Felicitas Genze

Targeting XIAP bypasses Bcl-2-mediated resistance to TRAIL and cooperates with TRAIL to suppress pancreatic cancer growth in vitro and in vivo.

Resistance to apoptosis is a hallmark of pancreatic cancer, a leading cause of cancer deaths. Therefore, novel strategies are required to target apoptosis resistance. Here, we report that the combination of X-linked inhibitor of apoptosis (XIAP) inhibition and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an effective approach to trigger apoptosis despite Bcl-2 overexpression and to suppress pancreatic cancer growth in vitro and in vivo. Knockdown of XIAP by RNA interference cooperates with TRAIL to induce caspase activation, loss of mitochondrial membrane potential, cytochrome c release, and apoptosis in pancreatic carcinoma cells. Loss of mitochondrial membrane potential and cytochrome c release are extensively inhibited by a broad range or caspase-3 selective caspase inhibitor and by RNAi-mediated silencing of caspase-3, indicating that XIAP inhibition enhances TRAIL-induced mitochondrial damage in a caspase-3-dependent manner. XIAP inhibition combined with TRAIL even breaks Bcl-2-imposed resistance by converting type II cells that depend on the mitochondrial contribution to the death receptor pathway to type I cells in which TRAIL-induced activation of caspase-3 and caspase-9 and apoptosis proceeds irrespective of high Bcl-2 levels. Most importantly, XIAP inhibition potentiates TRAIL-induced antitumor activity in two preclinical models of pancreatic cancer in vivo. In the chicken chorioallantoic membrane model, XIAP inhibition significantly enhances TRAIL-mediated apoptosis and suppression of tumor growth. In a tumor regression model in xenograft-bearing mice, XIAP inhibition acts in concert with TRAIL to cause even regression of established pancreatic carcinoma. Thus, this combination of XIAP inhibition plus TRAIL is a promising strategy to overcome apoptosis resistance of pancreatic cancer that warrants further investigation.

Small Molecule XIAP Inhibitors Enhance TRAIL-Induced Apoptosis and Antitumor Activity in Preclinical Models of Pancreatic Carcinoma

Evasion of apoptosis is a characteristic feature of pancreatic cancer, a prototypic cancer that is refractory to current treatment approaches. Hence, there is an urgent need to design rational strategies that counter apoptosis resistance. To explore X-linked inhibitor of apoptosis (XIAP) as a therapeutic target in pancreatic cancer, we analyzed the expression of XIAP in pancreatic tumor samples and evaluated the effect of small molecule XIAP inhibitors alone and in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) against pancreatic carcinoma in vitro and in vivo. Here, we report that XIAP is highly expressed in pancreatic adenocarcinoma samples compared with normal pancreatic ducts. Small molecule XIAP inhibitors synergize with TRAIL to induce apoptosis and to inhibit long-term clonogenic survival of pancreatic carcinoma cells. In contrast, they do not reverse the lack of toxicity of TRAIL on nonmalignant cells in vitro or normal tissues in vivo, pointing to a therapeutic index. Most importantly, XIAP inhibitors cooperate with TRAIL to trigger apoptosis and suppress pancreatic carcinoma growth in vivo in two preclinical models, i.e., the chorioallantoic membrane model and a mouse xenograft model. Parallel immunohistochemical analysis of tumor tissue under therapy reveals that the XIAP inhibitor acts in concert with TRAIL to cause caspase-3 activation and apoptosis. In conclusion, our findings provide, for the first time, evidence in vivo that XIAP inhibitors prime pancreatic carcinoma cells for TRAIL-induced apoptosis and potentiate the antitumor activity of TRAIL against established pancreatic carcinoma. These findings build the rationale for further (pre)clinical development of XIAP inhibitors and TRAIL against pancreatic cancer.

