The pan-deacetylase inhibitor panobinostat affects angiogenesis in hepatocellular carcinoma models via modulation of CTGF expression.

Post-translational modifications of chromatin components are significantly involved in the regulation of tumor suppressor gene and oncogene expression. Connective tissue growth factor (CTGF) is an epigenetically regulated growth factor with functions in angiogenesis and cell-matrix interactions and plays a pivotal role in hepatocellular carcinoma (HCC). The pharmacologic inhibition of histone and protein deacetylases represents a new approach to interfere with pathways of apoptosis and angiogenesis. We investigated the effect of the pan-deacetylase inhibitor panobinostat (LBH589) on human HCC cell lines HepG2 (p53wt) and Hep3B (p53null) and in a subcutaneous xenograft model and explored the influence on angiogenesis. Specimens were characterized by quantitative real-time PCR. Protein was separated for western blotting against CTGF, VEGF, VEGF receptor-1 (VEGFR-1/FLT-1), VEGF receptor-2 (VEGFR-2/KDR), MAPK and phospho-MAPK. In vivo, HepG2 cells were xenografted to NMRI mice and treated with daily i.p. injections of 10 mg/kg panobinostat. After 1, 7 and 28 days, real-time PCR was performed. Immunohistochemistry and western blotting were examined after 28 days. An increased significant expression of CTGF was only seen after 24 h treatment with 0.1 µM panobinostat in HepG2 cells and Hep3B cells, whereas after 72 h treatment CTGF expression clearly decreased. In the xenografts, treatment with panobinostat showed a minimal CTGF expression after 1 day and 4 weeks, respectively. In vitro as well as in vivo, VEGF was not affected by panobinostat treatment at any time. In conclusion, panobinostat influences extracellular signaling cascades via CTGF-dependent pathways.

Activated hedgehog pathway is a potential target for pharmacological intervention in biliary tract cancer.

Hedgehog (Hh) signalling contributes to carcinogenesis and represents a valid druggable target in human cancers, possibly also in biliary tract cancer (BTC). We analysed the expression of Hh components in BTC using eight heterogeneously differentiated cell lines, xenograft tumours and a human tissue microarray. The dose-, time- and cell line-dependent effects of two Hh inhibitors (cyclopamine and Gant-61) were analysed in vitro for survival, apoptosis, cell cycle distribution and possible synergism with conventional chemotherapeutic agents. In human BTC samples, the sonic Hh ligand and the Gli1 transcription factor showed increased expression in tumours compared to normal adjacent tissue and were significantly associated with high tumour grade and positive lymph node status. In BTC cell lines, we could confirm the Hh component expression at varying extent within the employed cell lines in vitro and in vivo indicating non-canonical signalling. Both Hh inhibitors showed dose-dependent cytotoxicity above 5 µM with a stronger effect for Gant-61 inducing apoptosis whereas cyclopamine rather inhibited proliferation. Cytotoxicity was associated with low cytokeratin expression and higher mesenchymal marker expression such as vimentin. Additionally, drug combinations of Gant-61 with conventional chemotherapy (cisplatin) exerted synergistic effects. In conclusion, Hh pathway is significantly activated in human BTC tissue compared to normal adjacent tissue. The current data demonstrate for the first time an effective anticancer activity of especially Gant-61 in BTC and suggest second generation Hh pathway inhibitors as a potential novel treatment strategy in BTC.

The pan-deacetylase inhibitor panobinostat modulates the expression of epithelial-mesenchymal transition markers in hepatocellular carcinoma models.

