Extrinsic Death Receptor Pathway Research Articles

Therapeutic Potential of Plant Metabolites in Bone Apoptosis: A Review.

Osteoporosis is one of the skeletal diseases of major health concern worldwide. Homeostasis of bone occurs with the help of cells, namely, osteoblasts and osteoclasts. Physiological and pathological conditions involve the death of the cells by apoptosis, autophagy, and necrosis. Apoptosis is a key factor in the growth, development, and maintenance of the skeleton. Apoptosis is generated by two pathways: the intrinsic (mitochondria) and extrinsic (death receptor) pathways. Osteoblast apoptosis is governed by the factors like B cell lymphoma 2 (Bcl-2) family proteins, extracellular signal-regulated kinase (ERK), mitogen-activated protein kinases (MAPK), phosphoinositide-3-kinase/ protein kinase B (PI3-K/Akt), Janus kinase 2 (JAK2), bone morphogenetic protein (BMP), and bone matrix protein. Cytokines interact with osteocytes and induce apoptosis. A pro-inflammatory signal stimulates osteocyte apoptosis and increases osteocyte cytokines production. Current therapies have adverse effects which limit their applications. Various plant metabolites have shown beneficial effects on bone. The present review converses about normal bone metabolism and the mechanism of apoptosis leading to bone deterioration. Furthermore, it discusses the role of plant metabolites on bone apoptosis with related indications of efficacy in various experimental models.

Research Status of Death Domain-associated Protein

Overexpression of death domain-associated protein (DAXX) is a common feature of many cancers, and it structurally has binding sites for many interactors (eg, ARTX, HDAC3). According to functional studies, DAXX interacts with a number of DNA-binding transcription factors (TFs), epigenetic regulators, core histones, and proteins involved in chromatin to induce apoptosis via the extrinsic death receptor pathway. Functions as a co-activator or transcriptional inhibitor of cobalt diplus to regulate gene expression. Current studies have found that DAXX is expressed upregulated in ovarian cancer. Additionally, research has demonstrated that DAXX increases the tumorigenicity of prostate cancer by preventing autophagy pathways. In contrast, patients with pancreatic neuroendocrine tumors have lower survival rates when their DAXX is lost. A powerful breast tumor-initiating cell (TIC) inhibitor, DAXX also inhibits the production of pluripotent and EMT genes via promoters that may bind to pluripotent TIC-related genes. As a result, DAXX has powerful carcinogenic properties and potential new therapeutic targets. This article will introduce the protein from the aspects of DAXX's structure, function, and relationship with cancer.

Hsa-miR-766-5p as a new regulator of mitochondrial apoptosis pathway for discriminating of cell death from cardiac differentiation

Dispose of unnecessary cells in multicellular organism take place through apoptosis as a mode of programmed cell death (PCD). This process is triggered through two main pathway including extrinsic pathway or death receptor pathway and intrinsic or mitochondrial pathway. An alternative role for mitochondrial pathway of cell death is its involvement in cell differentiation. Biochemistry of cell differentiation indicates a common origin for differentiation and apoptosis. miRNAs are a group of small non coding mediator RNAs in regulation of many routes such as apoptosis and differentiation. By using bioinformatics tools hsa-miR-766-5p was predicted to target the BAX, BAK and BOK genes involved in mitochondrial apoptosis pathway. RT-qPCR and dual luciferase assay showed targeting of BAX, BAK and BOK 3′UTRs via hsa-miR-766, detected in SW480 and HEK293T cell lines. Caspases 3/7 and 9 activity assay revealed the involvement of hsa-miR-766-5p in mitochondrial apoptosis pathway regulation detected following overexpression and downregulation of this miRNA, detected in SW480 cells treated with 1 μM doxorubicin. Flow cytometry and MTT assay indicated cell death reduction and viability elevation effect of hsa-miR-766 in SW480 cells after its overexpression. Endogenous expression of hsa-miR-766 during the course of human embryonic stem cells (hESCs) differentiation into cardiomyocytes revealed an inverse expression status of this miRNA with BOK. However, the expression of this miRNA was inversely related to BAX and BAK for some time points of differentiation. Overall this results show the involvement of hsa-miR-766 in regulation of mitochondrial apoptosis pathway.

