The MTT viability assay yields strikingly false-positive viabilities although the cells are killed by some plant extracts

The MTT assay is one of the often used cell viability/cytotoxicity assays. However, when the methanol extracts of plants are used to test their cytotoxic potential, interference may occur, resulting in false-positive viability results. Therefore, in this study, the reliability of the MTT assay was investigated in the case of plant use. The methanol extracts of three different plants (Hypericum adenotrichum, Salvia kronenburgii, and Pelargonium quercetorum) were tested in breast cancer cell lines (MCF-7 and MDA-MB-231) using the MTT assay and the results were compared to the ATP assay, which is a much more sensitive and reliable assay due to its interference-free feature. Additionally, decreased cell density was confirmed with phase-contrast microscopy and fluorescence staining (Hoechst 33342 dye). Although both of the viability/cytotoxicity assays are considered as metabolic assays, viabilities (in %) in the MTT assay were found to be strikingly higher when compared to the results with the ATP assay. Even in the case of total death, the MTT assay still produced artificial/false increases in viability. The morphology-based evaluation of viability/cytotoxicity by phase-contrast microscopy and Hoechst 33342 staining were greatly compatible with the ATP assay results. Overestimated (false) viabilities in the MTT assay suggests a serious interference between the MTT assay itself and the extracts used. Some ingredients of plants may have reducing activity (like the dehydrogenase activity of the cells) that converts the MTT compound into the colored formazan that is the principle of the assay. Therefore, the MTT assay may not be a suitable assay for some plant extracts, urging great caution when plants are use

Targeting pancreatic cancer and tumor microenvironment through eukaryotic elongation factor 2 kinase (EF2K) inhibition