Deacetylase inhibitors (DACis) represent a novel therapeutic option for human cancers by classically affecting proliferation or apoptosis. Since transdifferentiation and dedifferentiation play a key role in carcinogenesis, we investigated the epigenetic influence on the molecular differentiation status in human hepatocellular carcinoma (HCC) models. Markers of differentiation, including cytokeratin (Ck) 7, Ck8, Ck18, Ck19, Ck20, vimentin, sonic hedgehog homolog (SHH), smoothened (Smo), patched (Ptc), glioma-associated oncogene homolog 1 (Gli1), CD133, octamer-binding transcription factor 4 (Oct4) and ß-catenin, were examined in the human HCC cell lines HepG2 and Hep3B in vitro and in vivo (xenograft model) using quantitative real-time PCR and immunohistochemistry following treatment with the pan-DACi panobinostat (LBH589). Compared to untreated controls, treated HepG2 xenografts, and to a lesser extent cell lines, demonstrated a significant increase of differentiation markers Ck7 and Ck19 (classical cholangiocellular type) and Ck8 and Ck18 (classical HCC type), and a decreased level of dedifferentiation markers vimentin (mesenchymal) and SHH/Ptc (embryonic), paralleled with a more membranous expression of ß-catenin. These findings were dose-dependently correlated with tumor size, necrosis rate, microvessel density and mitosis/Ki-67-associated proliferation rate. Our results demonstrate that the differentiation status of human HCC cells is influenced by the pan-DACi panobinostat, indicating that this treatment may influence the epithelial-mesenchymal transition (EMT) status related to metastasis and aggressiveness.

Pancreatic cancer cells surviving gemcitabine treatment express markers of stem cell differentiation and epithelial-mesenchymal transition.

Objective response rates to standard chemotherapeutic regimens remain low in pancreatic cancer. Subpopulations of cells have been identified in various solid tumors which express stem cell-associated markers and are associated with increased resistance against radiochemotherapy. We investigated the expression of stem cell genes and markers of epithelial-mesenchymal transition in pancreatic cancer cells that survived high concentrations of gemcitabine treatment. Capan-1 and Panc-1 cells were continuously incubated with 1 and 10 µM gemcitabine. Surviving cells were collected after 1, 3 and 6 days. Expression of PDX-1, SHH, CD24, CD44, CD133, EpCAM, CBX7, OCT4, SNAIL, SLUG, TWIST, Ki-67, E-cadherin, ß-catenin and vimentin were quantified by qPCR or immunocytochemistry. Migration was assessed by wound­healing assay. SHH was knocked down using RNA interference. Five primary pancreatic cancer cell lines were used to validate the qPCR results. All investigated genes were upregulated after 6 days of gemcitabine incubation. Highest relative expression levels were observed for OCT4 (13.4-fold), CD24 (47.3-fold) and EpCAM (15.9-fold) in Capan-1 and PDX-1 (13.3­fold), SHH (24.1-fold), CD44 (17.4-fold), CD133 (20.2-fold) and SLUG (15.2-fold) in Panc-1 cells. Distinct upregulation patterns were observed in the primary cells. Migration was increased in Panc-1 cells and changes in the expression of E-cadherin and ß-catenin were typical of epithelial-mesenchymal transition in both cell lines. SHH knockdown reduced IC(50) from 30.1 to 27.6 nM in Capan-1 while it strongly inhibited proli-feration in Panc-1 cells. Cells surviving high-dose gemcitabine treatment express increased levels of stem cell genes, show characteristics associated with epithelial-mesenchymal transition and retain their proliferative capacity.

Inhibition of DNA methyltransferase activity and expression by treatment with the pan-deacetylase inhibitor panobinostat in hepatocellular carcinoma cell lines.

BACKGROUND: Hepatocellular carcinoma (HCC) still represents an unmet medical need. Epigenetic inactivation of tumor suppressor genes like RASSF1A or APC by overexpression of DNA methyltransferases (DNMTs) has been shown to be common in HCC and to be linked to the overall prognosis of patients. Inhibitors of protein and histone deacetylases (DACi) have been demonstrated to possess strong anti-tumor effects in HCC models. METHODS: We therefore investigated whether DACi also has any influence on the expression and activity of DNMTs and methylated target genes in HepG2 and Hep3B cell culture systems and in a xenograft model by immunohistochemistry, westernblotting, RT-qPCR and methylation-specific PCR. RESULTS: Our findings demonstrate a rapid inhibition of DNMT activity 6 h after treatment with 0.1 µM of the pan-DACi panobinostat. A downregulation of DNMT mRNAs and protein were also observed at later points in time. This loss of DNMT activity and expression was paralleled by a diminished methylation of the target genes RASSF1A and APC and a concomitant re-expression of APC mRNA and protein. Analysis of HepG2 xenograft specimens confirmed these results in vivo. CONCLUSION: We suggest a dual mode of action of DACi on DNA methylation status: a rapid inhibition of enzyme activity due to interference with posttranslational acetylation and a delayed effect on transcriptional control of DNMT genes by HDAC or miRNA mechanisms.

