Human amniotic membrane derived-mesenchymal stem cells induce C6 glioma apoptosis in vivo through the Bcl-2/caspase pathways

High-grade gliomas are difficult to treat. We examined the therapeutic effect of intratumoral administration of human amniotic membrane derived-mesenchymal stem cells (hAMCs) on the growth of gliomas. Tumor volume of the control group was 1632 ± 316 mm³ on day 30, and the group treated with a single intratumoral dose of hAMCs had a tumor volume of 1128 ± 269 mm³ (P < 0.05). Thus, administration of hAMCs significantly reduced tumor size. In rat glioma tissues treated with single and multiple dosages of hAMCs, there was a reduction in tumor volume of approximately 30.9 and 49.5%, respectively. We further evaluated the glioma tissue using Western blotting analysis and observed that the expression of Bax, caspase-8 and caspase-3 were greatly increased and the expression of Bcl-2 was greatly decreased in tumors treated with hAMCs. Sections of nude mice treated with hAMCs clearly showed the presence of an increase in apoptotic cells. The data collected herein confirms for the first time that hAMCs can inhibit C6 glioma growth and induce apoptosis of C6 gliomas in vivo. This demonstrates that hAMCs are a potential new therapeutic agent for the treatment of gliomas.     

p53-Independent ceramide formation in human glioma cells during gamma-radiation-induced apoptosis

Although the p53 tumor-suppressor gene product plays a critical role in apoptotic cell death induced by DNA-damaging chemotherapeutic agents, human glioma cells with functional p53 were more resistant to gamma-radiation than those with mutant p53. U-87 MG cells with wild-type p53 were resistant to gamma-radiation. U87-W E6 cells that lost functional p53, by the expression of type 16 human papillomavirus E6 oncoprotein, became susceptible to radiation-induced apoptosis. The formation of ceramide by acid sphingomyelinase (A-SMase), but not by neutral sphingomyelinase, was associated with p53-independent apoptosis. SR33557 (2-isopropyl-1-(4-[3-N-methyl-N-(3,4-dimethoxybphenethyl)amino]propyloxy)benzene-sulfonyl) indolizine, an inhibitor of A-SMase, suppressed radiation-induced apoptotic cell death. In contrast, radiation-induced A-SMase activation was blocked in glioma cells with endogenous functional p53. The expression of acid ceramidase was induced by gamma-radiation, and was more evident in cells with functional p53. N-oleoylethanolamine, which is known to inhibit ceramidase activity, unexpectedly downregulated acid ceramidase and accelerated radiation-induced apoptosis in U87-W E6 cells. Moreover, cells with functional p53 could be sensitized to gamma-radiation by N-oleoylethanolamine, which suppressed radiation-induced acid ceramidase expression and then enhanced ceramide formation. Sensitization to gamma-radiation was also observed in U87-MG cells depleted of functional p53 by retroviral expression of small interfering RNA. These results indicate that ceramide may function as a mediator of p53-independent apoptosis in human glioma cells in response to gamma-radiation, and suggest that p53-dependent expression of acid ceramidase and blockage of A-SMase activation play pivotal roles in protection from gamma-radiation of cells with endogenous functional p53.     

