Risk of high grade gliomas is lower in young females and its incidence enhances after menopause suggesting likely protective roles of female hormones. Hormone replacement therapy (HRT) was widely ...employed to treat osteoporosis and some epidemiological studies showed that HRT regimes including progesterone analogs such as medroxyprogesterone acetate (MPA) decreased risk of glial tumors. Tibolone is a unique progesterone analog employed in HRT with tissue specific estrogenic effects and stimulates gene expressions very similar to those induced by MPA. Tibolone's pro-estrogenic effects occur particularly in bone and brain and both MPA and tibolone inhibit AKR1C enzymes, which involve in temozolomide chemoresistance. Hence, we aimed to investigate interactions between MPA, tibolone and temozolomide in modification of glioma cell growth and fine structure.
For our studies, we have particularly chosen C6 rat glioma cell line due to several reasons: i) We previously showed that MPA reduced growth and induced procarbazine-sensitization in C6 cells; ii) temozolomide has a triazene-type molecular structure like procarbazine; iii) other groups previously showed that C6 glioma cell line is more resistant to temozolomide than human glioma cells; hence it may provide a native model of chemoresistance. Monolayer plating efficacy, soft agar colony growth, 3D-spheroid S-phase (as determined by BrdU-labeling) and electron microscopical analyses were performed to assess mutual interactions between MPA, tibolone and temozolomide.
MPA inhibited clonogenic growth of C6 glioma and this effect is augmented by both tibolone and temozolomide. MPA and tibolone inhibited DNA synthesis in C6 glioma spheroids to similar levels which can be achieved with temozolomide. Electron microscopical analyses revealed synergisms between MPA, tibolone and temozolomide involved mitochondrial proliferation, condensation, mitophagy and autophagy.
MPA and tibolone shall be studied in further experimental models of glioblastoma in vitro and in vivo.
•DNA Synthesis Inhibitory Agent Gemcitabine Acts Strongly Antitumoral on C6 Glioma Via Autophagy and Depletion of Mitochondria.•Microtubule Inhibitory Agent Vinorelbine Induces Mitotic Catastrophe ...and Apoptosis in C6 Glioma Cells.•Cyclooxygenase Inhibitors Did Not Reduce Monolayer Growth of C6 Glioma but Reduced S-phase in Glioma Spheroids.•Cyclooxygenase Inhibitors Enhanced Gemcitabine Induced Autophagy in C6 Glioma.•Cyclooxygenase Inhibitors Significantly Attenuated Antitumoral Efficacies of Vinorelbine.
To define ultrastructural features accompanying to antitumor effects of gemcitabine, vinorelbine and cyclooxygenase inhibitors in C6 glioma cells in vitro. Vinorelbine is a semisynthetic vinca alkaloid and recent studies showed its antitumor activity in pediatric optic and pontine gliomas. Vinorelbine infusion induces a severe tumor site-pain in systemic cancers, but it is unknown whether algesia and inflammation contribute to its antitumor effects. Gemcitabine is a nucleoside-chemotherapeutic which was recently shown to act as a radiosensitizer in high-grade glioma. Some studies showed synergism of anti-inflammatory cyclooxygenase-inhibitors with microtubule inhibitors and gemcitabine. DMSO is a solvent and blocks both cylooxygenase and ribonucleotide reductase, another target of gemcitabine. Rofecoxib is withdrawn from the market, yet we used it for investigational purposes, since it blocks cylooxygenase-2 1000-times more potently than cylooxygenase -1 and is also a selective inhibitor of crinophagy.
Plating efficacy, 3D-spheroid S-phase analysis with BrdU labelling and transmission electron microscopical analyses were performed.
