Proliferation Of Jurkat Cells Research Articles

Trefoil factor 3 can stimulate Th17 cell response in the development of type 2 diabetes mellitus

This study aims to evaluate the role of trefoil factor 3 (TFF3) peptides in type 2 diabetes mellitus (T2DM) from an inflammatory perspective. The focus was on exploring how TFF3 affects the function of T cells. TFF3 overexpression model was constructed using lentivirus in Jurkat cell lines. We evaluated the impact of TFF3 on the proliferation, apoptosis, and IL-17A levels of Jurkat cells cultured in high glucose. The T2DM model was induced in TFF3 knockout (KO) mice through streptozotocin combined with high-fat diet. The measurements included glucose tolerance, insulin tolerance, inflammation markers, Th17 cell proportion, and pancreatic pathological changes. The T2DM modeling led to splenomegaly in mice, and increased expression of TFF3 in their spleens. Overexpression of TFF3 increased the proportion of IL-17+ T cells and the levels of Th17-related cytokines in Jurkat cells. There was no difference in body weight and blood glucose levels between wild-type and TFF3 KO mice. However, T2DM mice lacking the TFF3 gene showed improved glucose utilization, ameliorated pancreatic pathology, decreased inflammation levels, and reduced Th17 cell ratio. TFF3 may be involved in the chronic inflammatory immune response in T2DM. Its mechanism may be related to the regulation of the RORγt/IL-17 signaling pathway and its impact on T cell proliferation and apoptosis.

Construction of ceRNA network mediated by circRNAs screening from microarray and identification of novel biomarkers for myasthenia gravis

BackgroundRecent studies have revealed that circRNA can serve as ceRNA to participate in multiple autoimmune diseases. Our study aims to explore the key circRNA as ceRNA and biomarker for MG. Methods: We used circRNA microarray to explore differentially expressed circRNAs (DECs) from MG and compare with control. Then, we predicted the target miRNA associated with DECs and screened miRNAs by the algorithm of random walk with restart (RWR). Next, we constructed the circRNA-miRNA-mRNA ceRNA regulated network (CMMC) to identify the hub objects. Following, we detected the expression of hub-circRNAs by RT-PCR. We verify has_circ_0004183 (circFRMD4) sponging miR-145-5p regulate cells proliferation using luciferase assay and CCK-8. Results: We found that the expression level of circFRMD4 and has_circ_0035381 (circPIGB) were upregulated and has_circ_0089153(circ NUP214) had the lowest expression level in MG. Finally, we proved circFRMD4 sponging miR-145-5p regulate Jurkat cells proliferation. CircFRMD4 take part in the genesis and development of MG via circFRMD4/miR145-5p axis. Conclusions: We found that circFRMD4, circPIGB and circNUP214 can be considered as valuable potential novel biomarkers for AchR + MG. CircFRMD4 participate in the development of AchR + MG via targeting binding with miR-145-5p.

HBV DNA polymerase upregulates the transcription of PD-L1 and suppresses T cell activity in hepatocellular carcinoma

BackgroundIn HBV-associated HCC, T cells often exhibit a state of functional exhaustion, which prevents the immune response from rejecting the tumor and allows HCC to progress. Moreover, polymerase-specific T cells exhibit more severe T-cell exhaustion compared to core-specific T cells. However, whether HBV DNA polymerase drives HBV-specific CD8+ T cell exhaustion in HBV-related HCC remains unclear.MethodsWe constructed a Huh7 cell line stably expressing HA-HBV-DNA-Pol and applied co-culture systems to clarify its effect on immune cell function. We also examined how HBV-DNA-Pol modulated PD-L1 expression in HCC cells. In addition, HBV-DNA-Pol transgenic mice were used to elucidate the underlying mechanism of HBV-DNA-Pol/PD-L1 axis-induced T cell exhaustion.ResultsBiochemical analysis showed that Huh7 cells overexpressing HBV-DNA-Pol inhibited the proliferation, activation, and cytokine secretion of Jurkat cells and that this effect was dependent on their direct contact. A similar inhibitory effect was observed in an HCC mouse model. PD-L1 was brought to our attention during screening. Our results showed that the overexpression of HBV-DNA-Pol upregulated PD-L1 mRNA and protein expression. PD-L1 antibody blockade reversed the inhibitory effect of Huh7 cells overexpressing HBV-DNA-Pol on Jurkat cells. Mechanistically, HBV-DNA-Pol interacts with PARP1, thereby inhibiting the nuclear translocation of PARP1 and further upregulating PD-L1 expression.ConclusionsOur findings suggest that HBV-DNA-Pol can act as a regulator of PD-L1 in HCC, thereby directing anti-cancer immune evasion, which further provides a new idea for the clinical treatment of liver cancer.

The expression and significance of PD-L1 in condyloma acuminatum.

