Decreased circulating microRNA-21 and microRNA-143 are associated to pulmonary hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by maladaptation of pulmonary vasculature which is leading to right ventricular hypertrophy and heart failure. miRNAs play a crucial role in the regulation of many diseases such as viral infection, cancer, cardiovascular diseases, and pulmonary hypertension (PH). In this study, we aimed to investigate the expression pattern of eight human plasma miRNAs (hsa-miR-21-3p, hsa-miR-143- 3p, hsa-miR-138-5p, hsa-miR-145-3p, hsa-miR-190a, hsa-miR-204-3p, hsamiR-206, hsa-miR-210-3p) in mild-to-severe PH patients and healthy controls. METHODS: : miRNAs were extracted from the peripheral plasma of the PH patients (n: 44) and healthy individuals (n: 30) by using the miRNA Isolation Kit. cDNA was synthesized using All in-One First strand cDNA Synthesis Kit. Expression of the human plasma hsa-miR- 21-3p, hsa-miR-143-3p, hsa-miR-138-5p, hsa-miR-145-3p, hsa-miR-190a, hsa-miR-204- 3p, hsa-miR-206, hsa-miR210-3p, and miRNAs were analyzed by qRT-PCR. RESULTS: According to our results, in PH patients hsa-miR-21-3p and hsa-miR-143-3p expression levels were decreased by 4.7 and 2.3 times, respectively. No significant changes were detected in hsa-miR-138-5p, hsa-miR-145-3p, hsa-miR-190a, hsa-miR-204-3p, hsamiR-206, and hsa-miR-210-3p expression levels between PH and control groups. In addition, considering the severity of the disease, it was observed that the decrease in miR-138, miR-143, miR-145, miR-190, mir-204, mir-206 and miR-208 expressions was significant in patients with severe PH. DISCUSSION: : In the early diagnosis of PAH, hsa-miR-21-3p and especially hsa-miR-143-3p in peripheral plasma can be considered as potential biomarkers.

Molecular dynamic simulation and functional analysis of pathogenic PTEN mutations in glioblastoma.

PTEN, a dual-phosphatase and scaffold protein, is one of the most commonly mutated tumour suppressor gene across various cancer types in human. The aim of this study therefore was to investigate the stability, structural and functional effects, and pathogenicity of 12 missense PTEN mutations (R15S, E18G, G36R, N49I, Y68H, I101T, C105F, D109N, V133I, C136Y, R173C and N276S) found by next generation sequencing of the PTEN gene in tissue samples obtained from glioblastoma patients. Computational tools and molecular dynamic simulation programs were used to identify the deleterious effects of these mutations. Furthermore, PTEN mRNA and protein expression levels were evaluated by qRT-PCR, Western Blot, and immunohistochemistry staining methods. Various computational tools predicted strong deleterious effects for the G36R, C105F, C136Y and N276S mutations. Molecular dynamic simulation revealed a significant decrease in protein stability for the Y68H and N276S mutations when compared with the wild type protein; whereas, C105F, D109N, V133I and R173C showed partial stability reduction. Significant residual fluctuations were observed in the R15S, N49I and C136Y mutations and radius of gyration graphs revealed the most compact structure for D109N and least for C136Y. In summary, our study is the first one to show the presence of PTEN E18G, N49I, D109N and N276S mutations in glioblastoma patients; where, D109N is neutral and N276S is a damaging and disease-associated mutation.Communicated by Ramaswamy H. Sarma.

The effects of PIKfyve inhibitor YM201636 on claudins and malignancy potential of nonsmall cell cancer cells.

PIKfyve is an evolutionarily conserved lipid and protein kinase enzyme that has pleiotropic cellular functions. The aim of the present study was to investigate the effects of phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) inhibitor, YM201636, on nonsmall cell lung cancer (NSCLC) cells growth, tumorigenicity, and claudin (CLDN) expressions. Three NSCLC cell lines (Calu-1, H1299 and HCC827) were used to compare the effects of YM201636. Cytotoxic effects of YM201636 were analysed using XTT assay. Malignancy potential of cells assesses with wound healing and soft agar colony-forming assays. mRNA and protein expressions of claudins were analysed by qRT-PCR and immunofluorescence staining. Our results revealed that YM201636 inhibited the proliferation and malignancy potential of Calu-1, H1299, and HCC827 cells in a dose-dependent manner. After YM201636 treatment CLDN1, -3 and -5 expressions increased significantly in HCC827 cells. CLDN3 and -5 expressions also significantly increased in Calu1 cell line. YM201636 treatment significantly reduced the CLDN1 and increased the CLDN5 expression in H1299 cells. Immunofluorescence staining of CLDN1, -3 and -5 proteins showed a significant increase after YM201636 treatment. Besides, YM201636 induced EGFR mRNA expression in all NSCLC cell lines. Our results have shown that YM201636 inhibits tumorigenicity of NSCLC cells. Furthermore, estimated glomerular filtration rate (EGFR) pathway is important signalling involved in the regulation of claudins. Understanding the mechanisms of PIKfyve inhibitors may improve cancer treatment particularly for EGFR overactivated NSCLC.

