IL-33 enhances Siglec-8 mediated apoptosis of human eosinophils

IL-33 activates eosinophils directly via the ST2 receptor. Like IL-5, IL-33 induces eosinophilia and eosinophilic airway inflammation in mouse models and primes human eosinophil responses. Previously, we reported that IL-5 priming enhances Siglec-8 mediated mitochondrial and reactive oxygen species (ROS)-dependent eosinophilic apoptosis and eliminates caspase dependence of this cell death process. Whether IL-33, like IL-5, augments pro-apoptotic pathways involving receptors such as Siglec-8 and in a similar manner has not been explored. Annexin-V labeling was performed to detect apoptosis in human eosinophils pre-incubated with or without a range of concentrations of IL-33 and/or IL-5 in the presence or absence of Siglec-8 monoclonal antibody (mAb) 2C4 and inhibitors of caspases. Tetramethyl-rhodamine staining was used as a marker of mitochondrial membrane potential loss and injury. ROS production was determined by measuring the superoxide dismutase-inhibitable reduction of cytochrome c. Cleavage of poly(ADP-ribose) polymerase (PARP) was assessed using Western blotting. Eosinophils cultured alone or with mAb 2C4 underwent low levels of apoptosis at 24 h. 2C4-induced eosinophil apoptosis was markedly and equally enhanced after culture for 24 h with either IL-33 or IL-5, although IL-5 was more potent. Effects on apoptosis with IL-33 and IL-5 were synergistic. In contrast, percentages of cells exhibiting reduced mitochondrial membrane potential were greater with IL-33 than IL-5 and effects of these cytokines were also synergistic. Antimycin, an inhibitor of mitochondrial electron transport, almost completely inhibited 2C4-induced apoptosis with either IL-33 or IL-5. Surprisingly, 2C4-induced eosinophil ROS production was significantly enhanced with IL-5 but not IL-33. Siglec-8-mediated apoptosis in the presence of IL-33 was more sensitive in magnitude than IL-5 to inhibition by the pan-caspase inhibitor Z-VAD-FMK, yet both cytokine conditions were associated with PARP cleavage. These data demonstrate that IL-33 is as effective but less potent than IL-5 in enhancing Siglec-8-mediated eosinophil apoptosis, and can synergize with IL-5. Eosinophils primed by IL-33 and/or IL-5 in vivo would be expected to display enhanced susceptibility to undergoing Siglec-8-induced apoptosis.     

Mechanisms of Siglec-F-Induced Eosinophil Apoptosis: A Role for Caspases but Not for SHP-1, Src Kinases,NADPH Oxidase or Reactive Oxygen

Background

Siglec-F and Siglec-8 are functional paralog proapoptotic cell surface receptors expressed on mouse and human eosinophils, respectively. Whereas Siglec-8 mediated death involves caspases and/or reactive oxygen species (ROS) generation and mitochondrial injury, very little is known about Siglec-F-mediated signaling and apoptosis. Therefore the objective of the current experiments was to better define apoptosis pathways mediated by Siglec-F and Siglec-8. Given that Siglec-F-induced apoptosis is much less robust than Siglec-8-induced apoptosis, we hypothesized that mechanisms involved in cell death via these receptors would differ.

Methods

Consequences of engagement of Siglec-F on mouse eosinophils were studied by measuring ROS production, and by performing apoptosis assays using eosinophils from normal, hypereosinophilic, NADPH oxidase-deficient, src homology domain-containing protein tyrosine phosphatase (SHP)-1-deficient, and Lyn kinase-deficient mice. Inhibitors of caspase and Src family kinase activity were also used.

Results

Engagement of Siglec-F induced mouse eosinophil apoptosis that was modest in magnitude and dependent on caspase activity. There was no detectable ROS generation, or any role for ROS, NADPH oxidase, SHP-1, or Src family kinases in this apoptotic process.

