17Alpha-estradiol arrests cell cycle progression at G2/M and induces apoptotic cell death in human acute leukemia Jurkat T cells

A pharmacological dose (2.5-10 μM) of 17α-estradiol (17α-E₂) exerted a cytotoxic effect on human leukemias Jurkat T and U937 cells, which was not suppressed by the estrogen receptor (ER) antagonist ICI 182,780. Along with cytotoxicity in Jurkat T cells, several apoptotic events including mitochondrial cytochrome c release, activation of caspase-9, -3, and -8, PARP degradation, and DNA fragmentation were induced. The cytotoxicity of 17α-E₂ was not blocked by the anti-Fas neutralizing antibody ZB-4. While undergoing apoptosis, there was a remarkable accumulation of G₂/M cells with the upregulatoin of cdc2 kinase activity, which was reflected in the Thr56 phosphorylation of Bcl-2. Dephosphorylation at Tyr15 and phosphorylation at Thr161 of cdc2, and significant increase in the cyclin B1 level were underlying factors for the cdc2 kinase activation. Whereas the 17α-E₂-induced apoptosis was completely abrogated by overexpression of Bcl-2 or by pretreatment with the pan-caspase inhibitor z-VAD-fmk, the accumulation of G₂/M cells significantly increased. The caspase-8 inhibitor z-IETD-fmk failed to influence 17α-E₂-mediated caspase-9 activation, but it markedly reduced caspase-3 activation and PARP degradation with the suppression of apoptosis, indicating the contribution of caspase-8; not as an upstream event of the mitochondrial cytochrome c release, but to caspase-3 activation. In the presence of hydroxyurea, which blocked the cell cycle progression at the G₁/S boundary, 17α-E₂ failed to induce the G₂/M arrest as well as apoptosis. These results demonstrate that the cytotoxicity of 17α-E₂ toward Jurkat T cells is attributable to apoptosis mainly induced in G₂/M-arrested cells, in an ER-independent manner, via a mitochondria-dependent caspase pathway regulated by Bcl-2.     

Goniothalamin-induced oxidative stress,DNA damage and apoptosis via caspase-2 independent and Bcl-2 independent pathways in Jurkat T-cells

Goniothalamin (GTN) isolated from Goniothalamus sp. has been demonstrated to induce apoptosis in a variety of cancer cell lines including Jurkat T leukemia cells. However, the mechanism of GTN-induced apoptosis upstream of mitochondria is still poorly defined. In this study, GTN caused a decrease in GSH with an elevation of reactive oxygen species as early as 30 min and DNA damage as assessed by Comet assay. Analysis using topoisomerase II processing of supercoiled pBR 322 DNA showed that GTN caused DNA damage via a topoisomerase II-independent pathway suggesting that cellular oxidative stress may contribute to genotoxicity. A 12-fold increase of caspase-2 activity was observed in GTN-treated Jurkat cells after 4 h treatment and this was confirmed using Western blotting. Although the caspase-2 inhibitor Z-VDVAD-FMK inhibited the proteolytic activity of caspase-2, apoptosis ensued confirming that caspase-2 activity was not crucial for GTN-induced apoptosis. However, GTN-induced apoptosis was completely abrogated by N-acetylcysteine further confirming the role of oxidative stress. Since cytochrome c release was observed as early as 1 h without any appreciable change in Bcl-2 protein expression, we further investigated whether overexpression of Bcl-2 confers resistance in GTN-induced cytotoxicity. Using a panel of Jurkat Bcl-2 transfectants, GTN cytotoxicity was not abrogated in these cells. In conclusion, GTN induces DNA damage and oxidative stress resulting in apoptosis which is independent of both caspase-2 and Bcl-2.     