Injury Pathways That Lead to AKI in a Mouse Kidney Transplant Model.

Prolonged cold ischemia (CI) is a risk factor for acute kidney injury after kidney transplantation. We endeavored to determine the pathways involved in the development of tubular cell injury and death before and after transplantation. We hypothesized that ex vivo cold storage before transplant would produce a different injury phenotype to that seen after engraftment in kidney transplants with or without CI. Four groups of mouse donor kidneys were studied: (1) nontransplanted control kidneys; (2) donor kidneys subjected to ex vivo cold ischemia (CI); (3) donor kidneys subjected to kidney transplant without CI (Txp); and (4) donor kidneys subjected to CI followed by transplantation (CI+Txp). Acute kidney injury only occurred in the CI+Txp group, which had significantly increased sCr versus the Txp group and the control mice. Histologically, the CI group demonstrated significantly increased tubular cell apoptosis and caspase-9 expression, whereas the Txp group demonstrated only mild brush border injury without apoptosis or necrosis. In contrast, the CI+Txp group had tubular cell apoptosis associated with expression of caspase-8, TNFR1, and increased serum TNF-α. CI+Txp also led to significantly higher ATN scores in association with increased RIP1, RIP3, pMLKL, and TLR4 expression. Our results suggest distinct therapies are needed at different times during organ preservation and transplantation. Prevention of apoptosis during cold storage is best achieved by inhibiting intrinsic pathways. In contrast, prevention of cell death and innate immunity after CI+Txp requires inhibition of both the extrinsic death receptor pathway via TNFR1 and caspase-8 and inhibition of programmed necrosis via TLR4 and TNFR1.

Ginsenoside Rg5 induces G2/M phase arrest, apoptosis and autophagy via regulating ROS-mediated MAPK pathways against human gastric cancer

Ginsenoside Rg5, a rare saponin belonging to the family of protopanaxadiol ginsenosides, has been demonstrated to have potential anti-tumor effects in various cancers. However, the effect of Rg5 on human gastric cancer and the underlying molecular mechanisms remain to be elucidated. In this study, Rg5 could suppress cell proliferation by causing G2/M phase arrest. Treatment with Rg5 could induce apoptosis through the extrinsic death receptor and intrinsic mitochondrial pathways. Autophagy induction was demonstrated by the formation of autophagosomes and autophagy-related proteins. Rg5-induced cell death was inhibited by the autophagy inhibitor 3-MA and apoptosis inhibitor Z-VAD-FMK. Moreover, the suppression of apoptosis weakened Rg5-induced autophagy, while the inhibition of autophagy attenuated Rg5-induced apoptosis. Further studies revealed that Rg5 induced ROS production and activated MAPK signaling pathways. The ROS scavenger NAC markedly diminished G2/M arrest, apoptosis, autophagy and activation of MAPK pathways induced by Rg5. The p38 inhibitor SB203580 or knockdown of p38 by siRNA clearly reversed Rg5-induced apoptosis and G2/M arrest. The JNK inhibitor SP600125 or knockdown of JNK by siRNA markedly attenuated Rg5-induced G2/M arrest, apoptosis and autophagy. The inhibition of ERK inhibitor U0126 or knockdown of ERK by siRNA clearly restored Rg5-induced apoptosis and autophagy. Finally, Rg5 significantly suppressed the growth of xenograft gastric tumors with fewer side effects. Overall, the evidence suggested that Rg5 is a novel and promising strategy for the treatment of gastric cancer owing to its high efficacy, multiple mechanisms and fewer side effects.

Programmed necrosis in cardiomyocytes: mitochondria, death receptors and beyond.

