Apoptosis In EL-4 Cells Research Articles

Antitumor activity of α-pinene in T-cell tumors.

T-cell acute leukemia and lymphoma have a poor prognosis. Although new therapeutic agents have been developed, their therapeutic effects are suboptimal. α-Pinene, a monoterpene compound, has an antitumor effect on solid tumors; however, few comprehensive investigations have been conducted on its impact on hematologic malignancies. This report provides a comprehensive analysis of the potential benefits of using α-pinene as an antitumor agent for the treatment of T-cell tumors. We found that α-pinene inhibited the proliferation of hematologic malignancies, especially in T-cell tumor cell lines EL-4 and Molt-4, induced mitochondrial dysfunction and reactive oxygen species accumulation, and inhibited NF-κB p65 translocation into the nucleus, leading to robust apoptosis in EL-4 cells. Collectively, these findings suggest that α-pinene has potential as a therapeutic agent for T-cell malignancies, and further investigation is warranted.

The Role of MicroRNA 143 and MicroRNA 206 in The Regulation of Apoptosis in Mouse Lukemia Cancer Cells and Spermatogonial Cells.

Objective In cancer treatments, smart gene delivery via nanoparticles (NPs) can be targeted for cancer cells, while concurrently minimizing damage to healthy cells. This study assessed the efficiency of poly lactic-co-glycolic acid (PLGA)-miR 143/206 transfection on apoptosis in mouse leukemia cancer cells (El4) and spermatogonial stem cells (SSCs). Materials and Methods In this experimental study, neonatal mouse spermatogonia cells and EL4 cancer cell lines were used. MicroRNA-PLGA NPs were prepared, characterized, and targeted with folate. Several doses were evaluated to obtain a suitable miR dose that can induce appropriate apoptosis in EL4 cells, while not harming SSCs. Cells were treated separately at 3 doses of each miR (for miR 143, doses of 25, 50 and 75 nmol and for miR 206, doses of 50, 100 and 150 nmol). The experiments were performed at 24, 48 and 72 hours. Viability and apoptosis were investigated by MTT and Annexin Kits. Results Based on MTT assay results, the optimal dose of miR 143 was 75 nmol (59.87 ± 2.85 % SSC and 35.3 ± 0.78 % EL4) (P≤0.05), and for miR 206, the optimal dose was 150 nmol (54.82 ± 6.7 % SSC and 33.92 ± 3.01% EL4) (P≤0.05). The optimal time was 48 hours. At these doses, the survival rate of the EL4 cells was below the half maximal inhibitory concentration (IC50) and SSC survival was above 50%. Annexin V staining also confirmed the selected doses (for miR 143 total apoptosis was 6.62% ± 1.8 SSC and 37.4% ± 4.2 EL4 (P≤0.05), and miR 206 was (10.98% ± 1.5 SSC and 36.4% ± 3.7 EL4, P≤0.05). ConclusionUsing intelligent transfection by NPs, we were able to induce apoptosis on EL4 cells and maintain acceptable SSC survival rates.

Calcium plays a key role in paraoxon-induced apoptosis in EL4 cells by regulating both endoplasmic reticulum- and mitochondria-associated pathways

Context and objective: Paraoxon (POX) is one of the most toxic organophosphorus pesticides, but its toxic mechanisms associated with apoptosis remain unclear. The aim of this study was to investigate calcium-associated mechanisms in POX-induced apoptosis in EL4 cells.Materials and methods: EL4 cells were exposed to POX for 0-16 h. EGTA was used to chelate Ca2+ in extracellular medium, and heparin and procaine were used to inhibit Ca2+ efflux from the endoplasmic reticulum (ER). Z-ATAD-FMK was used to inhibit caspase-12 activity. The apoptotic rate assay, western blotting and immunocytochemistry (ICC) were used to reveal the mechanisms of POX-induced apoptosis.Results and discussion: POX significantly increased the expression and activation of caspase-12 and caspase-3, enhanced expression of calpain 1 and calpain 2, and induced the release of cyt c, but did not change the expression of Grp 78. Inhibiting caspase-12 activity alleviated POX-induced upregulation of calpain 1 and caspase-3, promoted POX-induced upregulation of calpain 2, and reduced POX-induced cyt c release, suggesting that there was a cross-talk between the ER-associated pathway and mitochondria-associated apoptotic signals. Attenuating intracellular calcium concentration with EGTA, heparin or procaine decreased POX-induced upregulation of calpain 1, calpain 2, caspase-12 and caspase-3, and reduced POX-induced cyt c release. After pretreatment with EGTA or procaine, POX significantly promoted expression of Grp 78.Conclusions: Calcium played a key role in POX-induced apoptosis in EL4 cells by regulating both ER- and mitochondria-associated pathways. The cross-talk of ER- and mitochondria-associated pathways was accomplished through calcium signal.

