Bid Cleavage Research Articles

Abstract 4222: Induction of apoptosis and cell cycle arrest in renal cancer cells by phenethyl isothiocyanate (PEITC) and the mechanisms involved

Abstract Background: Renal Cell Carcinoma (RCC) has low 5 year survival rate and is resistant to radiation and chemotherapy. Phenethyl Isothiocyanate (PEITC) is a naturally occurring phytochemical that has a variety of anti-cancer properties. Here we explore two anti-cancer properties of PEITC: induction of apoptosis and induction of cell cycle arrest in RCC cell lines and the underlying mechanisms. Methods: We used two human RCC cell lines Caki-1 and Caki-2. Survival and cell proliferation was assayed using Calcein AM. Annexin V staining was used to measure apoptosis. Caspase-3/7 induction was measured using a fluorescent substrate. Cell Cycle was studied using Propidium Iodide staining. DNA damage was determined using H2AX-γ antibody. Protein expression and phosphorylation was determined using immunoblotting. Results: PEITC significantly reduced survival of Caki-1 and Caki-2 cells and inhibited their proliferation as determined by Calcein AM. 15 and 20 μM PEITC induced apoptosis (revealed by Annexin V staining) in both cell lines and induced caspase-3/7 activity. Western blot analysis revealed caspase-8, caspase-9 and Bid cleavage suggesting that the apoptosis is mediated through the extrinsic pathway. Lower doses (up to 10 μM) arrested Caki-1 cells in G2/M phase, and this was associated with an increase p38 and MK2 (Thr334) phosphorylation (as revealed by Western Blot). The p38 inhibitor SB203850 inhibited this G2 arrest induced by PEITC. In order to determine if DNA damage was responsible for the apoptosis and G2 arrest, we used an antibody to H2AX-γ phosphorylation. 15 and 20 µM treatment resulted in increased H2AX-γ phosphorylation, but this was completely blocked by caspase-3 inhibitor. Conclusion: PEITC induces apoptosis in Caki-1 and Caki-2 cells by a mechanism that likely involves the extrinsic pathway. We will use immunoblotting and siRNA to determine which of the extrinsic receptors (DR4, DR5, TRAIL or FAS) is responsible for this apoptosis induction. PEITC does not cause direct DNA damage to the cells; the observed DNA damage is a result of the apoptotic process and is blocked by caspase-3 inhibitors. PEITC induces G2/M arrest in Caki-1 cells and the mechanism is dependent on p38 phosphorylation which activates MK2. Inducing cell cycle arrest and apoptosis may play an important role in the anti-cancer properties of PEITC. Fully understanding the mechanism by which PEITC induces apoptosis and cell cycle arrest in RCC cells may lead to development of novel chemotherapeutic drugs against RCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4222. doi:10.1158/1538-7445.AM2011-4222

Delayed-onset caspase-dependent massive hepatocyte apoptosis upon fas activation in bak/bax-deficient mice

The proapoptotic Bcl-2 family proteins Bak and Bax serve as an essential gateway to the mitochondrial pathway of apoptosis. When activated by BH3-only proteins, Bak/Bax triggers mitochondrial outer membrane permeabilization leading to release of cytochrome c followed by activation of initiator and then effector caspases to dismantle the cells. Hepatocytes are generally considered to be type II cells because, upon Fas stimulation, they are reported to require the BH3-only protein Bid to undergo apoptosis. However, the significance of Bak and Bax in the liver is unclear. To address this issue, we generated hepatocyte-specific Bak/Bax double knockout mice and administered Jo2 agonistic anti-Fas antibody or recombinant Fas ligand to them. Fas-induced rapid fulminant hepatocyte apoptosis was partially ameliorated in Bak knockout mice but not in Bax knockout mice, and was completely abolished in double knockout mice 3 hours after Jo2 injection. Importantly, at 6 hours, double knockout mice displayed severe liver injury associated with repression of XIAP, activation of caspase-3/7 and oligonucleosomal DNA breaks in the liver, without evidence of mitochondrial disruption or cytochrome c-dependent caspase-9 activation. This liver injury was not ameliorated in a cyclophilin D knockout background nor by administration of necrostatin-1, but was completely inhibited by administration of a caspase inhibitor after Bid cleavage. Whereas either Bak or Bax is critically required for rapid execution of Fas-mediated massive apoptosis in the liver, delayed onset of mitochondria-independent, caspase-dependent apoptosis develops even in the absence of both. The present study unveils an extrinsic pathway of apoptosis, like that in type I cells, which serves as a backup system even in type II cells.