Excessive death of cardiac myocytes leads to many cardiac diseases, including myocardial infarction, arrhythmia, heart failure and sudden cardiac death. For the last several decades, most work on cell death has focused on apoptosis, which is generally considered as the only form of regulated cell death, whereas necrosis has been regarded to be an unregulated process. Recent findings reveal that necrosis also occurs in a regulated manner and that it is closely related to the physiology and pathophysiology of many organs, including the heart. The recognition of necrosis as a regulated process mandates a re‐examination of cell death in the heart together with the mechanisms and therapy of cardiac diseases. In this study, we summarize the regulatory mechanisms of the programmed necrosis of cardiomyocytes, that is, the intrinsic (mitochondrial) and extrinsic (death receptor) pathways. Furthermore, the role of this programmed necrosis in various heart diseases is also delineated. Finally, we describe the currently known pharmacological inhibitors of several of the key regulatory molecules of regulated cell necrosis and the opportunities for their therapeutic use in cardiac disease. We intend to systemically summarize the recent progresses in the regulation and pathological significance of programmed cardiomyocyte necrosis along with its potential therapeutic applications to cardiac diseases.Linked ArticlesThis article is part of a themed section on Mitochondrial Pharmacology: Featured Mechanisms and Approaches for Therapy Translation. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.22/issuetoc

PDK1 inhibitor GSK2334470 synergizes with proteasome inhibitor MG‑132 in multiple myeloma cells by inhibiting full AKT activity and increasing nuclear accumulation of the PTEN protein.

Phosphoinositide‑dependent kinase1 (PDK1) is generally active in multiple myeloma (MM) and higher expression than other hematopoietic cells, which is associated with the drug resistance and the disease progression. Previous studies have demonstrated that PDK1 can be targeted therapeutically in MM. In the present study, we examined the combination effect of GSK2334470 (GSK‑470), a novel and highly specific inhibitor of PDK1, with proteasome inhibitor MG‑132 in MM cell lines. GSK‑470 monotherapy significantly inhibited growth of MM cell lines and induced apoptosis that was associated with the activation of both the intrinsic mitochondrial pathway and the extrinsic death receptor pathway. Moreover, GSK‑470 demonstrated synergistic growth inhibitory effects with MG‑132. Notably, treatment with these inhibitors resulted in an almost complete inhibition of phosphorylation of mammalian target of rapamycin on Ser2448 and Ser2481 and full activation of AKT. The combination therapy also caused an upregulation of PTEN and an increased nuclear accumulation of PTEN protein. Collectively, our results provide the rationale for novel combination treatment with PDK1 inhibitor and proteasome inhibitors to improve outcomes in patients with MM.

Structure-dependent induction of apoptosis by hepatotoxic pyrrolizidine alkaloids in the human hepatoma cell line HepaRG: Single versus repeated exposure

Pyrrolizidine alkaloids (PA) are secondary plant compounds. PA intoxication in humans causes severe acute and chronic hepatotoxicity. However, the molecular mechanisms of PA hepatotoxicity in humans are not well understood yet.Therefore, we investigated cell death parameters in human HepaRG cells following either single (24 h) or repeated dose treatment (14 d) with structurally different PA of the retronecine (echimidine, senecionine), heliotridine (heliotrine), and otonecine type (senkirkine). After 24 h of exposure only retronecine-type PA were cytotoxic in HepaRG cells and induced apoptosis indicated by a loss of membrane asymmetry, disruption of the mitochondrial membrane potential, and increased pro-caspase and PARP cleavage. In contrast, after 14 d all four PA exerted the aforementioned effects. Furthermore, the apoptotic events caspase 3, 8 and 9 activation as well as nuclear condensation and DNA fragmentation were only detected for the retronecine-type PA after single exposure (6 h).Overall, our studies revealed a time- and structure-dependent apoptosis after PA exposure, suggesting that retronecine-type PA seem to be more potent apoptosis inducers than heliotridine- or otonecine-type PA. Furthermore, our results suggest that PA-induced apoptosis in HepaRG cells occur most probably by involving both, the extrinsic death receptor pathway as well as the intrinsic mitochondrial pathway.