Genetic and epigenetic factors associated with increased severity of Covid-19.

Since December 2019, a new form of severe acute respiratory syndrome (SARS) from a novel strain of coronavirus (SARS coronavirus 2 [SARS-CoV-2]) has been spreading worldwide. The disease caused by SARS-CoV-2 was named Covid-19 and declared as a pandemic by the World Health Organization in March 2020. Clinical symptoms of Covid-19 range from common cold to more severe disease defined as pneumonia, hypoxia, and severe respiratory distress. In the next stage, disease can become more critical with respiratory failure, sepsis, septic shock, and/or multiorgan failure. Outcomes of Covid-19 indicate large gaps between the male-female and the young-elder groups. Several theories have been proposed to explain variations, such as gender, age, comorbidity, and genetic factors. It is likely that mixture of genetic and nongenetic factors interplays between virus and host genetics and determines the severity of disease outcome. In this review, we aimed to summarize current literature in terms of potential host genetic and epigenetic factors that associated with increased severity of Covid-19. Several studies indicated that the genetic variants of the SARS-CoV-2 entry mechanism-related (angiotensin-converting enzymes, transmembrane serine protease-2, furin) and host innate immune response-related genes (interferons [IFNs], interleukins, toll-like receptors), and human leukocyte antigen, ABO, 3p21.31, and 9q34.2 loci are critical host determinants related to Covid-19 severity. Epigenetic mechanisms also affect Covid-19 outcomes by regulating IFN signaling, angiotensin-converting enzyme-2, and immunity-related genes that particularly escape from X chromosome inactivation. Enhanced understanding of host genetic and epigenetic factors and viral interactions of SARS-CoV-2 is critical for improved prognostic tools and innovative therapeutics.

Effect of SIRT1 activators and inhibitors on CD44+/CD133+­enriched non­small cell lung cancer cells.

Lung cancer is one of the most commonly diagnosed cancers and it is associated with high rates of morbidity and mortality. Metastasis and relapse of the tumor depend on the survival and proliferation of lung cancer stem cells (LCSCs). The ability to identify CSCs may prevent recurrence and lead to more effective treatments. Sirtuins are a group of deacetylases that include seven variants (SIRT1­7), with sirtuin 1 (SIRT1) being the most intensively investigated. Evidence suggests that SIRT1 is both a tumor­suppressor gene and an oncogene. SIRT1 can deacetylate the tumor­suppressor protein p53 to decrease its activity. SIRT1 activators increase the deacetylation of p53, whereas SIRT1 inhibitors can stimulate p53 by inhibiting deacetylation. In the present study, CD44+ and CD133+­enriched A549 (non­small cell lung cancer) cells collected using the CD44 and CD133 CSC surface markers by fluorescence­activated cell sorting method were treated with SIRT1 inhibitors (tenovin­6 and sirtinol) and SIRT1 activators (resveratrol and SRT1720), and their effects on apoptosis, as well as the mRNA and protein expression of SIRT1 and p53 were investigated. Of these agents, it was found that resveratrol increased p53 expression by 4.1­fold, decreased SIRT1 expression by 0.2­fold, and it was the most potent inducer of apoptosis.

Is there a causal link between PTEN deficient tumors and immunosuppressive tumor microenvironment?

The PTEN tumor suppressor is the second most commonly inactivated gene across cancer types. While it's role in PI3K/AKT and DNA damage pathways are clear, increasing evidences suggest that PTEN may also promote anti-tumor immunity. PTEN-deficient tumors are characterized by (i) reduced levels of cytotoxic T cells, helper T cells and NK cells, (ii) elevated pro-oncogenic inflammatory cytokines like CCL2 and (iii) increased levels of immunosuppressive cells such as MDSCs and Tregs. An intriguing possibility is that link between PTEN and anti-tumor immunity is mediated by the interferon signaling pathway. In this review, we summarize the evidences for the mechanistic link between PTEN deficiency and immunosuppressive tumor microenvironment and the interferon signaling pathway. We further discuss how the link between these pathways can be exploited for development of personalized immunotherapy for patients with PTEN deficient tumors.

