Sevinc Inan

Immunolocalization of VEGF, VEGF receptors, EGF-R and Ki-67 in leiomyoma, cellular leiomyoma and leiomyosarcoma.

Angiogenic factors, such as vascular endothelial growth factor (VEGF), its receptors and epidermal growth factor receptor (EGF-R), are involved in increased progression in many carcinomas. The aim of this study was to investigate the role of angiogenesis and immunolocalization of VEGF, its receptors, EGF-R and Ki 67 in leiomyomas and leiomyosarcomas using an indirect immunohistochemical method. Samples from patients with leiomyoma, cellular leiomyoma and cellular leiomyosarcoma (n=20 per group) were fixed in 10% formalin and processed using routine paraffin protocols. Following initial histological analysis, samples were immunostained with primary antibodies for VEGF, VEGFR-1, VEGFR-2, EGF-R and Ki-67 using an indirect avidin-biotin peroxidase method. Immunostaining intensities were evaluated as mild, moderate or strong and a semi-quantitative method (H-Score) was used to compare the samples. While mild/moderate EGF-R immunostaining and moderate immunostaining for VEGF and its receptors were observed in samples of leiomyomas, much less immunoreactivity was observed in cellular leiomyomas. All immunoreactivities and immune-stained cells increased in leiomyosarcomas. When scores of intensity and percentage of positive staining cells were compared, all immunoreactivities were shown to be significantly increased in leiomyosarcomas compared to leiomyomas. These results suggest that in leiomyosarcoma, angiogenic factors, such as VEGF, its receptors and EGF-R, may be involved in tumor angiogenesis. Active tumor cells can trigger angiogenesis, interaction with surrounding tissue and in the tissue itself initiating angiogenic activity. Angiogenic growth factors play an important role and induce malignant transformation through both autocrine and paracrine mechanisms. Anti-angiogenic agents may provide a novel therapeutic approach for the treatment of leiomyosarcoma.

Thymoquinone accelerates new bone formation in the rapid maxillary expansion procedure

OBJECTIVE The aim of this study was to evaluate the effect of systemic thymoquinone (TQ) in a rat rapid maxillary expansion (RME) study. DESIGN Thirty-two Wistar albino rats were divided into 4 equal groups: only-expansion (OE), expansion plus TQ (TQ1 group, TQ given to the rats during their nursery phase and during the expansion and retention period), expansion plus TQ (TQ2 group, TQ given to the rats only during the retention period), and control group (no procedure done). Expansion appliances were placed on the maxillary incisors of all animals for 5days. The appliance was deactivated during the 12day retention period. The rats were sacrificed at the end of the retention period. Histomorphometric evaluation was carried out in order to compare the number of osteoclasts, osteoblasts, and capillaries, as well as the intensities of inflammatory cells, and new bone formation amongst the groups. RESULTS New bone formation, number of capillaries and the ratio of intensities of inflammatory cells in maxillary sutures was higher in the TQ groups than in the other groups. Statistical analysis also demonstrated that osteoblast and osteoclast numbers were also highest in the TQ1 group. CONCLUSION Histomorphometric analysis demonstrated that systemic use of thymoquinone may be effective in accelerating new bone formation in the RME procedure and that TQ may be beneficial in preventing relapse following the RME procedure.

Systemic propolis stimulates new bone formation at the expanded suture A histomorphometric study

OBJECTIVE To investigate the effects of systemically given propolis on the expanded premaxillary suture in a rat study model. MATERIALS AND METHODS The 24 rats were randomly divided into three groups-only expansion (OE), expansion plus propolis (PRO), and nonexpansion (control) groups. After the 5-day expansion period was completed, the OE and PRO groups underwent 12 days of mechanical retention. At the end of this period, the animals were euthanatized and their pre-maxillae were dissected and fixed. Histomorphometric examination was performed to determine the number of osteoclasts, osteoblasts, and capillaries as well as the intensity of inflammatory cells and amount of new bone formation. RESULTS Statistical analysis showed that the intensities of inflammatory cells, number of osteoblasts, and amount of new bone formation were greater in the PRO group than in the other groups. The PRO group also had more osteoclasts and new capillaries. CONCLUSION Systemic use of propolis may hasten new bone formation at the expanded suture in rats.

