Modulation of inflammatory and adrenergic pathways in hypertension: effects of β-blockers on cytokine release in Jurkat T cells

Background: Natural polyphenols are naturally used in traditional medicine to treat various diseases. Despite their healthful properties, ingesting phenolic compounds in food form does not provide a sufficient concentration for systemic therapeutic effects due to their low solubility in water, poor absorption, and fast metabolism. This problem has been solved by creating various composite pharmaceuticals from phenolic compounds using different methods to stabilize polyphenols. Objective: The purpose of this study was to assess the effects of the DPPA (1,2-palmitoyl phosphatidic acid) and DPPC (dipalmitoyl phosphatidylcholine) liposomes on the protective effects of a quercetin-rich flavonoid fraction extracted from French Marigold (Tagetes patula L.) on the viability of Jurkat cells. The study will examine both intact cells and cells that have been incubated under oxidative stress conditions. Materials and Methods: Quercetin-rich flavonoid fraction was extracted from a French Marigold (Tagetes patula L.) by thin-layer chromatography (TLC), High-pressure liquid chromatography (HPLC), and Liquid Chromatography-Mass Spectrometry (LC-MS) methods. Extract alone and in complex with dipalmitoyl phosphatidylcholine (DPPC) and 1,2- dipalmitoyl phosphatidic acid (DPPA) liposomes were added to the Jurkat cells culture at a rate of 2 mg/mL−1.The 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) test assayed Cell viability by evaluating cellular dehydrogenase activity. Results: Flavonoids were separated and identified in the marigold extracts by TLC, HPLC, and LC-MS methods. The spectrophotometric absorption spectrum of the quercetin-rich flavonoid fraction extracted from French Marigold (Tagetes patula L.) shows two peaks corresponding to benzoyl (254nm) and cinnamyl (375nm) aromatic rings. In the complex of quercetin-rich flavonoid fraction with DPPC and DPPA liposomes, the spectrophotometric absorption peak at 254nm was not detected, while the absorption intensity of the peak at 375nm was sharply reduced. The quercetin-rich flavonoid fraction alone and in combination with DPPC liposome increased intact and incubated under low- and high-intensity oxidative stress conditions Jurkat cells’ viability but did not reveal effect in combination with DPPA liposome. Conclusions: The quercetin-rich flavonoid fraction extracted from French Marigold (Tagetes patula L.) forms stable complexes with DPPC and DPPA liposomes that allow the storage of high content of phenolic compounds in lipid nanocapsules. The use of the liposomal system in the pharmaceutical and food industry allows for carried and controlled bioactive-compound release, which is considered one of the main strategies to improve and enhance the quality of food, providing preventative healthcare for the population and decreasing the risk of disease. Keywords: bioactive-compound, polyphenols, Quercetin, Liposomes, Jurkat cells, therapeutic effects, pharmaceuticals

Gelsolin is an actin-regulatory protein that modulates actin assembly and disassembly, and is believed to regulate cell motility in vivo through modulation of the actin network. In addition to its actin-regulatory function, gelsolin has also been proposed to affect cell growth. Our present experiments have tested the possible involvement of gelsolin in the regulation of apoptosis, which is significantly affected by growth. When overexpressed in Jurkat cells, gelsolin strongly inhibited apoptosis induced by anti-Fas antibody, C2-ceramide or dexamethasone, without changing the F-actin morphology or the levels of Fas or Bcl-2 family proteins. Upon the induction of apoptosis, an increase in CPP32(-like) protease activity was observed in the control vector transfectants, while it was strongly suppressed in the gelsolin transfectants. Pro-CPP32 protein, an inactive form of CPP32 protease, remained uncleaved by anti-Fas treatment in the gelsolin transfectants, indicating that gelsolin blocks upstream of this protease. The tetrapeptide inhibitor of CPP32(-like) proteases strongly inhibited Fas-mediated apoptosis, but only partially suppressed both C2-ceramide- and dexamethasone-induced apoptosis. These data suggest that the critical target responsible for the execution of apoptosis may exist upstream of CPP32(-like) proteases in Jurkat cells and that gelsolin acts on this target to inhibit the apoptotic cell death program.

