Characterization of N,N,-dimethyl-D-erythro-sphingosine-induced apoptosis and signaling in U937 cells: independence of sphingosine kinase inhibition

In the present study, we studied N,N-dimethyl-D-erythro-sphingosine (DMS)-induced cell death and its signaling mechanism in U937 human monocytes. We found that DMS induced cell death in a concentration-dependent manner, while sphingosine 1-phosphate did not. DMS also induced DNA fragmentation, nuclear disruption, and cytochrome c release from mitochondria in a concentration- and time-dependent manner, implying apoptotic cell death. DMS was found to increase mitochondrial membrane potential (MMP) immediately after addition of DMS and to decrease MMP at 2h after addition. However, sphingosine kinase inhibitors and PKC inhibitors did not induce cell death in U937 cells, a result that appears to exclude sphingosine kinase and PKC as target molecules of DMS in the cell death induction process. Furthermore, DMS modulated the activity of several signaling molecules. DMS induced activation of JNK and p38 MAP kinase, while it decreased the activity of ERK and Akt kinase. However, decrease of MMP, inhibition of JNK, p38 MAP kinase, ERK, or Akt with specific inhibitors could not mimic the DMS-induced cell death, implying multiple concerted processes are involved in DMS-induced cell death. In summary, DMS induced apoptotic cell death via modulation of MMP, JNK, p38 MAP kinase, ERK, and Akt kinase, but not through inhibition of sphingosine kinase or PKC in U937 cells.     

Bradykinin enhances reactive oxygen species generation, mitochondrial injury, and cell death induced by ATP depletion--a role of the phospholipase C-Ca(2+) pathway

This study aimed to study the effect of bradykinin on reactive oxygen species (ROS) generation, mitochondrial injury, and cell death induced by ATP depletion in cell culture. Renal tubular cells were subjected to ATP depletion. Cell death was evaluated with LDH release, sub-G0/G1 fraction, Hoechst staining, and annexin V binding assay. ROS generation, mitochondrial membrane potential (DeltaPsi(m)), and intramitochondrial calcium were evaluated with flow cytometry. Translocation of cytochrome c and activation of apoptotic protein were analyzed with cell fractionating and Western blotting. Intracellular calcium was measured with a spectrofluorometer. Bradykinin enhanced cellular LDH release, apoptosis, generation of superoxide, and hydrogen peroxide induced by ATP depletion. Bradykinin also enhanced the loss of DeltaPsi(m), translocation of cytochrome c into cytosol, and activation of apoptotic protein. The intracellular/mitochondrial calcium was higher in bradykinin-treated cells. All these effects were reversed by coadministration with bradykinin B2 receptor (B2R) antagonist. Besides, blocking the phospholipase C (PLC) could reverse the synergistic effect of bradykinin with ATP depletion on ROS generation, mitochondrial damage, accumulation of intracellular/mitochondrial calcium, and apoptosis. Activation of B2R aggravates ROS generation, mitochondrial damage, and cell death induced by ATP depletion. These effects may act through the PLC-Ca(2+) signaling pathway.     

Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line

The primary objective of this study was to determine the sequence of biochemical signaling events that occur after modulation of the cellular redox state in the B cell lymphoma line, PW, with emphasis on the role of mitochondrial signaling. L-Buthionine sulphoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (gammaGCS), was used to modulate the cellular redox status. The sequence and role of mitochondrial events and downstream apoptotic signals and mediators was studied. After BSO treatment, there was an early decline in cellular glutathione (GSH), followed by an increase in reactive oxygen species (ROS) production, which induced a variety of apoptotic signals (detectable at different time points) in the absence of any external apoptotic stimuli. The sequence of biochemical events accompanying apoptosis included a 95% decrease in total GSH and a partial (25%) preservation of mitochondrial GSH, without a significant increase in ROS production at 24h. Early activation and nuclear translocation of the nuclear factor kappa B subunit Rel A was observed at approximately 3h after BSO treatment. Cytochrome c release into the cytosol was also seen after 24h of BSO treatment. p53 protein expression was unchanged after redox modulation for up to 72 h, and p21waf1 independent loss of cellular proliferation was observed. Surprisingly, a truncated form of p53 was expressed in a time-dependent manner, beginning at 24h after BSO incubation. Irreversible commitment to apoptosis occurred between 48 and 72 h after BSO treatment when mitochondrial GSH was depleted, and there was an increase in ROS production. Procaspase 3 protein levels showed a time-dependent reduction following incubation with BSO, notably after 48 h, that corresponded with increasing ROS levels. At 96 h, caspase 3 cleavage products were detectable. The pan-caspase inhibitor zVADfmk, partially blocked the induction of apoptosis at 48 h, and was ineffective after 72 h. PW cells could be rescued from apoptosis by removing them from BSO after up to 48, but not 72 h incubation with BSO. Mitochondrial transmembrane potential (DeltaPsi(m)) remained intact in most of the cells during the 72 h observation period, indicating that DeltaPsi(m) dissipation is not an early signal for the induction of redox dependent apoptosis in PW cells. These data suggest that a decrease in GSH alone can act as a potent early activator of apoptotic signaling. Increased ROS production following mitochondrial GSH depletion, represents a crucial event, which irreversibly commits PW cells to apoptosis.     

