H. Seda Vatansever

Propolis from Turkey induces apoptosis through activating caspases in human breast carcinoma cell lines

Propolis is a sticky substance that is collected from plants by honeybees that has anti-mutagenic and anti-carcinogenic properties with biological and therapeutic effects. The target of this study was to investigate the anti-apoptotic effect of propolis extracts (PE) on the caspase pathway in the human breast cell line MCF-7 in culture. Seven different propolis extracts, numbered PE 1-7, produced in their natural ecological environment, were collected from the Hacettepe University Beytepe Campus area in Ankara, Turkey. Individual extracts at 0.5, 0.25, 0.125 and 0.063mg/ml were incubated with MCF-7 cells during 2 days culture. Cell growth and cytotoxicity were measured colorimetrically by MTT assay. Apoptotic cell death was determined by the TUNEL method (terminal deoxynucleotidyltransferase-biotin nick end-labelling) and caspase activity was investigated by immunocytochemistry using antibodies directed against caspase 6, caspase 8 and caspase 9. The results showed that the PE 5 and 6 extracts at 0.125mg/ml dilution induced apoptosis in association with increased number of TUNEL positive cells. MTT results showed that cultures exposed to the same extracts and at the same dilution experienced better cell growth compared to those cultures exposed to the other extracts. Immunpositivity for all caspases was detected after treatment with all the extracts and at all dilutions, with stronger immunoreactivity for caspase 6 than caspases 8 and 9. Caspase 6 labelling was especially strong in PE 5 and PE 6. We conclude that propolis may have anti-tumour effects by increasing apoptosis through the caspase pathway. Such propolis extracts may be important economically and allow development of a relatively inexpensive cancer treatment.

Effect of thermal energy produced by drilling on the facial nerve: histopathologic evaluation in guinea pigs

The effect of thermal energy due to drilling around the facial nerve canal on the facial nerve was histopathologically evaluated in four guinea pigs. The bony canal of the facial nerve was drilled using a 3 mm diamond burr for one minute. The temperature changes on the facial nerve canal were noted before and after dissection. The temporal bones of the animals were histopathologically examined under light microscopy using haematoxylin & eosin (H&E) and solochrome cyanine staining for myelin, and immunohistochemical staining for neuronal nitric oxide synthase (nNOS). Compared to the control group, it was observed with H&E staining that there was oedema among the axonal fibres and with solochrome cyanine staining that the thickness of the myelin fibres was decreased, and that the severity and extent of nNOS activity was decreased in the axonal fibres. It was concluded that a temperature increase on the facial canal may potentially lead to inflammation of the nerve, and may also cause deterioration of nerve conduction to some extent.

Blood brain barrier in right- and left-pawed female rats assessed by a new staining method.

The asymmetrical breakdown of the blood-brain barrier (BBB) was studied in female rats. Paw preference was assessed by a food reaching test. Adrenaline-induced hypertension was used to destroy the BBB, which was evaluated using triphenyltetrazolium (TTC) staining of the brain slices just after giving adrenaline for 30 s. In normal rats, the whole brain sections exhibited complete staining with TTC. After adrenaline infusion for 30 s, there were large unstained areas in the left brain in right-pawed animals, and vice versa in left-pawed animals. Similar results were obtained in seizure-induced breakdown of BBB. These results were explained by an asymmetric cerebral blood flow depending upon the paw preference in rats. It was suggested that this new method and the results are consistent with contralateral motor control that may be important in determining the dominant cerebral hemisphere in animals.

Analysis of transferred keratinocyte-like cells derived from mouse embryonic stem cells on experimental surgical skin wounds of mouse

Autologous/allogenic skin grafts constituted from differentiated adult or embryonic stem cells can be used in treatment of skin disorders. In our study we aimed to differentiate keratinocytes from mouse embryonic stem cells and the transfer of viable keratinocyte-like cells to a model of surgical skin wound of mouse. Embryoid bodies, derived from mouse embryonic stem cells, were cultured on basement membrane matrix with added BMP-4 for 10 days. The identification of differentiated keratinocyte-like cells was done by detection of cytokeratin-8 and cytokeratin-14 localization using an indirect immunoperoxidase technique and transmission electron microscopy evaluation. Distribution of BrdU, cytokeratin-8 and cytokeratin-14 were evaluated using an indirect immunoperoxidase technique from the experimental (dressing including BrdU labelled cells applied after the surgical wound was created on mouse), control (only the surgical wound was created on mouse) and sham (only the dressing applied after the surgical wound was created on mouse) in groups after 3, 5 and 7 days. Immunohistochemically and ultrastructurally, cells derived from mouse embryonic stem cells were similar to differentiated keratinocyte-like cells. Differentiated keratinocyte-like cells were demonstrated by positive BrdU, cytokeratin-8 and cytokeratin-14 staining after transfer to the wound area. In the experimental group wound healing was better after transferring differentiated keratinocytes when compared to the sham and control groups. In vivo continuity and usability of derived cells are very important issues. In wound repair mechanisms, keratinocyte-like cells could provide positive effects during the wound healing and could be used in clinical treatments of wound repair process.