Conclusions

These data suggest that Siglec-F-mediated apoptosis is different in both magnitude and mechanisms when compared to published data on Siglec-8-mediated human eosinophil apoptosis. One likely implication of this work is that models targeting Siglec-F in vivo in mice may not provide identical mechanistic predictions for consequences of Siglec-8 targeting in vivo in humans.     

Effect of Dicycloplatin,a Novel Platinum Chemotherapeutical Drug,on Inhibiting Cell Growth and Inducing Cell Apoptosis

Dicycloplatin, a new supramolecular platinum-based antitumor drug, has been approved by the State Food and Administration (SFDA) of China. In this study, we investigated the anticancer activity of dicycloplatin in cancer cells and signaling pathways involved in dicycloplatin-induced apoptosis. Dicycloplatin inhibited the proliferation of cancer cells and increased the percentage of apoptosis in a concentration-dependent manner. Besides, some apoptosis related events were observed after treatment with dicycloplatin, including increase of reactive oxygen species (ROS), collapse of mitochondrial membrane potential (Δψm), release of cytochrome c from the mitochondria to the cytosol, upregulation of p53, which were accompanied by activation of caspase-9, caspase-3, caspase-8, and poly (ADP-ribose) polymerase cleavage in a concentration-dependent manner. The role of apoptosis in dicycloplatin-mediated cell death was further confirmed by the concomitant treatment with caspase-8 or caspase-9 inhibitors, which inhibited apoptosis and PARP cleavage. Intracellular glutathione (GSH) was also found to inhibit the cytotoxic effect of dicycloplatin. In conclusion, these findings suggest that dicycloplatin induces apoptosis through ROS stress-mediated death receptor pathway and mitochondrial pathway which is similar to carboplatin.     

Caudatin induces caspase-dependent apoptosis in human glioma cells with involvement of mitochondrial dysfunction and reactive oxygen species generation

Caudatin as one species of C-21 steroidal from Cynanchum bungei decne displays potential anticancer activity. However, the underlying mechanisms remain elusive. In the present study, the growth suppressive effect and mechanism of caudatin on human glioma U251 and U87 cells were evaluated in vitro. The results indicated that caudatin significantly inhibited U251 and U87 cell growth in both a time- and dose-dependent manner. Flow cytometry analysis revealed that caudatin-induced cell growth inhibition was achieved by induction of cell apoptosis, as convinced by the increase of Sub-G1 peak, PARP cleavage and activation of caspase-3, caspase-7 and caspase-9. Caudatin treatment also resulted in mitochondrial dysfunction which correlated with an imbalance of Bcl-2 family members. Further investigation revealed that caudatin triggered U251 cell apoptosis by inducing reactive oxygen species (ROS) generation through disturbing the redox homeostasis. Moreover, pretreatment of caspase inhibitors apparently weakens caudatin-induced cell killing, PARP cleavage and caspase activation and eventually reverses caudatin-mediated apoptosis. Importantly, caudatin significantly inhibited U251 tumour xenografts in vivo through induction of cell apoptosis involving the inhibition of cell proliferation and angiogenesis, which further validate its value in combating human glioma in vivo. Taken together, the results described above all suggest that caudatin inhibited human glioma cell growth by induction of caspase-dependent apoptosis with involvement of mitochondrial dysfunction and ROS generation.     

Motexafin gadolinium induces mitochondriallymediated caspase-dependent apoptosis

Motexafin gadolinium (MGd, Xcytrin^®) is a tumor-localizing redox mediator that catalyzes the oxidation of intracellular reducing molecules including NADPH, ascorbate, protein and non-protein thiols, generating reactive oxygen species (ROS). MGd localizes to tumors and cooperates with radiation and chemotherapy to kill tumor cells in tissue culture and animal models. In this report, we demonstrate that MGd triggers the mitochondrial apoptotic pathway in the HF-1 lymphoma cell line as determined by loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, activation of caspase-9 prior to caspase-8, cleavage of PARP and annexin V binding. There was minimal effect on MGd-induced apoptosis by the caspase inhibitor z-VAD-fmk, even though caspase-3 activity (as measured by DEVD-cleavage) was completely inhibited. However, MGd-induced apoptosis was reduced to baseline levels by the more potent caspase inhibitor Q-VD-OPh, demonstrating that MGd-induced apoptosis is indeed caspase-dependent. Apoptosis induced by dexamethasone, doxorubicin and etoposide (mediated through the mitochondrial pathway) was also more sensitive to inhibition by Q-VD-OPh than z-VAD-fmk. Our results demonstrating differential sensitivity of drug-induced apoptosis to caspase inhibitors suggest that the term “caspase-independent apoptosis” cannot be solely defined as apoptosis that is not inhibited by z-VAD-fmk as has been utilized in some published studies.     