Long non-coding RNA HOXA-AS2 promotes tumorigenesis and predicts poor prognosis in ovarian cancer

// Changsheng Yan 1, 2, * , Mi Gong 1, * , Yi Jiang 1, * , Huijian Li 1, 3, 4, 5 , Yicong Wan 1 , Lin Zhang 6 , Shulin Zhou 1 and Wenjun Cheng 1 1 Department of Gynecology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China 2 Institute for Microbiota Ecology, Medical College of Xiamen University, Xiamen 361000, China 3 State Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, Nanjing 211166, China 4 Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing 211166, China 5 Wuxi Maternal and Child Health Hospital, Nanjing Medical University, Wuxi 214002, China 6 Department of Obstetrics and Gynecology, Zhongda Hospital Affiliated to Southeast University, Medical School, Southeast University, Nanjing 210029, China * These authors have contributed equally to this work Correspondence to: Wenjun Cheng, email: wenjunchengdoc@163.com Keywords: epithelial ovarian cancer (EOC); IncRNA-HOXA-AS2; apoptosis; caspase activation; Bcl-2 protein family Received: June 14, 2017 Accepted: January 02, 2018 Published: January 02, 2018 ABSTRACT Recent reports confirms that there are some long non-coding RNAs involved in the different biological regulatories in cancer, including proliferation and apoptosis. However, the correlation between the HOXA-AS2 expression and proliferation in epithelial ovarian cancer was not completely investigated. In vivo expression of HOXA-AS2 in 73 epithelial ovarian cancer tissues and 57 normal tissues was determined by qRT-PCR. Results showed that not only HOXA-AS2 was overexpressed in epithelial ovarian cancer tissues, but also had a relationship with the tumor size and FIGO stage of patients. Further, the overall survival in epithelial ovarian cancer patients presenting high expression of HOXA-AS2 was lower to those presenting low expression. Next, the functional effect of HOXA-AS2 was explored via GSEA analysis in ovarian cancer datasets. The results confirmed that HOXA-AS2 was enriched in cell apoptosis and JAK-STAT pathway. Then, EOC cell lines were transfected with HOXA-AS2 siRNA. Cell proliferation was significantly inhibited after knockdown of HOXA-AS2 via promoting apoptosis both in HO-8910 and SKOV3 cells. Reversely, overexpression of HOXA-AS2 in normal ovarian cell line could promote proliferation and inhibit apoptosis. Animal study consistently indicated that lower expression of HOXA-AS2 inhibited proliferation of HO-8910 cells. Finally, Western blot results showed that expressions of procaspase-9, -8, -3, and PARP were decreased after knockdown of HOXA-AS2, while cleaved Caspase-9, -8 and -3 were increased. Meanwhile, expressions of Bcl-2 and Bid were decreased and Bax was increased. In addition, overexpression of HOXA-AS2 increased the level of Bcl-2, Bid, JAK2 and Stat3 while Bax was decreased in HO8910 and SKOV3. Besides, the IHC assay also showed the same expression changes of Bcl-2 and Bax. To sum up, this study suggested that lncRNA HOXA-AS2 acted as an oncogene in EOC that partly inhibited the intrinsic mitochondria pathway and extrinsic death receptor pathway, which was associated with Bcl-2 protein family; HOXA-AS2 would be regarded as a new biological prognosis and potential therapeutic target of epithelial ovarian cancer.

Apoptosis in human hepatoma HepG2 cells induced by the phenolics of Tetrastigma hemsleyanum leaves and their antitumor effects in H22 tumor-bearing mice

Tetrastigma hemsleyanum leaves (THL), a popular anticancer functional food in China, are used as nutrient or dietary supplement. However, the anticancer effects and the underlying mechanism of THL are still unknown. Thus, the phenolic profiles and anticancer activities of THL purified extract were investigated by a combination of chemical assays, LC-QTOF-MS/LC-QqQ-MS techniques, cell cultures and tumor-bearing mice model. It has been indicated that the TPC/TFC (421.63 and 343.38 mg/g DW) of THL extract were obviously increased after SPE purification, and the main phenolic components were apigenin and luteolin glycosides. Besides, THL could induce apoptosis by both of extrinsic death receptor pathway and intrinsic mitochondria pathway in HepG2 cells as well as inhibit the tumor growth, regulate Bcl-2 family proteins, and active the caspase family proteins in H22 tumor-bearing mice. Summarily, the findings clearly demonstrated that THL could be a potential functional food for the treatment and prevention of liver cancer.

