Sphingosine­1­phosphate analogue FTY720 exhibits a potent anti­proliferative effect on glioblastoma cells.

Sphingosine­1­phosphate (S1P) plays a key role in cell survival, growth, migration, and in angiogenesis. In glioma, it triggers the activity of the S1P­receptor 1 and of the sphingosine kinase 1; thus influencing the survival rate of patients. The aim of the present study was to investigate the anti­proliferative effect of the S1P analogue FTY720 (fingolimod) in glioblastoma (GBM) cells. A172, G28, and U87 cells were incubated with micromolar concentrations of FTY720 or temozolomide (TMZ) for 24 to 72 h. Proliferation and half maximal inhibitory concentration (IC50) were determined by using the xCELLigence system. FACS analysis was performed to check the cell cycle distribution of the cells after a 72­h incubation with FTY720. This was then compared to TMZ­incubated and to untreated cells. Gene expression was detected by RT­qPCR in A172, G28, U87 and three primary GBM­derived cell lines. FTY720 was able to reduce the number of viable cells. The IC50 value was 4.6 µM in A172 cells, 17.3 µM in G28 cells, and 25.2 µM in U87 cells. FTY720 caused a significant arrest of the cell cycle in all cells and stabilized or over­expressed the level of AKT1, MAPK1, PKCE, RAC1, and ROCK1 transcripts. The TP53 transcript level remained stable or was downregulated after treatment with FTY720. FTY720 may be a promising target drug for the treatment of GBM, as it has a strong anti­proliferative effect on GBM cells.

Individualized multimodal treatment strategy for anaplastic thyroid carcinoma-Case report of long-term remission and review of literature.

INTRODUCTION: The prognosis of anaplastic thyroid cancer (ATC) is poor with a mean survival time of six months following diagnosis. Despite various attempts to modify common treatment modalities including surgery, external beam radiation and chemotherapy, an effective treatment is not available yet. We report, here, a patient who achieved long-term survival based on multimodal treatment, including in vitro evaluation of drug response of his tumor cells. PRESENTATION OF CASE: A 42 years old male patient underwent total thyroidectomy with central and lateral neck dissection for ATC (pT4b, pN0 (0/36), L0, V0, Pn1, R0 cM0 - UICC-Stage: IV b). From the tumor tissue a primary cell culture was established. While the patient received a combined radio-chemotherapy cell viability assays were performed using Sorafenib, Vandetanib und MLN8054 (Aurora kinase inhibitor) as inhibitors. Cell viability was determined by MTT-assay after 72 and 144h of treatment. DISCUSSION: All the three compounds affected cell viability in a time- and dose dependent manner. These effects were most pronounced by Sorafenib. Based on in vitro findings, the patient was treated daily with 400mg Sorafenib for 75days. 43 months after initial diagnosis, the patient had no evidence of disease as shown by MRI, CT and FDG-PET-CT imaging. CONCLUSION: In the setting of multimodal treatment, in vitro drug evaluation of individual tumor cells of patients might be a promising tool to ameliorate the fatal prognosis of selected ATC patients.

Roscovitine has anti-proliferative and pro-apoptotic effects on glioblastoma cell lines: A pilot study.

Purine analogue roscovitine, a cyclin-dependent kinase (CDK) inhibitor, has shown strong anti-proliferative and pro-apoptotic effects in solid and hematologic cancers such as non small-cell lung cancer and lymphomas. It targets CDK2, 7 and 9 preferentially, which are also overexpressed in glioblastoma. Τherefore, the biological effects of roscovitine in glioblastoma cell lines were investigated. Glioblastoma A172 and G28 cell lines were incubated with serial concentrations of roscovitine for 24-120 h. Proliferation was measured using the xCELLigence Real-Time Cell Analyzer, an impedance­based cell viability system. Cell cycle distribution was assessed by flow cytometry and gene expression was quantified by quantitative RT-PCR and western blot analysis. Roscovitine exhibited a clear dose-dependent anti­proliferative and pro­apoptotic effect in the A172 cell line, while G28 cells showed a anti-proliferative effect only at 100 µM. The results of the flow cytometric (FACS) analysis revealed a dose-dependent increase of the G2/M and sub-G1 fractions in A172 cells, while G28 cells responded with an elevated sub-G1 fraction only at the highest concentration. Roscovitine led to a dose­dependent decrease of transcripts of p53, CDK 7 and cyclins A and E and an increase of >4-fold of p21 in A172 cells. In G28 cells, a dose­dependent induction of CDK2, p21 and cyclin D was observed between 10 and 50 µM roscovitine after 72 h, however, at the highest concentration of 100 µM, all investigated genes were downregulated. Roscovitine exerted clear dose-dependent anti-proliferative and pro-apoptotic effects in A172 cells and less distinct effects on G28 cells. In A172 cells, roscovitine led to G2/M arrest and induced apoptosis, an effect accompanied by induced p21 and a reduced expression of CDK2, 7 and 9 and cyclins A and E. These effects requre further studies on a larger scale to confirm whether roscovitine can be used as a therapeutic agent against glioblastoma.

