Didem Keles

Background and 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. 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.

Background Collagenases, 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. Objectives We aimed to develop a new “cell in situ collagen zymography” technique to enhance the efficiency of studying local collagenase activities in vitro. Methods We 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. Results Our optimization process revealed that that the best visualization of collagenase activity in 8505 C was provided by the “sandwich model” of gel, containing 25 µg/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. Conclusion The innovative “cell in situ collagen zymography” technique offers an efficient, cost-effective, and rapid method for analyzing local collagenase activities in vitro.

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 μM did not significantly affect cell viability for up to 72 hours, whereas higher doses (400-600 μM) reduced viability in a time-dependent manner. Aconitine at 200 μM 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-MB-231 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.

Objectives: Virtual laboratory simulations (VLSs) are computer-based tools that offer unlimited application options in scientific, medical, and engineering fields. The aim of this study was to evaluate whether VLSs are efficient learning tools and how these simulations can be integrated into laboratory practice in medical laboratory education. Methods: In this pre-test/post-test control group study, 32 volunteers were randomly assigned to either experimental or control groups. The experimental group performed laboratory simulations based on biochemistry and microbiology and then completed a self-report survey to evaluate their satisfaction and beliefs about simulations. Results: In the experimental group, post-test scores of each simulation were significantly elevated compared to pre-test scores; however, pre- and post-test scores of control group were statistically the same. The experimental group agreed that these simulations should be applied before theoretical lectures and laboratory practices. They also highlighted that translating from English to their native language creates difficulties in applying and understanding the simulation. Conclusions: We emphasized that VLSs are excellent learning tools that increase not only the knowledge but also the self-motivation and focus of the students. Based on feedbacks, native language options are necessary to enable the students to achieve equality of opportunity in education.

Objective: The aim of this study was to explore the alterations in levels of pro-inflammatory and catabolic mediators following vertebral fusion in a rabbit model of intervertebral disc degeneration. Methods: In this study, 24 female New Zealand albino rabbits (aged 4 to 5 months and weighing 3 to 3.5 kg) were used. All the animals were randomly categorized into four groups, and dorsal spinal exposure of all lumbar vertebrae was routinely performed in each group. While disc degeneration was created in groups B, C, and D, spinal fusion was added to disc degeneration in groups C and D. Disc degeneration was typically created by puncturing the discs with an 18-gauge needle under the guidance of C-arm imaging. Fusion was achieved with posterior/posterolateral decortication and iliac bone grafts. The rabbits in groups A, B, and C were euthanized, and the discs were removed in the first week after the surgery. The rabbits in Group D were sacrificed, and the discs were harvested at 5 weeks after the surgery. The levels of Interleukin (IL)-1β, IL-6, Nitric Oxide (NO), Matrix Metalloproteinase (MMP)-3, MMP-13, and Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) in the discs were analyzed using enzyme-linked immunosorbent assay kits. Results: Significant increase was observed in the protein levels of both pro-inflammatory and catabolic mediators in disc degeneration groups (Group B, C, and D) compared to Group A. In the fusion groups (Group C and D), these increased mediators decreased, compared to non-fusion group (Group B), (IL1-β P = 0.017, TIMP-1 P = 0.03, NO P = 0.03). However, there was no statistically significant difference in mediator levels between the short- and long-term fusion (Group C versus D). Conclusion: The results of this study have shown that a significant decrease in pro-inflammatory and catabolic mediators may be expected after vertebral fusion whereas there may be no significant difference between the first and fourth week of fusion surgery. These findings may contribute to clarifying the mechanism of action of vertebral fusion in the treatment of low back pain. Cite this article as: Dumanlıdağ D, Keleş D, Oktay G, Koşay C. Effects of vertebral fusion on levels of pro-inflammatory and catabolic mediators in a rabbit model of intervertebral disc degeneration. Acta Orthop Traumatol Turc 2021; 55(3): 246-52.

Objective: To assess the effects of Metformin, Dichloroacetate (DCA) and their combination on cell viability in oral squamous cell carcinoma, UPCI-SCC-131 cell line. Methods: UPCI-SCC-131 cells were plated in 96 E-plate (1x10 4 cells/well) and were treated with Metformin (1-16mM) and/or DCA (15-120mM) for 24-48-72h. xCELLigence SP system was used to monitor real time cell viability. In addition, drug combination index was analyzed with CompuSyn software according to Chou-Talalay method. Results: Half-maximal inhibitory concentrations (IC 50 ) of Metformin and DCA were found to be 3mM and 23mM, respectively, for 72 hours. CI values (0.76-0.80) in all combination groups below 1 indicated that Metformin/DCA combination had a moderate synergistic effect on cell viability in UPCI-SCC-131 cells. Discussion: Metformin/DCA combination synergistically decreased the cell viability of UPCI-SCC-131 cells. Therefore, a combined application of Metformin and DCA may be considered as a candidate therapy for the " drug repositioning " of the treatment of oral cavity cancer.

Lupus erythematosus is a chronic autoimmune disease characterized by remissions and exacerbations. Accumulated evidence indicated that matrix metalloproteinases (MMPs) are upregulated in inflammatory cells of cutaneous lupus erythematosus (CLE); however, the activity levels of these proteases have remained uncharacterized. To elucidate the significance of MMP-2, MMP-9, and TIMP-1 in CLE pathogenesis, gelatin zymography was used to investigate pro and active levels of MMP-2 and MMP-9 in lesional and perilesional skin biopsies obtained from twenty-two CLE patients. TIMP-1 protein levels were detected by ELISA in the biopsy specimens. The correlation between biochemical parameters and clinical characteristics of the disease was also evaluated. Significantly higher levels of active MMP-2, active MMP-9, proMMP-9, active/proMMP-2, and TIMP-1 were detected in lesional skin samples. Besides, the active/proMMP-9 was elevated in female and smoking patients. Active MMP-9 levels and active/proMMP-9 were also increased in elderly patients. Active MMP-9 levels were lower in patients who had smaller total damage score. Consistently, active/proMMP-9 and active/proMMP-2 were positively correlated with CLASI. Interestingly, in hydroxychloroquine or topical corticosteroid-treated patients, MMP-2/-9 activity levels were found to be higher compared to untreated patients. These findings suggest that increased MMP-2 and MMP-9 activities may contribute to the pathogenesis of CLE and cutaneous disease severity.