Effects of ocreotide on intestinal mucosa in rats with portal hypertensive enteropathy.

To clarify the effects of long-term ocreotide (a long-acting somatostatin analogue) treatment on mucosal changes in a rat model of portal hypertensive enteropathy, groups of male Swiss albino rats (n=15 each) were randomly assigned to one of three treatment arms. These were: sham laparotomy+twice daily subcutaneous saline 0.5 mL (Group 1); portal hypertension induction+twice daily subcutaneous saline 0.5 mL (Group 2); and portal hypertension induction+subcutaneous ocreotide 100 microg/kg/12h (Group 3). After 12 weeks of treatment, jejunal and ileal tissue specimens were obtained and evaluated histopathologically (villus/crypt ratio, mean diameter of dilated vessels, mucosal edema, and fibromuscular proliferation in the lamina propria) and immunohistochemically (vascular endothelial growth factor (VEGF), von Willebrand factor (F8), and cluster of differentiation 34 (CD34) labelling). In jejunal specimens, the villus/crypt ratio was markedly lower in Group 2 (2.38+/-0.46 microm) than in Group 1 (5.07+/-2.25 microm) or Group 3 (4.97+/-2.19 microm); mean diameter of dilated vessels was markedly higher in Group 2 (43.30+/-5.71 microm) than in Group 1 (33.53+/-4.00 microm) or Group 3 (36.76+/-3.96 microm); mucosal edema and fibromuscular proliferation were universally absent in Group 1 when compared with the other groups. There were statistically significant differences (p<0.05) between Groups 1 and 2 for villus/crypt ratio, mean diameter of dilated vessels, VEGF immunolabelling intensity, and CD34 immunolabelling intensity; between Groups 1 and 3 for mean diameter of dilated vessels, VEGF immunolabelling intensity, and CD34 immunolabelling intensity; and between Groups 2 and 3 for villus/crypt ratio, mean diameter of dilated vessels, and VEGF immunolabelling intensity. In ileal tissue specimens, the villus/crypt ratio was markedly lower in Group 2 (5.51+/-0.67 microm) than in either Group 1 (7.19+/-2.18 microm) or Group 3 (7.62+/-2.58 microm); mean diameter of dilated vessels was markedly higher in Group 2 (46.36+/-4.77 microm) than in either Group 1 (36.43+/-4.57 microm) or Group 3 (41.31+/-4.70 microm); while mucosal edema was absent in Group 1, it was present in Group 2 and Group 3; and fibromuscular proliferation was universally absent. There were statistically significant differences (p<0.05) between Groups 1 and 2 for villus/crypt ratio and mean diameter of dilated vessels; between Groups 1 and 3 for mean diameter of dilated vessels; and between Groups 2 and 3 for villus/crypt ratio, mean diameter of dilated vessels, and VEGF immunolabelling intensity. Together, these findings indicate that ocreotide treatment ameliorates histomorphological changes in a rat model of portal hypertensive enteropathy.

Immunolocalization of αV, α3 and β1 integrins in the human placenta with pre-eclampsia

Summary Signs of pre-eclampsia are considered to be caused by maternal endothelial dysfunction due to circulating factors of placental origin. Integrins are a large family of cell surface proteins that serve as receptors involved in cell-cell and cell-matrix interactions during placentation. Therefore, low expression of integrins or the lack of it may be encountered during pre-eclampsia. In the present study, we investigated the immunolocalisation of integrins αV, α3 and β1 in placentas of normal and pre-eclamptic women. Thirty-two placentas from pre-eclamptic (n = 14) and normotensive (n = 18) women were used. Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue specimens, using anti-αV, anti-α3 and anti-β1 antibodies and the indirect immunoperoxidase technique. A semi-quantitative grading system (HSCORE) was used to compare immunohistochemical staining intensities. Distribution patterns of αV, α3 and β1 integrins were detected in cytotrophoblasts and Hofbauer cells in normal and pre-eclamptic placentas. Immunostaining of αV and β1 integrins was slightly decreased in pre-eclamptic samples but α3 integrin immunostaining was similar in pre-eclamptic and normal placentas. Decreased immunostaining of integrins in the cytotrophoblasts may considered to be a structural basis for decreased placental perfusion in pre-eclampsia.