Pankreatik kanser ortalama 6 aylık sağ kalım süresi ile en agresif ve ölümcül kanser türlerinden biridir. Kullanılan geleneksel tedavi yöntemleri sadece kanser hücrelerini hedeflemektedir. Ancak son yıllardaki çalışmalar ile tümör mikroçevresinin de tümör gelişimi, invazyonu, kemorezistans ve metastaz süreçlerinde aktif rol oynadığı bilinmektedir. Bu nedenle etkin bir tedavi için hem tümör hücrelerinin, hem de tümör mikroçevresinin hedeflenmesi gerekmektedir. Ökaryotik elongasyon faktörü 2 kinaz (EF2K), kanser hücrelerinde artmış aktivite gösteren ve stres koşullarında kanser hücrelerinin hayatta kalmasında rol alan bir enzimdir. EF2K pankreas kanserinde invazyon sürecinde rol almaktadır ve bu enzimin inhibisyonu kanser hücrelerinde apoptozisi tetiklemektedir. Literatürde EF2K ile tümör mikroçevresi arasındaki ilişkiyi gösteren bir çalışma bulunmamaktadır. Bu nedenle bu çalışmada tümör mikroçevresinin önemli hücre gruplarından biri olan makrofajlar ile PANC1 pankreatik kanser hücreleri arasındaki etkileşimin EF2K ekspresyonuna etkileri incelendi. Çalışmada PANC1 hücreleri ile monositik THP-1 hücreleri ve bu hücrelerden polarize edilen makrofajlar indirekt olarak kültüre edildi. Ardından PANC1 hücrelerinde EF2K ekspresyonu, hücre migrasyon ve invazyonundaki değişimler sırasıyla western blot, migrasyon ve invazyon testleri ile incelendi. PANC1 hücreleri ile monosit ve makrofajların kokültürü sonrası, tümör mikroçevresinin önemli kemokinlerinden olan monosit kemoatraktan-1 proteini (MCP-1) düzeyleri ELISA testi ile ölçüldü. MCP-1'in PANC1 hücrelerinde EF2K ekspresyonu ve migrasyon yetenekleri üzerine etkileri de incelendi. MCP-1'in monosit-makrofaj dönüşümü üzerindeki etkisi western blot ile incelendi. MCP-1 ile EF2K arasındaki etkileşimi incelemek amacıyla, EF2K overeksprese eden PANC1 hücreleri kullanılarak MCP-1 düzeyi western blot ile incelendi. Ayrıca, EF2K siRNA'sı kullanılarak PANC1 hücrelerinin koloni oluşturma, migrasyon ve invazyon yeteneklerinde ve MCP-1 ekspresyonundaki değişimler incelendi. EF2K inhibisyonunun in vivo etkilerini değerlendirmek için pankreatik ortotopik tümör modeli kullanıldı ve EF2K inhibisyonu sonrası MCP-1 düzeyi western blot ile incelendi. Tümöre infiltre olan protümörijenik makrofajlar mmünohistokimyasal boyama ile gösterildi. Sonuç olarak, makrofajlar ile PANC1 hücreleri arasındaki etkileşimin EF2K ve MCP-1 ekspresyonunu arttırdığı ve hücre migrasyon ve invazyonunda artışa neden olduğu bulundu. Ayrıca EF2K ile MCP-1 arasında iki yönlü bir etkileşim olduğu ve MCP-1'in monositlerin protümörijenik makrofajlara farklılaşmasına yol açtığı bulundu. EF2K'nin in vitro susturulması MCP-1 protein düzeyinde, hücre invazyon ve migrasyonunda azalmaya yol açtı. Ayrıca, EF2K inhibisyonu sonucu in vivo olarak tümör hacmi ve proliferasyonunda ve MCP-1 protein ekspresyonunda azalma olduğu gösterildi. EF2K inhibisyonu tümöre infiltre olan protümörijenik makrofaj sayısında da azalmaya yol açtı. Sonuç olarak, EF2K inhibisyonu ile hem kanser hücreleri hem de makrofajların hedeflenmesinin pankreatik kanser tedavisi için umut verici bir yaklaşım olabileceği sonucuna varıldı.Pancreatic cancer is one of the most aggressive and deadliest cancer with 6 months average survival rate. Traditional therapies only target cancer cells. However, recent studies indicate that tumor microenvironment plays an important role during tumor progression, invasion, chemoresistance and metastasis. Therefore, both tumor cells and their microenvironment should be targeted for an effective treatment. Eukaryotic elongation factor 2 kinase (EF2K) is an enzyme which is overexpressed in cancer cells that plays a key role in cancer cell survival under stress conditions. EF2K participate in invasion of pancreatic cancer cells and inhibition of this enzyme triggers apoptosis in cancer cells. There is no study that have shown the relationship between EF2K and tumor microenvironment. Therefore, in this study it has been investigated that the interaction between macrophage (one of the most abundant cell type in tumor microenvironment) and PANC1 pancreatic cancer cells on EF2K expression. In this study, PANC1 cells were cultured with monocytic THP-1 cells and macrophages which were polarized from THP-1 cells. The changes in EF2K expression, cell migration and invasion were analyzed using western blot, migration and invasion assays, respectively. The effects of coculture of PANC1 cells with monocytes and macrophages on monocyte chemoattractant protein-1 (MCP-1) levels, which is one of the most important chemokines in tumor microenvironment were analyzed using ELISA. The role of MCP-1 on both EF2K and migration of PANC1 cells were investigated. The role of MCP-1 on monocyte-macrophage differentiation was also investigated. To determine the relationship between EF2K and MCP-1, EF2K was stably overexpressed in PANC1 cells and MCP-1 levels were measured using western blot. EF2K was silenced using siRNA and then the changes in MCP-1 expression levels, the ability of colony formation, migration and invasion of PANC1 cells were investigated. Pancreatic orthotopic tumor model was used to determine the in vivo effects of EF2K inhibition. Following EF2K inhibition, the changes in expression levels of MCP-1 was measured using western blot. The presence of tumor-infiltrated pro-tumorigenic macrophages were shown using immunohistochemical staining. As a result, it was found that the interaction between PANC1 cells and macrophages caused an increase in EF2K and MCP-1 proteins and this interaction accelarated cell migration and invasion. In addition, there was a bidirectional interaction between MCP-1 and EF2K. MCP-1 also caused differentiation of monocytes to pro-tumorigenic macrophages. In vitro silencing of EF2K decreased MCP-1 expression, cell invasion and migration. In vivo inhibition of EF2K also decreased MCP-1 expression, tumor volume and the number of tumor-infiltrated pro-tumorigenic macrophages. Consequently, targeting both tumor cells and macrohages through EF2K inhibition might be a promising strategy for pancreatic cancer treatment

Investigation of the responses of lung cancer cells to different anticancer drugs in the presence of fibroblasts grown in co-culture

