Proliferation Of Jurkat Cells Research Articles

MicroRNA-101 regulates T-cell acute lymphoblastic leukemia progression and chemotherapeutic sensitivity by targeting Notch1.

The present study aimed to investigate the role of microRNA (miR)-101 in acute lymphoblastic leukemia progression and chemoresistance. Furthermore, a novel target gene of miR-101 was identified. Here, we confirmed that miR-101 was significantly downregulated in the blood samples of patients with T-cell acute lymphoblastic leukemia (T-ALL) compared with the healthy controls, as determined by reverse transcription quantitative polymerase chain reaction (RTqPCR) analysis. The in vitro experiments demonstrated that miR-101 significantly repressed the proliferation and invasion, and induced potent apoptosis in Jurkat cells, as determined by CCK-8, flow cytometer and cell invasion assays. Luciferase assay confirmed that Notch1 was a target gene of miR-101, and western blotting showed that miR-101 suppressed the expression of Notch1 at the protein level. Moreover, functional restoration assays revealed that Notch1 mediates the effects of miR-101 on Jurkat cell proliferation, apoptosis and invasion. miR-101 enhanced the sensitivity of Jurkat cells to the chemotherapeutic agent adriamycin. Taken together, our results show for the first time that miR-101 acts as a tumor suppressor in T-cell acute lymphoblastic leukaemia and it could enhance chemotherapeutic sensitivity. Furthermore, Notch1 was identified to be a novel target of miR-101. This study indicates that miR-101 may represent a potential therapeutic target for T-cell acute lymphoblastic leukemia intervention.

Baicalein suppresses the proliferation of acute T-lymphoblastic leukemia Jurkat cells by inhibiting the Wnt/β-catenin signaling.

Although the response rates of chemotherapy in patients with acute T-lymphoblastic leukemia (T-ALL) have improved significantly, the outcome of these patients is still poor. Previous studies suggested that baicalein could inhibit the growth of several cancers, while its effect on T-ALL cells remains unclear. We used Jurkat cells as an in vitro model of T-ALL. Cell counting kit-8 assay and cytometric analysis with Annexin V-FITC/PI double staining were used to investigate the proliferation and apoptosis of Jurkat cells treated with increasing concentration of baicalein for indicated time. RT-PCR and western blotting was used to test the expression of Wnt/β-catenin associated genes and proteins. In cell viability assay, baicalein could inhibit the proliferation of Jurkat cells both in dose- and time-dependent manners. In cell apoptosis assay, baicalein could stimulate apoptosis of Jurkat cells both in dose- and time-dependent manners. Moreover, we demonstrated that baicalein could down-regulated the mRNA and protein levels of β-catenin and its widely accepted downstream targets (c-Myc, cyclin D1, and Axin2) in dose-dependent manners. These results proved that baicalein might be a potential choice for the treatment of T-ALL.

Effects of valproic acid and pioglitazone on cell cycle progression and proliferation of T-cell acute lymphoblastic leukemia Jurkat cells.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignant tumor. Administration of chemical compounds influencing apoptosis and T cell development has been discussed as promising novel therapeutic strategies. Valproic acid (VPA) as a recently emerged anti-neoplastic histone deacetylase (HDAC) inhibitor and pioglitazone (PGZ) as a high-affinity peroxisome proliferator-activated receptor-gamma (PPARγ) agonist have been shown to induce apoptosis and cell cycle arrest in different studies. Here, we aimed to investigate the underlying molecular mechanisms involved in anti-proliferative effects of these compounds on human Jurkat cells. Treated cells were evaluated for cell cycle progression and apoptosis using flowcytometry and MTT viability assay. Real-time RT-PCR was carried out to measure the alterations in key genes associated with cell death and cell cycle arrest. Our findings illustrated that both VPA and PGZ can inhibit Jurkat E6.1 cells in vitro after 24 hr; however, PGZ 400 μM presents the most anti-proliferative effect. Interestingly, treated cells have been arrested in G2/M with deregulated cell division cycle 25A (Cdc25A) phosphatase and cyclin-dependent kinase inhibitor 1B (CDKN1B or p27) expression. Expression of cyclin D1 gene was inhibited when DNA synthesis entry was declined. Cell cycle deregulation in PGZ and VPA-exposed cells generated an increase in the proportion of aneuploid cell population, which has not reported before. These findings define that anti-proliferative effects of PGZ and VPA on Jurkat cell line are mediated by cell cycle deregulation. Thus, we suggest PGZ and VPA may relieve potential therapeutic application against apoptosis-resistant malignancies.