Vinorelbine induced frequent mitotic slippage/apoptosis and autophagy. Despite both DMSO and rofecoxib induced autophagy alone and in synergy, they reduced mitotic catastrophe and autophagy triggered by vinorelbine, which was also reflected by reduced inhibition of spheroid S-phase. Gemcitabine induced karyolysis and margination of coarse chromatin towards the nuclear membrane, abundant autophagy, gutta adipis formation and decrease in mitochondria, which were enhanced by DMSO and rofecoxib.
Detailed ultrastructural analysis of the effects of chemotherapeutic drugs may provide a broader insight about their actions and pave to develop better strategies in treatment of glioblastoma.
Although doxorubicin (DOX) is a commonly used chemotherapeutic agent its clinical use is restricted due to its organ toxicities. The present investigation relates to reducing DOX induced side effects ...to the liver, kidney and ileum by usage of the antioxidant, anti-inflammatory agent, resveratrol (RES) and to investigate the role of nitric oxide synthase (NOS) in the process. Wistar rats were divided into four groups: control (saline i.p); DOX (20mg/kg i.p), RES (20mg/kg i.p) and DOX (20mg/kg i.p)+RES (20mg/kg i.p). Immunohistochemical activity of both iNOS and eNOS were evaluated after DOX treatment and ultrastructural changes such as cellular damage and mitochondrial degeneration were evaluated. Degenerative ultrastructural changes were demonstrated especially in the DOX treated group. Variations in biochemical marker levels of oxidative stress on ischemia in tissues were not observed. Our data indicate that RES may prevent cellular damage in the early phase of DOX induced toxicity. RES could be used with its beneficial effects during early cellular damage in organ toxicity after DOX treatment in cancer patients.
Purpose
To identify expression of Notch signaling proteins and its ligands in human cumulus cells which were obtained by follicle aspiration and to compare the differences of this protein expression ...between the normal and poor responder patients.
Methods
47 patients who applied to the assisted reproductive treatments with various infertility problems were included to the study. Controlled ovarian hyperstimulation was performed by using GnRH agonist and gonadotropins. Serum hormon levels were measured by using Chemilluminescent Microparticle Immunoassay method for each patient. After ultrasonographic ovarian follicle screening, oocytes were retrievaled. Cumulus cells obtained from the follicles were cultured for 72 h and immunuhistochemistry were performed for Notch1, Notch2, Notch3, Notch4, Jagged1 and Jagged2 proteins. Histological score (HSCORE) were applied to all of the samples. The association between Notch and its ligands protein expressions and the oocyte-embryo quality and fertilization rates were investigated.
Results
Significant differences were observed between the mean values of age, AMH and FSH in the 2 groups, respectively (
p
< 0.05). However, the mean female infertility duration and total gonadotropin dose did not differ significantly between normal and poor responder groups. All the patients cumulus cells expressed Notch1, Notch2, Notch3, Notch4, Jagged1 and Jagged2. There was a significant difference (
p
< 0.05) only for Notch2 between the 2 groups and a positive correlation between Notch2 and Notch3 (
r
= 547,
p
= 0.00) expressions were noted. Furthermore, no correlations were observed between the following: Notch1, Notch2, Notch3, Notch4, Jagged1, and Jagged2 expression; mature oocyte number; fertilization rates, and embryo quality percentage in both of the groups.
Conclusion
Notch signalling proteins can be an indicator for understanding the ovarian response in ovulation induction.
The aim of the present study was to determine the prognostic relevance of thymidine labeling index (TLI) in patients with breast cancer.
TLI of the primary tumor was measured in 268 patients at the ...time of the surgical biopsy by an in vitro method.
Fifty-four patients had stage I disease, and 138 patients had stage II disease, and 76 patients had stage III disease. One hundred-four patients were found to have low TLI-index (<3%), and 164 patients had high TLI-index (>/=3%). The median follow-up was 71.5 months (range, 6-138 months). The 5-year overall survival (OS) and disease free survival (DFS) rates was 84% and 74%, respectively. Lymph node involvement, tumor size more than 2 cm, high nuclear grade and estrogen receptor negativity were found to be associated with poorer DFS and OS rates. On subgroup analysis, however, the 5-year OS rate was significantly higher in the low TLI-group than in the high TLI-group in patients with stage I disease (100% vs 76%, p = 0.05).
