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Farklı tip propolis ekstrelerinin antiviral aktivitelerinin değerlendirilmesi
In this thesis, the effect of 4 different propolis extracts were investigated against Herpes Simplex Type 1 (HSV-1) and Herpes Simplex Type 2 (HSV-2) viruses. In-vitro cytotoxic effect of each extracts were determined by the Mitochondrial Dehydrogenase Enzyme Activity (MTS) method on Human Immortalized Keratinocyte (HaCaT) cell line and the cytotoxic concentration (CC50) values that killed 50% of the uninfected cells were calculated. The CC50 values were determined as 593 µg/mL, 375 µg/mL, 1723 µg/mL and 1664 µg/mL for the extracts of Propylene, Ethanol, Glycerol and Soya, respectively. Effective concentration (EC50) values that kill 50% of cells infected with HSV-1 and HSV-2 were analyzed by quantitative Real Time PCR method. The EC50 values calculated for HSV-1 were 86.64 µg/mL, 90.86 µg/mL, 768.6 µg/m L and 501 µg/mL for the extracts of Propylene, Ethanol, Glycerol, and Soya, respectively. The EC50 values calculated for HSV-2 were 92.05 µg/mL, 48.99 µg/mL, 904.1 µg/mL and 396.1 µg/mL for the extracts of Propylene, Ethanol, Glycerol and Soya, respectively. In vitro antiviral efficacy of an extract is determined by high of Selective Index values (SI:CC50/EC50). The SI values for HSV-1 were calculated as 6.84, 4.12, 2.24, 3.32 and 6.14 for the extracts of Propylene, Ethanol, Glycerol, Soya and Acyclovir, respectively. The SI values for HSV-2 were calculated as 6.44, 7.65, 1.90, 4.40 and 2.86 for the extracts of Propylene, Ethanol, Glycerol, Soya and Acyclovir, respectively. In comparison with the Acyclovir, Propylene extract showed significantly higher SI values while SI values for Ethanol, Soya and Glycerol extracts were lower than the values of Acyclovir for HSV-1. The SI values for Ethanol, Propylene and Soya extracts were significantly higher than the value of Acyclovir while no significant results were obtained for glycerol extract, in comparison with Acyclovir for HSV-2. As a conclusion it was revealed that in particular Propylene, Ethanol and Soya extracts of propolis having antiviral activity potantial against HSV-2.
Temozolomidin glioma hücrelerindeki eksozomal stres proteinleri üzerine etkileri
Brain tumors are one of the cancer types with lethal consequences worldwide. Gliomas are the most frequent brain tumors originated from glial cells of the central nervous system and most common and aggressive primary brain malignancy is called glioblastoma multiform (GBM grade IV astrocytoma). The median survival time for GBM patients is only 8-12 months. Despite the surgery, radiotherapeutic and chemotherapeutic interventions, the survival rates do not change remarkably. Thus, scientists across the world are still seeking for innovative therapies for gliomas. The presence of the blood-brain barrier (BBB) is an important factor in reducing the efficacy of chemotherapy. Temozolomide (TMZ), a DNA alkylating agent, which can penetrate the BBB due to its structure used as the most promising treatment for recurrent glioma. In recent cancer researches, treatment of brain tumors has been shaped to reduce Hsp levels. The reasons for this are to show that there is a relationship between stress proteins and brain tumor malignancy, and that those proteins are effective in the proliferation and progression of tumor cells and that cancerous cells increasingly show resistance to treatment due to the increment in the expression levels of these proteins. Stress proteins have become an important focal point in developing new therapies due to the contribution to the suppression of these proteins' expression to the treatment. There are many studies in the literature that suppress the expression of these proteins using distinct methods including targeting the TMZ agent and reducing the expression level of heat shock proteins. Recent research shows that cells can communicate through extracellular vesicles (EVs). Exosomes with spherical structures of 30-150 nm in diameter and surrounded by lipid bilayers have become the most studied group of the EV family. Exosomes have been indicated to have important roles in cancer improvement and can alter