Anti-MS4a4B treatment abrogates MS4a4B-mediated protection in T cells and ameliorates experimental autoimmune encephalomyelitis

Recent data show that anti-CD20 therapy is effective for some autoimmune diseases, including multiple sclerosis (MS). However, the efficacy of anti-CD20 therapy for MS is largely limited because anti-CD20 antibodies target only B cells. In previous studies, we have investigated the function of MS4a4B, a novel CD20 homologue, in T cell proliferation. Here, we found that MS4a4B regulates not only T cell proliferation but also T cell apoptosis. Knockdown of MS4a4B by MS4a4B-siRNA or MS4a4B-shRNA-expressing vector promoted apoptosis in primary T cells and T32 cell line. In contrast, vector-driven over-expression of MS4a4B reduced apoptosis in EL-4 cells. Machinery analysis showed that MS4a4B-mediated T cell survival was associated with decreased activity of caspases 3, 8 and 9. Interestingly, binding of anti-MS4a4B antibodies to T cells induced activated T cells to undergo apoptosis. To test whether anti-MS4a4B antibody interferes with MS4a4B-mediated protection of T cells, we injected anti-MS4a4B antibodies into mice with experimental autoimmune encephalomyelitis (EAE). The results show that anti-MS4a4B treatment ameliorated the severity of EAE, accompanied by decreased Th1 and Th17 cell responses and reduced levels of pro-inflammatory cytokines in the central nervous system, suggesting that MS4a4B may serve as a target of antibody-based therapy for T cell-mediated diseases.

The inhibition of p21 on irradiation-induced apoptosis in EL-4 cells.

e13558 Background: To elucidate the effect of p21 on ionizing radiation-induced apoptosis in EL-4 cells. Methods: In EL-4 cells, RT-PCR, immunocytochemistry and flow cytometry (FCM) were used to analyze the changes in the expression of p21 with time and doses caused by ionizing radiation; RNAi was used to silence the expression of p21 in EL-4 cells; FCM was applied to analyze the distribution of cell cycle,the number of the chromosome and the apoptosis in wild or p21-silencing EL-4 cells exposed to X-rays. Results: In EL-4 cells, p21 protein level was markedly increased at 8 ~ 72 h after exposure in 4.0 Gy of X-ray in vitro (p<0.01), as well as exposed in 1.0 ~ 4.0 Gy of X-ray at 24 h in vitro (p<0.05). Results of time-course experiments showed that, in EL-4 cells exposed to 4.0 Gy X-rays, the percentage in G0/G1 phase increased while that in S phase decreased, significantly different with the sham-irradiated groups (p<0.05), during 8-48h after exposure; the percentage of diploid cells increased significantly during 8-48 h (p<0.01), and the percentage of tetraploid and octoploid cells decreased significantly at 2 h and 8 h (p<0.05); the percentage of apoptosis during 2-72 h increased significantly (p<0.05-p<0.01). Compared with wild EL-4 cells, in p21-silencing EL-4 cells, the distribution of cell cycle had no apparent difference; the percentage of diploid cells decreased significantly and the percentage of octoploid cells increased significantly (p<0.01); the percentage of apoptosis increased significantly (p<0.05). Conclusions: p21 inhibited the apoptosis induced by ionizing radiation in EL-4 cells.