Our findings suggest that the prognostic significance of TLI appears to be limited to early breast cancer that might help to distinguish patients who need more aggressive adjuvant treatment.
The aim of this work is to investigate whether clomipramine (CIM) and lithium chloride (LiCl) potentiate the cytotoxicity of vinorelbine (VNR) on SH-SY5Y human neuroblastoma cells in vitro and ...whether midkine (MK) can be a resistance factor for these treatments. Four groups of experiments were performed for 96 h using both monolayer and spheroid cultures of SH-SY5Y cells: (1) control group, (2) singly applied VNR, CIM, and LiCl, (3) VNR with CIM, and (4) VNR with LiCl. Their effects on monolayer and spheroid cultures were determined by evaluating cell proliferation, bromodeoxyuridine labeling index (BrdU-LI), apoptosis, cyclic adenosine monophosphate (cAMP) and midkine levels, colony-forming efficiency, spheroid size, and ultrastructure. In comparison with the control group, single and combination drug treatments significantly reduced the proliferation index (PI) for 96 h. The most potent reduction of PI was observed with VNR in combination with CIM and LiCl for all time intervals. VNR with CIM and LiCl seemed to be ineffective in reducing BrdU-LI of both monolayer cell and spheroid cultures, spheroid size, and cAMP level. VNR with LiCl increased apoptosis at 24 h, however VNR with CIM increased apoptosis at 96 h. VNR was the most potent drug in inhibiting colony-forming efficiency. The combination of VNR with CIM was the most potent in reducing midkine levels among all groups. Interestingly, the combination of VNR with LiCl led to both nuclear membrane breakdown and disappearance of the cellular membranes inside the spheroids. Both CIM and LiCl seemed to potentiate VNR-induced cytotoxicity, and MK was not a resistance factor for VNR, LiCl, and CIM.
Epigenetic changes have major role in the normal development and programming of gene expression. Aberrant methylation results in carcinogenesis. The primary objective of our study is to determine ...whether primary tumor tissue and cultured tumor cells in 2D and 3D tissue culture systems have the same methylation signature for PAX5, TMPRSS2, and SBDS. These findings will play an important role in developing in vitro model system to understand the effect of methylation inhibitors on primary tumor tissue. In a previous study PAX5, TMPRSS2, and SBDS genes that we are investigating were reported to be methylated more than 60% in breast cancer and malignant melanoma cell lines. However, these genes have never been studied in primary tumor tissues. Thus, primary tumor tissues of breast cancer and malignant melanoma were first grown in 2D and 3D cultures. Then these two types of tumor tissues and their 2D and 3D cultures were investigated for changes considering methylation levels in PAX5, TMPRSS2, and SBDS genes using real-time polymerase chain reaction. No differences were observed in the primary tissues and culture systems for both PAX5 and TMPRSS2 in malignant melanoma tissues. We found that PAX5 gene was an efficient marker to measure the effects of methylation inhibitors for in vitro systems for malignant melanoma tissue.
Fat injection for soft tissue augmentation is a common procedure in plastic surgery. Because the limitation of fat injection is its resorption, understanding how different handling techniques affect ...adipocyte survival is crucial to optimizing long-term results.
In this study; we sought to determine the effect of aspiration and injection cannula diameters on adipocyte viability.
Fat aspiration samples were obtained from 6 female patients undergoing abdominoplasty. Viable adipocytes were counted at fat suspension, which was obtained with 2-, 3-, and 4-mm–diameter aspiration cannulas and injected with 1.6-, 2-, and 2.5-mm–diameter injection cannulas.
A greater number of viable adipocytes were detected using a 4-mm–diameter aspiration cannula (419 × 10
4 cell/1 mL,
P < .05) and a 2.5-mm–diameter injection cannula (410 × 10
4 cell/1 mL,
P < .05).