Trichostatin A sensitizes HBx-expressing liver cancer cells to etoposide treatment

Agents commonly used in cancer chemotherapy rely on the induction of cell death via apoptosis, mitotic catastrophe, premature senescence and autophagy. Chemoresistance is the major factor limiting long-term treatment success in patients with hepatocellular carcinoma (HCC). Recent studies have revealed that the hepatitis B virus X protein (HBx) exerts anti-apoptotic effects, resulting in an increased drug resistance in HCC cells. In this study, we showed that etoposide treatment activated caspase-8 and caspase-3, leading to cleavages of p53, Bid and PARP, which subsequently induced apoptosis. Furthermore, p53 and Bid were accumulated in cytoplasm following etoposide treatment. However, HBx significantly attenuated etoposide-induced cell death. In HBx-expressing cells, despite the translocation of p53 and Bid to cytoplasm, the activation of caspases was inhibited. Furthermore, the phosphorylation of extracellular-signal-regulated kinase (ERK) was markedly increased in HBx-expressing cells. Moreover, the pretreatment with trichostatin A (TSA, a histone deacetylase inhibitor) or TSA in combination with etoposide significantly sensitized HCC cells to apoptosis by inhibiting ERK phosphorylation, reactivating caspases and PARP, and inducing translocation of p53 and Bid to cytoplasm. Collectively, HBx reduces the sensitivity of HCC cells to chemotherapy. TSA in combination with etoposide can significantly overcome the increased resistance of HBx-expressing HCC cells to chemotherapy.

Wogonin induces apoptosis by activation of ERK and p38 MAPKs signaling pathways and generation of reactive oxygen species in human breast cancer cells.

Wogonin is a one of the bioactive compounds of Scutellaria baicalensi Georgi which has been shown to have antiinflammatory, anticancer, antiviral and neuroprotective effects. However, the underlying mechanisms by which wogonin induces apoptosis in cancer cells still remain speculative. Here we investigated the potential activation of MAPKs and generation of reactive oxygen species (ROS) by wogonin on MCF-7 human breast cancer cells. These results showed that wogonin induced mitochondria and death-receptor-mediated apoptotic cell death, which was characterized by activation of several caspases, induction of PARP cleavage, change of antiapoptotic/proapoptotic Bcl-2 family member ratios and cleavage of Bid. We also found that generation of ROS was an important mediator in wogonin-induced apoptosis. Further investigation revealed that wogonin activated ERK and p38 MAPKs, which was inhibited by N-acetyl cysteine (NAC), a ROS scavenger, indicating that wogonin-induced ROS are associated with MAPKs activation. These data demonstrate that wogonin may be a novel anticancer agent for treatment of breast cancer.

Pro-inflammatory cytokines intensify the activation of NO/NOS, JNK1/2 and caspase cascades in immature neurons exposed to elevated levels of unconjugated bilirubin

