Theses supervised by Prof. Dr. Fikrettin Şahin
47 theses · Yeditepe University
Ocimum basilicum derived extracellular vesicles: Novel promising approach for type 1 diabetes mellitus
Pancreatic β-cells are crucial for regulating glucose metabolism, and their dysfunction is a key factor in both type 1 (T1DM) and type 2 (T2DM) diabetes mellitus. Hyperglycemia, a common feature of diabetes, increases the production of reactive oxygen species (ROS), leading to oxidative stress and further β-cell impairment. Plant-derived extracellular vesicles (EVs) offer new therapeutic possibilities to counteract the detrimental consequences of hyperglycemia. This study investigates the impact of Ocimum basilicum (basil) derived EVs (BEVs) on pancreatic β-TC-6 cell lines under hyperglycemic conditions. Basil is known for its antioxidant properties and generates EVs that contain bioactive molecules. These EVs play a role in the plant's therapeutic effects. In this study, the effects of BEVs on β-TC-6 cells were evaluated in terms of cell proliferation, apoptosis, cell cycle distribution, insulin secretion, ROS production, antioxidant enzyme activity, mitochondrial function, and gene expression. BEVs were isolated from basil using sucrose cushion with ultracentrifugation and characterized by nanoparticle tracking analysis (NTA), uptake analysis, and transmission electron microscopy (TEM) following MISEV criteria. β-TC-6 cells were treated with different range of concentrations of BEVs (1010, 109, 108, 107, and 106 particles) for 24 and 48 hours. Cell proliferation was measured using the MTS assay, showing no cytotoxic effects relative to the control. Under hyperglycemic conditions (33 mM glucose), β-TC-6 cells showed a remarkable decrease in cell viability relative to control, in which BEVs significantly improved at 24 hours and slightly at 48 hours relative to hyperglycemia group. Further studies were done to provide a more detailed understanding of the effect of BEVs under hyperglycemic conditions including, cell cycle analysis, Annexin V & Dead Cell Assay, live-cell imaging, Rhodamine staining, Griess Reagent System, insulin-ELISA assay and SOD assay. Overall, these findings suggest that BEVs enhance pancreatic β-TC-6 cell proliferation, reduce oxidative stress, and improve insulin secretion and glucose metabolism under hyperglycemic conditions. These results highlight BEVs as a promising therapeutic approach for hyperglycemia. However, it should be emphasized that advanced evaluations are needed.
Investigating the role of NRF2 pathway on T cell & Endothelial cell based in vitro autoimmune disease model
Autoimmune diseases occur when the immune system attacks the body's own tissues mistakenly, resulting in tissue damage and chronic inflammation. These conditions are prevalent worldwide and can lead to a range of complications. A crucial factor in progression of autoimmune diseases is the interaction between leukocytes and endothelial cells. This interaction is fundamental to the inflammatory process. This study investigates antiinflammatory impacts of NRF2 pathway on the activated T cells that nonspecifically induce endothelial inflammation. It also aims to elucidate the role of oxidative stress and NRF2- mediated responses in modulating T cell interactions with endothelial cells, as well as to provide insights into the potential mechanisms underlying autoimmune diseases characterized by aberrant T cell binding and endothelial damage. In the current study, the NRF2 pathway was induced in Human Umbilical Vein Endothelial cells (HUVEC) utilizing NRF2 gene overexpression via lentiviral systems and the synthetic NRF2 inducer, tert-butyl hydroquinone (TBHQ). The effect of the NRF2 pathway on activated Jurkat cells were examined by direct co-culture with HUVEC in vitro. Underlying mechanisms were demonstrated with qPCR, human inflammatory cytokine cytometric bead array (CBA) and Immunocytochemistry (ICC). The NRF2-Overexpressed (NRF2OE) cell line demonstrated a 3-fold change of NRF2 gene compared to TBHQ group. In the NRF2OE HUVEC cells, only the SOD2 gene exhibited a significant fold change. Co-culture of NRF2OE HUVEC cells with activated Jurkat cells induced a phenotype shift from Th1 to Treg in Jurkat cells. Overall, these findings suggest that NRF2 pathway influences the interaction between endothelial cells and activated T cells, potentially altering T cell phenotypes and preventing firm adhesion of T cells. Further exploration could significantly advance the development of novel therapeutic strategies for autoimmune diseases and inflammatory conditions. By promoting the generation of Treg cells, NRF2 pathway has the potential to enhance immune tolerance, reduce autoimmune tissue damage, and improve outcomes in various inflammatory disorders.
