Abstract: The prognosis of pancreatic cancer remains disappointing due to a high intrinsic resistance against chemotherapeutic agents. Standard gemcitabine therapies have improved overall survival only marginally and recently, inhibition of the proteasome by the boronic acid derivative bortezomib has been introduced as a novel therapeutic strategy for solid and hematological malignancies including pancreatic cancer. The mucus-producing pancreatic cancer cell line Capan-1 was cultured under standard conditions and treated with different concentrations of gemcitabine or bortezomib. Mucus production was suppressed by siRNA-mediated silencing of apomucin genes. Cell proliferation was determined by 3H-thymidine incorporation and apoptosis was quantified after propidium iodide staining by flow cytometry. Apoptotic cell death was confirmed by TUNEL staining, determination of mitochondrial transmembrane potential and assessment of caspase 3/7 activity. NFκB-activity was determined by EMSA. The unfolded protein response (UPR) was further investigated by PCR, Western blotting and caspase 12 activity assays. Silencing of MUC4 significantly reduced expression of mucins for up to 5 days after transfection. While native cells showed an increased sensitivity to bortezomib treatment, silenced cells were more sensitive to gemcitabine treatment. Bortezomib induces mitochondrial damage in native cells and also activates the UPR by splicing of Xbp-1 and induction of CHOP, which is significantly reduced by silencing of MUC4. Our data suggest that mucinous pancreatic cancers are more sensitive towards proteasome inhibition by bortezomib and that alternative pathways of apoptosis are involved in cell death induction, while tumor cells with a low secretory activity show a better response to gemcitabine.
Abstract: 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.
Abstract: Up-regulation of phosphatidylinositol-3-kinase (PI3K)-AKT signaling facilitates tumor cell growth and inhibits cell demise. The AKT-pathway also plays an important role in cytostatic therapy resistance and response to hypoxia and angiogenesis. Using real-time cell proliferation assay we examined the potency of triciribine in three distinct neuroendocrine gastrointestinal tumor cell lines. Also we investigated triciribine's induction of apoptosis and effects on a broad range of cancer-associated gene products. Furthermore, we characterized the role of PTEN as a possible predictor of sensitivity to triciribine in GEP-NETs. We also looked for additive anti-neoplastic effects of triciribine when combined with conventional cytostatic drugs or other targeted drugs, affecting different molecules of the PI3K-AKT-pathway and we assessed the potency of triciribine to inhibit tumor growth in vivo, by using the chick chorioallantoic membrane assay. Treatment of insulinoma (CM) or gut neuroendocrine tumor cells (STC-1) with triciribine significantly reduced tumor cell growth by 59% and 65%, respectively. By contrast, the highly expressing PTEN carcinoid cell line BON did not respond, even at higher doses. Combinations of triciribine with classic cytostatic drugs as well as drugs targeting other molecules of the PI3K-AKT-pathway led to synergistic anti-proliferative effects. Additional in vivo-evaluations confirmed the anti-neoplastic potency of triciribine. Thus, our data show that inhibition the AKT-pathway potently reduces the growth of GEP-NET cells alone or in combination therapies. AKT inhibition may provide a rationale for future evaluations.
Abstract: A metal complex is identified in which the metal fulfills two independent functions: as a structural scaffold for the specific molecular recognition of protein kinases resulting in antiangiogenic properties, together with a visible-light-induced photoreactivity triggering apopotosis in cancer cells.
Abstract: Treatment of gastroenteropancreatic neuroendocrine tumors (GEP-NET) is still unsatisfactory and innovative therapeutic approaches are urgently needed. Heat shock protein 90 (Hsp90) is overexpressed in a wide range of tumor types and is an emerging target for the treatment of cancer. However, the potential activity of Hsp90 inhibitors in GEP-NET has not yet been investigated. We studied the antineoplastic activity of the Hsp90 inhibitor IPI-504 on GEP‑NET cells, and characterized its mechanism of action. In human insulinoma (CM) and pancreatic carcinoid (BON) cells IPI-504 induced a dose-dependent growth inhibition by almost 70%. The antiproliferative effect of IPI-504 correlated with a reduction in protein levels of the IGF-1 receptor. Additionally, several proteins of the PI3K/AKT/mTOR pathway, downstream of IGF-1 receptor activation in GEP-NETs, were downregulated as a consequence of Hsp90 inhibition. Combination treatment of IPI-504 with mTOR- or AKT-inhibitors led to additive antiproliferative effects. In addition, effects of IGF-1 receptor tyrosine kinase inhibition were strongly enhanced by IPI-504. Cancer gene expression profiling and FACS analysis revealed that IPI-504 antiproliferative effects were due to both induction of cell cycle arrest and apoptosis. A modified chick chorioallantoic membrane (CAM) assay confirmed the antineoplastic activity of IPI-504 in GEP-NETs in vivo. In conclusion, this study showed that Hsp90 inhibition may be an attractive target for innovative GEP-NET treatment alone or in combination with either IGF-1R or mTOR inhibitors.
Abstract: Introduction : Inhibition of protein kinases has become a standard of modern clinical oncology. PIM1 belongs to a novel class of serine/threonine kinases with distinct molecular and biochemical features regulating various oncogenic pathways, for example hypoxia response, cell cycle progression and apoptosis resistance. PIM1 is overexpressed in human cancer diseases and has been associated with metastasis and overall treatment response; in experimental models, inhibition of PIM1 suppressed cell proliferation and migration, induced apoptotic cell death and synergized with other chemotherapeutic agents. Areas covered : A PubMed literature search was performed to review the currently available data on PIM1 expression, regulation and targets; its implication in different types of cancer and its impact on prognosis are described. We present ATP-competitive PIM1 inhibitors and the state of the art of PIM1 inhibitor design. Finally, we highlight the development of the unusual class of highly selective and potent organometallic PIM1 inhibitors. Expert opinion : As PIM1 possesses oncogenic functions and is overexpressed in various kinds of cancer diseases, its inhibition provides a new option in cancer therapy. Based on the ability of highly selective organometallic PIM1 inhibitors, promising in vivo applicability is expected.
Abstract: Resistance to cell death induction has been recognized as a hallmark of cancer. Increasing understanding of the underlying molecular events regulating different cell death mechanisms like apoptosis, endoplasmic reticulum stress, autophagy, necroptosis and others has opened new possibilities for targeted interference with these pathways. While conventional chemotherapeutic agents usually inhibit cell cycle progression, DNA replication or mitosis execution, novel agents like small molecule kinase inhibitors also target survival-related kinases and signaling pathways and contribute to overcome resistance to chemotherapy and apoptosis. Additionally, antibodies targeting cellular death receptors have been described to specifically target tumor cells only. This review briefly highlights the pathways involved in (apoptotic) cell death and summarizes the current state of development of specific modulators of cell death and how they can help to improve the tolerability of chemotherapy regimens and increase survival rates in patients with advanced cancer diseases.
Abstract: 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.
Abstract: Advanced hepatocellular carcinoma still represents an unmet medical need that has only a limited overall survival despite the introduction of the multi-kinase inhibitor sorafenib. Recently, inhibitors of histone and other protein deacetylases have been established as novel therapeutic approaches to cancer diseases. We here review the molecular rationale for combining these two novel targeted therapies and report a patient with metastasized hepatocellular carcinoma who showed a partial remission of primary and metastatic lesions for five months after a combination therapy with sorafenib and the orally available pan-deacetylase inhibitor panobinostat.
Abstract: The multi-kinase-inhibitor Sorafenib has been shown to prolong survival of patients suffering from hepatocellular carcinoma (HCC). We investigated effects of the serine/threonine kinase inhibitor 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) on experimental HCC growth, and identified mechanisms and target kinases of DMAT. Our results show that DMAT application in vivo reduced tumor growth in a xenotransplant model by interference with tumor cell proliferation. Biochemical parameters and histology following DMAT administration revealed no alterations in liver tissue. Similar to Sorafenib, DMAT interfered with NFκB activation and Wnt-signaling. Of the kinases inhibited by DMAT at almost equimolar IC50, CK2 and PIM-3 were found to be over-expressed or more active in hepatoma cells and human HCC tissue. Knockdown of PIM-3 or CK2 by shRNA revealed that both kinases are important for hepatoma cell proliferation and survival. In conclusion, DMAT reduces HCC growth by interference with NFκB- and Wnt-signaling. PIM-3 and CK2 seem to be important target kinases. Inhibition of these kinases by application of inhibitors, e.g., DMAT, might represent a promising therapeutic approach in future HCC therapy.
Abstract: 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.
Abstract: Discovery of small molecules able to induce several cellular self-killing mechanisms improved cancer therapy in the last years. Research focused on canonical apoptotic (mitochondria or death receptor related) pathways to induce cell death in several hematologic and solid malignancies, showing that treatment with different synthetic and natural compounds reactivates the cell death machinery previously silenced in resistant cancer cells. Besides the canonical apoptotic pathways, alternative pathways of cell death induction have recently been rediscovered as potential new targets for cancer therapy. Under certain conditions, protein folding can be disturbed causing an accumulation of unfolded proteins inside the endoplasmic reticulum (ER). This situation leads to stress ER, involving the transcriptional and translational machinery to induce the expression and post-transcriptional modifications of many factors involved in ER stress response mediated cell death. In this scenario, some apoptotic players like caspase 4 or caspase 12 start to control cell fate by inducing downstream cell death proteins. Recently, inhibitors of protein deacetylases have been demonstrated to potently induce this alternative cell death pathway and will be reviewed here.
Abstract: Signal transduction processes (communication from extra- to intracellular compartments) are involved in a plethora of physiologic and pathologic processes. Molecular analyses identified a variety of molecules that have currently led to novel therapy strategies in solid and hematologic malignancies. This increased understanding of signal transduction cascades has now been applied successfully with Fingolimod (FTY720) in two phase III trials of multiple sclerosis. Fingolimod is a sphingosine-1-phosphate receptor-modulator
and a functional antagonist of sphingosine-1-phosphate (S1P), which binds to different G-protein coupled transmembrane receptors and modulates immunologic, vascular and cell biologic processes. In the CNS, S1P also regulates the egress of lymphocytes from blood vessels and promotes proliferation of astrocytes, which is associated with the pathogenesis of neurodegenerative diseases with astrogliosis. In this article, the basic underlying molecular and biochemical processes of Fingolimod will be reviewed and the
therapeutic effect in multiple sclerosis will be discussed.
