Simultaneous Inhibition of Mcl-1 and Bcl-2 Induces Synergistic Cell Death in Hepatocellular Carcinoma.

Despite the recent approval of new therapies, the prognosis for patients with hepatocellular carcinoma (HCC) remains poor. There is a clinical need for new highly effective therapeutic options. Here, we present a combined application of BH3-mimetics as a potential new treatment option for HCC. BH3-mimetics inhibit anti-apoptotic proteins of the BCL-2 family and, thus, trigger the intrinsic apoptosis pathway. Anti-apoptotic BCL-2 proteins such as Bcl-2 and Mcl-1 are frequently overexpressed in HCC. Therefore, we analyzed the efficacy of the two BH3-mimetics ABT-199 (Bcl-2 inhibitor) and MIK665 (Mcl-1 inhibitor) in HCC cell lines with differential expression levels of endogenous Bcl-2 and Mcl-1. While administration of one BH3-mimetic alone did not substantially trigger cell death, the combination of two inhibitors enhanced induction of the intrinsic apoptosis pathway. Both drugs acted synergistically, highlighting the effectivity of this specific BH3-mimetic combination, particularly in HCC cell lines. These results indicate the potential of combining inhibitors of the BCL-2 family as new therapeutic options in HCC.

Liver stiffness assessed by shear-wave elastography declines in parallel with immunoregulatory proteins in patients with chronic HCV infection during DAA therapy.

BACKGROUND: A rapid decline of liver stiffness (LS) was detected by non-invasive methods in patients with chronic hepatitis C (HCV) infection during treatment with direct-acting antivirals (DAA). OBJECTIVE: To investigate the influence of inflammation on LS. METHODS: We prospectively examined LS by sonographic shear-wave elastography in 217 patients during DAA therapy from treatment initiation (BL) to 12 weeks after end of therapy (SVR12). Demographic data, laboratory findings and serum levels of cytokines were determined. RESULTS: Values of LS decreased from 1.86 m/s to 1.68 m/s (p = 0.01) which was most pronounced in patients who had F4 fibrosis at BL (3.27 m/s to 2.37 m/s; p < 0.001). Initially elevated values of aminotransferases, ferritin, IgG (p < 0.001 each) and international normalized ratio (p < 0.003) declined, thrombocyte count (p = 0.007) increased. Correlations of these laboratory parameters with BL levels of LS measurement (LSM) were most apparent in patients with F1-F3 fibrosis. Tumor necrosis factor (TNF)-α (p = 0.031), interleukin (IL)-10 (p = 0.005) and interferon y inducible protein (IP)-10 (p < 0.001) decreased in parallel with LSM under DAA therapy and corelated with BL values. CONCLUSION: Decrease of systemic inflammatory parameters correlated with LSM under DAA therapy. We conclude that regression of LSM is attributable to the decline of inflammation rather than reflecting fibrosis.

Galectin-3 Modulates Experimental Colitis.

BACKGROUND: We recently identified galectin-3 (gal-3) as a new and strong fibroblast activator produced by colonic epithelial cells. Very little is known about the influence of gal-3 in inflammatory bowel disease. We, therefore, investigated the impact of gal-3 on dextran sodium sulfate (DSS)-induced colitis in a mouse model. METHODS: Colonic lamina propria fibroblasts of healthy controls were used for co-incubation studies of gal-3 with gal-1, TGF-ß1, IFNγ, IL-4 and IL-10. Acute and chronic DSS colitis was induced by 3% DSS in drinking water in female Balb/c mice weighing 20-22 g. Recombinant gal-3 was expressed by the pET vector system and used for a 5-day treatment in different concentrations intraperitoneally. The distal third of the colon was used for histologic analysis. Colonic cytokine expression was determined by quantitative RT-PCR. RESULTS: In vitro, gal-3 induced IL-8 secretion was significantly reduced by co-incubation with IL-10 (5 ng/ml) and IL-4 (10 ng/ml). Acute DSS-induced colitis was ameliorated by gal-3 treatment as indicated by increased colonic length and reduced weight loss compared to that of controls. In acute and chronic colitis, gal-3 treatment resulted in a significant suppression of colonic IL-6. CONCLUSION: Gal-3 significantly reduces inflammation in acute and chronic DSS colitis in mice indicating a potential role in intestinal inflammation.

Expression of growth factor receptors and targeting of EGFR in cholangiocarcinoma cell lines.

BACKGROUND: Cholangiocarcinoma (CC) is a malignant neoplasm of the bile ducts or the gallbladder. Targeting of growth factor receptors showed therapeutic potential in palliative settings for many solid tumors. The aim of this study was to determine the expression of seven growth factor receptors in CC cell lines and to assess the effect of blocking the EGFR receptor in vitro. METHODS: Expression of EGFR (epithelial growth factor receptor), HGFR (hepatocyte growth factor receptor) IGF1R (insulin-like growth factor 1 receptor), IGF2R (insulin-like growth factor 2 receptor) and VEGFR1-3 (vascular endothelial growth factor receptor 1-3) were examined in four human CC cell lines (EGI-1, HuH28, OZ and TFK-1). The effect of the anti-EGFR-antibody cetuximab on cell growth and apoptosis was studied and cell lines were examined for KRAS mutations. RESULTS: EGFR, HGFR and IGFR1 were present in all four cell lines tested. IGFR2 expression was confirmed in EGI-1 and TFK-1. No growth-inhibitory effect was found in EGI-1 cells after incubation with cetuximab. Cetuximab dose-dependently inhibited growth in TFK-1. Increased apoptosis was only seen in TFK-1 cells at the highest cetuximab dose tested (1 mg/ml), with no dose-response-relationship at lower concentrations. In EGI-1 a heterozygous KRAS mutation was found in codon 12 (c.35G>A; p.G12D). HuH28, OZ and TFK-1 lacked KRAS mutation. CONCLUSION: CC cell lines express a pattern of different growth receptors in vitro. Growth factor inhibitor treatment could be affected from the KRAS genotype in CC. The expression of EGFR itself does not allow prognoses on growth inhibition by cetuximab.

