Browsing by Author "Paunović, Verica (24342012700)"

Introduction: Beta cell dysfunction by loss of beta cell identity, dedifferentiation, and the presence of polyhormonal cells are main characteristics of diabetes. The straightforward strategy for curing diabetes implies reestablishment of pancreatic beta cell function by beta cell replacement therapy. Aristaless-related homeobox (Arx) gene encodes protein which plays an important role in the development of pancreatic alpha cells and is a main target for changing alpha cell identity. Results: In this study we used CRISPR/dCas9-based epigenetic tools for targeted hypermethylation of Arx gene promoter and its subsequent suppression in mouse pancreatic αTC1-6 cell line. Bisulfite sequencing and methylation profiling revealed that the dCas9-Dnmt3a3L-KRAB single chain fusion constructs (EpiCRISPR) was the most efficient. Epigenetic silencing of Arx expression was accompanied by an increase in transcription of the insulin gene (Ins2) mRNA on 5th and 7th post-transfection day, quantified by both RT-qPCR and RNA-seq. Insulin production and secretion was determined by immunocytochemistry and ELISA assay, respectively. Eventually, we were able to induce switch of approximately 1% of transiently transfected cells which were able to produce 35% more insulin than Mock transfected alpha cells. Conclusion: In conclusion, we successfully triggered a direct, transient switch of pancreatic alpha to insulin-producing cells opening a future research on promising therapeutic avenue for diabetes management. Copyright © 2023 Đorđević, Stepper, Feuerstein-Akgoz, Gerhauser, Paunović, Tolić, Rajić, Dinić, Uskoković, Grdović, Mihailović, Jurkowska, Jurkowski, Jovanović and Vidaković.

We investigated the ability of graphene quantum dot (GQD) nanoparticles to protect SH-SY5Y human neuroblastoma cells from oxidative/nitrosative stress induced by iron-nitrosyl complex sodium nitroprusside (SNP). GQD reduced SNP cytotoxicity by preventing mitochondrial depolarization, caspase-2 activation, and subsequent apoptotic death. Although GQD diminished the levels of nitric oxide (NO) in SNP-exposed cells, NO scavengers displayed only a slight protective effect, suggesting that NO quenching was not the main protective mechanism of GQD. GQD also reduced SNP-triggered increase in the intracellular levels of hydroxyl radical (•OH), superoxide anion (O2•−), and lipid peroxidation. Nonselective antioxidants, •OH scavenging, and iron chelators, but not superoxide dismutase, mimicked GQD cytoprotective activity, indicating that GQD protect cells by neutralizing •OH generated in the presence of SNP-released iron. Cellular internalization of GQD was required for optimal protection, since a removal of extracellular GQD by extensive washing only partly diminished their protective effect. Moreover, GQD cooperated with SNP to induce autophagy, as confirmed by the inhibition of autophagy-limiting Akt/PRAS40/mTOR signaling and increase in autophagy gene transcription, protein levels of proautophagic beclin-1 and LC3-II, formation of autophagic vesicles, and degradation of autophagic target p62. The antioxidant activity of GQD was not involved in autophagy induction, as antioxidants N-acetylcysteine and dimethyl sulfoxide failed to stimulate autophagy in SNP-exposed cells. Pharmacological inhibitors of early (wortmannin, 3-methyladenine) or late stages of autophagy (NH4Cl) efficiently reduced the protective effect of GQD. Therefore, the ability of GQD to prevent the in vitro neurotoxicity of SNP depends on both •OH/NO scavenging and induction of cytoprotective autophagy. © 2021