Abstract: Histone deacetylases (HDAC) are responsible for the transcriptional control of genes through chromatin remodeling and control tumor suppressor genes. In several tumors, their expression has been linked to clinicopathological factors and patient survival. This study investigates HDACs 1, 2, 3, and 7 expressions in hepatocellular carcinoma (HCC) and their correlation with clinical data and patient survival. Tissue microarrays of 170 surgically resected primary HCCs and adjacent uninvolved tissue were evaluated immunohistochemically for the expression of HDACs 1, 2, 3, 7, and Ki-67 and were analyzed with respect to clinicopathological data and patient survival. HDACs 1, 2, 3, and Ki-67 were expressed significantly higher in cancer cells compared to normal tissue (HDAC1: p = 0.034, HDACs 2 and 3 and Ki-67: p < 0.001), while HDAC7 expression did not differ between HCC and non-cancerous liver tissue. In tumor tissue HDACs 1-3 expression levels showed high concordance with each other, Ki-67 and tumor grade (p < 0.001). High HDAC2 expression was associated with poor survival in low-grade and early-stage tumors (p < 0.05). The expression of the HDACs 1, 2, and 3 (but not HDAC7) isoenzymes correlates with clinicopathological factors, and HDAC2 expression has an impact on patient survival.
Abstract: The cannabinoid system (CS) is implicated in the regulation of hepatic fibrosis, steatosis and inflammation, with cannabinoid receptors (CB) 1 and 2 being involved in regulation of pro- and anti-fibrogenic effects. Daily cannabis smoking is an independent risk factor for the progression of fibrosis in chronic hepatitis C and a mediator of experimental alcoholic steatosis. However, the role and function of CS in alcoholic liver fibrosis (ALF) is unknown so far. Thus, human liver samples from patients with alcoholic liver disease (ALD) were collected for analysis of CB1 expression. In vitro, hepatic stellate cells (HSC) underwent treatment with acetaldehyde, H(2)O(2), endo- and exocannabinoids (2-arachidonoylglycerol (2-AG) and Δ9-tetrahydrocannabinol (THC)), and CB1 antagonist SR141716 (Rimonabant). In vivo, CB1 knock-out (KO) mice received thioacetamide (TAA)/ethanol (EtOH) to induce fibrosis. As a result, in human ALD CB1 expression was restricted to areas with advanced fibrosis only. In vitro, acetaldehyde, H(2)O(2), as well as 2-AG and THC, alone or in combination with acetaldehyde, induced CB1 mRNA expression, whereas CB1 blockage with SR141716 dose-dependently inhibited HSC proliferation, downregulated mRNA expression of fibrosis-mediated genes PCα1(I), TIMP-1 and MMP-13. This was paralleled by marked cytotoxicity of SR141716 at high doses (5-10μM). In vivo, CB1 knockout mice showed marked resistance to alcoholic liver fibrosis. In conclusion, CB1 expression is upregulated in human ALF which is at least in part triggered by AA and oxidative stress. Inhibition of CB1 by SR141716, or via genetic knock-out protects against alcoholic-induced fibrosis in vitro and in vivo.
Abstract: Signalling pathways such as Hedgehog (Hh), Wnt, Notch, bone morphogenetic protein (BMP) and transforming growth factor-b (TGF-b) 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-b 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.
Abstract: The HIV/SIV accessory protein Nef is known to down-modulate cell surface receptors that are required for virus entry such as CD4, CCR5 and CXCR4 to block lethal viral superinfection of the infected cell. The chemokine receptor CXCR4 also plays an important role in promoting cell proliferation, metastasis and tumor angiogenesis. Therefore it was of interest to evaluate if Nef can down-regulate CXCR4 in tumor cells since this could affect these critical prognostic parameters. The CXCR4-expressing cell line ACC3 that was derived from a salivary gland adenoid cystic carcinoma (ACC) of the head and neck was transfected with Nef from SIV(mac239) and cell surface expression of the receptor was monitored by FACS analysis. Real time proliferation of cells was measured with the xCELLigence system (Roche, Mannheim, Germany). Cell migration was detected by an in vitro scratch assay. Similarly, COS-7 cells were co-transfected with CXCR4 and Nef and were treated as described for ACC3. In vitro tube formation was deployed to assess the effect of Nef on angiogenesis. siRNA was used for CXCR4 knockdown. Cell surface down-modulation of endogenous CXCR4 could be observed in ACC3 cells after Nef-transfection as well as in COS-7 cells after co-transfection of CXCR4 and Nef. Proliferation as well as migration of Nef-transfected ACC3 tumor cells appeared significantly reduced. In vitro tube formation was significantly lowered after Nef-transfection or CXCR4 knockdown with siRNA. SIV-Nef could serve as an interesting tool to study the biologic behavior of CXCR4-expressing tumors such as ACC. Deploying SIV-Nef thereby could help in the discovery of new therapeutic approaches for the treatment of ACC and other CXCR4-expressing tumors.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: Thymoquinone (TQ), a component of black seed essential oil, is known to induce apoptotic cell death and oxidative stress, however, the direct involvement of oxidants in TQ-induced cell death has not been established yet. Here, we show that TQ inhibited the proliferation of a panel of human colon cancer cells (Caco-2, HCT-116, LoVo, DLD-1 and HT-29), without exhibiting cytotoxicity to normal human intestinal FHs74Int cells. Further investigation in DLD-1 revealed that apoptotic cell death is the mechanism for TQ-induced growth inhibition as confirmed by flow cytometry, M30 cytodeath and caspase-3/7 activation. Apoptosis was induced via the generation of reactive oxygen species (ROS) as evidenced by the abrogation of TQ apoptotic effect in cells preincubated with the strong antioxidant N-acetyl cysteine (NAC). TQ increased the phosphorylation states of the mitogen-activated protein kinases (MAPK) JNK and ERK, but not of p38. Their activation was completely abolished in the presence of NAC. Using PD98059 and SP600125, specific ERK and JNK inhibitors, the two kinases were found to possess pro-survival activities in TQ-induced cell death. These data present evidence linking the pro-oxidant effects of TQ with its apoptotic effects in colon cancer and prove a protective role of MAPK.
Abstract: Three new oxazole-bridged combretastatin A analogues with additional functional groups at the B-ring [-SMe, -OH, p-quinone] were tested for antiproliferative activity and specificity on human HL-60 leukemia, 518A2 melanoma, and colon carcinomas HCT-116 (wt)/(p53(-/-)) and HT-29 cells. While all oxazoles, except quinone 8, were efficacious against HCT-116 cells at submicromolar IC(50) values (48 h incubation), only thioanisole 5 achieved this potency in combretastatin-refractory HT-29 cells by significant upregulation of p21(cip1/waf1) associated with an S/G(2) cell-cycle arrest.
Abstract: 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.
Abstract: Thymoquinone (TQ), the major compound of black seed oil, has been shown to induce pro-apoptotic signaling pathways in various human cancer models. Although TQ is commonly used in traditional medicine, its use in humans is limited due to its chemical properties and poor membrane penetration capacity. We therefore attached saturated and unsaturated fatty acid residues to TQ and evaluated the effect on cell proliferation, apoptosis and underlying signaling pathways in HCT116 and HCT116p53â’/â’ colon cancer and HepG2 hepatoma cells in vitro. Treatment with thymoquinone-4-α-linolenoylhydrazone (TQ-H-10) or thymoquinone-4-palmitoylhydrazone (TQ-H-11) induced a cytostatic effect, particularly in p53-competent HCT116 cells, mediated by an up-regulation of p21cip1/waf1 and a down-regulation of cyclin E, and associated with an S/G2 arrest of the cell cycle. Cells lacking p53 (HCT116p53â’/â’) or HepG2 liver cancer cells showed only a minor response to TQ-H-10. These findings demonstrate that derivatives of TQ inhibit cell proliferation dependent on p53 status by activating the cell cycle inhibitor p21cip1/waf1 at lower concentrations than unmodified TQ. Structural modifications can therefore contribute to the further clinical development of TQ.
Abstract: The anti-inflammatory and antiapoptotic heme degrading enzyme heme oxygenase-1 (HO-1) has been shown recently to interfere with replication of hepatitis C virus (HCV). We investigated the effect of HO-1 products carbon monoxide (CO), iron and biliverdin on HCV replication using the replicon cell lines Huh-5-15 and LucUbiNeo-ET, stably expressing HCV proteins NS3 through NS5B. Incubation of these cell lines in the presence of the CO donor methylene chloride transiently reduced HCV replication, whereas an increase of iron in cell culture by administration of FeCl(3) or iron-saturated lactoferrin did not interfere with HCV replication. Likewise, depletion of iron by deferoxamine during induction of HO-1 by cobalt-protoporphyrin IX did not restore HCV replication. The most prominent effect was observed after incubation of replicon cell lines in the presence of biliverdin. Biliverdin seems to interfere with HCV replication-mediated oxidative stress by inducing expression of antiviral interferons, such as interferon alpha2 and alpha17. CONCLUSION: The antioxidant biliverdin reduces HCV replication in vitro by triggering the antiviral interferon response and might improve HCV therapy in the future.
Abstract: The overall survival for patients with advanced hepatocellular carcinoma (HCC) is still limited. Although the multi-kinase inhibitor sorafenib has recently been approved for this disease, response rates are still low and patients often face dose-limiting toxicities which lead to a reduction in prognosis and treatment success. We here report a patient with metastasized HCC who shows a sustained response for more than 30 mo to sorafenib therapy after failure of a first line therapy with gemcitabine, oxaliplatin and bevacizumab.