It has been reported that programmed death-ligand 1 (PD-L1) is highly expressed in cells during viral infection, which helps the virus escape host immunity. However, the relationship between human papillomavirus (HPV) and PD-L1 in condyloma acuminatum and whether they participate in immunosuppression have not been reported. In this paper, we aimed to explore the expression and significance of PD-L1 in condyloma acuminatum. The expression of PD-L1 in the wart of condyloma acuminatum patients and the foreskin of healthy individuals was evaluated. Lentivirus transfection was used to introduce the HPV11-E7 gene into HaCaT cells to investigate whether HPV infection could affect the expression of PD-L1. The successfully constructed HPV11-E7 HaCaT cells were cocultured with Jurkat cells, and Jurkat cell apoptosis and proliferation as well as the Jurkat cell cycle were evaluated by flow cytometry and cell counting kit-8 (CCK-8) assays. PD-L1 was highly expressed in keratinocytes of genital warts. Through the construction of a cell model, we found that HPV11-E7 could upregulate the expression of PD-L1 in HaCaT cells. Furthermore, HPV11-E7 HaCaT cells can promote the apoptosis of Jurkat cells, inhibit the proliferation of Jurkat cells and mediate the cell cycle arrest of Jurkat cells through the PD-1/PD-L1 signalling pathway. HPV infection may upregulate PD-L1 expression in the keratinocytes of genital warts and participate in the inhibition of local T-cell function.

Genetic and Pharmacological Interruption of the SIX1/EYA2 Axis Impairs Leukemia Proliferation

Background: The CALM-AF10 translocation is found in 5-10% of T-cell acute lymphoblastic leukemias (T-ALL), and a subset of acute myeloid leukemias (AML). CALM-AF10 leukemias are characterized by elevated expression of proleukemic HOXA genes. Since HOXA genes are difficult to target, we sought to identify non- HOXA effector genes through RNA sequencing and Microarray analyses, and showed that SIX1 expression is increased in CALM-AF10 leukemia cells. Like HOXA genes, SIX1 is a homeobox gene involved in cell proliferation, embryogenesis, and apoptosis. SIX1 interacts with the cofactor Eyes Absent 2 (EYA2), a protein phosphatase, to transcriptionally activate developmental genes, the TGFβ pathway, and the epithelial-mesenchymal transition (EMT). SIX1 expression is elevated during development but decreases with maturation. Intriguingly, overexpression of SIX1 has been observed in numerous malignancies including breast cancer, ovarian cancer, gliomas and esophageal cancer, and SIX1 is involved in accelerating the epithelial mesenchymal transition (EMT) and metastasis. Knockdown of either SIX1 or EYA2 has been shown to reverse these effects, and two novel inhibitors - one of the SIX1/EYA2 complex (Compound 8430, “8430”) and one of the EYA2 phosphatase (Compound 9987, “9987”) - have replicated the effects of SIX1 or EYA2 knockdown. Methods: SIX1 gene and protein expression were assessed in leukemia cell lines via RT-qPCR and Western Blot. SIX1 expression vectors were transduced into fetal liver hematopoietic stem cells (HSC) and immortalization was assessed using colony assays. CALM-AF10 leukemia cells lines were derived from irradiated mice transplanted with HSC retrovirally transfected with CALM-AF10 plasmids created in our lab. The established leukemia cell lines Jurkat, MOLT16, OCI-M2, TF-1, and SHI-1 were obtained from ATCC or DSMZ. shRNAs targeting SIX1 were lentivirally transduced into CALM-AF10 and SHI1leukemia cell lines. Cell-Titer-Glo assays and liquid culture were used to assess the effects of shRNAs, 8430, or 9987 on cell proliferation and cell growth. SIX1 gene expression in leukemias was determined from The Cancer Dependency Map from The Broad Institute using R. Results: We first validated RNAseq and Microarray studies, showing that CALM-AF10 induces SIX1 expression in murine fibroblasts and murine HSC, and localizes to the SIX1 locus. Furthermore, SIX1 overexpression was sufficient to immortalize murine fetal liver HSC (FL-mHSCs), implying a role in leukemic transformation. Multiple murine derived CALM-AF10 cell lines displayed increased SIX1 expression compared to fresh murine bone marrow and isolated HSCs. shRNA knockdown of SIX1 decreased viability of murine CALM-AF10 cells in culture. In addition, both the SIX1/EYA2 complex inhibitor (8430) and the EYA2 inhibitor (9987) decreased CALM-AF10 leukemia cell proliferation in vitro. To expand the relevance of SIX1 to other leukemias, we then queried The Cancer Dependency Map from the Broad Institute and identified other leukemia cell lines with increased SIX1 expression, including Jurkat and Molt16 (T-ALL), and SHI-1, OCIM2, and TF-1 (AML). SIX1 expression in these cells was validated by immunoblot and shRNA knockdown of SIX1 slowed the proliferation of SHI-1 cells. Finally, the 8430 and 9987 inhibitors reduced the proliferation of Jurkat, OCI-M2, and SHI-1 cells at doses similar to those used in CALM-AF10 leukemias. Conclusions: We have identified involvement of the SIX1/EYA2 axis in several different leukemias, including T-ALL and AML. Furthermore, we have shown that inhibitors of the SIX1/EYA2 interaction and of EYA2 reduce the proliferation of SIX1 expressing leukemias, suggesting a potential new therapeutic target in these leukemias.