Implication of calpain in caspase activation during B cell clonal deletion.

In the absence of costimulating signals, B cell receptor (BCR) crosslinking on immature B cells triggers the apoptotic cell death program. In the WEHI-231 B cell lymphoma model, anti-IgM crosslinking triggers activation of caspase-7 independently of caspase-8, followed by apoptosis. Two main mechanisms for caspase-7 activation have been proposed: (i) caspase-8 recruitment to death receptors (Fas or tumour necrosis factor); and (ii) changes in mitochondrial membrane permeability and cytochrome c release, which activate caspase-9. Here we report that caspase-7 activation induced by BCR crosslinking is independent of caspase-8 and cytochrome c translocation from mitochondria to the cytosol, as well as of mitochondrial depolarization. In addition, in a cell-free system, the S-100 fraction of anti-IgM-treated WEHI-231 cells induces a caspase activation pattern different from that activated by cytochrome c and dATP. We demonstrate that calpain specifically triggers activation and processing of caspase-7 both in vitro and in vivo, and that both processes are inhibited by calpain inhibitors. Furthermore, calpain activation is associated with decreased expression levels of calpastatin, which is upregulated by CD40 ligation. These data confirm a role for calpain during BCR crosslinking, which may be critical for cell deletion by apoptosis during B cell development and activation.     

Molecular mechanism of diclofenac-induced apoptosis of promyelocytic leukemia: dependency on reactive oxygen species, Akt, Bid, cytochrome and caspase pathway

Nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in a variety of cells, but the mechanism of this effect has not been fully elucidated. We report that diclofenac, a NSAID, induces growth inhibition and apoptosis of HL-60 cells through modulation of mitochondrial functions regulated by reactive oxygen species (ROS), Akt, caspase-8, and Bid. ROS generation occurs in an early stage of diclofenac-induced apoptosis preceding cytochrome c release, caspase activation, and DNA fragmentation. N-Acetyl-L-cysteine, an antioxidant, suppresses ROS generation, Akt inactivation, caspase-8 activation, and DNA fragmentation. Cyclic AMP, an inducer of Akt phosphorylation, suppresses Akt inactivation, Bid cleavage, and DNA fragmentation. LY294002, a PI3 kinase inhibitor, enhances Akt inactivation and DNA fragmentation. Ac-IETD-CHO, a caspase-8 inhibitor, suppresses Bid cleavage and DNA fragmentation. z-VAD-fmk, a universal caspase inhibitor, but not cyclosporin A (CsA), an inhibitor of mitochondrial membrane permeability transition, suppresses DNA fragmentation. These results suggest the sequential mechanism of diclofenac-induced apoptosis of HL-60 cells: ROS generation suppresses Akt activity, thereby activating caspase-8, which stimulates Bid cleavage and induces cytochrome c release and the activation of caspase-9 and-3 in a CsA-insensitive mechanism. Furthermore, we found that 2-methoxyestradiol (2-ME), a superoxide dismutase inhibitor, significantly enhances diclofenac-induced apoptosis; that is, diclofenac combined with 2-ME may have therapeutic potential in the treatment of human leukemia.     