Downregulation of HMGA2 by the pan-deacetylase inhibitor panobinostat is dependent on hsa-let-7b expression in liver cancer cell lines.

Inhibitors of protein deacetylases represent a novel therapeutic option for cancer diseases due to their effects on transcriptional regulation by interfering with histones acetylation and on several other cellular pathways. Recently, their ability to modulate several transcription factors and, interestingly, also co-factors, which actively participate in formation and modulation of transcription complexes was shown. We here investigate whether HMGA2 (High Mobility Group AT-2 hook), a nuclear non-histone transcriptional co-factor with known oncogenic properties, can be influenced by the novel pan-deacetylase inhibitor panobinostat (LBH589) in human hepatocellular carcinoma models. Panobinostat strongly downregulated HMGA2 in HepG2 and Hep3B cells; this effect was mediated by transcriptional upregulation and promotion of the maturation of the tumorsuppressor miRNA hsa-let-7b, which could inhibit HMGA2 expression via RNA interference pathways. siRNA knockdown of HMGA2 or transfection of hsa-let-7b mimicking oligonucleotides confirmed the role of HMGA2 in regulating cell proliferation and apoptosis in liver cancer cell lines. Co-incubation with panobinostat showed an additive effect on inhibition of cell proliferation using an impedance-based real-time cell analyzer. Treatment of HepG2 xenografts with panobinostat also led to a downregulation of HMGA2 in vivo. These findings show that pan-deacetylase inhibitors also modulate other signaling pathways and networks than histone modifications to influence cell fate.

Association of stem cell marker expression pattern and survival in human biliary tract cancer.

The aim of this study was to investigate the molecular and protein expression pattern of markers of stemness phenotype and its clinicopathological significance in human biliary tract cancer (BTC). Human BTC cell lines (CCLP-1, Egi-1, MzChA-1, MzChA-2, SkChA-1, TFK-1 and GBC) were analyzed in vitro and in xenotransplanted animals for expression of markers of stemness and compared to tissue microarrays (TMA) of 34 cases of human BTC with complete pathomorphological and clinical data (survival). Molecular analyses on the mRNA and protein level included makers of stemness and progenitor (Bmi-1, Sox-2, Nestin, CD133, CD44 and Nanog), proliferation and differentiation (cell cycle proteins, intermediate filaments). The investigated BTC samples showed a low to moderate and partially significantly different expression pattern of the stem cell markers in vitro, in vivo and in TMA. Hierarchical cluster analysis identified subgroups with homogenous expression of stem cell markers significantly differing with respect to cytokeratin expression in xenografts and Ki67 proliferation marker in human TMA, respectively - thus indicating possible heterogeneous carcinogenesis pathways in BTC. Additionally, these stem cell markers could be linked to morphology and molecular markers of proliferation and differentiation on the mRNA and protein level. Finally, survival analysis identified the combination of CD133 and CD44 as an independent prognostic factor yet their value as prognostic factors need testing in prospective study design.

Influence of five potential anticancer drugs on wnt pathway and cell survival in human biliary tract cancer cells.