Abstract: BACKROUND: Testicular germ cell tumour (TGCT) is the most common cause of death from solid tumours in young men and especially for platinum-refractory patients novel treatment approaches are urgently needed. Using an in silico screening approach for the detection of novel cancer drugs with inhibitory effects on the tyrosine kinase activity of growth factors (e.g., VEGFR, PDGFR), we identified two compounds (HP-2 and HP-14) with antiangiogenic and antiproliferative potency, which were evaluated in endothelial cell models and TGCT cells. RESULTS: HP-2 and HP-14 effectively inhibited the growth of VEGFR-2-expressing TGCT cell lines (Tera-1, Tera-2 and 2102EP) and endothelial cell models, while they failed to supress the growth of VEGFR-2-lacking tumour cells. cDNA-microarrays revealed an inhibition of the expression of several growth factor receptors and related signal transduction molecules. Vascular endothelial growth factor (VEGF)-induced cell migration was also potently inhibited. Cell cycle-regulating proteins such as p21 and p27 were upregulated, leading to an S-phase arrest. Additional in vivo evaluations confirmed the antiangiogenic potency and good tolerability of the novel substances. CONCLUSION: Our data show that the identified novel compounds inhibit the growth of TGCT cells and decrease angiogenic microvessel formation. The mode of action involves cell cycle arresting effects and changes in the expression pattern of several angiogenic genes. The novel compounds may qualify as new candidates for targeted treatment of TGCT and merit further evaluation.
Abstract: Oxazole-bridged combretastatin A-4 analogues bind to tubulin and exert anti-vascular and anti-angiogenic effects. When linked to Ru(η(6)-arene) complex fragments, conjugates with additional cytotoxic activity result which can ruthenate bionucleophiles such as DNA and proteins. For instance, the Ru(II)(p-cymene)(isonicotinate)Cl(2) complex 6a of the known 4-(3,4,5-trimethoxyphenyl)-5-(3-hydroxy-4-methoxyphenyl)-oxazole 4a was far more active than the latter against cells of the p53-competent wild-type form of HCT-116 colon carcinoma at low 0.01 μM concentrations. A fast reaction of 6a with nucleophilic N-acetyl-L-cysteine was observed in NMR studies. The Ru(arene) complexes 6a-c were also more efficacious against combretastatin-refractory p53(+) cells of human HT-29 colon carcinoma when compared to their parent 4-(3,4-dimethoxy-5-methoxy/halo-phenyl)-5-(3-hydroxy-4-methoxyphenyl)-oxazoles 4a-c. These cells are rich in ABC-transporters which are responsible for their multi-drug resistance and for which conjugates 6 are less good substrates than the phenols 4. Unlike 4a, its complex 6a also diminished the motility of human 518A2 melanoma cells in a wound-healing assay which is indicative of anti-metastatic activity in solid tumors. Overall, the Ru(arene) complex conjugates 6 broaden the anti-tumoral spectrum of the combretastatin A-4 analogues 4 considerably.
Abstract: Inhibitors of protein deacetylases have recently been established as a novel therapeutic principle for several human diseases, including cancer. The original notion of the mechanism of action of these compounds focused on the epigenetic control of transcriptional processes, especially of tumor suppressor genes, by interfering with the acetylation status of nuclear histone proteins, hence the name histone deacetylase inhibitors was coined. Yet, this view could not explain the high specificity for tumor cells and recent evidence now suggests that non-histone proteins represent major targets for protein deacetylase inhibitors and that the post-translational modification of the acetylome is involved in various cellular processes of differentiation, survival and cell death induction.
Abstract: Psychological stress is correlated with and may even cause DNA damage, which contributes to the etiology of various diseases. Recent studies point to the role of micro-RNA (miRNA), small molecules that regulate gene expression, in health and disease. This study investigated the relationship between transient stress and two cancer-related miRNAs, and determined whether health-behaviour moderated these relationships. Using a pre-post design, 37 German students completed measures on health-behaviour and perceived stress, the latter after a study break (low stress) and after an exam (high stress). On both occasions, students underwent blood tests to determine the expression of let-7b and miR-21, two miRNAs recently found to be related to cancer. The students reported significantly higher stress after the exam than in the study break period. The levels of let-7b and miR-21 expression significantly declined from low- to high-stress periods. Importantly, baseline health-behaviour interacted with time in relation to miR-21, such that the expression of this marker decreased only in students with inadequate health-behaviour, while it did not change in students with adequate health-behaviour. This is the first study showing that brief academic stress can alter the expression of two cancer-related miRNA molecules, and that health-behaviour may moderate these effects for miR-21.
Abstract: To investigate the involvement of transdifferentiation and dedifferentiation phenomena inside atherosclerotic plaques, we analyzed the differentiation status of vascular smooth muscle cells (VSMC) in vitro and in vivo. Forty normal autoptic and 20 atherosclerotic carotid endarterectomy specimens as well as 20 specimens of infrarenal and suprarenal aortae were analyzed for the expression of cytokeratins 7 and 18 and beta-catenin as markers (epithelial transdifferentiation) as well as CD31 and CD34 (embryonic dedifferentiation) by conventional and double fluorescence immunohistochemistry and reverse transcription polymerase chain reaction. Looking at these markers, additional cell culture experiments with human aortic (HA)-VSMC were done under stimulation with IL-1beta, IL-6, and TNF-alpha. Cytokeratins and beta-catenin were expressed significantly higher in atherosclerotic than in normal carotids primarily localized in VSMC of the shoulder/cap region of atherosclerotic lesions. Additionally, heterogeneous cellular coexpression of CD31 and/or CD34 was observed in subregions of progressive atherosclerotic lesions by VSMC. The expression of those differentiation markers by stimulated HA-VSMC showed a time and cytokine dependency in vitro. Our findings show that (1) VSMC of progressive atheromas have the ability of differentiation, (2) that transdifferentiation and dedifferentiation phenomena are topographically diverse localized in the subregions of advanced atherosclerotic lesions, and (3) are influenced by inflammatory cytokines like IL-1beta, IL-6, and TNF-alpha.
Abstract: BACKGROUND: Quantitative testing of liver function (QTLF) is one way to show the efficacy of antiviral treatment of Hepatitis C. Data on liver function in patients with chronic Hepatitis C during antiviral therapy are lacking. We therefore investigated if and to what extent antiviral therapy influenced quantitative testing of liver function (QTLF). METHODOLOGY: One hundrend seven patients with chronic Hepatitis C (genotype 1) were treated with pegylated-interferon 2alpha/ribavirin for 48 weeks. Quantitative testing of liver function, including aminopyrine breath test (ABT), galactose elimination capacity (GEC), sorbitol clearance (SCl) and indocyanine green clearance (ICG) was performed before and 12 weeks after initiation of antiviral therapy. QTLF was repeated at the end of the therapy (week 48) and 6 months after therapy. RESULTS: After 3 months of treatment, 97 patients showed normal transaminases and were negative for HCV-RNA. ABT and GEC as parameters of microsomal and cytosolic liver function were reduced in all patients before therapy initiation and returned to normal values in the therapy responders after 3 months. Parameters of liver perfusion (SCl and ICG) require one year of treatment before returning to normal levels. In non-responders, QTLF did not change during therapy, in relapsers, QTLF results deteriorated after ending the therapy. CONCLUSION: All liver tests return to normal within one year after eradication of the Hepatitis C virus. Parameters measuring the liver plasma flow (SCI and ICG) require more time to become normal, most likely due to tissue remodelling processes.
Abstract: Regulation of the profibrotic and angiogenesis modulating cytokine connective tissue growth factor (CTGF) occurs primarily at the transcriptional level. Therefore, we hypothesized that histone deacetylating enzymes (HDAC), which modulate the accessibility of transcriptionally active promoter regions, might play a role in the regulation of CTGF gene expression. We analyzed microvascular endothelial cells, which showed immunoreactivity for acetylated histone in kidney sections, and compared them with renal tubular epithelial cells. Treatment of cultured endothelial cells with different HDAC inhibitors up-regulated CTGF mRNA and protein. Pre-treatment with HDAC inhibitors facilitated induction of CTGF by transforming growth factor-beta (TGF-beta) or lysophosphatidic acid. Transcription factors of the FoxO family were involved in the up-regulation of CTGF as shown at protein level and by reporter gene analyses. In tubular epithelial cells, up-regulation of CTGF was only observed when these cells were cultured as subconfluent cells. Dense cells, which are more likely to resemble tubular cells in vivo, showed no up-regulation upon treatment with HDAC inhibitors and were protected against CTGF induction by TGF-beta. Taken together, our data indicate that the effect of HDAC inhibitors on CTGF expression is largely cell dependent in non-tumour cells. Different cell type-specific transcription factors seem to determine whether CTGF expression is reduced or increased in cells exposed to HDAC inhibitors.
Abstract: Tumor cells have the capability to trans- and to dedifferentiate, for example by reactivating embryonic development genes and stem cell characteristics. The aim of our study was to show the differential expression of stem- and progenitor cell markers in human hepatocellular carcinoma cell lines (HCC). Different human HCC cell lines (HUH7, HUH7 5-15, HUH7 pcDNA3.1, Hep3B and HepG2) were cultured under standard conditions in vitro or implanted subcutaneously (5x10(6) cells) in male NMRI mice. Specimens were characterized by quantitative real-time PCR, Western blotting, methylation-specific PCR and immunohistochemistry for markers of differentiation (cytokeratins, vimentin), embryonic development or stem cells (PTC, PDX-1, SHH, Thy1, c-kit, CD34, beta-catenin, Ki-67). The investigated HCC cell lines showed different patterns of marker expression allowing to distinguish four distinct groups: the classical cholangiocellular type (Huh-7, Huh-7 pcDNA3.1, Hep3B) with expression of CK7/19, beta-catenin and CD34; a dedifferentiated mesenchymal-proliferative type (Huh-7 5-15) characterized by CK19, Vimentin and Ki-67; a dedifferentiated embryonic-development type (Hep3B implanted in matrigel) with expression of CK19, beta-catenin and PTC and a classical HCC type (HepG2) showing CK18/19 and beta-catenin expression. HCC cell lines showed significantly different expression patterns of differentiation markers in a xenograft model. Furthermore, direct association of some markers was observed. The groups differ from each other in expression patterns, but also show that environmental factors play an important role in the behaviour of cells.