We investigated the ability of graphene quantum dot (GQD) nanoparticles to protect SH-SY5Y human neuroblastoma cells from oxidative/nitrosative stress induced by iron-nitrosyl complex sodium nitroprusside (SNP). GQD reduced SNP cytotoxicity by preventing mitochondrial depolarization, caspase-2 activation, and subsequent apoptotic death. Although GQD diminished the levels of nitric oxide (NO) in SNP-exposed cells, NO scavengers displayed only a slight protective effect, suggesting that NO quenching was not the main protective mechanism of GQD. GQD also reduced SNP-triggered increase in the intracellular levels of hydroxyl radical (•OH), superoxide anion (O2•−), and lipid peroxidation. Nonselective antioxidants, •OH scavenging, and iron chelators, but not superoxide dismutase, mimicked GQD cytoprotective activity, indicating that GQD protect cells by neutralizing •OH generated in the presence of SNP-released iron. Cellular internalization of GQD was required for optimal protection, since a removal of extracellular GQD by extensive washing only partly diminished their protective effect. Moreover, GQD cooperated with SNP to induce autophagy, as confirmed by the inhibition of autophagy-limiting Akt/PRAS40/mTOR signaling and increase in autophagy gene transcription, protein levels of proautophagic beclin-1 and LC3-II, formation of autophagic vesicles, and degradation of autophagic target p62. The antioxidant activity of GQD was not involved in autophagy induction, as antioxidants N-acetylcysteine and dimethyl sulfoxide failed to stimulate autophagy in SNP-exposed cells. Pharmacological inhibitors of early (wortmannin, 3-methyladenine) or late stages of autophagy (NH4Cl) efficiently reduced the protective effect of GQD. Therefore, the ability of GQD to prevent the in vitro neurotoxicity of SNP depends on both •OH/NO scavenging and induction of cytoprotective autophagy. © 2021

Polyclonal T regulatory cells (Treg - CD4+CD25+CD127lowFoxp3+)are used in several protocols for the treatment of type 1 diabetes (T1D), multiple sclerosis and graft-versus host disease in clinical trials. However, general opinion is that autoantigen-specific Treg could be more efficient in autoimmunity suppression due to their direct effect on pathogenic autoantigen-specific effector T cells. This study describes isolation and expansion of insulin-specific Treg in vitro. Insulin-specific Treg are uniformly distributed in lymphoid tissues however their number is extremely low. To enrich the proportion of insulin-specific Treg, pure CD4+ cells were co-cultured with insulin B chain peptide-loaded dendritic cells, isolated from mice that develop T1D spontaneously – NOD mice. Insulin-specific CD4+ cell expansion peaked after 48 h of incubation and was in favour of Treg. These cells were then sorted using insulin peptide-loaded MHC class II tetramers and cultured in vitro for 48 h in the presence of TCR stimulators, TGF-β and IL-2. The proportion of gained insulin-specific cells with T regulatory phenotype (CD4+CD25highCD127lowGITR+FoxP3+)was in average between 93% and 97%. These cells have shown potent in vitro suppressive effect on T effector cells, produced IL-10 and TGF-β and expressed PD-1 and CD39. Further proliferation of these insulin-specific Treg required the presence of dendritic cells, anti-CD3 antibody and IL-2. This study provides new, reproducible experimental design for the enrichment and expansion of insulin-specific Treg that can be used for the cell-based therapy of autoimmunity. © 2019 Elsevier B.V.

Polyclonal T regulatory cells (Treg - CD4+CD25+CD127lowFoxp3+)are used in several protocols for the treatment of type 1 diabetes (T1D), multiple sclerosis and graft-versus host disease in clinical trials. However, general opinion is that autoantigen-specific Treg could be more efficient in autoimmunity suppression due to their direct effect on pathogenic autoantigen-specific effector T cells. This study describes isolation and expansion of insulin-specific Treg in vitro. Insulin-specific Treg are uniformly distributed in lymphoid tissues however their number is extremely low. To enrich the proportion of insulin-specific Treg, pure CD4+ cells were co-cultured with insulin B chain peptide-loaded dendritic cells, isolated from mice that develop T1D spontaneously – NOD mice. Insulin-specific CD4+ cell expansion peaked after 48 h of incubation and was in favour of Treg. These cells were then sorted using insulin peptide-loaded MHC class II tetramers and cultured in vitro for 48 h in the presence of TCR stimulators, TGF-β and IL-2. The proportion of gained insulin-specific cells with T regulatory phenotype (CD4+CD25highCD127lowGITR+FoxP3+)was in average between 93% and 97%. These cells have shown potent in vitro suppressive effect on T effector cells, produced IL-10 and TGF-β and expressed PD-1 and CD39. Further proliferation of these insulin-specific Treg required the presence of dendritic cells, anti-CD3 antibody and IL-2. This study provides new, reproducible experimental design for the enrichment and expansion of insulin-specific Treg that can be used for the cell-based therapy of autoimmunity. © 2019 Elsevier B.V.