BACKGROUND: The role of Wnt signalling in carcinogenesis suggests compounds targeting this pathway as potential anti-cancer drugs. Several studies report activation of Wnt signalling in biliary tract cancer (BTC) thus rendering Wnt inhibitory drugs as potential candidates for targeted therapy of this highly chemoresistant disease. METHODS: In this study we analysed five compounds with suggested inhibitory effects on Wnt signalling (DMAT, FH535, myricetin, quercetin, and TBB) for their cytotoxic efficiency, mode of cell death, time- and cell line-dependent characteristics as well as their effects on Wnt pathway activity in nine different BTC cell lines. RESULTS: Exposure of cancer cells to different concentrations of the compounds results in a clear dose-dependent reduction of viability for all drugs in the order FH535 > DMAT > TBB > myricetin > quercetin. The first three substances show high cytotoxicity in all tested cell lines, cause a direct cytotoxic effect by induction of apoptosis and inhibit pathway-specific signal transduction in a Wnt transcription factor reporter activity assay. Selected target genes such as growth-promoting cyclin D1 and the cell cycle progression inhibitor p27 are down- and up-regulated after treatment, respectively. CONCLUSIONS: Taken together, these data demonstrate that the small molecular weight inhibitors DMAT, F535 and TBB have a considerable cytotoxic and possibly Wnt-specific effect on BTC cell lines in vitro. Further in vivo investigation of these drugs as well as of new Wnt inhibitors may provide a promising approach for targeted therapy of this difficult-to-treat tumour.

Current status of therapeutic targeting of developmental signalling pathways in oncology.

Signalling pathways such as Hedgehog (Hh), Wnt, Notch, bone morphogenetic protein (BMP) and transforming growth factor-ß (TGF-ß) hold a central position in regulation of vertebrate development by controlling vital processes such as migration, differentiation and proliferation. Insights into the mechanistic aspects of cancer initiation and progression have pointed to striking similarities between tumourigenesis and embryonic development. These observations can partly be explained by the fact that similar cellular signalling mechanisms are employed in both situations. This review focuses on the role and therapeutic potential of Hh, Wnt, Notch and BMP/TGF-ß signalling and discusses i) their signal transduction mechanisms during development and tumourigenesis, ii) evidence of pathway activation in different types of cancers, and, iii) strategies for pharmacological targeting. Numerous studies have demonstrated a crucial role of developmental signalling in a variety of tumours, where their signalling mechanisms contribute to oncogenic properties such as tumour cell proliferation, apoptosis inhibition and / or metastatic migration. From the literature available, it is obvious that the relative importance and the oncogenic mechanisms of developmental pathways vary with the tumour type, the stage of the disease as well as the interaction with the tumour microenvironment, thus highlighting the complexity of cellular signalling strategies employed during tumourigenesis. Intensive research activities are devoted to identification of drugs that interfere with oncogenic signalling by developmental pathways. First clinical data for such compounds--e.g. GDC-0449 for the Hh pathway--are promising and indicate that targeted therapy of developmental signalling pathways has potential for future anti-cancer therapies.

pS6 Expression in normal renal parenchyma, primary renal cell carcinomas and their metastases.

In cancer therapy novel concepts focus on phosphoinositide-3-kinase/protein kinase B/mammalian target of rapamycin (mTOR) inhibitors. In this context, phosphorylated S6 protein of the 40S ribosomal subunit (pS6) overexpression was previously shown to be associated with sensitivity to inhibitors of mTOR. The present study therefore evaluated pS6 expression in normal renal parenchyma (NRP), primary renal cell carcinomas (PRCC) and their metastases. pS6 and pmTOR expression was immunohistochemically analyzed in a tissue microarray (TMA) from localized primary renal cell carcinoma (lPRCC) (n = 35), metastasized primary renal cell carcinoma (mPRCC) (n = 45), their metastases (n = 45), and NRP (n = 45). pS6 expression was stronger in mPRCCs and metastases than in NRP and lPRCCs (p < 0.05). In mPRCCs high-grade and high-stage tumors showed higher pS6 levels. pS6 overexpression was more frequently found in metastases (40/45; 88.9%) than in mPRCC (24/45; 53.3%) (p < 0.05). Overexpression of pS6 in metastases without concomitant overexpression in their primary tumors was found in 16/45 (35.56%) cases. Patients with pS6 overexpression in mPRCCs but also in metastases showed a tendency to shorter overall survival. pS6 score and pmTOR score correlated positively in NRP and in tumorous tissue (mPRCC and metastases). In conclusion, the present study showed stronger pS6 expression and more frequent overexpression in metastases than in corresponding PRCCs. In approximately one-third of the cases pS6 overexpression was found exclusively in metastases, which is interesting with regard to the association between high pS6 expression and sensitivity to mTOR inhibitor therapy.