Significance of apoptosis related proteins on malignant transformation of ovarian tumors: A comparison between Bcl-2/Bax ratio and p53 immunoreactivity

In this study, we compared the immunoreactivities of Bcl-2, Bax and p53 proteins in ovarian tumors and related the immunohistochemical findings to the histological type of the tumors. Formalin-fixed, paraffin wax-embedded tissue sections from 40 patients who had serous-mucinous borderline tumors and serous-mucinous adenocarcinoma of the ovary (n=10 each) were stained with hematoxylin-eosin (H&E). After histopathological examination, serial sections were stained immunohistochemically with primary antibodies to Bcl-2, Bax and p53 using an avidin-biotin-peroxidase method. A semi-quantitative grading system was used to compare the immunohistochemical staining intensities. The nuclear DNA fragmentation of apoptosis was determined using TUNEL method. As a result of immunohistochemical staining, increased immunoreactivity of Bcl-2 was observed in adenocarcinomas when compared to borderline tumors (P<0.001). Strong immunoreactivity of Bcl-2 and mild immunoreactivities of Bax and p53 were detected in ovarian adenocarcinomas. There were no significant statistical differences in the immunoreactivity of Bax among the histological type of ovarian tumors. Whereas a balance was observed between the immunoreactivities of Bcl-2 and Bax in the borderline cases, and this balance was strongly changed toward the anti-apoptotic Bcl-2 protein in patients with adenocarcinoma. TUNEL staining of sections indicated apoptotic cells in the serous borderline tumors were about 8-fold higher than in the serous adenocarcinoma. The results of this study on apoptosis-related factors might help to develop novel protective and therapeutic approaches, such as isoflavonoids and isothiocyanates, which were associated with decreased Bcl-2/Bax ratio, against the malignant epithelial ovarian tumors.

Jak-Stat signaling pathway may play a role in the pathogenesis of cholesteatoma

PURPOSE Jak-Stat signaling pathway is one of the major signal transduction cascades which regulates most of the cellular events such as cell proliferation, differentiation, cell migration and apoptosis. This study aims to determine the activity of Jak-Stat signaling pathway in the pathogenesis of cholesteatoma. MATERIALS AND METHODS Cholesteatoma and skin samples were obtained from 10 patients who underwent tympanomastoidectomy for chronic otitis media with cholesteatoma. Immunohistochemical analysis of cholesteatoma and skin was performed using anti-Jak1, anti-Jak2, anti-Jak3, anti-Stat1, anti-Stat2, anti-Stat3, anti-Stat4 and anti-Stat5 antibodies. The immunoreactivities in cholesteatoma and skin were quantified using H-score measurement and statistical comparison was performed. RESULTS Jak1, Jak2, Jak3, Stat1 and Stat3 immunoreactivities were not detected in cholesteatoma; in contrast to the skin (129.8; 226.7; 33.0; 66.4;115.9). In addition, when H-score measurements of Stat2, Stat4 and Stat5 immunoreactivities were compared between cholesteatoma (172.8; 166.7; 120.0) and skin (400.0; 284.9; 292.0), statistically significant differences were found (p<0.0001, p<0.0001, p<0.0001). CONCLUSIONS A remarkable deficiency in the family members of Jak-Stat signaling pathway was demonstrated in cholesteatoma. Therefore, perturbations in Jak-Stat signaling pathway may play a role in the pathogenesis of cholesteatoma.