The RIP-like kinase, RIP3, induces apoptosis and NF-kappaB nuclear translocation and localizes to mitochondria

A RIP-like protein, RIP3, has recently been reported that contains an N-terminal kinase domain and a novel C-terminal domain that promotes apoptosis. These experiments further characterize RIP3-mediated apoptosis and NF-kappaB activation. Northern blots indicate that rip3 mRNA displays a restricted pattern of expression including regions of the adult central nervous system. The rip3 gene was localized by fluorescent in situ hybridization to human chromosome 14q11.2, a region frequently altered in several types of neoplasia. RIP3-mediated apoptosis was inhibited by Bcl-2, Bcl-x(L), dominant-negative FADD, as well as the general caspase inhibitor Z-VAD. Further dissection of caspase involvement in RIP3-induced apoptosis indicated inhibition by the more specific inhibitors Z-DEVD (caspase-3, -6, -7, -8, and -10) and Z-VDVAD (caspase-2). However, caspase-1, -6, -8 and -9 inhibitors had little or no effect on RIP3-mediated apoptosis. Mutational analysis of RIP3 revealed that the C-terminus of RIP3 contributed to its apoptotic activity. This region is similar, but distinct, to the death domain found in many pro-apoptotic receptors and adapter proteins, including FAS, FADD, TNFR1, and RIP. Furthermore, point mutations of RIP3 at amino acids conserved among death domains, abrogated its apoptotic activity. RIP3 was localized by immunofluorescence to the mitochondrion and may play a key role in the mitochondrial disruptions often associated with apoptosis.     

5-Phenylselenyl- and 5-methylselenyl-methyl-2′-deoxyuridine induce oxidative stress, DNA damage, and caspase-2-dependent apoptosis in cancer cells

In the present study, we investigated the signaling pathways implicated in the induction of apoptosis by two modified nucleosides, 5-phenylselenyl-methyl-2′-deoxyuridine (PhSe-T) and 5-methylselenyl-methyl-2′-deoxyuridine (MeSe-T), using human cancer cell lines. The induction of apoptosis was associated with proteolytic activation of caspase-3 and -9, PARP cleavage, and decreased levels of IAP family members, including c-IAP-1 and c-IAP-2, but had no effect on XIAP and survivin. PhSe-T and MeSe-T also enhanced the activities of caspase-2 and -8, Bid cleavage, and the conformational activation of Bax. Additionally, nucleoside derivative-induced apoptosis was inhibited by the selective inhibitors of caspase-2, -3, -8, and -9 and also by si-RNAs against caspase-2, -3, -8, and -9; however, inhibition of caspase-2 and -3 was more effective at preventing apoptosis than inhibition of caspase-8 and -9. Moreover, the inhibition of caspase-2 activation by the pharmacological inhibitor z-VDVAD-fmk or by the knockdown of protein expression using siRNA suppressed nucleoside derivative-induced caspase-3 activation, but not vice versa. PhSe-T and MeSe-T also induced a Δψ_m loss via a CsA-insensitive mechanism, ROS production, and DNA damage, including strand breaks. Moreover, ROS scavengers such as NAC, tiron, and quercetin inhibited nucleoside derivative-induced ROS generation and apoptosis by blocking the sequential activation of caspase-2 and -3, indicating the role of ROS in caspase-2-mediated apoptosis. Taken together, these results indicate that caspase-2 acts upstream of caspase-3 and that caspase-2 functions in response to DNA damage in both PhSe-T- and MeSe-T-induced apoptosis. Our results also suggest that ROS are critical regulators of the sequential activation of caspase-2 and -3 in nucleoside derivative-treated cancer cells.     

Mitochondrial mechanism of microvascular endothelial cells apoptosis in hyperhomocysteinemia

