Search

Angiogenesis is a central component of vital biological processes such as wound healing, tissue nourishment, and development. Therefore, angiogenic activities are precisely maintained with secreted factors such as angiopoietin‐1 (Ang1), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF). As an element of intracellular communication, extracellular vesicles (EVs)—particularly EVs of vascular origin—could have key functions in maintaining angiogenesis. However, the functions of EVs in the control of angiogenesis have not been fully studied. In this study, human umbilical vein endothelial cell line (HUVEC)‐derived small EVs (

Ex vivo dermal toxicology analyses are crucial for replacement of in vivo test methods and have been of interest in recent years for testing cosmetics, drugs, and chemicals. Development of an appropriate reconstructed epidermis model might overcome the limitations of monolayer culture systems. In the current study, we used the immortalized human keratinocyte cell line (HaCaT) to develop an ex vivo 3D cell culture system for keratinocyte-based toxicity analysis. Mouse embryonic fibroblast-conditioned medium and Matrigel matrix-based 3D HaCaT cell culture systems expressed skin-related genes and proteins in culture. The 3D HaCaT cultures demonstrated a skin-like phenotype and response against selected test compounds. Reliable results were obtained compared to monolayer HaCaT cells which were exposed to selected chemicals for 1 h and 24 h. Gene expression profiles of 3D HaCaT cell cultures and monolayer cultures were completely different after administration of the test compounds.Overall, our results showed that a 3D HaCaT cell culture system generated in Matrigel matrix exerted a skin epidermis-like phenotype. Consequently, 3D HaCaT cell cultures may be an acceptable test method for conducting in vitro toxicology experiments.

Through the process of cryopreservation, cells are stored at very low temperature for a long time to decrease the biological and chemical reactions in viable cells. In this process, the administration of cryoprotective agents is crucial since cryopreservation is regarded as a leading process in various research fields such as biotechnology, clinical medicine and maintenance of both animal and plant cells. Even after a long time of storage in very low temperatures, a recovery is achieved by cryo-preservative agents that act on cellular metabolism and biophysiology of cells. In the current study, the effect of boron on cryopreservation of human lung cancer cell line, A549, and murine fibroblast cell line, L929, was investigated with the help of cell viability assay, colony forming unit assay and RT-PCR analysis. 15 µg/ml boron supplemented freezing medium was found to indicate a positive effect on cell viability. Moreover, gene expression profiles of A549 and L929 cell lines have been altered. The levels of apoptosis related genes decreased while proliferation related gene levels increased significantly after repeated freeze-thaw cycles or long period of freezing. As indicated through our results, sodium pentaborate pentahydrate, as a boron source, might be a crucial cryoprotective agent for cryo-protection and bio-banking of cancer and healthy cells while keeping their viability and functionality.

Cyclooxygenase enzymes play a vital role in inflammatory pathways in the human body. Apart from their relation with inflammation, the additional involvement of COX-2 enzyme with cancer activity was recently discovered. In some cancer types the level of COX-2 enzyme is increased indicating that this enzyme could be a suitable target for cancer therapy. Based on these findings, we have synthesized some new diflunisal thiosemicarbazides and 1,2,4-triazoles and tested them against androgen-independent prostate adenocarcinoma (PC-3), colon carcinoma (HCT-116), human breast cancer (T47D), breast carcinoma (MCF7) and human embryonic kidney (HEK-293) cell lines. Specifically, the diflunisal and thiosemicarbazide functionality are combined during the synthesis of original compounds anticipating a potency enhancement. Compounds 6, 10, 15 and 16 did not show cytotoxic effects for the HEK293 cell line. Among them, compounds 15 and 16 demonstrated anticancer activity for the breast cancer cell line T47D, whereas compounds 6 and 10 which are thiosemicarbazide derivatives displayed anti-tumourigenic activity against the PC-3 cell line, consistent with the literature. However, no activity was observed for the HCT-116 cancer cell line with the tested thiosemicarbazide derivatives. Only compound 16 displayed activity against the HCT-116 cell line. Therefore, it was speculated that the diflunisal and thiosemicarbazide functionalities potentiate anticancer activity on prostate cancer and the thiosemicarbazide functionality decreases the anticancer activity of diflunisal on colon cancer cell lines. In order to gain insight into the anticancer activity and COX-2 inhibition, molecular docking studies were carried out for COX-1 and COX-2 enzymes utilizing the newly synthesized compounds 15, and 16. Both 15 and 16 showed high selectivity and affinity toward COX-2 isozyme over COX-1, which is in agreement with the experimental results.