The Nd:YAG LIMAX® 120 high-output laser: local effects and resection capacity on liver parenchyma.

Various technical options are available for the resection of liver metastases, including CUSA, Ultracision, water-jet, and stapler devices. It has been shown that new generation high-output lasers are suitable for the resection of lung metastases. The goal of the present study was to evaluate the local effects of laser application on liver parenchyma. Livers of freshly slaughtered pigs (N = 6) were analyzed. The handheld laser was vertically held in the clamp of a hydraulic machine and sharply focused on the liver surface. The diode pumped Nd:YAG laser LIMAX® 120 (Gebrüder Martin GmbH & Co. KG, Tuttlingen, Germany) moved evenly over the liver surface at speeds of 5, 10, and 20 mm/s. Laser outputs of 60 and 120 W were applied at every speed. Histological sections (hematoxylin and eosin (HE) staining) of the extension area of vaporization and coagulation were analyzed by the use of the ImageJ software. In addition, the area of the liver parenchyma cut by the laser within 1 min was measured. The vaporized zone appeared wedge-shaped after histological section, whereas the area of coagulation appeared radiated outward. At 10 mm/s and 60 W, the mean vaporization of the measured zone was 356.6 ± 3.9 µm in length. Superficial coagulation was observed at 20 mm/s laser speed, without effective resection. At 120 W and 5 mm/s working speed, the mean vaporization zone and the average width of coagulation were largest with 664.6 ± 5.9 and 375.6 ± 2.3 µm, respectively. The laser output power of 120 W allowed resection of an area of 6 ± 0.4 cm(2) of liver parenchyma within 1 min. The Nd:YAG Laser LIMAX® 120 might be an effective tool for liver parenchyma dissection when it is applied at maximum output (120 W) and at a constant working speed of 5 mm/s.

Embryonic transcription factors CDX2 and Oct4 are overexpressed in neuroendocrine tumors of the ileum: a pilot study.

BACKGROUND: Neuroendocrine tumors (NETs) of the ileum are rare submucosal tumors that are often diagnosed at advanced stages with metastatic spread to the liver causing a carcinoid syndrome. They present as solitary or multiple tumors. In NETs, loss of sequences on chromosomes 11, 16, 18 and 22 or gain of sequences on chromosomes 17 and 19 has been described. In this study we explored the expression of two novel candidate genes, CDX2 and Oct4, in NETs of the ileum and analyzed whether the molecular expression pattern correlates with the clinical phenotype (solitary/multiple tumors). METHODS: Data from all patients who underwent surgery for a NET of the ileum between 2000 and 2010 were retrieved from a prospective database. For each patient, frozen normal and tumor tissue was used for the comparison of gene expression levels of two putative cancer stem cell markers, CDX2 and Oct4, using real-time PCR (rtPCR). Serial slides from paraffin blocks were used for immunohistochemistry. Gene expression was compared between normal and tumor tissue as well as between solitary and multiple tumors. RESULTS: 78 patients were identified. In rtPCR, a statistically significant higher expression of CDX2 in tumor tissue (p < 0.001) compared to normal tissue was found. The expression of Oct4 was elevated in the tumors, but did not reach the level of significance (p = 0.155). The expression of both candidate genes was confirmed immunohistochemically and showed a nuclear expression pattern. There was no difference in expression between solitary and multiple tumors or between tumors that had already spread to the liver. CONCLUSION: CDX2 is overexpressed in ileum NETs, thus playing a role in the tumorigenesis of these rare tumors. Since expression does not correlate with clinical stage or phenotype, it might be an early event in tumor development.

Histone deacetylase inhibitors induce in human hepatoma HepG2 cells acetylation of p53 and histones in correlation with apoptotic effects.

This report shows that histone deacetylase inhibitors (HDACIs) induced apoptosis in human hepatoma HepG2 cells in a dose- and time-dependent manner. Trichostatin A (TSA), ITF2357 and suberoylanilide hydroxamic acid (SAHA), which were very effective agents, caused apoptotic effects after a lag phase of 12-16 h. In order to elucidate the mechanism of HDACIs action in HepG2 cells we have studied the effects of TSA, ITF2357 and SAHA on acetylation of p53 and histones H2A, H2B, H3 and H4. It was observed that HDACIs rapidly induced acetylation of these proteins, being the effects clearly visible already at 30 min of treatment at the same doses which caused apoptosis. Analysis of the immunocomplexes, obtained from nuclear extracts using an antibody against p53, revealed the presence of acetylated p53 together with acetylated forms of histones and histone acetyltransferases p300 and PCAF. Experiments performed using pifithrin-alpha, a reversible inhibitor of p53, showed a correlation between acetylation of p53 and induction of apoptosis. In addition treatment with siRNA against p53 indicated that p53 is involved in the acetylation of histones. In conclusion, this report suggests that complexes constituted by acetylated p53, acetylated histones and coactivators can play a central role in HDACI-induced apoptosis in HepG2 cells.

SAHA induces apoptosis in hepatoma cells and synergistically interacts with the proteasome inhibitor Bortezomib.

