Browsing by Author "Sipahi, Murat"

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1,25-Dihydroxyvitamin D3 Induces N-Myc Downstream Regulated Gene-2 Expression in Papillary Thyroid Carcinoma Cells
(Dokuz Eylul Univ Inst Health Sciences, 2020) Sipahi, Murat; Bartik, Didem Keles; Doruk, Mehmet; Bayraktar, Firat; Oktay, Gulgun
Purpose: In addition to its role in serum calcium homeostasis, the anti-tumor function of 1,25-dihydroxyvitamin D-3 (calcitriol) in cancer development is well established. N-myc Downstream Regulated Gene 2 which functions as a tumor suppressor gene has recently been shown to be downregulated in various cancer leading to increased tumor incidence, progression and metastasis. The goal of this study was to investigate the possible effects of calcitriol treatment on NDRG2 expression in BCPAP papillary thyroid carcinoma cells. Methods: The experiments were carried on human primary thyroid follicular epithelial cells (Nthy-ori-3-1), and human papillary thyroid carcinoma cells (BCPAP). The half maximal inhibitory concentration (IC 50) of calcitriol on BCPAP cells was determined by WST-1 assay. BCPAP cells were treated with 15 and 30 mu M calcitriol for 24, 48, and 72 hours, respectively. Basal NDGR2 expression in Nthy-ori-3-1 and BCPAP cells as well as the alterations on NDRG2 expression in calcitriol treated BCPAP cells were evaluated with western blot. Results: A significant downregulation of NDRG2 was observed in BCPAP cells when compared to Nthy-ori-3-1 cells (p<0.01). IC50 dose of calcitriol was found to be 64, 54 and 43 mu M for 24, 48 and 72 hours, respectively. NDRG2 protein expression levels were significantly increased in 30 mu M calcitriol treated BCPAP cells after 48 hours (p<0.05). Conclusions: Calcitriol induced NDRG2 protein expression in BCPAP cells. We predict that calcitriol increased NDRG2 protein levels in BCPAP cells via c-Myc repression, which is upregulated by aberrant Wnt/beta-catenin signaling. Further investigation is required to enlighten the possible effect mechanisms of calcitriol in BCPAP cells.
Aconitine Impedes Cell Motility in Mda-Mb Breast Cancer Cells
(Dokuz Eylul Univ Inst Health Sciences, 2024) Keles, Didem; Sipahi, Murat; Surer, Seniz Inanc; Oktay, Gulgun
Purpose: Aconitine, a potent alkaloid from Aconitum plants, has shown promising anticancer properties. The aim of the study is to investigate the effects of aconitine on lateral migration, and matrix metalloproteinase (MMP) activity in MDA-MB-231 triple-negative breast cancer cells. Material and Methods: A WST-1 viability assay was conducted to determine the effect of aconitine on the viability of MDA-MB-231 cells. Following treatment with non-cytotoxic doses of aconitine, lateral migration was evaluated through wound healing assays. Additionally, gelatin zymography was conducted to analyze MMP-2 and MMP-9 activity and secretion levels. Results: Aconitine concentrations up to 200 mu M did not significantly affect cell viability for up to 72 hours, whereas higher doses (400-600 mu M) reduced viability in a time-dependent manner. Aconitine at 200 mu M showed a trend towards decreased lateral motility, with a significant reduction at 9 hours post-treatment. Gelatin zymography revealed no alterations in MMP-2 and MMP-9 activity or secretion levels following aconitine treatment. Conclusion: Aconitine demonstrates limited efficacy in modulating the migratory capacity of MDA-MB231 cells and does not affect gelatinase activity. Further investigation into underlying mechanisms is necessary, potentially leading to novel therapeutic strategies for triple-negative breast cancer.