Boric Acid Affects Cell Proliferation, Apoptosis, and Oxidative Stress in ALL Cells.

T-cell acute lymphoblastic leukemia (T-ALL) is a type of acute lymphoblastic leukemia from early T-cell progenitors. Interest grows in creating less toxic agents and therapies for chemo-resistant T-ALL cancer. Recently, elemental boron has special properties useful in the creation of new drugs. Studies have revealed the cytotoxic properties of boric acid (BA) on cancer, but not fully understood. We aimed to investigate the effect of BA on cell proliferation, apoptosis, and oxidative stress in the Jurkat cells. The effects of BA on cell viability were determined by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay for 24-48-72 h. The impact of BA on apoptosis was analyzed by acridine orange/ethidium bromide. Expression of apoptosis regulatory genes (Bcl-2, Bax, Caspase-3-8-9) and apoptotic miRNA (miR-21) was used by real-time quantitative polymerase chain reaction (RT-qPCR). The total oxidant status (TOS), total antioxidant status (TAS), and the oxidative stress index (OSI) value were calculated for oxidative stress. We determined the cytotoxic activity of BA on Jurkat cells by using XTT and defined the IC50 concentration (802.7 μg/mL) of BA. The findings clearly show that BA inhibited Jurkat cell proliferation dose-dependently. BA induced apoptosis through downregulated anti-apoptotic genes, and upregulated pro-apoptotic genes. Additionally, we found that BA significantly reduced the expression of miR-21 (p<0.001). Our findings demonstrated that different doses of BA increased TAS levels while decreasing TOS levels in Jurkat cells. Our study suggests that BA might be potential anti-cancer agent candidate in ALL via inhibition of cell proliferation, induced apoptosis, and reducing the amounts of anti-oxidants in cells.

CircPTPN22 modulates T-cell activation by sponging miR-4689 to regulate S1PR1 expression in patients with systemic lupus erythematosus

BackgroundCircular RNAs are involved in autoimmune disease pathogenesis. Our previous study indicated that circPTPN22 is involved in autoimmune diseases, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis, but the underlying mechanisms remain unclear.MethodsFirst, the expression of circPTPN22 was detected by real-time PCR and western blotting. After overexpression or knockdown of circPTPN22, the proliferation of Jurkat cells was detected by the CCK-8 assay, and the apoptosis of Jurkat cells was detected by flow cytometry. In addition, the relationship between circPTPN22-miR-4689-S1PR1 was confirmed by bioinformatic analyses, fluorescence in situ hybridization assays, RNA-binding protein immunoprecipitation, and dual luciferase reporter assays.ResultsWe found that circPTPN22 expression was downregulated in the PBMCs of SLE patients compared to those of healthy controls. Overexpression of circPTPN22 increased proliferation and inhibited apoptosis of Jurkat T cells, whereas knockdown of circPTPN22 exerted the opposite effects. CircPTPN22 acts as a miR-4689 sponge, and S1PR1 is a direct target of miR-4689. Importantly, the circPTPN22/miR-4689/S1PR1 axis inhibited the secretion of TNF-α and IL-6 in Jurkat T cells.ConclusionsCircPTPN22 acts as a miR-4689 sponge to regulate T-cell activation by targeting S1PR1, providing a novel mechanism for the pathogenesis of SLE.

Effects of Methionine Restriction on Proliferation, Cell Cycle, and Apoptosis of Human Acute Leukemia Cells

To investigate the effects of methionine restriction on proliferation, cell cycle and apoptosis of human acute leukemia cells. Cell Counting Kit-8 (CCK-8) assay was used to detect the effect of methionine restriction on HL-60 and Jurkat cells proliferation. The effect of methionine restriction on cell cycle of HL-60 and Jurkat cells was examined by PI staining. Annexin V-FITC / PI double staining was applied to detect apoptosis of HL-60 and Jurkat cells following methionine restriction. The expression of cell cycle-related proteins cyclin B1, CDC2 and apoptosis-related protein Bcl-2 was evaluated by Western blot assay. Methionine restriction significantly inhibited the proliferation of HL-60 and Jurkat cells in a time-dependent manner (HL-60: r =0.7773, Jurkat: r =0.8725), arrested the cells at G2/M phase (P < 0.001), and significantly induced apoptosis of HL-60 and Jurkat cells (HL-60: P < 0.001; Jurkat: P < 0.05). Furthermore, Western blot analysis demonstrated that methionine restriction significantly reduced the proteins expression of Cyclin B1 (P < 0.05), CDC2 (P < 0.01) and Bcl-2 (P < 0.001) in HL-60 and Jurkat cells. Acute leukemia cells HL-60 and Jurkat exhibit methionine dependence. Methionine restriction can significantly inhibit the proliferation, promote cell cycle arrest and induce apoptosis of HL-60 and Jurkat cells, which suggests that methionine restriction may be a potential therapeutic strategy for acute leukemia.