Differential bone marrow homing capacity of VLA-4 and CD38 high expressing chronic lymphocytic leukemia cells.

BACKGROUND: VLA-4 and CD38 predict a poor clinical outcome in chronic lymphocytic leukemia (CLL). We used CLL samples with discordant VLA-4/CD38 risk to address their individual roles in human bone marrow infiltration (BM), CLL cell homing to murine BM, and in supportive CLL cell-stromal cell interactions. METHODS: VLA-4, CD38, and Ki-67 expression was measured in CLL cells from peripheral blood (PB) and bone marrow (BM) aspirates. CLL BM infiltration rates, routinely determined by Pathology, were correlated to VLA-4 and CD38 expression. Short-term homing capacity of CLL cells was evaluated by adoptive transfer experiments. CLL cell viability and adhesion in stromal cell co-culture was determined. RESULTS: About 20% of CLL samples in our cohort displayed discordant VLA-4 and CD38 risk, with either high VLA-4 and low CD38 risk or vice versa. Using particularly such samples, we observed that VLA-4, and not CD38, was responsible for recirculation of CLL cells to murine BM. Human BM infiltration was also significantly higher in patients with high VLA-4 risk but not high CD38 risk. However, both molecules acted as independent prognostic markers. While both VLA-4 and CD38 expression were increased in BM-derived CLL cells, and VLA-4+ and CD38+ subpopulations showed enriched Ki-67 expression, VLA-4 did not contribute to CLL cell protection by stromal cells in vitro. CONCLUSIONS: Our data argue for a prominent role of VLA-4 but not CD38 expression in the homing of CLL cells to BM niches and in human BM infiltration, but only a limited role in their protection by stromal cells.

Myelodysplastic syndrome and histone deacetylase inhibitors: "to be or not to be acetylated"?

Myelodysplastic syndrome (MDS) represents a heterogeneous group of diseases with clonal proliferation, bone marrow failure and increasing risk of transformation into an acute myeloid leukaemia. Structured guidelines are developed for selective therapy based on prognostic subgroups, age, and performance status. Although many driving forces of disease phenotype and biology are described, the complete and possibly interacting pathogenetic pathways still remain unclear. Epigenetic investigations of cancer and haematologic diseases like MDS give new insights into the pathogenesis of this complex disease. Modifications of DNA or histones via methylation or acetylation lead to gene silencing and altered physiology relevant for MDS. First clinical trials give evidence that patients with MDS could benefit from epigenetic treatment with, for example, DNA methyl transferase inhibitors (DNMTi) or histone deacetylase inhibitors (HDACi). Nevertheless, many issues of HDACi remain incompletely understood and pose clinical and translational challenges. In this paper, major aspects of MDS, MDS-associated epigenetics and the potential use of HDACi are discussed.

Role of cannabinoid receptors and RAGE in inflammatory bowel disease.