Bcl-2 blocks 2-methoxyestradiol induced leukemia cell apoptosis by a p27-dependent G1/S cell cycle arrest in conjunction with NF-κB activation

2-Methoxyestradiol (2-ME2) induces leukemia cells to undergo apoptosis in association with Bcl-2 inactivation but the mechanisms whereby Bcl-2 contributes to protection against programmed cell death in this context remain unclear. Here we showed that 2-ME2 inhibited the proliferation of Jurkat leukemia cells by markedly suppressing the levels of cyclins D3 and E, E2F1 and p21^Cip1/Waf1 and up-regulating p16^INK4A. Further, 2-ME2 induced apoptosis of Jurkat cells in association with down-regulation and phosphorylation of Bcl-2 (as mediated by JNK), up-regulation of Bak, activation of caspases-9 and -3 and PARP-1 cleavage. To determine the importance and mechanistic role of Bcl-2 in this process, we enforced its expression in Jurkat cells by retroviral transduction. Enforcing Bcl-2 expression in Jurkat cells abolished 2-ME2-induced apoptosis and instead produced a G1/S phase cell cycle arrest in association with markedly increased levels of p27^Kip1. Bcl-2 and p27^Kip1 were localized mainly in the nucleus in these apoptotic resistant cells. Interestingly, NF-κB activity and p50 levels were increased by 2-ME2 and suppression of NF-κB signaling reduced p27^Kip1 expression and sensitized cells to 2-ME2-induced apoptosis. Importantly, knocking-down p27^Kip1 in Jurkat Bcl-2 cells sensitized them to spontaneous and 2-ME2-induced apoptosis. Thus, Bcl-2 prevented the 2-ME2-induced apoptotic response by orchestrating a p27^Kip1-dependent G1/S phase arrest in conjunction with activating NF-κB. Thus, we achieved a much better understanding of the penetrance and mechanistic complexity of Bcl-2 dependent anti-apoptotic pathways in cancer cells and why Bcl-2 inactivation is so critical for the efficacy of apoptosis and anti-proliferative inducing drugs like 2-ME2.     

T cell selective apoptosis by a novel immunosuppressant,FTY720, is closely regulated with Bcl-2

1. A novel immunosuppressant FTY720 caused a significant decrease in peripheral T lymphocytes, but not in B lymphocytes upon oral administration. This decrease was mainly a result of FTY720-induced apoptosis. In this study, we confirmed FTY720-induced T cell selective apoptosis using lymphoma cell lines in vitro. 2. Viability loss, DNA fragmentation, Annexin V binding, and caspases activation (caspase-3, -8, and -9) were observed in Jurkat cells (T lymphoma cells), but not significantly in BALL-1 cells (B lymphoma cells). These results indicated that FTY720 selectively induced apoptosis in T cell lymphoma to a greater extent than in B cell lymphoma, a finding that is similar to the result observed when FTY720 was treated with T lymphocytes and B lymphocytes in vitro. 3. FTY720 released cytochrome c from mitochondria in Jurkat intact cells as well as from isolated Jurkat mitochondria directly, but not from mitochondria in BALL-1 cells nor from isolated BALL-1 mitochondria. 4. BALL-1 cells and B cells had more abundant mitochondria-localized anti-apoptotic protein Bcl-2 than did Jurkat cells and T cells. 5. FTY720-induced apoptosis is inhibited by the overexpression of Bcl-2, suggesting that the cellular Bcl-2 level regulates the sensitivity to FTY720.     

Mitogenic activity of <Emphasis Type="Italic">Artocarpus lingnanensis</Emphasis> lectin and its apoptosis induction in Jurkat T cells

Lectins are a class of carbohydrate-binding proteins or glycoproteins and used in the purification and characterization of glycoproteins according to their specificity to carbohydrates. In the present study, the mitogenic activity of Artocarpus lingnanensis lectin (ALL) and its apoptosis induction in Jurkat T cells were explored. MTT assay revealed strong mitogenic potential of ALL. Meanwhile, the anti-cancer activity of ALL was also explored using the human leukemic Jurkat T cell line. ALL exhibited strong binding affinity (97%) to the cell membrane, which could be effectively inhibited by N-acetyl-d-galactosaminide (NAD). ALL induced time- and dose-dependent growth inhibition in Jurkat T cells. ALL could induce morphologic change and increase the hypodiploid cell population with the decreased population of S and G2/M phases. The induction of phosphatidylserine externalization and PARP cleavage further confirmed its apoptosis-inducing activity due to the activation of caspase-8 and -9. The inhibition of caspase-9 but not caspase-8 could rescue ALL-induced apoptotic cells. Further studies showed that ALL enhanced the cleavage of Bid, the release of cytochrome C, the depolarization of mitochondria and the activation of caspase-3. ALL downregulated the expression of Bcl-xl and Bcl-2 without impact on Bax and Bad. In addition, the activation of p38/JNK MAPK signaling pathways was observed to be a requisite for ALL apoptotic activity. In contrast, ALL could not induce apoptosis of normal T cells. These findings present the differential effect of ALL on Jurkat and normal T lymphocytes, suggesting its therapeutic value in leukemia.     

