Daniela Haase

Sunitinib targets PDGF-receptor and Flt3 and reduces survival and migration of human meningioma cells

The multitargeted tyrosine-kinase inhibitor sunitinib is a highly effective anti-angiogenic and cytostatic agent in the therapy of various tumours. While malignant gliomas have been shown to be responsive to sunitinib, detailed studies analysing human meningiomas are missing. We therefore analysed the effects of sunitinib in two benign (BenMen-1, HBL52) and two malignant (IOMM-Lee, KT21MG) human meningioma cell lines and found that DNA synthesis was significantly (p ≤ 0.001) inhibited following 1, 2 or 5 μM sunitinib, with IC(50) values between 2 and 5 μM in all cell lines. This effect was associated with a G(2)M-arrest at 10 μM for BenMen-1, HBL52 and IOMM-Lee, and 20 μM in KT21MG cells. Nuclear bisbenzimide staining revealed chromatin condensation following treatment with sunitinib concentrations of 10 μM or higher. Corresponding, cell viability assays showed a significant (p ≤ 0.001) short term decrease of viable cells (24h) only for high sunitinib concentrations with IC(50)-values between 10 and 20 μM. However, pre-irradiated meningioma cells (5 Gy) showed a sensitivity shift towards IC(50)-values around 5 μM sunitinib. We also found that 5 μM strongly reduced meningioma cell migration in vitro. Western blot analyses showed abolished platelet derived growth factor receptor (PDGFR)-autophosphorylation after sunitinib. Interestingly, the drug also inhibited the autophosphorylation of the receptor tyrosine kinase fms-like tyrosine kinase 3 (Flt3) in a dose-dependent manner. Taken together, the present data show that micromolar sunitinib has strong cytostatic and anti-migratory effects on human meningioma cells.

Identification and validation of selective upregulation of ventricular myosin light chain type 2 mRNA in idiopathic dilated cardiomyopathy

the etiology of idiopathic dilated cardiomyopathy (IDCM) is unknown, methods such as suppression subtractive hybridization (SSH) and DNA microarray technology can help to identify genes which might be involved in the pathogenesis of this disease.

Fatty acid synthase as a novel target for meningioma therapy

High levels of fatty acid synthase (FAS) expression have been reported in hormone receptor-positive tumors, including prostate, breast, and ovarian cancers, and its inhibition reduces tumor growth in vitro and in vivo. Similar to other hormone receptor-positive tumor types, meningiomas are progesterone receptor- and estrogen receptor-immunoreactive brain tumors. To define the role of FAS in human meningioma growth control, we first analyzed the FAS expression using a tissue microarray containing 38 meningiomas and showed increased FAS expression in 70% of atypical WHO grade II and anaplastic WHO grade III meningiomas compared with 10% of benign WHO grade I tumors. We next confirmed this finding by real-time PCR and Western blotting. Second, we demonstrated that treatment with the FAS inhibitor, cerulenin (Cer), significantly decreased meningioma cell survival in vitro. Third, we showed that Cer treatment reduced FAS expression by modulating Akt phosphorylation (activation). Fourth, we demonstrated that Cer treatment of mice bearing meningioma xenografts resulted in significantly reduced tumor volumes associated with increased meningioma cell death. Collectively, our data suggest that the increased FAS expression in human meningiomas represents a novel therapeutic target for the treatment of unresectable or malignant meningioma.

Reduced Activity of CD13/Aminopeptidase N (APN) in Aggressive Meningiomas Is Associated with Increased Levels of SPARC

Meningiomas are the second most common brain tumors in adults, and meningiomas exhibit a tendency to invade adjacent structures. Compared with high‐grade gliomas, little is known about the molecular changes that potentially underlie the invasive behavior of meningiomas. In this study, we examined the expression and function of the membrane alanyl‐aminopeptidase [mAAP, aminopeptidase N (APN), CD13, EC3.4.11.2] zinc‐dependent ectopeptidase in meningiomas and meningioma cell lines, based on its prior association with tumor invasion in colorectal and renal carcinomas. We found a significant reduction of APNmRNA and protein expression, as well as enzymatic activity, in high‐grade meningiomas. While meningioma tumor cell proliferation was not affected by either pharmacologic APN inhibition or siRNA‐mediated APN silencing, APN pharmacologic and siRNA knockdown significantly reduced meningioma cell invasion in vitro. Next, we employed pathway‐specific cDNA microarray analyses to identify extracellular matrix and adhesion molecules regulated by APN, and found that APN‐siRNA knockdown substantially increased the expression of secreted protein, acidic and rich in cysteine (SPARC)/osteonectin. Finally, we demonstrated that SPARC, which has been previously associated with meningioma invasiveness, was increased in aggressive meningiomas. Collectively, these results suggest that APN expression and enzymatic function is reduced in aggressive meningiomas, and that alterations in the balance between APN and SPARC might favor meningioma invasion.