The primary role of the parathyroid glands is to maintain calcium homeostasis through the secretion of parathyroid hormone (PTH). The limited proliferative capacity and differentiation of parathyroid cells hinder the generation of cell therapy options. In this study, parathyroid organoids are successfully generated from human‐induced pluripotent stem cells (hiPSCs). At the end of the 20 days of differentiation, the parathyroid organoids exhibited distinct parathyroid morphology. Stereomicroscope, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) analysis demonstrated the 3D arrangement of the cell layers in which intracellular structures of parathyroid cells resemble human parathyroid cellular morphology. Comprehensive molecular analyses, including RNA sequencing (RNA‐Seq) and liquid chromatography/mass spectrometry (LC‐MS/MS), confirmed the expression of key parathyroid‐related markers. Protein expression of CasR, CxCr4, Gcm2, and PTH are observed in parathyroid organoids. Parathyroid organoids secrete PTH, demonstrate active intercellular calcium signaling, and induce osteogenic differentiation via their secretome. The tissue integration potential of parathyroid organoids is determined by transplantation into parathyroidectomized rats. The organoid transplanted animals showed significant elevations in PTH‐related markers (CasR, CxCr4, Foxn1, Gcm2, and PTH). PTH secretion is detected in organoid‐transplanted animals. The findings represent a significant advancement in parathyroid organoid culture and may offer a cellular therapy for treating PTH‐related diseases, including hypoparathyroidism. [ABSTRACT FROM AUTHOR]

Angiogenesis is a central component of vital biological processes such as wound healing, tissue nourishment, and development. Therefore, angiogenic activities are precisely maintained with secreted factors such as angiopoietin‐1 (Ang1), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF). As an element of intracellular communication, extracellular vesicles (EVs)—particularly EVs of vascular origin—could have key functions in maintaining angiogenesis. However, the functions of EVs in the control of angiogenesis have not been fully studied. In this study, human umbilical vein endothelial cell line (HUVEC)‐derived small EVs (<200 nm; HU‐sEVs) were investigated as a potential pro‐angiogenic agent. Treating mesenchymal stem cells (MSCs) and mature HUVEC cells with HU‐sEVs induced their tube formation under in vitro conditions and significantly increased the expression of angiogenesis‐related genes, such as Ang1, VEGF, Flk‐1 (VEGF receptor 2), Flt‐1 (VEGF receptor 1), and vWF (von Willebrand Factor), in a dose‐dependent manner. These results indicate that HU‐sEVs take part in angiogenesis activities in physiological systems, and suggest endothelial EVs as a potential therapeutic candidate for the treatment of angiogenesis‐related diseases. [ABSTRACT FROM AUTHOR]

Chordoma as a malignant bone tumor, occurs along the axial skeleton and does not have an effective therapy. Brachyury, which is a crucial player for the formation of early embryonic notochord, is abundantly found in both sporadic and familial chordoma. During embryonic development, Brachyury expression was reported to be regulated by the Wnt pathway. The objective of the study is to investigate the role of Wnt signaling in a human chordoma cell line in terms of proliferation, survival, and invasiveness. We tried to elucidate the signaling events that regulate Chordoma cancer. In this regard, Wnt pathway was activated or inhibited using various strategies including small molecules, siRNA-based knockdown and overexpression applications. The results indicated the negative regulatory effect of Wnt signaling activity on proliferation and migration capacity of the chordoma cells. It was revealed that when GSK3β was inhibited, the Wnt pathway was activated and negatively regulated T/Bra expression. Activity of the Wnt pathway caused cell cycle arrest, reduced migration potential of the cells, and led to cell death. Therefore, the present study suggests that the Wnt pathway plays a key role in suppressing the proliferation and invasive characteristics of human chordoma cells and has a great potential as a therapeutic target in further clinical studies. [ABSTRACT FROM AUTHOR]