Novel celastrol derivatives with improved selectivity and enhanced antitumour activity: Design, synthesis and biological evaluation

Celastrol is one of the most active antitumour compounds among the natural triterpenoids. It has been reported to be highly active against a wide variety of tumours and to affect multiple cellular pathways. A series of new celastrol derivatives, including compounds bearing a urea group, have been synthesised and analysed for their biological activity against human cancer cell lines. Several compounds presented a stronger growth inhibition effect than celastrol on the cell lines studied. Among them, compound 24 was the most promising derivative, as it exhibited both a remarkable antiproliferative activity and an improved selectivity in tumour versus non-tumour cells. The anticancer molecular mechanism of compound 24 in the human ovary cancer cell line SKOV-3 was further studied and the results showed that compound 24 induced apoptosis through the activation of the extrinsic death receptor pathway. Interestingly, the results revealed that compound 24 might be able to decrease the levels of dysfunctional p53. The assays also suggested that compound 24 is an Hsp90 inhibitor, and that the Akt/mTOR pathway might be involved in the downstream regulation that leads to its antiproliferative activity. Moreover, a synergistic anticancer effect was evidenced when SKOV-3 cells were simultaneously treated with compound 24 and cisplatin. Taken together, these results suggest that compound 24 may be a promising lead for the development of new cancer therapies.

Potential Toxicity of Polymyxins in Human Lung Epithelial Cells.

Inhaled polymyxins are of considerable utility in achieving optimal exposure in the respiratory tract for the treatment of lung infections caused by multidrug-resistant Gram-negative pathogens. Current inhaled polymyxin therapy is empirical, and often large doses are used that may lead to potential pulmonary adverse effects. This study aimed to investigate the effect of polymyxins on human lung epithelial (A549) cells. The viability of A549 cells was examined after treatment with polymyxins by flow cytometry. Activation of caspases 3, 8, and 9, expression of Fas ligand (FasL), loss of mitochondrial membrane potential, and mitochondrial oxidative stress induced by polymyxin B were evaluated. The concentration of polymyxin B required to induce 50% of maximal cell death was 1.74 mM (95% confidence interval, 1.60 to 1.90 mM). Colistin was at least 2-fold less toxic than polymyxin B, while colistimethate was nontoxic. With 2.0 mM polymyxin B, 30.6% ± 11.5% (mean ± standard deviation) of the cells were apoptotic at 8 h and this increased to 71.3% ± 3.72% at 24 h. Concentration- and time-dependent activation of caspases 3, 8, and 9 was evident, while the activation of caspase 9 was more dramatic. Furthermore, polymyxin B caused concentration- and time-dependent FasL expression, production of mitochondrial reactive oxygen species, and changes in mitochondrial membrane potential. This is the first study to demonstrate that both extrinsic death receptor and intrinsic mitochondrial pathways are involved in polymyxin-induced toxicity in A549 cells. This knowledge base is critical for the development of novel strategies for the safe and effective inhalation therapy of polymyxins against Gram-negative "superbugs."

AG36 Inhibits Human Breast Cancer Cells Proliferation by Promotion of Apoptosis In vitro and In vivo.

AG36 is the biotransformation product of triterpenoid saponin from Ardisia gigantifolia stapf. In this study, the antitumor activity and underlying molecular mechanisms of AG36 against human breast MCF-7, MDA-MB-231, and SK-BR-3 cancer cells were investigated. AG36 inhibited the viability of MCF-7, MDA-MB-231, and SK-BR-3 cells in a dose and time-dependent manner, with an IC50 of approximately 0.73, 18.1, and 23.4 μM at 48 h, respectively. AG36 obviously induced apoptosis and G2/M arrest of all the three breast cancer cells. Moreover, AG36 decreased the protein expression of cycle regulatory proteins cyclin B1 or cyclin D1. In MCF-7 and MDA-MB-231 cells, AG36 strongly increased the cleaved caspase-3 and -8 protein expressions, while in SK-BR-3 cells, AG36 only increased the protein expression of cleaved caspase-3. In all the three breast cancer cells, the ratio of Bax/Bcl-2 and cytosolic cytochrome c content increased significantly compared with control group. The death receptor-related proteins Fas/FasL, TNFR1, and DR5 were detected by Western blot, it showed that different breast cancer cells activated the death receptor-mediated extrinsic caspase-8 pathway through different receptors. In addition, the caspase-8 inhibitor z-IETD-fmk could significantly block AG36-triggered MCF-7 cells apoptosis. The in vivo studies showed that AG36 significantly inhibited the growth of MCF-7 xenograft tumors in BALB/c nude mice comparing with control. In conclusion, AG36 inhibited MCF-7, MDA-MB-231, and SK-BR-3 cells proliferation by the intrinsic mitochondrial and the extrinsic death receptor pathways and AG36 might be a potential breast cancer therapeutic agent.