Molecular action mechanisms of two novel and selective calcium release-activated calcium channel antagonists

The prototypical calcium release-activated calcium (CRAC) channel, composed of STIM1 and Orai1, is a sought-after drug target for treating autoimmune disorders. Herein, we identified two novel and selective CRAC channel inhibitors, the indole-like compound C63368 and pyrazole core-containing compound C79413, potently and reversibly inhibiting the CRAC channel with low micromolar IC50s and sparing various off-target ion channels. These two compounds did not inhibit STIM1 activation or its coupling with Orai1, nor did they affect the channel's calcium-dependent fast inactivation. Instead, they directly acted on the Orai1 protein, with the channel's pore geometry profoundly affecting their potencies. In vitro, C63368 and C79413 effectively inhibited Jurkat cell proliferation and cytokines production in human T lymphocytes. Intragastric administration of C63368 and C79413 to mice yielded great therapeutic benefits in psoriasis and colitis animal models of autoimmune disorders, reducing serum cytokines production and significantly relieving pathological symptoms. It's worth noting, that this study provided the first insight into the characterization and mechanistic investigation of an indole-like CRAC channel antagonist. Altogether, the identification of these two highly selective CRAC channel antagonists, coupled with the elucidation of their action mechanisms, not only provides valuable template molecules but also offers profound insights for drug development targeting the CRAC channel.

Plasma-Derived Small Extracellular Vesicles From VKH Patients Suppress T Cell Proliferation Via MicroRNA-410-3p Modulation of CXCL5 Axis.

Circulating exosomes regulate immune responses and induce immune tolerance in immune-mediated diseases. This study aimed to investigate the role of circulating small extracellular vesicles (sEVs) derived from patients with Vogt-Koyanagi-Harada (VKH) syndrome, in T-cell responses. The sEVs were isolated from the plasma of healthy controls, patients with VKH, and other uveitis patients. The effects of autologous and allogeneic sEVs on the proliferation of circulating CD4+ T cells were evaluated. Microarray analysis of sEVs was performed to determine their differential miRNA expression profiles. The target genes of the candidate miRNA were predicted and verified. The role of both the candidate miRNA and target genes in T-cell proliferation was tested. Plasma-derived sEVs from patients with VKH inhibited the proliferation of autologous CD4+ T cells. Among all the miRNAs that might be associated with inflammatory activity, we found that miR-410-3p had the largest number of T-cell proliferation target genes. MiR-410-3p mimics inhibited the proliferation of Jurkat cells and CD4+ T cells. C-X-C motif chemokine ligand 5 (CXCL5) was confirmed to be a potential target gene of miR-410-3p, and siRNA-mediated CXCL5 knockdown inhibited cell proliferation. Circulating sEVs exert an inhibitory effect on autologous CD4+ T cells mediated by miR-410-3p by targeting CXCL5, supporting the possibility of using autogenic sEVs to inhibit ocular inflammation.

Tracking intracellular nuclear targeted-chemotherapy of chidamide-loaded Prussian blue nanocarriers by SERS mapping

The novel histone deacetylase drug chidamide (CHI) has been proven to regulate gene expression associated with oncogenesis via epigenetic mechanisms. However, huge side effects such as non-targeting, poor intracellular accumulation and low nuclear entry efficiency severely restrict its therapeutic efficacy. Dual-targeted nanodrug delivery systems have been proposed as the solution. Herein, we developed a CHI-loaded drug delivery nanosystem based on Prussian blue (PB) nanocarrier, which combines surface-enhanced Raman scattering (SERS) tracking function with cancer cell/nuclear-targeted chemotherapy capability. With the property of background-free SERS mapping, PB nanocarriers can serve as tracking agents to localize intracellular CHI. The incorporation of targeted molecules specifically enhances the cancer cell/nuclear internalization and chemotherapeutic effects of CHI-loaded PB nanocarriers. In vitro cytotoxicity assay clearly shows that the constructed CHI-loaded PB nanocarriers have significant inhibitory on Jurkat cell proliferation. Furthermore, SERS spectral analysis of Jurkat cells incubated with the CHI-loaded PB nanocarriers reveals obvious features of cellular apoptosis: DNA skeleton fragmentation, chromatin depolymerization, histone acetylation, and nucleosome conformation change. Importantly, this CHI-loaded PB nanocarrier will provide a new insight for lymphoblastic leukemia targeted chemotherapy.