Mesoderm-derived cells, including bone, muscle, and mesenchymal stem/stromal cells (MSCs), constitute various parts of vertebrate body. Cell therapy with mesoderm specification in vitro may be a promising treatment for diseases affecting organs of mesodermal origin. Repair and regeneration of damaged organs with in vitro generation of mesoderm-derived tissues and MSCs hold a great potential for regenerative therapy. Therefore, understanding the signaling pathways involving mesoderm and mesoderm-derived cellular differentiation is important. Previous findings indicated the importance of Apelin receptor (Aplnr) signaling, during embryonic development, in gastrulation, cell migration, and differentiation. Nevertheless, regulatory role of Aplnr pathway in differentiation of mesoderm and mesoderm-derived MSCs remains unclear. In the current study, we tried to elucidate the role of Aplnr signaling during mesoderm cell migration and differentiation from mouse embryonic stem cells (mESCs). By activating and suppressing Aplnr signaling pathway via peptide, small molecule, and genetic modifications including siRNA- and shRNA-mediated knockdown and CRISPR-Cas9-mediated knockout (KO), we revealed that Aplnr signaling not only induces migration of cells during germ layer formation but also enhances mesoderm differentiation through FGF/MAPK pathway. Antibody array and LC/MS protein profiling data demonstrated that Apelin-13 treatment enhanced cell cycle, EGFR, FGF, Wnt, and Integrin signaling pathway proteins. Furthermore, Aplelin-13 treatment improved MSC characteristics, with mesenchymal phenotype and high expression of MSC markers, and silencing Aplnr signaling components resulted in significantly reduced expression of MSC markers. Also, Aplnr signaling activity enhanced proliferation and survival of the cells during MSC derivation from mesoderm. [ABSTRACT FROM AUTHOR]

Pluripotent stem cells as a promising cell source with unlimited proliferation and differentiation capacity hold great promise for cell-based therapies in regenerative medicine. Establishment of appropriate culture conditions might enable the control of cellular fate decision in cell culture. Transfer of three-dimensional (3D) embryoid bodies to two-dimensional (2D) monolayer culture systems for initiation of cell differentiation and specialization requires an adaptation of cells which can be managed by extracellular matrix (ECM) materials. Here we compare the characteristics of four different cell culture coating materials and their effect on attachment and differentiation of cells spreading from mouse embryonic stem cell (mESC) derived embryoid bodies (EBs) in mesoderm inducing culture conditions. Atomic force microscope (AFM) and scanning electron microscope (SEM) analysis along with Water Contact Angle technique were used to analyze physical properties of ECM materials and to evaluate cellular behavior on surfaces. Cell migration and differentiation were performed initially by using mesoderm inducing culture conditions and then three germ layer specification conditions. We investigated properties of coating materials such as roughness and wettability control cell attachment, migration and differentiation of mESCs. Matrigel-Gelatin combination is suitable for cell attachment and migration of cells spreading from 3D EBs followed by transfer onto coated surfaces. Matrigel-Gelatin coating enhanced differentiation of cells into mesoderm like cells via EMT process. Our data demonstrated that the Matrigel-Gelatin combination as a cell culture coating matrix might serve as a suitable platform to transfer EBs for differentiation and might influence pluripotent stem cell fate decision into mesoderm and further mesoderm derivative cell populations. [ABSTRACT FROM AUTHOR]