The CRISPR/Cas9 technology is a prominent genome‐editing tool as an adaptable nuclease capable of producing a double strand break at almost any genomic loci. However, the modification of hematopoietic stem and progenitor cells (HSPC) via the homology directed repair (HDR) pathway is still inefficient. We hypothesize that histone deacetylase inhibitors (HDACi), such as Valproic Acid (VPA) and Sodium Butyrate (NaB), could enhance HDR efficiency by increasing the accessibility of the genome‐editing machinery. To address the potential utilization of HDACi therapeutically, we began by assessing the effect of VPA and NaB on cell growth and viability. No statistically significant effect on cell growth or viability was observed at concentrations as high as 50mM in a myeloid‐erythroid leukemic cell line (K562 cells). However, K562 cells only represent one lineage of the HSPC progeny. Therefore, we also treated an immortalized T lymphocyte cell line (Jurkat cells), representative of the lymphoid lineage, with either VPA or NaB. No effect on the growth pattern or viability of Jurkat cells was evident at concentrations as high as 5mM VPA and NaB. However, Jurkat cells did appear to be more susceptible to HDACi. Therefore, we ascertained that concentrations of HDACi lower than 5mM would be tolerated in HSPCs. At concentrations as low as 0.0005mM HDACi an enhancement in CRISPR cutting efficiency was evidenced as assessed in both K562 cells and Jurkat cells. This enhancement did not appear to be locus specific, in that two sets of CRISPR guide RNAs were analyzed revealing similar trends of enhancement. If the same trend follows, treatment with HDACi can be expanded to virtually any genome editing experiment utilizing the CRISPR‐Cas9 system to enhance cutting and thus therapeutic gene editing.Support or Funding InformationCSUPERBEffect of Valproic Acid and Butyrate on the Growth Curve of Hematopoietic Cell Lines A) Schematic representation of the experimental design utilized to test the effect of HDACi on the division of two hematopoietic cell lines. B) K562 cells treated with 300mM and 100mM VPA were not observed to divide. C) K562 cells treated with 500mM NaB were not observed to divide. D) Jurkat cells appear to be more susceptible to VPA. E) Jurkat cells treated with 50mM NaB proliferated at a much slower rate.Figure 1

αB-crystallin/sHSP protects cytochrome c and mitochondrial function against oxidative stress in lens and retinal cells

Our research aimed to establish the effectiveness of various β-adrenergic receptor blockers in the regulation of T cell proliferation in the model system of the Jurkat cells.Material and Methods. The research was conducted on the human leukemic mature T cells (Jurkat cells) (DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Germany)) activated by phytohemagglutinin (PHA). Viability and proliferative activity of intact and PHA-stimulated Jurkat cells under the influence of Nebilet, Egilok, Betalok Zok, and Propranolol were studied with the MTT test. Results. Study results show that β-adrenergic receptors blockers selectively influence the activity of mitochondrial dehydrogenases, and therefore viability in both intact and mitogen-stimulated Jurkat cells. In particular, Nebilet, Betalok Zok, and Propranolol induced a statistically significant decrease of intact Jurkat cells viability by 63%, 20%, and 32%, respectively; Egilok and Betalok Zok (25mg) didn’t statistically significantly affect the PHA- activated Jurkat cells viability. Nebilet, Betalok Zok (50 mg), and Propranolol decreased the viability of the mitogen (PHA) activated Jurkat cells (for PHA dose 20μg/ml) and didn’t affect at a PHA dose of 50μg/ml.