Chlorpyrifos induces apoptosis in human T cells

It was found previously that organophosphorus pesticides (OPs) significantly inhibited cytotoxic T lymphocyte (CTL) activity. To explore the mechanism of OP-induced inhibition of CTL activity, the present study investigated whether OPs can induce cell death/apoptosis in T cells. Jurkat human T cells were treated with chlorpyrifos at 0-100 ppm for 2, 4, and 6h at 37 degrees C in vitro. It was found that chlorpyrifos induced cell death of Jurkat human T cells in a dose- and time-dependent manner, as shown by MTT and LDH assays. Then, it was investigated if chlorpyrifos-induced cell death consisted of apoptosis, as determined by analysis of Annexin-V staining and the intracellular level of active caspase-3 by flow cytometry, and DNA fragmentation analysis. It was found that chlorpyrifos induces apoptosis in Jurkat T cells in a dose- and time-dependent manner, as determined by analysis of Annexin-V staining. DNA fragmentation was detected when cells were treated with 50 or 100 ppm chlorpyrifos for 4 and 6h. Chlorpyrifos also induced an increase in intracellular active caspase-3 in Jurkat T cells in a dose- and time-dependent manner, and a caspase-3 inhibitor, Z-DEVD-FMK, significantly inhibited chlorpyrifos-induced apoptosis. These findings indicate that chlorpyrifos can induce apoptosis in human Jurkat T cell cells, and this effect is partially mediated by the activation of intracellular caspase-3.     

Mechanism underlying cytotoxicity of thialysine, lysine analog, toward human acute leukemia Jurkat T cells

We first report the mechanism for the inhibitory effect of the lysine analog, thialysine on human acute leukemia Jurkat T cells. When Jurkat T cells were treated with thialysine (0.32-2.5 mM), apoptotic cell death along with several biochemical events such as mitochondrial cytochrome c release, caspase-9 activation, caspase-3 activation, degradation of poly (ADP-ribose) polymerase, and DNA fragmentation was induced in a dose- and time-dependent manner. However, these thialysine-induced apoptotic events were significantly abrogated by an ectopic expression of Bcl-xL, which is known to block mitochondrial cytochrome c release. Decylubiquinone, a mitochondrial permeability transition pore inhibitor, also suppressed thialysine-induced apoptotic events. Comparison of the thialysine-induced alterations in the cell cycle distribution between Jurkat T cells transfected with Bcl-xL gene (J/Bcl-xL) and Jurkat T cells transfected with vector (J/Neo) revealed that the apoptotic cells were mainly derived from the cells accumulated in S and G2/M phases following thialysine treatment. The interruption of cell cycle progression in the presence of thialysine was accompanied by a significant decline in the protein level of cdk4, cdk6, cdc2, cyclin A, cyclin B1, and cyclin E. These results demonstrate that the cytotoxic activity of thialysine toward Jurkat T cells is attributable to not only apoptotic cell death mediated by a mitochondria-dependent death signaling pathway, but also interruption of cell cycle progression by a massive down-regulation in the level of cdks and cyclins.     

