Ras-association domain family protein 6 induces apoptosis via both caspase-dependent and caspase-independent pathways

The Ras-association domain family (RASSF) comprises six members (RASSF1-6) that each harbors a RalGDS/AF-6 (RA) and Sav/RASSF/Hippo (SARAH) domain. The RASSF proteins are known as putative tumor suppressors but RASSF6 has not yet been studied. We have here characterized human RASSF6. Although RASSF6 has RA domain, it does not bind Ki-Ras, Ha-Ras, N-Ras, M-Ras, or TC21 under the condition that Nore1 (RASSF5) binds these Ras proteins. The message of RASSF6 is detected by RT-PCR in several cell lines including HeLa, MCF-7, U373, A549, and HepG2 cells, but the protein expression is low. The enhanced expression of RASSF6 causes apoptosis in HeLa cells. RASSF6 activates Bax and induces cytochrome C release. Caspase-3 activation is also induced, but the caspase inhibitor, Z-VAD-FMK, does not block RASSF6-mediated apoptosis. Apoptosis-inducing factor and endonuclease G are released from the mitochondria upon expression of RASSF6 and their releases are not blocked by Z-VAD-FMK. The knock down of RASSF6 partially blocks tumor necrosis factor-alpha-induced cell death in HeLa cells. These findings indicate that RASSF6 is implicated in apoptosis in HeLa cells and that it triggers both caspase-dependent and caspase-independent pathways.     

Thai Acanthamoeba isolate (T4) induced apoptotic death in neuroblastoma cells via the Bax-mediated pathway

A Thai Acanthamoeba isolate named AS recovered from a corneal scraping of a keratitis patient was genotypically determined as T4. AS trophozoites were used for studying Acanthamoeba-induced apoptosis in mouse neuroblastoma NA cells during in vitro co-cultivation. The Acanthamoeba-exposed NA cells showed signs of apoptosis including cell shrinkage, nuclear condensation and DNA fragmentation. The effect was confirmed by DNA laddering electrophoresis. Involvement of caspase enzymes and mitochondrial pro- and anti-apoptotic proteins (Bax and Bcl-2) in AS-induced apoptosis was determined. The use of Z-VAD-FMK, a pan-caspase inhibitor, significantly reduced the apoptotic effect, while Bax/Bcl-2 ratio analysis showed a significant increase in the expression of apoptotic proteins in AS-exposed NA cells. These results strongly indicated that apoptosis induced by AS trophozoites is caspase-dependent and is mediated by over-expression of pro-apoptotic proteins in the mitochondrial pathway. This is the first report on the role of Bax in mediating apoptosis induced by Acanthamoeba.     

The apoptosis inhibitory domain of FE65-like protein 1 regulates both apoptotic and caspase-independent programmed cell death mediated by tumor necrosis factor

Tumor necrosis factor (TNF) can induce caspase-dependent (apoptotic) and caspase-independent pathways to programmed cell death (PCD). Here, we demonstrate that stable transfection of a cDNA encompassing the C-terminal apoptosis inhibitory domain (AID) of FE65-like protein 1 into mouse L929 fibrosarcoma cells protects from caspase-independent as well as from apoptotic PCD induced by TNF. We show that the AID does not protect from caspase-independent PCD elicited by 1-methyl-3-nitro-1-nitrosoguanidine, suggesting that the AID might prevent cell death by affecting assembly of the death inducing signaling complex of the 55 kDa TNF receptor or clustering of the receptor itself. Interference with caspase-independent PCD mediated by the sphingolipid ceramide further increases protection conferred by the AID, as does the antioxidant butylated hydroxyanisole, implicating ceramide and reactive oxygen species as potential factors interacting with caspase-independent PCD regulated by the AID.     