FLIP: A Targetable Mediator of Resistance to Radiation in Non-Small Cell Lung Cancer.

Resistance to radiotherapy due to insufficient cancer cell death is a significant cause of treatment failure in non-small cell lung cancer (NSCLC). The endogenous caspase-8 inhibitor FLIP is a critical regulator of cell death that is frequently overexpressed in NSCLC and is an established inhibitor of apoptotic cell death induced via the extrinsic death receptor pathway. Apoptosis induced by ionizing radiation (IR) has been considered to be mediated predominantly via the intrinsic apoptotic pathway; however, we found that IR-induced apoptosis was significantly attenuated in NSCLC cells when caspase-8 was depleted using RNA interference (RNAi), suggesting involvement of the extrinsic apoptosis pathway. Moreover, overexpression of wild-type FLIP, but not a mutant form that cannot bind the critical death receptor adaptor protein FADD, also attenuated IR-induced apoptosis, confirming the importance of the extrinsic apoptotic pathway as a determinant of response to IR in NSCLC. Importantly, when FLIP protein levels were downregulated by RNAi, IR-induced cell death was significantly enhanced. The clinically relevant histone deacetylase (HDAC) inhibitors vorinostat and entinostat were subsequently found to sensitize a subset of NSCLC cell lines to IR in a manner that was dependent on their ability to suppress FLIP expression and promote activation of caspase-8. Entinostat also enhanced the antitumor activity of IR in vivo Therefore, FLIP downregulation induced by HDAC inhibitors is a potential clinical strategy to radiosensitize NSCLC and thereby improve response to radiotherapy. Overall, this study provides the first evidence that pharmacological inhibition of FLIP may improve response of NCSLC to IR. Mol Cancer Ther; 15(10); 2432-41. ©2016 AACR.