Abstract: Cancer is the second leading cause of death worldwide. Conventional cancer therapies cause serious
side effects and, at best, merely extend the patient’s lifespan by a few years. Cancer control may therefore
benefit from the potential that resides in alternative therapies. The demand to utilize alternative concepts
or approaches to the treatment of cancer is therefore escalating. There is compelling evidence from epidemiological
and experimental studies that highlight the importance of compounds derived from plants
“phytochemicals” to reduce the risk of colon cancer and inhibit the development and spread of tumors in
experimental animals. More than 25% of drugs used during the last 20 years are directly derived from
plants, while the other 25% are chemically altered natural products. Still, only 5-15% of the approximately
250,000 higher plants have ever been investigated for bioactive compounds. The advantage of using such
compounds for cancer treatment is their relatively non-toxic nature and availability in an ingestive form.
An ideal phytochemical is one that possesses anti-tumor properties with minimal toxicity and has a defined
mechanism of action. As compounds that target specific signaling pathways are identified, researchers can
envisage novel therapeutic approaches as well as a better understanding of the pathways involved in disease
progression. Here, we focus on 4 classes of natural anticancer drugs: methyltransferase inhibitors, DNA
damaging/pro-oxidant drugs, HDAC inhibitors (HDACi), and mitotic disrupters, and we will focus on the
mode of action for one promising example per group.
Abstract: PURPOSE: To asses if laser-induced thermotherapy (LITT) induces a specific cytotoxic T cell response in patients treated with LITT for colorectal cancer liver metastases. METHODS: Eleven patients with liver metastases of colorectal cancer underwent LITT. Blood was sampled before and after LITT. Peripheral T cell activation was assessed by an interferon gamma (IFNg) secretion assay and flow cytometry. Test antigens were autologous liver and tumor lysate obtained from each patient by biopsy. T cells were stained for CD3/CD4/CD8 and IFNg to detect activated T cells. The ratio of IFNg positive to IFNg negative T cells was determined as the stimulation index (SI). To assess cytolytic activity, T cells were co-incubated with human colorectal cancer cells (CaCo) and cytosolic adenylate kinase release was measured by a luciferase assay. RESULTS: IFNg secretion assay: before LITT SI was 12.73 (+/-4.83) for CD3+, 4.36 (+/-3.32) for CD4+ and 3.64 (+/-1.77) for CD8+ T cells against autologous tumor tissue. Four weeks after LITT SI had increased to 92.09 (+/-12.04) for CD3+ (P < 0.001), 42.92 (+/-16.68) for CD4+ (P < 0.001) and 47.54 (+/-15.68) for CD8+ T cells (P < 0.001) against autologous tumor tissue. No increased SI was observed with normal liver tissue at any time point. Cytotoxicity assay: before LITT activity against the respective cancer cells was low, with RLU = 1,493 (+/-1,954.68), whereas after LITT cytolytic activity had increased to RLU = 7,260 [+/-3,929.76 (P < 0.001)]. CONCLUSION: Patients with liver metastases of colorectal cancer show a tumor-specific cytotoxic T cell stimulation and a significantly increased cytolytic activity of CD3+, CD4+ and CD8+ T cells after LITT against an allogenic tumor (CaCo cell line).
Abstract: Increasing insights into molecular alterations of signalling pathways have led to the development of specific targeted therapies for cancer. Due to the high specificity of monoclonal antibodies or small molecule inhibitors, identification of patients who will benefit from these therapeutics is crucial for treatment success. Furthermore, as classical endpoints of clinical trials are not fully applicable to targeted therapies, biomarkers for monitoring treatment response have to be identified. The recent introduction of a multi-kinase inhibitor for the treatment of liver cancer has accelerated efforts in the field of biomarker research. As further novel targeted therapies are on the horizon for liver cancer therapy, we will here review candidate markers for new hepatocellular carcinoma therapies, with a focus on EGF- and VEGF-receptor related pathways.
Abstract: 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.
Abstract: A simplified procedure for the isolation of gram quantities of illudin M from culture broths of basidiomycete Omphalotus olearius is described. Esters of illudin M with docosahexaenoic acid, chlorambucil, demethylcantharidinic acid (endothall) and 2,2'-bipyridyl-5,5'-dicarboxylic acid were synthesised and tested for cytotoxicity and induction of apoptosis in two clinically relevant tumour cell lines (Panc-1 pancreas carcinoma and HT-29 colon carcinoma) and in non-malignant human foreskin fibroblasts. The demethylcantharidin and the bipyridine conjugates retained the cytotoxicity of the parent illudin M while displaying an improved specificity for the tumour cells over the fibroblasts.
Abstract: 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.
Abstract: 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.
Abstract: We have shown that thymoquinone (TQ) is a potent antitumor agent in human colorectal cancer cells. In this study, we evaluated TQ's 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 (40 microM) 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.
Abstract: Our interesting case deals with the clinical and morphological aspects of a chronic neutrophilic leukemia and the critical evaluation of differential diagnosis of leukemoid reaction in bone marrow biopsies.
Abstract: Effective therapies for advanced stages of hepatocellular carcinoma (HCC) have yet to be developed. We investigated how far a combination of the HDAC inhibitor MS-275 and the CDK inhibitor CYC-202 synergizes to inhibit proliferation and promotes apoptosis of hepatoma cells in vitro. Human hepatoma cell lines Hep3B and HepG2 as well as primary human foreskin fibroblasts as non-malignant controls were cultured under standardized conditions and incubated with increasing concentrations of CYC-202 and MS-275 as single agents and in combination. After 24 to 72 h, apoptosis was analyzed by flow cytometry (propidium iodide, JC-1) and by immunocytochemistry for cytokeratin 18 fragmentation. DNA synthesis was assessed using bromodeoxyuridine incorporation. Protein was separated for Western blotting against p21, bax and bcl-2 and fluorimetric activity assays against caspase 3 and 8. The results showed that the combination of CYC-202 and MS-275 leads to better pro-apoptotic effects than the employment of single substances. Apoptosis was induced via the mitochondrial pathway as evidenced by a shift in the bax/bcl-2 ratio and breakdown of mitochondrial transmembrane potentials. Caspase assays revealed a strong induction of caspase 3 but not of the extrinsic initiator caspase 8. In conclusion, combination therapy with the biomodulators MS-275 and CYC-202 is a promising treatment option for HCC.
Abstract: Endogenous overexpression of the antiapoptotic protein heme oxygenase 1 (HO-1) has been shown to occur in various cancer diseases and might contribute to cancer progression. We compared the expression levels of HO-1 in human liver to expression levels in hepatocellular carcinoma (HCC), as well as the effect of HO-1 inhibition by small interfering RNA (siRNA) on cellular survival and apoptosis in the mouse hepatoma cell lines Hepa129 and Hepa1-6 and on orthotopic tumor growth in immune-competent C3H/HeN mice. Our results show that HO-1 is frequently overexpressed in human HCC. Downmodulation of HO-1 by siRNA resulted in increased cellular damage and apoptosis, reduced proliferation, reduced growth of orthotopic HCC and reduced angiogenesis. Livers and kidneys of treated animals did not reveal signs of damage by this treatment. In conclusion, a specific knockdown of HO-1 might represent a novel therapeutic approach in HCC therapy.
Abstract: Nach Schätzungen der WHO sind weltweit ca. 170 Millionen Menschen chronisch mit dem Hepatitis C-Virus (HCV) infiziert. Nach einem häufig milden oder subakuten Verlauf wird die Infektion bei der Mehrzahl der Patienten erst nach einer Latenzzeit von mehreren Jahrzehnten klinisch manifest. Die aktuelle Therapie
besteht aus einer Kombination aus pegyliertem Interferon α und dem Nukleosidanalogon Ribavirin. Diese Therapie führt zu einer Ausheilung der Erkrankung bei 80% der Patienten, die mit den HCV-Genotypen 2 oder
3 infiziert sind, ist aber bei weniger als 55% der Genotyp 1 Patienten erfolgreich. In Europa und den USA sind jedoch über 60% der Patienten Träger des HCV-Genotyps 1, so dass die aktuell verfügbare Therapie unbefriedigend ist. Hinzu kommen hohe Kosten der Therapie durch Interferon/Ribavirin und die häufig
nicht tolerierbaren Nebenwirkungen (Depression, Myelosuppression) dieser Kombination, so dass dringend neue Therapieformen für die chronische Hepatitis C-Infektion etabliert werden müssen (Stauber et al., J Clin
Virol 2006).
Abstract: Gemcitabine has been shown to ameliorate disease related symptoms and to prolong overall survival in pancreatic cancer.Yet, resistance to Gemcitabine is commonly observed in this tumour entity and has been linked to increased expression of anti-apoptotic bcl-2. We therefore investigated if and to what extend silencing of bcl-2 by specific siRNAs (siBCL2) might enhance Gemcitabine effects in human pancreatic carcinoma cells. siBCL2 was transfected into the pancreatic cancer cell line YAP C alone and 72 hrs before co-incubation with different concentrations of Gemcitabine. Total protein and RNA were extracted for Western-blot analysis and quantitative polymerase chain reaction. Pancreatic cancer xenografts in male nude mice were treated intraperitoneally with siBCL2 alone, Gemcitabine and control siRNA or Gemcitabine and siBCL2 for 21 days. Combination of both methods lead to a synergistic induction of apoptosis at otherwise ineffective concentrations of Gemcitabine. Tumour growth suppression was also potentiated by the combined treatment with siBCL2 and Gemcitabine in vivo and lead to increased TUNEL positivity. In contrast, non-transformed human foreskin fibroblasts showed only minor responses to this treatment. Our results demonstrate that siRNA-mediated silencing of anti-apoptotic bcl-2 enhances chemotherapy sensitivity in human pancreatic cancer cells in vitro and might lead to improved therapy responses in advanced stages of this disease.
Abstract: OBJECTIVE: Pancreatic cancer continues to be an urgent clinical problem. We used the novel DNA methyltransferase inhibitor Zebularine and the histone deacetylase inhibitor SAHA to investigate the epigenetic influence on viability and differentiation of the pancreatic cancer cell lines YAP C, DAN G and Panc-89 in vitro and in vivo. MATERIAL AND METHODS: Cell vitality, proliferation and expression of PDX-1, cytokeratin 7 and 20, chromogranin A, vimentin, bax and bcl-2 were determined on the protein and mRNA level in vitro and in a subcutaneous xenograft model. RESULTS: A time- and dose-dependent increase of apoptosis, paralleled by decreased proliferation, was observed after incubation with single agents or a combination therapy with lower concentrations. This was associated with up-regulation of pro-apoptotic bax and a phenotypic stabilization by the enhanced expression of cytokeratin 7. In vivo, growth of xenografts was delayed with the most pronounced effect in Panc-89 after 1 week of daily intraperitoneal injections of Zebularine paralleled with CK7 up-regulation and down-regulation of dedifferentiation markers. CONCLUSIONS: Epigenetic modulation via inhibition of DNA methyltransferase and histone deacetylase induces apoptosis in human pancreatic cancer cells in vitro and delays xenograft growth in vivo, which is associated with a morphological/molecular phenotypic stabilization. These compounds may therefore be suitable as adjunctive therapeutic agents in the treatment of pancreatic cancer.