Histone deacetylase (HDAC) inhibitors represent a promising group of anticancer agents. This paper shows that the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) stimulated at 5-10 microM apoptosis in human hepatoma HepG2 and Huh6 cells, but was ineffective in primary human hepatocytes (PHH). In HepG2 cells SAHA induced the extrinsic apoptotic pathway, increasing the expression of both FasL and FasL receptor and causing the activation of caspase-8. Moreover, SAHA enhanced the level of Bim proteins, stimulated alternative splicing of the Bcl-X transcript with the expression of the proapoptotic Bcl-Xs isoform, induced degradation of Bid into the apoptotic factor t-Bid and dephosphorylation and inactivation of the anti-apoptotic factor Akt. Consequently, SAHA caused loss of mitochondrial transmembrane potential, release of cytochrome c from mitochondria, activation of caspase-3 and degradation of PARP. Interestingly, a combination of suboptimal doses of SAHA (1 microM) and bortezomib (5-10 nM), a potent inhibitor of 26S proteasome, synergistically induced apoptosis in both HepG2 and Huh6 cells, but was ineffective in PHH. Combined treatment increased with synergistic effects the expression levels of c-Jun, phospho-c-Jun and FasL and the production of Bcl-Xs. These effects were accompanied by activation of Bid, caspase-8 and 3. In conclusion, SAHA stimulated apoptosis in hepatoma cells and exerted a synergistic apoptotic effect when combined with bortezomib. In contrast, these treatments were quite ineffective in inducing apoptosis in PHH. Thus, our results suggest the potential application of the SAHA/bortezomib combination in clinical trials for liver cancer.

In vivo visualization of pituitary dopaminergic receptors by iodine-123 methoxybenzamide (IBZM) correlates with sensitivity to dopamine agonists in two patients with macroprolactinomas.

We performed in two patients with macroprolactinoma, pituitary scintigraphy with 123 iodine-methoxybenzamide (IBZM), a dopaminergic antagonist that specifically binds to the D2 dopaminergic receptors. In a 34-yr-old woman with basal PRL levels of about 2000 ng/mL, 7.5 mg/day of Bromocriptine (Br) for a month neither reduced PRL levels nor affected tumor size; in this patient single photon emission tomography SPECT failed to show any pituitary accumulation of the tracer. In the other patient, a 27-yr-old man presenting with cerebrospinal fluid rhinorrhea, basal PRL levels were at 5000 ng/mL; magnetic resonance imaging (MRI) demonstrated a huge pituitary tumor, and SPECT showed a very intense concentration of IBZM at the level of the adenoma. PRL levels fell dramatically to 530 ng/mL with only 2.5 mg/day of Br after 4 days; after 6 days with 7.5 mg/day Br, PRL levels were 63 ng/mL, and the patient underwent surgery to correct cerebrospinal fluid leakage. We conclude that, in these two patients, the pituitary scintigraphy with IBZM has given information on the density of dopamine receptors on the adenoma and has correlated with the inhibitory effect of Br on PRL secretion. Whether this tool might be of value in identifying patients with pituitary tumors potentially responsive to Br treatment is still to be investigated.

Tc-99m HMPAO SPECT in a cerebral gunshot wound.

The authors examined regional cerebral blood flow by using Tc-99m HMPAO SPECT studies in a patient who received a gunshot wound to the brain. Although the presence of the retained bullet's fragments adversely affected the quality of CT images and contraindicated MRI studies, the SPECT examination did not have the same constraints and allowed both therapy assessment and prognostic evaluation. The repair of the cortical defect could also be assessed.

Bombesin/gastrin releasing peptide levels of peripheral mononuclear cells, monocytes and alveolar macrophages in chronic bronchitis.

Bombesin-related peptides (BRP), a family of neuropeptides showing carboxy-terminal homology with the amphibian bombesin, are present in humans in many body systems (CNS, lung, gastro-intestinal tract) with a variety of biological activities. In the lung, BRP are mitogens for normal bronchial epithelial cells and fibroblasts, chemoattractant for monocytes and exert bronchoconstrictive activity. Increased levels of BRP have been described in the lung of cigarette smokers and in smoking-related diseases. Moreover appreciable quantities of BRP have been recently found in lysates of peripheral monocytes and alveolar macrophages of man and guinea pig. It has therefore been inferred that these peptides may play a role in the immunological function of lung tissue. The aim of this study was to determine the amount of BRP present in peripheral-blood mononuclear cells (PBMNC), monocytes and alveolar macrophages (AM) of normal subjects (n = 36) and chronic bronchitis patients (n = 36). Patients with chronic bronchitis showed a significant increase in BRP levels in all cell types (PBMNC, monocytes and AM) (p < 0.005) in comparison with normal subjects. In addition levels of BRP in monocytes and AM were found to be nearly four times higher than in PBMNC in both groups of subjects. We can therefore confirm previous observations concerning the presence of BRP in human cells of the monocyte-macrophage lineage. Furthermore our results demonstrate that BRP levels are increased in monocytes of chronic bronchitis patients and imply a potential role for these neuropeptides in lung immunological response in smoking-related diseases.