Activation of Phosphatidylinositol 3-Kinase and Extracellular Signal-Regulated Kinase Is Required for Glial Cell Line-Derived Neurotrophic Factor-Induced Migration and Invasion of Pancreatic Carcinoma Cells

Pancreatic carcinoma cells exhibit a pronounced tendency to invade along and into intra- and extrapancreatic nerves, even at early stages of the disease. The neurotrophic factor glial cell line-derived neurotrophic factor (GDNF) has been shown to promote pancreatic cancer cell invasion. Here, we demonstrate that pancreatic carcinoma cell lines, such as PANC-1, expressed the RET and GDNF family receptor α receptor components for GDNF and that primary pancreatic tumor samples, derived from carcinomas with regional lymph node metastasis, exhibited marked expression of the mRNA encoding the RET51 isoform. Moreover, GDNF was an efficacious and potent chemoattractant for pancreatic carcinoma cells as examined in in vitro and in vivo model systems. Treatment of PANC-1 cells with GDNF resulted in activation of the monomeric GTPases N-Ras, Rac1, and RhoA, in activation of the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) and in activation of the phosphatidylinositol 3-kinase/Akt pathway. Both inhibition of the Ras-Raf-MEK (mitogen-activated protein/ERK kinase)-ERK cascade by either stable expression of dominant-negative H-Ras(N17) or addition of the MEK1 inhibitor PD98059 as well as inhibition of the phosphatidylinositol 3-kinase pathway by LY294002 prevented GDNF-induced migration and invasion of PANC-1 cells. These results demonstrate that pancreatic tumor cell migration and possibly perineural invasion in response to GDNF is critically controlled by activation of the Ras-Raf-MEK-ERK and the phosphatidylinositol 3-kinase pathway.

Sodium butyrate and tributyrin induce in vivo growth inhibition and apoptosis in human prostate cancer

Histone deacetylase inhibitors (HDACs) are known to exhibit antiproliferative effects on various carcinoma cells. In this study, the in vivo efficiency of two HDACs, sodium butyrate and tributyrin, on prostate cancer growth inhibition were investigated. To gain an insight into the possible underlying pathways, cell culture experiments were performed focusing on the expression of p21, Rb and c-myc. For in vivo testing, prostate cancer cell lines (PC3 and TSU-Pr1) were seeded on the chorioallantois membrane (CAM) and implanted in a xenograft model using nude mice. Standard Western blot analysis was performed for protein expression of p21, Rb and c-myc in HDAC-treated vs untreated prostate cancer cells. Both sodium butyrate and tributyrin had a considerable treatment effect on microtumours on the chicken egg at already very low concentrations of 0.1 mM. Tributyrin-treated tumours showed the strongest effect with 38% apoptotic nuclei in the prostate cancer cell line PC3. In the mouse model, there was almost no difference between sodium butyrate and tributyrin. In untreated animals the tumours were almost double the size 4 weeks after implantation. Tumours of the treatment groups had a significantly lower percentage of Ki-67-positive-stained nuclei. As demonstrated by Western blot analysis, these effects seem to be independent of p53 status and a pathway via p21–Rb–c-myc is possibly involved. In this study we have demonstrated a substantial in vivo treatment effect, which can be induced by the application of sodium butyrate or the orally applicable tributyrin in human prostate cancer. The given results may provide the rationale to apply these drugs in well-controlled clinical trials in patients being at high risk of recurrence after specific therapy or in patients with locally or distant advanced prostate cancer.

Antiinflammatory and Antiatherogenic Effects of the NF-κB Inhibitor Acetyl-11-Keto-β-Boswellic Acid in LPS-Challenged ApoE−/− Mice

Objective—In this article, we studied the effect of acetyl-11-keto-β-boswellic acid (AKβBA), a natural inhibitor of the proinflammatory transcription factor NF-&kgr;B on the development of atherosclerotic lesions in apolipoprotein E–deficient (apoE−/−) mice. Methods and Results—Atherosclerotic lesions were induced by weekly LPS injection in apoE−/− mice. LPS alone increased atherosclerotic lesion size by ≈100%, and treatment with AKβBA significantly reduced it by ≈50%. Moreover, the activity of NF-&kgr;B was also reduced in the atherosclerotic plaques of LPS-injected apoE−/− mice treated with AKβBA. As a consequence, AKβBA treatment led to a significant downregulation of several NF-&kgr;B–dependent genes such as MCP-1, MCP-3, IL-1α, MIP-2, VEGF, and TF. By contrast, AKβBA did not affect the plasma concentrations of triglycerides, total cholesterol, antioxidized LDL antibodies, and various subsets of lymphocyte-derived cytokines. Moreover, AKβBA potently inhibited the I&kgr;B kinase (IKK) activity immunoprecipitated from LPS-stimulated mouse macrophages and mononuclear cells leading to decreased phosphorylation of I&kgr;Bα and inhibition of p65/NF-&kgr;B activation. Comparable AKβBA-mediated inhibition was also observed in LPS-stimulated human macrophages. Conclusion—The inhibition of NF-&kgr;B activity by plant resins from species of the Boswellia family might represent an alternative for classical medicine treatments for chronic inflammatory diseases such as atherosclerosis. (Arterioscler Thromb Vasc Biol. 2008;28:272-277)

Methylene blue mediated photodynamic therapy in experimental colorectal tumors in mice.