Abstract: Chromatin-modifying enzymes such as histone deacetylases (HDAC) facilitate a closed chromatin structure and hence transcriptional repression. HDAC are commonly affected in human cancer diseases. Thus, inhibition of HDAC represents a novel therapeutic approach. Several studies have shown that HDAC inhibitors strongly activate the expression of the cyclin-dependent kinase inhibitor p21(cip1/waf1) through (i) enhanced histone acetylation around the p21(cip1/waf1) promoter and (ii) the Sp1 sites on the p21(cip1/waf1) promoter releasing the repressor HDAC1 from its binding. p21(cip1/waf1) expression is regulated in a p53-dependent and p53-independent manner. The decision if p21(cip1/waf1) up-regulation results in cell cycle arrest or apoptosis, decides about the therapeutic efficacy of an anti-cancer treatment with HDAC inhibitors.
Abstract: The past decades have brought major breakthroughs in the identification of distinct genetic alterations (eg, mutations, chromosomal aberrations) in various human tumors, leading to the development and clinical use of novel target-specific antibodies and small molecules. Recently, variable modifications of chromatin elements have become the focus of cell biology research. Compounds that inhibit the key chromatin-modifying enzyme classes of histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are currently being evaluated in various preclinical studies and clinical trials. The overexpression of both HDAC and DNMT has been demonstrated to be associated with the epigenetic inactivation of tumor suppressor genes, as well as cell cycle and apoptosis regulators. In addition, inhibitors of HDAC and DNMT possess direct cytotoxic properties, and can sensitize tumor cells to conventional radiotherapy and chemotherapy.
Abstract: The prognosis of advanced pancreatic cancer is poor. Established chemotherapy shows only limited efficacy and significant side effects. We investigated how far a combination of trichostatin A (TSA) and gemcitabine synergizes to inhibit proliferation and promotion of apoptosis of pancreatic adenocarcinoma cells in vitro. The human pancreatic carcinoma cells YAPC, DANG and Panc-89 and primary human foreskin fibroblasts as non-malignant controls were cultured under standardized conditions and incubated with gemcitabine und TSA alone (10(-4) to 10(-8) M) or together (10(-6) to 10(-7) M). After 24-72 h the apoptotic rate was analyzed by flow cytometry (propidium iodide, FACS). DNA-synthesis was assessed using bromodeoxyuridine (BrdU) incorporation. Protein was separated for Western blotting against caspase-3 and -8, p21, bax and bcl-2. The combination of TSA und gemcitabine leads to better pro-apoptotic effects than the employment of single substances. Bcl-2, a mitochondrial protein, which protects against apoptosis, was not expressed. Bax, an apoptosis inducing protein, which destabilizes the mitochondrial membrane potential, was increasingly expressed. Combination of TSA and gemcitabine shows promise for treatment of pancreatic cancer in vivo.
Abstract: We investigated the effect of a novel histone deacetylase inhibitor, A-423378.0, on the colon carcinoma cell line HCT116 and genetically modified derivatives lacking either p21(cip1/waf1) or p53. HCT116 cell lines were incubated with A-423378.0 at different concentrations for 3-120 h. Cell viability, proliferation and apoptosis rates were determined and verified by western blot, detection of mitochondrial membrane potential breakdown DeltaPsi(m), activation of caspases-3, -8 and cytokeratin 18 cleavage. A subcutaneous xenograft model was established in NMRI mice with daily intraperitoneal injections of 10 mg/kg for 14 days. All three HCT116 cell lines responded to A-423378.0 treatment in a dose- and time-dependent manner via induction of apoptosis as measured by breakdown of DeltaPsi(m) and BrdU incorporation. We identified that A-423378.0 induced the expression of TRAIL and TRAIL receptor, especially TRAIL-R2/hDR5, which was up-regulated in HCT116 cells after treatment with A-423378.0. In vivo, a growth inhibitory effect was observed with HDAC-I treatment, which was paralleled by a down-regulation of PCNA and a concomitant induction of apoptosis. Treatment of wild-type or knock-out HCT116 cells with A-423378.0 exerts potent anti-proliferative and pro-apoptotic effects in vitro and in vivo. A-423378.0 was able to induce apoptosis in both p21(WAF1) and p53 deficient tumour cells, which appeared to be mediated by the intrinsic cell death pathway. Interestingly, the effects of A-423378.0 on the extrinsic cell death pathway through activation of TRAIL and its signalling pathway indicate that A-423378.0 may be a potent new therapeutic compound for the treatment of advanced colorectal cancer.
Abstract: Recent research on embryonic and adult stem cells questions the currently accepted models of multi-step carcinogenesis in solid cancer. Accordingly, differentiated epithelial cells are considered to be the main target for mutational steps, leading to a growth and survival advantage of malignantly transformed cells. In contrast, the stem cell model of carcinogenesis emphasizes the role of stem cells as the initiating structure for tumor development. Yet, it is unclear if tumors contain dysregulated (embryonic) stem cells or if tumors consist of differentiated adult cells that obtained a de-differentiated stem cell-like phenotype. Here, we review the current knowledge on the roles of stem cells in gastrointestinal cancer formation and the implication on future diagnostic and therapeutic strategies.
Abstract: BACKGROUND/AIMS: The induction of liver fibrosis is difficult in mice. Here, we intended to improve fibrosis induction by combination of thioacetamide (TAA) injections and ethanol (EtOH) feeding and to characterize features of liver damage in this model. Most experimental therapeutic studies are performed in mice without pre-damaged livers. METHODS: C3H mice were injected three times/week (0.15 mg/g body weight) and fed with EtOH. Tissue and serum samples were collected and analysed. RESULTS: Portal fibrosis was verified by van Gieson staining showing a mild fibrosis (score F2) in TAA-treated mice and liver fibrosis (score F4) in the combination group using TAA/EtOH. Consonant with the histological results, the fibrosis marker MMP-2 and alpha 1 procollagen (I) were elevated at week 10 and 15 after treatment initiation in the combination group, whereas tissue protective proteinase, TIMP-1, was 18.5-fold increased only at week 10 but normalized until week 15. Fibrosis development was associated with elevated ICAM-1 expression. CONCLUSIONS: Taken together, TAA/EtOH application was suitable to induce liver fibrosis characterized by typical bio-markers in C3H/He.
Abstract: Tissue remodeling by matrix metalloproteinases (MMPs) and plasminogen activators such as tissue factor (TF) is postulated to be involved in the pathogenesis of atherosclerosis. The in situ expression of MMP9 and TF in unstable atherosclerotic plaques has not been examined in detail. Moreover, interference of tissue remodeling by vascular inflammation, apoptosis, and Chlamydia pneumoniae inside plaque subregions is unclear. A total of 40 autopsy carotid arteries (controls) and 20 atherosclerotic carotid endarterectomy specimens (with type VI lesions, according to the American Heart Association classification) from stroke patients were analyzed for expression of MMP9 and TF using in situ techniques. The data on tissue remodeling were correlated with the presence of inflammatory cells (T cells, B-cells, macrophages), apoptosis, and the presence of C. pneumoniae using immunohistochemistry and Western blot analyses. We found a significant overexpression of MMP9 and TF in progressive atherosclerotic carotid arteries, especially in the shoulder and cap subregions (both p < 0.05). Expression of MMP9 and TF correlated significantly with T-cell and macrophage infiltrates as well as with apoptosis (p < 0.05). C. pneumoniae infection was significantly associated with elevated TF expression (p < 0.01) but not with MMP9. MMP9 and TF are thus significantly overexpressed in progressive atherosclerotic plaques, and their relevant subregions (shoulder and cap) are involved in plaque instability. This process is associated with local inflammatory cell infiltrates and apoptosis, which might be influenced by infectious agents such as C. pneumoniae.
Abstract: Treatment for advanced stages of hepatocellular carcinoma (HCC) remains unsatisfactory. While 5-fluorouracil (5-FU) and irinotecan are first-line treatment options for other gastrointestinal tumors, their effect on HCCs is low. Histone-deacetylase inhibitors such as suberoylanilide hydroxamic acid (SAHA) have shown antitumoral activity at micromolar concentrations in a variety of human cancers in vitro and in vivo. Here, we investigated the effects of a combination of 5-FU, irinotecan and SAHA on growth inhibition and apoptosis induction in HCC cell lines. HepG2, Hep1B and MH-7777A hepatoma cell lines and human foreskin fibroblasts as non-transformed controls were incubated with 5-FU, irinotecan and SAHA either alone or in combination. While the single agents did not show any effects on growth of the cell lines, the combination of 5-FU and irinotecan (both 10 microM) led to a moderate increase in apoptosis and proliferation inhibition. Adding 1 microM SAHA increased the apoptosis rate in hepatoma cell lines up to 92% after 72 h, while fibroblasts showed no response (5.5% apoptosis). Induction of apoptosis was paralleled by loss of the mitochondrial transmembrane potential, downregulation of bcl-2 expression and activation of caspase 3 but not caspase 8. In summary, SAHA sensitized HCC cell lines for treatment with an otherwise ineffective combination of 5-FU and irinotecan and led to mitochondrial apoptosis induction. The use of the triple combination could optimize treatment results in vivo and needs further evaluation.