the tumor microenvironment with materials, which they can cargo (DNA, RNA, and proteins-some Hsps). Therefore, it is thought that cancer-associated exosomes and exosome surface HSPs can be a therapeutic target. The main objective of this study was to evaluate the effects of TMZ on the expression of heat shock proteins in exosomes released from U87MG cells. At the beginning of the study, the effect of TMZ on cell viability was analyzed. According to the cytotoxicity analyzes, the agent was treated to the cells at concentrations that did not show any toxic or proliferative effect on the cells (100% cell viability) or that affected cell viability by less than 20% and 50% cell viability. After the exosome isolation was performed, the effect of TMZ application on both exosomal and cell-derived soluble protein profiles was demonstrated by 1-dimensional electrophoresis technique. The proteins were separated on the gels then transferred to the membrane by Western blotting technique and incubated with specific antibodies for target proteins. The most common exosomal marker proteins demonstrated the yield of the isolation by Western Blot and Scanning Electron Microscopy (SEM) images. Cell migration assay was carried out to explain the role of exosomes in metastasis. According to the results of this study, it has been seen that TMZ at various concentrations in dose dependent manner treated to glioma cells increased the expression level of intracellular Hsp70, while the expression level of Hsp70 that is secreted by extracellular-exosomal pathway was decreased. It was determined that TMZ concentration was 750 μM, which the most increased Hsp70 expression in U87MG glioma cells and decreased expression of exosomal Hsp70. The results could be considered a promising approach in slowing cell migration and consequently slowing angiogenesis by decreasing the amount of extracellular Hsp70 by TMZ treatment. These results indicate that the chemotherapy agent was administered to cells, perhaps becomes an effective therapeutic option for the brain tumors treatments. The data from this study are promising to the development of innovative therapies for the treatment of aggressive brain tumors with high fatality rates.
Kemoterapi uygulamalarında kullanılabilir nitelikte nanoboyutlu ilaç taşıyıcı sistemlerin geliştirilmesi ve meme kanseri tedavisinde kullanabilirliğinin ın vitro olarak araştırılması
Meme kanseri tedavisinde kullanılan geleneksel yöntemlerden kemoterapi ve radyoterapi direkt veya dolaylı olarak tümör bölgesini spesifik olarak hedef alan stratejiler olmadığı için sağlıklı doku ve hücreler üzerinde meydana getirdikleri ağır yan etkiler kanser kaynaklı ölümlerin ardında yatan ana sebebi oluşturmaktadır. Son yıllarda, bu ağır yan etkilerin üstesinden gelmek için ilaçların bir kapsül içerisinde hedeflendirilerek tümör bölgesine gönderilmesi yeni ve umut verici bir yaklaşım olarak ilgi çekmiştir. Meme kanseri tedavisinde kullanılan ve kemoterapötik bir ajan olan doksorubisini enkapsüle edecek polimerik ilaç taşıyıcı sistemler bu tez kapsamında geliştirilmiştir. Burada, polyester grubu olan poliadipatların, pPAd, pBAd ve pEAd, mPEG-550 kullanılarak PEGilenmesi sonucu sentezlenen kopolimerlerden metot optimizasyonu gerçekleştirilerek, çift emülsiyon çözücü buharlaştırma yöntemi ile üç farklı yapıda ilaç taşıyıcı sistem, PANPs, BANPs ve EANPs, ilk kez bu tez kapsamında üretilmiştir. Optimize edilmiş metot ile üretilen bu nanotaşıyıcılara DOX yüklenmiş ve enkapsülasyon verimlilikleri sırasıyla %57, %58 ve %44.5 olarak hesaplanmıştır. Serbest formdaki DOX, ilaç yüklü olmayan nanotaşıyıcılar ve ilaç yüklü nanotaşıyıcıların in vitro sitotoksisite analizleri meme kanseri hücre hatlarında (MCF-7 ve MDA-MB-231'de) gerçekleştirilmiştir. Sitotoksite analiz sonuçları, üretilen nanopartiküllerin serbest formdaki DOX'a kıyasla düşük toksisite sergilediklerini ve ilaç taşıyıcı sistem olarak kullanılabileceklerini ortaya koymuştur. Fiziksel, kimyasal ve hücre kültürü verileri değerlendirildiğinde, tez kapsamında üretilen PANPs, BANPs ve EANPs nanotaşıyıcıların, meme kanseri tedavisinde kullanılabilecek yeni ilaç taşıyıcı sistemler olabilme potansiyeline sahip oldukları belirlenmiştir.