T cell acute lymphoblastic leukemia (T-ALL), an aggressive and heterogeneous malignancy originating from T cell precursors (thymocytes), accounts for ~15% of all ALL cases in children and for ~25% in adults. The present study aimed to investigate the role of microRNA-221 (miR-221) in the regulation of cell viability and apoptosis of human T-ALL cells and its related regulatory mechanisms. To perform this investigation, miR-221 was upregulated or knocked down in human T-ALL cells (Jurkat cells) using miR-221 mimic or inhibitor, respectively. Then, cell viability was determined using a 3-(4,5-dimethylthiahiazol-2-y1)-2,5-diphenytetrazolium bromide assay, cell invasion and migration were analyzed via Transwell assays, and cell apoptosis was detected using flow cytometry. It was found that transfection with a miR-221 inhibitor significantly inhibited Jurkat cell viability, migration and invasion, and induced Jurkat cell apoptosis. Whereas, transfection with the miR-221 mimic resulted in the opposite effects. Besides, the results showed that phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was a target of miR-221. Moreover, it was observed that the effects of the miR-221 inhibitor on Jurkat cell viability, migration and invasion, and cell apoptosis were significantly eliminated by PTEN-small interfering RNA. In addition, it was shown that the phosphatidylinositol 3-kinase/AKT pathway was involved in the effect of miR-221 on Jurkat cells. In conclusion, the data indicated that miR-221 existed as an oncogene in T-ALL, and its downregulation could inhibit the development of ALL by targeting PTEN. Therefore, miR-221 may be a novel potential therapeutic target for ALL.

Introduction: The vascular endothelial glycocalyx, crucial for blood vessel integrity and homeostasis, is vulnerable to oxidative stress, leading to endothelial dysfunction, which strongly correlates with cardiovascular disease (CVD). This study investigates the protective effects of rivaroxaban, a factor X inhibitor, on the glycocalyx under oxidative stress condition. Methods: We examined the impact of rivaroxaban on human umbilical vein endothelial cells exposed to acute and chronic H2O2-induced oxidative stress. Results: Rivaroxaban dose-dependently suppressed syndecan-1, a key component of the glycocalyx, shedding from cell surface, and enhanced protease-activated receptor (PAR)1-PAR2/phosphatidylinositol-3-kinase (PI3K)-dependent cell viability after acute induction of H2O2. This protective effect was linked to the translocation of IQGAP1, a scaffold protein that modulates the actin cytoskeleton, to the perinucleus from the cell membrane. Under chronic H2O2 treatments, rivaroxaban improves cell viability accompanied by an increase in hyaluronidase activities, aiding the turnover and remodeling of hyaluronic acid within the glycocalyx. Conclusion: We identify that rivaroxaban protects against oxidative stress-induced endothelial glycocalyx damage and cell viability through IQGAP1/PAR1-2/PI3K/Akt pathway, offering a potential to be a therapeutic target for CVD prevention. Introduction: The vascular endothelial glycocalyx, crucial for blood vessel integrity and homeostasis, is vulnerable to oxidative stress, leading to endothelial dysfunction, which strongly correlates with cardiovascular disease (CVD). This study investigates the protective effects of rivaroxaban, a factor X inhibitor, on the glycocalyx under oxidative stress condition. Methods: We examined the impact of rivaroxaban on human umbilical vein endothelial cells exposed to acute and chronic H2O2-induced oxidative stress. Results: Rivaroxaban dose-dependently suppressed syndecan-1, a key component of the glycocalyx, shedding from cell surface, and enhanced protease-activated receptor (PAR)1-PAR2/phosphatidylinositol-3-kinase (PI3K)-dependent cell viability after acute induction of H2O2. This protective effect was linked to the translocation of IQGAP1, a scaffold protein that modulates the actin cytoskeleton, to the perinucleus from the cell membrane. Under chronic H2O2 treatments, rivaroxaban improves cell viability accompanied by an increase in hyaluronidase activities, aiding the turnover and remodeling of hyaluronic acid within the glycocalyx. Conclusion: We identify that rivaroxaban protects against oxidative stress-induced endothelial glycocalyx damage and cell viability through IQGAP1/PAR1-2/PI3K/Akt pathway, offering a potential to be a therapeutic target for CVD prevention.