The role of plant-derived extracellular vesicles on the in vitro Alzheimer's disease model
Alzheimer's disease is a progressive and irreversible neurodegenerative disorder, in which the accumulation of amyloid beta (Aβ) peptides and the hyperphosphorylation of tau proteins play a pivotal role in its pathogenesis. Current treatment methods mostly aim to alleviate symptoms, but are insufficient in stopping disease progression or targeting the underlying mechanisms. Extracellular vesicles (EVs) are nanovesicles that provide intercellular communication, have a lipid bilayer membrane structure, and can carry biomolecules such as proteins, lipids, and RNA. Lately, EVs have gained significant interest as therapeutic tools due to their effects such as regulating gene expression, suppressing inflammation, and triggering cellular repair processes in disease models. Plant-derived EVs (PDEVs) stand out as important biotherapeutic candidates due to their natural origin, low immunogenicity, economical production, and cross-species biological activity. In addition, their anti- inflammatory and antioxidant properties make EVs attractive candidates for targeting neurodegenerative pathways. This study aims to investigate the effects of extracellular vesicles isolated from tobacco (Nicotiana tabacum) (TEV) suspension culture and celery (Apium graveolens) extract (CEV) in an in vitro AD model. In the study, Aβ accumulation and hyperphosphorylation of tau proteins were induced in differentiated SH-SY5Y cells and an AD-like model was created by treating neurons with TEV, while astrocytes were treated with CEV in a gliotic model created by applying Aβ peptide. Molecular and cellular analyses performed after the application showed that TEV application reduced neuronal degeneration, while CEV application suppressed Aβ-induced astrogliosis responses. In particular, a significant decrease was observed in Aβ and phospho-tau accumulations and protein aggregate load. The findings suggest that PDEVs may affect AD pathophysiology and have neuroprotective potential on both neuronal and glial targets. This study is the first to explore the therapeutic potential of PDEVs in AD, laying an important foundation for future research in this field.
Discovery of functional characteristics of plant cell suspension-derived extracellular vesicles on the in vitro cerebrovascular inflammation
Extracellular vesicles (EVs) are essential biomolecule carriers responsible for intercellular communication. They play vital roles in many physiological events. Thanks to their bilayer lipid membrane, biomolecules can be protected from the harsh extracellular environment. One of living organisms in which EVs consistently function is plants. Plant-derived EVs (PDEVs) have a role in physiological events and defense against pathogens. Besides, these nanovesicles showed therapeutic potential in antioxidant, anti-inflammatory, and immunomodulatory activities. On the other hand, cerebrovascular inflammation (CVI) is a chronic inflammatory disorder characterized by cerebrovascular dysfunction, proinflammatory phenotype, and vascular hyperpermeability. Based on their therapeutic activities, PDEVs can become a promising tool for cerebrovascular disorders. Our study aims to investigate the functionality of plant cell suspension extracellular vesicles (PCSEVs) and their therapeutic effects on the in vitro CVI model. To that end, these nanovesicles were purified from the media of Stevia rebaudiana, Vaccaria hispanica and characterized. The CVI pathology was modelled and the effects of PCSEVs were investigated. In the results, first, the PCSEVs were successfully purified and characterized in the sense of physical and molecular properties. Besides, the CVI pathology was successfully modelled in the cells and it exhibited critical features of its pathologies. Lastly, it was observed that PCSEVs led to a protective response in the CVI model, and these effects mainly occurred by modulating adhesion molecule expression via the NRF2 pathway. In addition, further analysis revealed that these effects of PCSEVs were mediated through the phosphorylation of key signaling pathways. However, further studies are needed to transform these in vitro results into clinical applications. Future studies should include in vivo experiments in animal models to confirm the efficacy and safety of the PCSEVs. By demonstrating the therapeutic effects of PCSEVs on cerebrovascular inflammation, our study paves the way for innovative, plant-derived nanomedicine solutions for vascular diseases.
Anti-cancer effect of postbiotics of novel probiotic strains isolated from fermented food
Kolorektal kanser, dünya genelinde en yaygın ve ölümcül kanser türlerinden biri olarak kabul edilmektedir. Son yıllarda, bağırsak mikrobiyotası ve bu mikroorganizmaların metabolitlerinin hem kanserin gelişiminde hem de önlenmesinde oynadığı rol üzerine artan bir ilgi gözlemlenmektedir. Bu tez çalışması iki ana aşamada gerçekleştirilmiştir. İlk aşamada, çeşitli probiyotik suşları izole edilerek ayrıntılı bir şekilde karakterize edilmiştir. Bu karakterizasyon sürecinde morfolojik ve biyokimyasal özellikler, enzim aktiviteleri (örneğin, proteaz, ve lipaz) ve antioksidan kapasiteleri değerlendirilmiştir. Özellikle Lactobacillus ve Bacillus türlerine odaklanılmış olup, bu türlerin gastrointestinal sistemde sağlık üzerinde olumlu etkiler gösterdiği bilinmektedir. İkinci aşamada ise bu probiyotik suşlardan elde edilen postbiyotiklerin HCT116 ve HT29 kolon kanseri hücreleri üzerindeki biyolojik etkileri değerlendirilmiştir. Postbiyotikler, canlı olmayan bakteri metabolitleri ve hücre bileşenleri olarak tanımlanmakta ve potansiyel terapötik etkileri ile dikkat çekmektedir. Hücre kültürü tabanlı deneylerde, çeşitli hücresel süreçler incelenmiştir. MTS testi ile hücre canlılığı ve metabolik aktivite değerlendirilmiş, akış sitometrisi ile hücre döngüsü ve apoptoz analizleri yapılmıştır. Ayrıca, apoptoz (Caspase 3, 7, 8, BAX), hücre döngüsü düzenlenmesi (CDK1, CDK2, CDK4, CDK6) ile ilişkili genlerin ekspresyon düzeyleri RT-PCR ile analiz edilmiştir. Bu araştırma, seçilen probiyotik suşların ürettiği postbiyotiklerin kanser hücrelerinin çoğalmasını baskılayabildiğini, hücre ölümünü indükleyebildiğini ve hücre döngüsüne müdahale edebildiğini göstermektedir. Elde edilen veriler, probiyotik kaynaklı postbiyotiklerin kolorektal kanser hücrelerinde kritik hücresel ve moleküler süreçleri modüle edebileceğini ortaya koyarak, yeni önleyici ve tedavi edici stratejilere kapı aralamaktadır. Bu kapsamda, bağırsak sağlığını destekleyici ve kanser riskini azaltıcı fonksiyonel gıda ürünleri veya biyoterapötikler geliştirilmesi mümkün görünmektedir. Bu çalışmada, laboratuvar ortamında elde edilen sonuçların klinik uygulamalara dönüştürülmesinde karşılaşılabilecek zorluklara da dikkat çekilmektedir. Bu etkilerin fizyolojik koşullar altında doğrulanması için ileri düzey in vivo çalışmalar ve klinik araştırmalar gereklidir.