Hyperbilirubinemia may lead to encephalopathy in neonatal life, particularly in premature infants. Although the mechanisms were never established, clinicians commonly consider sepsis as a risk factor for bilirubin-induced neurological dysfunction (BIND). Our previous studies showed that elevated levels of unconjugated bilirubin (UCB) have immunostimulant effects, which are potentiated by lipopolysaccharide (LPS), and that immature neural cells are more vulnerable to UCB. The present study was undertaken to explore the role of nitric oxide (NO)/NO synthase (NOS), c-Jun N-terminal kinases (JNK) 1/2 and caspase activation in BIND, as well as the additional effects of inflammation, in immature neurons, incubated from 1 h to 24 h, at 37 °C. UCB, at conditions mimicking those of jaundiced newborns (UCB/serum albumin = 0.5), induced NO production, neuronal NOS (nNOS) expression and JNK1/2 activation in 3 days in vitro neuron cultures. As a consequence of these events, mitochondrial and extrinsic pathways of apoptosis were initiated, ultimately leading to neuronal dysfunction. Co-incubation with TNF-α + IL-1β intensified the activation of NO/NOS, JNK1/2, caspase-8, caspase-9 and caspase-3 by UCB. Cleavage of Bid into truncated Bid ( tBid), as well as increased cytotoxic potential, were also observed. Interestingly, both l-NAME (NOS inhibitor) and SP600125 (JNK1/2 inhibitor) reversed the effects produced by UCB either alone, or in association with pro-inflammatory cytokines. Taken together, our data reveal not only that activation of NO/NOS, JNK1/2 and caspase cascades are important determinants of BIND, but also that the association of TNF-α + IL-1β have cumulative effects. These events provide a reason for the risk of sepsis in BIND and point to potential targets for therapeutic intervention.

Antiatherogenic Effect of Bisvanillyl-Hydralazone, a New Hydralazine Derivative with Antioxidant, Carbonyl Scavenger, and Antiapoptotic Properties

Reactive oxygen species (ROS) generated within the vascular wall trigger low-density lipoprotein (LDL) oxidation, lipid peroxidation, and carbonyl stress that are involved in atherogenesis. We recently reported that the antihypertensive drug, hydralazine, exhibits carbonyl scavenger and antiatherogenic properties, but only moderate antioxidant activity, so that high concentrations are required for inhibiting LDL oxidation. We aimed to develop agents sharing both antioxidant and carbonyl scavenger properties. We have synthesized a new hydralazine derivative, the bisvanillyl-hydralazone (BVH). BVH strongly inhibited LDL oxidation induced by copper and by human endothelial cells (HMEC-1), and prevented the formation of macrophagic foam cells. BVH reduced both the extracellular generation of ROS (superoxide anion and hydrogen peroxide) induced by oxidized LDL (oxLDL), as well as intracellular oxidative stress and proteasome activation, NFkappaB activation, and oxLDL-mediated proinflammatory signaling. In parallel, BVH prevented the carbonyl stress induced by oxLDL on cellular proteins, and blocked the apoptotic cascade as assessed by the inhibition of Bid cleavage, cytochrome C release, and DEVDase activation. Lastly, BVH prevented atherogenesis and carbonyl stress in apoE(-/-) mice. In conclusion, BVH is the prototype of a new class of antioxidant and carbonyl scavenger agents designed for new therapeutical approaches in atherosclerosis.

Destabilization of CARP mRNAs by aloe‐emodin contributes to caspase‐8‐mediated p53‐independent apoptosis of human carcinoma cells

Using short hairpin RNA against p53, transient ectopic expression of wild-type p53 or mutant p53 (R248W or R175H), and a p53- and p21-dependent luciferase reporter assay, we demonstrated that growth arrest and apoptosis of FaDu (human pharyngeal squamous cell carcinoma), Hep3B (hepatoma), and MG-63 (osteosarcoma) cells induced by aloe-emodin (AE) are p53-independent. Co-immunoprecipitation and small interfering RNA (siRNA) studies demonstrated that AE caused S-phase cell cycle arrest by inducing the formation of cyclin A-Cdk2-p21 complexes through extracellular signal-regulated kinase (ERK) activation. Ectopic expression of Bcl-X(L) and siRNA-mediated Bax attenuation significantly inhibited apoptosis induced by AE. Cyclosporin A or the caspase-8 inhibitor Z-IETD-FMK blocked AE-induced loss of mitochondrial membrane potential and prevented increases in reactive oxygen species and Ca(++). Z-IETD-FMK inhibited AE-induced apoptosis, Bax expression, Bid cleavage, translocation of tBid to mitochondria, ERK phosphorylation, caspase-9 activation, and the release of cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G from mitochondria. The stability of the mRNAs encoding caspase-8 and -10-associated RING proteins (CARPs) 1 and 2 was affected by AE, whereas CARP1 or 2 overexpression inhibited caspase-8 activation and apoptosis induced by AE. Collectively, our data indicate AE induces caspase-8-mediated activation of mitochondrial death pathways by decreasing the stability of CARP mRNAs in a p53-independent manner.