The Scorpion Toxin Analogue BmKTX-D33H as a Potential Kv1.3 Channel-Selective Immunomodulator for Autoimmune Diseases.

The Kv1.3 channel-acting scorpion toxins usually adopt the conserved anti-parallel β-sheet domain as the binding interface, but it remains challenging to discover some highly selective Kv1.3 channel-acting toxins. In this work, we investigated the pharmacological profile of the Kv1.3 channel-acting BmKTX-D33H, a structural analogue of the BmKTX scorpion toxin. Interestingly, BmKTX-D33H, with its conserved anti-parallel β-sheet domain as a Kv1.3 channel-interacting interface, exhibited more than 1000-fold selectivity towards the Kv1.3 channel as compared to other K+ channels (including Kv1.1, Kv1.2, Kv1.7, Kv11.1, KCa2.2, KCa2.3, and KCa3.1). As expected, BmKTX-D33H was found to inhibit the cytokine production and proliferation of both Jurkat cells and human T cells in vitro. It also significantly improved the delayed-type hypersensitivity (DTH) responses, an autoreactive T cell-mediated inflammation in rats. Amino acid sequence alignment and structural analysis strongly suggest that the “evolutionary” Gly11 residue of BmKTX-D33H interacts with the turret domain of Kv1 channels; it appears to be a pivotal amino acid residue with regard to the selectivity of BmKTX-D33H towards the Kv1.3 channel (in comparison with the highly homologous scorpion toxins). Together, our data indicate that BmKTX-D33H is a Kv1.3 channel–specific blocker. Finally, the remarkable selectivity of BmKTX-D33H highlights the great potential of evolutionary-guided peptide drug design in future studies.

Apoptosis-inducing effects of jujube (Zǎo) seed extracts on human Jurkat leukemia T cells.

BackgroundJujube (Zǎo) seeds exhibited anticancer effects and used in Chinese medicine for many years. This study aims to investigate the apoptosis-inducing effects of seed extracts from eight different cultivated species (‘Apple’, ‘Bombay’, ‘Jumbo’, ‘Kaew’, ‘Nomsod’, ‘Rianthong’, ‘Samros’, and ‘Taiwan’) on human Jurkat leukemia T cells.MethodsWe evaluated the effects of seed extracts from eight jujube cultivated species on human Jurkat leukemia T cells. The crude seed extracts were prepared sequentially by using water, 95 % ethanol, dichloromethane, ethyl acetate, chloroform or hexane. The antiproliferative effects of the jujube seed extracts relative to that of melphalan were evaluated by neutral red assays. Apoptotic cell death induced by the ethanolic extracts at 1 × IC50 and 2 × IC50 concentrations was demonstrated by DAPI staining, gel electrophoresis, flow cytometry with Annexin V/propidium iodide staining, and caspase-3, -8, and -9 enzyme activities.ResultsEthanolic extracts of ‘Taiwan’, ‘Jumbo’, ‘Nomsod’, ‘Rianthong’, ‘Samros’, and ‘Bombay’, significantly inhibited the proliferation of Jurkat cells compared with untreated cells (all P < 0.001), while the extracts of ‘Kaew’ and ‘Apple’ were inactive. The six active extracts preferentially induced apoptotic cell death in a concentration-dependent manner with DNA fragmentation (2 × IC50). Increased caspase-3 activity was detected after treatment with the six extracts. The ‘Taiwan’, ‘Nomsod’, ‘Jumbo’, and ‘Rianthong’ extracts (2 × IC50) induced both the extrinsic and intrinsic apoptosis pathways by increasing caspase-8 and caspase-9 activity, respectively. Alkaloids (Dragendorff’s method) and reducing sugars (Fehling’s test) were mainly identified in the apoptosis-inducing extracts.ConclusionsThe tested of six active extracts (‘Taiwan’, ‘Jumbo’, ‘Nomsod’, ‘Rianthong’, ‘Samros’ and ‘Bombay’) contained alkaloids or reducing sugars, and induced caspase-dependent apoptosis in human Jurkat leukemia T cells.

Cutting off the power: inhibition of leukemia cell growth by pausing basal ATP release and P2X receptor signaling?