Chewing the fat about death with the neighbours

Pointed threats, they bluff with scorn Suicide remarks are torn From the fool's gold mouthpiece the hollow horn Plays wasted words, proves to warn That he not busy being born is busy dying (Bob Dylan, 'It's Alright, Ma (I'm Only Bleeding)') There are as many ways to interpret Bob Dylan's lyrics as there are ways that a cell can die. Like Dylan's music, the cell death field has undergone several transformations over the years. In the early days cell death could be divided into three categories based primarily on morphology: Type 1 (programmed cell death or apoptosis), Type 2 (cell death with autophagy), and Type 3 (necrosis).1, 2 Genetic approaches in model organisms like Caenorhabditis elegans and Drosophila uncovered the apoptotic cell death genes and ordered them into pathways,3, 4 while biochemical approaches, primarily in cell lines, established the molecular mechanisms by which apoptosis proteins function. Chemical approaches were instrumental in the subsequent identification and study of non-apoptotic forms of cell death, such as necroptosis and ferroptosis.5, 6 The two reviews in this issue of CDD cover the roads less travelled on the journey to death. They address how signals from neighbouring cells license and fine-tune the apoptotic machinery, and detail the role of lipids in non-apoptotic cell death. Apoptotic cell death can be activated through either the intrinsic (mitochondrial) or extrinsic (death receptor) pathways, and over the years much effort has been focused on understanding the detailed molecular mechanisms by which the proteins in these pathways function. However, until recently, much less attention has been paid to the influence of neighbouring cells on the death process. It is well known that apoptosis can be regulated by exogenous factors, such as cytokines and nerve growth factor, but there are many differences in how cells die in vivo versus in vitro. Genetic studies in worms have taught us that the engulfing cell can help promote the execution of a neighbour fated to die, and secreted factors can license the execution machinery.7, 8, 9, 10 Work in Drosophila has shown that secreted factors collaborate with the core apoptosis pathway to fine-tune its activity, and dying cells (or cells primed for apoptotic death) can stimulate proliferation of their neighbours in a process called compensatory proliferation.11 The review by Eroglu and Derry focuses on recent advances in cell non-autonomous mechanisms that regulate apoptosis, a topic that has important implications in development and disease. While we still have much to learn about apoptosis, a series of papers have recently described alternate ways in which cells can die. These forms of non-apoptotic death, referred to as regulated cell death, involve distinct effector molecules and unique morphological changes. One might even say that we have entered a new renaissance in the cell death field, as we begin to embrace the many forms of cell death that can occur. The review by Matanong, Ko and Dixon tackles the mysterious roles of lipids in regulated cell death. Not only do lipids assist in the formation of pores in cellular membranes that cause their contents to bleed out, but they also serve as signalling molecules in apoptosis and regulated cell death. The plasma membrane is comprised of many different lipid molecules that can be called into action to initiate or assist in various forms of cell death, but we really do not understand how cells utilize these lipids (or 'chew the fat') in these processes. Decoding the logic of how lipids function has important implications in the treatment of numerous diseases that have defects in both apoptotic and regulated cell death (Figure 1). Figure 1 Cells use a variety of molecules, including fatty acids, to assist their neighbours with suicide There are many questions remaining to be answered about non-autonomous apoptosis and the molecular mechanisms of regulated cell death. The intense debate in these areas is reminiscent of the early days of apoptosis research. Autophagy was originally shown to prolong cell survival under nutrient starvation through organelle cannibalism, but more recent studies have shown that it can also trigger the rapid demise of a cell under specific conditions.12 This reinforces the old paradigm that life hangs in the balance with death, and the words of Bob Dylan remind us 'that he not busy being born is busy dying'.

Quantitative antibody-free LC-MS/MS analysis of sTRAIL in sputum and saliva at the sub-ng/mL level.

Soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) induces apoptosis via the extrinsic death receptor pathway and may be a biomarker in the pathogenesis of a broad range of diseases. To investigate the role of sTRAIL in asthma, we developed a quantitative LC-MS/MS method with a lower limit of quantitation (LLOQ) of ≈3pM in induced sputum (174pg/mL) and saliva (198pg/mL) without the use of antibodies. sTRAIL was enriched by immobilized metal affinity chromatography (IMAC) solid-phase extraction (SPE) followed by tryptic digestion and subsequent enrichment of a signature peptide by strong cation exchange (SCX) SPE. The method was validated with respect to stability, accuracy and precision using the standard addition approach and fully metabolically (15)N-labelled hrTRAIL as internal standard. Our results indicate that it is possible to quantify cytokines like sTRAIL at the pM level by LC-MS/MS without the use of antibodies, which has, to our knowledge, never been shown before.

Traditional Chinese medicine targeting apoptotic mechanisms for esophageal cancer therapy.

Esophageal cancer is one of the most common types of cancer in the world, and it demonstrates a distinct geographical distribution pattern in China. In the last decade, inducing apoptosis with traditional Chinese medicine (TCM) has become an active area in both fundamental and clinical research on cancer therapy. In this review, we summarize the molecular mechanisms by which TCM induces apoptosis in esophageal cancer cells. These mechanisms are generally related but not limited to targeting the extrinsic death receptor pathway, the intrinsic mitochondrial pathway, and the endoplasmic reticulum (ER) stress pathway. By using different monomers and composite prescriptions of TCM, it is possible to modulate the ratio of Bcl-2/Bax, regulate the expression of caspase proteases and mitochondrial transmembrane potential, increase the expression of Fas and p53, down-regulate NF-κB pathway and the expression of Chop and survivin, and block cell cycle progression.

BA-j as a novel CDK1 inhibitor selectively induces apoptosis in cancer cells by regulating ROS.