The sphingolipid degradation product trans-2-hexadecenal induces cytoskeletal reorganization and apoptosis in a JNK-dependent manner

The bioactive signaling molecule d- erythro-sphingosine-1-phosphate (S1P) is irreversibly degraded by the enzyme S1P lyase (SPL). The reaction of SPL with C18-S1P generates ethanolamine phosphate and a long-chain fatty aldehyde, trans-2-hexadecenal. Modulation of SPL expression in cells and organisms produces significant phenotypes, most of which have been attributed to corresponding changes in S1P-dependent signaling. However, the physiological functions of SPL products are not well understood. In the present study, we explored the biological activities of trans-2-hexadecenal in human and murine cells. We demonstrate that trans-2-hexadecenal causes cytoskeletal reorganization leading to cell rounding, detachment and eventual cell death by apoptosis in multiple cell types, including HEK293T, NIH3T3 and HeLa cells. Trans-2-hexadecenal stimulated a signaling pathway involving MLK3 and the respective phosphorylation of MKK4/7 and JNK, whereas ERK, AKT and p38 were unaffected. Trans-2-hexadecenal-induced apoptosis was accompanied by activation of downstream targets of JNK including c-Jun phosphorylation, cytochrome c release, Bax activation, Bid cleavage and increased translocation of Bim into mitochondria. The antioxidant N-acetylcysteine prevented JNK activation by trans-2-hexadecenal. Further, inhibition of JNK abrogated the cytoskeletal changes and apoptosis caused by trans-2-hexadecenal, whereas Rac1 and RhoA were not involved. In conclusion, our studies provide a new paradigm of sphingolipid signaling by demonstrating for the first time that S1P metabolism generates a bioactive product that induces cellular effects through oxidant stress-dependent MAP kinase cell signaling.

English

Andrographolide (AGP), the major phytoconstituent isolated from Andrographis paniculata was found to exhibit growth inhibition and cytotoxicity against the hormone-independent (PC-3 and DU-145) and hormone-dependent (LNCaP) prostate cancer cell lines via the microculture tetrazolium (MTT) assay. Due to its greater cytotoxic potency and selectivity towards PC-3 cells, flow cytometry was used to analyze the cell cycle distribution of control and treated PC-3 cells whereas Annexin V-FITC/PI flow cytometry analysis was carried out to confirm apoptosis induced by AGP in PC-3 cells. Cell cycle and apoptotic regulatory proteins were determined by western blot analysis. AGP was found to induce G2/M cell cycle arrest which led to predominantly apoptotic mode of cell death. Mechanistically, AGP was found to downregulate CDK1 without affecting the levels of CDK4 and cyclin D1. Induction of apoptosis was associated with an increase in activation and expression of caspase 8 which then is believed to have induced cleavage of Bid into tBid. In addition, activation and enhancement of executioner caspase 9 and Bax proteins without affecting Bcl-2 protein levels were observed.   Key words: Andrographolide, prostate cancer, cell cycle, cyclin-dependent kinases, apoptosis, caspase 8, caspase 9, Bcl-2 family.