SET8 localization to chromatin flanking DNA damage is dependent on RNF168 ubiquitin ligase.

The DNA damage response (DDR) associated post-translational modifications recruit chromatin remodelers, signaling proteins such as 53BP1 and repair factors to chromatin flanking DNA double strand breaks (DSBs) to promote its repair. Although localization of both RNF168 ubiquitin ligase and SET8 methyltransferase at DSBs is essential for 53BP1's recruitment to DSBs, it is unclear if they do so via the same pathways. Here we report that RNF168 mediates SET8's recruitment to DSBs. Depletion of cellular pool of ubiquitin through proteasome inhibition abolished RNF168 and SET8's localization to DNA damage. Knockdown of RNF8 or RNF168 abolished SET8's recruitment to DNA damage. Moreover, RNF168 and SET8 form stable complexes in vivo. Based on these results we propose a model in which SET8, which despite being a pan-chromatin binding protein, can accumulate several folds at chromatin flanking DSBs through tethering to other proteins that specifically localize to chromatin regions with specific modifications.

Effects of New Generation All-Ceramic and Provisional Materials on Fibroblast Cells.

PURPOSE: The purpose of this in vitro study was to evaluate the cytotoxic and apoptotic effects of seven new-generation all-ceramic materials for CAD/CAM (Lava Ultimate [LU], VITA Mark II [VM], InCoris TZI [IC], IPS e.max CAD [EM], VITA Suprinity [VS], Cerasmart [CS], IPS Empress CAD [EC]) and six provisional materials (Protemp 4 [PT], Telio CAD [TC], CAD-Temp [CT], Telio Lab [TL], Temdent Classic [TD], Telio CS C&B [TS]) on L929 mouse fibroblast cells. MATERIALS AND METHODS: 24 disc-shaped specimens (∅ = 5 mm, h = 2 mm) were prepared from each test material. Medium extracts were collected at the 1st, 3rd, and 7th days for each group and tested using the L929 cell line. Cytotoxicity was evaluated using XTT assay, and apoptosis was determined by Annexin-V/PI staining. Data were analyzed using one-way ANOVA, Tukey's multiple comparison tests at a significance level of p < 0.05. RESULTS: The cell viability results among all-ceramic material groups after the 1st and 7th days of incubation periods showed statistically significant differences (p < 0.05). There were significant differences within the ceramic groups in different incubation periods regarding apoptosis rate (p < 0.05). Throughout the entire test period, LU and VM from the CAD/CAM all-ceramic materials and PT and TC from the provisional restoration materials showed cell viability higher than 90%. EC and TD showed the lowest cell viability and highest apoptosis rates in their own groups. For the provisional materials, there were significant differences in cell viability and apoptosis rate in all the incubation periods for each material (p < 0.05). CONCLUSIONS: Although some new-generation CAD/CAM and provisional restoration materials display slight cytotoxicity values, the results are still within the reliable range, and they can safely be used in clinical conditions.

Case-control study on PCSK9 R496W (rs374603772) and D374Y (rs137852912) mutations in Turkish patients with primary dyslipidemia.

OBJECTIVE: The aim of this study was to investigate the relationships between F216L (rs28942112), R496W (rs374603772), S127R (rs28942111), and D374Y (rs137852912) PCSK9 gain-of-function (GOF) mutations and primary dyslipidemia and serum lipid levels in patients with primary dyslipidemia. METHODS: In this case-control study, DNA was isolated from blood samples collected from patients diagnosed with primary dyslipidemia in cardiology outpatient clinic of Ege University (n=200) and healthy individuals (n=201). F216L, R496W, S127R, and D374Y GOF mutations in the PCSK9 gene were evaluated and genotyped according to the results of melting curve analysis performed in a real-time polymerase chain reaction (PCR) 480 instrument using specific primers for each mutation. RESULTS: There were statistically significant differences between the patient and individuals in control groups in the R496W and D374Y mutations (x2=10.742 p=0.005; x2=6.078 p=0.048, respectively). In addition, triglyceride levels in patients with primary dyslipidemia heterozygous for R496W and D374Y mutations were 12.8-fold (p=0.015) and 3.4-fold (p=0.03) higher than that in mutant and wild-type genotype, respectively. Additionally, in the entire study group (n=401), PCSK9 R496W and D374Y mutation carriers had increased total cholesterol (p=0.021), triglycerides (p=0.0001), HDL cholesterol (p=0.028), and low-density lipoproteins (LDL) cholesterol (p=0.028) levels. However, F216L (rs28942112) and S127R (rs28942111) mutations were not detected in patients with primary dyslipidemia and healthy controls. CONCLUSION: We conclude that the PCSK9 R496W (rs374603772) and D374Y (rs137852912) GOF mutations may be significant risk factors in the development of primary dyslipidemia and may have significant impact on lipid parameters in general population.