Hypothalamic–pituitary–adrenal (HPA) axis regulates stress response in the body and abnormal increase in oxidative stress contributes to the various disease pathogenesis. Although hypothalamic distribution of Apelin receptor (APLNR) has been studied, the potential regulatory role in hormone releasing function of hypothalamus in response to stress is not well elucidated yet. To determine whether APLNR is involved in the protection of the hypothalamus against oxidative stress, gonadotropin-releasing hormone (GnRH) cells were used as an in vitro model system. GT1-7 mouse hypothalamic neuronal cell line was subjected to H2O2 and hypoxia induced oxidative stress under various circumstances including APLNR overexpression, knockdown and knockout. Overexpression and activation of APLNR in GnRH producing neurons caused an increase in cell proliferation under oxidative stress. In addition, blockage of APLNR function by siRNA reduced GnRH release. Activation of APLNR initiated AKT kinase pathway as a proliferative response against hypoxic culture conditions and blocked apoptosis. Although expression and activation of APLNR have not been related to GnRH neuron differentiation during development, positive contribution of activated APLNR signaling to GnRH release in mouse embryonic stem cell derived GnRH neurons was observed in the present study. Sustained overexpression and complete deletion of APLNR in mouse embryonic stem cell derived GnRH neurons reduced GnRH release in vitro. The present findings suggest that expression and activation of APLNR in GnRH releasing GT1-7 neurons might induce a protective mechanism against oxidative stress induced cell death and APLNR signaling may play a role in GnRH neurons. [ABSTRACT FROM AUTHOR]

Cancer is a complex and multistage disease that causes suffering worldwide. Several mutations in tumor suppressor proteins are mostly responsible for tumorigenic development. Thus, determination of the mutations and developing a mutation targeted therapy are crucial in order to cure cancer. Moreover, since healthy cells do not have mutations in their tumor suppressor genes, mutation-specific treatment is responsible for selective treatment without harming a healthy tissue in the body. In this current study, lead borate nanoparticles (LB-Np) have been synthesized, and their effects on P53 mutant cancer cells were investigated. The synthesis method includes steps of mixing a borate buffer solution with the lead nitrate solution, washing the resulting precipitate with distilled water and eventually preparing stable LB-Np solutions. Cell viability analysis was conducted to identify the toxicity of LB-Np in HaCaT, A549, MCF7, and T47D cell lines. The changes in morphologies of breast cancer cell lines were demonstrated by using microscopical analysis. Additionally, alterations in gene expressions were determined in breast cancer cell lines after LB-Np treatment. This multidisciplinary study also identified the selective effect of LB-Np in cancer cell lines, in vitro. MTS and quantitative polymerase chain reaction assays demonstrated the effect of LB-Np were specific for p53 mutation cell line, T47D. Breast cancer cell line T47D has 580 C/T mutation which affects the activation of p53 tumor suppressor protein. However, LB-Np treatment effectively killed T47D cell lines and did not affect any other cell lines that have no p53 mutations such as MCF7, A549, and healthy HaCaT. Overall, synthesized LB-Np were found to be effective in p53-mutated cell lines and showed a remarkable selective anti-cancer activity. [ABSTRACT FROM AUTHOR]

Embryonic stem cells (ESCs), which are derived from the undifferentiated inner cell mass of the embryo, can differentiate every cell type of the body regarding their pluripotency. Therefore, human or mouse ESCs can be used as an unlimited cell source for numerous researches or therapeutical approaches. However, pluripotency maintenance of ESCs during in vitro culture is challenging because of their endless differentiation capacity. In the current study, the effect of USP7 on pluripotency maintenance of mouse ESCs (mESCs) has been investigated with the help of cell viability assay, morphological analysis, alkaline phosphatase (ALP) staining, qPCR analysis, and Western Blotting. 600 nM P5091 application, which showed no significant toxicity in mESCs, increased the total ubiquitinated protein amount as a proof of the accomplishment of proper USP7 inhibition. Morphological analysis and ALP activity evaluation indicated that dual inhibition of GSK3 and MEK together with leukemia inhibitory factor (LIF) treatment protects the pluripotency in presence of active USP7 enzyme. Yet, inactivation of USP7 reduced the ALP activity and altered the cell morphology in each treatment group. This morphological change and decreased ALP activity refer to differentiated mESCs. These findings were supported by gene expression and protein analysis. Gene expressions and protein amounts of pluripotency related Oct4, Nanog, c-Myc, Sox2 and Klf4 transcription factors decreased significantly after USP7 inhibition. Together with this observation, a remarkable reduction in ß-Catenin expression was also noticed. It was also observed that USP7 inactivation shortens the half-live of ß-Catenin and GSK3ß proteins. This study demonstrates that USP7 activation is crucial for proper pluripotency maintenance, which is provided through ß-Catenin stabilization. [ABSTRACT FROM AUTHOR]