Comparison of two-dimensional and three-dimensional triple negative breast cancer cell culture models on treatment response
Triple negative breast cancer (TNBC) has a dismal prognosis and few available treatments, making it a major clinical challenge. This study assesses the effectiveness of cannabidiol (CBD), which has pro-apoptotic and anti-proliferative qualities, and olaparib (OLAP), a PARP inhibitor that targets DNA repair, as single and combination therapies in TNBC and estrogen receptor positive (ER+) breast cancer cell lines. In two-dimensional (2D) and three- dimensional (3D) culture systems, analyses were conducted on MDA-MB-231 (TNBC), HCC-70 (TNBC), and MCF-7 (ER+) cell lines. The results demonstrated that in 3D culture models, increased efficacy was attained with lower doses, and that OLAP and CBD caused synergistic cytotoxic effects, particularly in TNBC cells. In MDA-MB-231 cells with G2/M phase arrest, increased apoptosis was seen, as demonstrated by flow cytometry and caspase 3/7 tests. DNA damage response and cell cycle-related genes (ATM, ATR, BRCA1/2, RAD51, and CDK1/2/4/6) were down-regulated by the combined treatment, according to gene expression analysis. Specifically, 3D spheroid models under metabolic and hypoxic stress demonstrated heightened drug sensitivity. These results imply that 3D culture methods provide more physiologic and sensitive models for evaluating medication efficacy, and that OLAP and CBD together offer a possible therapeutic approach for aggressive breast cancer subtypes.
An alternative and therapeutic approach for hepatocellular carcinoma
Hepatocellular carcinoma is among of the most frequently forms of liver cancer, with a high death rate, emphasizing the need for improved treatment techniques. Current research has focused on the anti-cancer effects of natural compounds. The most common therapy for HCC varies according to stage: patients in the early stages may require surgical resection or liver transplantation, whereas intermediate-stage patients may get Transarterial chemoembolization (TACE). In advanced stages, immunotherapy combined with targeted therapies, especially Atezolizumab-Bevacizumab, has emerged as the standard. CBD (cannabidiol), a non-psychoactive cannabinoid, has gained attention in cancer research because of its anti-inflammatory, anti-proliferative, anti-angiogenic, and apoptosis-inducing properties. Studies suggest CBD may inhibit tumor growth, induce cell death, and reduce metastasis. Additionally, CBD is being explored as a complementary treatment for alleviating chemotherapy side effects. Vitamin D is also recognized for its potential function in HCC treatment, given its impact on immune regulation and cell growth control. Vitamin D insufficiency has been associated to HCC progress, emphasizing its potential therapeutic value. The current research tested the anti-cancer impacts of single and combination treatments of Vitamin D and CBD in HEP3B and HUH7 cell lines, focusing on apoptosis, autophagy, cell cycle regulation, and DDR. The combination treatment of Vitamin D and CBD demonstrated a synergistic effect, significantly reducing cell survival in both HEP3B and HUH7 cell lines. Notably, the combination induced apoptosis and autophagy in HEP3B cells and inhibited the cell cycle in HUH7 cells, suggesting anti-tumorigenic and anti-cancer effects. These findings indicate the possibility of Vitamin D and CBD as a novel treatment approach against HCC, requiring additional in vivo studies and clinical trials to assess efficacy, safety, and mechanisms in more complex biological systems.
Mechanisms in biomining of magnesite and its scale up production
Magnesite, that is called magnesium carbonate (MgCO3) mineral, is one of the important ores of Turkey among countries rich in terms of ore resources. Since magnesium found in magnesite is a valuable raw material at the pure state or various compound states for present-day technology, developing new approaches on obtaining and enrichment of this material are very beneficial on account of national economics. In order to trade magnesite economically, SiO2 and CaCO3 rates of raw material needs to be less than 2 percent and 1 percent, respectively. A number of different enrichment methods have been developed to increase purity of magnesite. The aim of the present study is to develop a biomining process which will be used for enrichment of magnesite from different ores in Turkey by dissolving carbonate compounds, to scale up this process and to determine Ca dissolving factors. Previously and newly isolated and identified bacterial strains, Enterococcus spp., Lactococcus spp. Staphylococcus spp., and fungi isolate Neurospora spp. were used. Optimum working parameters were identified for all microorganisms. Results indicated that, microorganisms show effective dissolving capacity at 30ºC, pH between 3 and 7, in the presence of glucose as carbon source. The subcronic toxicology tests performed with Wistar rats, showed that Enterococcus spp., and Lactococcus spp. strains are not pathogenic and toxic for mammalian. Lactococcus spp. was used at scale up studies and 3 L and 30 L fermenter processes were performed successfully. Consequently, genomic, transcriptomic and proteomic analysis of Enterococcus spp. and Lactococcus spp. strains were performed to illustrate the molecular mechanisms of magnesite biomining. This is the first study to demonstrate Enterococcus spp. and Lactococcus spp. strains can be used for enrichment of magnesite in industrial scale. A further study needs to be done to identify other microorganisms from nature that have potential to be used in magnesite biomining and formulation of them for industrial scale.