Matrine induced G0/G1 arrest and apoptosis in human acute T-cell lymphoblastic leukemia (T-ALL) cells.

Matrine, a natural product extracted from the root of Sophora flavescens, is a promising alternative drug in different types of cancer. Here, we aimed to investigate the therapeutic effects and underlying molecular mechanisms of matrine on human acute lymphoblastic leukemia (ALL) cell line, CCRF-CEM. Cell viability and IC50 values were determined by WST-1 cell cytotoxicity assay. Cell cycle distribution and apoptosis rates were analyzed by flow cytometry. Expression patterns of 44 selected miRNAs and 44 RNAs were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using the Applied Biosystems 7500 Fast Real-Time PCR System. Matrine inhibited cell viability and induced apoptosis of CCRF-CEM cells in a dose-dependent manner. Cell cycle analysis demonstrated that matrine-treated CCRF-CEM cells significantly accumulated in the G0/G1 phase compared with the untreated control cells. hsa-miR-376b-3p (-37.09 fold, p = 0.008) and hsa-miR-106b-3p (-16.67 fold, p = 0.028) expressions were decreased, whereas IL6 (95.47 fold, p = 0.000011) and CDKN1A (140.03 fold, p = 0.000159) expressions were increased after matrine treatment. Our results suggest that the downregulation of hsa-miR-106b-3p leads to the upregulation of target p21 gene, CDKN1A, and plays a critical role in the cell cycle progression by arresting matrine-treated cells in the G0/G1 phase.

Preparation and characterization of non-viral gene delivery systems with pEGFP-C1 Plasmid DNA

ABSTRACT In recent years, non-viral delivery systems for plasmid DNA have become particularly important. They can overcome the disadvantages of viral systems such as insertional mutagenesis and unpredicted immunogenicity. Some additional advantages of non-viral gene delivery systems are; good stability, low cost, targetability, delivery of a high amount of genetic materials. The aim of the study was to develop novel non-viral nanosystems suitable for gene delivery. Two formulations were developed for this purpose: water-in-oil microemulsion (ME) and solid lipid nanoparticles (SLN). The microemulsion was composed of Peceol, Tween 80, Plurol oleique, ethanol and water. The SLN was consisting of Precirol, Esterquat-1 (EQ1), Tween 80, Lecithin, ethanol and water. Characterization studies were carried out by measuring particle size, zeta potential, viscosity and pH. TEM imaging was performed on SLN formulations. Protection against DNase I degradation was examined. Cytotoxicity and transfection efficacy of selected formulations were tested on L929 mouse fibroblast cells. Particle sizes of complexes were below 100 nm and with high positive zeta potential. TEM images revealed that SLNs are spherical. The SLN:DNA complexes have low toxicity and good transfection ability. All results showed that the developed SLN formulations can be considered as suitable non-viral gene delivery systems.

Cytotoxicity of Dental Implants: The Effects of Ultrastructural Elements.