The use of wider-diameter cannulas can potentially improve fat graft survival and reduce fat graft resorption.
Background. Alterations in collagen synthesis and metabolism have previously been reported in patients with pelvic organ prolapse (POP) and/or urodynamic stress incontinence (USI). Since urinary ...incontinence does not always associate with POP, the objective of this study was to examine connective tissues from patients with USI plus POP, and patients with prolapse only. Methods. Biopsies from the uterosacral ligaments were obtained during the operation from POP patients (n =28), and from continent women (control group, n =12) who underwent surgery for other benign reasons. POP patients were classified following urodynamic tests and symptom questionnaire with respect to the presence (n =14) or absence (n =14) of USI. N-terminal propeptides of collagen (PINP and PIIINP), TGF- and leptin were measured in plasma. Hydroxyproline and glycosaminoglycan (GAGs) concentrations and total hexosaminidase activity were measured in tissue samples. Histological sections were prepared using Masson's trichrome technique, and digitised solutions were used for imaging provided by Soft Imaging System GmBh. Statistical evaluations were made by the Kruskal-Wallis test. Results. A significant decrease in hydroxyproline content was found in USI+POP women in comparison to controls (p<0.05). In contrast, histopathological examination revealed an increased density of collagen in USI+POP patients. Hexosaminidase activity was decreased in both groups with POP, but no change in the amount of GAGs was observed. Markers of collagen synthesis (PINP, PIIINP), and factors related to the collagen synthesis (TGF- , leptin) remained unaltered. Conclusion. Our biochemical and morphological findings suggest a different organisation of collagen fibres in tissues of patients with USI+POP, when compared with both the controls and the POP patients.
Abstract
Tumor mass contains a complex and heterogeneous phenotypic population including a rare group of cancer cells that are capable of serving cancer- initiating cells or cancer stem cells (CSCs). ...Established cancer cell lines contain CSCs, which can be propagated, to form in vitro 3D tumor spheroids. Spheroids reflect differentiation properties of CSCs in serum contained medium and more accurately reflect the complex in vitro microenvironment than simple two-dimensional monolayer. Aberrant activation of Wnt signaling is strongly implicated in the progression of cancer and controls CSCs properties. In this study we hypothesize that when cells maintained as spheroids the structure of CSCs could be show differentiation between CSCs and non- CSCs counterpart. It is possible to determine target molecules for CSCs eradication and specify a clue in therapeutic strategies of cancer. With this aim, CD133+/CD44+ cancer-initiating cells were isolated from DU-145 human prostate cancer cell line monolayer cultures and propagated as tumor spheroids and compared with remained heterogeneous cancer cells bulk population. Wnt1, Fzd1, Adar, Apc, Axin, Btrc, Frat1 gene expression analysis was applied and protein expression levels of Wnt1, Fzd and Axin were shown by immunohistochemistry. Results show that Wnt1 expression significantly higher in non cancer stem cells compared to CSCs. Nevertheless Fzd1, Adar, Apc, Axin, Btrc, Frat1 gene expressions were higher in CSCs group than the other population. Increased Wnt1 immunoreactivity was demonstrated in the non-cancer stem cells. It is possible to assume that intracellular signaling of Wnt pathway and related molecules in the nucleus and/or cytoplasm might play an important role independent of Wnt ligand. This unexpected expression could be important for CSCs behavior and targeting of this pathway could have therapeutic value in cancer.
Citation Format: Gamze Goksel, Ayhan Bilir, Ruchan Uslu, Hakan Akbulut, Ummu Guven, Gulperi Oktem. Wnt1 gene expression alters heterogeneous population of prostate cancer cells; decreased expression pattern observed in CD133+/CD44+ prostate cancer stem cells. abstract. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4072. doi:10.1158/1538-7445.AM2013-4072
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