Hygenic adult/patient diaper development and production
Microorganisms can easily reproduce in humid environments containing nutrients and cause diseases in humans. For this reason, diapers used by patients or adults, who are unable to control fecal or urinary excretory, create a suitable growing medium for opportunistic pathogens. In the current study, it is aimed to design and develop patient or adult diapers which have antimicrobial properties using boron compounds and Pluronic polymers (Poloxamer). For this purpose, Diaper dermatitis related pathogenic microorganisms (bacteria, yeast and fungi) were used to conduct antimicrobial activity tests. Boron, Pluronic polymers (F68 and F127), prepared at different doses, and their combinations were tested for their antimicrobial activities against a broad range of microorganisms. As a result of in-vitro tests, diapers containing boron and Pluronic combination were found to display remarkable antimicrobial properties. The use of adult or patient diapers is expected to grow due to increasing population of old people in the future. Therefore, the objective of this project was to develop disposable adult and/or patient diapers providing higher comfort compared to commercially available products, prevent problems related to diaper usage, have high leakage barrier and posses antimicrobial properties. This study will create a competition within national and international arena and access to market with the antimicrobial adult and/or patient diaper developed as a consequence of this study.
Characterization of stem like cells in a new astroblastoma cell line
Astroblastomas are uncommon neuroepithelial tumors of glial origin. It predominantly affects younger people mainly teenagers and children with a high female proportion. Only a single study so far reported that astroblastoma contains a high amount of neural stem like cells which had only partial proliferation capacity and differentiation. In our study a patient derived astroblastoma cell line was generated and presence of cancer stem cells was investigated. Primary cell lines are powerful tools for clarifying the biological characteristics of a cancer and for developing effective treatment strategies. Characterization for astroblastoma and stem cell markers through flow cytometric and gene expression analyses, capacity to differentiate into other lineages and recapitulation of the original tumor has been studied through several methods. The presence of stem cells in this heteregenous cell population might allow is to learn about the initiation, progression and chemoresistance of this specific astroblastoma cell line. This new cell line might be used as a model cell line for further studies that scientific people might have an interest in astroblastoma.
Boron promotes the survival and the maintenance of β-cells in type i diabetes mellitus
Diabetes mellitus is worldwide disease. The life of diabetic patients are dependent on exogeneous insulin. Pancreas or particularly islet transplantations are performed for treatment to heal this disease. External substances are also used to achieve improvements in the healing process of the disease. In the current study, two different boron derivatives (boric acid-BA and sodium pentaborate pentahydrate-NaB) were investigated for their effect on pancreatic cells in terms of pro-apoptotic and anti-apoptotic markers, genes related to insulin production mechanism, pancreatic development and glucose metabolism, some antioxidant enzymes, and genes for the initiation of diabetes, insulin secretion and antioxidant enzyme activities in vitro. The results revealed that boron derivatives did not lead to apoptosis. On the contrary, they increased cell viability, antioxidant enzyme activities and insulin secretion in the gene level. Overall evaluation, data in the current study showed that boron derivatives might be promising therapeutic agents for type I diabetes. However, additional investigations are strictly needed to elucidate molecular mechanisms of boron derivatives.
Analyzing inhibitory and disruptive effect of boron derivatives onpseudomonas aeruginosa (ATCC- 27853) and Escherichia coli(MG1655/K12) biofilms on surfaces in water supplies
Biofilm is a multi-stage composition of one or more microorganisms and extracellular matrix which adhere to the wet surfaces and supported by matrix against any external perturbation. Every year, people get serious sicknesses or even die because of chronic infections typically induced by biofilm. Hygienic water is the basis for healthy society. It is impossible to continue life without hygienic water. Most people use swimming pools or take showers in their own houses as well as public places. Additionally, water tanks are being used for washing or as commodity water,in particular in poor countries. Numerous studies have shown bacterial growth and biofilm formation on water tanks, swimming pools, etc. Moreover, the bacterial growth can seriously cause diseases and harm the society. Pseudomonas aeruginosa and Escherichia coli are microorganisms which are gram negative and opportunist can proliferate easily and directly cause illness like sepsis, dysentery and even can cause death. Preventing or at least controlling biofilm formation is of great value from not only environmental but also social health perspectives. Biocidal agents such as boron derivatives are promising ways to control biofilm formation without harming human body. In this project, it was aimed to prevent biofilm formation and inhibit bacterial growth in places such swimming pools, bathrooms etc with special biocidal compounds which are non-toxic and environmentally friendly. In addition, the disruptive effect of these kinds of chemicals were be analyzed and tested on biofilm to make safe and healthy places for a human. It is expected to find a new way to prevent biofilm formation from these two bacteria inplaces such as swimming pool and water tanks that the water would be still for a long time. Therefore, disruption and inhibitory effects of different boron derivatives including boric acid (BA), sodium tetraborate decahydrate (STD) and disodium octaborate tetrahydrate (DOT) on biofilm formation by two bacterial species, Pseudomonas aeruginosa and Escherichia coli, were investigated in-vitro micro-well bioassay in this study.The results showed that 25 mg/ml concentration of STD and DOT have better potential to disrupt and/or inhibit both of bacterial biofilm formation tested. Further studies are needed to confirm that boron derivatives may be beneficial to use in the drug industry and construction material such as ceramic in order to control biofilm formation and related health problems in the future.