PURPOSE: In this in vitro study, the purpose was to assess the cytotoxicity profiles of seven commercial dental implant materials by using cell culture methods on an osteoblastic cell line. MATERIALS AND METHODS: The microstructure of seven commercial dental implants (each given a letter code) was investigated via scanning electron microscopy and energy-dispersive x-ray analysis. Medium extracts were collected on the first and fifth days for each group and tested using MC3T3-E1 cell line. Cytotoxicity was evaluated with Xcelligance System and XTT reagent, and apoptosis was determined by Annexin-V staining. One-way analysis of variance (ANOVA) and Tukey's multiple range tests were used for statistical analyses. In all tests, P was set as .05. RESULTS: ANOVA results disclosed that Ti (P = .001), Na (P = .001), Ca (P = .019), Al (P = .024), and P (P = .020) amounts were significantly different between test materials. Cytotoxicity and apoptosis analyses revealed that implant materials (C) and (E) were the materials with the lowest cell vitality and the highest apoptosis rates among the test materials. Phosphorus was the only element that presented the highest amount in C and E (14.23% and 12.29%, respectively) compared with the other implant materials tested. (F) and (G) had favorable results for all experiments. CONCLUSION: The results suggest that pure dental implant materials with a lower number of additional elements may possess fewer cytotoxic effects than the other implant materials tested in this study.

Current updates on microRNAs as regulators of chemoresistance.

Resistance to chemotherapeutics including platinum agents, nucleoside analogues, topoisomerase and microtubule inhibitors is the most important problem in the treatment of many cancer. Several resistance mechanisms has been described for each group and researchers have been trying to develop new approaches to overcome chemoresistance. miRNAs are the regulators of gene transcription at the post-transcriptional level. Dysregulated miRNAs have been reported in cancer tumorigenesis, diagnosis and prognosis and they are promising new therapeutic approaches to improve diagnosis and predictions of outcomes and response to chemotherapy. In this article, we summarized the results from 2015 January to 2017 June which explore the roles of miRNAs in the development of the chemotherapy resistance.

Preparation and characterization of lipid nanoparticle/pDNA complexes for STAT3 downregulation and overcoming chemotherapy resistance in lung cancer cells.

Developments in the field of molecular oncology have revealed that resistance to chemotherapeutics is acqured through several mechanisms including overexpression of common oncogenic proteins. Signal Transducer and Activator of Transcription 3 (STAT3) is one of these oncogenes that is overexpressed in many cancer types. RNA interference (RNAi) is proven powerful tool for downregulating STAT3, allowing re-sensitization of resistant cancer cells. However, delivery of RNA interference-mediating molecules for STAT3 downregulation in lung cancer cells is limited to a small number of studies most of which employ commercially available transfection kits. The aim of this study was to develop and evaluate cationic solid lipid nanoparticles for delivery of RNAi-mediating plasmid DNA in order to down regulate STAT3 in cisplatin resistant lung cancer cells. We focused on obtaining cSLN:plasmid DNA complexes with size below or equal to 100nm, and a positive zeta potential. Two successful candidate cSLN:plasmid DNA complexes (K2 and K3) were selected for in vitro tests and cell culture studies. These formulations have particle sizes of 98 and 93nm, and zeta potential values of 10.5 and 8.9mV, respectively. Plasmid DNA in these complexes was protected against DNaseI and serum-mediated degradation. Substantial part of DNA retained its supercoiled and circular conformation. TEM images showed nearly spherical complex structure. Both formulations reduced STAT3 expression by approx. 5-fold in cisplatin resistant Calu1 cell line and increased the sensitivity of cells to cisplatin.

Polymorphisms of lipid metabolism enzyme-coding genes in patients with diabetic dyslipidemia.

OBJECTIVE: The polymorphisms/mutations of genes encoding proteins and enzymes involved in lipoprotein metabolism play important roles in the development of diabetic dyslipidemia. The aim of our study was to investigate the effects of LPL (rs320), LIPC (rs2070895), SCARB1 (rs5888), LCAT (rs2292318), CETP (rs708272), ADIPOQ (rs1501299), RETN (rs3745367), PON1 (rs662), and MNSOD (rs4880) gene polymorphisms on lipid metabolism and diabetic dyslipidemia. METHODS: This case-control study included 217 patients with diabetic dyslipidemia and 212 healthy age- and gender-matched individuals. Genomic DNA isolation was performed from blood samples, and genotype analysis was performed using melting curve analysis on a LightCycler® 480 Instrument. The chi-square test was used to compare genotype distribution and allele frequencies between the groups. RESULTS: Significant associations were observed between LPL (rs320) (p<0.001), LIPC (rs2070895) (p<0.001), SCARB1 (rs5888) (p<0.001), LCAT (rs2292318) (p<0.001), CETP (rs708272) (p<0.001), ADIPOQ (rs1501299) (p=0.01), RETN (rs3745367) (p<0.001), and MNSOD (rs4880) (p<0.001) polymorphisms and diabetic dyslipidemia. However, no association was observed between PON1 (rs662) polymorphisms and diabetic dyslipidemia (p=0.611). CONCLUSION: LPL (rs320), LIPC (rs2070895), SCARB1 (rs5888), LCAT (rs2292318), CETP (rs708272), ADIPOQ (rs1501299), RETN (rs3745367), and MNSOD (rs4880) polymorphisms play an important role in basic molecular metabolism in diabetic dyslipidemia. Therefore, these polymorphisms may be used as a predictive marker for diabetic dyslipidemia in high-risk patients.