Cadmium-induced apoptosis in C6 glioma cells: Mediation by caspase 9-activation

The induction of apoptotic cell death by cadmium was investigated in eight mammalian cell lines. Great differences in the cytotoxicity of cadmium were found with different cell lines: Rat C6 glioma cells turned out to be most sensitive with an IC₅₀-value of 0.7 M, while human A549 adenocarcinoma cells were relatively resistant with an IC₅₀-value of 164 M CdCl₂. The mode of cadmium-induced cellular death was identified to involve apoptotic DNA fragmentation in three cell lines, i.e., in C6 glioma cells, E367 neuroblastoma cells and NIH3T3 fibroblasts. In C6 glioma cells, this process was investigated in detail. Internucleosomal DNA-fragmentation occurred 40 h after application of CdCl₂ and was concentration-dependent between 1–100 M CdCl₂, followed by a decrease at higher concentrations due to necrotic processes. Apoptotic chromatin-condensation and nuclear fragmentation was observed 48 h after application of 2.5 M CdCl₂. Furthermore, cadmium (1 M, 48 h) caused a breakdown of the mitochondrial membrane potential as shown by the decline in mitochondrial uptake of rhodamine 123. Also, we found an activation of caspase 9, a protease known to be activated in apoptotic processes following mitochondrial damage. Besides Cd²⁺, other toxic heavy metal ions (Hg²⁺, Pb²⁺, Ni²⁺, Fe²⁺, CrO₄ ^2–, Cu²⁺ or Co²⁺) did not induce apoptotic DNA fragmentation in C6 cells. The only exception was Zn²⁺ which caused apotosis at high concentrations (>150 M) whereas it protected against cadmium-induced apoptosis at low concentrations (10–50 M).     

Bcl-2 rescues ceramide- and etoposide-induced mitochondrial apoptosis through blockage of caspase-2 activation

Recent studies indicate that caspase-2 is involved in the early stage of apoptosis before mitochondrial damage. Although the activation of caspase-2 has been shown to occur in a large protein complex, the mechanisms of caspase-2 activation remain unclear. Here we report a regulatory role of Bcl-2 on caspase-2 upstream of mitochondria. Stress stimuli, including ceramide and etoposide, caused caspase-2 activation, mitochondrial damage followed by downstream caspase-9 and -3 activation, and cell apoptosis in human lung epithelial cell line A549. When A549 cells were pretreated with the caspase-2 inhibitor benzyloxycarbonyl-Val-Asp(-OMe)-Val-Ala-Asp(-OMe)-fluoromethyl ketone or transfected with caspase-2 short interfering RNA, both ceramide- and etoposide-induced mitochondrial damage and apoptosis were blocked. Overexpression of Bcl-2 prevented ceramide- and etoposide-induced caspase-2 activation and mitochondrial apoptosis. Furthermore, caspase-2 was activated when A549 cells were introduced with Bcl-2 short interfering RNA or were treated with Bcl-2 inhibitor, which provided direct evidence of a negative regulatory effect of Bcl-2 on caspase-2. Cell survival was observed when caspase-2 was inhibited in Bcl-2-silencing cells. Blockage of the mitochondrial permeability transition pore and caspase-9 demonstrated that Bcl-2-modulated caspase-2 activity occurred upstream of mitochondria. Further studies showed that Bcl-2 was dephosphorylated at serine 70 after ceramide and etoposide treatment. A protein phosphatase inhibitor, okadaic acid, rescued Bcl-2 dephosphorylation and blocked caspase-2 activation, mitochondrial damage, and cell death. Taken together, ceramide and etoposide induced mitochondria-mediated apoptosis by initiating caspase-2 activation, which was, at least in part, regulated by Bcl-2.     

Cobalt chloride-induced apoptosis and extracellular signal-regulated protein kinase 1/2 activation in rat C6 glioma cells

Brain ischemia brings about hypoxic insults. Hypoxia is one of the major pathological factors inducing neuronal injury and central nervous system infection. We studied the involvement of mitogen-activated protein (MAP) kinase in hypoxia-induced apoptosis using cobalt chloride in C6 glioma cells. In vitro cytotoxicity of cobalt chloride was tested by MTT assay. Its IC(50) value was 400 microM. The DNA fragment became evident after incubation of the cells with 300 microM cobalt chloride for 24 h. We also evidenced nuclear cleavage with morphological changes of the cells undergoing apoptosis with electron microscopy. Next, we examined the signal pathway of cobalt chloride-induced apoptosis in C6 cells. The activation of extracellular signal-regulated protein kinase 1/2 (ERK 1/2) started to increase at 1 h and was activated further at 6 h after treatment of 400 M cobalt chloride. In addition, pretreatment of PD98059 inhibited cobalt chloride-induced apoptotic cell morphology in Electron Microscopy. These results suggest that cobalt chloride is able to induce the apoptotic activity in C6 glioma cells, and its apoptotic mechanism may be associated with signal transduction via MAP kinase (ERK 1/2).     