Investigation of cancer development on human newborn foreskin cells and human melanocytes via exosomes containing Rab27a and Met oncogene isolated from human malignant melanoma cancer cell lines
Exosomes are small membrane derived vesicles originating from endocytic core with 40100 nm. Exosomes transmit DNA constituents, mRNAs, microRNAs and proteins that are relocated from a donor cells to a receiver cell. Many divergent cells comprising mesenchymal cells, immune cells and cancer cells discharge exosomes. Studies show that cancer cell exosomes create the entry and reprogramming of essentials connected to tumor environment. A reference study established that melanoma-derived exosomes convey diverse proteins such as c-Met and Rab27a, which indicate a melanoma mark. Increased Met expressions in serum exosomes have thought to be a predictor of disease progression. Meanwhile, Rab27a has identified to be important in exosome discharge. Decreased expressions of Rab27a in human melanoma cells determined to diminish exosome release. This project aims to examine the effects of downregulation and upregulation of Rab27a and c-Met in human melanocyte and recently identified stem cells isolated from human newborn foreskin tissue by utilizing the isolated exosomes from malignant melanoma cell lineages. Throughout the analysis of cancer-like formation; different protocols covering gene transfections, flow cytometry analysis of the transfection efficiency, Annexin-V apoptosis assays, cell migration assays, tube formation assays, ELISA assays and gene expression profiling were performed. According to the results, exosomes derived from cancer cells conveyed information to healthy fibroblastic like stem cells from human newborn prepuce and healthy melanocytes/keratinocyte and induce cellular reaction with Met and Rab27a overexpression and silencing may be beneficial approaches for future treatment possibilities. The developing molecular contextual of melanoma exosomes and their implications for improved management of melanoma patients can be an astonishing therapeutic methodology for future actions.
Molecular consequences of EMT silencing on pancreas cancer cell lines
Pancreatic cancer is known to spread rapidly and rarely detected in early stages. Not until quite advanced stages, no signs and symptoms are observed and in advanced stages, it is nearly impossible to remove the tumor. This type of cancer is known to be aggressive and migrate to different sections of the body quickly. If metastasis of pancreatic cancer could be stopped it would be possible to manage the tumor without searching for additional tumor spread and if this treatment could be combined with drug resistance decrease it could be used as a good treatment option for pancreas cancer. Epithelial to Mesenchymal Transition (EMT) is counted as one of the possible routes for initiating metastasis and drug resistance. Movement of epithelial cells is limited. Hence it must convert itself into a more motile cell type "mesenchymal" by using EMT pathways. In this study, important EMT transcription factors "Snail, Slug, and Twist" were silenced by short hairpin technology on different pancreas cancer (adenocarcinoma) cell lines, Panc-1, MIA PaCa-2, BxPc-3, AsPc-1. It was shown that this silencing causes the cell to turn back into the epithelial stage; being less mobile and invasive, having decreased stem cell characteristics. After these favorable results from gene silencing, AsPc-1, metastatic pancreas cancer cell line, was subjected to chemotherapeutic agents and drug resistance was evaluated. EMT downregulated AsPc-1 cells have shown less resistance to chemotherapeutics compared to the control group. Combinational therapy (both gene therapy and drug induction) is a rising trend for cancer. During this study, together with gene therapy small molecule inhibitors of EMT were used to enhance the effect of gene downregulation. EMT downregulated AsPc-1 cells were treated with two EMT inhibitors, SD-208 and CX-4945, to understand the cumulative effect of gene therapy and small molecule inhibitors on migration and invasion. The results have shown that it is possible to increase the effect of gene therapy by using chemical support but gene therapy is irreplaceable with small molecule inhibitors alone. Overall it was found that inhibition of EMT, either by gene therapy alone or with the enhancement by small molecules, is a good candidate for the treatment of pancreas cancer since it simultaneously minimize metastasis, stem cell properties and drug resistance.