Helenalin bypasses Bcl-2-mediated cell death resistance by inhibiting NF-κB and promoting reactive oxygen species generation

Evasion of cell death by overexpression of anti-apoptotic proteins, such as Bcl-2, is commonly observed in cancer cells leading to a lack of response to chemotherapy. Hence, there is a need to find new chemotherapeutic agents that are able to overcome chemoresistance mediated by Bcl-2 and to understand their mechanisms of action. Helenalin, a sesquiterpene lactone (STL), induces cell death and abrogates clonal survival in a highly apoptosis-resistant Bcl-2 overexpressing Jurkat cell line as well as in two other Bcl-2 overexpressing solid tumor cell lines (mammary MCF-7; pancreatic L6.3pl). This effect is not achieved by directly affecting the mitochondria-protective function of Bcl-2 in the intrinsic pathway of apoptosis since Bcl-2 overexpressing Jurkat cells do not show cytochrome c release and dissipation of mitochondrial membrane potential upon helenalin treatment. Moreover, helenalin induces an atypical form of cell death with necrotic features in Bcl-2 overexpressing cells, neither activating classical mediators of apoptosis (caspases, AIF, Omi/HtrA2, Apaf/apoptosome) nor ER-stress mediators (BiP/GRP78 and CHOP/GADD153), nor autophagy pathways (LC3 conversion). In contrast, helenalin was found to inhibit NF-κB activation that was considerably increased in Bcl-2 overexpressing Jurkat cells and promotes cell survival. Moreover, we identified reactive oxygen species (ROS) and free intracellular iron as mediators of helenalin-induced cell death whereas activation of JNK and abrogation of Akt activity did not contribute to helenalin-elicited cell death. Our results highlight the NF-κB inhibitor helenalin as a promising chemotherapeutic agent to overcome Bcl-2-induced cell death resistance.     

Pentoxifylline augments TRAIL/Apo2L mediated apoptosis in cutaneous T cell lymphoma (HuT-78 and MyLa) by modulating the expression of antiapoptotic proteins and death receptors

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a promising anticancer agent but cutaneous T lymphoma cells (CTCL) are less sensitive to TRAIL-induced apoptosis. Here, we report that pentoxifylline (PTX), a phosphodiesterase inhibitor, augments TRAIL-mediated apoptosis in HuT-78 and MyLa cells through modulating extrinsic death receptors and intrinsic mitochondria dependent pathways. Our results clearly show that PTX augments TRAIL-mediated activation of caspase-8 and induces cleavage of Bid, although PTX alone cannot activate caspase-8. This is followed by cytochrome c release and subsequent, activation of caspase-9 and caspase-3 and cleavage of poly (ADP ribose) polymerase (PARP). Combined treatment downregulates the expression of various antiapoptotic proteins including c-FLIP, Bcl-xl, cIAP-1, cIAP-2 and XIAP. PTX induces the expression of death receptors DR4 and DR5 on cell surface of both the cell types where c-Jun NH2-terminal kinase (JNK) pathway plays an important role. Moreover, combined silencing of DR4 and DR5 by small interfering RNA abrogates the ability of PTX to induce TRAIL-mediated apoptosis. Thus, this is the first demonstration that PTX can potentiate TRAIL-mediated apoptosis through downregulation of cell survival gene products and upregulation of death receptors.     

Dependency of 2-methoxyestradiol-induced mitochondrial apoptosis on mitotic spindle network impairment and prometaphase arrest in human Jurkat T cells