Bcl-2 regulates chondrocyte morphology and aggrecan gene expression independent of caspase activation and full apoptosis.

Bcl-2 is widely expressed in a variety of cell types and is known to block apoptosis through a conserved pathway. However, recent reports have demonstrated that Bcl-2 regulates cell behavior independent of its control of apoptosis. Chondrocytes express a unique set of matrix proteins, including the proteoglycan aggrecan, and have been widely used to study the relationship between trophic factors and apoptosis. In this article, we report that Bcl-2 affects the morphology and regulates the expression of aggrecan in a rat chondrocyte cell line (IRC). Endogenous Bcl-2 and aggrecan mRNA were both down-regulated in response to serum withdrawal in parental IRC cells, while constitutive expression of Bcl-2 maintained aggrecan levels under conditions of serum withdrawal. In addition, expression of anti-sense Bcl-2 resulted in decreased aggrecan mRNA and produced a fibroblastic morphology compared with parental cells. The caspase inhibitor ZVAD-fmk effectively blocked full apoptosis of IRC cells in response to serum withdrawal or anti-sense Bcl-2 but did not prevent the down-regulation of aggrecan expression from either signal. These results suggest a novel role for Bcl-2 in regulating the differentiated phenotype of chondrocytes and the expression of a differentiation-specific gene independent of its control of apoptosis.     

Erucylphosphocholine-induced apoptosis in glioma cells: involvement of death receptor signalling and caspase activation

Erucylphosphocholine (ErPC) is a promising anti-neoplastic drug for the treatment of malignant brain tumours. It exerts strong anti-cancer activity in vivo and in vitro and induces apoptosis even in chemoresistant glioma cell lines. The purpose of this study was to expand on our previous observations on the potential mechanisms of ErPC-mediated apoptosis with a focus on death receptor activation and the caspase network. A172 and T98G glioma cells were treated with ErPC for up to 48 h. ErPC effects on the expression of the tumour necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL) receptor system, and on caspase activation were determined. ErPC had no effect on the expression of TNFalpha or TRAIL. Inhibition of the TNF or TRAIL signalling pathway with antagonistic antibodies or fusion proteins did not affect apoptosis induced by ErPC, and a dominant-negative FADD construct did not abolish ErPC-induced effects. Western blot analysis indicated that ErPC-triggered apoptosis resulted in a time-dependent processing of caspases-3, -7, -8 and -9 into their respective active subunits. Co-treatment of A172 cells with different caspase inhibitors prevented apoptosis but did not abrogate cell death. These data suggest that A172 cells might have an additional caspase-independent pathway that insures cell death and guarantees killing of those tumour cells whose caspase pathway is incomplete.     

Ordering of ceramide formation and caspase-9 activation in CD95L-induced Jurkat leukemia T cell apoptosis

Ceramide, a biologically active sphingolipid in cell death signaling, accumulates upon CD95L treatment, concomitantly to apoptosis induction in Jurkat leukemia T cells. Herein, we show that ceramide did not increase in caspase-8 and -10-doubly deficient Jurkat cells in response to CD95L, indicating that apical caspases are essential for CD95L-triggered ceramide formation. Jurkat cells are typically defined as type 2 cells, which require the activation of the mitochondrial pathway for efficient apoptosis induction in response to CD95L. Caspase-9-deficient Jurkat cells significantly resisted CD95L-induced apoptosis, despite ceramide accumulation. Knock-down of sphingomyelin synthase 1, which metabolizes ceramide to sphingomyelin, enhanced (i) CD95L-triggered ceramide production, (ii) cytochrome c release from the mitochondria and (iii) caspase-9 activation. Exogenous ceramide-induced caspase-3 activation and apoptosis were impaired in caspase-9-deficient Jurkat cells. Conversely, caspase-9 re-expression in caspase-9-deficient Jurkat cells restored caspase-3 activation and apoptosis upon exogenous ceramide treatment. Collectively, our data provide genetic evidence that CD95L-triggered endogenous ceramide increase in Jurkat leukemia T cells (i) is not a mere consequence of cell death and occurs mainly in a caspase-9-independent manner, (ii) is likely involved in the pro-apoptotic mitochondrial pathway leading to caspase-9 activation.     