T cells respond to antigen stimulation with the rapid release of cellular ATP, which stimulates an autocrine feedback mechanism that regulates calcium influx through P2X receptors. This autocrine purinergic feedback mechanism plays an essential role in the activation of T cells resulting in cell proliferation and clonal expansion. We recently reported that increases in mitochondrial ATP production drive this stimulation-induced purinergic signaling mechanism but that low-level mitochondrial ATP production fuels basal T cell functions required to maintain vigilance of unstimulated T cells. Here we studied whether defects in these purinergic signaling mechanisms are involved in the unwanted proliferation of leukemia T cells. We found that acute leukemia T cells (Jurkat) possess a larger number and more active mitochondria than their healthy counterparts. Jurkat cells have higher intracellular ATP concentrations and generat more extracellular ATP than unstimulated T cells from healthy donors. As a result, increased purinergic signaling through P2X1 and P2X7 receptors elevates baseline levels of cytosolic Ca(2+) in Jurkat cells. We found that pharmacological inhibition of this basal purinergic signaling mechanism decreases mitochondrial activity, Ca(2+) signaling, and cell proliferation. Similar results were seen in the leukemic cell lines THP-1, U-937, and HL-60. Combined treatment with inhibitors of P2X1 or P2X7 receptors and the chemotherapeutic agent 6-mercaptopurine completely blocked Jurkat cell proliferation. Our results demonstrate that increased mitochondrial metabolism promotes autocrine purinergic signaling and uncontrolled proliferation of leukemia cells. These findings suggest that deranged purinergic signaling can result in T cell malignancy and that therapeutic targeting aimed at purinergic signaling is a potential strategy to combat T cell leukemia.

TRAIL-CM4 fusion protein shows in vitro antibacterial activity and a stronger antitumor activity than solo TRAIL protein

A TRAIL-CM4 fusion protein in soluble form with tumor selective apoptosis and antibacterial functions was expressed in the Escherichia coli expression system and isolated through dialysis refolding and histidine-tag Nickel-affinity purification. Fresh Jurkat cells were treated with the TRAIL-CM4 fusion protein. Trypan blue staining and MTT analyses showed that, similar to a TRAIL positive control, Jurkat cell proliferation was significantly inhibited. Flow cytometry analyses using Annexin V-fluorescein revealed that Jurkat cells treated with the TRAIL-CM4 fusion protein exhibited increased apoptosis. Laser confocal microscopy showed that APB-CM4 and the fusion protein TRAIL-CM4 can bind to Jurkat cell membranes and initiate their destruction. ABP-CM4 enhances the antitumor activity of TRAIL by targeting and damaging the tumor cell membrane. In antibacterial experiments, agar well diffusion and bacterial growth inhibition curve assays revealed concentration-dependent TRAIL-CM4 antibacterial activity against Escherichia coli K12D31. The expressed TRAIL-CM4 fusion protein exhibited enhanced antitumor and antibacterial activities. Fusion protein expression allowed the two different proteins to function in combination.

Low toxic and high soluble camptothecin derivative 2–47 effectively induces apoptosis of tumor cells in vitro

The cytotoxic activity of camptothecin derivatives is so high that these compounds need to be further modified before their successful application as anti-cancer agents clinically. In this study, we reported the synthesis and biological evaluation of a novel camptothecin derivative called compound 2–47. The changes in structure did not reduce its activity to inhibit DNA topoisomerase I. Compound 2–47 induced apoptosis of many tumor cells including leukemia cells K562, Jurkat, HL-60, breast cancer cell BT-549, colon cancer cell HT-29 and liver cancer cell HepG2 with a half maximal inhibitory concentration (IC50) of 2- to 3-fold lower than HCPT as a control. In particular, 2–47 inhibited the proliferation of Jurkat cells with an IC50 of as low as 40 nM. By making use of Jurkat cell as a model, following treatment of Jurkat cells, compound 2–47 activated caspase-3 and PARP, resulting in a decreased Bcl-2/Bax ratio. These data showed that compound 2–47 induces Jurkat cell death through the mitochondrial apoptotic pathway. In addition, compound 2–47 showed a decreased cytotoxic activity against normal cells and an improved solubility in low-polar solvent. For example, compound 2–47 solutes in CHCl3 130-fold higher than HCPT. Taken together, our data demonstrated that camptothecin derivative 2–47 notably inhibits the tumor cell proliferation through mitochondrial-mediated apoptosis in vitro.