The roles of Transforming growth Factor Type β3(TGF-β3) and mast cells in the pathogenesis of scleroderma

Scleroderma is a connective tissue disorder characterised by excessive accumulation of collagen in the skin and internal organs. The most likely explanation for this process is local activation of collagen synthesis from fibroblasts. Our intention was to elucidate whether TGF-β3 and mast cells play a pathogenic role in abnormal connective tissue formation in scleroderma. In this study, skin biopsies from 20 patients with scleroderma and five from healthy individuals were studied by an indirect immunoperoxidase technique to determine the immunoreactivity of TGF-β3 in the dermis. In addition, skin samples were stained with toluidine blue to count the number of mast cells in scleroderma, and tissues were examined under the electron microscope to evaluate the ultrastructural changes. Increased TGF-β3 immunoreactivities were detected in the dermis in the patients skin, suggesting the presence of a subpopulation responsible for the increased collagen production. Mast cell counts in the skin of patients with scleroderma were significantly greater (19.2 ± 4.1/unit) than those of normal controls (4.4 ± 1.2/unit). Ultrastructural observations indicated that there is a close relationship between the mast cells and fibroblasts. These results suggest that fibrosis in scleroderma could evolve through the activation of fibroblasts and the regulatory mechanisms that appear to modulate the behavior of these cells with respect to collagen production.

Expression profiling of stem cell signaling alters with spheroid formation in CD133high/CD44high prostate cancer stem cells

Cancer stem cells (CSC) isolated from multiple tumor types differentiate in vivo and in vitro when cultured in serum; however, the factors responsible for their differentiation have not yet been identified. The first aim of the present study was to identify CD133high/CD44high DU145 prostate CSCs and compare their profiles with non-CSCs as bulk counterparts of the population. Subsequently, the two populations continued to be three-dimensional multicellular spheroids. Differentiation was then investigated with stem cell-related genomic characteristics. Polymerase chain reaction array analyses of cell cycle regulation, embryonic and mesenchymal cell lineage-related markers, and telomerase reverse transcriptase (TERT) and Notch signaling were performed. Immunohistochemistry of CD117, Notch1, Jagged1, Delta1, Sox2, c-Myc, Oct4, KLF4, CD90 and SSEA1 were determined in CSC and non-CSC monolayer and spheroid subcultures. Significant gene alterations were observed in the CD133high/CD44high population when cultured as a monolayer and continued as spheroid. In this group, marked gene upregulation was determined in collagen type 9 α1, Islet1 and cyclin D2. Jagged1, Delta-like 3 and Notch1 were respectively upregulated genes in the Notch signaling pathway. According to immunoreactivity, the staining density of Jagged1, Sox2, Oct4 and Klf-4 increased significantly in CSC spheroids. Isolated CSCs alter their cellular characterization over the course of time and exhibit a differentiation profile while maintaining their former surface antigens at a level of transcription or translation. The current study suggested that this differentiation process may be a mechanism responsible for the malignant process and tumor growth.

Comparison of the effects of collagenase and extract of Centella asiatica in an experimental model of wound healing : An immunohistochemical and histopathological study

In this study, we compared the effects of collagenase and Centella asiatica in the rat model. Twenty‐seven female rats were divided into three groups, and two full‐thickness wounds were made for each animal. Collagenase ointment was applied topically to Group I and C. asiatica ointment to Group II rats. In Group III, no treatment was applied. On the third day of treatment, wounds on the left side of three animals of each group were excised. On the fifth and eighth day of the treatments, the same procedure was performed for the remaining animals. Indirect immunohistochemical examination was performed to detect transforming growth factor beta (TGF)‐β, endothelial and inducible nitric oxide synthase (eNOS and iNOS), vascular endothelial growth factor, TGF‐α, laminin, fibronectin, collagen I, and interleukin‐1β. According to the measurements of the wound areas and wound healing periodo, collagenase was superior to the control group. Immunohistochemical examinations showed strong (+++) iNOS and TGF‐β immunoreactivities in C. asiatica group. eNOS immunoreactivity was moderate (++) in this group. For the collagenase group, iNOS, eNOS, and TGF‐β immunoreactivities were moderate (++). In the collagenase group, while TGF‐β and iNOS immunoreactivities were weaker, laminin and fibronectin reactivities were stronger than in C. asiatica and control groups. Collagenase was superior to C. asiatica according to the immunohistochemical findings. Collagenase ointment significantly improves the quality of wound healing and scar formation and is a more appropriate treatment choice than extract of C. asiatica in the early stages of the wound healing process.

Effects of 5-fluorouracil and gemcitabine on a breast cancer cell line (MCF-7) via the JAK/STAT pathway.

Aberrant activation of the JAK/STAT pathway may predispose to malignancy as a consequence of the deregulation of cell proliferation, differentiation or apoptosis such as in cancer of the blood, head and neck, and breast. In our study we aimed to investigate the effects of 5-fluorouracil (5-FU) and gemcitabine on a breast cancer cell line (MCF-7 cells) via the JAK/STAT pathway. Distribution of JAK1, JAK2, JAK3 and STAT2, STAT3, STAT4, STAT5 were evaluated on MCF-7 cells following gemcitabine and 5-FU treatment and in the absence of drug treatment by an indirect immunohistochemical method. It was observed that JAK1, JAK3, STAT5 and particularly STAT2 activation were more effective than the other JAK/STATs in breast cancer progression. Following treatment with 5-FU, JAK1 and STAT5 immunoreactivities were decreased in MCF-7 cells in comparison with both gemcitabine-treated and non-treated groups. These results suggest that the JAK/STAT pathway plays an important role in breast cancer pathogenesis and may be more affected after 5-FU treatment rather than gemcitabine. Drugs which block STAT5 may provide a novel therapeutic approach for the treatment of breast cancer.