Effects of flavopiridol on critical regulation pathways of CD133high/CD44high lung cancer stem cells.

BACKGROUND: Flavopiridol a semisynthetic flavone that inhibits cyclin-dependent kinases (CDKs) and has growth-inhibitory activity and induces a blockade of cell-cycle progression at G1-phase and apoptosis in numerous human tumor cell lines and is currently under investigation in phase II clinical trials. Cancer stem cells (CSCs) are comprised of subpopulation of cells in tumors that have been proposed to be responsible for recurrence and resistance to chemotherapy. The aim of the present study was to investigate the effects of flavopiridol in cancer stem cell cytoskeleton, cell adhesion, and epithelial to mesenchymal transition in CSCs. METHODS: The cells were treated with flavopiridol to determine the inhibitory effect. Cell viability and proliferation were determined by using the WST-1 assay. Caspase activity and immunofluorescence analyses were performed for the evaluation of apoptosis, cell cytoskeleton, and epithelial-mesenchymal transition (EMT) markers. The effects of flavopiridol on the cell cycle were also evaluated. Flow cytometric analysis was used to detect the percentages of CSCs subpopulation. We analyzed the gene expression patterns to predict cell cycle and cell cytoskeleton in CSCs by RT-PCR. RESULTS: Flavopiridol-induced cytotoxicity and apoptosis at the IC50 dose, resulting in a significant increase expression of caspases activity. Cell cycle analyses revealed that flavopiridol induces G1 phase cell cycle arrest. Flavopiridol significantly decreased the mRNA expressions of the genes that regulate the cell cytoskeleton and cell cycle components and cell motility in CSCs. CONCLUSION: Our results suggest that Flavopiridol has activity against lung CSCs and may be effective chemotherapeutic molecule for lung cancer treatment.

Comparison of cell cycle components, apoptosis and cytoskeleton-related molecules and therapeutic effects of flavopiridol and geldanamycin on the mouse fibroblast, lung cancer and embryonic stem cells.

Similarities and differences in the cell cycle components, apoptosis and cytoskeleton-related molecules among mouse skin fibroblast cells (MSFs), mouse squamous cell lung carcinomas (SqCLCs) and mouse embryonic stem cells (mESCs) are important determinants of the behaviour and differentiation capacity of these cells. To reveal apoptotic pathways and to examine the distribution and the role of cell cycle-cell skeleton comparatively would necessitate tumour biology and stem cell biology to be assessed together in terms of oncogenesis and embryogenesis. The primary objectives of this study are to investigate the effects of flavopiridol, a cell cycle inhibitor, and geldanamycin, a heat shock protein inhibitor on mouse somatic, tumour and embryonic stem cells, by specifically focusing on alterations in cytoskeletal proteins, cell polarity and motility as well as cell cycle regulators. To meet these objectives, expression of several genes, cell cycle analysis and immunofluorescence staining of intracellular cytoskeletal molecules were performed in untreated and flavopiridol- or geldanamycin-treated cell lines. Cytotoxicity assays showed that SqCLCs are more sensitive to flavopiridol than MSFs and mESCs. Keratin-9 and keratin-2 expressions increased dramatically whereas cell cycle regulatory genes decreased significantly in the flavopiridol-treated MSFs. Flavopiridol-treated SqCLCs displayed a slight increase in several cell cytoskeleton regulatory genes as well as cell cycle regulatory genes. However, gene expression profiles of mESCs were not affected after flavopiridol treatment except the Cdc2a. Cytotoxic concentrations of geldanamycin were close to each other for all cell lines. Cdkn1a was the most increased gene in the geldanamycin-treated MSFs. However, expression levels of cell cytoskeleton-associated genes were increased dramatically in the geldanamycin-treated SqCLCs. Our results revealing differences in molecular mechanisms between embryogenesis and carcinogenesis may prove crucial in developing novel therapeutics that specifically target cancer cells.

miR-15a enhances the anticancer effects of cisplatin in the resistant non-small cell lung cancer cells.