The cell cycle inhibitor p57(Kip2) promotes cell death via the mitochondrial apoptotic pathway

The p57(Kip2) gene belongs to the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors and has been suggested to be a tumor suppressor gene, being inactivated in various types of human cancers. However, little is known concerning p57(Kip2) possible interplay with the apoptotic cell death machinery and its possible implication for cancer. Here, we report that selective p57(Kip2) expression sensitizes cancer cells to apoptotic agents such as cisplatin, etoposide and staurosporine (STS) via a mechanism, which does not require p57(Kip2)-mediated inhibition of CDK. Translocation of p57(Kip2) to mitochondria occurs within 20 min after STS application. In fact, p57(Kip2) primarily promotes the intrinsic apoptotic pathways, favoring Bax activation and loss of mitochondrial transmembrane potential, consequent release of cytochrome-c into cytosol, caspase-9 and caspase-3 activation. In accordance, Bcl2 overexpression or voltage-dependent anion channel (VDAC) inhibition is able to inhibit p57(Kip2) cell death promoting effect. Thus, in addition to its established function in control of proliferation, these results reveal a mechanism whereby p57(Kip2) influences the mitochondrial apoptotic cell death pathway in cancer cells.     

Activation of intrinsic and extrinsic pathways in apoptotic signaling during UV-C-induced death of Jurkat cells: the role of caspase inhibition

We have examined UV irradiation-induced cell death in Jurkat cells and evaluated the relationships that exist between inhibition of caspase activity and the signaling mechanisms and pathways of apoptosis. Jurkat cells were irradiated with UV-C light, either with or without pretreatment with the pan-caspase inhibitor, z-VAD-fmk (ZVAD), or the more selective caspase inhibitors z-IETD-fmk (IETD), z-LEHD-fmk (LEHD), and z-DEVD-fmk (DEVD). Flow cytometry was used to examine alterations in viability, cell size, plasma membrane potential (PMP), mitochondrial membrane potential (DeltaPsi(mito)), intracellular Na(+) and K(+) concentrations, and DNA degradation. Processing of pro-caspases 3, 8, and 9 and the pro-apoptotic protein Bid was determined by Western blotting. UV-C irradiation of Jurkat cells resulted in characteristic apoptosis within 6 h after treatment and pretreatment of cells with ZVAD blocked these features. In contrast, pretreatment of the cells with the more selective caspase inhibitors under conditions that effectively blocked DNA degradation and inhibited caspase 3 and 8 processing as well as Bid cleavage had little protective effect on the other apoptotic characteristics examined. Thus, both intrinsic and extrinsic pathways are activated during UV-induced apoptosis in Jurkat cells and this redundancy appears to assure cell death during selective caspase inhibition.     

Autocrine prostaglandin E2 signaling promotes promonocytic leukemia cell survival via COX-2 expression and MAPK pathway

The COX-2/PGE₂ pathway has been implicated in the occurrence and progression of cancer. The underlying mechanisms facilitating the production of COX-2 and its mediator, PGE₂, in cancer survival remain unknown. Herein, we investigated PGE₂-induced COX-2 expression and signaling in HL-60 cells following menadione treatment. Treatment with PGE₂ activated anti-apoptotic proteins such as Bcl-2 and Bcl-xL while reducing pro-apoptotic proteins, thereby enhancing cell survival. PGE₂ not only induced COX-2 expression, but also prevented casapse-3, PARP, and lamin B cleavage. Silencing and inhibition of COX-2 with siRNA transfection or treatment with indomethacin led to a pronounced reduction of the extracellular levels of PGE₂, and restored the menadione-induced cell death. In addition, pretreatment of cells with the MEK inhibitor PD98059 and the PKA inhibitor H89 abrogated the PGE₂-induced expression of COX-2, suggesting involvement of the MAPK and PKA pathways. These results demonstrate that PGE₂ signaling acts in an autocrine manner, and specific inhibition of PGE₂ will provide a novel approach for the treatment of leukemia. [BMB Reports 2015; 48(2): 109-114]     

Exposure of insect midgut cells to Sambucus nigra L. agglutinins I and II causes cell death via caspase-dependent apoptosis