The effects of bisphosphonates on osteonecrosis of jaw bone: aA stem cell perspective
Bisphosphonates (BPs) are commonly used drugs for the treatment of osteoporosis in routine clinical practice. Although BPs are successfully used for osteoporosis treatment, they may also cause bisphosphonate-induced osteonecrosis of the jaw (BIONJ). Up to present, the researchers have proposed several factors but the effects of BPs on dental stem cells' proliferation, differentiation or maintenance capacity have not been evaluated yet. In current study, the stem cells isolated from dental pulp, periodontal ligament and tooth germ were characterized by specific cell surface marker expression, differentiation and immunostaining assays. Osteogenic differentiation of the Dental Pulp Stem Cells (DPSCs), Periodontal Stem Cells (PDLSCs) and Human Tooth Germ Stem Cells (hTGSCs) treated with Zoledronate (ZOL), Alendronate (ALE) and Risedronate (RIS) were evaluated by mineral staining assays and gene expression analysis. BPs did not cause any negative effect on the osteogenic differentiation of DPSCs, PDLSCs and hTGSCs. Moreover, scratch assay was performed to detect the effects of ZOL, ALE and RIS on migration capacity of these dental stem cells, and migration related genes, extracellular matrix proteins and cytokines were analyzed. The results revealed that all BPs tested significantly inhibited migration ability of the dental tissue-derived stem cells. To evaluate the effects of ZOL, ALE and RIS on angiogenesis, two experimental models, aortic ring and tube formation assays, were performed with human umbilical vein endothelial cells (HUVECs). The results showed that BPs robustly inhibited angiogenic potential of the endothelial cells. The drastic effects of BPs on migration of DPSCs, PDLSCs and TGSCs, and angiogenesis of endothelial cells were determined. Inhibition of the migration capacities of the stem cells localized in proximity to the jaw bone, and interference with angiogenesis process might be the possible explanation for BIONJ observed after BP treatment. Further in vivo studies are highly warranted to investigate dental stem cell conditions in BP treated animals to elucidate the importance of these cells in BIONJ formation.
Anti cancer effect of garlic exosomes on different cancertypes
Exosomes are nano-sized vesicles that are recognized as the messengers of cell-to-cell signalling. These vesicles, ranging in size from 30-100 nm, are responsible for transporting molecules -such as protein, RNA, DNA, mRNA, and miRNA- from a given cell to another. Studies show that cells have a special mechanisms for releasing exosomes. Cells have the potential to engulf all exosomes, regardless of which cell they are secreted from. That said, current study aims to understand the effect of plant-derived exosomes on mammalian cells. Various malignant cell lines MDA-MB231 (metastatic breast cancer), DMS114 (small-cell lung cancer), and HCT 116 (colon cancer cells) were treated with plant derived exosomes isolated from garlic. Effects of garlic exosomes was examined in terms of inducing apoptosis, cell proliferation, cell viability. At the same time, potential cytotoxic effects of these agents on normal cells were analysed by using human umbilical vein endothelial cells (HUVEC). Exosomes obtained from garlic were visualized by scanning electron microscope (SEM) and characterized based on the surface markers CD69, HSP70, CD9, and CD81 by flow cytometry. Cytotoxicity assays were performed and the most appropriate doses were selected for treatment. In order to detect apootpsis, the annexin V and TUNEL assays were performed. VEGF and caspase 3 protein expression were analyzed by ELISA. p53, caspase3, caspase 9, Bcl-2 and Bax in gene expression levels were determined with RTPCR. Finally, effect of garlic exosomes on cell cycle was analysed by the Edu assay.
The effect of exosomes isolated from human foreskin stem cells on wound healing
Wound healing is one of the important processes in the body. The wounds are separated into two groups when considering the healing time and process: acute wounds and chronic wounds. Side effects of natural and chemical wound healing compounds to the human body are well known. Due to these side effects, attempts to create new drugs are of interest. Therefore, the use of stem cells has augmented. However, both difficulties in collection and risks such as rejection and teratoma in the recipient body have limited the use of stem cells. The extracellular vesicles secreted by stem cells have recently become prominent agents. With the use of these agents, the potential content of the stem cells can be transferred to the recipient cells without an immune response, and since it is easier to obtain than the stem cells, it has been used as a new generation wound healing agent. In this study, the wound healing effect of exosomes derived from foreskin stem cells was investigated on both keratinocyte and endothelial cells in vitro. Cell migration, new vessel formation, inflammation response, quantitative polymerase chain reaction and western blot experiments were performed to investigate the potential positive contribution to gene and protein levels and the underlying mechanisms. Overall, cell proliferation, cell invasion and cell migration were accelerated in exosome treated groups compared to the control group and new vessel formation was inducted in exosome treated groups. Increase in gene levels of growth factors related to proliferation and inflammation, and change in related signaling pathway proteins in exosome treated groups compared to the control group were shown. Possible molecular mechanisms underlying cell movements were associated with mitogen-activated protein kinase (MAPK) pathway. We found that foreskin stem cell exosomes can heal the wound in a short period of time so that it could be a promising wound healing agent. The current study was the first study in the literature representing the wound healing activity of foreskin stem cell-derived exosomes.
Role of ananas comosus derived exosomes in scarless cutaneous wound repair
Wound healing is a complex and dynamic process involving a great number of cellular interactions based on growth factors, cytokine, and chemokine release. Although many new therapeutic agents are being introduced to the market for their remarkable wound healing capabilities, the problem of why some organisms heal perfectly while others heal with a pathological connective tissue known as a scar remains a mystery. Scar formation is a natural process that occurs after the healing process due to dysfunctionalities in the remodeling phase that may occur either in the epithelial epidermis or the dermis. Reducing scar formation is lately gaining interest as a new area of scientific research. Following the discovery of scarless healing of the fetal skin, an explosive number of studies have highlighted striking differences between fetal and adult human skin in terms of the differences in their extracellular matrix, growth factor, and cytokine characteristics. Despite of the accumulating evidence to mitigate the scar formation, gain or loss of function of no single pathway or cell type has been shown to be responsible for scar formation therefore mimicking the scarless healing remains as a challenging issue. Nonetheless, findings from recent studies have shed light to certain aspects of scarless healing. In this respect, the use of plant-based products in the treatment medical conditions is becoming a matter of preference due to their lower side effects. Taking a parallel approach, this study aimed to evaluate the potential of scar reducing effects of Ananas comosus derived exosomes by determining their proliferative, migratory, and angiogenetic activities on the gene and protein expression levels of key factors of wound healing. Our results indicate that these exosomes activate ERK/MAPK and PI3K/AKT pathways and induce cellular proliferation and migration while increasing the ratio of transforming growth factor-β3 (TGF-β3) to TGF-β1, and certain MMPs, which is an indication for suppression of scar formation. Additionally, it has been shown that these exosomes also promote extracellular matrix reconstruction. Based on these results A. comosus exosomes emerge as a promising agent that facilitate cutaneous wound healing suppressing formation of scars.