Sphingosine kinase: a point of convergence in the action of diverse neutrophil priming agents

Neutrophils are a vital component of the early acute inflammatory response, but can cause profound tissue damage when activated to excess or prevented from undergoing apoptosis. However, much remains unknown about the intracellular signaling pathways regulating neutrophil activity. The structurally diverse neutrophil-priming agents platelet-activating factor, TNF-alpha, and the substance P analog [D-Arg(6), D-Trp(7,9),N(me)Phe(8)]-substance P(6-11) (SP-G) stimulated a rapid increase in sphingosine kinase activity in freshly isolated human neutrophils. This activity was blocked by preincubation with the sphingosine kinase inhibitor N,N-dimethylsphingosine (DMS). DMS also inhibited the increase in intracellular calcium concentration stimulated by platelet-activating factor, fMLP, and SP-G. This suggests that the increase in intracellular calcium concentration by these agents is dependent on sphingosine kinase activation and the generation of sphingosine-1-phosphate. Changes in cell polarization and the augmentation of the fMLP-induced superoxide anion generation, by all priming agents were also inhibited by DMS, while only the superoxide anion release was blocked by the phosphatidylinositol 3-kinase inhibitor LY294002. Moreover, SP-G and GM-CSF inhibited constitutive neutrophil apoptosis which was completely blocked by DMS. These results suggest a novel role for sphingosine kinase in the regulation of neutrophil priming.     

Endoplasmic reticulum stress-mediated apoptosis of EBV-transformed B cells by cross-linking of CD70 is dependent upon generation of reactive oxygen species and activation of p38 MAPK and JNK pathway

CD70 is expressed in normal activated immune cells as well as in several types of tumors. It has been established that anti-CD70 mAb induces complement-dependent death of CD70(+) tumor cells, but how anti-CD70 mAb affects the intrinsic signaling is poorly defined. In this report, we show that ligation of CD70 expressed on EBV-transformed B cells using anti-CD70 mAb induced production of reactive oxygen species (ROS) and subsequent apoptosis. We observed an early expression of endoplasmic reticulum (ER) stress response genes that preceded the release of apoptotic molecules from the mitochondria and the cleavage of caspases. CD70-induced apoptosis was inhibited by pretreatment with the ER stress inhibitor salubrinal, ROS quencher N-acetylcysteine, and Ca(2+) chelator BAPTA. We supposed that ROS generation might be the first event of CD70-induced apoptosis because N-acetylcysteine blocked increases of ROS and Ca(2+), but BAPTA did not block ROS generation. We also found that CD70 stimulation activated JNK and p38 MAPK. JNK inhibitor SP600125 and p38 inhibitor SB203580 effectively blocked upregulation of ER stress-related genes and cleavage of caspases. Inhibition of ROS generation completely blocked phosphorylation of JNK and p38 MAPK and induction of ER stress-related genes. Taken together, we concluded that cross-linking of CD70 on EBV-transformed B cells triggered ER stress-mediated apoptosis via ROS generation and JNK and p38 MAPK pathway activation. Our report reveals alternate mechanisms of direct apoptosis through CD70 signaling and provides data supporting CD70 as a viable target for an Ab-based therapy against EBV-related tumors.     

TNF-alpha-induced sphingosine 1-phosphate inhibits apoptosis through a phosphatidylinositol 3-kinase/Akt pathway in human hepatocytes.