Cyclin-dependent kinase 1 (CDK1) is the only necessary CDK in cell proliferation and a novel target in the development of anticancer drugs. 8-Hydroxypiperidinemethyl-baicalein (BA-j) is a novel selective CDK1 inhibitor with broad spectrum anti-cancer activity (IC50 12.3 μM) and 2 tumor xenografts. Because of the differential mechanisms controlling redox-states in normal and cancer cells, BA-j can capture oxygen free radicals (·O2−) and selectively increase the level of H2O2 in cancer cells, thereby specifically oxidize and activate the intrinsic apoptosis pathway bypassing the extrinsic death receptor pathway, thus inducing apoptosis in cancer cells rather than in normal cells. BA-j is different from cytotoxic anticancer drugs which can activate both the intrinsic apoptosis pathway and the extrinsic death receptor pathway, and therefore harm normal cells while killing cancer cells. The molecular and biochemical mechanisms of reactive oxygen species (ROS) regulation suggest that BA-j may be developed into a novel anticancer agent.

Pennogenyl Saponins from Paris quadrifolia L. Induce Extrinsic and Intrinsic Pathway of Apoptosis in Human Cervical Cancer HeLa Cells.

Pennogenyl saponins are the active compounds of large number of plant species and consequently many polyherbal formulations. Hence, great interest has been shown in their characterization and in the investigation of their pharmacological and biological properties, especially anticancer. This present study reports on the evaluation of cytotoxic effects and explanation of the molecular mechanisms of action of the two pennogenyl saponins (PS 1 and PS 2) isolated from Paris quadrifolia L. rhizomes on human cervical adenocarcinoma cell line HeLa. To determine the viability of the cells treated with the compounds we used real-time cell proliferation analysis and found that the pennogenyl saponins PS 1 and PS 2 strongly inhibited the tumor cells growth with IC50 values of 1.11 ± 0.04 μg/ml and 0.87 ± 0.05 μg/ml, respectively. The flow cytometry analysis indicated that the two compounds induced apoptosis in a dose-dependent manner and decreased mitochondrial membrane potential in HeLa cells in the early stage of apoptosis. Quantitative PCR and Western Blot analysis showed that the two saponins significantly increased mRNA expression of FADD and BID as well as induced caspase-8 via increased of procaspase-8 processing in the treated cells. The results of this study suggest that both the extrinsic death receptor and intrinsic mitochondrial pathways are involved in the programmed cell death.

Caspase-dependent Proteolysis of Human Ribonucleotide Reductase Small Subunits R2 and p53R2 during Apoptosis

Ribonucleotide reductase (RnR) is a key enzyme synthesizing deoxyribonucleotides for DNA replication and repair. In mammals, the R1 catalytic subunit forms an active complex with either one of the two small subunits R2 and p53R2. Expression of R2 is S phase-specific and required for DNA replication. The p53R2 protein is expressed throughout the cell cycle and in quiescent cells where it provides dNTPs for mitochondrial DNA synthesis. Participation of R2 and p53R2 in DNA repair has also been suggested. In this study, we investigated the fate of the RnR subunits during apoptosis. The p53R2 protein was cleaved in a caspase-dependent manner in K-562 cells treated with inhibitors of the Bcr-Abl oncogenic kinase and in HeLa 229 cells incubated with TNF-α and cycloheximide. The cleavage site was mapped between Asp(342) and Asn(343). Caspase attack released a C-terminal p53R2 peptide of nine residues containing the conserved heptapeptide essential for R1 binding. As a consequence, the cleaved p53R2 protein was inactive. In vitro, purified caspase-3 and -8 could release the C-terminal tail of p53R2. Knocking down these caspases, but not caspase-2, -7, and -10, also inhibited p53R2 cleavage in cells committed to die via the extrinsic death receptor pathway. The R2 subunit was subjected to caspase- and proteasome-dependent proteolysis, which was prevented by siRNA targeting caspase-8. Knocking down caspase-3 was ineffective. Protein R1 was not subjected to degradation. Adding deoxyribonucleosides to restore dNTP pools transiently protected cells from apoptosis. These data identify RnR activity as a prosurvival function inactivated by proteolysis during apoptosis.