Purpose: Anaplastic thyroid carcinoma (ATC) is an aggressive, chemo-resistant malignancy. Chemo-resistance is often associated with changes in activity of the RAS/MAPK/ERK and PI3K/AKT/mTOR pathways and/or a high expression of ATP binding cassette (ABC) transporters, such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). To assess the therapeutic efficacy in ATC of a combination of the dual mTOR kinase inhibitor vistusertib (AZD2014) and paclitaxel (PTX), we generated a new cell line (Rho-) via the selection of human thyroid carcinoma 8505C cells that exhibit a low accumulation of rhodamine 123, which serves as a P-gp and BCRP substrate. Methods: Immunohistochemistry was used for P-gp and BCRP expression analyses in primary ATC patient samples. Spheroid formation and immunodeficient NSG mice were used for performing in vitro and in vivo tumorigenicity assays, respectively. MTT, flow-cytometry, fluorescent microscopy, cell death and proliferation assays, as well as migration, invasion and gelatin degradation assays, were used to assess the potential of AZD2014 to enhance the effects of PTX. ATC xenografts in SCID mice were used for evaluating in vivo treatment efficacies. Results: Rho- cells were found to be 10-fold more resistant to PTX than 8505C cells and, in addition, to be more tumorigenic. We also found that AZD2014 sensitized Rho- cells to PTX by inhibiting proliferation and by inducing autophagy. The combined use of AZD2014 and PTX efficiently inhibited in vitro ATC cell migration and invasion. Subsequent in vivo xenograft studies indicated that the AZD2014 and PTX combination effectively suppressed ATC tumor growth. Conclusions: Our data support results from recent phase I clinical trials using combinations of AZD2014 and PTX for the treatment of solid tumors. Such combinations may also be employed for the design of novel targeted ATC treatment strategies. © 2018, International Society for Cellular Oncology.

Purpose: Anaplastic thyroid carcinoma (ATC) is an aggressive, chemo-resistant malignancy. Chemo-resistance is often associated with changes in activity of the RAS/MAPK/ERK and PI3K/AKT/mTOR pathways and/or a high expression of ATP binding cassette (ABC) transporters, such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). To assess the therapeutic efficacy in ATC of a combination of the dual mTOR kinase inhibitor vistusertib (AZD2014) and paclitaxel (PTX), we generated a new cell line (Rho-) via the selection of human thyroid carcinoma 8505C cells that exhibit a low accumulation of rhodamine 123, which serves as a P-gp and BCRP substrate. Methods: Immunohistochemistry was used for P-gp and BCRP expression analyses in primary ATC patient samples. Spheroid formation and immunodeficient NSG mice were used for performing in vitro and in vivo tumorigenicity assays, respectively. MTT, flow-cytometry, fluorescent microscopy, cell death and proliferation assays, as well as migration, invasion and gelatin degradation assays, were used to assess the potential of AZD2014 to enhance the effects of PTX. ATC xenografts in SCID mice were used for evaluating in vivo treatment efficacies. Results: Rho- cells were found to be 10-fold more resistant to PTX than 8505C cells and, in addition, to be more tumorigenic. We also found that AZD2014 sensitized Rho- cells to PTX by inhibiting proliferation and by inducing autophagy. The combined use of AZD2014 and PTX efficiently inhibited in vitro ATC cell migration and invasion. Subsequent in vivo xenograft studies indicated that the AZD2014 and PTX combination effectively suppressed ATC tumor growth. Conclusions: Our data support results from recent phase I clinical trials using combinations of AZD2014 and PTX for the treatment of solid tumors. Such combinations may also be employed for the design of novel targeted ATC treatment strategies. © 2018, International Society for Cellular Oncology.