal rectum resection is becoming increasingly popular as a surgical option for the treatment of obstructive defecation syndrome. However, details about the anatomical changes produced by stapled transanal rectum resection and its correlation with success or failure is poorly understood. The aim of this study was to correlate the defecographical and clinical patterns in patients treated with stapled transanal rectum resection. PATIENTS AND METHODS: Based on a multi-institutional stapled transanal rectum resection registry composed of a total of 182 patients, correlation analysis of clinical and radiological parameters was prospectively obtained from 51 patients with a completed 12-month follow-up. RESULTS: Postoperative defecography shows significant changes in the following parameters: intussusception (89%–19%; P < .0001), enterocele (38%–18%; P = .038), rectocele (mean ± SD: 27.1 ± 7.4 mm to 16.5 ± 9.7 mm; P < .0001), rectal lumen (mean ± SD: 46 ± 11.4 mm to 35 ± 9.9 mm; P < .0001), anorectal angle (mean ± SD: 146.4 ± 10.6° to 132.4 ± 11.1°; P = .002), pelvic floor descent (mean ± SD: 59 ± 18 mm to 47 ± 1.3 mm; P = .0001), and, as a dynamic parameter, dynamic pelvic floor descent (mean ± SD: 30 ± 0.8 mm to 17 ± 0.4 mm; P < .0001). Of these parameters, reduction of intussusception (r = 0.433, 95% CI 0.15–0.61; P = .003), rectocele (r = 0.507, 95% CI 0.26–0.67; P = .001), and dynamic pelvic floor descent (r = 0.427, 95% CI 0.31–0.64; P = .001) correlated with a significant improvement in constipation. Reduction of intussusception positively affected postoperative continence (r = 0.524, 95% CI 0.29–0.70; P = .001), whereas reduced rectal lumen size correlated with incontinence and fecal urgency (r = −0.557, 95% CI −0.69 to −0.28; P = .001). CONCLUSIONS: Improved constipation after stapled transanal rectum resection is associated with improvement of intussusception, rectocele, and dynamic pelvic floor descent. Postoperative continence is determined by 2 parameters, reduction of intussusception and rectal lumen size, which have opposing effects. Reduction of rectal lumen size may be responsible for new-onset fecal urgency, which is occasionally seen after stapled transanal rectum resection.