Naringenin up‐regulates the expression of death receptor 5 and enhances TRAIL‐induced apoptosis in human lung cancer A549 cells

While TRAIL is relatively non-toxic to normal cells, it can selectively induce apoptosis in many types of transformed cells. Nevertheless, some non-small cell lung cancer (NSCLC) cells are particularly resistant to the effects of TRAIL. Here, we report that in combination with naringenin exposure to TRAIL induced apoptosis in TRAIL-resistant NSCLC A549 cells with no detectable inhibitory effects on cell proliferation of normal lung fibroblast cells. Cytotoxicity was evaluated by MTT assay. Apoptosis was detected using DAPI staining, and flow cytometry. The protein levels were determined by Western blot analysis. Caspase activity was measured using a colorimetric assay. For knockdown of Bid and DR5 expression, Bid and DR5 siRNAs were transfected into cells via lipofection. We could show that following exposure to naringenin, DR5 proteins were up-regulated and knockdown of DR5 expression by siRNA attenuated naringenin plus TRAIL-induced apoptosis. Naringenin and TRAIL effectively induced Bid cleavage and siRNA-mediated silencing of Bid reduced the sensitizing effect of naringenin. Furthermore, co-treatment with naringenin and TRAIL resulted in reduction of the clonogenic capacity of A549 cells, and surviving clones could be re-sensitized for repeated TRAIL treatment. Our results indicate that treatment with a combination of TRAIL and naringenin may be a safe strategy for treatment of resistant NSCLC.

Axl Promotes Cutaneous Squamous Cell Carcinoma Survival through Negative Regulation of Pro-Apoptotic Bcl-2 Family Members

Expression of Axl, a receptor tyrosine kinase, is increased in cutaneous squamous cell carcinoma (SCC). Examination of a series of cutaneous SCC tumors revealed positive phospho-Akt (P-Akt) staining accompanied by weak TUNEL staining in Axl-positive tumors, suggesting an anti-apoptotic role for Axl in SCC survival. The role of Axl in UV-induced apoptosis was investigated in a cutaneous SCC cell line using retroviral short hairpin RNA sequences enabling stable Axl knock-down. We show that, although Axl knock-down has no effect on cell proliferation, it sensitizes cells to UV-induced apoptosis through increased activation of the pro-apoptotic protein Bad, a change in the conformation of Bax and Bak, release of cytochrome c into the cytosol, and activation of caspases. These events are accompanied by faster Akt dephosphorylation in UV-treated Axl knock-down cells and correlate with the degree of Axl knock-down. Treatment with the pan-caspase inhibitor zVAD-fmk partially rescued cells from UV-induced apoptosis but did not affect Bid cleavage or cytochrome c release, suggesting that cells die via the mitochondrial-mediated pathway. Thus, Axl confers resistance of SCC cells to apoptosis and displays potential as a target for therapeutic intervention in cutaneous SCC.

Artesunate Activates Mitochondrial Apoptosis in Breast Cancer Cells via Iron-catalyzed Lysosomal Reactive Oxygen Species Production

The antimalarial agent artesunate (ART) activates programmed cell death (PCD) in cancer cells in a manner dependent on the presence of iron and the generation of reactive oxygen species. In malaria parasites, ART cytotoxicity originates from interactions with heme-derived iron within the food vacuole. The analogous digestive compartment of mammalian cells, the lysosome, similarly contains high levels of redox-active iron and in response to specific stimuli can initiate mitochondrial apoptosis. We thus investigated the role of lysosomes in ART-induced PCD and determined that in MCF-7 breast cancer cells ART activates lysosome-dependent mitochondrial outer membrane permeabilization. ART impacted endolysosomal and autophagosomal compartments, inhibiting autophagosome turnover and causing perinuclear clustering of autophagosomes, early and late endosomes, and lysosomes. Lysosomal iron chelation blocked all measured parameters of ART-induced PCD, whereas lysosomal iron loading enhanced death, thus identifying lysosomal iron as the lethal source of reactive oxygen species upstream of mitochondrial outer membrane permeabilization. Moreover, lysosomal inhibitors chloroquine and bafilomycin A1 reduced ART-activated PCD, evidencing a requirement for lysosomal function during PCD signaling. ART killing did not involve activation of the BH3-only protein, Bid, yet ART enhanced TNF-mediated Bid cleavage. We additionally demonstrated the lysosomal PCD pathway in T47D and MDA-MB-231 breast cancer cells. Importantly, non-tumorigenic MCF-10A cells resisted ART-induced PCD. Together, our data suggest that ART triggers PCD via engagement of distinct, interconnected PCD pathways, with hierarchical signaling from lysosomes to mitochondria, suggesting a potential clinical use of ART for targeting lysosomes in cancer treatment.