Human hepatocytes usually are resistant to TNF-alpha cytotoxicity. In mouse or rat hepatocytes, repression of NF-kappaB activation is sufficient to induce TNF-alpha-mediated apoptosis. However, in both Huh-7 human hepatoma cells and Hc human normal hepatocytes, when infected with an adenovirus expressing a mutated form of IkappaBalpha (Ad5IkappaB), which almost completely blocks NF-kappaB activation, >80% of the cells survived 24 h after TNF-alpha stimulation. Here, we report that TNF-alpha activates other antiapoptotic factors, such as sphingosine kinase (SphK), phosphatidylinositol 3-kinase (PI3K), and Akt kinase. Pretreatment of cells with N,N-dimethylsphingosine (DMS), an inhibitor of SphK, or LY 294002, an inhibitor of PI3K that acts upstream of Akt, increased the number of apoptotic cells induced by TNF-alpha in Ad5IkappaB-infected Huh-7 and Hc cells. TNF-alpha-induced activations of PI3K and Akt were inhibited by DMS. In contrast, exogenous sphingosine 1-phosphate, a product of SphK, was found to activate Akt and partially rescued the cells from TNF-alpha-induced apoptosis. Although Akt has been reported to activate NF-kappaB, DMS and LY 294002 failed to prevent TNF-alpha-induced NF-kappaB activation, suggesting that the antiapoptotic effects of SphK and Akt are independent of NF-kappaB. Furthermore, apoptosis mediated by Fas ligand (FasL) involving Akt activation also was potentiated by DMS pretreatment in Hc cells. Sphingosine 1-phosphate administration partially protected cells from FasL-mediated apoptosis. These results indicate that not only NF-kappaB but also SphK and PI3K/Akt are involved in the signaling pathway(s) for protection of human hepatocytes from the apoptotic action of TNF-alpha and probably FasL.     

Induction of apoptosis by nitric oxide in macrophages is independent of apoptotic volume decrease

Apoptosis occurs through a sequence of specific biochemical and morphological alterations that define the progress of cell death. The changes of the mitochondrial inner membrane potential (DeltaPsi(m)), the release of cytochrome c to the cytosol, the apoptotic volume decrease (AVD) and the activation of caspases have been measured in RAW 264.7, HeLa and Jurkat T cells incubated with molecules that induce apoptosis through the mitochondrial pathway. Our data show that NO, staurosporine, etoposide and camptothecin increased DeltaPsi(m) in macrophages but not in HeLa and Jurkat cells, that exhibited a DeltaPsi(m) decrease. Moreover, the apoptosis induced by NO in macrophages, but not that promoted by staurosporine, might occur in the absence of AVD. Analysis of the sequence of apoptotic manifestations shows that DeltaPsi(m) precedes AVD and caspase activation in RAW 264.7 cells. Inhibition of AVD abrogates apoptosis in HeLa and Jurkat T cells regardless of the stimuli used. These data suggest that the changes of DeltaPsi(m) are cell-type dependent and that AVD is dispensable for apoptosis in macrophages.     

Autophagy-Regulated ROS from Xanthine Oxidase Acts as an Early Effector for Triggering Late Mitochondria-Dependent Apoptosis in Cathepsin S-Targeted Tumor Cells

Cathepsin S (CTSS), which is highly expressed in various malignant tumor cells, has been proposed to promote tumor progression, migration, and invasion. CTSS inhibition not only blocks tumor cell invasion and endothelial tube formation but also induces cellular cytotoxicity. In our previous studies, we have observed that CTSS inhibition induces autophagy, which is responsible for up-regulating xanthine oxidase for early ROS generation and consequent cell death. However, whether the autophagy-regulated early ROS triggers apoptosis remains unclear. We conducted a long-term follow-up study to investigate the relationship between early autophagy and late mitochondria-dependent apoptosis. We demonstrated that early ROS generation is critical for mitochondria damage and the activation of intrinsic apoptotic pathway. Attenuating the early ROS level diminished later mitochondrial damage and downstream apoptotic signaling. Collectively, mitochondria-dependent apoptosis is regulated by autophagy-regulated early ROS, which serves as an early effector that triggers mitochondrial signaling for late apoptosis. The data emphasize the essential role of autophagy-regulated early ROS in triggering late apoptotic signaling.     