Methylene blue (MB+) is a well-known dye in medicine and has been discussed as an easily applicable drug for the topical treatment during photodynamic therapy (PDT). The therapeutic response of MB+ was investigated in vivo by local injection of MB+ in a xenotransplanted subcutanous tumor (adeno-carcinoma, G-3) in female nude mice. MB+ in a concentration of 1% was applied both undiluted and diluted to 0.1 and 0.01% with isotonic sodium chloride. Treatment with 1% MB+ and subsequent irradiation at 662 nm with 100 J/cm2 led to complete tumor destruction in 79% of the treated animals. A decrease of the fluence rate from 100 to 50 mW/cm2 increased the phototoxic response as well as fractionated light application. Small sensitizer concentrations reduced the PDT effect significantly. It seems that the light induced reaction of MB+ could be correlated with the rapid production of reactive oxygen species. Below a threshold dose of MB+ oxidative damage of the tissue is prevented. However, above this dose, as a point of no return, MB+ acts as an extremely potent oxidant.

CHORIOALLANTOIC MEMBRANE ASSAY: VASCULARIZED 3-DIMENSIONAL CELL CULTURE SYSTEM FOR HUMAN PROSTATE CANCER CELLS AS AN ANIMAL SUBSTITUTE MODEL

PURPOSE Chorioallantoic membranes have been used as a reliable biomedical assay system for many years. Chicken eggs in the early phase of breeding are between in vitro and in vivo systems but may provide an immunodeficient, vascularized test environment. We tested this model as an in vivo system for prostate cancer research. MATERIALS AND METHODS Single cell suspensions of LNCaP, PC-3 and Tsu-Pr1 human prostatic cancer cell lines as well as 2 immortalized normal human prostate epithelial cell lines were inoculated on the chorioallantoic membrane of fertilized chicken eggs on day 5 or 6 of breeding. Tumor growth and viability of the embryo was evaluated by stereo microscopy. At day 10 the membranes were removed and embedded in paraffin. Cell morphology was assessed after hematoxylin and eosin staining. Cellular expression of cytokeratin, prostate specific antigen and androgen receptor as well as apoptosis induction was confirmed by immunohistochemistry. RESULTS Three days after tumor cell inoculation on the extraembryonic vascular system of the chorioallantoic membrane cell growth and formation of 3-dimensional tumors became apparent in 100% of inoculated membranes. Strong neo-angiogenesis was detected next to the established tumors and tumor cells invading the stroma of the chorioallantoic membrane. Cytokeratin expression as well as prostate specific antigen and androgen receptor in LNCaP cells confirmed the human prostate tumor origin. Assessment of quantitative in vivo apoptosis induction in LNCaP cells after intravenous injection of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate confirmed the model as a versatile in vivo system. CONCLUSIONS The well vascularized chorioallantoic membrane of bred chicken eggs is a suitable system for early in vivo cancer research. Reliable growth of prostate cancer cell lines is feasible and allows the evaluation of proliferation and apoptosis induction after intravascular or topic application of anticancer drugs. Exploitation of this assay enables a substantial reduction in or substitution for subsequent animal experiments.

Expression of MTA1 promotes motility and invasiveness of PANC-1 pancreatic carcinoma cells

The human metastasis-associated protein 1 (MTA1) is a constituent of the nucleosome-remodelling and -deacetylation complex. Its expression has been correlated with the invasion and metastasis of epithelial neoplasms. To address the functional consequences of MTA1 expression in pancreatic carcinoma cells, we have established PANC-1 pancreatic carcinoma cells that stably express MTA1 as an enhanced green fluorescent fusion protein (EGFP–MTA1). Here, we demonstrate that heterologous expression of EGFP–MTA1 markedly enhanced the cellular motility and the invasive penetration of epithelial barriers by the cells. Expression of EGFP-MTA1 had no effect on substrate-independent growth, but reduced substrate-dependent cell proliferation. In addition, the organisation of the cytokeratin filament system and the localisation of the actin cytoskeleton-associated protein IQGAP1 were distinctly altered in EGFP–MTA1-expressing cells. These results indicate that enhanced expression of MTA1 promotes the acquisition of an invasive, metastatic phenotype, and thus enhances the malignancy of pancreatic adenocarcinoma cells by modulation of the cytoskeleton.