Insulin involved Akt/ERK and Bcl-2/Bax pathways against oxidative damages in C6 glial cells

Insulin, a hypoglycemic hormone, has multiple functions in the brain. The aim of this study to identify the mechanisms of insulin in hydrogen peroxide (H₂O₂)-induced toxicity in the C6 glial cells. Cytotoxicity, lactate dehydrogenase, nitric oxide, reactive oxygen species and calcium ion, lipid peroxidation, protein oxidation and glutathione levels were determined. Signaling pathway molecules were assessed by western blotting and RT-PCR. The results showed that treatment with insulin reduced the cell death and cell membrane damages against H₂O₂-induced toxicity. Furthermore, insulin interfered H₂O₂-induced intracellular generation of reactive oxygen species and calcium-ion transport, apoptosis, including lipid and protein oxidation products. Cells treated with insulin reverted H₂O₂-induced suppression of reduced glutathione levels by blocking oxidized glutathione. Moreover, insulin treatment activates Akt, restores ERK1/2 and Bcl-2 by preventing Bax and Bax/Bcl-2 ratio. Our results suggest that treatment of insulin exerts potential role against 24?h of H₂O₂-induced toxicity in C6 cells.     

Dexamethasone inhibits apoptosis in C6 glioma cells through increased expression of Bcl-XL

The glucocorticoid dexamethasone (Dex) has been reported to modulate a number of signaling pathways and physiological processes, including apoptosis. This study was carried out to investigate the cytoprotective mechanism of Dex in C6 glioma cells. Pre-treatment of cells with Dex inhibited apoptosis induced by staurosporine, etoposide and thapsigargin. Apoptosis inhibition correlated with blockade of mitochondrial cytochrome c release, abolition of caspase-3 activity along with inhibition of caspase-9 and PARP cleavage. Dex-mediated cytoprotection coincided with the induction of the anti-apoptotic protein, Bcl-X_L. The specific glucocorticoid receptor antagonist, RU486, reversed the anti-apoptotic effect of Dex and prevented Bcl-X_L induction. Here, we show for the first time that knockdown of Bcl-X_L expression with siRNA reversed the protective effects of the glucocorticoid in glioma cells. We conclude that Dex-mediated inhibition of apoptosis in C6 glioma cells is through induction of Bcl-X_L.     

C6 ceramide sensitizes pemetrexed-induced apoptosis and cytotoxicity in osteosarcoma cells

Chemotherapy has significantly improved the prognosis of high-grade osteosarcoma (OS), but over 30% of OS patients can still not be cured. Pemetrexed, the newly-developed anti-folate chemotherapy drug, exerted lower efficacy against OS cells. Here, we aimed to increase pemetrexed efficiency, and found that the cell-permeable short-chain ceramide (C6) significantly enhanced pemetrexed-induced viability reduction and death in cultured OS cell lines (U2OS and MG-63). Pemetrexed induced moderate apoptosis in OS cells, which was dramatically augmented by C6 ceramide. The apoptosis inhibitor z-VAD-fmk largely inhibited C6 ceramide plus pemetrexed-induced cytotoxicity and apoptosis in OS cells. By using pharmacological and siRNA-knockdown strategies, we showed that Akt–mammalian TOR (mTOR) over-activation was an important pemetrexed resistance factor in OS cells, and C6 ceramide-mediated pemetrexed sensitization effect was mediated, at least in part, by Akt–mTOR inhibition. Finally, we found that Akt–S6 Kinase 1 (S6K1, an indicator of mTOR activation) was over-activated in human OS tissues. On the other hand, the osteoblastic MC3T3-E1 cells, which expressed lower Akt–S6K1 phosphorylation, were resistant to pemetrexed and/or C6 ceramide. Together, we conclude that C6 ceramide sensitizes pemetrexed-induced apoptosis and cytotoxicity in OS cells probably through in-activation of Akt–mTOR signaling.     