Sambucus nigra agglutinins I and II, further referred to as SNA-I and SNA-II, are two ricin-related lectins from elderberry. SNA-I is a chimeric lectin composed of an A-chain with enzymatic activity and a B-chain with carbohydrate-binding activity, and therefore belongs to the group of type 2 ribosome-inactivating proteins. In contrast, SNA-II consists only of carbohydrate-binding B-chains. The physiological effect of SNA-I was tested on different insect cell lines (midgut, ovary, fat body, embryo). In sensitive midgut CF-203 cells, SNA-I induced cell death with typical characteristics such as cell shrinkage, plasma membrane blebbing, nuclear condensation and DNA fragmentation. The effect was dose-dependent with 50% death of 4-day-exposed cells at 3 nM. SNA-I exposure induced caspase-3 like activities, suggesting that SNA-I can induce the apoptotic pathway. Interestingly, the hololectin SNA-II also induced apoptosis in CF-203 cells at similar doses with the same physiological events. SNA-I and SNA-II both induced caspase-dependent apoptosis at low concentrations (nM order), leading to typical symptoms of cell death in sensitive cells. This effect seems independent from the catalytic activity of the A-chain, but depends on the carbohydrate-binding B-chain.     

Death ligand concentration and the membrane proximal signaling module regulate the type 1/type 2 choice in apoptotic death signaling

Apoptotic death pathways are frequently activated by death ligand induction and subsequent activation of the membrane proximal signaling module. Death receptors cluster upon binding to death ligands, leading to formation of a membrane proximal death-inducing-signaling-complex (DISC). In this membrane proximal signalosome, initiator caspases (caspase 8) are processed resulting in activation of both type 1 and type 2 pathways of apoptosis signaling. How the type 1/type 2 choice is made is an important question in the systems biology of apoptosis signaling. In this study, we utilize a Monte Carlo based in silico approach to elucidate the role of membrane proximal signaling module in the type 1/type 2 choice of apoptosis signaling. Our results provide crucial mechanistic insights into the formation of DISC signalosome and caspase 8 activation. Increased concentration of death ligands was shown to correlate with increased type 1 activation. We also study the caspase 6 mediated system level feedback activation of apoptosis signaling and its role in the type 1/type 2 choice. Our results clarify the basis of cell-to-cell stochastic variability in apoptosis activation and ramifications of this issue is further discussed in the context of therapies for cancer and neurodegenerative disorders.     

Amyloid precursor protein promotes endoplasmic reticulum stress-induced cell death via C/EBP homologous protein-mediated pathway

The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) is known to activate the ER, which is termed ER stress. Here, we demonstrated that amyloid precursor protein (APP) is a novel mediator of ER stress-induced apoptosis through the C/EBP homologous protein (CHOP) pathway. Expression of APP mRNA was elevated by tunicamycin- or dithiothreitol-induced ER stress. The levels of C83 and APP intracellular domain (AICD) fragments, which are cleaved from APP, were significantly increased under ER stress, although the protein level of full-length APP was decreased. Cellular viability was reduced in APP-over-expressing cells, which was attenuated by treatment with a γ-secretase inhibitor, N -[ N -(3,5-difluorophenacetyl)-L-alanyl]- S -phenylglycine t -butyl ester (DAPT). Cellular viability was also reduced in AICD-FLAG-over-expressing cells. The mRNA and protein levels of CHOP, an ER stress-responsive gene, were remarkably increased by APP over-expression, which was attenuated by treatment with DAPT. CHOP mRNA induction was also found in AICD-FLAG-over-expressing cells. Cell death and CHOP up-regulation by ER stress were attenuated by APP knockdown. Data obtained with a luciferase assay and chromatin immunoprecipitation assay indicated that AICD associates with the promoter region of the CHOP gene. In conclusion, ER stress-induced APP undergoes α- and γ-secretase cleavage and subsequently induces CHOP-mediated cell death.     