Cytochrome c is released in a single step during apoptosis

Release of cytochrome c from mitochondria is a central event in apoptotic signaling. In this study, we utilized a cytochrome c fusion that binds fluorescent biarsenical ligands (cytochrome c-4CYS (cyt. c-4CYS)) as well as cytochrome c-green fluorescent protein (cyt. c-GFP) to measure its release from mitochondria in different cell types during apoptosis. In single cells, the kinetics of cyt. c-4CYS release was indistinguishable from that of cyt. c-GFP in apoptotic cells expressing both molecules. Lowering the temperature by 7 degrees C did not affect this corelease, but further separated cytochrome c release from the subsequent decrease in mitochondrial membrane potential (DeltaPsi(m)). Cyt. c-GFP rescued respiration in cells lacking endogenous cytochrome c, and the duration of cytochrome c release was approximately 5 min in a variety of cell types induced to die by various forms of cellular stress. In addition, we could observe no evidence of caspase-dependent amplification of cytochrome c release or changes in DeltaPsi(m) preceding the release of cyt. c-GFP. We conclude that there is a general mechanism responsible for cytochrome c release that proceeds in a single step that is independent of changes in DeltaPsi(m).     

DeltaPsi(m)-Dependent and -independent production of reactive oxygen species by rat brain mitochondria.

Mitochondria are widely believed to be the source of reactive oxygen species (ROS) in a number of neurodegenerative disease states. However, conditions associated with neuronal injury are accompanied by other alterations in mitochondrial physiology, including profound changes in the mitochondrial membrane potential DeltaPsi(m). In this study we have investigated the effects of DeltaPsi(m) on ROS production by rat brain mitochondria using the fluorescent peroxidase substrates scopoletin and Amplex red. The highest rates of mitochondrial ROS generation were observed while mitochondria were respiring on the complex II substrate succinate. Under this condition, the majority of the ROS signal was derived from reverse electron transport to complex I, because it was inhibited by rotenone. This mode of ROS generation is very sensitive to depolarization of DeltaPsi(m), and even the depolarization associated with ATP generation was sufficient to inhibit ROS production. Mitochondria respiring on the complex I substrates, glutamate and malate, produce very little ROS until complex I is inhibited with rotenone, which is also consistent with complex I being the major site of ROS generation. This mode of oxidant production is insensitive to changes in DeltaPsi(m). With both substrates, ubiquinone-derived ROS can be detected, but they represent a more minor component of the overall oxidant signal. These studies demonstrate that rat brain mitochondria can be effective producers of ROS. However, the optimal conditions for ROS generation require either a hyperpolarized membrane potential or a substantial level of complex I inhibition.     

Bcl-x(L) prevents the initial decrease in mitochondrial membrane potential and subsequent reactive oxygen species production during tumor necrosis factor alpha-induced apoptosis

The Bcl-2 family of proteins are involved in regulating the redox state of cells. However, the mode of action of Bcl-2 proteins remains unclear. This work analyzed the effects of Bcl-x(L) on the cellular redox state after treatment with tumor necrosis factor alpha (TNF-alpha) or exogenous oxidants. We show that in cells that undergo TNF-alpha-induced apoptosis, TNF-alpha induces a partial decrease in mitochondrial membrane potential (DeltaPsi(m)) followed by high levels of reactive oxygen species (ROS). ROS scavengers delay the progression of mitochondrial depolarization and apoptotic cell death. This indicates that ROS are important mediators of mitochondrial depolarization. However, ROS scavengers fail to prevent the initial TNF-alpha-induced decrease in DeltaPsi(m). In contrast, expression of Bcl-x(L) prevents both the initial decrease in DeltaPsi(m) following TNF-alpha treatment and the subsequent induction of ROS. Bcl-x(L) itself does not act as a ROS scavenger. In addition, Bcl-x(L) does not block the initial decrease in DeltaPsi(m) following treatment with the oxidant hydrogen peroxide. However, unlike control-transfected cells, Bcl-x(L)-expressing cells can recover their mitochondrial membrane potential following the initial drop in DeltaPsi(m) induced by hydrogen peroxide. These data suggest that Bcl-x(L) plays a regulatory role in controlling the membrane potential of and ROS production by mitochondria rather than acting as a direct antioxidant.     