BACKGROUND: The endocannabinoid system is involved in many inflammatory diseases, such as Crohn's disease (CD) and ulcerative colitis (UC). The distribution and expression of cannabinoid receptors 1 (CNR1) and 2 (CNR2) in combination with inflammatory cytokines and RAGE (receptor of advanced glycation end products), which is also overactive in these diseases, in dependency of the extent of inflammation and alteration of the colon barrier is still unclear and needs to be elucidated. MATERIAL AND METHODS: 10 specimens of CD patients who underwent colectomy and 14 colectomy specimens of patients suffering from UC were investigated histologically for inflammatory infiltrate, extent of fibrosis and for disturbance of the intestinal barrier. Immunohistochemistry was carried out to examine the distribution and localization of CNR1, CNR2 and RAGE. Additionally, qRT-PCR was performed to study the expression of CNR1, CNR2, RAGE and inflammatory cytokines (TNFα, TGFß, CTGF, IL12, IFNγ). 35 morphological and histological normal specimens of colectomy cases served as controls. RESULTS: The expression level of CNR2 did not differ between the control group and the group of patients with IBD, while CNR1 displayed a significant up regulation, especially in cases of CD. A differential association between the expression of CNR1/CNR2 and RAGE with morphological changes and expression of molecular markers of inflammation could be established. CONCLUSION: We showed that cannabinoid receptors are expressed differentially in inflammatory bowel disease and that the expression seems to be influenced by the underlying disease and by localized inflammation.

Uptake and phototoxicity of meso-tetrahydroxyphenyl chlorine are highly variable in human biliary tract cancer cell lines and correlate with markers of differentiation and proliferation.

Photodynamic therapy (PDT) using Photofrin and, recently, Foscan has gained broad acceptance for palliative treatment of non-resectable cholangiocarcinoma (CC). No information, however, is available whether the phenotype of CC tumour cells has an effect on the efficiency of the treatment. Using a well-characterised set of n = 9 biliary tract cancer cell lines this study investigated the uptake, phototoxicity, and release of meso-tetrahydroxyphenyl chlorine (mTHPC, Foscan) after incubation with 200 or 400 ng ml(-1) mTHPC. For uptake of mTHPC we found great variations between the individual cell lines (up to a factor 2), resulting in even more pronounced differences in phototoxicity. Based on statistical classification by hierarchical cluster analysis, two groups of cell lines can be distinguished which are characterised by either high or low susceptibility towards mTHPC-based photodynamic treatment. Correlation analysis with previously established immunochemical parameters showed that cells with a low cytokeratin-19 (ductal differentiation), high vimentin (mesenchymal marker), and high proliferative phenotype preferentially show higher uptake of mTHPC and subsequent phototoxicity. These results demonstrate high variability of biliary tract cancer cells when subjected to mTHPC-based photodynamic treatment and identify possible markers that could be used in the clinical setting in order to predict the efficiency of PDT and adjust the dose for complete tumour elimination.

The pan-deacetylase inhibitor panobinostat inhibits growth of hepatocellular carcinoma models by alternative pathways of apoptosis.

Inhibition of deacetylases represents a new treatment option for human cancer diseases. We applied the novel and potent pan-deacetylase inhibitor panobinostat (LBH589) to human hepatocellular carcinoma models and investigated by which pathways tumor cell survival is influenced. HepG2 (p53wt) and Hep3B (p53null) responded to panobinostat treatment with a reduction of cell proliferation and a significant increase in apoptotic cell death at low micromolar concentrations. Apoptosis was neither mediated by the extrinsic nor the intrinsic pathway but quantitative RT-PCR showed an upregulation of CHOP, a marker of the unfolded protein response and endoplasmic reticulum stress with subsequent activation of caspase 12. Dependent on the p53 status, a transcriptional upregulation of p21(cip1/waf1), an increased phosphorylation of H2AX, and an activation of the MAPK pathway were observed. In a subcutaneous xenograft model, daily i.p. injections of 10 mg/kg panobinostat lead to a significant growth delay with prolonged overall survival, mediated by reduced tumor cell proliferation, increased apoptosis and reduced angiogenesis in tumor xenografts. Panobinostat increased the acetylation of histones H3 and H4. Panobinostat is a well tolerated new treatment option for HCC that activates alternative pathways of apoptosis, also in p53-deficient tumors.

Active Wnt signalling is associated with low differentiation and high proliferation in human biliary tract cancer in vitro and in vivo and is sensitive to pharmacological inhibition.