Detection and localization of calpain 3-like protease in a neuronal cell line: possible regulation of apoptotic cell death through degradation of nuclear IkappaBalpha

Calpains are a family of calcium-dependent cysteine proteases involved in major cellular processes including cell death. Their intracellular localization is essential to the understanding of their biological functions. In a previous confocal microscopy study, we observed the presence of a calpain 3-like protein in the mammalian brain. We thus first identified and confirmed the presence of a calpain 3-like protease in a neuronal cell model (NGF-differentiated PC12 cells). The goal of this study was to determine, for the first time in non-muscular cells, the relation between the subcellular localization, activation and function of this protease. We thus investigated its ability to regulate nuclear IkappaBalpha and therefore NF-kappaB activation after cell death stimulation. The IkappaBalpha/NF-kappaB signalling pathway indeed influences the neurodegenerative process by directly affecting gene expression in neurons. In the present study, we found that calpain 3 is present in the cytoplasm and nucleus of neuron-like PC12 cells and could be activated through autolysis in the nuclei of cells undergoing apoptosis after ionomycin treatment. Moreover, in these conditions, we demonstrated formation of the IkappaBalpha/calpain 3 complex and an increase in calpain-dependent IkappaBalpha cleavage products in cell nuclei. Stimulation of calpain-dependent cell death in neuron activated nuclear calpain 3-like protease and IkappaBalpha proteolysis resulted in the regulation of NF-kappaB activation. These data suggest a new mechanism by which calpain 3 activation is able to regulate the IkappaBalpha/NF-kappaB pathway and thus neurodegenerative processes.     

Molecular mechanisms of luteolin-7-O-glucoside-induced growth inhibition on human liver cancer cells: G2/M cell cycle arrest and caspase-independent apoptotic signaling pathways

Luteolin-7-O-glucoside (LUT7G), a flavone subclass of flavonoids, has been found to increase anti-oxidant and anti-inflammatory activity, as well as cytotoxic effects. However, the mechanism of how LUT7G induces apoptosis and regulates cell cycles remains poorly understood. In this study, we examined the effects of LUT7G on the growth inhibition of tumors, cell cycle arrest, induction of ROS generation, and the involved signaling pathway in human hepatocarcinoma HepG2 cells. The proliferation of HepG2 cells was decreased by LUT7G in a dose-dependent manner. The growth inhibition was due primarily to the G2/M phase arrest and ROS generation. Moreover, the phosphorylation of JNK was increased by LUT7G. These results suggest that the anti-proliferative effect of LUT7G on HepG2 is associated with G2/M phase cell cycle arrest by JNK activation. [BMB Reports 2013; 46(12): 611-616]     

Hypothermia attenuates ischemia/reperfusion-induced endothelial cell apoptosis via alterations in apoptotic pathways and JNK signaling

Hypothermia is the most effective means of protecting the brain, heart and other organs during ischemia/reperfusion (I/R) injury. However, the precise mechanisms for hypothermia to inhibit I/R-induced endothelial cell apoptosis are not fully understood. In the present study, human umbilical endothelial cells (HUVECs) were exposed to ischemia followed by reperfusion under normothermia (37 °C) or hypothermia (33 °C). Our results showed that hypothermia markedly reduced I/R-induced endothelial cell apoptosis, the expression of cleaved caspase-3 and PARP. Moreover, hypothermia markedly reversed I/R-induced activation of Fas/caspase-8, the increase of Bax and decrease of Bcl-2. Furthermore, hypothermia inhibited JNK1/2 activation via MKP-1 induction. Together, these data demonstrate that hypothermia represses I/R-induced endothelial cell apoptosis by inhibiting both extrinsic- and intrinsic-dependent apoptotic pathways and activation of JNK1/2.     

Ginsenoside RH-2 induces apoptotic cell death in rat C6 glioma via a reactive oxygen- and caspase-dependent but Bcl-X(L)-independent pathway.