An elevated level of homocysteine (Hcy) limits the growth and induces apoptosis. However, the mechanism of Hcy-induced programmed cell death in endothelial cells is largely unknown. We hypothesize that Hcy induces intracellular reactive oxygen species (ROS) production that leads to the loss of transmembrane mitochondrial potential (Deltapsi(m)) accompanied by the release of cytochrome-c from mitochondria. Cytochrome-c release contributes to caspase activation, such as caspase-9, caspase-6, and caspase-3, which results in the degradation of numerous nuclear proteins including poly (ADP-ribose) polymerase (PARP), which subsequently leads to the internucleosomal cleavage of DNA, resulting cell death. In this study, rat heart microvascular endothelial cells (MVEC) were treated with different doses of Hcy at different time intervals. Apoptosis was measured by DNA laddering and transferase-mediated dUTP nick-end labeling (TUNEL) assay. ROS production and MP were determined using fluorescent probes (2,7-dichlorofluorescein (DCFH-DA) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzamidazolocarbocyanin iodide (JC-1), respectively, by confocal microscopy. Differential gene expression for apoptosis was analyzed by cDNA array. The results showed that Hcy-mediated ROS production preceded the loss of MP, the release of cytochrome-c, and the activation of caspase-9 and -3. Moreover the Hcy treatment resulted in a decrease in Bcl(2)/Bax ratio, evaluated by mRNA levels. Caspase-9 and -3 were activated, causing cleavage of PARP, a hallmark of apoptosis and internucleosomal DNA fragmentation. The cytotoxic effect of Hcy was blocked by using small interfering RNA (siRNA)-mediated suppression of caspase-9 in MVEC. Suppressing the activation of caspase-9 inhibited the activation of caspase -3 and enhanced the cell viability and MP. Our data suggested that Hcy-mediated ROS production promotes endothelial cell death in part by disturbing MP, which results in subsequent release of cytochrome-c and activation of caspase-9 and 3, leading to cell death.     

Pre-processed caspase-9 contained in mitochondria participates in apoptosis.

As shown here, mitochondria purified from different organs (liver, brain, kidney, spleen and heart) contain both pro-caspase-9 and the processed, mature form of caspase-9. Purified liver mitochondria release mature caspase-9 upon induction of permeability transition in vitro. This is accompanied by a discrete increase in the enzymatic cleavage of pro-caspase-9 substrates. We found that SHEP neuroblastoma cells constitutively contain pre-processed caspase-9 in their mitochondria, using a combination of subcellular fractionation and immunofluorescence with an antibody specific for the processed caspase. This is a cell type-specific phenomenon since HeLa cells mitochondria mainly contain pro-caspase-9 and comparatively little processed caspase-9. Upon introduction of apoptosis, mitochondrial pro-caspase-9 translocates to the cytosol and to the nucleus. This phenomenon is inhibited by transfection with Bcl-2. In synthesis, we report the unexpected finding that mitochondria can contain a pre-processed caspase isoform in non-apoptotic cells. Bcl-2-mediated regulation of mitochondrial membrane permeabilization may contribute to apoptosis control by preventing mitochondrial, pre-processed caspase-9 from interacting with its cytosolic activators.     

Modulation of ROS/MAPK signaling pathways by okadaic acid leads to cell death via,mitochondrial mediated caspase-dependent mechanism

Okadaic acid (OA) is a specific and potent protein phosphatase inhibitor and tumor promoter. The present study establishes the role of reactive oxygen species (ROS) and mitogen activated protein kinases in cell death induced by okadaic acid. The study showed that okadaic acid is cytotoxic at 10 nM with an IC50 of 100 nM in U-937 cells. The CVDE assay and mitochondrial dehydrogenase assay showed a time dependent cytotoxicity. The phase contrast visualization of the OA treated cells showed the apoptotic morphology and was confirmed with esterase staining for plasma membrane integrity. OA activated caspases-7, 9 and 3, PARP cleavage and induced nuclear damage in a time and dose dependent manner. Compromised mitochondrial membrane potential, release of cytochrome-c and apoptosis inducing factor confirms the involvement of mitochondria. A time dependent decrease in glutathione levels and a dose dependent increase in ROS with maximum at 30 min were observed. ROS scavenger-N-acetyl cysteine, mitochondrial stabilizer-cyclosporin-A, and broad spectrum caspase inhibitor Z-VAD-FMK inhibited the OA induced caspase-3 activation, DNA damage and cell death but caspase-8 inhibitor had no effect. OA activated p38 MAPK and JNK in a time dependent manner, but not ERK½. MAP kinase inhibitors SB203580, SP600125 and PD98059 confirm the role of p38 MAPK and JNK in OA induced caspase-3 activation and cell death. Over all, our results indicate that OA induces cell death by generation of ROS, and activation of p38 MAPK and JNK, and executed through mitochondrial mediated caspase pathway.     