PurposeTo evaluate the effects of bevacizumab on expression of B-cell leukemia/lymphoma (Bcl)-2 and apoptosis in retinal pigment epithelial (RPE) cells under oxidative stress conditions.MethodsRPE cells were treated with H2O2 (0, 100, 200, 300, and 400 µM) and bevacizumab at or above the doses normally used in clinical practice (0, 0.33, 0.67, 1.33, and 2.67 mg/mL). Cell apoptosis was measured using flow cytometry with annexin V-fluorescein isothiocyanate. The expression of Bcl-2 mRNA was determined using reverse transcription polymerase chain reaction.ResultsUnder low oxidative stress conditions (H2O2 100 µM), cell apoptosis was not significantly different at any concentration of bevacizumab, but Bcl-2 mRNA expression decreased with increasing concentration of bevacizumab (0.33, 0.67, 1.33, and 2.67 mg/mL). Under moderate oxidative stress conditions (H2O2 200 µM), Bcl-2 mRNA expression decreased with increasing concentration of bevacizumab (0.33, 0.67, 1.33, and 2.67 mg/mL), but cell apoptosis increased only at 2.67 mg/mL of bevacizumab. Under high oxidative stress (300 µM) conditions, cell apoptosis increased at high concentrations of bevacizumab (1.33 and 2.67 mg/mL), but it did not correlate with Bcl-2 expression.ConclusionsWithdrawal of vascular endothelial growth factor can lead to RPE cell apoptosis and influences the expression of anti-apoptotic genes such as Bcl-2 under oxidative stress conditions. Since oxidative stress levels of each patient are unknown, repeated injections of intravitreal bevacizumab, as in eyes with age-related macular degeneration, might influence RPE cell survival.

Zinc plays a dual role, as an integral part of metabolic machinery and in defense against reactive oxygen species. Hepatocytes are important sites for zinc metabolism for synthesis of zinc metalloproteins and maintaining its homeostasis. However, the factors influencing post absorptive zinc metabolism under normal and oxidative stress (OS) conditions are not well understood. Using rat liver slices, we conducted a series of four in vitro zinc uptake experiments to study influence of ascorbic acid and folic acid in normal and oxidative stress conditions with Zn concentrations representing deficient to excess states (7.7-30.7 millimole/L). Zinc uptakes under OS at these four zinc levels were lower than the normal conditions. Folic acid showed significant inhibitory effect on zinc uptake under both normal and OS conditions in a dose response manner. Nevertheless, dose response of ascorbic acid at four zinc levels indicated its marked enhancing effect under OS condition. Differences in zinc uptake trend lines between the normal and OS conditions for interaction of both the vitamins narrowed down as the zinc levels increased. Our results suggest that folic acid causes inhibitory effect, while ascorbic acid may be protective in OS with reference to zinc uptake.

Cellular accumulation of Cys326-OGG1 protein complexes under conditions of oxidative stress

Acute lymphocytic leukemia (ALL) is a type of childhood leukemia with the highest incidence; T-acute lymphocytic leukemia (T-ALL) is far more difficult to treat than B-acute lymphocytic leukemia (B-ALL) and has a poor long-term prognosis. Therefore, there is an urgent requirement to develop effective drugs for the treatment of T-ALL. Hirsutanol A is a natural sesquiterpenoid compound. The aim of the present study was to evaluate the in vitro anticancer activity of hirsutanol A against T-acute lymphocytic leukemia Jurkat cells and investigate the mechanism of action. A Cell Counting Kit-8 assay demonstrated that hirsutanol A inhibited the viability of Jurkat cells in a dose- and time-dependent manner. In addition, hirsutanol A induced cell cycle arrest at the G2 phase as determined via flow cytometry. Furthermore, Hoechst staining, Annexin V-FITC/propidium iodide double staining, mitochondrial membrane potential detection using JC-1 and western blot analysis of apoptotic proteins indicated that the inhibitory effect of hirsutanol A on Jurkat cells was associated with the induction of apoptosis. Of note, hirsutanol A induced the expression of the tumor suppressor p53, whereas simultaneous treatment with pifithrin-α, an inhibitor of p53, significantly reduced Jurkat cell apoptosis induced by hirsutanol A. In summary, the present study suggested that hirsutanol A inhibited Jurkat cell viability through induction of cell cycle arrest and p53-dependent initiation of apoptosis, thus hirsutanol may serve as a promising compound for the treatment of T-ALL.