Differential Effects of Sertraline and Penfluridol on EMT and ECM Remodeling in Glioblastoma Cell Lines
(Dokuz Eylul Univ inst Health Sciences, 2025) Keleş Bartık, Didem; Oktay, Gulgun; Surer,Seniz Inanc; Sipahi, Murat; Keles, Didem
Purpose: Glioblastoma multiforme (GBM) is an aggressive brain tumor with poor prognosis due to rapid recurrence, chemoresistance, and limited efficacy of standard therapies. Epithelial-to-mesenchymal transition (EMT) and matrix metalloproteinase (MMP)-mediated extracellular matrix (ECM) remodeling are critical processes in GBM progression and metastasis. The aim of this study is to examine the potential effects of sertraline and penfluridol on the EMT process and gelatinase activity in human glioblastoma cell lines. Material and Methods: U87 and U251 human glioblastoma cells were treated with sertraline and penfluridol at previously identified IC50 doses. Protein levels of EMT markers, E-cadherin, vimentin, Snail, Slug, Twist1, phospho-Akt (p-Akt), and tissue inhibitor of metalloproteinases-2 (TIMP-2), were evaluated using Western blotting. Additionally, the impact of sertraline and penfluridol on the release and activity of MMP-2 and MMP-9 were assessed through gelatin zymography. Results: Both sertraline and penfluridol significantly reduced vimentin expression in U251 cells, indicating inhibition of the mesenchymal phenotype. Conversely, these drugs increased vimentin levels in U87 cells, highlighting cell line-specific differences. Sertraline and penfluridol also increased TIMP-2 levels in U251 cells but not in U87 cells. Neither drug altered MMP-2 or MMP-9 activity in either cell line, suggesting that their effects on ECM remodeling may be mediated through TIMP-2 upregulation rather than direct modulation of gelatinase activity. Conclusion: These findings suggest that sertraline and penfluridol potentially inhibit EMT and reduce ECM degradation in U251 cells but exert contrasting effects in U87 cells. This highlights the heterogeneity of GBM tumors and the importance of personalized therapeutic approaches.
A Simple and Efficient "Cell in Situ Collagen Zymography" Technique To Evaluate Cellular Collagenase Activities in Thyroid Cancer Cell Lines
(Springer, 2025) Savas, Ege Gokce; Surer, Seniz Inanc; Sipahi, Murat; Keles, Didem; Oktay, Gulgun
BackgroundCollagenases, a subgroup of matrix metalloproteinases (MMPs), play crucial roles in local invasion and metastasis in cancer. While substrate zymography and in situ zymography are commonly used to analyze the collagenases, traditional techniques have limitations in determining their local activities in vitro.ObjectivesWe aimed to develop a new "cell in situ collagen zymography" technique to enhance the efficiency of studying local collagenase activities in vitro.MethodsWe utilized human thyroid cancer cell lines (8505 C, B-CPAP, FTC-133) and normal follicular thyroid cell line (Nhty-ori-3-1). We compared collagenase levels across these cell lines and selected 8505 C as a model due to its highest collagenase activity. We optimized factors including (i) fixation method (methanol, ethanol and zinc), (ii) dye-quenched (DQ) collagen concentration and (iii) collagen gel configuration. For gel configuration, cells were seeded under, on the top of, or between (sandwich) collagen gel layers. As controls, enzymatic activity was suppressed in the presence of EDTA, piroxicam and matrix metalloproteinase 8 inhibitor I. The optimized method was also applied to BCPAP, FTC-133, and Nthy-ori-3-1.ResultsOur optimization process revealed that that the best visualization of collagenase activity in 8505 C was provided by the "sandwich model" of gel, containing 25 mu g/mL of DQ-collagen with 100% cold methanol fixation. We confirmed the optimized method's applicability in other thyroid cell lines. The use of inhibitors validated the specificity of the fluorescent signal to MMP activity.ConclusionThe innovative "cell in situ collagen zymography" technique offers an efficient, cost-effective, and rapid method for analyzing local collagenase activities in vitro.