Synthesis and anti-cancer activity evaluation of 5-(2-carboxyethenyl)-isatin derivatives

A series of novel di- or trisubstituted isatin derivatives were designed and synthesized in 5–6 steps in 25–45% overall yields. Their structures were confirmed by 1H NMR and 13C NMR as well as LC-MS. The anticancer activity of the fourty-three new isatin derivatives against human T lymphocyte cells Jurkat was evaluated by MTT assay in vitro. SAR study suggested that the combination of 1-benzyl and 5-[trans-2-(methoxycarbonyl)ethen-1-yl] substitution greatly enhanced their cytotoxic activity. Among them, compound 2h was shown to have a significant cytotoxic activity with an IC50 value of 0.03 μM, more than 330-fold higher than that of it's mother molecule isatin. Investigation of the cell morphology changes and annexin-V/PI staining study demonstrated that compound 2h inhibited the proliferation of Jurkat cells by inducing apoptosis. Since compound 2h induced the dissipation of mitochondrial membrane potential and the activation of caspase-3, it was obvious that compound 2h inhibited the proliferation of Jurkat cells through the mitochondrial apoptotic pathway. Other than this, compound 2h exerted inhibition effect to many other tumor cells and only showed weak cytotoxic to human normal cells suggesting that compound 2h possessed a broad range of anticancer spectrum and high safety to normal cells.

The expression and function of E3 ligase SIAH2 in acute T lymphoblastic leukemia

IntroductionThe seven in absentia homolog 2 (SIAH2) protein plays a significant role in human cancer by regulating hypoxia-inducible factor-a (HIF-1α); however, its role in T-cell acute lymphoblastic leukemia (T-ALL) is less clear. MethodsImmunofluorescence evaluation of SIAH2 protein expression and location were conducted in Jurkat cell (a T-ALL cell line) as well as in bone marrow mononuclear cells (BMMNCs) from T-ALL and idiopathic thrombocytopenic purpura (ITP) patients. The expression of SIAH2 mRNA was also examined by quantitative real-time PCR (qRT-PCR) in these cells. Lentivirus-packed shRNA targeting on SIAH2 (Lv-shSIAH2) was used to knock down SIAH2 expression in Jurkat cells. Cell proliferation, apoptosis, invasion and protein levels were then determined by CCK-8 assay, annexin V-PI assay, transwell and Western blotting, respectively. ResultsThe mRNA expression of SIAH2 in BMMNCs from primary T-ALL patients was significantly higher than cells from ITP patients (P=0.0312); There were significant positive associations between SIAH2 expression and the extramedullary infiltration (EMI) (P=0.0003), especially with the mediastinal lymph node metastasis (P=0.0168) and the pleural effusion (P=0.014). However, SIAH2 expression in T-ALL BMMNCs was not correlated with age, gender, white cell count or the clinical risk classification. SIAH2 knockdown by shRNA led to increased apoptosis and decreased proliferation, migration and invasion of Jurkat cells. Moreover, Prolyl Hydroxylase (PHD), P27 and Caspase3 were upregulated and HIF-1α, VEGF, VEGF Receptor 2, MMP-13, CyclinE1, C-myc and BCL2 were downregulated in SIAH2 knockdown Jurkat cells. ConclusionsOur results suggest that SIAH2 regulates multi processes in T-ALL and may be an attractive therapeutic target.

Promoter methylation status of SFRP genes and induced apoptosis by demethylation in Jurkat cells

目的探讨人T淋巴细胞白血病（T-ALL）细胞株Jurkat细胞分泌性卷曲相关蛋白（SFRP）基因甲基化及去甲基化诱导细胞凋亡对Wnt/β-catenin信号通路的影响。方法以不同浓度（1.0、2.0、4.0 µmol/L）5-杂氮-2′-脱氧胞苷（5-Aza-CdR）对Jurkat细胞进行去甲基化处理，采用MTT法观察5-Aza-CdR对Jurkat细胞增殖的抑制作用，流式细胞术检测细胞凋亡率，甲基化特异性PCR(MSP)法检测药物处理前后SFRP基因的甲基化状态，实时荧光定量PCR检测SFRP基因以及RT-PCR检测survivin、c-myc和cyclin D1基因mRNA的表达改变，Western blot鉴定处理前后β-catenin的蛋白表达。结果1.0、2.0、4.0 µmol/L 5-Aza-CdR对Jurkat细胞的增殖有明显抑制作用，呈时间-剂量依赖性（P<0.05）；流式细胞术检测显示5-Aza-CdR作用Jurkat细胞48 h后，不同浓度5-Aza-CdR处理组与对照组比较细胞早期凋亡率明显升高（P<0.05）；SFRP1、SFRP2、SFRP4、SFRP5基因甲基化水平随5-Aza-CdR浓度升高而下降，呈剂量依赖性（P<0.05），同时mRNA表达水平较对照组明显上调（P<0.05）；Jurkat细胞总蛋白中β-catenin的蛋白表达随5-Aza-CdR浓度的升高而逐渐下降，呈剂量依赖性（P<0.05）；凋亡相关基因survivin、c-myc和cyclin D1的mRNA表达随5-Aza-CdR浓度的增高而降低，呈剂量依赖性（P<0.05）。结论逆转Jurkat细胞SFRP基因的甲基化，可以恢复SFRP基因转录表达，通过阻断β-catenin蛋白抑制Wnt/β-catenin信号通路的激活而诱导细胞凋亡。