This study was purposed to investigate the effect of phenylhexyl isothiocyanate (PHI) on Wnt/β-catenin signaling pathway, histone acetylation, histone methylation and cell apoptosis in Jurkat cell line. The viability of Jurkat cells after treatment with PHI was tested by MTT. Apoptotic rate of Jurkat cells was measured by flow cytometry. The levels of Wnt/β-catenin related proteins including β-catenin, TCF, c-myc, and cyclinD1, histone acetylated H3 and H4, histone methylated H3K9 and H3K4 were detected by Western blot. The results showed that PHI inhibited the cell growth and induced apoptosis in Jurkat cells in time-and dose-dependent manners. Its IC50 at 48 h was about 20 µmol/L. Expression of histone acetylated H3, H4 and histone methylated H3k4 increased after exposure to PHI for 3 h, while histone methylated H3K9 decreased. Expression of β-catenin was not changed after exposure to PHI for 3 h, but expression of β-catenin, and its cell cycle-related genes such as TCF, c-myc and cyclinD1 decreased after exposure to PHI for 7 h. It is concluded that PHI regulates acetylation and methylation of histone, inhibits Wnt/β-catenin signal pathway, and is able to induce apoptosis and inhibits growth of Jurkat cells.

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive hematologic malignancy. Immune tolerance induced by CD4+CD25+ regulatory T cells (Tregs) with high expression of Foxp3 is an important hypothesis for poor therapy response. Notch1 signaling is thought to be involved in the pathogenesis of this disease. Crosstalk between Notch and Foxp3+Tregs induced immune tolerance is unknown in T-ALL. We studied Foxp3 and Notch1 expression in vivo and in vitro, and analyzed the biological characteristics of T-ALL cell line systematically after Notch inhibition and explored the crosstalk between Notch signaling and Foxp3 expression.MethodsIn vivo, we established T-ALL murine model by Jurkat cells transplantation to severe combined immunodeficiency (SCID) mice. Notch1 and Foxp3 expression was detected. In vitro, we used γ-secretase inhibitor N-S-phenyl-glycine-t-butyl ester (DAPT) to block Notch1 signaling in Jurkat cells. Notch1, Hes-1 and Foxp3 genes and protein expression were detected by PCR and western blotting, respectively. The proliferation pattern, cell cycle and viability of Jurkat cells after DAPT treatment were studied. Protein expression of Notch1 target genes including NF-κB, p-ERK1/2 and STAT1 were determined.ResultsWe show that engraftment of Jurkat cells in SCID mice occurred in 8 of 10 samples (80%), producing disseminated human neoplastic lymphocytes in PB, bone marrow or infiltrated organs. Notch1 and Foxp3 expression were higher in T-ALL mice than normal mice. In vitro, Jurkat cells expressed Notch1 and more Foxp3 than normal peripheral blood mononuclear cells (PBMCs) in both mRNA and protein levels. Blocking Notch1 signal by DAPT inhibited the proliferation of Jurkat cells and induced G0/G1 phase cell cycle arrest and apoptosis. Foxp3 as well as p-ERK1/2, STAT1 and NF-κB expression was down regulated after DAPT treatment.ConclusionsThese findings indicate that regulation of Foxp3 expression does involve Notch signaling, and they may cooperatively regulate T cell proliferation in T-ALL.

Single- and repeated-dose toxicity study of bevacizumab, ranibizumab, and aflibercept in ARPE-19 cells under normal and oxidative stress conditions

Cytotoxic effect of inositol hexaphosphate and its Ni(II) complex on human acute leukemia Jurkat T cells