We used the rat C6 gliomal cell line to investigate the potential role of ginsenoside Rh2 (G-Rh2) in brain tumor. G-Rh2 induced many apoptotic manifestations in C6 gliomal cells as evidenced by changes in cell morphology, generation of DNA fragmentation, activation of caspase and production of reactive oxygen species (ROS). As a result, cotreatment with antioxidants or a broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone effectively attenuated G-Rh2-induced cell death. However, specific cleavage of poly(ADP-ribose)polymerase into 85 kDa protein was not detected as demonstrated in many other apoptotic paradigms. Expression levels of Bcl-2 and Bax remained unchanged following G-Rh2 treatment. Furthermore, G-Rh2-induced cell death in C6 gliomal cells overexpressing antiapoptotic protein, Bcl-X(L), was comparable to that in parental cells. Taken together, our data indicate that G-Rh2-induced cell death is mediated by the generated ROS and the activation of caspase pathway in a Bcl-X(L)-independent manner.     

ALG-2 knockdown in HeLa cells results in G2/M cell cycle phase accumulation and cell death

ALG-2 (apoptosis-linked gene-2 encoded protein) has been shown to be upregulated in a variety of human tumors questioning its previously assumed pro-apoptotic function. The aim of the present study was to obtain insights into the role of ALG-2 in human cancer cells. We show that ALG-2 downregulation induces accumulation of HeLa cells in the G2/M cell cycle phase and increases the amount of early apoptotic and dead cells. Caspase inhibition by the pan-caspase inhibitor zVAD-fmk attenuated the increase in the amount of dead cells following ALG-2 downregulation. Thus, our results indicate that ALG-2 has an anti-apoptotic function in HeLa cells by facilitating the passage through checkpoints in the G2/M cell cycle phase.     

FOXO-independent suppression of programmed cell death by the PI3K/Akt signaling pathway in Drosophila

Signaling through the PI3K/Akt/FOXO pathway plays an important role in vertebrates in protecting cells from programmed cell death. PI3K and Akt have been similarly shown to be involved in survival signaling in the invertebrate model organism Drosophila. However, it is not known whether PI3K and Akt execute this function by controlling a pro-apoptotic activity of Drosophila FOXO. In this study, we show that elevated signaling through PI3K and Akt can prevent developmentally controlled death in the salivary glands of the fruit fly. We further show that Drosophila FOXO is not required for normal salivary gland death and that the rescue of salivary gland death by PI3K occurs independent of FOXO. These results give support to the notion that FOXOs have acquired pro-apoptotic functions after separation of the vertebrate and invertebrate lineages.     

Pak2 inhibition promotes resveratrol-mediated glioblastoma A172 cell apoptosis via modulating the AMPK-YAP signaling pathway

As a polyphenolic compound, resveratrol (Res) is widely present in a variety of plants. Previous studies have shown that Res can inhibit various tumors. However, its role in c remains largely unexplored. In the present study, we first demonstrated that Res inhibited cell viability and induced apoptosis of glioblastoma A172 cell. Further experiments showed that Res induced mitochondrial dysfunction and activated the activity of caspase-9. Functional studies have found that Res treatment is associated with an increase in the expression of Pak2. Interestingly, inhibition of Pak2 could further augment the proapoptotic effect of Res. Mechanistically, Pak2 inhibition induced reactive oxygen species overproduction, mitochondria-JNK pathway activation, and AMPK-YAP axis suppression. However, overexpression of YAP could abolish the anticancer effects of Res and Pak2 inhibition, suggesting a necessary role played by the AMPK-YAP pathway in regulating cancer-suppressive actions of Res and Pak2 inhibition. Altogether, our results indicated that Res in combination with Pak2 inhibition could further enhance the anticancer property of Res and this effect is mediated via the AMPK-YAP pathway.     

Mycobacterium tuberculosis acyl carrier protein inhibits macrophage apoptotic death by modulating the reactive oxygen species/c-Jun N-terminal kinase pathway