The developments of nanoparticle-based treatments that benefit from novel discoveries have an essential place in the regeneration of acute and chronic wounds. Furthermore, research about the treatment methods which attempt to swiftly and scarless wound recovery has increased over time. In recent years, it has been shown that metallic-based nanoparticles, especially silver and gold derived, have an accelerating effect on chronic and contaminated wound healing. The crucial factors of inducing and completion of regeneration of wound are enhanced epithelialization rate and neovascularization in the tissue. In our study, the main purpose is the investigation of the boosting effects of erbium borate nanoparticles on the wound healing process, especially scarless ones. Newly syntesized erbium borate nanoparticles (ErB-Nps) were characterized by their concentration and particle size using nanoparticle tracking analysis (NTA). In order to examine the effect of ErB-Np on wound closure, scratch assay for dermal epithelial cells and tube formation assay for endothelial cells were performed. In addition, in order to examine the effect of the ErB-Np at a molecular level, the levels of genes related to both wound healing, inflammation, and scarless wound closure were determined with the RT-PCR experiment. Consequently, it has been shown that erbium borate nanoparticles have increased the melioration speed of scar tissue and have given clues about scarless healing potential. The investigation of the regeneration potential of erbium borate nanoparticles was done via MTS assay, quantitative PCR analysis, reactive oxygen species assay, and scratch assay. Our results show that ErB-Np is a proper agent that can be used for scarless wound healing. [ABSTRACT FROM AUTHOR]

Because breast cancer is complicated at the pathological, histological, clinical, and molecular levels, identification of new genetic targets against carcinogenic pathways is required to generate clinically relevant treatment options. In the current study, ubiquitinspecific protease 7 (USP7), which regulates various cellular pathways including Mdm2, p53, and NF–κB, was selected as a potential gene editing strategy for breast cancer in vitro. Anticancer activity of USP7 gene suppression has been evaluated through cell proliferation, gene expression, cell cycle, sphere dissemination, and cell migration analysis. Here, siRNA and shRNA strategies and an allosteric small-molecule inhibitor of USP7 were used to define potential anticancer activity against MCF7 and T47D human breast cancer cell lines. Both blockage of deubiquitination by p5091 and knockdown of USP7 reduced cell proliferation, cell migration, colony formation, and sphere dissemination for both MCF7 and T47D breast cancer cell lines. Restriction of USP7 activity strongly enhanced apoptotic gene expression and reduced metastatic ability of breast cancer cell lines. This study describes one potential molecular target for the suppression of breast cancer proliferation and metastasis. Identification of USP7 as a promising gene editing candidate might open up the possibility of new molecular drug research in targeting the ubiquitination pathway in cancer. [ABSTRACT FROM AUTHOR]