The present study sought to determine the correlation between 2-methoxyestradiol (2-MeO-E₂)-induced cell cycle arrest and 2-MeO-E₂-induced apoptosis. Exposure of Jurkat T cell clone (JT/Neo) to 2-MeO-E₂ (0.5–1.0 μM) caused G₂/M arrest, Bak activation, Δψm loss, caspase-9 and -3 activation, PARP cleavage, intracellular ROS accumulation, and apoptotic DNA fragmentation, whereas none of these events except for G₂/M arrest were induced in Jurkat T cells overexpressing Bcl-2 (JT/Bcl-2). Under these conditions, Cdk1 phosphorylation at Thr-161 and dephosphorylation at Tyr-15, up-regulation of cyclin B1 expression, histone H1 phosphorylation, Cdc25C phosphorylation at Thr-48, Bcl-2 phosphorylation at Thr-56 and Ser-70, Mcl-1 phosphorylation at Ser-159/Thr-163, and Bim phosphorylation were detected irrespective of Bcl-2 overexpression. Concomitant treatment of JT/Neo cells with 2-MeO-E₂ and the G₁/S blocking agent aphidicolin resulted in G₁/S arrest and abrogation of all apoptotic events, including Cdk1 activation, phosphorylation of Bcl-2, Mcl-1 and Bim, and ROS accumulation. The 2-MeO-E₂-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by a Cdk1 inhibitor, but not by an Aurora A kinase (AURKA), Aurora B kinase (AURKB), JNK, or p38 MAPK inhibitor. Immunofluorescence microscopic analysis revealed that 2-MeO-E₂-induced mitotic arrest was caused by mitotic spindle network impairment and prometaphase arrest. Whereas 10–20 μM 2-MeO-E₂ reduced the proportion of intracellular polymeric tubulin to monomeric tubulin, 0.5–5.0 μM 2-MeO-E₂ increased it. These results demonstrate that the apoptogenic effect of 2-MeO-E₂ (0.5–1.0 μM) was attributable to mitotic spindle defect-mediated prometaphase arrest, Cdk1 activation, phosphorylation of Bcl-2, Mcl-1, and Bim, and activation of Bak and mitochondria-dependent caspase cascade.     

Apoptosis induced by a new member of saponin family is mediated through caspase-8-dependent cleavage of Bcl-2

OSW-1 is a new member of cholestane saponin family, which is cytotoxic against several types of malignant cells. We reported herein that OSW-1 induced apoptosis of mammalian cells in a concentration- and time-dependent manner. The drug-induced apoptosis was mediated through the mitochondrial pathway, involving the cleavage of Bcl-2. This drug-induced Bcl-2 cleavage in Chinese hamster ovary (CHO) cells could be suppressed either by dominant-negative caspase-8 or by a caspase-8 inhibitor, suggesting that the Bcl-2 cleavage is dependent on caspase-8. In contrast, the Bcl-2 cleavage was independent of caspase-3 activity. The inhibition of caspase-8 activity also resulted in the reduction of apoptotic cells, indicating that Bcl-2 cleavage induced by caspase-8 promotes the progression of apoptosis. The involvement of the caspase-8 activity in the processes of the OSW-1-induced apoptosis was further examined by using caspase-8-deficient Jurkat T cells. It was found that the caspase-8-deficient cells were resistant to OSW-1-induced Bcl-2 cleavage or apoptosis. Furthermore, the small subunit of caspase-8 was found to interact with Bcl-2 as determined by yeast two-hybrid and coimmunoprecipitation assays. Overexpression of caspase-8 small subunit reduced the cleavage of Bcl-2 and inhibited the apoptosis induced by OSW-1. Taken together, these results demonstrate that OSW-1 is capable of inducing apoptosis in mammalian cells, in which the caspase-8-dependent cleavage of Bcl-2 plays an important role.     

γ-Tocotrienol induced cell cycle arrest and apoptosis via activating the Bax-mediated mitochondrial and AMPK signaling pathways in 3T3-L1 adipocytes

This study aimed to examine the anti-proliferative effects of α-, γ- and δ-tocotrienols (αT3, γT3 and δT3), and α-tocopherol on 3T3-L1 adipocytes. Results showed that compared with other vitamin E analogues, γT3 demonstrated the most potent anti-proliferative effect on 3T3-L1 cells. It significantly caused a reduction in mitochondrial membrane potential (Δψm) and an increase in ROS formation, as well as inducing cell apoptosis and cell cycle arrest at S phase. Further studies showed that it down-regulated Bcl-2 and PPAR-γ expression, suppressed Akt and ERK activation and phosphorylation, and caused cytochrome c release from mitochondria to cytosol, whereas it up-regulated CD95 (APO-1/CD95) and Bax expression, and caused caspase-3 and JNK activation, PARP cleavage and AMPK phosphorylation. Pretreatments with caspase-3 (z-DEVD-fmk) and AMPK (CC) inhibitors significantly suppressed the γT3-induced ROS production and cell death. Caspase-3 inhibitor also efficiently blocked CD95 (APO-1/CD95) and Bax expression, caspase-3 activation and PARP cleavage, whereas antioxidant N-acetyl-l-cysteine, AMPK inhibitor and AMPK siRNA effectively blocked the AMPK phosphorylation. Taken together, these results conclude that the potent anti-proliferative and anti-adipogenic effects of γT3 on 3T3-L1 adipocytes could be through the Bax-mediated mitochondrial and AMPK signaling pathways.     