Allicin induces apoptosis in EL-4 cells in vitro by activation of expression of caspase-3 and -12 and up-regulation of the ratio of Bax/Bcl-2

Garlic (Allium sativum L.; Liliaceae) has been widely demonstrated in the role of cancer prevention, but the specific compound in garlic corresponding to this effect and its mechanisms are not clearly known. Allicin is one of the organic sulphur compounds derived from garlic. In the present study we investigated the anti-proliferative and pro-apoptotic activities of allicin in murine T-lymphocytes (EL-4) and the mechanism of inducing apoptosis in vitro. The results showed that allicin was effective in inhibiting the proliferation of EL-4 cells in vitro in a concentration-dependent manner. Further, allicin could induce the formation of apoptotic bodies, nuclear condensation, DNA spallation, and even activated the expression of caspase-3, -12 and cytochrome C (cyt C). Finally, allicin up-regulated the ratio of Bax/Bcl-2 and induced a mitochondrion membrane potential (MMP) decrease. Allicin induced apoptosis in EL-4 cells in a time- and concentration-dependent manner, in which the mitochondrial pathway might play a central role.

Resveratrol (trans‐3,5,4′‐trihydroxystilbene) suppresses EL4 tumor growth by induction of apoptosis involving reciprocal regulation of SIRT1 and NF‐κB

Understanding the molecular mechanisms through which natural products and dietary supplements exhibit anticancer properties is crucial and can lead to drug discovery and chemoprevention. The current study sheds new light on the mode of action of resveratrol (RES), a plant-derived polyphenolic compound, against EL-4 lymphoma growth. Immuno-compromised NOD/SCID mice injected with EL-4 tumor cells and treated with RES (100 mg/kg body weight) showed delayed development and progression of tumor growth and increased mean survival time. RES caused apoptosis in EL4 cells through activation of aryl hydrocarbon receptor (AhR) and upregulation of Fas and FasL expression in vitro. Blocking of RES-induced apoptosis in EL4 cells by FasL mAb, cleavage of caspases and PARP, and release of cytochorme c, demonstrated the participation of both extrinsic and intrinsic pathways of apoptosis. RES also induced upregulation of silent mating type information regulation 2 homolog, 1 (SIRT1) and downregulation of nuclear factor kappa B (NF-κB) in EL4 cells. siRNA-mediated downregulation of SIRT1 in EL4 cells increased the activation of NF-κB but decreased RES-mediated apoptosis, indicating the critical role of SIRT1 in apoptosis via blocking activation of NF-κB. These data suggest that RES-induced SIRT1 upregulation promotes tumor cell apoptosis through negative regulation of NF-κB, leading to suppression of tumor growth.

Role of MS4a4B in regulation of T cell apoptosis (104.20)

Abstract MS4a4B is a membrane protein that highly expressed in T cells. We have demonstrated previously that MS4a4B has an inhibitory role in T cell proliferation. Interestingly, a decreased level of MS4a4B is associated with apoptotic T cells. In this study, we further analyzed the role of MS4a4B in T cell proliferation and apoptosis. We co-cultured MS4a4B+ EL4 cells (GFP+) with MS4a4B- EL4 cells (GFP-) and compared cell proliferation by flow cytometry. We found that MS4a4B inhibited EL4 cell proliferation during the early stage of co-culture. However, the percentage of MS4a4B+ EL4 cells was enhanced, suggesting that MS4a4B prolonged EL4 cell survival. To determine if MS4a4B expression reduced apoptosis in EL4 cells, we analyzed apoptotic cells during MS4a4B+/MS4a4B- cell co-culture. The results showed that the apoptosis percentage in MS4a4B+ EL4 cells was markedly lower than in MS4a4B- EL4 cells. To determine whether MS4a4B protected T cells from activation-induced cell death (AICD), we knocked down MS4a4B expression in mouse T cell line (T32 cells) and primary T cells by MS4a4B shRNA vector and MS4a4B siRNA. Knockdown of MS4a4B promoted anti-CD3/anti-CD28-induced apoptosis in T32 cells and primary T cells. Machinery analysis showed that over-expression of MS4a4B decreased the activity of caspase 3/7, caspase 8 and caspase 9. Our data suggest that MS4a4B not only has an inhibitory role in T cell proliferation but also plays a role in regulation of T cell apoptosis.