Novel thiourea (5a, 5b) and thiazolidinone derivatives (6a, 6b) were synthesized by hybridizing molecules starting from the compound 6‐(4‐phenylpiperazin‐1‐yl)pyridin‐3‐amine (4) which is known to show anticancer activity. The synthesis of the leading compound was carried out by using 1‐(5‐nitropyridin‐2‐yl)‐4‐phenylpiperazine (3) which was obtained by a novel method of the reaction of 2‐chloro‐5‐nitropyridine (1) and N‐phenylpiperazine (2). The structures of the compounds were confirmed using FTIR, 1H NMR, 13C NMR, HRMS spectroscopic methods and elemental analysis. The organic molecules were tested for their anticancer activities against prostate cancer (PC) cell lines: DU 145, PC‐3 and LNCaP. As the compound 5a exerted the highest cytotoxic activity, IC50 concentrations of compound 5a were further investigated in terms of morphology, colony‐forming ability, RNA expression, fragmented DNA and cell cycle distributions of PC cell lines. Overall data revealed that compound 5a treatment induces apoptosis and DNA fragmentation in PC cell lines and inhibits cell cycle progression resulting in the accumulation of cells in either the G1 or the S phases. [ABSTRACT FROM AUTHOR]

Purvalanol A is a specific CDK inhibitor which triggers apoptosis by causing cell cycle arrest in cancer cells. Although it has strong apoptotic potential, the mechanistic action of Purvalanol A on significant cell signaling targets has not been clarified yet. Polyamines are crucial metabolic regulators affected by CDK inhibition because of their role in cell cycle progress as well. In addition, malignant cells possess impaired polyamine homeostasis with high level of intracellular polyamines. Especially induction of polyamine catabolic enzymes spermidine/spermine N1-acetyltransferase (SSAT), polyamine oxidase (PAO) and spermine oxidase (SMO) induced toxic by-products in correlation with the induction of apoptosis in cancer cells. In this study, we showed that Purvalanol A induced apoptosis in caspase- dependent manner in MCF-7 ER(+) cells, while MDA-MB-231 (ER−) cells were less sensitive against drug. In addition Bcl-2 is a critical target for Purvalanol A, since Bcl-2 overexpressed cells are more resistant to Purvalanol A-mediated apoptosis. Furthermore, exposure of MCF-7 cells to Purvalanol A triggered SSAT and PAO upregulation and the presence of PAO/SMO inhibitor, MDL 72,527 prevented Purvalanol A-induced apoptosis. [ABSTRACT FROM AUTHOR]

Cyclooxygenase enzymes play a vital role in inflammatory pathways in the human body. Apart from their relation with inflammation, the additional involvement of COX-2 enzyme with cancer activity was recently discovered. In some cancer types the level of COX-2 enzyme is increased indicating that this enzyme could be a suitable target for cancer therapy. Based on these findings, we have synthesized some new diflunisal thiosemicarbazides and 1,2,4-triazoles and tested them against androgen-independent prostate adenocarcinoma (PC-3), colon carcinoma (HCT-116), human breast cancer (T47D), breast carcinoma (MCF7) and human embryonic kidney (HEK-293) cell lines. Specifically, the diflunisal and thiosemicarbazide functionality are combined during the synthesis of original compounds anticipating a potency enhancement. Compounds 6, 10, 15 and 16 did not show cytotoxic effects for the HEK293 cell line. Among them, compounds 15 and 16 demonstrated anticancer activity for the breast cancer cell line T47D, whereas compounds 6 and 10 which are thiosemicarbazide derivatives displayed anti-tumourigenic activity against the PC-3 cell line, consistent with the literature. However, no activity was observed for the HCT-116 cancer cell line with the tested thiosemicarbazide derivatives. Only compound 16 displayed activity against the HCT-116 cell line. Therefore, it was speculated that the diflunisal and thiosemicarbazide functionalities potentiate anticancer activity on prostate cancer and the thiosemicarbazide functionality decreases the anticancer activity of diflunisal on colon cancer cell lines. In order to gain insight into the anticancer activity and COX-2 inhibition, molecular docking studies were carried out for COX-1 and COX-2 enzymes utilizing the newly synthesized compounds 15, and 16. Both 15 and 16 showed high selectivity and affinity toward COX-2 isozyme over COX-1, which is in agreement with the experimental results. [ABSTRACT FROM AUTHOR]