Hexahydro-β-acids induce apoptosis through mitochondrial pathway, GADD153 expression, and caspase activation in human leukemia cells

Hexahydro-β-acids (HBA) and β-acids (BA) displayed strong growth inhibitory effects against human leukemia HL-60 cells and were able to induce apoptosis in a concentration- and time-dependent manner and the morphological changes associated with apoptotic cell death; however, BA was less effective. Treatment with HBA caused a rapid loss of mitochondrial trans-membrane potential, release of mitochondrial cytochrome c into cytosol. The levels of Bad and Bax were dramatically increased in cells treated with HBA. In addition, the results showed that HBA promoted the up-regulation of Fas prior to the processing and activation of pro-caspase-8 and cleavage of Bid, suggesting the involvement of a Fas-mediated pathway in HBA-induced cells. Moreover, the changes occurred after single breaks in DNA were detected, suggesting that HBA induced irreparable DNA damage, which in turn triggered the process of apoptosis. HBA markedly enhanced the growth arrest DNA damage-inducible gene 153 (GADD153) protein in a concentration- and time-dependent manner. These findings suggest that HBA creates an oxidative cellular environment that induces DNA damage and GADD153 gene activation, which in turn triggers apoptosis in HL-60 cells. Our study identified the novel mechanisms of HBA-induced apoptosis and indicated that HBA may be used as a potential chemopreventive and chemotherapeutic agent.

An apoptosis targeted stimulus with nanosecond pulsed electric fields (nsPEFs) in E4 squamous cell carcinoma

Stimuli directed towards activation of apoptosis mechanisms are an attractive approach to eliminate evasion of apoptosis, a ubiquitous cancer hallmark. In these in vitro studies, kinetics and electric field thresholds for several apoptosis characteristics are defined in E4 squamous carcinoma cells (SCC) exposed to ten 300 ns pulses with increasing electric fields. Cell death was >95% at the highest electric field and coincident with phosphatidylserine externalization, caspase and calpain activation in the presence and absence of cytochrome c release, decreases in Bid and mitochondria membrane potential (Δψm) without apparent changes reactive oxygen species levels or in Bcl2 and Bclxl levels. Bid cleavage was caspase-dependent (55–60%) and calcium-dependent (40–45%). Intracellular calcium as an intrinsic mechanism and extracellular calcium as an extrinsic mechanism were responsible for about 30 and 70% of calcium dependence for Bid cleavage, respectively. The results reveal electric field-mediated cell death induction and progression, activating pro-apoptotic-like mechanisms and affecting plasma membrane and intracellular functions, primarily through extrinsic-like pathways with smaller contributions from intrinsic-like pathways. Nanosecond second pulsed electric fields trigger heterogeneous cell death mechanisms in E4 SCC populations to delete them, with caspase-associated cell death as a predominant, but not an unaccompanied event.

Proteasome inhibitor sensitizes oral squamous cell carcinoma cells to TRAIL-mediated apoptosis.

Oral squamous cell carcinoma (OSCC) cells are relatively resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis during culture. We investigated the role of a proteaosome inhibitor in the survival and apoptosis of these cells. We found that the proteasome inhibitor MG132 markedly accelerated TRAIL-mediated apoptosis in OSCC cell lines HSC-2 and HSC-3. Addition of TRAIL to MG132-treated cells resulted in Bid cleavage. Furthermore, the inhibitors of caspase-3, caspase-8 and caspase-9 reduced the accelerative effect of MG132 on TRAIL-mediated apoptosis. These results suggest that the pro-apoptotic effect of a proteasome inhibitor on TRAIL-mediated apoptosis may contribute to both extrinsic and intrinsic pathways. MG132 enhanced the expression of the TRAIL receptors DR4 and DR5, and neutralization of DR5 receptors showed a marked reduction of TRAIL-mediated apoptosis, whereas that of DR4 was a partial reduction. MG132 also markedly reduced cellular FLICE-inhibitory protein (c-FLIP), cellular inhibitor of apoptosis protein-1 (cIAP-1), X-linked IAP (XIAP) and survivin. Therefore, MG132 provides partial regulation of TRAIL-mediated apoptosis in OSCC cells via modulation of DR5, c-FLIP, cIAP-1, XIAP and survivin. The proteasome inhibitor MG132 may therefore represent a novel strategy for overcoming resistance to TRAIL-mediated apoptosis in OSCC cells.