Nanotubes and their functions in cancer

A a noninvasive liquid biopsy, plasma cell-free DNA (cfDNA) was studied as a potentially valuable surrogate specimen for detecting tumor-specific aberrations. Non-small cell lung cancer (NSCLC) is the common type of lung cancer, which is the leading cause of cancer death throughout the world. Most patients were diagnosed too late for curative treatment. So, it is necessary to develop a minimal invasive method to identify NSCLC at an early stage. Here, we studied cfDNA collected from subjects with advanced NSCLC by performing droplet digital PCR on serial cfDNA specimens collected from patients and healthy control. Our findings demonstrated that the ctDNA could serve as a viable tool to monitor NSCLC and prompted us to find more sensitive and specific biomarkers for clinical practice, especially monitor these cases with at least one known gene abnormality.D to abnormal expression of integrins αvβ3 in various disease conditions, this integrin pair has been a focus as targets for drug development. Studies yield a few successful examples. Among them are various antibodies against the integrins, and most recently, Cilengitide, a RGD-based peptidomimetic.Nevertheless, most of current approaches focus on ligand-binding with goal of inhibition of integrin functions.A major draw-back of targeting ligand-binding of integrins is activation of integrin signaling by the developed agent, which largely limit the clinical success of the integrin ligand based antagonist/agonist. We report here development of a new class of therapeutically protein agent (Ref to as ProAgio) by rational protein design using a stable host protein. ProAgio is designed to target integrins αvβ3 at a novel site. ProAgio exhibits a strong in vitro activity in induction of apoptosis of integrin αvβ3 expressing cells. ProAgio induces apoptosis by recruiting and activating caspase 8 to the cytoplasmic domain of the targeted integrins. Tests with tumor xenografts show that ProAgio strongly inhibits tumor growth. Histology analyses indicate that tumor vessels are reduced, while the established vasculatures are not affected. The results confirm targeting of integrin αvβ3 as an anti-angiogenic agent. Toxicity analyses demonstrate that ProAgio is not toxic to mouse at very high doses. Our study develops an effective integrin targeting agent via a novel mechanism of action. Our approach provides a new platform for development of therapeutics by targeting integrins.Purpose: While anecdotal evidence underscores the positive impact of therapy dogs for children with cancer and their families, rigorous studies of efficacy are currently lacking, even as animal-assisted interventions (AAIs) occur daily in today’s pediatric oncology settings. This national, multi-site study is the first of its kind to rigorously measure the psychosocial effects of AAIs for this population. Specifically, researchers are interested in whether or not AAIs have positive effects on patient stress, anxiety and health-related quality of life and on parent stress and anxiety, as well as whether or not therapy dogs experience distress during AAI sessions.Introduction: Nucleophosmin1 (NPM1) gene mutation is the most frequently occurring gene mutation in Acute Myeloid Leukemia (AML) accounting for 35 to 50% of the AML patients. It encodes the nucleophosmin which is found in the cytoplasm of AML patients with variable prevalence and proven to have prognostic significance. It is involved in several activities at cellular level such as ribosomal biosynthesis, maintenance of genome stability and molecular chaperon functions. It causes inactivation of tumor suppressor gene p53. This mutation seem to identify patients that respond better to chemotherapy hence this study will help to identify patients with good prognosis. Its association with FLT3 is related to poor outcome.I 2013, the number of new cases of breast cancer in the US was 234, 580 and ~1.7 million worldwide. Despite the advances in early detection and treatment of breast cancer, about 30% of patients with early stage breast cancer have recurrent disease where resistance to therapy is common and expected. HER2 is overexpressed in 20-40% of invasive breast cancer, and 12-24% of these patients develop resistance within 6 months for the widely used therapy trastuzumab. HER2 oncogenic variant HER2 16 has been shown to promote receptor dimerization, cell invasion and also trastuzumab resistance of NIH3T3 and MCF-7 tumor cells lines. We have identified several naphthoquinones that show growth inhibition of the MCF-7 HER216 cell lines with a significantly higher potency than the present clinically used tyrosine kinase inhibitor lapatinib. These compounds also inhibit auto-phosphorylation of the Y1248 and Y1068 residues of HER2 and EGFR, respectively.Methodology: Eighty weanling female fisher (F344) rats were divided into five groups of 16 each and fed with synthetic diets containing partially-hydrogenated vegetable oil / PHVO (transfat), palmolein (saturated fatty acids) , sunflower oil (n-6 PUFA), soyabean oil (α-linoleic acid) and sunflower + fish oil(LC n-3 PUFA) for 4 months after which 8 rats from each group were administered DMBA orally, once a week, for 4 weeks and continued on same diet for 8 months, while remaining 8 rats of each group were continued on respective diets.W mammalian blood and normal tissues are usually maintained at pH around 7.4, the extracellular pH drops below 6.5 in solid cancer nests. Enzymes which function preferentially at acidic pH may be the target molecules of anti-cancer drugs. Our group have