Growth inhibitory effects and molecular mechanisms of crotoxin treatment in esophageal Eca-109 cells and transplanted tumors in nude mice

Aim:

To investigate the antitumor actions of the Crotalus durissus neurotoxin (crotoxin) on human esophageal carcinoma (Eca-109) cells in vitro and transplanted esophageal Eca-109 tumors in nude mice.

Methods:

The growth-inhibitory effect was analyzed in Eca-109 cells using MTT assay. Cell morphology changes in nuclei were observed using Hoechst 33342 staining, while apoptosis and cell cycle distribution were examined by flow cytometry. RT-PCR was used to measure the Bcl-2, p15, and caspase-3 p17 gene expression levels. A tumor transplantation model was established by inoculation of Eca-109 cells were into female Balb/c nude mice. Crotoxin (25, 50, and 100 mg/kg) was subcutaneously injected into the transplanted tumors every 2 d for a total of 10 injections. Tumor size and weight were measured. Bcl-2, p15, and caspase-3 p17 protein expression in transplanted tumors was analyzed using Western blotting.

Results:

Crotoxin (25, 50, and 100 μg/mL) inhibited the growth of Eca-109 cells in a dose-dependent manner with inhibition rates of 22.9%, 35.8%, and 57.2%, respectively. Hoechst 33342 staining revealed apoptotic cells with pyknotic nuclear chromatin after crotoxin treatment. In Eca-109 cells, crotoxin induced apoptosis and G1 block, significantly upregulated the expression of p15 and caspase-3 p17 genes and downregulated the expression of Bcl-2 gene. Furthermore, crotoxin inhibited the growth of Eca-109 tumors in nude mice in a dose-dependent manner. Western blotting showed that crotoxin increased p15 and caspase-3 p17 protein levels and reduced Bcl-2 protein level in tumor specimens.

Conclusion:

Crotoxin inhibits the growth of Eca-109 cells in vitro via apoptosis induction and G1 block. Local administration of crotoxin inhibits the growth of subcutaneously transplanted Eca-109 cells in nude mice, possibly via increasing p15 and caspase-3 p17 protein expression and reducing Bcl-2 protein expression.     

Rutin from Lonicera japonica inhibits myocardial ischemia/reperfusion-induced apoptosis in vivo and protects H9c2 cells against hydrogen peroxide-mediated injury via ERK1/2 and PI3K/Akt signals in vitro

We investigated pharmacological effects of rutin isolated form Lonicera japonica on H₂O₂-induced cell death in H9c2 cells in vitro and rat myocardial ischemia-reperfusion injury model in vivo. Western blot analysis showed that H₂O₂ increased expression of cleaved form of caspase-3 and proapoptotic Bax protein, but decreased antiapoptotic Bcl-2 protein in H9c2 cell. However, treatment with rutin decreased expression of both cleaved from of caspase-3 and increased Bcl-2/Bax ratio in H9c2 cells. The protective effect of rutin was inhibited not by JNK inhibitor or p38 MAPK inhibitor but by PI3K inhibitor or ERK inhibitor. Rutin increased phosphorylation of ERK and Akt in H9c2 cells. These anti-apoptotic effects of rutin were confirmed both by annexin-V and TUNEL assay. Furthermore, rutin improved I/R-induced myocardial contractile function and reduced infarct size. Rutin administration also inhibited apoptosis in myocardial tissues in I/R rats by increasing Bcl-2/bax ratio and decreasing active caspase-3 expression. These results suggest that rutin reduced oxidative stress-mediated myocardial damage in vitro model and in vivo model, which might be useful in treatment of myocardial infarction.     