The differential effect of USP7 on breast cancer cell lines
THE DIFFERENTIAL EFFECT OF USP7 ON BREAST CANCER CELL LINES Breast cancer has the highest mortality rate among women compared to other various types of cancer. As a multi-stage and complicated cancer type, breast cancer might be initiated by various stimuli. Most of the breast cancer patients have been diagnosed with a familial predisposition due to the poor genetical heritage. Investigation of mutated proteins and affected pathways are bare essential to develop an effective cure for targeted therapy. In the current study, potential anti-cancer activity of USP7 gene supression has been evaluated by in vitro cell proliferation analysis, the examination of alterations in gene and protein expression profile, three dimensional sphere formation capacity and colony forming unit assays. The small molecule inhibitor of USP7 enzyme in addition to small interfering RNA (siRNA) and lentiviral short hairpin vector (shRNA) has been used as targeting strategies against USP7. MCF7 and T47D human breast cancer cell lines, which maintain different P53 gene status, have been used to determine whether the anti-cancer effect of USP7 enzyme is p53 dependent or in-dependent. Overall, a significant anticancer activity of USP7 inhibition has been detected in vitro for the breast cancer cell lines. Many molecular, physiological and morphological changes have been observed upon USP7 inactivation in all of the three different experimental strategy indicating the potential anti-cancer activity of USP7 blockage. USP7 inactivation inhibited the cancer cell proliferation, invasion and metastasis in MCF7 and T47D cell lines. However, further investigation should also be conducted with the purpose of comprehending the exact molecular mechanism which underlies in the total effect of USP7 on tumorigenic progression. This is the first study in literature which offers a critical examination of the differential effects of targeting USP7 enzyme by both at protein and gene levels in MCF7 and T47D cell lines.
Market and business potential analysis of wound healing products of dermobor
Acute and chronic wounds are critical medical problems throughout the world due to leading serious mortality and morbidity along with economic burden. Acute wounds do not always need professional treatment, but chronic wounds are mainly accompanied by infection resulted in continuous tissue degradation. There are a variety type of wound healing products performed according to the severity of the wound at the moment. Boron containing product Dermobor demonstrated the wound healing effect in vivo and in vitro wound model and it has been newly launched in Turkey. Dermobor enhances acute cutaneous wound healing by preventing infections in wound areas and increasing fibroblast migration, antioxidant enzyme activity, growth factor expression levels. Carbopol-based hydrogel formulated with boron and pluronic block copolymers is also effective for burn wound management with its enhanced epithelization, wound closure, and angiogenesis feature. Treatment with Dermobor also successfully diminished radiation-induced skin reactions. In this study, Dermobor business and market potential researched based on acute and chronic wound healing product sales that has been already in the market. Topical antibacterials, dermotolgical preparation, antiseptics and wound healing ATC classification has been analyzed in this study utilized from IQIVIA databases. The made-up market consisting from those products has increased %18 its value and reached 258 million Turkish Lira in 2018. Considerin the same market for the world data of those market costs 3 billion Euro in value. Consumer behaviour in the pharmacy to the selection a product from those market has been commented on the result of online survey which has 381 respondents. Dermobor positioned as a wound healing product which regenerates tissue and stimulates the healing of: ulcers, wounds, fistulas, burns, inflamations, surgery, grafting, radioepitelitis in various medical specialties.
Role of exosomes in malignant transformation of astrocytes
Glioblastoma multiforme (GBM) is the mainly common type of primary brain tumors with dismal prognosis. Every year, nearly 3 people out of 100.000 become affected by the disease. Glioblastomas belong to the group of astrocytomas which originate purely from astrocytic cells in the brain parenchyma and usually do not affect neurons. The World Health Organization (WHO) classifies the gliomas into four grades according to their morphologies and the severity of their malignant behavior. Exosomes are the small nanovesicles which are known to be released by all cell types and are present in all biological fluids. Proteins, lipids, mRNA and miRNA molecules can be transported with exosomes. They thereby have specialized functions in a variety of key biological processes like intercellular genetic and metabolic communication as well as modulation of immune responses. Recently, their role in cancer and other pathological conditions has gained more attention. Their content has been studied in GBM as well as other cancer cells and they are considered as potential diagnostic biomarkers. However, their role in diffuse glioblastoma invasion has been overlooked. In this study human astrocyte cells (NHA) cells were treated with exosomes harvested from commercially available cell lines of GBM which are U87-MG and A172. Phenotypic and behavioral changes of NHAs were observed and analyzed. These changes were determined by cell proliferation assays (like MTS and EDU), cell cycle analysis, real-time PCR, TG2 Elisa, Western Blotting and as well as direct monitoring via live cell imaging. The obtained results were shown that GBM-derived exosomes play important role in invasion and metastasis. Only a few studies have been published about the possible mechanism of exosomes in diffuse glioblastoma invasion. We think that understanding the intercellular communication of exosomes will open a new approach in the desperate search of therapeutic options for glioblastomas. The next step should include in vivo studies to shed light on the role of exosomes in GBM invasion in CNS.