Abstract: BACKGROUND/AIMS: Hepatocellular carcinoma, which usually develops in cirrhotic livers, is one of the most frequent cancers worldwide. If and how far hepatoma growth influences liver function is unclear. Therefore, we compared a broad panel of quantitative tests of liver function in cirrhotic patients with and without hepatocellular carcinoma. METHODOLOGY: Patients with (n=40) and without (n=40) hepatocellular carcinoma were matched according to Child-Pugh grade and subjected to testing of aminopyrine demethylation capacity, galactose elimination capacity, sorbitol clearance and indocyanine green clearance. RESULTS: Compared to healthy controls, patients with cirrhosis Child-Pugh grade B and grade C revealed reduced metabolic (aminopyrine demethylation capacity, galactose elimination capacity) and perfusion-dependent QTLF (sorbitol clearance, indocyanine green clearance). Comparing values of quantitative tests of liver function in matched patients with and without hepatocellular carcinoma, no differences in liver function parameters were observed. CONCLUSIONS: Quantitative tests of liver function correlated inversely with the Child-Pugh grade. Since these parameters are not affected by the occurrence of hepatocellular carcinoma, the emergence of hepatic neoplasia in cirrhotics does not appear to be determined by the degree of hepatic functional deterioration.
Abstract: AIMS: New concepts of tumorigenesis favor an unregulated process recapitulating different stages of embryogenic development with dysregulation of transition states. The aim of our study was to investigate the possibility of differentiation pathways of human pancreatic cancer cell lines in vivo. MATERIAL AND METHODS: Different human pancreatic cancer cell lines (YAPC, DAN-G, CAPAN-1, PANC-1 and MIA PaCa-2) were implanted subcutaneously (3 x 10(6) cells) for 28 days in nude mice. Xenotransplants were characterized with histochemistry (HE, PAS), immunohistochemistry (cytokeratin (CK)7, CK8, CK18, CK19, CK20, vimentin, chromogranin A (Chr-A), alpha1-antichymotrypsin (alpha1-chym), beta-catenin, laminin-5, pancreatic and duodenal homeobox gene 1 (pdx-1), sonic hedgehog protein (shh), Patched (ptc)), Western blotting and real-time PCR (CK7, CK8, CK20, Chr-A, pdx-1, shh, ptc). RESULTS: Depending on three major morphologic phenotypes of tumor cell xenotransplants (ductal (YAPC), ductal/solid (DAN-G, CAPAN-1), solid (PANC-1, MIA PaCa-2)), a decrease of CK7/CK19 was found, accompanied by an increase of CK8/18 and vimentin. Predominantly the CK7-positive ductal phenotype (YAPC and DAN-G) was associated with pdx-1 expression, whereas the CK8-positive solid phenotype was associated with shh/ptc expression on protein and mRNA level. Additionally, CK-20 expression was mainly linked to the ductal phenotype, co-localized with nuclear beta-catenin. The endocrine-exocrine transdifferentiation, as assessed by Chr-A and alpha1-chym, was on a constant low to moderate level in all xenotransplants. Finally, an intensive epithelial-mesenchymal interaction was observed by overexpression of laminin-5 at the invasion front. CONCLUSION: The observed patterns of morphology and molecular differentiation in human pancreatic cancer xenografts indicate that these cancer cell lines have different capabilities of pattern formation in vivo associated with molecular differentiation markers, especially of embryonic pancreatic development.
Abstract: AIM: To investigate if and to what extent antiviral therapy influenced a broad panel of quantitative testing of liver function (QTLF). METHODS: Fifty patients with chronic hepatitis C were either treated with interferon (n = 8), interferon/ribavirin (n = 19) or peg-interferon/ribavirin (n = 23). Quantitative testing of liver function, including aminopyrine breath test (ABT), galactose elimination capacity (GEC), sorbitol clearance (SCl) and indocyanine green clearance (ICG) was performed before and 3 mo after initiation of antiviral therapy. RESULTS: After 3 mo of antiviral treatment, 36 patients showed normal transaminases and were negative for HCV-RNA, 14 patients did not respond to therapy. ABT and GEC as parameters of microsomal and cytosolic liver function were reduced in all patients before therapy initiation and returned to normal values in the 36 therapy responders after 3 mo. Parameters of liver perfusion (SCl and ICG) were not affected by antiviral therapy. In the 14 non-responders, no changes in QTLF values were observed during the treatment period. CONCLUSION: ICG and SCl remained unaffected in patients with chronic hepatitis C, while ABT and GEC were significantly compromised. ABT and GEC normalized in responders to antiviral therapy. Early determination of ABT and GEC may differentiate responders from non-responders to antiviral treatment in hepatitis C.
Abstract: BACKGROUND AND AIMS: Pancreatic cancer remains a devastating diagnosis with only limited therapeutic options. Specific inhibition of expression of target genes has become possible using small interfering (si) RNAs. We therefore investigated how far siRNA specific for bcl-2 may serve as a therapeutic option for pancreatic cancer in vitro and in vivo. METHODS: siRNAs targeting two different regions in the bcl-2 gene were transfected to YAP C and DAN G pancreatic carcinoma cells and human foreskin fibroblasts. Permutations were generated by changing 3' and 5' overhangs and varying the length of the paired RNA duplex. Transfection efficacy was determined using FITC labelled siRNAs and fluorescence microscopy. Cell survival and apoptosis were quantified at 24-120 hours. Pancreatic cancer xenografts in male nude mice were treated intraperitoneally with siRNAs daily for 24 days. siRNA pharmacokinetics in vivo were assessed using radioactively labelled siRNAs. Total protein and RNA were extracted for western Blot analysis and quantitative polymerase chain reaction. RESULTS AND CONCLUSIONS: Bcl-2 specific siRNAs specifically inhibited expression of the target gene in vitro and in vivo. Antiproliferative and proapoptotic effects were observed in tumour cells but not in fibroblasts or non-malignant tissues. siRNA permutations and diverse overhangs influenced gene silencing efficacy. siRNA was quickly distributed to all organs and excreted via the kidney and liver. Bcl-2 specific siRNA is a promising adjunctive treatment for pancreatic carcinoma.
Abstract: Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-dependent transcription factor involved in various processes including the inflammation and carcinogenesis. The aim of the present study was 1) to examine the mRNA and protein expression of PPARgamma in gastric cancer (GC); 2) to evaluate the effect of PPARgamma ligand (ciglitazone) on the proliferation and apoptosis of GC cell line; and 3) to assess the levels of gastric tissue proinflammatory cytokines, IL-1beta and IL-8, and plasma gastrin in GC patients before and after Helicobacter pylori (H. pylori) eradication. The trial material included 30 H. pylori-negative controls and 30 sex- and age-matched GC patients without or with H. pylori before and after its eradication. Expression of tissue PPARgamma, tissue levels of IL-1beta and IL-8, and plasma concentration of gastrin were significantly higher in H. pylori-positive GC compared to controls, but H. pylori eradication significantly reduced these parameters. Kato III cells incubated with alive H. pylori upregulated PPARgamma expression and ciglitazone inhibited cell proliferation and induced apoptosis. PPARgamma, proinflammatory cytokines and plasma gastrin appear to be implicated in H. pylori-related gastric carcinogenesis and PPARgamma agonists may have potential in cancer therapy.
Abstract: BACKGROUND/AIMS: Medical treatment for hepatocellular carcinoma (HCC) remains elusive. While an acyclic retinoid improved tumor-free survival after hepatoma resection, tamoxifen finally proved ineffective. Combination therapy of both agents has not been investigated in vitro and in vivo. METHODS: MH7777A hepatoma cells were incubated with tamoxifen (TAM) and 9-cis retinoic acid (CRA) alone or in combination. Proliferation rate and apoptosis were assessed by BrdU incorporation and flow cytometry. In vivo efficacy was studied using the Morris hepatoma model in immunocompetent rats. End points were macroscopic tumor growth, metastasis and immunohistochemistry for proliferative and apoptotic tumor cells (PCNA and TUNEL staining). RESULTS: In vitro, CRA and TAM monotherapy was effective only in the highest concentration. Combination therapy significantly enhanced apoptosis rate and growth inhibition in hepatoma cells. While in vivo monotherapy did not reduce tumor growth or metastasis, their combination reduced tumor size after 28 days by 64.5+/-28%. This was paralleled by an increase in TUNEL positive and a decrease in PCNA positive cells. CONCLUSIONS: The combination of TAM and CRA enhances their anti-tumoral efficacy in vitro as well as in vivo, while monotherapy is ineffective. This combination could be a promising adjunctive therapy of HCC.
Abstract: Retinoids can block cell proliferation and induce apoptosis in tumor cells. The antitumoral effect of synthetic retinoids like Adapalene (ADA) on hepatoma cells (HepG2, Hep1B) was investigated. Cell proliferation was assessed by measuring DNA synthesis and apoptosis by flow cytometry and immunocytochemistry. Cell cycle- and apoptosis-associated proteins were semi-quantified by Western Blotting and breakdown of mitochondrial membrane potentials was detected by JC-1 staining. ADA at 10(-4)M efficiently induced apoptosis, reaching 61.7% in HepG2 and 79.1% in Hep1B after 72 h incubation. This was accompanied by up-regulation of pro-apoptotic bax and caspase 3, while bcl-2 was down-regulated, shifting the bax/bcl-2 ratio to >2.3 in hepatoma cells. ADA inhibits hepatoma cell growth in vitro and is a powerful inducer of hepatoma cell apoptosis.
Abstract: Effective therapy for advanced hepatocellular carcinoma (HCC) is lacking. Conventional chemotherapy was judged to be ineffective. We previously demonstrated that the histone deacetylase inhibitor Trichostatin A (TSA) blocks growth of HCC cells in vitro. The anti-tumoral effect of a combination of more than 2 classes of drugs remains unexplored. Four hepatoma cell lines were incubated with increasing concentrations of Tamoxifen (TAM), 9-cis retinoic acid (CRA), the methioninaminopeptidase inhibitor TNP-470 and TSA as single agents and in combination. Anti-proliferative and pro-apoptotic effects were assessed using BrdU-incorporation, FACS analysis and immunocytochemistry. Central pro- and anti-apoptotic proteins were measured by semi-quantitative Western blotting and substrate assays. All single substances inhibited proliferation and induced apoptosis in HCC cells only at high concentrations. The combination of TAM/CRA/TNP/TSA multiplied the anti-tumoral effects, reaching up to 93% inhibition of proliferation and 63% induction of apoptosis after 24 h in Hep1B cells. Pro-apoptotic factors bax and caspase 3 were highly increased with quadruple therapy, while anti-apoptotic bcl-2 decreased to undetectable levels. Fibroblasts remained largely unaffected. While the single substances were not effective on hepatoma cells in tolerable doses, their combination significantly increases anti-tumoral efficacy. Combination therapy with biomodulators is a promising treatment option for HCC.