Activation of developmental pathways has been recognized as a key mechanism for tumourigenesis and, hence, might be a valuable target for otherwise difficult to treat tumour entities such as biliary tract cancer (BTC). Therefore, we performed a comprehensive analysis of the Wnt signalling pathway in 9 BTC cell lines on cell blocks, xenograft tumours and on human tissue microarrays by real-time reverse transcription PCR and by immunochemistry. Furthermore, the effects of pharmacological pathway inhibition were investigated. As a result we found a significant positive correlation of Wnt pathway activation with cyclin D1 expression and the proliferation parameters Ki67, cell cycle distribution, and growth kinetics as well as the mesenchymal marker vimentin and an inverse correlation with E-cadherin in BTC cell lines in vitro and in vivo. In human BTC samples loss of membranous beta-catenin, an indicator of active Wnt signalling, correlated with vimentin expression and advanced tumour stage or metastasis, whereas membranous localisation of beta-catenin was associated with the differentiation marker cytokeratin-8/18 and differentiated tumour morphology (ductal or mixed type BTC). In addition, Wnt pathway inhibition by DMAT effectively reduced viability in all cancer cell lines, most effectively in those showing cytoplasmatic beta-catenin localisation, i.e. active Wnt signalling. In summary, activation of the Wnt pathway is associated with high proliferation, dedifferentiation and a solid morphology in human biliary tract cancer cell lines both in vitro and in vivo, and in human BTC tissues. Further investigation of the mechanism(s) of Wnt pathway activation and its inhibition may provide new molecular treatment strategies for biliary tract cancer.

Hedgehog signaling is involved in differentiation of normal colonic tissue rather than in tumor proliferation.

The Hedgehog (Hh) pathway is a main regulation cascade in embryonic differentiation. It is also present in adult tissues and unusual expression has been associated with formation of benign and malignant lesions. We examined the presence of the Hedgehog pathway in normal and pathological human colon tissue. Components investigated include Sonic (Shh), Indian (Ihh), and Desert Hedgehog (Dhh), Gli1, Gli2, Gli3, and Patched (Ptch). Pathological tissue samples comprised 23 benign and 20 malignant lesions of human colon. The influence of the Hedgehog pathway on differentiation and proliferation has been investigated by analyzing the effect of the pathway inhibitor Cyclopamine on human colon cancer cell lines HT29 and CaCo2. In normal colon, we detected expression of Shh and Dhh within the lining epithelium and Patched, Gli1, and Gli2 along the whole crypts. Within all benign lesions, positive staining of Shh, Dhh, Gli1, Gli2, and Ptch was detected. Expression of Shh and Dhh was restricted to single cell aggregates. Malignant lesions also displayed focal staining pattern for Shh and Dhh but to a much lesser extent. We conclude that Hedgehog signaling is involved rather in constant differentiation and renewing of the colonic lining epithelium than in cancer formation, growth, or proliferation.

The expression pattern of PDX-1, SHH, Patched and Gli-1 is associated with pathological and clinical features in human pancreatic cancer.

