Activation of Wnt Pathway Suppresses Growth of MUG-Chor1 Chordoma Cell Line.

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 GSK3ß 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.

Human ESC-derived Neuromesodermal Progenitors (NMPs) Successfully Differentiate into Mesenchymal Stem Cells (MSCs).

Mesenchymal Stem Cells (MSCs), as an adult stem cell type, are used to treat various disorders in clinics. However, derivation of homogenous and adequate amount of MSCs limits the regenerative treatment potential. Although mesoderm is the main source of mesenchymal progenitors during embryonic development, neuromesodermal progenitors (NMPs), reside in the primitive streak during development, is known to differentiate into paraxial mesoderm. In the current study, we generated NMPs from human embryonic stem cells (hESC), subsequently derived MSCs and characterized this cell population in vitro and in vivo. Using a bFGF and CHIR induced NMP formation protocol followed by serum containing culture conditions; here we show that MSCs can be generated from NMPs identified by not only the expression of T/Bra and Sox 2 but also FLK-1/PDGFRα in our study. NMP-derived MSCs were plastic adherent fibroblast like cells with colony forming capacity and trilineage (osteo-, chondro- and adipo-genic) differentiation potential. In the present study, we demonstrate that NMP-derived MSCs have an endothelial tendency which might be related to their FLK-1+/PDGFRα + NMP origin. NMP-derived MSCs displayed a protein expression profile of characterized MSCs. Growth factor and angiogenesis related pathway proteins were similarly expressed in NMP-derived MSCs and characterized MSCs. NMP-derived MSCs keep characteristics after short-term and long-term freeze-thaw cycles and localized into bone marrow followed by tail vein injection into NOD/SCID mice. Together, these data showed that hESC-derived NMPs might be used as a precursor cell population for MSC derivation and could be used for in vitro and in vivo research.

Apelin Receptor Signaling Protects GT1-7 GnRH Neurons Against Oxidative Stress In Vitro.

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.

Ubiquitin-specific protease 7 downregulation suppresses breast cancer in vitro.

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, ubiquitin-specific 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.

Effect of a novel bioceramic root canal sealer on the angiogenesis-enhancing potential of assorted human odontogenic stem cells compared with principal tricalcium silicate-based cements.

OBJECTIVE: This study evaluated the angiogenesis-enhancing potential of a tricalcium silicate-based mineral trioxide aggregate (ProRoot MTA), Biodentine, and a novel bioceramic root canal sealer (Well-Root ST) in human dental pulp stem cells (hDPSCs), human periodontal ligament stem cells (hPLSCs), and human tooth germ stem cells (hTGSCs). METHODOLOGY: Dulbecco's modified Eagle's medium was conditioned for 24 h by exposure to ProRoot MTA, Biodentine, or Well-Root ST specimens (prepared according to the manufacturers' instructions). The cells were cultured in these conditioned media and their viability was assessed with 3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H tetrazolium (MTS) on days 1, 3, 7, 10, and 14. Angiogenic growth factors [platelet-derived growth factor (PDGF), basic fibroblast growth factor (FGF-2), and vascular endothelial growth factor (VEGF)] were assayed by sandwich enzyme-linked immunosorbent assay (ELISA) on days 1, 7, and 14. Human umbilical vein endothelial cell (HUVEC) migration assays were used to evaluate the vascular effects of the tested materials at 6-8 h. Statistical analyses included Kruskal-Wallis, Mann-Whitney U, and Friedman and Wilcoxon signed rank tests. RESULTS: None of tricalcium silicate-based materials were cytotoxic and all induced a similar release of angiogenic growth factors (PDGF, FGF-2, and VEGF) (p>0.05). The best cell viability was observed for hDPSCs (p<0.05) with all tricalcium silicate-based materials at day 14. Tube formation by HUVECs showed a significant increase with all tested materials (p<0.05). CONCLUSION: The tricalcium silicate-based materials showed potential for angiogenic stimulation of all stem cell types and significantly enhanced tube formation by HUVECs.