Caspase-8 and caspase-7 sequentially mediate proteolytic activation of acid sphingomyelinase in TNF-R1 receptosomes

We previously demonstrated that tumour necrosis factor (TNF)-induced ceramide production by endosomal acid sphingomyelinase (A-SMase) couples to apoptosis signalling via activation of cathepsin D and cleavage of Bid, resulting in caspase-9 and caspase-3 activation. The mechanism of TNF-mediated A-SMase activation within the endolysosomal compartment is poorly defined. Here, we show that TNF-induced A-SMase activation depends on functional caspase-8 and caspase-7 expression. The active forms of all three enzymes, caspase-8, caspase-7 and A-SMase, but not caspase-3, colocalize in internalized TNF receptosomes. While caspase-8 and caspase-3 are unable to induce activation of purified pro-A-SMase, we found that caspase-7 mediates A-SMase activation by direct interaction resulting in proteolytic cleavage of the 72-kDa pro-A-SMase zymogen at the non-canonical cleavage site after aspartate 253, generating an active 57 kDa A-SMase molecule. Caspase-7 down modulation revealed the functional link between caspase-7 and A-SMase, confirming proteolytic cleavage as one further mode of A-SMase activation. Our data suggest a signalling cascade within TNF receptosomes involving sequential activation of caspase-8 and caspase-7 for induction of A-SMase activation by proteolytic cleavage of pro-A-SMase.

Involvement of mitochondrial dysfunction in human islet amyloid polypeptide-induced apoptosis in INS-1E pancreatic beta cells: An effect attenuated by phycocyanin

Misfolded human islet amyloid polypeptide (hIAPP) in pancreatic islets is associated with the loss of insulin-secreting beta cells in type 2 diabetes. Insulin secretion impairment and cell apoptosis can be due to mitochondrial dysfunction in pancreatic beta cells. Currently, there is little information about the effect of hIAPP on mitochondrial function. In this study, we used INS-1E rat insulinoma beta cells as a model to investigate the role of mitochondria in hIAPP-induced apoptosis and the protective effects of phycocyanin (PC). We demonstrated that hIAPP induced apoptosis in INS-1E cells was associated with the disruption of mitochondrial function, as evidenced by ATP depletion, mitochondrial mass reduction, mitochondrial fragmentation and loss of mitochondrial membrane potential (Δ Ψ m). Further molecular analysis showed that hIAPP induced changes in the expression of Bcl-2 family members, release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria into cytosol, activation of caspases and cleavage of poly (ADP-ribose) polymerase. Interestingly, the hIAPP-induced mitochondrial dysfunction in INS-1E cells was effectively restored by co-treatment of PC. Moreover, there was crosstalk between the extrinsic and intrinsic apoptotic pathways as demonstrated by cleavage of Bid by caspase-8 in the apoptotic process triggered by hIAPP. Taken together, we demonstrated for the first time the involvement of mitochondrial dysfunction in hIAPP-induced INS-1E cell apoptosis. Attenuation of mitochondrial dysfunction provides a mechanism for the protective effects of PC.