Calcium signals and caspase-12 participated in paraoxon-induced apoptosis in EL4 cells

In order to investigate whether calcium signals participate in paraoxon (POX)-induced apoptosis in EL4 cells, real-time laser scanning confocal microscopy (LSCM) was used to detect Ca 2+ changes during the POX application. Apoptotic rates of EL4 cells and caspase-12 expression were also evaluated. POX (1–10 nM) increased intracellular calcium concentration ([Ca 2+]i) in EL4 cells in a dose-dependent manner at early stage (0–2 h) of POX application, and apoptotic rates of EL4 cells after treatment with POX for 16 h were also increased in a dose-dependent manner. Pre-treatment with EGTA, heparin or procaine attenuated POX-induced [Ca 2+]i elevation and apoptosis. Additionally, POX up-regulated caspase-12 expression in a dose-dependent manner, and pre-treatment with EGTA, heparin or procaine significantly inhibited POX-induced increase of caspase-12 expression. Our results suggested that POX induced [Ca 2+]i elevation in EL4 cells at the early stage of POX-induced apoptosis, which might involve Ca 2+ efflux from the endoplasmic reticulum (ER) and Ca 2+ influx from extracellular medium. Calcium signals and caspase-12 were important upstream messengers in POX-induced apoptosis in EL4 cells. The ER-associated pathway possibly operated in this apoptosis.

Cell death induced by vaccine adjuvants containing surfactants

Many vaccine adjuvants contain surface-active agents, but the immunological roles played by these components have been essentially ignored. The objective of this study was to examine possible apoptotic and necrotic effects of the surface-active agents, Pluronic L121 and Tween 80, which are components of L121-adjuvant (a formulation we synthesized with the aim of representing several commercially produced adjuvants), on EL4 lymphoma cells. Cell viability and cytolytic effects were analyzed using the MTT and LDH release assays, and the distribution of cells in different stages of the cell cycle after treatment with these agents was analyzed by propidium iodide (PI) staining and flow cytometry. L121-adjuvant was shown to induce cell cycle arrest and inhibit cell proliferation. Treatment of EL4 cells with surface-active agents resulted in a concentration-dependent increase in the apoptotic/necrotic cell populations. Fluorescence microscopy using Hoechst 33342 staining demonstrated chromosome condensation and DNA fragmentation in cells treated with surfactants or adjuvant. The apoptotic and necrotic effects of vaccine adjuvant containing surface-active agents were confirmed by Annexin V/propidium iodide staining and flow cytometric analysis. Pretreatment of EL4 cells with zVAD-fmk, a broad range caspase inhibitor, partially prevented apoptosis induced by Pluronic L121, but did not prevent the cell death induced by Tween 80 or L121-adjuvant. These findings suggested that Tween 80 and L121-adjuvant induced apoptosis in EL4 cells via a “non-classical” caspase-independent pathway. Results presented in this study suggest mechanisms of elicitation of CD8 +, class I-restricted CTL response by soluble antigens mediated by the vaccine adjuvant containing surface-active agents.