Cytotoxicity of alkaline serine protease (ASPNJ) on Jurkat cells and its correlation with changes in the expression of membrane-associated proteins.

We aim to study the inhibitory effect of alkaline serine protease (ASPNJ) on lymphocytic leukemia Jurkat cells and its related mechanism through examining the expression of membrane proteins or membrane-associated proteins. MTT assay and trypan blue staining were used to detect the inhibitory effect of ASPNJ on the proliferation and growth of Jurkat cells. Wright-Giemsa staining was used to observe the effect of ASPNJ on the morphology of Jurkat cells. The effect of ASPNJ on Jurkat cell apoptosis was detected by flow cytometry. Two-dimensional electrophoresis-mass spectrometry (2-DE-MS) was used to detect and identify the differentially expressed proteins of Jurkat cells treated with ASPNJ (4 μg/mL, 3 h), of which three were selected and verified by Western blot. ASPNJ significantly inhibited the proliferation of leukemia cells (Raji, U937, and Jurkat), caused obvious morphological changes, and induced apoptosis of Jurkat cells. ASPNJ also increased the sensitivity of Jurkat cells to vincristine (VCR). Seven differentially expressed proteins were obtained through 2DE-MS, of which Peroxiredoxin-6 (PRDX6), Calcium-binding protein (CHP1), and 40S ribosomal protein SA (RPSA) were validated. ASPNJ can cause significant toxic effects on Jurkat cells and enhance the effects of VCR. The mechanism of action of ASPNJ on Jurkat cells may be related to differentially expressed proteins such as PRDX6. This study provides a new experimental basis and direction for antileukemia research.

Novel Protease-Mediated Double Antigen Recognizing Chimeric Antigen Receptor (CAR) Enhances Directionality of CAR-T Cell Activity and Improves Target Cell Specificity

Recently, Chimeric Antigen Receptor (CAR)-T cell therapies show excellent therapeutic effect against various hematologic malignancies, including B-cell leukemia, B-cell lymphoma, and multiple myeloma. Expansion of target diseases of this attractive immunotherapy is one of the most important issues. To identify the novel specific tumor antigen as CAR-T cell target is the standard way to accomplish this goal. However, recent CAR-T cells recognize single tumor antigen, so normal tissues expressing the target antigen are possible to be attacked by CAR-T cells. For example, anti-CD19 CAR-T cells against B-cell malignancies kill not only tumor cells but also normal B-cells, so patients receiving the CAR-T cells showed severe immune deficiency. Improvement of target cell recognition system of CAR-T cells minimizes this adverse effect and contributes to expand target diseases. To improve target-cell-specificity of CAR-T cell, we established a protease-mediated regulatory CAR, named "Scissors-CAR" (panel A). This regulatory CAR is constituted with extracellular antigen recognition portion and cytoplasmic Human Immunodeficiency Virus protease (HIVPR) catalytic domain. When CAR-T cells expressing both anti-A Scissors-CAR and anti-B Counterpart-CAR that has HIVPR recognition polypeptide contact with target tumor expressing both antigen-A and B, these two CARs interact on cell membrane. This interaction induces Scissors-CAR-mediated cleavage of target polypeptide and structural change of Counterpart-CAR. Previously, we reported double antigen recognition system using Scissors-CAR. Previous Counterpart-CAR named "Signal-CAR" had HIVPR recognition polypeptide upstream of CD3-zeta, so Scissors-CAR-mediated cleavage broke the CAR protein structure and suppressed CAR-T cell activity (panel B, named "Negative system"). This system allowed CAR-T cells to directional attack against target cells expressing only antigen-A and protect the cells expressing both antigen-A and B. Here, we established another double-antigen recognition system using Scissors-CAR. In many cases including both hematologic malignancies and solid tumors, additional ectopic antigens are expressed on malignant cells. If CAR-T cells empowered to directional attack against target cells expressing both antigen-A and B, much more tumors can be treated with this tumor immunotherapy (panel C, named "Positive system"). To establish this directional CAR-T cells, we constructed novel Counterpart-CAR named as "Activating-CAR" constituted with target recognition portion, co-stimulatory domain, CD3-zeta, and protein destabilization motif known as degron. Between CD3-zeta and degron motif, HIVPR recognition polypeptide was inserted. In vitro analysis revealed that this degron motif destabilized Activating-CAR and HIVPR-mediated cleavage and that release of degron motif increased Activating-CAR stability. Furthermore, cell proliferation of Jurkat cells expressing both anti-CD19 Activating-CAR and anti-HER2 Scissors-CAR was enhanced co-cultivated with target cells expressing both CD19 and HER2. From these results, combined use of Activating-CAR and Scissors-CAR allowed CAR-T cells to directionally activate against target cells expressing two antigens recognized by each CAR. By choosing the Counterpart-CAR from "Signal-CAR" or "Activating-CAR", this protease-mediated double antigen recognition system could optimize target-cell-specificity of CAR-T cells for the individual tumor and contribute to expand the target tumors of CAR-T cell therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Alpha-Ketoglutarate or 5-HMF: Single Compounds Effectively Eliminate Leukemia Cells via Caspase-3 Apoptosis and Antioxidative Pathways.