Visualizing common deletion of mitochondrial DNA-augmented mitochondrial reactive oxygen species generation and apoptosis upon oxidative stress

Common deletion (CD) 4977 bp of mitochondrial DNA (mtDNA) disrupt specifically mitochondrial complex I, IV and V on the electron transport chain (ETC) and is closely associated with wide spectrums of clinical manifestations. To quantitatively investigate how CD-induced ETC defect alters mitochondrial reactive oxygen species (mROS) generation as well as down stream apoptotic signaling, we employed an established array of human CD cytoplasmic hybrids (cybrids) harboring 0%-80% of CD. Pathological effects of CD on the mitochondria were visualized at single cell level by the application of fluorescent probes coupled with conventional and multiphoton imaging microscopy. Intriguingly, we observed CD-augmented mROS generation omitted "threshold effect". CD-augmented mROS generation was associated with depolarized mitochondrial membrane potential (DeltaPsi(m)). Upon oxidative stress, the amount of CD-augmented mROS generation was greatly enhanced to cause pathological apoptotic deterioration including opening of the mitochondrial permeability transition, cytochrome c release, phosphatidylserine externalization and DNA fragmentation. In addition, heterogeneous mitochondrial dysfunctions were found in cybrids containing 80% of CD (D cybrids), i.e., low sensitive-D (LS-D, roughly 80%) and a super sensitive-D (SS-D, 20%). As compared to LS-D, SS-D had higher resting mROS level but slightly hyperpolarized DeltaPsi(m). Upon H2O2 treatment, much faster generation of mROS was observed which induced a faster depolarization of DeltaPsi(m) and later apoptotic deterioration in SS-D. We proposed a dose-dependent, feed-forward and self-accelerating vicious cycle of mROS production might be initiated in CD-induced ETC defect without threshold effect. As CD-augmented mROS generation is obligated to cause an enhanced pathological apoptosis, precise detection of CD-augmented mROS generation and their degree of heterogeneity in single cells may serve as sensitive pathological indexes for early diagnosis, prognosis and treatment of CD-associated diseases.     

Apoptosis induced by capsaicin in prostate PC-3 cells involves ceramide accumulation,neutral sphingomyelinase,and JNK activation

Numerous studies have recently focused on the anticarcinogenic, antimutagenic, or chemopreventive activities of the main pungent component of red pepper, capsaicin (N-vanillyl-8-methyl-1-nonenamide). We have previously shown that, in the androgen-independent prostate cancer PC-3 cells, capsaicin inhibits cell growth and induces apoptosis through reactive oxygen species (ROS) generation [Apoptosis 11 (2006) 89–99]. In the present study, we investigated the signaling pathways involved in the antiproliferative effect of capsaicin. Here, we report that capsaicin apoptotic effect was mediated by ceramide generation which occurred by sphingomyelin hydrolysis. Using siRNA, we demonstrated that N-SMase expression is required for the effect of capsaicin on prostate cell viability. We then investigated the role of MAP kinase cascades, extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, in the antiproliferative effect of capsaicin, and we confirmed that capsaicin could activate ERK and JNK but not p38 MAPK. Pharmacological inhibition of JNK kinase, as well as inhibition of ROS by the reducing agent N-acetylcysteine, prevented ceramide accumulation and capsaicin-induced cell death. However, inhibition of ceramide accumulation by the SMase inhibitor D609 did not modify JNK activation. These data reveal JNK as an upstream regulator of ceramide production. Capsaicin-promoted activation of ERK was prevented with all the inhibitors tested. We conclude that capsaicin induces apoptosis in PC-3 cells via ROS generation, JNK activation, ceramide accumulation, and second, ERK activation.     

Benzyl isothiocyanate targets mitochondrial respiratory chain to trigger reactive oxygen species-dependent apoptosis in human breast cancer cells