BACKGROUND AND AIMS: Pancreatic cancer cells have been shown to possess stem-cell-like properties, especially by reactivating embryonic transcription factors involved in tissue differentiation. We therefore investigated if and to what extent developmental genes of the human pancreas are expressed in pancreatic ductal adenocarcinomas and precursor lesions, pancreatic intraepithelial neoplasia (PanIN), and if this correlates or predicts response to treatment and overall survival. MATERIAL AND METHODS: Invasive ductal adenocarcinomas of the pancreas [UICC pT3pN0 (n = 13) vs. pT3pN1 (n = 25)] and tumors after neoadjuvant chemotherapy [5-fluorouracil (FU)/folic-acid and gemcitabine; UICC ypN0 (n = 7) vs. ypN1 (n = 6)] resected between 1997 and 2003 were characterized histochemically and immunohistochemically [pancreas duodenum homeobox 1 (PDX-1), Sonic hedgehog protein (SHH), Patched (Ptc) and Gli-1]. Gene distribution was compared with morphological patterns of the pancreatic carcinoma and PanIN as well as with peritumorous reactions of normal pancreas. RESULTS: The overall expression of PDX-1, SHH, Ptc and Gli-1 was low, but showed a distinctive and topographic linkage inside pancreatic carcinomas as well as inside PanINs. Additionally, a topographic and significant association of these markers with nodal status (PDX-1, Ptc, Gli-1), tumor size (PDX-1, Gli-1) and R status (PDX-1) was found. After stratification with the strongest outcome predictor, grading, survival analysis revealed that Ptc expression in grade 2 and PDX-1 expression in grade 3 carcinomas are independent survival factors. CONCLUSIONS: Markers of pancreas development are reexpressed in invasive ductal adenocarcinomas and their expression is essentially associated with general clinical and pathological features such as survival or nodal status.

The dual EGF/VEGF receptor tyrosine kinase inhibitor AEE788 inhibits growth of human hepatocellular carcinoma xenografts in nude mice.

We investigated the effect of AEE788, a novel dual receptor tyrosine kinase inhibitor of the EGF and the VEGF receptor, for treatment of human HCC cell lines and in a subcutaneous xenograft model. Cell viability and apoptosis of HepG2 and Hep3B cells incubated with 0.1-100 microM AEE788 were quantified. In vivo, HepG2 cells were xenografted to NMRI mice and animals were treated orally with 50 mg/kg AEE788 3x/week. Immunohistochemistry and quantitative Western blotting was performed for pathway analysis in vitro and in vivo. AEE788 reduced growth and induced apoptosis of HCC cells by disrupting mitochondrial transmembrane potentials and inhibiting MAPK phosphorylation. In the xenografts, AEE788 lead to a reduced tumor growth by reducing proliferation and vascularisation. Except for a reversible skin reaction and weight loss, no signs of toxicity were observed. AEE788 is a promising new option for the treatment of HCC.

Gene gun immunization with clinically relevant allergens aggravates allergen induced pathology and is contraindicated for allergen immunotherapy.

Gene gun immunization has been associated with the induction of a heterologous type of immune response characterized by a T(H)1-like immune reaction on the cellular level, i.e. generation of IFN-gamma secreting CD8(+) T-cells, yet a T(H)2 biased serology as indicated by high IgG1:IgG2a ratios and induction of IgE. Nevertheless, gene gun immunization using the model molecule beta-galactosidase has been argued to prevent IgE induction and to promote T(H)1 cells with respect to allergy DNA immunization. In our current study, we evaluated the potential of gene gun immunization to prevent type I allergic reactions comparing beta-galactosidase with two clinically relevant allergens, and further investigated the effect of gene gun immunization on relevant lung parameters. BALB/c mice were immunized with plasmids encoding the birch pollen allergen Bet v 1, the grass pollen allergen Phl p 5, or the model molecule beta-galactosidase, either by gene gun or intradermal injection followed by sensitization and intranasal provocation with the respective allergen. IgG1 and IgG2a antibody titers were determined by ELISA. IgE levels were evaluated in a rat basophil release assay. The severity of eosinophilia was determined in bronchoalveolar lavages, and the overall infiltrate was analyzed by histology on lung paraffin sections. Gene gun immunization induced a T(H)2-biased immune reaction, which did not prevent from production of IgE after subsequent sensitization. This T(H)2 effect was influenced by the nature of the antigen, with a more pronounced T(H)2-bias for the allergens Bet v 1 and Phl p 5 compared to beta-galactosidase. Gene gun immunization with all three antigens promoted eosinophil influx into the lung and did not alleviate lung pathology after intranasal provocation. In contrast to needle injection of plasmid DNA, which triggers a clearly T(H)1-biased and allergy-preventing immune response, gene gun application fails to induce anti-allergic reactions with all tested antigens and is therefore contraindicated for allergen-specific immunotherapy.