found that the inhibition of protein prenylation attenuates proliferation of cancer cells at acidic pH, suggesting that an enzyme(s) to prenylate proteins is functioning under acidic conditions. In addition to the enzyme for protein prenylation, there may be other enzymes functioning under acidic conditions. To find such enzymes, the expression of 24,000 genes was examined using a DNA array chip in mesothelioma cells, and the expression of approximately 700 genes was elevated at acidic pH. The elevated expression of these genes was found in other cancer cells grown under acidic conditions and in human specimens from cancer patients. These results suggest that mammalian cells have many enzymes which function preferentially in acidic cancer nests, and that drugs inhibiting these enzymes could be potent candidates for anticancer chemotherapeutics with less side-effect, especially on immune systems in blood and normal tissues, because acidosisdependent drugs are expected to be less effective in tissues whose pH is alkaline. In fact, inhibitors of protein prenylation had little effect on proliferation and cytokine production of immune cells at alkaline pH. The screening under acidic conditions may be a useful way to find new anti-cancer drugs which are effective in acidic cancer nests.A R1R2NOR3 are able to undergo homolysis upon chemical activation to release a stable nitroxide R1R2NO•, which can be used for DNP-MRI, and a transient alkyl radical R3•, which can be used for killing tumor cells. By combining diagnostic and therapeutic activities into a single low-molecular weight molecule, alkoxyamines are new theranostic tools. We have developed this concept recently, and proved that they are reliable and controllable sources of in-situ generated radicals able to exhibit interesting biological and imaging properties.Multiple spinal tumors are known to occur in association with phacomatosis like neurofibromatosis. Rarely, sporadic occurrences have been described in the absence of a clear underlying pathogenesis. The majority of intradural extramedullary spinal tumors are meningiomas or nerve sheath tumors like schwannoma and neurofibroma. Ependymomas, which are the most common intramedullary spinal tumors in adults, can also occur in the extramedullary intradural space and impose a diagnostic challenge. Only a few such cases have been described in the literature. We report the first sporadic occurrence of an intradural extramedullary ependymoma synchronously presenting with a different neoplasm, a schwannoma. A 43-year-old woman presented to the authors’ institution with lumbosacral pain and neurological deficits lasting a decade. On magnetic resonance imaging, she was found to have two stable, contrast-enhancing intradural extramedullary spinal lesions in the L2-3 and L4-5 vertebral areas. She subsequently underwent L2-L5 laminectomy with total resection of the former lesion and subtotal resection of the later lesion. Histological and immunohistochemical analysis revealed two distinct tumors: a cellular ependymoma and a schwannoma at the two respective locations. Ependymomas are low grade, slowly growing tumors that mimic other, benign tumors clinically as well as radiologically. However, the management of these tumors differs since ependymomas have a potential for recurrence and metastasis. The underlying etiology and pathogenesis of these tumors is not clearly established. However, an association with an underlying phacomatosis needs to be considered. Our purpose is to create awareness that an intradural extramedullary ependymoma can coexist with other spinal tumors and should be included in the differential diagnosis of a multifocal spinal lesion presentation. Owing to difficulties in differentiating these closely mimicking spinal tumors clinically and radiologically, early identification with surgical resection, when possible, might be a recommended strategyA monoclonal antibody (Mab) designated as GHR106 was generated against the extracellular domain (N1-29 synthetic peptide) of human gonadotropin releasing hormone (GnRH) receptor. It is a first-in-class GnRH analog and can serve as a drug candidate for potential applications in the treatment of human cancers and/or fertility regulations. Both Mabs in murine (mGHR106) or humanized (hGHR106) forms were shown to have comparable specificity and affinity to intact GnRH receptor on cancer cells or to N1-29 synthetic peptides from humans and monkeys. Similar to decapeptide GnRH analogs, both Mabs were shown to induce apoptosis to cultured cancer cells of various tissue origins, including those from the ovary, breast, prostate, and lung. However, both Mabs were also found to induce complement-dependent cytotoxicity (CDC) reaction for lysis of cancer cells, an immune property which is not shared by peptide analogs of GnRH. By using semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR), both GHR106 Mabs and the GnRH decapeptide antagonist, Antide, were shown to be bioequivalent in terms of their respective effects on genes involved in the proliferation and apoptosis of cancer cells. In addition, GHR106 Mabs have a much longer circulating half-life than GnRH peptide analogs (days versus hours). Based on the results of these studies, it can be concluded that both mGHR106 and hGHR106 are bioequivalent to the GnRH decapeptide antagonist, Antide, in many biological and functional properties. Therefore, hGHR106 can serve as a long-acting alternative to the current decapeptide GnRH antagonists for therapeutic treatments in the immunotherapy of human cancers, including those of gynecologic origin.