Background: We recently showed that a combined solution containing alpha-ketoglutarate (aKG) and 5-hydroxymethyl-furfural (5-HMF) has a solid antitumoral effect on the Jurkat cell line due to the fact of its antioxidative, caspase-3 and apoptosis activities, but no negative effect on human fibroblasts was obtained. The question arises how the single compounds, aKG and 5-HMF, affect peroxynitrite (ONOO−) and nitration of tyrosine residues, Jurkat cell proliferation and caspase-activated apoptosis. Methods: The ONOO− luminol-induced chemiluminescence reaction was used to measure the ONOO− scavenging function of aKG or 5-HMF, and their protection against nitration of tyrosine residues on bovine serum albumin was estimated with the ELISA technique. The Jurkat cell line was cultivated in the absence or presence of aKG or 5-HMF solutions between 0 and 3.5 µM aKG or 0 and 4 µM 5-HMF. Jurkat cells were tested for cell proliferation, mitochondrial activity and caspase-activated apoptosis. Results: aKG showed a concentration-dependent reduction in ONOO−, resulting in a 90% elimination of ONOO− using 200 mM aKG. In addition, 20 and 200 mM 5-HMF were able to reduce ONOO− only by 20%, while lower concentrations of 5-HMF remained stable in the presence of ONOO−. Nitration of tyrosine residues was inhibited 4 fold more effectively with 5-HMF compared to aKG measuring the IC50%. Both substances, aKG and 5-HMF, were shown to cause a reduction in Jurkat cell growth that was dependent on the dose and incubation time. The aKG effectively reduced Jurkat cell growth down to 50% after 48 and 72 h of incubation using the highest concentration of 3.5 µM, and 1, 1.6, 2, 3 and 4 µM 5-HMF inhibited any cell growth within (i) 24 h; 1.6, 2, 3 and 4 µM 5-HMF within 48 h (ii); 2, 3 and 4 µM 5-HMF within 72 h (iii). Furthermore, 4 µM was able to eliminate the starting cell number of 20,000 cells after 48 and 72 h down to 11,233 cells. The mitochondrial activity measurements supported the data on aKG or 5-HMF regarding cell growth in Jurkat cells, in both a dose- and incubation-time-dependent manner: the highest concentration of 3.5 µM aKG reduced the mitochondrial activity over 24 h (67.7%), 48 h (57.9%) and 72 h (46.8%) of incubation with Jurkat cells compared to the control incubation without aKG (100%). 5-HMF was more effective compared to aKG; the mitochondrial activity in the presence of 4 µM 5-HMF decreased after 24 h down to 68.4%, after 48 h to 42.9% and after 72 h to 32.0%. Moreover, 1.7 and 3.4 µM aKG had no effect on caspase-3-activated apoptosis (0.58% and 0.56%) in the Jurkat cell line. However, 2 and 4 µM 5-HMF increased the caspase-3-activated apoptosis up to 22.1% and 42.5% compared to the control (2.9%). A combined solution of 1.7 µM aKG + 0.7 µM 5-HMF showed a higher caspase-3-activated apoptosis (15.7%) compared to 1.7 µM aKG or 2 µM 5-HMF alone. In addition, 3.5 µM µg/mL aKG + 1.7 µM 5-HMF induced caspase-activated apoptosis up to 55.6% compared to 4.5% or 35.6% caspase-3 activity using 3.5 µM aKG or 4 µM 5-HMF. Conclusion: Both substances showed high antioxidative potential in eliminating either peroxynitrite or nitration of tyrosine residues, which results in a better inhibition of cell growth and mitochondrial activity of 5-HMF compared to aKG. However, caspase-3-activated apoptosis measurements revealed that the combination of both substances synergistically is the most effective compared to single compounds.

