Relevance of signaling molecules for apoptosis induction on influenza A virus replication

Apoptosis is an important mechanism to maintain homeostasis in mammals, and disruption of the apoptosis regulation mechanism triggers a range of diseases, such as cancer, autoimmune diseases, and developmental disorders. The severity of influenza A virus (IAV) infection is also closely related to dysfunction of apoptosis regulation. In the virus infected cells, the functions of various host cellular molecules involved in regulation of induction of apoptosis are modulated by IAV proteins to enable effective virus replication. The modulation of the intracellular signaling pathway inducing apoptosis by the IAV infection also affects extracellular mechanisms controlling apoptosis, and triggers abnormal host responses related to the disease severity of IAV infections. This review focuses on apoptosis related molecules involved in IAV replication and pathogenicity, the strategy of the virus propagation through the regulation of apoptosis is also discussed.     

FERM domain promotes resveratrol-induced apoptosis in endothelial cells via inhibition of NO production

Focal adhesion kinase (FAK) consists of an N-terminal band 4.1; ezrin, radixin, moesin (FERM) domain; tyrosine kinase domain; and C-terminal FA targeting domain. Here we show that ectopically expressed FERM is largely located in the cytosolic fraction under quiescent conditions. We further found that this ectopically expressed FERM domain aggravates endothelial cell apoptosis triggered by 100 μM resveratrol, whereas FERM had no effect on apoptosis induced by TNF-α. We determined that resveratrol at low doses (<20 μM) promotes phosphorylation (S1177) of eNOS via an AMPK-dependent pathway. The presence of the FERM domain blocked this resveratrol-stimulated eNOS phosphorylation and NO production. Thus, the pro-apoptotic activity of cytosolic FERM domain is at least partially mediated by down-regulation of NO, a critical cell survival factor. Consistently, we found that the apoptosis induced by cytosolic FERM in the presence of resveratrol was reversed by an NO donor, SNAP. In conclusion, FERM located in the cytosolic fraction plays a pivotal role in aggravating cell apoptosis through diminishing NO production.     

Prognostic significance of X-linked inhibitor of apoptosis protein in solid tumors: A systematic review and meta-analysis

X-linked inhibitor of apoptosis protein (XIAP) is aberrantly expressed in solid tumors. Considering conflicting data, we conducted this meta-analysis to investigate its prognostic role. Electronic databases were searched to collect studies about associations between XIAP expressions and survival outcomes. Hazard ratio (HR), odds ratio (OR), and 95% confidence interval (CI) were utilized as effect size estimates. A total of 3,794 patients from 21 published studies were included. The results revealed that high XIAP expressions correlated with age (OR = 2.02; 95% CI, 1.07–3.84), lymph node metastasis (OR = 1.69; 95% CI, 1.02–2.77), histological grade (OR = 2.04; 95% CI, 1.01–4.11), and tumor stage (OR = 2.18; 95% CI, 1.20–3.96). The combined HR revealed that high XIAP expressions associated with poor overall survival (OS) (HR = 1.60; 95% CI, 1.22–2.10). Our study suggested high XIAP expressions may be indicative of poor prognosis in solid tumors.     

Study of caspase inhibitors for limiting death in mammalian cell culture

Apoptosis in mammalian cell culture is associated with decreased bioproduct yields and can be inhibited through altering the intracellular signaling pathways mediating programmed cell death. In this study, we evaluated the capacity to inhibit caspases to maintain high viable cell numbers in CHO and 293 cultures. Two genetic caspase inhibitors, XIAP and CrmA, were examined along with a mutant of each, XIAP-BIR123NC, which contains three BIR domains but lacks the RING finger, and CrmA-DQMD, which has CrmA's pseudosubstrate site replaced with that of another caspase inhibitor, p35. Stable CHO pooled and 293 clonal cell lines expressing each protein were exposed to apoptotic insults, including spent medium, Sindbis virus, and etoposide. For each insult the mutated protein resulted in higher viabilities than its wild-type counterpart. However, the mutants provided different levels of protection, depending on the insult considered. CrmA-DQMD was the preferred inhibitor for spent medium-induced apoptosis, whereas XIAP-BIR123NC conferred better protection for etoposide-induced death. Addition of Z-VAD.fmk to the genetically engineered cells enhanced viabilities in the presence of spent medium or etoposide; however, the largest increases in viability were experienced by the control cells, indicating an overlap in caspase inhibition between the genetic and chemical inhibitors. Finally, parental 293 cells were treated with caspase-8 and -9 inhibitors, Z-IETD.fmk and Z-LEHD.fmk, in concert with spent medium or etoposide exposure. Spent medium-induced death was delayed more readily with the caspase-8 inhibitors, CrmA-DQMD and Z-IETD.fmk, and etoposide-induced death was stalled more so with XIAP-BIR123NC and Z-LEHD.fmk. These results suggest that the apoptosis pathways induced and the level of protection afforded by a particular caspase inhibitor may vary with the insult considered.     

Sustained release of Smac/DIABLO from mitochondria commits to undergo UVB-induced apoptosis

Apoptotic response of keratinocytes to UVB irradiation has physiological significance on photocarcinogenesis. Here, we show that the sustained release of Smac/DIABLO from mitochondria is an important event for the onset of apoptosis in keratinocytes exposed to UVB irradiation. In human keratinocyte HaCaT cells, UVB irradiation at 500 J/m², but not at 150 J/m², induces apoptosis. Significant activations of caspases-9 and -3, and slight activation of caspase-7 were observed only in 500 J/m² UVB irradiated HaCaT cells. Correspondingly, the cleavage of PARP, a substrate of caspases-3 and -7, was detected in cells irradiated at 500 J/m² UVB, but not at 150 J/m². However, with both 150 and 500 J/m² UVB irradiation, cytochrome c, an activator of caspase-9 via the formation of apoptosome, was released from mitochondria to the cytosol at the same extent. In contrast, significant amounts of Smac/DIABLO are released from mitochondria to the cytosol only with 500 J/m² UVB irradiation, and that the level of XIAP is decreased. These results suggest that the extent of Smac/DIABLO efflux from mitochondria is a determinant whether a cell will undergo apoptosis or survival.     