Benzyl isothiocyanate (BITC), a dietary cancer chemopreventive agent, causes apoptosis in MDA-MB-231 and MCF-7 human breast cancer cells, but the mechanism of cell death is not fully understood. We now demonstrate that the BITC-induced apoptosis in human breast cancer cells is initiated by reactive oxygen species (ROS) due to inhibition of complex III of the mitochondrial respiratory chain. The BITC-induced ROS production and apoptosis were significantly inhibited by overexpression of catalase and Cu,Zn-superoxide dismutase and pharmacological inhibition of the mitochondrial respiratory chain. The mitochondrial DNA-deficient Rho-0 variant of MDA-MB-231 cells was nearly completely resistant to BITC-mediated ROS generation and apoptosis. The Rho-0 MDA-MB-231 cells also resisted BITC-mediated mitochondrial translocation (activation) of Bax. Biochemical assays revealed inhibition of complex III activity in BITC-treated MDA-MB-231 cells as early as at 1 h of treatment. The BITC treatment caused activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), which function upstream of Bax activation in apoptotic response to various stimuli. Pharmacological inhibition of both JNK and p38 MAPK conferred partial yet significant protection against BITC-induced apoptosis. Activation of JNK and p38 MAPK resulting from BITC exposure was abolished by overexpression of catalase. The BITC-mediated conformational change of Bax was markedly suppressed by ectopic expression of catalytically inactive mutant of JNK kinase 2 (JNKK2(AA)). Interestingly, a normal human mammary epithelial cell line was resistant to BITC-mediated ROS generation, JNK/p38 MAPK activation, and apoptosis. In conclusion, the present study indicates that the BITC-induced apoptosis in human breast cancer cells is initiated by mitochondria-derived ROS.     

Synthesis,antiproliferative and apoptosis induction potential activities of novel bis(indolyl)hydrazide-hydrazone derivatives

In recent years, indole-indazolyl hydrazide-hydrazone derivatives with strong cell growth inhibition and apoptosis induction characteristics are being strongly screened for their cancer chemo-preventive potential. In the present study, N-methyl and N,N-dimethyl bis(indolyl)hydrazide-hydrazone analog derivatives were designed, synthesized and allowed to evaluate for their anti-proliferative and apoptosis induction potential against cervical (HeLa), breast (MCF-7 and MDA-MB-231) and lung (A549) cancer cell lines relative to normal HEK293 cells. The MTT assay in conjunction with mitochondrial potential assays and the trypan blue dye exclusion were employed to ascertain the effects of the derivatives on the cancer cells. Further, mechanistic studies were conducted on compound 14a to understand the biochemical mechanisms and functional interactions with various signaling pathways triggered in HeLa and MCF-7 cells. Compound 14a induced apoptosis via caspase independent pathway through the participation of mitogen-activated protein kinases (MAPK) such as extracellular signal related kinase (ERK) and p38 as well as p53 pathways. It originates the activation of pro-apoptotic proteins such as Bak and Mcl-1s and also strongly induced the generation of reactive oxygen species. In downstream signaling pathway, activated p53 protein interacted with MAPK pathways, including SAPK/c-Jun N-terminal protein kinase (JNK), p38 and ERK kinases resulting in apoptotic cell death. The involvement of MAPK cascades such as p38, ERK and p38 on compound 14a induced apoptotic cell death was evidenced by the fact that the inclusion of specific inhibitors of p38, ERK1/2 and JNK MAPK (SB2035809, PD98059 and SP600125) prevented the compound 14a towards induced apoptosis. The results clearly showed that MAP kinase cascades were crucial for apoptotic response in compound 14a induced cellular killing and were dependent on p53 activity. Based on the results, compound 14a was identified as a promising candidate for cancer therapeutics and these findings furnish a basis for further in vivo experiments on anti-proliferative activity.     

Arisostatins A induces apoptosis through the activation of caspase-3 and reactive oxygen species generation in AMC-HN-4 cells

A microbial secondary metabolite, arisostatins A (As-A), was originally discovered as a substance carrying the antibiotic activity against Gram-positive bacteria and shown to possess potent anti-tumor properties. The mechanism by which arisostatins A initiates apoptosis remains poorly understood. In the present report we investigated the effect of arisostatins A on activation of the apoptotic pathway in HN-4 cells. Arisostatins A was shown to be responsible for the inhibition of HN-4 cell growth by inducing apoptosis. Treatment with 4 microM arisostatins A for 24h produced morphological features of apoptosis and DNA fragmentation in HN-4 cells. Arisostatins A caused dose-dependent apoptosis and DNA fragmentation of HN-4 cells used as a model. Treatment with caspase inhibitor significantly reduced the arisostatins A-induced caspase 3 activation. In addition, arisostatins A-induced apoptosis was associated with the generation of reactive oxygen species (ROS), which was prevented by an antioxidant NAC (N-acetyl-cysteine). These data indicate that cytotoxic effect of arisostatins A on HN-4 cells is attributable to the induced apoptosis and that arisostatins A-induced apoptosis is mediated by caspase-3 activation pathway, loss of mitochondrial transmembrane potential (DeltaPsi(m)), and release of cytochrome c into cytosol.     