Abstract: BACKGROUND AND PURPOSE: Numerous seroepidemiological and pathological studies linked Chlamydia pneumoniae and Helicobacter pylori with atherosclerosis. However, analyses of these infectious agents in the pathogenesis of stroke are either lacking or contradictory. Therefore, we evaluated the detection rate of C. pneumoniae and H. pylori in normal carotids vs. atherosclerotic carotids and compared these findings with serology, plaque morphology, inflammatory cell infiltrates and apoptosis rate. METHODS: The study was performed on 40 morphological normal carotids from autopsy and 20 advanced atherosclerotic carotids from endarterectomy after stroke. Serum IgG antibody titre was measured by enzyme immunoassay (H. pylori) and microimmunofluorescence (MIF) technique (C. pneumoniae). Immunohistochemistry (IHC) and Western blotting were performed to identify C. pneumoniae, H. pylori, to characterize plaque morphology (macrophages and smooth muscle cells) and the inflammatory infiltrate (T- and B cells) and to detect apoptosis (TUNEL staining). RESULTS: C. pneumoniae was found significantly more frequently in atherosclerotic than in normal carotids (P=0.001), which correlated with elevated C. pneumoniae IgG-antibody titres (P=0.048). Although H. pylori was not detected in carotids, elevated H. pylori antibody titres were significantly associated with the degree of atherosclerosis (P=0.001). The C. pneumoniae infected carotids displayed a slightly enhanced infiltrate of T cells and apoptosis rate, but no morphological changes. CONCLUSION: C. pneumoniae but not H. pylori, was detected by IHC primarily in symptomatic carotids, without specific morphological differences. Correlation of C. pneumoniae in-situ-detection and IgG antibodies suggested a possible connection between respiratory-tract and endovascular infection. The C. pneumoniae associated T-lymphocytes and apoptosis rate indicate an immune-mediated inflammatory process, involving vascular walls.
Abstract: BACKGROUND: Several studies have shown a link between gastrin and gastric cancer, both in humans and animals, especially infected with Helicobacter pylori (H. pylori). However, the exact role of hypergastrinemia in gastric carcinogenesis remains still undetermined. The aim of the present study was to evaluate the interaction between gastrin, cyclooxygenase-2 (COX-2), hepatocyte growth factor (HGF) and apoptosis-related proteins (Bax, Bcl-2, caspase-3, survivin) in cultured gastric epithelial cancer cells. MATERIAL AND METHODS: In the present study, gastric cultured cancer cells (KATO III cells) were exposed to increasing concentrations of gastrin (1-1000 nM). Cells incubated with culture medium alone, without added gastrin, served as controls. Using RT-PCR and Western blot, we examined the mRNA and protein expression for COX-2, HGF and apoptosis-related proteins (Bax, Bcl-2, caspase-3 and survivin). In addition, the gene expression of gastrin and gastrin receptor (CCK-2) as well as the release of gastrin in culture medium in the unstimulated cells were examined by RT-PCR and RIA, respectively. The apoptosis rate in cells was measured by flow cytometric analysis. RESULTS: The present study shows that the gastric cultured epithelial cells exhibit the expression of gastrin and CCK-2 receptors and release of gastrin into the culture medium. The epithelial gastric cancer cells incubated with gastrin showed a concentration-dependent increase of COX-2 and HGF expression. Although no significant changes in apoptosis rate were observed, the exposure of these cells was associated with a dose-dependent increase in the expression of antiapoptotic proteins Bcl-2 and survivin. CONCLUSIONS: This study demonstrates that 1) gastrin stimulates the gene and protein expression of COX-2 and HGF in human cultured gastric cancer cells and 2) gastrin shows antiapoptotic activity through the upregulation of Bcl-2 and survivin.
Abstract: BACKGROUND AND AIM: Quantitative tests of liver function (QTLF) can be modulated by enzyme-inducing agents. The objective of the study was to examine changes in QTLF after treatment with Phenobarbital, a potent cytochrome P450-inducing agent. METHODS: Forty-six consecutive patients with liver cirrhosis Child-Pugh score B and C (34 alcoholic, 12 hepatitis C) and a compromised aminopyrine breath test (ABT) were included. Thirty-six patients (group I) received phenobarbitone (150 mg) for 7 days. Ten patients (group II) received a placebo. The QTLF, which included ABT (microsomal liver function), galactose elimination capacity (GEC, cytosolic liver function), sorbitol clearance (SCl, liver plasma flow) and indocyanine green clearance (ICG, liver perfusion) was carried out before and after pharmacological induction. RESULTS: Group I showed a basal ABT of 0.18 +/- 0.11% dose.kg/mmol CO2 (normal >0.6%), which increased significantly after induction (172%, P < 0.05), whereas in group II the ABT values did not change. In group IB, a subgroup of group I which exceeded the basal threshold value of 0.30% after stimulation (n = 22), the ABT values increased significantly to 0.44 +/- 0.17% (244%, P < 0.01). The GEC, SCl and ICG remained unchanged before and after induction. CONCLUSIONS: After pharmacological induction, microsomal liver function increased significantly in a subgroup of patients with liver cirrhosis, whereas the GEC, SCl and ICG remained unchanged. Inducibility of the microsomal liver function could be used as a novel parameter of functional hepatic reserve and prognosis in cirrhosis.
Abstract: Chemotherapy of advanced stages of colorectal carcinoma is unsatisfactory. Retinoids inhibit cell growth and induce apoptosis in a variety of human malignancies. We compared the effect of the synthetic retinoid adapalene (ADA) and 9-cis-retinoic acid (CRA) on carcinoma cell lines in vitro. Colon carcinoma cell lines CC-531, HT-29 and LOVO as well as human foreskin fibroblasts were exposed to different concentrations of ADA and CRA for 3-72 hr. Proliferation was assessed by BrdU incorporation and apoptosis by FACS analysis. Breakdown of DeltaPsi(m) was determined by JC-1 staining and activity of caspases 3 and 8, by a colorimetric assay. Quantitative Western blots were performed to detect changes in bax, bcl-2 and caspase-3. Both retinoic derivatives suppressed DNA synthesis and induced apoptosis in all tested cell lines time- and dose-dependently. While the natural retinoid CRA showed moderate antiproliferative and proapoptotic effects only at the highest concentration (10(-4) M), the synthetic retinoic ADA was significantly more effective, showing remarkable effects even at 10(-5) M. ADA and CRA disrupt DeltaPsi(m) and induce caspase-3 activity in responsive tumor cells. Quantitative Western blots showed a shift of the bax:bcl-2 ratio toward proapoptotic bax in ADA-treated cells. Our results clearly indicate the superiority of ADA compared to CRA. Therefore, we suggest that ADA may be far more suitable as an adjunctive therapeutic agent for treatment of colon cancer in vivo.
Abstract: The prognosis of advanced colorectal carcinoma (CC) is poor. Established chemotherapy shows only limited efficacy but significant side effects. We investigated how far a combination of tamoxifen (TAM), 9-cis-retinoic acid (CRA) and the fluoroquinolone ciprofloxacin (CIP) synergize to inhibit proliferation and promote apoptosis of CC cells in vitro. The CC cell lines LOVO, CC-531 and SW-403 were incubated with TAM, CRA and CIP (10(minus;4)-10(minus;6) M) as single agents and in combination. Cell proliferation was assessed by bromodeoxyuridin incorporation. Apoptosis was quantified immunohistochemically and by FACS analysis after staining with propidium iodide. Changes in the expression of caspase 3, bax, bcl-2 and p21cip/waf were assessed by quantitative Western blotting. CRA and TAM monotherapy was moderately effective. Their combination enhanced apoptosis from 60% to more than 80% in all cell types. Apoptosis was paralleled by inhibition of proliferation and further potentiated by addition of CIP. The combination effectively up-regulated caspase 3 and bax and down-regulated bcl-2 and p21cip/waf. Combinations of biomodulaters act synergistically to inhibit proliferation and promote apoptosis in CC cells. Due to their known safety profile, this justifies clinical trials for colorectal cancer using combinations of these biological response modifiers.
Abstract: Chemotherapy does not have a prominent role in the treatment of hepatoma. However, an acyclic retinoid prevented tumor recurrence post-hepatectomy, and tamoxifen (TAM) induced apoptosis in tumor cells. Combination therapy of these agents on proliferation and apoptosis of hepatoma cells has not been explored. HepG2, Hep1B, Hepa1-6 and MH1C1 hepatoma cells were incubated with TAM, 9-cis- and all-trans retinoic acid (CRA, ATRA, respectively) alone or in combination. Proliferation rate was assessed and apoptosis was analyzed by flow cytometry, immunostaining, caspase activity assays and the expression of apoptosis- and/or cell cycle-related molecules. CRA and TAM, but not ATRA monotherapy were moderately effective. Apoptosis was accompanied by upregulation of caspase 3 and 8 activity, and increased p27, bax, caspase 3 expression, while the levels of p21cip/waf and bcl-2 were unchanged or decreased. Combination therapy enhanced apoptosis from a maximum of 60% after monotherapy to more than 90% after 96 h in all cell types. Pro-apoptotic effects were paralleled by inhibition of proliferation. Combination of TAM and CRA, but not ATRA, have an additive to synergistic anti-proliferative and pro-apoptotic effect on HCC cells. This justifies trials for HCC using combinations of these biological response modifiers.