Solid-phase synthesis of libraries of ethynylated aminosteroid derivatives as potential antileukemic agents

Steroids possessing an ethynyl group at position 17α (tertiary alcohols) are well known to be more stable than their non-ethynyl analogs (secondary alcohols). To facilitate the development of new drugs with better metabolic stability, we developed a new diethylsilyl acetylenic linker allowing us to rapidly synthesize libraries of ethynylated steroid derivatives using a solid-phase strategy. To illustrate its usefulness, this linker was used to expand the molecular diversity of a lead compound having a hydroxy acetylenic pattern and to potentially find new compounds with interesting cytotoxic activity against leukemia cell lines. Herein, we report the chemical synthesis and the characterization of three libraries of ethynylated aminosteroid derivatives using the diethylacetylenic linker. We discuss their antiproliferative activities obtained in 2 leukemia cell lines (HL-60 and Jurkat), which results provided new structure–activity relationships. We also identified a new promising aminosteroid derivative with an azetidine moiety (compound B1) inhibiting 60% and 75% of HL-60 and Jurkat cell proliferation, respectively, at 1μM. More generally, these results validate the use of a diethylsilyl acetylenic linker for researchers interested in generating libraries of alcohol derivatives with better stability and drug profile.

Identification of Novel Drug Targets in T-Cell Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and one of the leading causes of death in children with tumors. Genetic changes in T-cell acute lymphoblastic leukemia (T-ALL) are relatively well known, but the underlying molecular processes driving the disease remain insufficiently understood. Better knowledge of molecular events in T-ALL would improve our understanding of the development and maintenance of the disease and could also lead to the development of targeted and more effective treatments.We have compiled a large gene expression data-set from microarray studies of various hematological and lymphoid malignancies and healthy tissues that use a uniform technical platform (Affymetrix HG U133 Plus 2.0) (see abstract by Liuksiala et al). Data-set includes 1302 healthy samples and 4418 leukemia samples: 1713 acute myeloid leukemias, 1648 precursor B-ALLs, 801 chronic lymphocytic leukemias, 385 T-ALLs, and 215 chronic myeloid leukemias. From this data-set, we identified a number of transcription factors (TFs) that were differentially expressed in T-ALL, including high expression of NOTCH1 and BCL11B as previously reported (Weng et al Science 2004; Gutierrez et al Blood 2011). Several novel candidate TFs with specific expression in T-ALLs were also discovered, including strong expression of two poorly characterized TFs, SIX6 and PCBP3. These findings were validated using real-time quantitative PCR (RT-qPCR) in a cell line panel consisting of T-ALL and pre-B-ALL cell lines as well as healthy controls.We next sought to identify novel drug targets in T-ALL by comparing our leukemia expression data-set with the therapeutic target database (TTD). TTD is a database providing information about the known and explored therapeutic protein and nucleic acid targets, and the corresponding drugs aimed at these targets. We identified high expression of a nicotinic acetylcholine receptor (nAChR) subunit CHRNA3 (cholinergic receptor, nicotinic, alpha 3), which is a target of nAChR inhibitor bupropion. RT-qPCR confirmed the high expression of CHRNA3 in T-ALL cell lines but not in pre-B-ALL cells or healthy controls. The effect of bupropion was tested in Jurkat cells which represent T-ALL cell line with high expression of CHRNA3. Increasing concentrations of bupropion (1-100µM) resulted in dose-dependent decrease in proliferation of Jurkat cells as measured by cell viability assay AlamarBlue. As a control, cell lines with low level of CHRNA3 expression (CCRF-CEM and REH) were treated as well but these cells did not show any changes in the rate of proliferation.In summary, we have identified several candidate transcription factors which could have a leukemic role in T-ALL. Furthermore, we identified high expression of CHRNA3 in T-ALL, suggesting a role for the cholinergic system in T-cell leukemia, and thus a novel avenue in search of putative therapeutic options. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of the JAK/STAT and Bcl-2 Pathways Enhances Anti-Tumor Effects in an in Vitro model of T-Cell Acute Lymphoblastic Leukemia (T-ALL)

T cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive hematologic malignancy comprising 15% of pediatric and 25% of adult cases of ALL. With current treatment options, T-ALL survival rates have reached 50-60% in adults and 85% in children. Despite great strides in the treatment of this subset of ALL, T-ALL still shows resistance to first-line therapies in over 50% of adults and 25% of children, and relapse is often chemorefractory. Mutations in the Janus Activating Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathways and overexpression of the B cell lymphoma-2 (Bcl-2) protein are highly linked to the progression of T-ALL, and to the resistance of a number of available treatments for lymphoid malignancies. JAK/STAT is one of the main cytokine signaling pathways involved in hematopoietic cell growth and development. Indeed, the JAK/STAT pathway is often constitutively activated by T-ALL. The Bcl-2 pathway also plays an important role in cell survival. Overexpression of Bcl-2, an anti-apoptotic member of the Bcl-2 family, promotes cell survival by binding and neutralizing pro-apoptotic members. Inhibition of key proteins in both of these pathways has been greatly explored individually, but little is known about their combined effects on T-ALL. It is hypothesized that T-ALL manipulates both of these pathways as a means of escaping individual inhibition of either JAK/STAT or Bcl-2. Our hypothesis is that inhibiting both the JAK/STAT and Bcl-2 pathways with two small molecule inhibitors; Ruxolitinib (JAK 1/2 inhibitor) and Venetoclax (BH3 mimetic targeting Bcl-2), will inhibit T-ALL growth and survival. Proliferation of T-ALL was assessed by MTT assay and viability was measured by trypan blue and flow cytometry at 24, 48 and 72-hour time points post-treatment. Single-drug dose responses were conducted for both inhibitors. Six doses of both Ruxolitinib and Venetoclax were tested from a range of 0.156uM-5uM for Ruxolitinib and 1.56nM-50nM of Venetoclax. A response was seen for the three highest doses of both inhibitors (1.25uM, 2.5uM, and 5uM for Ruxolitinib and 12.5nM, 25nM, 50nM for Venetoclax). However, a synergistic effect was only achieved when combining 1.25uM Ruxolitinib with 25nM Venetoclax or 2.5uM Ruxolitinib with 12.5nM or 25nM Venetoclax. The combination dose of 1.25uM Ruxolitinib and 25nM Venetoclax demonstrated the greatest combined synergistic effect (CI<1) for all three assays at both 48 hours and 72 hours post-treatment. This optimal in vitro dose of 1.25uM Ruxolitinib and 25nM Venetoclax significantly lowered proliferation and viability of jurkat cells compared to no treatment (P<0.0001), vehicle control (P<0.0001) and the single-drug dose control groups (P<0.0001). Targeting both the JAK/STAT and Bcl-2 pathway with orally available FDA approved small molecule inhibitors could provide a novel alternative treatment for patients who relapse, fail or are resistant to first-line chemotherapeutic regiments. DisclosuresNo relevant conflicts of interest to declare.

Downregulation of SATB1 Increase the Invasiveness of Jurkat Cell Via Activation of the WNT/Beta-Catenin Signaling Pathway in Vitro

Backgroud: Special AT-rich sequence-binding protein-1 (SATB1) is critical for genome organizer that reprograms chromatin organization and transcription profiles, and associated with tumor growth and metastasis in several cancer types. Many studies suggest that SATB1 overexpression is an indicator of poor prognosis in various cancers, such as breast cancer, malignant cutaneous melanoma, liver cancer, etc. However, their expression patterns and function values for adult T-cell leukemia (ATL) are still largely unknown. Objective: The aim of this study is to examine the levels of SATB1 in ATL and to explore its function and mechanisms in ATL.METHODS: 20 ATL peripheral blood samples and 20 normal controls were collected. Expressions of SATB1 in both groups were evaluated by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Cellular proliferation and invasion of SATB1-knockdown Jurkat cells and cells in control group were evaluated by manually count and transwell matrigel invasion assay, respectively.RESULTS: SATB1 expressions were decreased in ATL peripheral blood mononuclear cells (p<0.001) compared with normal controls. Knockdown of SATB1 gene might increase Jurkat cell invasiveness through the activation of Wnt/β-Catenin signaling pathway.CONCLUSIONS: SATB1 expression is down-regulated in ATL and decreased expression of SATB1 increase the invasiveness of Jurkat cell through the activation of Wnt/β-Catenin signaling pathway in vitro.Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (81200399) and Key Clinical Disciplines of Guangdong Province (20111219) DisclosuresNo relevant conflicts of interest to declare.