(E)-2,4-bis(p-hydroxyphenyl)-2-butenal has an antiproliferative effect on NSCLC cells induced by p38 MAPK-mediated suppression of NF-κB and up-regulation of TNFRSF10B (DR5)

Background And Purpose

The Maillard Reaction Products (MRPs) are known to be effective in chemoprevention. Here we focused on the anticancer effects of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal (a MRP) on human non-small-cell lung cancer (NSCLC) cells and its mechanism of action.

Experimental Approach

We analysed the activity of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal on NSCLC cells (NCI-H460 and A549) by use of Western blot analysis for major apoptotic proteins, MAPK, NF-κB and death receptor expression. We also used RT-PCR to determine its effects on death receptor mRNA expression, EMSA for effects on NF-κB DNA binding activity and colony formation assay for effects of inhibitors on (E)-2,4-bis(p-hydroxyphenyl)-2-butenal''s actions.

Key Results

(E)-2,4-bis(p-hydroxyphenyl)-2-butenal induced a concentration (10–40 μg·mL⁻¹)- and time (30 min–72 h)-dependent inhibitory effect on the growth of NSCLC cells due to induction of apoptosis. Concomitantly, it significantly increased the expression of apoptotic proteins such as cleaved caspase-3, cleaved caspase-9, Bax and p53, but down-regulated the expression of anti-apoptotic proteins Bcl-2, cIAP1 and cIAP2. This effect was induced by up-regulation of MAPK and death receptor proteins TNFRSF12, TNFRSF10B and TNFRSF21, but suppression of NF-κB. Of the death receptors activated, only TNFRSF10B knock down with siRNA reversed the effect of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal. Even though all the MAPKs were activated, only pretreatment with a p38 MAPK inhibitor reversed (E)-2,4-bis(p-hydroxyphenyl)-2-butenal-induced cell growth inhibition, increase in cleaved caspase-3, -9 and TNFRSF10B expression, and NF-κB inactivation.

Conclusions And Implications

(E)-2,4-bis(p-hydroxyphenyl)-2-butenal induces apoptosis in NSCLC cells by p38 MAPK-mediated suppression of NF-κB and activation of TNFRSF10B, which then activates the caspase-3 and caspase-9 pathways.     

Naringenin-induced apoptosis is attenuated by Bcl-2 but restored by the small molecule Bcl-2 inhibitor,HA 14-1, in human leukemia U937 cells

Naringenin, a naturally occurring citrus flavonone, has shown cytotoxicity in various human cancer cell lines as well as inhibitory effects on tumor growth and there is increasing interest in its therapeutic applications. In this study, the effect of ectopic Bcl-2 expression on naringenin-induced apoptosis was investigated. We found that Bcl-2 overexpression markedly protected human leukemia U937 cells from time- and dose-dependent induction of apoptosis by naringenin, as did caspase-3 and caspase-9 inhibitors. Additionally, Bcl-2 overexpression attenuated naringenin-induced Bax translocation and cytosolic release of cytochrome c. Our results also indicated that co-administration of HA14-1 and naringenin increased apoptosis in Bcl-2 overexpressing U937 cells by restoring mitochondrial dysfunction and activation of caspase-9 and caspase-3, as well as by cleavage of poly (ADP-ribose) polymerase. Taken together, these observations indicate that Bcl-2 confers apoptosis resistance to naringenin by inhibiting a mitochondrial amplification step in U937 cells.     

Ghrelin protects H9c2 cells from hydrogen peroxide-induced apoptosis through NF-κB and mitochondria-mediated signaling