Protein kinase D2 is a crucial regulator of tumour cell–endothelial cell communication in gastrointestinal tumours

Background Tumour angiogenesis is crucially dependent on the communication between the tumour and the associated endothelium. Protein kinase D (PKD) isoenzymes mediate vascular endothelial growth factor-A (VEGF-A) induced endothelial cell proliferation and migration and are also highly expressed in various tumours. Aim To examine the role of PKDs for tumour proliferation and angiogenesis selectively in pancreatic and gastric tumours and in tumour-associated endothelium in vitro and in vivo. Methods PKD2 expression in human tumours was determined by immunohistochemistry. The effect of PKD2 depletion in endothelial cells by siRNAs was examined in sprouting assays, the chorioallantois model (CAM) and tumour xenografts. In murine endothelium in vivo PKD2 was knocked-down by splice switching oligonucleotides. Human PKD2 was depleted in xenografts by siRNAs and PKD2-miRs. PKD2 activation by hypoxia and its role for hypoxia-induced NR4/TR3- and VEGF-A promoter activity, expression and secretion was investigated in cell lines. Results PKD2 is expressed in gastrointestinal tumours and in the tumour-associated endothelium. Tumour growth and angiogenesis in the CAM and in tumour xenografts require PKD expression in endothelial cells. Conversely, hypoxia activates PKD2 in pancreatic cancer cells and PKD2 was identified as the major mediator of hypoxia-stimulated VEGF-A promoter activity, expression and secretion in tumour cells. PKD2 depletion in pancreatic tumours inhibited tumour-driven blood vessel formation and tumour growth in the CAM and in orthotopic pancreatic cancer xenografts. Conclusion PKD2 regulates hypoxia-induced VEGF-A expression/secretion by tumour cells and VEGF-A stimulated blood vessel formation. PKD2 is a novel, essential mediator of tumour cell–endothelial cell communication and a promising therapeutic target to inhibit angiogenesis in gastrointestinal cancers.

Thioredoxin-1 Promotes Anti-Inflammatory Macrophages of the M2 Phenotype and Antagonizes Atherosclerosis

Objective—Oxidative stress is believed to play a key role in cardiovascular disorders. Thioredoxin (Trx) is an oxidative stress-limiting protein with anti-inflammatory and antiapoptotic properties. Here, we analyzed whether Trx-1 might exert atheroprotective effects by promoting macrophage differentiation into the M2 anti-inflammatory phenotype. Methods and Results—Trx-1 at 1 &mgr;g/mL induced downregulation of p16INK4a and significantly promoted the polarization of anti-inflammatory M2 macrophages in macrophages exposed to interleukin (IL)-4 at 15 ng/mL or IL-4/IL-13 (10 ng/mL each) in vitro, as evidenced by the expression of the CD206 and IL-10 markers. In addition, Trx-1 induced downregulation of nuclear translocation of activator protein-1 and Ref-1, and significantly reduced the lipopolysaccharide-induced differentiation of inflammatory M1 macrophages, as indicated by the decreased expression of the M1 cytokines, tumor necrosis factor-&agr; and monocyte chemoattractant protein-1. Consistently, Trx-1 administered to hyperlipoproteinemic ApoE2.Ki mice at 30 &mgr;g/30 g body weight challenged either with lipopolysaccharide at 30 &mgr;g/30 g body weight or with IL-4 at 500 ng/30 g body weight significantly induced the M2 phenotype while inhibiting differentiation of macrophages into the M1 phenotype in liver and thymus. ApoE2.Ki mice challenged once weekly with lipopolysaccharide for 5 weeks developed severe atherosclerotic lesions enriched with macrophages expressing predominantly M1 over M2 markers. In contrast, however, daily injections of Trx-1 shifted the phenotype pattern of lesional macrophages in these animals to predominantly M2 over M1, and the aortic lesion area was significantly reduced (from 100%±18% to 62.8%±9.8%; n=8; P<0.01). Consistently, Trx-1 colocalized with M2 but not with M1 macrophage markers in human atherosclerotic vessel specimens. Conclusion—The ability of Trx-1 to promote differentiation of macrophages into an alternative, anti-inflammatory phenotype may explain its protective effects in cardiovascular diseases. These data provide novel insight into the link between oxidative stress and cardiovascular diseases.