Paraoxon induces apoptosis in EL4 cells via activation of mitochondrial pathways

The toxicity of organophosphorus compounds, such as paraoxon (POX), is due to their anticholinesterase action. Recently, we have shown that, at noncholinergic doses (1 to 10 nM), POX (the bioactive metabolite of parathion) causes apoptotic cell death in murine EL4 T-lymphocytic leukemia cell line through activation of caspase-3. In this study, by employing caspase-specific inhibitors, we extend our observations to elucidate the sequence of events involved in POX-stimulated apoptosis. Pretreatment of EL4 cells with the caspase-9-specific inhibitor zLEHD-fmk attenuated POX-induced apoptosis in a dose-dependent manner, whereas the caspase-8 inhibitor zIETD-fmk had no effect. Furthermore, the activation of caspase-9, -8, and -3 in response to POX treatment was completely inhibited in the presence of zLEHD-fmk, implicating the involvement of caspase 9-dependent mitochondrial pathways in POX-stimulated apoptosis. Indeed, under both in vitro and in vivo conditions, POX triggered a dose- and time-dependent translocation of cytochrome c from mitochondria into the cytosol, as assessed by Western blot analysis. Investigation of the mechanism of cytochrome c release revealed that POX disrupted mitochondrial transmembrane potential. Neither this effect nor cytchrome c release was dependent on caspase activation, since the general inhibitor of the caspase family zVAD-fmk did not influence both processes. Finally, POX treatment also resulted in a time-dependent up-regulation and translocation of the proapoptotic molecule Bax to mitochondria. Inhibition of this event by zVAD-fmk suggests that the activation and translocation of Bax to mitochondria is subsequent to activation of the caspase cascades. The results indicate that POX induces apoptosis in EL4 cells through a direct effect on mitochondria by disrupting its transmembrane potential, causing the release of cytochrome c into the cytosol and subsequent activation of caspase-9. Inhibition of this specific pathway might provide a useful strategy to minimize organophosphate-induced poisoning.

Inactivation of the small GTP binding protein Rho induces multinucleate cell formation and apoptosis in murine T lymphoma EL4.

The small G-protein Rho regulates the actin microfilament-dependent cytoskeleton. Exoenzyme C3 of Clostridium botulinum ADP-ribosylates Rho at Asn41, a modification that functionally inactivates Rho. Using a Sindbis virus-based transient gene expression system, we studied the role of Rho in murine EL4 T lymphoma cells. We generated a double subgenomic infectious Sindbis virus (dsSIN:C3) recombinant which expressed C3 in >95% of EL4 cells. This intracellular C3 resulted in modification and inactivation of virtually all endogenous Rho. dsSIN:C3 infection led to the formation of multinucleate cells, likely by inhibiting the actin microfilament-dependent step of cytokinesis. Intriguingly, in spite of the inhibition of cytokinesis, karyokinesis continued, with the result that cells containing a nuclear DNA content as high as 16N (eight nuclei) were observed. In addition, dsSIN:C3-mediated inactivation of Rho was a potent activator of apoptosis in EL4 cells. To discern whether the formation of multinucleate cells was responsible for the activation of apoptosis, 5-fluorouracil (5-FUra) was used to induce cell cycle arrest. As expected, EL4 cells treated with 5-FUra were prevented from forming multinucleate cells upon infection with dsSIN:C3. dsSIN:C3 infection, however, still caused marked apoptosis in 5-FUra-treated cells, indicating that this activation of apoptosis was independent of multinucleate cell formation.

Modification of the aerobic cytotoxicity of etanidazole

Purpose : To determine the feasibility of modifying the aerobic cytotoxicity of etanidazole without interfering with the tumoricidal action of radiation plus etanidazole. Methods and Materials : The aerobic cytotoxicity of etanidozole was studied using two different models: (1) Induction of apoptosis in EL4 cells: apoptotic DNA fragmentation was analyzed by agarose gel electrophoresis following 24 h treatment with etanidazole alone or in combination with various modifiers. (2) Spinal cord neuronal loss in organotypic roller tube cultures: Survival of acetylcholinesterase positive ventral horn neurons was analyzed morphometrically following 72 h treatment with etanidazole alone or in combination with vitamin E succinate. Results : Etanidazole (10 mM) induced apoptosis in EL4 cells. This effect was suppressed by 24 h treatment with TPA, IBMX, the free radical scavenger TEMPOL or vitamin E succinate. Vitamin E succinate also protected spinal cord cultures from etanidazole-induced neuronal loss. Conclusion : These results suggest that it might be possible to modify the neurotoxicity of etanidazole with agents that would not be expected to interfere with the tumoricidal action of radiation plus etanidazole.