Loss of caspase-9 provides genetic evidence for the type I/II concept of CD95-mediated apoptosis

The death receptor CD95 triggers apoptosis upon formation of a death-inducing signaling complex and the activation of caspase-8. Two types of CD95-mediated apoptosis have been distinguished that differ in their efficiency of death-inducing signaling complex formation and the requirement of mitochondria for caspase activation. The validity of the type I/II model, however, has been challenged, as Bcl-2 expression or the use of various CD95 agonists resulted in different apoptosis effects. By identifying a caspase-9-deficient T cell line, we now provide genetic evidence for the two-pathway model of CD95-mediated apoptosis and demonstrate that type II cells strongly depend on caspase-9. Caspase-9-deficient cells revealed strongly impaired apoptosis, caspase activation, and mitochondrial membrane depolarization upon CD95 triggering, whereas, surprisingly, activation of Bak and cytochrome c release were not inhibited. Furthermore, caspase-9-deficient cells did not switch to necrosis, and reconstitution of caspase-9 expression restored CD95 sensitivity. Finally, we also show that different death receptors have a distinct requirement for caspase-9.     

Bleomycin initiates apoptosis of lung epithelial cells by ROS but not by Fas/FasL pathway

Epithelial cells are considered to be a main target of bleomycin-induced lung injury, which leads to fibrosis in vivo. We studied the characteristics of in vitro bleomycin-induced apoptosis in a mouse lung epithelial (MLE) cell line. Bleomycin caused an increase of reactive oxygen species (ROS) resulting in oxidative stress, mitochondrial leakage, and apoptosis. These were associated with elevated caspase-8 and resultant caspase-9 activity and with upregulation of Fas expression. Glutathione and inhibitors of caspase-8 or caspase-9, but not of FasL, inhibited these effects, suggesting their dependence on ROS, caspase-8 and -9, in a Fas/FasL-independent pathway. However, postbleomycin-exposed MLE cells were more sensitive to Fas-mediated apoptosis. These results demonstrate that the initial bleomycin-induced oxidative stress causes a direct apoptotic effect in lung epithelial cells involving a regulatory role of caspase-8 on caspase-9. Fas represents an amplification mechanism, and not a direct trigger of bleomycin-induced epithelial cell apoptosis.     

Zebularine inhibits the growth of HeLa cervical cancer cells via cell cycle arrest and caspase-dependent apoptosis

Zebularine (Zeb) as a DNA methyltrasferase (DNMT) inhibitor has various cellular effects such as cell growth inhibition and apoptosis. In the present study, we evaluated the effects of Zeb on the growth and death of HeLa cervical cancer cells. Zeb inhibited the growth of HeLa cells with an IC(50) of approximately 130?μM at 72?h in a dose-dependent manner. DNA flow cytometric analysis indicated that Zeb induced an S phase arrest of the cell cycle, which was accompanied by the increased levels of cdk2 and cyclin A proteins. This agent also induced apoptosis, which was accompanied by the loss of mitochondrial membrane potential (Ψ(m)), PARP-1 cleavage and the activation of caspase-3, -8 and -9. All of the tested caspase inhibitors significantly rescued some cells from Zeb-induced HeLa cell death. In relation to reactive oxygen species (ROS) and glutathione (GSH) levels, O (2) (?-) level was significantly increased in 100?μM Zeb-treated HeLa cells and caspase inhibitors reduced O (2) (?-) level in these cells. Zeb induced GSH depletion in HeLa cells, which was attenuated by caspase inhibitors. In conclusion, this is the first report that Zeb inhibited the growth of HeLa cells via cell cycle arrest and apoptosis.     