Cleavage of Survivin by Granzyme M Triggers Degradation of the Survivin-X-linked Inhibitor of Apoptosis Protein (XIAP) Complex to Free Caspase Activity Leading to Cytolysis of Target Tumor Cells

Granzyme M (GzmM) is a chymotrypsin-like serine protease that preferentially cuts its substrates after Met or Leu. GzmM is constitutively expressed in activated innate effector natural killer (NK) cells. GzmM-induced cell death is consistent with the kinetics of cytotoxicity of NK cells. These suggest that GzmM may play an important role in innate immunity. Our previous work demonstrated that GzmM induces caspase-dependent apoptosis. However, it is unknown about how GzmM causes caspase activation. Here, we showed that the inhibitor of the apoptosis gene family member Survivin is a physiological substrate for GzmM. GzmM hydrolyzes Survivin at Leu-138 to remove the last four C-terminal residues. The truncated form (sur-TF) is more rapidly hydrolyzed through proteasome-mediated degradation. In addition, Survivin is in complex with X-linked inhibitor of apoptosis protein (XIAP) to inhibit caspase activation as an endogenous inhibitor. Survivin cleavage by GzmM abolishes the stability of the Survivin-XIAP complex and enhances XIAP hydrolysis, which amplifies caspase-9 and 3 activation of target tumor cells. The noncleavable L138A Survivin overexpression can significantly inhibit GzmM-mediated XIAP degradation, caspase activation, and GzmM- and NK cell-induced cytotoxicity. Moreover, Survivin silencing promotes XIAP degradation and enhances GzmM-induced caspase activation as well as GzmM- and NK cell-induced cytolysis of target tumor cells.     

Pioglitazone, a synthetic ligand for PPARγ, induces apoptosis in RB-deficient human colorectal cancer cells

No published data are available about the expression of peroxisome proliferator-activated receptor γ (PPARγ) and the role of PPARγ in retinoblastoma protein (RB)-deficient human colorectal cancer (CRC) cells (SNU-C4 and SNU-C2A). Our aim was to investigate whether PPARγ is expressed in SNU-C4 and SNU-C2A cells and to elucidate possible molecular mechanisms underlying the effect of pioglitazone, a synthetic ligand for PPARγ, on cell growth in these cell lines. RT-PCR and Western blot analysis showed that both human CRC cell lines expressed PPARγ mRNA and protein. Pioglitazone inhibited the cell growth of both cell lines through G2/M phase block and apoptosis. In addition, pioglitazone caused a down-regulation of the X chromosome-linked inhibitor of apoptosis (XIAP), Bcl-2, and cyclooxygenase-2 (COX-2) under conditions leading to PPARγ down-regulation. These results suggest that pioglitazone may have therapeutic relevance or significance in the treatment of human CRC, and the down-regulation of XIAP, Bcl-2, and COX-2 may contribute to pioglitazone-induced apoptosis in these and other RB-deficient cell lines and tumors.     

Identification of a novel splice variant of X-linked inhibitor of apoptosis-associated factor 1

XAF1 (XIAP-associated factor 1) binds to XIAP and blocks its anti-apoptotic activity. It has been reported that XAF1 is mainly expressed in normal tissues but is missing or present at low levels in most cancer cell lines, which implies a tumor-suppressing function. In the present study we describe the identification of a novel splice variant of human XAF1, designated XAF1C, which contains a cryptic exon. Incorporation of this exon (exon 4b) into the mRNA introduces an in-frame stop codon, resulting in a shortened open-reading frame (ORF) of 495 nucleotides. This ORF is predicted to encode a 164 amino acid (AA) protein lacking the C-terminal domain of the previously described XAF1(A), but containing a unique 24 AA carboxy terminus. Like XAF1(A), XAF1C mRNA expression was detected in a variety of human cancer cell lines and also in normal human tissues. The ratio of XAF1(A) and XAF1C mRNA expression differs amongst the cell lines tested, suggesting differential mRNA stabilities and/or the existence of tissue- or cell type-specific splicing regulation. In transfected cells, xaf1c encodes a truncated protein of 18kDa, which is distributed primarily in the nucleus.     

Caspase-8 acts as a key upstream executor of mitochondria during justicidin A-induced apoptosis in human hepatoma cells

Justicia procumbens is a traditional Taiwanese herbal remedy used to treat fever, pain, and cancer. Justicidin A, isolated from Justicia procumbens, has been reported to suppress in vitro growth of several tumor cell lines as well as hepatoma cells. In this study, justicidin A activated caspase-8 to increase tBid, disrupted mitochondrial membrane potential (Delta psi(m)), and caused the release of cytochrome c and Smac/DIABLO in Hep 3B and Hep G2 cells. Justicidin A also reduced Bcl-x(L) and increased Bax and Bak in mitochondria. Caspase-8 inhibitor (Z-IETD) attenuated the justicidin A-induced disruption of Delta psi(m). Growth of Hep 3B implanted in NOD-SCID mice was suppressed significantly by oral justicidin A (20 mg/kg/day). These results indicate that justicidin A-induced apoptosis in these cells proceeds via caspase-8 and is followed by mitochondrial disruption.