Activation of Tax protein by c-Jun-N-terminal kinase is not dependent on the presence or absence of the early growth response-1 gene product.

The Tax protein of human T cell leukemia virus type 1 plays a major role in the pathogenesis of adult T cell leukemia (ATL), an aggressive neoplasia of CD4+ T cells. In the present study, we investigated whether the EGR-1 pathway is involved in the regulation of Tax-induced JNK expression in human Jurkat T cells transfected to express the Tax protein in the presence or absence of PMA or ionomycin. Overexpression of EGR-1 in Jurkat cells transfected to express Tax, promoted the activation of several genes, with the most potent being those that contained AP-1 (Jun/c-Fos), whereas knockdown of endogenous EGR-1 by small interfering RNA (siRNA) somewhat reduced Tax-mediated JNK-1 transcription. Additionally, luciferase-based AP-1 and NF-κB reporter gene assays demonstrated that inhibition of EGR-1 expression by an siRNA did not affect the transcriptional activity of a consensus sequence of either AP-1 or NF-κB. On the other hand, the apoptosis assay, using all-trans retinoic acid (ATRA) as an inducer of apoptosis, confirmed that siRNA against EGR-1 failed to suppress ATRA-induced apoptosis in Jurkat and Jurkat-Tax cells, as noted by the low levels of both DEVDase activity and DNA fragmentation, indicating that the induction of apoptosis by ATRA was Egr-1-independent. Finally, our data showed that activation of Tax by JNK-1 was not dependent on the EGR-1 cascade of events, suggesting that EGR-1 is important but not a determinant for the activity for Tax-induced proliferation of Jurkat cells.

Analysis of the expression of PHTF1 and related genes in acute lymphoblastic leukemia.

BackgroundPrevious study showed that downregulated BCL11B expression in T cell acute lymphoblastic leukemia (T-ALL) cell line Molt-4 inhibited cell proliferation and induce apoptosis, which may be related to PHTF1 gene overexpression. The objective of this study was to investigate the expression of PHTF1 and related genes in ALL and further explore its function in T-ALL cell lines.MethodsReal-time PCR was used to determine the gene expression level of PHTF1 in hematologic malignancies. The PHTF1, BCL11B, FEM1B and Apaf-1 gene expression levels and correlations were analyzed in patients with primary ALL (including T-ALL and B-ALL) and healthy individuals (HIs). Inhibition and overexpression of PHTF1 by lentiviral transduction were performed using the Molt-4 and Jurkat cell lines. Cell growth and apoptosis were measured by the Cell Counting Kit-8 assay and flow cytometry, respectively. Upon PHTF1 overexpression, the BCL11B, FEM1B and Apaf-1 gene expression levels were determined by real-time PCR.ResultsPHTF1 overexpression was found in both T-ALL (p = 0.004) and B-ALL (p < 0.001) groups compared with HIs group. A trend toward a negative correlation between the PHTF1 and BCL11B genes was detected for the T-ALL group, while positively correlated expression was found for the PHTF1 and BCL11B genes in HIs (P = 0.001). FEM1b and Apaf-1 overexpression was found in recently diagnosed ALL patients compared with HIs (p < 0.05). Positively correlated expression was found for the PHTF1, FEM1b and Apaf-1 genes in patients with ALL (p < 0.05) and HIs (p < 0.05). Direct up-regulation of PHTF1 expression inhibited the proliferation of Jurkat and Molt-4 cells and effectively induced apoptosis in Molt-4 cells. Direct inhibition of PHTF1 expression had no significant effect on the proliferation or apoptosis of Jurkat and Molt-4 cells. FEM1b and Apaf-1 overexpression, which did not obviously alter the BCL11B expression level, was detected in PHTF1-transduced T-ALL cell lines.ConclusionsPHTF1 overexpression is responsible for regulating cell proliferation and apoptosis in T-ALL cell lines. PHTF1 may be a tumor-suppressor like gene and a therapeutic target for triggering the PHTF1-FEM1b-Apaf-1 apoptosis pathway.