Gene Editing in Human Pluripotent Stem Cells: Recent Advances for Clinical Therapies.

The identification of human embryonic stem cells and reprogramming technology to obtain induced pluripotent stem cells from adult somatic cells have provided unique opportunity to create human disease models, gene editing strategies and cell therapy options.Development of pluripotent stem cells from somatic cells and genomic manipulation tools enabled to use site specific nucleases in the cell therapy research. Identification of efficient gene manipulation, safe differentiation and use will provide a novel strategy to treat many diseases in the near future. Current available registered clinical trials clearly indicate the need for pluripotent stem cell and gene therapy treatment options. Although gene editing based pluripotent stem cell research is a popular field for research worldwide, improvement of clinical approaches for treatment still remains to be investigated. In this review, we summarized the current situation of gene editing based pluripotent cell therapy developments and applications in clinics.

Effect of a novel bioceramic root canal sealer on the angiogenesis-enhancing potential of assorted human odontogenic stem cells compared with principal tricalcium silicate-based cements

Abstract Objective: This study evaluated the angiogenesis-enhancing potential of a tricalcium silicate-based mineral trioxide aggregate (ProRoot MTA), Biodentine, and a novel bioceramic root canal sealer (Well-Root ST) in human dental pulp stem cells (hDPSCs), human periodontal ligament stem cells (hPLSCs), and human tooth germ stem cells (hTGSCs). Methodology: Dulbecco's modified Eagle's medium was conditioned for 24 h by exposure to ProRoot MTA, Biodentine, or Well-Root ST specimens (prepared according to the manufacturers' instructions). The cells were cultured in these conditioned media and their viability was assessed with 3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H tetrazolium (MTS) on days 1, 3, 7, 10, and 14. Angiogenic growth factors [platelet-derived growth factor (PDGF), basic fibroblast growth factor (FGF-2), and vascular endothelial growth factor (VEGF)] were assayed by sandwich enzyme-linked immunosorbent assay (ELISA) on days 1, 7, and 14. Human umbilical vein endothelial cell (HUVEC) migration assays were used to evaluate the vascular effects of the tested materials at 6-8 h. Statistical analyses included Kruskal-Wallis, Mann-Whitney U, and Friedman and Wilcoxon signed rank tests. Results: None of tricalcium silicate-based materials were cytotoxic and all induced a similar release of angiogenic growth factors (PDGF, FGF-2, and VEGF) (p>0.05). The best cell viability was observed for hDPSCs (p<0.05) with all tricalcium silicate-based materials at day 14. Tube formation by HUVECs showed a significant increase with all tested materials (p<0.05). Conclusion: The tricalcium silicate-based materials showed potential for angiogenic stimulation of all stem cell types and significantly enhanced tube formation by HUVECs.

Design and synthesis of phenylpiperazine derivatives as potent anticancer agents for prostate cancer.

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, 1 H NMR, 13 C 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.

Mesenchymal Stem Cells as Regulators of Carcinogenesis.

Mesenchymal Stem Cells (MSCs) are adult stem cells; isolated from various body parts including bone marrow, adipose tissue and dental tissue, have been characterized well and used in regenerative medicine applications. The promising potential of MSCs makes them great candidates in many disorders. It has been well known in the literature that MSCs interact with cancer cells and regulate the carcinogenesis process at different stages. The dual role of MSCs in cancer progression should be clearly identified at the physiological and molecular level to identify clinical potential in cancer treatment. The promoting or suppressive role of MSCs in cancer is controlled by various growth factors, cytokines and chemokines which affect the cell proliferation, angiogenesis and metastasis. Although many studies have been conducted to explore MSC-cancer cell interactions, it is still unclear how MSCs communicate with cancer cells and tumor microenvironment. Further studies are required to investigate secreted factors and paracrine effects, tumor stroma environment, molecular regulators and downstream pathways that are involved in MSC-cancer interaction loop. MSC type, cancer type and stage specific phenotypic and transcriptomic profile changes should be identified in detail to improve clinical use of MSCs in cancer either as a target or as a tool.In the current book chapter, we review the literature to summarize current information about the MSC-cancer cell interactions in terms of soluble factors, angiogenesis, metastasis and drug resistance. The role of MSCs in tumor progression or suppression was discussed based on the current literature.