Abstract: Efficacy of chemotherapy in advanced stages of colorectal tumours is limited. The quinolone antibiotic ciprofloxacin was recently shown to inhibit growth and to induce apoptosis in human bladder carcinomas cells. We investigated the effect of ciprofloxacin on colon carcinoma lines in vitro. CC-531, SW-403 and HT-29 colon carcinoma and HepG2 hepatoma cells (control cells) were exposed to ciprofloxacin. Proliferation was assessed by bromodeoxyuridine-incorporation into DNA and apoptosis was measured by flow cytometry after propidium iodide or JC-1 staining. Expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax was analyzed by semiquantitative Western blot analysis and activity of caspases 3, 8 and 9 by substrate-cleavage assays. Ciprofloxacin suppressed DNA synthesis of all colon carcinoma cells time- and dose-dependently, whereas the hepatoma cells remained unaffected. Apoptosis reached its maximum between 200 and 500 microg ml(-1). This was accompanied by an upregulation of Bax and of the activity of caspases 3, 8 and 9, and paralleled by a decrease of the mitochondrial membrane potential. Ciprofloxacin decreases proliferation and induces apoptosis of colon carcinoma cells, possibly in part by blocking mitochondrial DNA synthesis. Therefore, qualification of ciprofloxacin as adjunctive agent for colorectal cancer should be evaluated.
Abstract: BACKGROUND/AIMS: Effective treatment for hepatocellular carcinoma is urgently needed. The histone-deacetylase inhibitor Trichostatin A (TSA) was shown to induce apoptosis in non-hepatic cells at submicromolar concentrations. However, the effect of TSA on hepatoma cells is unknown. METHODS: The hepatoma cells HepG2, MH1C1, Hepa1-6 and Hep1B as well as human fibroblasts (control cells) were exposed to TSA (10(-6) to 10(-9)M). Cell proliferation was assessed by measuring DNA-synthesis and cell numbers. Apoptosis was quantified by flow cytometry and by the TdT-mediated dUTP nick-end labeling method. Expression patterns of cell cycle- and/or apoptosis-associated p27, p21(cip/waf), bax, bcl-2, cyclin A and (pro)-caspase 3 were studied using quantitative Western blotting. Activation of caspase 3 was analyzed via a colorimetric assay. RESULTS: 10(-6)M TSA inhibited DNA-synthesis by 46% (HepG2) to 64% (MH1C1) after 24h, inducing a G(2)/M-phase arrest and apoptosis. TSA increased activation of caspase 3 and expression of cyclin A, p2l(cip/waf), bax and (pro)-caspase 3, while bcl-2 was downregulated. Human fibroblasts remained unaffected. CONCLUSIONS: TSA inhibits hepatoma cell growth in vitro, which are otherwise particularly resistant to chemotherapy. Its anti-proliferative activity is paralleled by a comparable rate of apoptosis. TSA may be a promising agent for treatment of hepatocellular carcinoma in vivo.
Abstract: Studies comparing quantitative testing of liver function (QTLF) in large numbers of patients with defined etiology of cirrhosis are lacking. In all 316 patients with proven cirrhosis underwent QTLF, including aminopyrine breath test (ABT), galactose elimination capacity (GEC), sorbitol (SCI), and indocyanine green clearance (ICG). Values were correlated with the Child-Pugh classification (CP) and the etiology of liver cirrhosis. Fifty-five percent of the patients had alcoholic cirrhosis (ALC), 31% cirrhosis due to viral hepatitis (VIC), and 14% primary biliary cirrhosis (PBC). In all three groups there was a decrease of QTLF levels from CP grade A to C, which differed from normal values. QTLF was most compromised in patients with ALC and VIC compared to patients with PBC. In conclusion, QTLF in ALC and VIC patients was more reduced than in patients with PBC. This may be due to saturation of enzymes in ALC and ongoing inflammation in VIC.
Abstract: Little is known about the pathogenesis and etiology of benign tumors of the adrenal cortex. A variety of cellular oncogenes and tumor suppressor genes has been studied so far. The role of K-ras in this process is not yet clearly understood. Recent findings suggest a strong influence of mutated K-ras in the pathogenesis of adrenal adenomas (Lin et al., 1998). Therefore we studied 40 human adrenal tumors for mutations in the coding region of the cellular proto-oncogene K-ras by PCR-SSCP (Single-strand conformation polymorphism) analysis. We did not identify any activating mutation in the coding region of the K-ras gene. We conclude that activating mutations of the K-ras gene are not a major cause for the development of adrenal adenomas, if at all.
Abstract: Liver cancer is among the most common neoplastic diseases worldwide and its incidence is projected to increase dramatically in the next decade due to the increasing numbers of chronic viral hepatitis infections and fatty liver diseases. While conventional radio- or chemotherapy strategies are largely ineffective in the treatment of liver cancer, novel gene therapy approaches have entered the focus of research. Here, either the delivery of anti-angiogenic viral constructs or the direct silencing of angiogenic genes by RNA interference (RNAi) is commonly applied. As the liver is a preferential target organ for either adenovirus mediated gene transfer or
systemic application of short interfering RNAs (siRNA), these approaches have already shown convincing results in experimental models of liver cancer. The combination of these techniques, i.e. the viral delivery of siRNA encoding DNA plasmids, has increased the efficacy of gene silencing in the liver. Further targets are proliferation regulating genes or genes mediating the high intrinsic apoptosis resistance of liver tumor cells. To
enhance safety and efficacy of gene delivery to the liver, novel targeting constructs are currently under investigation.
Abstract: RNA interference (RNAi) through small interfering RNA (siRNA) is a recently described post-transcriptional gene silencing system and is regarded as one of the most effective and specific gene silencing techniques with therapeutic potential. Briefly, longer double-stranded RNA molecules (dsRNA, e.g. viral RNA or endogenous siRNA precursors like micro-RNA (miRNA) or short hairpin-RNA (shRNA)) are cut into siRNA, 21 nucleotide dsRNA, by a member of the RNAse III family referred to as Dicer. The siRNA is unwound by a helicase and associates with the RNA-induced silencing complex (RISC). Within RISC, the sense strand is removed and the antisense strand of the siRNA directs RISC to the target mRNA sequence, where it anneals complementarily. Finally, the target mRNA is degraded by RISC’s endonuclease activity. siRNA technology is also expected to be an invaluable treatment tool for viral infections, dominant disorders, neurological disorders and cancers. Here, novel and more effective therapies are urgently needed as conventional radio/chemotherapy is of limited efficacy in advanced stages. In some cancers, it is known that the resistance to chemotherapy is mainly attributed to increased expression of anti-apoptotic genes, e.g. members of the bcl-2 family which stabilize the mitochondrial membrane. The siRNAmediated knockdown of bcl-2 lead to pronounced anti-tumor effects in a pancreatic cancer model, especially in combination with chemotherapy even at otherwise ineffective concentrations. The future success of this approach will depend on the development of effective, specific and safe delivery systems.Additionally to therapeutic RNAi, endogenous RNAi processes may also contribute to cancer development. Here, miRNAs have been shown to suppress target gene expression through binding to the 3’-untranslated region (3’-UTR) of the target mRNA. miRNAs control many functions of cell viability including differentiation, proliferation and apoptosis especially during cancer progression. More investigations on miRNA will lead to new diagnostic and therapeutic approaches in the near future.
Abstract: The ability to induce and execute apoptosis is essential for the maintenance and regulation of tissue and organ homeostasis. A common feature of malignant cells is their
capability of evading apoptosis, which contributes to the high rate of resistance to radio or chemotherapy of tumor cells. Although some progress has been made in inhibiting
tumor growth by developing receptor-tyrosine-kinase antagonists that inhibit survival and growth promoting pathways, resistance against these and other compounds is mainly
due to defects in apoptosis regulation. In contrast, low apoptotic thresholds may sensitize tumors to well tolerated and clinical established treatment regimens. Therefore,
downstream targets of the apoptosis machinery, e.g. survivin, caspases or members of the bcl-2 family, seem to represent interesting new targets for cancer therapy. Modulating
apoptotic thresholds can be achieved by reconstituting genetically lost or epigenetically silenced pro-apoptotic factors, e.g. by viral gene transfer or inducing re-expression with
DNA methyltransferase or histone deacetylase inhibitors, or by inhibiting the activity or expression of anti-apoptotic factors. Here, RNAi-based approaches, e.g. against antiapoptotic bcl-2, seem to hold great promise to overcome treatment resistance of a variety of malignant tumor types. Overall, restoring apoptosis sensitivity or lowering apoptotic thresholds will lead to better tolerated and more potent treatment strategies.
Abstract: Current concepts of gastrointestinal tumor formation favor a multi-step model with the acquisition of several targeted mutations in oncogenes and tumor suppressor genes that finally lead to the acquisition of different “hallmarks of cancer” like self-sufficiency in growth or anti-growth signals, evasion of apoptosis, immortality and angiogenesis as has been highlighted by Hanahan and Weinberg in their outstanding review [1]. Recent evidences indicate that targeted genetic events alone (e.g. point mutations in oncogenes) are not sufficient to provide tumor cells with these growth advantages but that the process of carcinogenesis closely resembles and involves processes of trans- and dedifferentiation as have been observed during embryonic development. Comparative analyses of embryogenesis and tumorigenesis, especially of the gastrointestinal tract,
revealed immense analogies of morphological patterning as well as of expression of differentiation markers. Both processes of development and carcinogenesis as well as
integral processes of regeneration like reparation after acute or chronic inflammation are characterized by pattern maintenance which is essentially maintained by different
gradients of markers of embryonic differentiation. So called epigenetic modulations of chromatin and DNA elements lead to changes in phenotypic properties that can also favor the malignant conversion of progenitor cells. Furthermore, the role of cell-cell and cell-matrix contacts has gathered new attention as these signaling pathways strongly
influence survival and differentiation of embryonic and tumorigenic cells and is one of the key features of the still to be defined (cancer) stem cell niche, especially in the
gastrointestinal tract. The so far unclear factors that contribute to the maintenance and longevity of tumor stem cells may also contribute to relapsing tumor diseases after
successful first-line treatment and the outbreak of disseminated micro metastases. This novel concept yields great impact for the development of new diagnostic and therapeutic approaches, as highly chemo- and radioresistant long-living tumor progenitor or stem cells will enter the focus of future cancer medicine and research.
Abstract: The present invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of an anti-apoptotic gene, comprising a complementary RNA strand having a nucleotide sequence which is less than 25 nucleotides in length and which is substantially identical to at least a part of an apoptotic gene, such as a Bcl gene. The invention also relates to a pharmaceutical composition comprising the dsRNA together with a pharmaceutically acceptable carrier; methods for treating diseases caused by the expression of an anti-apoptotic gene using the pharmaceutical composition; and methods for inhibiting the expression of an anti-apoptotic gene in a cell.