Reduced Morg1 expression in ischemic human brain

The mitogen-activated protein kinase organizer 1 (Morg1) has been recently identified as modular scaffold regulating ERK signaling. Morg1 also attenuates expression of the hypoxia-inducible factor-1alpha (HIF-1alpha) by activating or stabilizing of prolyl-hydroxylase 3 (PHD3). Here we demonstrate for the first time that Morg1 is expressed in the human brain in neurons, glial cells, and blood vessel walls. Immunohistochemistry, RT real-time PCR and western blotting indicated that Morg1 expression is reduced in human brain tissue with ischemic damage. Moreover, reactive astrocytes in the surrounding brain tissue showed strong Morg1 expression. Since hypoxic adaptation with enhancing HIF-1alpha expression can engage a genetic program leading to profound sparing of brain tissue and enhanced recovery of function, down-regulation of Morg1 expression in the ischemic brain may be viewed as an intrinsic mechanism to stimulate this response. On the other hand, upregulation of Morg1 in astrocytes surrounding the penumbra may counteract this hypoxic adaptation.

Collagen prolyl hydroxylase 3 has a tumor suppressive activity in human lung cancer

Abstract Collagen prolyl hydroxylases (P3H) are required for proper collagen biosynthesis. One of the family members P3H3 was downregulated in breast cancer and lymphoma due to DNA methylation. However the role of P3H3 in lung cancer has not yet been elucidated. In this study, we analyzed P3H3 expression in a panel of lung cancer cell lines and primary lung tumors. Epigenetic regulation was explored and the function of P3H3 was investigated by stable transfection and RNA interference. We found that P3H3 was downregulated in 6 out of 10 lung cancer cell lines. A heterogeneous methylation pattern of P3H3 was found in the exon region. In primary lung tumors, immunohistochemistry on tissue microarray (TMA) showed that higher expression of P3H3 was significantly associated with lower tumor N stage and grade (p = 0.035 and p = 0.026, respectively). Ectopic expression of P3H3 inhibited cell proliferation, colony formation, migration as well as invasion, and induced apoptosis together with cell cycle arrest in the G2/M phase. Knockdown of P3H3 led to increased migratory and invasive potential. These Phenomena are accompanied by enhanced p21, decreased cyclin A1 levels and increased caspase 3/7 activities. Taken together, we feel that P3H3 is a novel tumor suppressor and its protein expression is inversely related to lymph node metastasis and tumor differentiation in lung cancer.

Development and characterization of an ex vivo arterial long-term proliferation model for restenosis research.

One of the main limitations of percutaneous coronary interventions is the restenosis, occurring in small-diameter arteries, and efforts are high to find improved intracoronary devices to prevent in-stent-restenosis. Aim of this study was to produce a new in vitro test platform for restenosis research, suitable for long-term cell proliferation and migration studies in stented vessels. Fresh segments of porcine coronary arteries were obtained for decellularization and were then reseeded with human coronary artery endothelial (HCAEC) and human coronary artery smooth muscle cells (HCASMC). Subsequently, bare metal stents (BMS) and drug eluting stents (DES), respectively, were implanted and the segments were reseeded with HCAEC and HCASMC for up to three months. The stented segments were examined at time zero and after 2, 4, 6, 8 and 12 weeks by histochemical and immunohistochemical characterization and the reseeded areas before and after stent implantation were measured. We have found that cells formed multiple layers after three months, and the detection with both CD31 and a-smooth muscle actin specific antibodies showed that HCAEC and HCASMC are adherent and growing in several layers. Furthermore, we could show a significantly smaller proliferation area in DES (70% ± 3.5%), compared to BMS (17% ± 2.3%). These data are similar to animal and human studies. Therefore, this vessel model might appear as an initial benchmark for testing new anti-proliferative endovascular therapies and consequently helps to reduce animal experiments in this research area.