Inhibition of Mitogen-Activated Kinase Signaling Sensitizes HeLa Cells to Fas Receptor-Mediated Apoptosis

The Fas receptor (FasR) is an important physiological mediator of apoptosis in various tissues and cells. However, there are also many FasR-expressing cell types that are normally resistant to apoptotic signaling through this receptor. The mitogen-activated protein kinase (MAPK) signaling cascade has, apart from being a growth-stimulating factor, lately received attention as an inhibitory factor in apoptosis. In this study, we examined whether MAPK signaling could be involved in protecting FasR-insensitive cells. To this end, we used different approaches to inhibit MAPK signaling in HeLa cells, including treatment with the MAPK kinase inhibitor PD 98059, serum withdrawal, and expression of dominant-interfering MAPK kinase mutant protein. All of these treatments were effective in sensitizing the cells to FasR-induced apoptosis, demonstrating that MAPK indeed is involved in the control of FasR responses. The MAPK-mediated control seemed to occur at or upstream of caspase 8, the initiator caspase in apoptotic FasR responses. Transfection with the constitutively active MAPK kinase abrogated FasR-induced apoptosis also in the presence of cycloheximide, indicating that the MAPK-generated suppression of FasR-mediated apoptotic signaling is protein synthesis independent. In cells insensitive to FasR-induced apoptosis, stimulation of the FasR with an agonistic antibody resulted in significant MAPK activation, which was inhibited by PD 98059. When different cell types were compared, the FasR-mediated MAPK activation seemed proportional to the degree of FasR insensitivity. These results suggest that the FasR insensitivity is likely to be a consequence of FasR-induced MAPK activation, which in turn interferes with caspase activation.     

Role of BI-1 (TEGT)-mediated ERK1/2 activation in mitochondria-mediated apoptosis and splenomegaly in BI-1 transgenic mice

Bax Inhibitor-1 (BI-1) is an evolutionally conserved apoptotic suppressor and belongs to the BI-1 family of proteins, which contain BI-1-like transmembrane domains. As their cellular functions and regulatory mechanisms remain incompletely understood, we compared their anti-apoptotic properties. Forced expression of BI-1 resulted in the most effective suppression of stress-induced apoptosis, compared with other family members, together with significant extracellular signal-regulated kinase (ERK)1/2 activation. BI-1-mediated ERK1/2 activation led to the suppression of mitochondria-mediated reactive oxygen species (ROS) production. Involvement of the ERK signaling pathway in BI-1-induced anti-apoptotic effects was confirmed by knockdown studies with ERK- or BI-1-specific siRNA. Moreover, we produced transgenic (TG) mice overexpressing BI-1, and the relationship between ERK1/2 activation and the suppression of ROS production or apoptosis was confirmed in mouse embryonic fibroblast (MEF) cells derived from these mice. Interestingly, we found that BI-1 TG mice showed splenomegaly and abnormal megakaryopoiesis. Taken together, our results suggest that BI-1-induced ERK1/2 activation plays an important role in the modulation of intracellular ROS generation and apoptotic cell death and may also affect autoimmune response.     

大黄素提高HeLa细胞对三氧化二砷促凋亡敏感性的研究

活性氧(reactive oxygen species,ROS)在三氧化二砷(arsenic trioxide,As2O3)诱导肿瘤细胞凋亡中扮演重要角色。本研究用一种天然蒽醌类物质——大黄素(emodin)作为提高HeLa细胞ROS水平的手段,考察其对As2O3促凋亡敏感性的影响,并探究可能涉及的信号传导机制。结果显示大黄素10μmol／L提高ROS并增加了HeLa细胞在As2O32μmol／L作用下的凋亡率,对正常成纤维细胞却无影响。该联合作用可以促进HeLa细胞线粒体跨膜电位降低;抑制转录因子NF-κB激活。本研究提示：大黄素通过提高ROS介导凋亡信号传导的增强和生存信号传导的抑制,增加HeLa细胞对As2O3促凋亡的敏感性。