Çeşitli ekstraksiyon ve fermentasyon işlemlerinin bal, propolis ve polen örneklerinin antioksidan kapasitesi üzerine etkileri
Various honeybee products attract great attention of food, pharmaceutical and cosmetics industries due to their nutritive properties and healthful effects such as antioxidant, antimicrobial, anticancer etc. activities. The aim of current study is to determine the effects of different extraction processes utilizing different solvents and ambient temperatures onto the antioxidant capacity of honey, propolis and bee pollen samples, and to evaluate whether fermentation process, which is known to enhance beneficial effects of some food products by increasing and/or transforming the active constituents both in quality and quantity, changes the antioxidant capacity of the most active sample. Oak honey sample, two (autumn and spring) propolis samples and pollen sample were obtained from a local beekeeper in Kırklareli province (Turkey). The samples were extracted individually with different solvents [100% (v/v) methanol, 70% (v/v) ethanol and distilled water] at three different temperature conditions (room temperature for 24 hour, 45oC for 24 hour and 45oC for 24 hour + boiling for 2 hour). DPPH, Folin-Ciocalteu and aluminum chloride methods were performed to assess the radical scavenging activity, total phenolic content (TPC) and total flavonoid content (TFC) of the samples, respectively. Since the highest radical scavenging activity was detected in the extract of autumn propolis obtained by the extraction method utilizing ethanol as a solvent and 45oC for 24 hour + boiling for 2 hour as temperature condition, this sample was fermented using Lactobacillus brevis, and antioxidant capacity of fermented propolis was measured. The results obtained for the same type of product as well as for different groups of products were compared and discussed. Among honey samples, the highest TPC was found in the aqueous extract prepared by boiling, as 0.90 ± 0.09 mg gallic acid equivalent (GAE)/g dry weight of extract (DWE) while the highest TFC was detected in methanol extract prepared in room temperature as 0.055 ± 0.002 mg quercetin equivalent (QUE)/g of DWE. This extract also showed the highest radical scavenging activity with the lowest IC50 (157.0 ± 9.8 mg/mL). Among propolis samples, the best TPC, TFC and IC50 values were found in the extract obtained by extraction of autumn propolis with ethanol at 45 oC for 24 hour + boiling for 2 hour, as 340.4 ± 34.5 mg GAE/g DWE, 103.5 ± 5.2 mg QUE/g DWE and 0.011 ± 0.001 mg/mL, respectively. In fact, this extract was also the most active one among all samples of three honeybee products, and hence the fermented one in this study. Among pollen samples, the best TPC, TFC and IC50 values were found in ethanol extract prepared at room temperature, as 22.8 ± 1.2 mg GAE/g DWE, 4.68 ± 0.30 mg QUE/g DWE and 5.8 ± 0.8 mg/mL, respectively. Fermentation of propolis was acheived in 6 days, and TPC, TFC and antioxidant capacity tests were carried out in various samples of second, fourth and sixth days of fermentation, before and after centrifugation. The best values for TPC, TFC and IC50 were determined in unfermented (no bacteria were added in assay conditions) pasteurized propolis sample, quite close each other at the second and sixth days, as a mean 268.15 mg GAE/g DWE, 23.9 mg QUE/g DWE, 0.047 mg/mL, respectively. The highest TPC, TFC and the lowest IC50 values in fermented propolis after centrifugation were found for the second day of fermentation as 46.7 ± 1.1 mg GAE/g DWE, 9.65 ± 1.5 mg QUE/g DWE and 0.220 ± 0.005 mg/mL, respectively. The data related to fermentation indicated that antioxidant capacity and principal active phenolic substances of propolis significantly decreased during the fermentation process day by day. Overall findings reveal that solvent type and temperature in extraction as well as fermentation may affect the antoxidant capacity and the chemical composition of honeybee products. Besides, particularly autumn propolis seems to be one of the the best bee products as a natural antioxidant, or as a source of new antioxidants, since its decoction in ethanol exhibits an effective radical scavenging activity because of its high phenolic and flavonoid content. This study is expected to be a guide for further studies targeting to exhibit or produce new honeybee products and related substances beneficial for human health and welfare, and to contribute to the attempts for increasing the industrial value of these products.