Mycobacterial acyl carrier protein (AcpM; Rv2244) is a meromycolate extension acyl carrier protein of Mycobacterium tuberculosis (Mtb), which participates in multistep mycolic acid biosynthesis. However, the function of AcpM in host–mycobacterium interactions during infection remains largely uncharacterized. Here we show that AcpM inhibits host cell apoptosis during mycobacterial infection. To examine the function of AcpM during infection, we generated a recombinant Mycobacterium smegmatis (M. smegmatis) strain overexpressing AcpM (Ms_AcpM) and a strain transformed with an empty vector (Ms_Vec). Ms_AcpM promoted intracellular survival of M. smegmatis and led to a significant decrease in the death rate of primary bone marrow-derived macrophages (BMDMs). Importantly, Ms_AcpM showed significantly decreased reactive oxygen species (ROS) generation and activation of c-Jun N-terminal kinase (JNK) signaling compared with Ms_Vec. In addition, treatment of BMDMs with recombinant AcpM significantly inhibited the apoptosis and ROS/JNK signaling induced by M. smegmatis. Moreover, recombinant AcpM enhanced intracellular survival of Mtb H37Rv. Taken together, these results indicate that AcpM plays a role as a virulence factor by modulating host cell apoptosis during mycobacterial infection.     

CD437, a synthetic retinoid, induces apoptosis in human respiratory epithelial cells via caspase-independent mitochondrial and caspase-8-dependent pathways both up-regulated by JNK signaling pathway

The synthetic retinoid-related molecule CD437-induced apoptosis in human epithelial airway respiratory cells: the 16HBE bronchial cell line and normal nasal epithelial cells. CD437 caused apoptosis in S-phase cells and cell cycle arrest in S phase. Apoptosis was abolished by caspase-8 inhibitor z-IETD-fmk which preserved S-phase cells but was weakly inhibited by others selective caspase-inhibitors, indicating that caspase-8 activation was involved. z-VAD and z-IETD prevented the nuclear envelope fragmentation but did not block the chromatin condensation. The disruption of mitochondrial transmembrane potential was also induced by CD437 treatment. The translocation of Bax to mitochondria was demonstrated, as well as the release of cytochrome c into the cytosol and of apoptosis-inducing factor (AIF) translocated into the nucleus. z-VAD and z-IETD did not inhibit mitochondrial depolarization, Bax translocation or release of cytochrome c and AIF from mitochondria. These results suggest that CD437-induced apoptosis is executed by two converging pathways. AIF release is responsible for chromatin condensation, the first stage of apoptotic cell, via a mitochondrial pathway independent of caspase. But final stage of apoptosis requires the caspase-8-dependent nuclear envelope fragmentation. In addition, using SP600125, JNK inhibitor, we demonstrated that CD437 activates the JNK-MAP kinase signaling pathway upstream to mitochondrial and caspase-8 pathways. Conversely, JNK pathway inhibition, which suppresses S-phase apoptosis, did not prevent cell cycle arrest within S phase, confirming that these processes are triggered by distinct mechanisms.     

Bcl2 at the endoplasmic reticulum protects against a Bax/Bak-independent paraptosis-like cell death pathway initiated via p20Bap31

Bap31 is an integral ER membrane protein which functions as an escort factor in the sorting of newly synthesized membrane proteins within the endoplasmic reticulum (ER). During apoptosis signaling, Bap31 is subject to early cleavage by initiator caspase-8. The resulting p20Bap31 (p20) fragment has been shown to initiate proapoptotic ER-mitochondria Ca2+ transmission, and to exert dominant negative (DN) effects on ER protein trafficking. We now report that ectopic expression of p20 in E1A/DNp53-transformed baby mouse kidney epithelial cells initiates a non-apoptotic form of cell death with paraptosis-like morphology. This pathway was characterized by an early rise in ER Ca2+ stores and massive dilation of the ER/nuclear envelope, dependent on intact ER Ca2+ stores. Ablation of the Bax/Bak genes had no effect on these ER/nuclear envelope transformations, and delayed but did not prevent cell death. ER-restricted expression of Bcl2 in the absence of Bax/Bak, however, delayed both ER/nuclear envelope dilation and cell death. This prosurvival role of Bcl2 at the ER thus extended beyond inhibition of Bax/Bak, and correlated with its ability to lower ER Ca2+ stores. Furthermore, these results indicate that ER restricted Bcl2 is capable of antagonizing not only apoptosis, but also a non-apoptotic, Bax/Bak independent, paraptosis-like form of cell death.