The course of etoposide-induced apoptosis in Jurkat cells lacking p53 and Bax

Jurkat T-lymphocytes lack p53 and Bax but contain p73 and Bid and are killed by etoposide (ETO). With ETO c-abl is phosphorylated and phosphorylated p73 increased. Translocation of full-length Bid to mitochondria follows, with induction of the mitochondrial permeability transition (MPT) and release of cytochrome c into the cytosol. Pronounced swelling of mitochondria was evident ultrastructurally, and the MPT inhibitor cyclosporin A prevented the release of cytochrome c. Overexpression of Bcl-2 prevented the translocation of Bid, the release of cytochrome c, and cell death. The pan-caspase inhibitor ZVAD-FMK prevented the cell killing, but not the initial release of cytochrome c. An accumulation of tBid occurred at later times in association with Bid degradation. A sequence is proposed that couples DNA damage to Bid translocation via activation of c-abl and p73. Bid translocation induces the MPT, the event that causes release of cytochrome c, activation of caspases, and cell death.     

Sphingosine kinase expression regulates apoptosis and caspase activation in PC12 cells

Sphingosine-1-phosphate (SPP), a bioactive sphingolipid metabolite, suppresses apoptosis of many types of cells, including rat pheochromocytoma PC12 cells. Elucidating the molecular mechanism of action of SPP is complicated by many factors, including uptake and metabolism, as well as activation of specific G-protein-coupled SPP receptors, known as the endothelial differentiation gene-1 (EDG-1) family. In this study, we overexpressed type 1 sphingosine kinase (SPHK1), the enzyme that converts sphingosine to SPP, in order to examine more directly the role of intracellularly generated SPP in neuronal survival. Enforced expression of SPHK1 in PC12 cells resulted in significant increases in kinase activity, with corresponding increases in intracellular SPP levels and concomitant decreases in both sphingosine and ceramide, and marked suppression of apoptosis induced by trophic factor withdrawal or by C(2)-ceramide. NGF, which protects PC12 cells from serum withdrawal-induced apoptosis, also stimulated SPHK1 activity. Surprisingly, overexpression of SPHK1 had no effect on activation of two known NGF-stimulated survival pathways, extracellular signal regulated kinase ERK 1/2 and Akt. However, trophic withdrawal-induced activation of the stress activated protein kinase, c-Jun amino terminal kinase (SAPK/JNK), and activation of the executionary caspases 2, 3 and 7, were markedly suppressed. Moreover, this abrogation of caspase activation, which was prevented by the SPHK inhibitor N,N-dimethylsphingosine, was not affected by pertussis toxin treatment, indicating that the cytoprotective effect was likely not mediated by binding of SPP to cell surface G(i)-coupled SPP receptors. In agreement, there was no detectable release of SPP into the culture medium, even after substantially increasing cellular SPP levels by NGF or sphingosine treatment. In contrast to PC12 cells, C6 astroglioma cells secreted SPP, suggesting that SPP might be one of a multitude of known neurotrophic factors produced and secreted by glial cells. Collectively, our results indicate that SPHK/SPP may play an important role in neuronal survival by regulating activation of SAPKs and caspases.     

Zerumbone induced apoptosis in liver cancer cells via modulation of Bax/Bcl-2 ratio

Background  

Zerumbone is a cytotoxic component isolated from Zingiber zerumbet Smith, a herbal plant which is also known as lempoyang. This new anticancer bioactive compound from Z. zerumbet was investigated for its activity and mechanism in human liver cancer cell lines.     