Increased vascular surface density in ovarian endometriosis.

Our goal in this study was to investigate the presence of angiogenesis-related factors in endometriomas by evaluating their vascular surface densities. Thirty ovarian samples were included in the study. Of these ,ten were histologically confirmed endometriomas ,ten were ovarian specimens in the follicular phase and ten were ovarian specimens in the luteal phase ,serving as controls. Histological specimens were immunostained for von Willebrand factor (vWF: factor VIII-related antigen) and CD34. The area with the highest microvessel density in endometriosis and in the normal ovary was evaluated by using an intercept grid. All microvessels in a specific field (× 100 magnification) were counted and vascular surface density was measured ,as 164.01 ± 21.26 vs. 125.15 ± 11.28 and 117.44 ± 9.27 by using vWF ,and as 172.97 ± 25.64 vs. 138.65 ± 32.21 and 120.34 ± 18.40 by using CD34 in endometriotic ,follicular and luteal ovarian samples ,respectively (p < 0.001). The mean vascular surface density was significantly higher in endometriosis than in the ovarian samples of the follicular phase or the luteal phase. No significant difference was seen between normal ovarian samples. Endometriosis was associated with angiogenic properties. Having demonstrated elevated angiogenic factors in endometriotic samples ,we concluded that activation of angiogenesis might be a key factor in the pathogenesis of endometriosis.

The effects of Gemcitabine and Vinorelbine on inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) distribution of MCF-7 breast cancer cells.

Gemcitabine, which induces S-phase arrest, and Vinorelbine, which arrests microtubule organization, are two agents that have demonstrate preferred anti-tumor activity. Nitric oxide acts in diverse functions including anti-tumor and anti-pathogenic activities. In this study, we aimed to examine the distribution of immunoreactivities of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in cells of the MCF-7 breast cancer cell line in response to treatment with Gemcitabine (G), Vinorelbine (V) and combination of Gemcitabine and Vinorelbine (G+V). The distributions of iNOS and eNOS were determined by using indirect immunoperoxidase or immunofluorescence methods and ELISA. Cells incubated with G, V and G+V for 24, 48 and 72h were immunolabelled with anti-eNOS and anti-iNOS primary antibodies. Apoptosis was determined by TUNEL assay. A significant increase of eNOS immunolabelling on MCF-7 cells treated with G and G+V was observed. Apoptotic cells were also detected in G, V and G+V treated MCF-7 cells. The immunolabelling of iNOS was detected in all groups but this immunoreactivity was not different among the groups. In conclusion, while G treatment, induced S-phase arrest, triggered the NOS pathway after treatment of MCF-7 cells, V treatment, arrested microtubule organization and did not change the NOS pathway. Detection of increased eNOS immunolabelling and apoptosis after G treatment of MCF-7 cells could be important to the treatment of human breast cancer.

The role of CAPE in PI3K/AKT/mTOR activation and oxidative stress on testis torsion

Ischemia reperfusion injury arises from testicular torsion resulting in a loss of spermatogenesis and significant germ cell apoptosis. This study evaluates the prooxidant/antioxidant effects of caffeic acid phenethyl ester (CAPE) through PI3K/AKT/mTOR signal pathways on testis torsion. A total of (28) male Wistar rats were divided randomly into 4 groups (n=7 for each group):group A (sham) group,group B torsion/detorsion group, group C (saturation group, during four days of CAPE, one dose (10 μmol/kg, i.p)) and group D (a single dose of CAPE 2h after torsion and before detorsion). At the end of the study, unilateral orchiectomies were performed for measurements of MDA and 8OHdG levels, histopathologic and immunohistochemical and TUNEL apoptotic cell examination. Testicular torsion-detorsion led to a significant decrease in the mean values of the Johnsens scores and a significant increase in the apoptotic cell values of group B. There were no significant differences between group D and group A. In addition, the MDA and 8OHdG levels increased significantly in group B. The MDA and 8OHdG values were lower in group D. However, the 8OHdG levels were higher in group C than the groups A and D. On the other hand, CAPE suppresses mTOR activation and reduces the apoptosis on ischemia/reperfusion damage in rat testis. These results demonstrate that CAPE suppresses mTOR activation and reduces the apoptosis on ischemia/reperfusion damage in rat testis.