Akciğer kanseri günümüzde kadın ve erkeklerde en fazla ölüme neden olan kanser türüdür. Akciğer kanseri hastalarında beş yıllık sağkalım oranı %15’ten daha azdır ve tedavisinde yeni yaklaşımlara rağmen etkin bir başarı sağlananamaktadır. Bu nedenle her geçen gün yeni tedavi yaklaşımları geliştirilmektedir. Bu yaklaşımlardan biri de, kanser hücreleri ile birlikte tümör mikro çevresini de hedeflemektedir. Tümör mikro çevresi, tümöre pozitif ya da negatif katkı sağlayan stromal hücrelerden oluşmaktadır. Bu çalışmada, tümör mikro çevresi bileşenlerinden biri olan fibroblastlar ile A549 insan akciğer kanseri hücre hattının ko-kültür yöntemi ile etkileşimi sağlanmış ve bu etkileşimin A549 hücrelerinin kemoterapi yanıtı üzerindeki etkileri incelenmiştir. Sonuç olarak, fibroblast varlığında A549 hücrelerinin kemoterapi ilaçlarına yanıtında bir değişiklik olmadığı belirlenmiştir.Lung cancer is the most common cause of cancer related death to date in both men and women. The overall five year survival rate for lung cancer is less than 15% and the outcome of patients is not satisfactory although new approaches introduced into the clinics. Thus, new treatment approaches are emerging. One of them is, targeting the tumor microenviroment besides the tumor cells. The tumor microenviroment consists of stromal cells that contributes positively or negatively to the tumor. In this study, the interaction between fibroblast, one of the major constituents of the microenviroment, and A549 human lung cancer cell line is provided by co-culture method and the effects of this interaction to the chemotherapeutic response is investigated in A549 cell line. As a result, it was determined that there is no differences in response of A549 cells to chemotherapy drugs in the presence of fibroblast

Production of Tomato Stalk Biochar and its Usage in Hydrophonic Agriculture

Tomato stalk is a waste remained after harvesting in agricultural productions. These wastes can be utilized as biochar for various purposes such as fuel, absorbent and soil improver. In this study, in order to waste utilization, biochar production from tomato stalk under different experimental conditions was carried out and effect of temperature, nitrogen gas flow and heating rate on yield of biochar was investigated. Characterization of biochar was performed with FTIR, TGA and SEM analysis. The highest biochar yield was obtained at the temperature of 773 K, heating rate of 5 K/min and inert gas flowrate of 500 ml/min. The highest yielded tomato stalk biochar was used as a supporting material for the tomato seedling planted in the hydroponic system of greenhouse. The tomato stalks obtained as a waste after harvesting in greenhouse were used for biochar production and biochars were applied as supporting material in greenhouse again, so, zero waste idea for a greenhouse was carried out. It has been observed that the supporting material provided a little more growth in the tomato seedling because of water and nutrient holding capacity

Erythema elevatum diutinum coexisting with ankylosing spondylitis

A 43-year-old woman presented to our hospital with the complaint of a reddish-purple rash on the extensor sides of her forearms. She had been diagnosed with ankylosing spondylitis 7 years ago. On physical examination, reddish-purple nodules were detected on the pretibial areas of both legs and extensor sides of both hands and forearms. Neutrophil, eosinophil, lymphocyte, and mixed-type leukocyte infiltration and erythrocyte extravasation were observed in skin biopsy. Erythema elevatum diutinum (EED) was diagnosed. For treatment, sulphasalazine, colchicine, and diclofenac were started. After 3 months of treatment, the lesions were healed. To the best of our knowledge, this is the first report of EED coexisting with ankylosing spondylitis

Erythema elevatum diutinum coexisting with ankylosing spondylitis

A 43-year-old woman presented to our hospital with the complaint of a reddish-purple rash on the extensor sides of her forearms. She had been diagnosed with ankylosing spondylitis 7 years ago. On physical examination, reddish-purple nodules were detected on the pretibial areas of both legs and extensor sides of both hands and forearms. Neutrophil, eosinophil, lymphocyte, and mixed-type leukocyte infiltration and erythrocyte extravasation were observed in skin biopsy. Erythema elevatum diutinum (EED) was diagnosed. For treatment, sulphasalazine, colchicine, and diclofenac were started. After 3 months of treatment, the lesions were healed. To the best of our knowledge, this is the first report of EED coexisting with ankylosing spondylitisA 43-year-old woman presented to our hospital with the complaint of a reddish-purple rash on the extensor sides of her forearms. She had been diagnosed with ankylosing spondylitis 7 years ago. On physical examination, reddish-purple nodules were detected on the pretibial areas of both legs and extensor sides of both hands and forearms. Neutrophil, eosinophil, lymphocyte, and mixed-type leukocyte infiltration and erythrocyte extravasation were observed in skin biopsy. Erythema elevatum diutinum (EED) was diagnosed. For treatment, sulphasalazine, colchicine, and diclofenac were started. After 3 months of treatment, the lesions were healed. To the best of our knowledge, this is the first report of EED coexisting with ankylosing spondyliti