Smac/DIABLO is required for effector caspase activation during apoptosis in human cells

Mitochondria play a pivotal role during stress-induced apoptosis as several proapoptotic proteins are released to the cytosol to activate caspases. Smac/DIABLO is one of the proapoptotic proteins released from the mitochondria and has been shown to inactivate IAPs. However, gene knockout studies in mice revealed a redundant role for Smac during development and cell death. By applying RNA interference-mediated loss of function approach, we demonstrate that Smac/DIABLO is required for the activation of effector but not initiator caspases during stress and receptor-mediated cell death in HeLa cells. Cells with reduced Smac resist apoptosis and retained clonogenicity. Our results suggest an obligatory role for Smac/DIABLO in these tumor cells during several pathways of apoptosis induction.     

重组人白细胞介素-6对缺氧-复氧后大鼠海马培养神经元Bcl-2、Bax 表达和神经元凋亡的影响

实验在培养的大鼠海马神经元中观察了重组人白细胞介素-6（recombinant human interleukin-6,rhIL-6）对缺氧-复氧后Bcl-2、Bax表达和神经元凋亡的影响。把培养12d的大鼠海马神经元分为对照组和rhIL-6组,同时于缺氧环境（90% N2 10% CO2）中培养2、4h后,再于常氧培养箱内复氧培养24和72h。于不同时间取出,分别用抗Bcl-2和Bax抗血清进行免疫组织化学染色,观察缺氧-复氧后大鼠海马培养神经元Bcl-2和Bax的表达,并用原位末端标记（TUNEL）法和流式细胞术分别检测缺氧-复氧对体外培养海马神经元凋亡的影响。结果可见,与缺氧前相比,缺氧-复氧后24和72h,海马神经元Bal-2表达明显减弱,Bax表达明显增强,凋亡神经元明显增多。经rhIL-6预处理的海马神经元与对照组相比,缺氧-复氧后24和72h,Bcl-2表达明显增强,Bax神经明显减弱,凋亡神经元明显减少。本实验结果提示,rhIL-6对海马神经元缺氧-复氧损伤具有一定的保护作用。     

Bcl-2 and Bax regulation of apoptosis in germ cells during prenatal oogenesis in the mouse embryo.

Apoptosis is the main cause of primordial germ cell and oocyte degeneration in the developing fetal ovary. In this study we examined by immunohistochemistry and immunoblotting the expression of the anti- and pro-apoptotic proteins Bcl-2 and Bax in primordial germ cells and fetal oocytes during pre natal oogenesis in the mouse embryo. While Bcl-2 and Bax were not detectable in primordial germ cells in vivo, both proteins were upregulated when they undergo apoptosis in culture. Treatment with the stem cell factor (SCF), a growth factor known to partially reduce primordial germ cell apoptosis, resulted in decreased Bax expression. Bcl-2 was barely detectable in oocytes entering into meiosis and its expression did not change during the stage of meiotic prophase I examined. On the contrary, high levels of Bax was expressed in degenerating oocytes while low levels of the protein was present in many apparently healthy oocytes between 15.5 days post coitum (d.p.c.) and birth, when Bax was downregulated. Oocytes isolated from 15.5 days post coitum (d.p.c.) ovaries that progress through prophase I and undergo a wave of apoptosis at the stage of pachytene/diplotene in vitro, showed a pattern of Bax expression similar to the in vivo condition. Although the addition of SCF to the culture medium reduced significantly apoptosis in oocytes at the pachytene/diplotene stages, it was not possible to directly correlate this effect with the downregulation of Bax in the surviving oocytes. These findings indicate that whereas a balance between Bcl-2 and Bax might regulate apoptosis of proliferating primordial germ cells under a partial control by SCF, Bax-mediated apoptosis in meiotic oocytes may be due to intrinsic meiotic checkpoints which act to monitor aberrant DNA recombination rather than to a growth factor-dependent process. Elimination of supernumerary oocytes might be a subsequent apoptotic phenomenon controlled by the availability of growth factors such as SCF within the ovary.