Effect of Bortezomib on Proliferation, Apoptosis and SHP-2 Gene Expression of Lymphoma Cells

To investigate the effects of Bortezomib on proliferation, apoptosis and SHP-2 gene expression of lymphoma Jurkat cells and Raji cells. Methylthiazoly tetrazolium assay (MTT) was used to observe the proliferation of Jurkat cells and Raji cells treated with bortezomib in different doses. Cell apoptosis was detected by morphological examination and flow cytometry. The level of SHP-2 mRNA expression before and after the treatment with bortezomib was measured by RT-PCR. Bortezomib could inhibit the proliferation of Jurkat and Raji cells and induce their apoptosis with time-and dose-dependent manner. After treatment with 5-100 nmol/L bortezomib, the expression of SHP-2 in Jurkat cells and Raji cells was upregulated. Bortezomib can inhibit the proliferation and induc the apoptosis of Jurkat and Raji cells obviously, upregulate the expression of SHP-2 mRNA, suggesting that the SHP-2 may participate in regulation of bortezomib induced apoptosis of Jurkat cells and Raji cells.

Effect of ADAM10 Inhibitor GI254023X on Proliferation and Apoptosis of Acute T-Lymphoblastic Leukemia Jurkat Cells In Vitro and Its Possible Mechanisms

To investigate the effect of ADAM10 inhibitor GI254023X on the proliferation and apoptosis of acute T-lymphoblastic leukemia Jurkat cells and its mechanisms. Jurkat cells were treated with different concentrations of GI254023X, the proliferation-inhibition curve was assayed and plotted by CCK-8 method, the cell viability and apoptosis was detected by flow cytometry with Annexin V and 7-AAD staining, the cleavage of Notch1 protein was determined by Western blot, the transcripts of anti-apoptotic genes BCL-2, MCL-1, BCL-xl and Notch1 target gene Hes-1 were detected by real-time PCR. The GI254023X obviously inhibited the proliferation of Jurkat cells in concentration-dependent manner. As compared with the control group, the apoptosis of cells increased along with increment of GI254023X concentration. Compared with control group, the expression of Cleaved Notch1 was down-regulated while the expression of Notch1 was up-regulated in a time-dependent manner after the treatment with GI254023X. The levels of MCL-1 and Hes-1 mRNA transcripts in Jurkat cells were reduced in GI254023X treated group, but did not show obvious effect on the level of BCL-2 and BCL-xl mRNA transcripts. GI254023X can remarkably inhibit proliferation and induce apoptosis of Jurkat cells. The inhibition of Notch1 activation and the down-regulation of apoptosis-related gene MCL-1 may be involved in the process of apoptosis.

Effects of tetramethylpyrazine injection on the proliferation and apoptosis of Jurkat cells and the relevant molecular mechanisms

Objective To study the effect of tetramethylpyrazine injection on proliferation and apoptosis of human acute lymphoblastic leukemia (ALL) cell line Jurkat and the relevant molecular mechanisms. Methods Cells were treated with tetramethylpyrazine injection at various concentrations (0, 0.25, 0.50, 0.75, 1.00, 1.25 and 1.50 mg/ml), CCK-8 method was used to detect the inhibition rates at 24, 48 and 72 h. After cells were treated with different concentrations of tetramethylpyrazine injection (0, 0.50, 1.00 and 1.50 mg/ml) for 48 h, the cell cycle and apoptosis were detected by flow cytometry, and the Aurora-B and Survivin protein expression were analyzed by Western blot. Results Compared with the control group (cells without tetramethylpyrazine injection treatment), various concentrations of tetramethylpyrazine injection could effectively inhibit Jurkat cells proliferation in a time-and dose-dependent manner (P < 0.05), and IC50 at 24 h,48 h and 72 h after treatment were (1.33±0.16), (0.91±0.10) and (0.67±0.11) mg/ml, respectively. After cells were treated with tetramethylpyrazine injection at different concentrations (0.5, 1.0 and 1.5 mg/ml) for 48 h, the number of treated cells in G2/M phase was increased, but that in S phase was decreased, and the apoptosis rates were significantly higher than that of control group, with a dose dependence (P < 0.05). The results of Western blot showed that the expression levels of Aurora-B and Survivin in treated cells were lower than that of control group, also with a dose dependence (P < 0.05). Conclusions Tetramethylpyrazine injection can effectively inhibit proliferation and induce apoptosis of Jurkat cells in vitro, and its underlying mechanisms involve with down-regulation of the Aurora-B and Survivin protein expression. Key words: Tetramethylpyrazine injection; Leukemia; Cell proliferation; Cell apoptosis; Aurora-B; Survivin