Evaluation of the effect of citrus sinensis derived exosomes on neurogenic differentiation of periodontal ligament stem cells
The nervous system is often irreversibly damaged by both genetic and acquired diseases throughout life. Nowadays it is determined that in a vast majority of neurodegenerative diseases, completion of tissue regeneration are extremely challenging. As a consequence, not only the differentiation of neuronal stem cells in certain regions of the brain to mature neurons but also the differentiation of adult stem cells with multipotent characteristics to functional and healthy nerve cells at various developmental stages have a considerable potential to be used in future therapies. On the other hand, in the tissues that are involved in neurons, the vesicular structures, which are about 100 nanometers in size, undertake the role of a messenger in the provision of intercellular communication. Moreover, studies have shown that exosomes have a significant effect on cellular mechanisms. However, there are no studies on the effect of plant-derived exosomes on the differentiation of adult stem cells into mature neurons. As a result, the differentiation of periodontal ligament stem cells to mature neurons and an investigation of the possible effects of Citrus sinensis derived exosomes (CSDE) on the model are aimed. Accordingly, the cells were differentiated for 10 days by utilizing multi-stage inductive media. At the end of the differentiation protocol, differentiation success and efficiency were analyzed by various experiments in terms of both morphological and transcriptional-translational characteristics. As a result of the experiments, it was determined that the induction protocol used successfully differentiated the periodontal ligament stem cells into mature neurons and CSDE increased the cell viability during the differentiation process. Accordingly, a new induction protocol that can be used for the differentiation of stem cells into neurons has emerged and it is observed that plant-derived exosomes have a positive role in the differentiation process for the first time. In the future, it is extremely important to use the designed differentiation protocol in the modeling of neuronal diseases and to investigate the effects of plant-derived exosomes in these diseases.
The investigation of diverse physiological and therapeutic impact of cell based products derived from human Cumulus cells
The mammalian oocyte is enveloped by particular somatic cells namely cumulus cells (CCs) that support the oocyte maturation, fertility, and viability by providing the nutrients and energy. CCs have a unique extracellular matrix rich in hyaluronic acid (HA), which is responsible for the cumulus cell expansion event having a part in ovulation, fertilization, and determination of oocyte quality. At in vitro fertilization laboratories, partial removal of CCs is required to evaluate egg maturity and to have a better visualization of the oocyte during an intracytoplasmic sperm injection procedure. In this study, discarded human cumulus tissues were used to reveal the value of HA-rich CCs. The usage of conditioned medium, recovered from the primary culture of CCs, was designed for repairing functionally distorted human nucleus pulposus cells (hNPCs) through the induction of enhanced chondrogenesis. Enlightening the impact of cumulus conditioned medium (CCM) on wound healing and angiogenesis was targeted. In line with these goals, differentiation of hNPCs into chondrocytes and osteocytes with CCM as the basal medium containing traditional differentiation agents were induced upon isolation and characterization of hCCs and hNPCs. The effects were detected by differentiation-specific cell stains and gene expression analysis. Scratch assay and tube formation assay was performed for discovering the effect of CCM on wound healing after detecting the presence of angiogenesis-related genes in hCCs. Our results showed that cumulus cell-conditioned media promoted the chondrogenesis and osteogenesis of hNPCs. Furthermore, a significant increase in the number of branches and wound closure area was detected only in groups cultured in CCM compared to the control. Remarkably, it was shown that the hCCs can be suggested as an alternative model to the widely used models in angiogenesis assays. When all these are considered, trashed cumulus cells are a very special and promising cell source that should be highlighted as a treasure.
Anti-cancer effects of boron derivatives on lung cancer cell lines
Lung cancer is the most common cancer type in the world, and it is the second leading cause of death in the world after cardiovascular diseases. The average 5-year survival rate of lung cancer, which is divided into two subtypes as small cell and non-small cell, is only 19 per cent. Although there are different treatment methods such as surgical intervention, radiotherapy, and chemotherapy, there is no effective method or drug that treats lung cancer, especially small cell type. In this study, the anticancer effect of sodium pentaborate pentahydrate, sodium perborate tetrahydrate, and boric acid against lung cancer have been studied and the effects of these substances on cancer cells metastasis, cell proliferation, cell migration, cell cycle, and cell viability have been demonstrated in detail at the molecular level. Although in vivo toxicology and efficacy experiments of sodium pentaborate pentahydrate and sodium perborate tetrahydrate are continuing, it has been observed that these substances show strong and selective anticancer effects against lung cancer via anti-proliferative, anti-migration, and cell cycle arrest effects in in vitro experiments. This study is the first study in the literature showing the anticancer effect of sodium pentaborate pentahydrate and sodium perborate tetrahydrate on lung cancer.