Bor türevlerinin meme kanseri hücrelerinin çoğalması ve tümöre özgü T hücreleri üzerindeki etkilerinin in vitro olarak farklı mekanizmalarla incelenmesi
Breast cancer is the most commonly identified cancer in women throughout the world. Despite the initial clinical response obtained with the widely used conventional chemotherapy, an improved prognosis for breast cancer individuals has been missing in the clinic because of the high toxicity to normal cells, induction of drug resistance, and the potential immunosuppressive effects of these agents. Therefore, our objective was to examine the potential anti-proliferative ability of some boron derivatives (SPP) and (SPT), which showed a promising effect on some types of cancers in the literature, on breast cancer cell lines as well as immuno-oncological side effects on tumor-specific T cell activity. These findings suggest that both SPP and SPT suppressed proliferation and induced apoptosis in MCF-7 and MDA-MB231 cancer cell lines through downregulation of the monopolar spindle-one-binder (MOB1) protein. Contrarily, these molecules increased the expression of CD274 protein through their effect on the phosphorylation level of Yes-associated protein. In addition, they reduced the concentrations of pro-inflammatory cytokines such as IFN-γ and cytolytic effector cytokines such as sFasL, perforin, granzyme A, Granzyme B, and granulysin and raised the level of CD279 surface protein in activated T cells. In conclusion, SPP, SPT, and their combination could have growth inhibitory (antiproliferative) effects and could be a therapeutic option for breast cancer. However, their stimulatory effects on the PD-1/PD-L1 signaling pathway and their effects on cytokines could ultimately account for the observed repression of the charging of effector T cells that have been particularly activated against breast cancer cells. This study paved the way for a future line of research to investigate the anti-carcinogenic effect of the boron derivatives in vivo and the synergistic effect of these boron derivatives with an optimal dose and modified T cells activated against breast cancer cells.
The effect of radısh (raphanus satıvus) derıved exosomes on osteoarthrıtıs
Osteoarthritis, characterized by articular cartilage loss, is a well-established cause of impairment. Given the underlying pathology of OA, it is clear that injured cartilage has a low ability for regeneration. This means the proposed action is essential. Deterioration of articular cartilage, alterations in subchondral bone, and synovitis are hallmarks of this complex illness. Current OA treatments focus mostly on alleviating symptoms like pain and inflammation rather than stopping the disease itself from progressing. There is a significant knowledge gap when it comes to the mechanism that contribute to the OA development. Articular cartilage's extracellular matrix homeostasis is kept in check by a delicate balancing act between anabolic and catabolic pathways; abnormalities in this equilibrium are linked to OA. Chondrocytes are the special cells that make up cartilage in joints. Collagens, proteoglycans, and non-collagen proteins are all synthesized by these cells to form the extracellular matrix. Chondrocyte responses to molecular cues vary between areas and stages of illness, suggesting that mechanical stress and biochemical variables play major roles in disease development. In this regard, the utilization of plant-based products in the treatment of medical disorders is gradually becoming a preferred option due to the fewer negative effects associated with these items. In this respect, objective of this study to examine potential effects of Raphanus sativus derived exosomes on osteoarthritis treatment by defining proliferative, anti-inflammation potential and upregulating cartilage specific activities on protein and gene expression levels for osteoarthritis. Our results demonstrated that, Raphanus sativus derived exosomes has anti-inflammation effect on OA while decreasing expression of IL-1β and IL-6 levels and inducing TGF-β. Also, these exosomes has proliferative effect on OA chondryocyte cells while promoting main extracellular matrix components. Based on these findings, Raphanus sativus derived exosomes appear as a potential therapeutic for osteoarthritis treatment.