Oxidative stress is a major mechanism underlying the pathogenesis of cardiovascular disease. Herein we investigate the protective effects of ghrelin in H₂O₂-induced apoptosis of H9c2 cells, as well as the possible molecular mechanisms involved. To study apoptosis, the cells were assessed by morphologic examination, MTS assay, Annexin V–propidium iodide dual staining and TUNEL analysis. Intracellular reactive oxygen species (ROS) production and mitochondrial membrane potential were also measured. To investigate the underlying molecular mechanisms, the expression of Bcl-2, Bax, active caspase-9 and NF-κB were assessed by Western blotting, and caspase-3 activity was determined by a colorimetric activity assay kit. After stimulation with H₂O₂ for 18 h, H9c2 cells viability decreased significantly; a large fraction of cells underwent apoptosis. We observed a dose-dependent rescue of H9c2 cells from H₂O₂-induced apoptosis in the presence of different ghrelin concentrations. Preincubation with ghrelin also restored the ROS and mitochondrial membrane potential levels that had been altered by H₂O₂ treatment. Moreover, ghrelin decreased H₂O₂-induced Bax production and caspase-9 activation, and increased Bcl-2 levels. NF-κB phosphorylation was also significantly inhibited by ghrelin in H₂O₂-treated cells. Caspase-3 activation was suppressed by ghrelin in H₂O₂-treated H9c2 cells in a dose-dependent manner. In summary, ghrelin protects H9c2 cells from oxidative stress-induced apoptosis through downregulation of Bax expression, caspase-9 activation and NF-κB phosphorylation, and upregulation of Bcl-2 expression. Caspase-3 activation was also reduced in a dose-dependent manner. These data suggest that ghrelin might protect against cardiovascular disease by protecting the mitochondria.     

Xanthoangelol, a major chalcone constituent of Angelica keiskei, induces apoptosis in neuroblastoma and leukemia cells

Xanthoangelol, a major chalcone constituent of the stem exudates of Angelica keiskei, was evaluated for cell toxicity and apoptosis-inducing activity in human neuroblastoma (IMR-32) and leukemia (Jurkat) cells. Xanthoangelol concentration-dependently reduced the survival rates of both cell lines as revealed by the trypan blue exclusion test. Early apoptosis induced by 4 h incubation with xanthoangelol was detected using flow cytometry after double-staining with annexin V and propidium iodide (PI). Western blot analysis showed that xanthoangelol markedly reduced the level of precursor caspase-3 and increased the level of cleaved caspase-3, but Bax and Bcl-2 proteins were not affected. These results suggest that xanthoangelol induces apoptotic cell death by activatation of caspase-3 in neuroblastoma and leukemia cells through a mechanism that does not involve Bax/Bcl-2 signal transduction. Therefore, xanthoangelol may be applicable as an effective drug for treatment of neuroblastoma and leukemia.     

Emodin induces human T cell apoptosis in vitro by ROS-mediated endoplasmic reticulum stress and mitochondrial dysfunction

Aim:

To elucidate the molecular mechanisms underlying the immunosuppressive effects of emodin isolated from Rheum palmatum L.

Methods:

Human T cells were isolated from the peripheral venous blood of 10 healthy adult donors. Cell viability was analyzed with MTT assay. AO/EB and Annexin V/PI staining and DNA damage assay were used to detect cell apoptosis. Fluorescence staining was used to detect the levels of ROS, the mitochondrial membrane potential and intracellular Ca²⁺. Colorimetry was used to detect the levels of MDA and total SOD and GSH/GSSG ratio. The expression and activity of caspase-3, -4, and -9 were detected with Western blotting and a fluorometric assay. Western blotting was also used to detect the expression of Bcl-2, Bax, cytochrome C, and endoplasmic reticulum (ER) markers.

Results:

Emodin (1, 10, and 100 μmol/L) inhibited the growth of human T cells and induced apoptosis in dose- and time dependent manners. Emodin triggered ER stress and significantly elevated intracellular free Ca²⁺ in human T cells. It also disrupted mitochondrial membrane potential, and increased cytosolic level of cytochrome C, and the levels of activated cleavage fragments of caspase-3, -4, and -9 in human T cells. Furthermore, emodin significantly increased the levels of ROS and MDA, inhibited both SOD level and GSH/GSSG ratio in human T cells, whereas co-incubation with the ROS scavenger N-acetylcysteine (NAC, 20 μmol/L) almost completely blocked emodin-induced ER stress and mitochondrial dysfunction in human T cells, and decreased the caspase cascade-mediated apoptosis.

Conclusion:

Emodin exerts immunosuppressive actions at least partly by inducing apoptosis of human T cells, which is triggered by ROS-mediated ER stress and mitochondrial dysfunction.