Platinum-based chemotherapies have long been used as a standard treatment in non-small cell lung cancer. However, cisplatin resistance is a major problem that restricts the use of cisplatin. Deregulated cell death mechanisms including apoptosis and autophagy could be responsible for the development of cisplatin resistance and miRNAs are the key regulators of these mechanisms. We aimed to analyse the effects of selected miRNAs in the development of cisplatin resistance and found that hsa-miR-15a-3p was one of the most significantly downregulated miRNAs conferring resistance to cisplatin in Calu1 epidermoid lung carcinoma cells. Only hsa-miR-15a-3p mimic transfection did not affect cell proliferation or cell death, though decreased cell viability was found when combined with cisplatin. We found that induced expression of hsa-miR-15a-3p via mimic transfection sensitised cisplatin-resistant cells to apoptosis and autophagy. Our results demonstrated that the apoptosis- and autophagy-inducing effects of hsa-miR-15a-3p might be due to suppression of BCL2, which exhibits a major connection with cell death mechanisms. This study provides new insights into the mechanism of cisplatin resistance due to silencing of the tumour suppressor hsa-miR-15a-3p and its possible contribution to apoptosis, autophagy and cisplatin resistance, which are the devil's triangle in determining cancer cell fate.

Extracellular Vesicles as Natural Nanosized Delivery Systems for Small-Molecule Drugs and Genetic Material: Steps towards the Future Nanomedicines.

A new platform for drug, gene and peptide-protein delivery is emerging, under the common name of "extracellular vesicles". Extracellular vesicles (EVs) are 30-1000 nm-sized cell-derived, liposome-like vesicles. Current research on EVs as nano-delivery systems for small-molecule drugs and genetic material, reveal that these tiny, biologically-derived vesicles carry a great potential to boost the efficacy of many therapeutic protocols. Several features of EVs; from efficacy to safety, from passive to active targeting ability, the opportunity to be biologically or chemically labelled, and most importantly, their eobiotic origin make them promising candidate for development of the next generation personalized nanomedicines. The aim of this article is to provide a view on the current research in which EVs are used as drug/genetic material delivery systems. Their application areas, drug loading and targeting strategies, and biodistribution properties are discussed.

An Association Study Between Gene Polymorphisms of Folic Acid Metabolism Enzymes and Biochemical and Hormonal Parameters in Acromegaly.

AIM: Folate metabolism is fundamental to several biological functions and required for cell replication, division, and survival. The mammalian folic acid cycle is highly complex and the enzymes, methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), and methionine synthase reductase (MTRR), have crucial roles in this metabolic pathway. The common polymorphisms of the MTHFR (C677T and A1298C), MTRR (A66G), and MTR (A2756G) enzymes are well documented as folate deficiency-related disorders, but their roles have not been examined in acromegalic patients. The aim of this study was to compare the genotypic distribution of these gene polymorphisms between patients with acromegaly and controls and explore whether these polymorphisms were associated with biochemical and hormonal parameters in acromegaly. We examined 91 acromegaly patients and 112 healthy subjects who were compared in terms of age and gender. Blood specimens of the subjects were collected in tubes containing ethylenediaminetetraacetic acid. Genomic DNA was isolated from peripheral blood leukocytes and genotyping of the MTHFR (C677T and A1298C) gene polymorphisms was assessed by melting temperature analyses after real-time polymerase chain reaction (PCR), whereas MTRR A66G and MTR A2756G gene polymorphism analyses were performed by PCR/restriction fragment length polymorphism from the isolated DNA of the subjects. RESULTS: MTHFR-677TT genotype frequency was significantly higher in the acromegaly group than the control group (p=0.017), and a significant increase was found in fibrinogen (p=0.032) levels in 677TT-carrying acromegaly patients. MTRR-66AA genotype was significantly higher in the control group than the acromegaly group (p=0.004). Total cholesterol (p=0.048) and C-reactive protein (p=0.046) levels decreased significantly in 66AA genotypes. Although MTR-2756AG genotype frequency was not different between the control and acromegaly groups, 2756AG genotype-carrying individuals have higher left carotid intima-media thickness levels within the patient group. CONCLUSION: Our results suggest that polymorphisms of the genes encoding the folic acid metabolism enzymes affect biochemical parameters in acromegaly and this may result in predispositions to some complications associated with folate metabolism and acromegaly.