Quercetin induces FasL-related apoptosis, in part, through promotion of histone H3 acetylation in human leukemia HL-60 cells

Quercetin, a naturally occurring flavonoid abundant in fruits and vegetables, has been demonstrated as a multipotent bioflavonoid with great potential for the prevention and treatment of cancer. Apoptosis is thought to be an important response to most chemotherapeutic agents in leukemia cells. However, the underlying mechanism of induction of apoptosis by quercetin involving epigenetic regulation is poorly understood. In the present study, by evaluation of fragmentation of DNA, poly (ADP-ribose) polymerase (PARP) and procaspases, we found that quercetin was able to induce apoptosis of human leukemia HL-60 cells in a dose-dependent manner. Quercetin triggered the extrinsic apoptosis pathway through activation of caspase-8 and induction of Bid cleavage, Bax conformation change and cytochrome c release. Furthermore, quercetin induced Fas ligand (FasL) expression involving activation of the extracellular signal-regulated kinase (ERK) and Jun N-terminus kinase (JNK) signaling pathways. In addition to activation of c-Jun, quercetin increased histone H3 acetylation which resulted in the promotion of the expression of FasL. Quercetin exhibited potential for the activation of histone acetyltransferase (HAT) and the inhibition of histone deacetyltransferase (HADC), both of which contributed to histone acetylation. However, only the activation effect on HAT was associated with the ERK and JNK pathway. These results demonstrated that quercetin induced FasL-related apoptosis by transactivation through activation of c-jun/AP-1 and promotion of histone H3 acetylation in HL-60 cells.

Celastrol isolated from Tripterygium regelii induces apoptosis through both caspase-dependent and -independent pathways in human breast cancer cells

The aim of the present study was to evaluate the underlying apoptotic mechanisms of celastrol, a major biologically active component of Tripterygium regelii, in human breast adenocarcinoma MCF-7 cells. Celastrol was isolated from T. regelii chloroform extract by silica gel column chromatography, and its chemical structure was identified via 1H NMR and 13C NMR. Celastrol significantly inhibited cell growth in dose- and time-dependent manners. Celastrol induced sub-G1 DNA accumulation, formation of apoptotic bodies, nuclear condensation, and a DNA ladder in MCF-7 cells. Celastrol triggered the activation of caspase family proteins. Celastrol caused activation of caspase-7, -8, and -9, PARP cleavage, caspase-8-mediated bid cleavage, and release of cytochrome c and AIF. In addition, celastrol decreased the expression of anti-apoptotic Bcl-2 protein and increased expression of pro-apoptotic Bax protein. These results suggest that celastrol inhibits the proliferation of MCF-7 cells through induction of apoptosis, which is mediated by a mitochondrial-dependent caspase pathway.

Mild thermotolerance induced at 40 °C protects HeLa cells against activation of death receptor-mediated apoptosis by hydrogen peroxide

Preexposure to mild temperatures such as 40 °C induces thermotolerance, whereby cells resist subsequent exposure to a toxic insult. This study investigates the protective effect of mild thermotolerance (3 h, 40 °C) against activation of death receptor-mediated apoptosis by H 2O 2 in HeLa cells. H 2O 2 (5–50 μM) caused rapid activation (1–3 h) of the Fas death receptor pathway of apoptosis, which was evident by up-regulation of the death ligand FasL and recruitment of the adaptor protein Fas-associated death domain to the plasma membrane. This resulted in activation of caspase-8 and caspase-2, which led to activation of the cross-talk pathway involving Bid cleavage, t-Bid translocation to mitochondria, and caspase-9 activation. These changes were all diminished in thermotolerant cells. Mild thermotolerance also protected cells against cytotoxicity from H 2O 2 as well as execution-phase events of apoptosis such as caspase-3 activation and chromatin condensation. The antioxidant polyethylene glycol–catalase abolished FasL induction and caspase-8 activation due to H 2O 2. FasL up-regulation; activation of caspases-8, -2, -9, and -3; and chromatin condensation were decreased by the p53 inhibitor pifithrin-α, implicating p53 as an upstream factor in the activation of death receptor-mediated apoptosis by H 2O 2. This study advances knowledge about the protective effect of adaptive responses induced by mild stresses, such as fever temperatures, against induction of apoptosis by oxidative stress.