Delineation of the caspase-9 signaling cascade

In the intrinsic apoptosis pathway, mitochondrial disruption leads to the release of multiple apoptosis signaling molecules, triggering both caspase-dependent and -independent cell death. The release of cytochrome c induces the formation of the apoptosome, resulting in caspase-9 activation. Multiple caspases are activated downstream of caspase-9, however, the precise order of caspase activation downstream of caspase-9 in intact cells has not been completely resolved. To characterize the caspase-9 signaling cascade in intact cells, we employed chemically induced dimerization to activate caspase-9 specifically. Dimerization of caspase-9 led to rapid activation of effector caspases, including caspases-3, -6 and -7, as well as initiator caspases, including caspases-2, -8 and -10, in H9 and Jurkat cells. Knockdown of caspase-3 suppressed caspase-9-induced processing of the other caspases downstream of caspase-9. Silencing of caspase-6 partially inhibited caspase-9-mediated processing of caspases-2, -3 and -10, while silencing of caspase-7 partially inhibited caspase-9-induced processing of caspase-2, -3, -6 and -10. In contrast, deficiency in caspase-2, -8 or -10 did not significantly affect the caspase-9-induced caspase cascade. Our data provide novel insights into the ordering of a caspase signaling network downstream of caspase-9 in intact cells during apoptosis.     

Interleukin-11 antagonizes Fas ligand-mediated apoptosis in IEC-18 intestinal epithelial crypt cells: role of MEK and Akt-dependent signaling

Interleukin-11 (IL-11) displays epithelial cytoprotective effects during intestinal injury. Antiapoptotic effects of IL-11 have been described, yet mechanisms remain unclear. Fas/CD95 death receptor signaling is upregulated in ulcerative colitis, leading to mucosal breakdown. We hypothesized that IL-11 inhibits Fas ligand (FasL)-mediated apoptosis in intestinal epithelia. Cell death was monitored in IEC-18 cells by microscopy, caspase and poly(ADP-ribose) polymerase cleavage, mitochondrial release of cytochrome c, and abundance of cytoplasmic oligonucleosomal DNA. RT-PCR was used to monitor Fas, cIAP1, cIAP2, XIAP, cFLIP, survivin, and Bcl-2 family members. Fas membrane expression was detected by immunoblot. Inhibitors of JAK2, phosphatidylinositol 3-kinase (PI3-kinase), Akt 1, MEK1 and MEK2, and p38 MAPK were used to delineate IL-11's antiapoptotic mechanisms. IL-11 did not alter Fas expression. Pretreatment with IL-11 for 24 h before FasL reduced cytoplasmic oligonucleosomal DNA by 63.2%. IL-11 also attenuated caspase-3, caspase-9, and poly(ADP-ribose) polymerase cleavage without affecting expression of activated caspase-8 p20 or cytochrome c release. IL-11 did not affect mRNA expression of the candidate antiapoptotic genes. The MEK1 and MEK2 inhibitors U-0126 and PD-98059 significantly attenuated the protection of IL-11 against caspase-3 and caspase-9 cleavage and cytoplasmic oligonucleosomal DNA accumulation. Although Akt inhibition reversed IL-11-mediated effects on caspase cleavage, it did not reverse the protective effects of IL-11 by DNA ELISA. We conclude that IL-11-dependent MEK1 and MEK2 signaling inhibits FasL-induced apoptosis. The lack of reversal of the IL-11 effect on DNA cleavage by Akt inhibition, despite antagonism of caspase cleavage, suggests that IL-11 inhibits caspase-independent cell death signaling by FasL in a MEK-dependent manner.     