Release of Exosomes Derived from Leukocyte-Depleted Red Cell Suspension and Its Regulation on Hematological Tumor Cells

To investigate the release of exosome (Exo) from leukocyte-depleted red cell suspension (LDRCS) at different storage time and its regulation on proliferation of hematological tumor cells and possible mechanism. The Exo (RBC-Exo) in LDRCS at different storage time was obtained by ultracentrifugation, and the morphology and immunological marker of RBC-Exo were detected by transmission electron microscopy and Western blot, respectively. The particle size distribution of RBC-Exo in LDRCS at different storage time was detected by Dynamic Light Scattering. CCK-8 assay was used to explore the effect of RBC-Exo on hematological tumor cell proliferation. Western blot was used to detect the expression of proliferation-related proteins in hematological tumor cells after co-culture with RBC-Exo. RBC-Exo was isolated, which was characterized by cup-like shape, particle size distribution ranged from 20 to 200 nm, CD63/TSG101 enriched, Calnexin negative, CD235a positive and CD41 negative. The particle size distribution of RBC-Exo from LDRCS between middle was not significantly different and late stored stage. But the particle size distribution of RBC-Exo at middle-late stored stage(>14 d) was larger than that at early stored stage (≤14 days). Compared with the control group, RBC-Exo could significantly promote the proliferation of HBL1, U2932 and Jurkat cells. Compared with the control group, the cycle-related protein P21 was significantly down-regulated in HBL1, U2932 and Jurkat cells after co-culture with RBC-Exo for 3 days, while the anti-apoptotic protein BCL-2 was not changed significantly. The morphology of RBC-Exo from LDRCS at middle-late stored stage was different from that at early stored stage. RBC-Exo could promote the proliferation of hematological tumor cells, possibly by regulating the expression of cycle-associated protein P21.

Kaempferol-3-O-Rhamnoside Inhibits the Proliferation of Jurkat Cells Through Jun Amino-Terminal Kinase Signaling

Background: Indonesian herbal medicine has become target of new drugs against diseases, including cancer. The high incidence and mortality rate of cancer, anticancer resistance, and side effects of chemotherapy contribute to the urgency of researching novel anticancer drugs. A natural product from Schima wallichii Korth., an Indonesian herbal medicine empirically used for many diseases, has shown anticancer activity in MCF-7 and LNCaP cells. Objective: In this study, we investigated the antiproliferative mechanism of the active compound of S. wallichii, kaempferol-3-O-rhamnoside, against Jurkat cells. Methods: Treated cells were analyzed using a proliferation assay and real time-reverse transcriptase polymerase chain reaction for IL-2 mRNA measurement. The mechanism of antiproliferative activity was assesed by western blotting analysis for Mitogen Activated Protein Kinases (MAPKs). Results: Kaempferol-3-O-rhamnoside has an antiproliferative activity at IC50 of 76.3 μM and slightly inhibited IL-2 mRNA expression. The mechanism to inhibit Jurkat cells proliferation was through the stimulation of phosphorylated Jun amino-terminal kinase. Conclusions: The present study observed the molecular mechanism of antiproliferative activity of kaempferol-3-O-rhamnoside.

Ganoderma lucidum polysaccharide inhibits the proliferation of leukemic cells through apoptosis.

To investigate the cytotoxic effect of polysaccharides derived from Ganoderma lucidum on T lymphocyte leukemia cells. Water-soluble polysaccharides were extracted from the fruit bodies of G. lucidum, purified, and characterized using HPGPC-MALLS and NMR. The cytotoxicity of G. lucidum polysaccharide fraction 5 (GLP5) to T lymphocyte leukemia cell line Jurkatand human immortalized epidermal cell line HaCat was assessed using MTT assay. Apoptosis was assessed using flow cytometry. Expressions of apoptosis-related genes in the cells after being exposed to GLP5 were detected using Western blot assay. GLP5 was a β-(1→3) and β-(1→6) linked glucan. It inhibited the proliferation of Jurkat cells in a concentration-dependent manner and the half-maximal inhibitory concentration (IC50) was 34.5 mg/L but did not suppress the growth of HaCat cells. Apoptotic cells in Jurkatcells were detected to increase with increasing GLP5 concentrations. The expression levels of cleaved caspase-3 were significantly higher after the cells were exposed to 25 and 50 mg/L GLP5 when compared to non-exposed cells (Control). In addition, the expression levels of BAX and Bcl2 were significantly up- and down-regulated after treatment with GLP5 at 25 and 50 mg/L when compared with control (P<0.05), respectively. GLP5 has antiproliferative activity against Jurkat cells and the activity is likely mediated through the activation of apoptosis pathways.

LncRNA LINC00680 Acts as a Competing Endogenous RNA and Is Associated With the Severity of Myasthennia Gravis.