The investigation of Helicobacter pylori in the dental biofilm and saliva samples of children with dyspeptic complaints.

BACKGROUND: The oral cavity can be an extra-gastric reservoir for Helicobacter pylori (H.pylori). This can play a role in the pathogenesis of halitosis, glossitis, recurrent aphthous stomatitis, and dental caries. The present study was conducted to detect the presence of H.pylori within the dental biofilm and in saliva samples collected from children suffering from dyspepsia and children without any gastrointestinal complaints. Associations with gastric infection, halitosis, and some oral parameters were also evaluated. METHODS: Seventy children (aged between 5-16) with dyspepsia were selected for the study group and control group composed of 30 healthy children without dyspepsia were also included in the study. After detailed oral and clinical examinations for oral parameters, saliva, and supragingival dental biofilm samples were collected for 16S rRNA and 23S rRNA genes detection by real-time polymerase chain reaction (RT-PCR). The presence of gastric H.pylori was evaluated in endoscopic biopsy specimens histopathologically. Halitosis was evaluated by benzoyl-DL-arginine-naphthylamid (BANA) test. Salivary S.mutans and Lactobacilli sp. counts were also carried out by commercial kits. RESULTS: H.pylori was histopathologically detected amongst 83% of the children with the dyspeptic condition. The detection rate of this bacteria in dental biofilm and saliva samples and halitosis were found relatively higher in the dyspeptic children rather than the control group (p < 0.01). Halitosis was not significantly different between dyspeptic children and those detected with H.pylori (p > 0.05). In the gastric H.pylori positive group with dyspepsia, DMFT/S and dmft/s numbers and plaque indices were found higher than the control group (p < 0.01). Only plaque indices of gastric H.pylori negative group with dyspepsia were found higher than the control group (p < 0.01). S.mutans and Lactobacilli sp. counts were not significantly different between gastric H.pylori positive and negative groups (p > 0.05). Comparing to those with negative for both genes, in children whose dental biofilm and saliva samples were positive for both 16S rRNA and 23S rRNA genes, significantly higher results for halitosis, and DMFS numbers and significantly lower results for dmfs numbers and pH values were found (p < 0.01). CONCLUSIONS: Helicobacter pylori can occur in the oral cavity aside and independently from the stomach. However, the high number of bacteria in the oral cavities of children with gastric H.pylori, an association between the presence of H.pylori and halitosis, DMFS, and pH were found.

Cytoglobin: a potential marker for adipogenic differentiation in preadipocytes in vitro.

Obesity, mainly characterized by the excess fat storage, is a global health problem resulting in serious morbidity and mortality. Identification of molecular mechanisms in adipogenic differentiation pathway might lead to development of new strategies for diagnosis, prevention and therapy of obesity and associated diseases. Discovery of new genes and proteins in the differentiation pathway could help to understand the key specific regulators of the adipogenesis. Cytoglobin (Cygb), identified as a new globin family member protein, is expressed in various tissues. Although its interaction with oxygen and nitric oxide indicates the potential role in antioxidant pathways, the exact role remains unclear. In the current study, expression level of Cygb was determined in proliferating and differentiating 3T3-F442A cells by gene expression and protein expression analysis. Results revealed that Cygb expression up-regulated in differentiated cells in parallel with adipogenic differentiation markers; PPARγ, CEBPα and FABP4 expressions. Besides, Cygb overexpression in preadipocytes contributed to the adipogenic differentiation as verified by detection of higher lipid droplets and increased PPARγ, CEBPα and FABP4 expressions with respect to control cells. These findings will shed light on the unknown roles of Cygb in adipogenesis and obesity.