Early redox imbalance mediates hydroperoxide-induced apoptosis in mitotic competent undifferentiated PC-12 cells

Our recent study has demonstrated that cellular redox imbalance can directly initiate apoptosis in a mitotic competent PC-12 cell line without the involvement of reactive oxygen species (ROS). However, whether cell apoptosis induced by ROS is, in fact, mediated by a loss of redox balance caused by the oxidant is unresolved. The linkage between oxidant-mediated apoptosis and the induction of cellular redox was examined in PC-12 cells using the oxidant, tert-butylhydroperoxide (TBH). TBH caused cell apoptosis in 24 h that was preceded by an early increase (30 min) in oxidized glutathione (GSSG). Pretreatment with N-acetyl cysteine prevented TBH-induced GSSG increases and cell apoptosis. Altered Bax/BcL-2 expression and release of mitochondrial cytochrome c occurred post-redox imbalance and was kinetically linked to caspase-3 activation and poly ADP-ribose polymerase cleavage. Moreover, cell apoptosis was attenuated by inhibition of caspase-9, but not caspase-8, and blockade of mitochondrial ROS generation and permeability transition pore attenuated caspase 3 activation and cell apoptosis. Collectively, these results show that TBH-induced GSSG elevation is associated with the disruption of mitochondrial integrity, activation of caspase-3 and cell apoptosis. This redox induction of the apoptotic cascade was dissociated from cellular GSH efflux.     

Synergistic induction of apoptosis in A549 cells by dihydroartemisinin and gemcitabine

This report is designed to study the ability of the combined treatment with gemcitabine (Gem) and dihydroartemisinin (DHA) to induce apoptosis in a non-small-cell lung cancer cell line (A549 cells). This combination treatment synergistically inhibited cell growth by inducing apoptosis, and this synergistic action was not associated with reactive oxygen species (ROS). Although either Gem or DHA induced a significant increase in ROS generation, the combination treatment did not further enhance ROS level. Compared with single drugs, the combination treatment significantly potentiated Bak activation, loss of mitochondrial membrane potential, caspase-9 and -3 activation, indicating the important role of the Bak-mediated intrinsic apoptosis pathway in the synergistic action, which was further verified by the significant prevention of the cytotoxicity of the combination treatment by inhibiting one of caspase-9, -3 and Bcl-xL or silencing Bak. In addition, the combination treatment also synergistically activated caspase-8, and inhibition of Fas and caspase-8 presented significant prevention on the cytotoxicity of the combination treatment, indicating that the Fas-caspase-8-mediated extrinsic apoptosis pathway partially participated in the synergistic action. Collectively, the present study demonstrates a strong synergistic action of the combined treatment with Gem and DHA in inducing apoptosis of A549 cells via both the Bak-mediated intrinsic pathway and the Fas-caspase-8-mediated extrinsic pathway.     

Induction of apoptosis by shikonin through a ROS/JNK-mediated process in Bcr/Abl-positive chronic myelogenous leukemia （CML） cells

This study examined the signaling events induced by shikonin that lead to the induction of apoptosis in Bcr/ Abl-positive chronic myelogenous leukemia （CML） cells （e.g., K562, LAMA84）. Treatment of K562 cells with shikonin （e.g., 0.5 pM） resulted in profound induction of apoptosis accompanied by rapid generation of reactive oxygen species （ROS）, striking activation of c-Jun-N-terminal kinase （JNK） and p38, marked release of the mitochondrial proteins cytochrome c and Smac/DIABLO, activation of caspase-9 and -3, and cleavage of PARP. Scavenging of ROS completely blocked all of the above-mentioned events （i.e., JNK and p38 phosphorylation, cytochrome c and Smac/DIABLO release, caspase and PARP cleavage, as well as the induction of apoptosis） following shikonin treatment. Inhibition of JNK and knock-down of JNK1 significantly attenuated cytochrome c release, caspase cleavage and apoptosis, but did not affect shikonin-mediated ROS production. Additionally, inhibition of caspase activation completely blocked shikonin-induced apoptosis, but did not appreciably modify shikonin-mediated cytochrome c release or ROS generation. Altogether, these findings demonstrate that shikonin-induced oxidative injury operates at a proximal point in apoptotic signaling cascades, and subsequently activates the stress-related JNK pathway, triggers mitochondrial dysfunction, cytochrome c release, and caspase activation, and leads to apoptosis. Our data also suggest that shikonin may be a promising agent for the treatment of CML, as a generator of ROS.