Background and PurposeMyasthenia gravis (MG) is a T cell-dependent antibody-mediated autoimmune disorder that can seriously affect patients' quality of life. However, few studies have focused on the severity of MG. Moreover, existing therapeutic efforts, including those targeting biomarkers for MG, remain unsatisfactory. Therefore, it is vital that we investigate the pathogenesis of MG and identify new biomarkers that can not only evaluate the severity of the disease but also serve as potential therapeutic targets. Long noncoding RNA LINC00680 has been found to be associated with the progression of a variety of diseases as a competing endogenous RNA (ceRNA). However, the specific role of LINC00680 in MG has yet to be clarified. Here, we aimed to investigate the association between LINC00680 and the severity of MG.MethodsBioinformatics tools, quantitative real-time PCR, Western blotting, and luciferase assays were selected to investigate key signaling pathways and RNA expression in patients with MG. The Quantitative MG Score scale and the MG Composite scale were used to evaluate the severity of MG in the included patients. Cell viability assays and flow cytometry analysis were selected to analyze cell proliferation and apoptosis.ResultsCompared with control subjects, the expression levels of LINC00680 and mitogen-activated protein kinase 1 (MAPK1) in peripheral blood mononuclear cells of patients with MG were both upregulated; the levels of miR-320a were downregulated. A positive correlation was detected between LINC00680 expression and the severity of MG. Luciferase reporter assays identified that LINC00680 acts as a target for miR-320a. The in vitro analysis confirmed that LINC00680 regulates the expression of MAPK1 by sponging miR-320a. Finally, the functional analysis indicated that LINC00680 promoted Jurkat cell proliferation and inhibited cellular apoptosis by sponging miR-320a.ConclusionLINC00680 may be associated with the severity of MG as a ceRNA by sponging miR-320a to upregulate MAPK1. These findings suggest that LINC00680 may represent a potential biomarker which evaluates the severity of MG and may serve as a therapeutic target.

LncRNA OIP5-AS1 modulates the proliferation and apoptosis of Jurkat cells by sponging miR-181c-5p to regulate IL-7 expression in myasthenia gravis.

BackgroundMyasthenia gravis (MG) is an antibody-mediated autoimmune disease. In recent years, accumulating evidence has indicated that long non-coding RNAs (lncRNAs) can function as competing endogenous RNAs (ceRNAs), contributing to the progression of various autoimmune diseases. Nevertheless, the regulatory roles of ceRNAs in MG pathogenesis remain unclear. In this study, we aimed to elucidate the role of lncRNA OIP5-AS1 as a ceRNA associated with MG progression.MethodsReal-time PCR was used to detect OIP5-AS1 levels in peripheral blood mononuclear cells (PBMCs) from patients with MG. Luciferase reporter assays were performed to validate the relationship between OIP5-AS1 and miR-181c-5p. CCK-8 and flow cytometry were performed to test the proliferation and apoptotic abilities of OIP5-AS1 in Jurkat cells. Furthermore, real-time PCR and Western blot assays were performed to explore the interactions between OIP5-AS1, miR-181c-5p, and IL-7.ResultsThe expression of OIP5-AS1 was up-regulated in patients with MG. Luciferase reporter assay indicated that OIP5-AS1 targeted the miR-181c-5p. Functional assays showed that OIP5-AS1 suppressed Jurkat cell apoptosis and promoted cell proliferation by sponging miR-181c-5p. Mechanistically, knockdown of OIP5-AS1 inhibited IL-7 expression at both the mRNA and protein levels in Jurkat cells, whereas the miR-181c-5p inhibitor blocked the reduction of IL-7 expression induced by OIP5-AS1 suppression.ConclusionsWe confirmed that OIP5-AS1 serves as an endogenous sponge for miR-181c-5p to regulate the expression of IL-7. Our findings provide novel insights into MG processes and suggests potential therapeutic targets for patients with MG.

The Potential Role of Nigella sativa Seed Oil as Epigenetic Therapy of Cancer.

Nigella sativa oil, commonly known as black seed oil (BSO), is a well-known Mediterranean food, and its consumption is associated with beneficial effects on human health. A large number of BSO’s therapeutic properties is attributed to its pharmacologically active compound, thymoquinone (TQ), which inhibits cell proliferation and induces apoptosis by targeting several epigenetic players, including the ubiquitin-like, containing plant homeodomain (PHD) and an interesting new gene, RING finger domains 1 (UHRF1), and its partners, DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1). This study was designed to compare the effects of locally sourced BSO with those of pure TQ on the expression of the epigenetic complex UHRF1/DNMT1/HDAC1 and the related events in several cancer cells. The gas chromatographs obtained from GC-MS analyses of extracted BSO showed that TQ was the major volatile compound. BSO significantly inhibited the proliferation of MCF-7, HeLa and Jurkat cells in a dose-dependent manner, and it induced apoptosis in these cell lines. BSO-induced inhibitory effects were associated with a significant decrease in mRNA expression of UHRF1, DNMT1 and HDAC1. Molecular docking and MD simulation showed that TQ had good binding affinity to UHRF1 and HDAC1. Of note, TQ formed a stable metal coordinate bond with zinc tom, found in the active site of the HDAC1 protein. These findings suggest that the use of TQ-rich BSO represents a promising strategy for epigenetic therapy for both solid and blood tumors through direct targeting of the trimeric epigenetic complex UHRF1/DNMT1/ HDAC1.