Differential protein expression profile in the intestinal epithelium from patients with inflammatory bowel disease.

The loss of intestinal epithelial cell (IEC) function is a critical component in the initiation and perpetuation of chronic intestinal inflammation in the genetically susceptible host. We applied proteome analysis (PA) to characterize changes in the protein expression profile of primary IEC from patients with Crohn's disease (CD) and ulcerative colitis (UC). Surgical specimens from 18 patients with active CD (N = 6), UC (N = 6), and colonic cancer (N = 6) were used to purify primary IEC from ileal and colonic tissues. Changes in protein expression were identified using 2D-gel electrophoreses (2D SDS-PAGE) and peptide mass fingerprinting via MALDI-TOF mass spectrometry (MS) as well as Western blot analysis. PA of primary IEC from inflamed ileal tissue of CD patients and colonic tissue of UC patients identified 21 protein spots with at least 2-fold changes in steady-state expression levels compared to the noninflamed tissue of control patients. Statistical significance was achieved for 9 proteins including the Rho-GDP dissociation inhibitor alpha that was up-regulated in CD and UC patients. Additionally, 40 proteins with significantly altered expression levels were identified in IEC from inflamed compared to noninflamed tissue regions of single UC (N = 2) patients. The most significant change was detected for programmed cell death protein 8 (7.4-fold increase) and annexin 2A (7.7-fold increase). PA in primary IEC from IBD patients revealed significant expression changes of proteins that are associated with signal transduction, stress response as well as energy metabolism. The induction of Rho GDI alpha expression may be associated with the destruction of IEC homeostasis under condition of chronic intestinal inflammation.

A new organotypic model to study cell interactions in the intestinal mucosa.

BACKGROUND: Recently, we demonstrated that freshly elutriated monocytes differentiate into macrophages with a phenotype similar to that of intestinal macrophages in a three-dimensional model of intestinal epithelial cells. Here we describe a more organotypic model to study cell interactions in the intestinal mucosa. METHODS: Primary intestinal fibroblasts and freshly elutriated blood monocytes (ratio 1:1) were embedded in collagen type I gels and cultured for 5 days. At day 5, intestinal epithelial cells (HT-29) were seeded on top of the collagen gels. After another 7 days collagen gels were harvested and fixed for immunohistochemical analysis. Cryosections of the aggregates were prepared and staining for monocyte/macrophage markers and basement membrane compounds was performed. Cell interactions inside the aggregates were examined by electron microscopy. RESULTS: Intestinal fibroblasts contracted the collagen gels which formed stable three-dimensional aggregates within the first 5 days of culture. Intestinal epithelial cells formed a monolayer on top of the gels about 3 days after seeding. Intestinal fibroblasts were distributed randomly over the aggregate. Monocytes inside aggregates were localized in the vicinity to epithelial cells by positive staining for CD68. Typical monocyte/macrophage specific markers such as CD14, CD16, CD11b, CD11c and the co-stimulatory molecules CD80 and CD86 were down-regulated or not detectable on these cells after co-culture in three-dimensional aggregates. Omission of epithelial cells from the model was followed by impaired differentiation of intestinal macrophages. CONCLUSION: In the three-dimensional organotypic cell culture model monocytes differentiate into intestinal-like macrophages when co-cultured with control intestinal fibroblasts and intestinal epithelial cells. Intestinal epithelial cells may be necessary for differentiation of intestinal macrophages.

Reduced migration of fibroblasts in inflammatory bowel disease: role of inflammatory mediators and focal adhesion kinase.

BACKGROUND & AIMS: Crohn's disease (CD) and ulcerative colitis (UC) are associated with chronic tissue damage and continuous tissue repair. A central, but not well-characterized, event during this process is the migration of activated fibroblasts to the wound. METHODS: Human colonic lamina propria fibroblasts (CLPF) were isolated from patients with CD and UC and from healthy controls and were characterized by immunocytochemistry. Migration assays of CLPF were performed in the modified 48-well Boyden chamber. Focal adhesion kinase (FAK) and FAK autophosphorylation in migrating CLPF were determined by Western blotting. FAK mRNA expression was investigated by Northern blotting. RESULTS: The migration of CD-CLPF and UC-CLPF was significantly reduced when compared with control-CLPF. This was correlated with a decrease in FAK phosphorylation, whereas, in migrating control-CLPF, an increase was found. Similarly, the presence of the inflammatory mediators interferon (IFN)-gamma (50 ng/mL) or tumor necrosis factor (TNF) (30 ng/mL) in conditioned medium significantly reduced the migration of control-CLPF to 41% +/- 4% or 30% +/- 7%, respectively. Preincubation of control-CLPF with TNF (20 ng/mL) and IFN-gamma (10 ng/mL) for 3 days reduced their migratory response to 10% of control (P < 0.001), which also was correlated with a decrease in FAK phosphorylation. Culture of IFN-gamma/TNF-treated CLPF for a further 7 days without cytokines did not restore the migratory potential and FAK phosphorylation, indicating a persistent functional change. CONCLUSIONS: CD- and UC-CLPF have a reduced migratory potential compared with normal CLPF. That may be caused by contact with IFN-gamma and TNF. This loss of migratory potential was correlated with diminished FAK phosphorylation.