Induction of the cell cycle arrest and apoptosis by flavonoids isolated from Korean Citrus aurantium L. in non-small-cell lung cancer cells

This study investigated the anti-proliferative and apoptotic effect of flavonoids isolated from Korean Citrus aurantium L. using A549 lung cancer cells. Flavonoids potently inhibited of A549 cells in a dose-dependent manner, whereas flavonoids had a weak inhibitory effect on proliferation of WI-38 cells. Flow cytometry and Western blot analysis showed that flavonoids induced cell cycle arrest at the G2/M checkpoint by controlling the proteins expression level of cyclin B1, cdc2, cdc25c and p21^WAF1/CIP1. Also, flavonoids induced apoptosis through the regulation of the expression of caspases, cleaved PARP and Bax/Bcl-xL ratio. The activity of caspase-3 on A549 cells increased in a dose-dependent manner. These results clearly indicated that the anti-cancer effect of flavonoids on A549 cells follows multiple cellular pathways through G2/M arrest and the induction of apoptosis.     

Regulatory Roles of Flavonoids on Inflammasome Activation during Inflammatory Responses

Inflammation is an innate immune response to noxious stimuli to protect the body from pathogens. Inflammatory responses consist of two main steps: priming and triggering. In priming, inflammatory cells increase expressions of inflammatory molecules, while in triggering, inflammasomes are activated, resulting in cell death and pro‐inflammatory cytokine secretion. Inflammasomes are protein complexes comprising intracellular pattern recognition receptors (PRRs) (e.g., nucleotide‐binding oligomerization domain‐like receptors (NLRs), absent in melanoma 2 (AIM2), and caspases‐4/5/11) and pro‐caspase‐1 with or without a bipartite adaptor molecule ASC. Inflammasome activation induces pyroptosis, inflammatory cell death, and stimulates caspase‐1‐mediated secretion of interleukin (IL)‐1b and IL‐18. Flavonoids are secondary metabolites found in various plants and are considered as critical ingredients promoting health and ameliorating various disease symptoms. Anti‐inflammatory activity of flavonoids and underlying mechanisms have been widely studied. This review introduces current knowledge on different types of inflammasomes and their activation during inflammatory responses and discusses recent studies regarding anti‐inflammatory roles of flavonoids as suppressors of inflammasomes in inflammatory conditions. Understanding the regulatory effects of flavonoids on inflammasome activation will increase our knowledge of flavonoid‐mediated anti‐inflammatory activity and provide new insights into the development of flavonoid preparations to prevent and treat human inflammatory diseases.     

Postmortem muscle cells die through apoptosis

Several reports suggested the activation of caspases in postmortem muscle implicating the onset of a caspase-dependent cell death process after animal bleeding. It has been further well established that apoptosis and necrosis are the two major cell death pathways. The questions addressed in the present work were as follows: (a) in postmortem muscle, do cells die as in vivo? and (b) if so, by which dying process this goal is achieved? Selected hallmarks of apoptosis (phosphatidylserine externalization (PS), cell shrinkage, actin degradation) were analyzed in postmortem rat muscles and compared to usual cell behavior in apoptotic and necrotic processes. Results presented clearly demonstrate a rapid PS externalization and cell shrinkage extending during the first 24 h postexsanguination together with a progressive degradation of cytoskeletal and thin filaments of actin. It was therefore concluded that, in postmortem muscle, cells commit suicide soon after animal bleeding through apoptosis.     

Caspase-mediated apoptosis in the vertebrate ovary

In the vertebrate ovary, apoptosis is the process by which excess or non-viable germ and granulosa cells are eliminated early in ontogeny (often beginning before birth), and thereafter continuously throughout reproductive life. Accordingly, an excessively high rate or abnormal triggering of such cell death (and, by implication, follicle atresia) can negatively affect fertility. Programmed cell death involves the integration of many pathways and intracellular proteins, and central among these at almost every stage are members of the caspase family. Relatively little attention has been focused upon the ovary with regards to elucidating initiator and effector members of the caspase family, and pathways by which they are activated and inactivated. The present review briefly describes vertebrate caspases and the regulation of their function in non-ovarian tissues. Subsequently, the status of caspase expression and function in orchestrating apoptotic cell death in ovarian germ and follicle somatic cells is considered. The most compelling results implicating specific caspases in ovarian function have been derived from mouse single and double knockout model systems. The final outcome of continued studies, in addition to providing information regarding understanding and management of infertility, will influence the development of strategies to treat ovarian cancers and ameliorate the adverse effects of their therapy (for example, chemotherapy).     

畜禽屠宰后肌肉细胞死亡机制研究进展

畜禽宰后肌肉变化为食肉是一个极为复杂的的过程。宰前应激、击晕方式(电击、CO2致晕等)以及宰后放血都会使畜禽肌肉细胞处于极端生存环境(缺氧、ATP消耗、H+积累等)中,不可避免地对细胞代谢产生重大影响,最终导致其死亡。凋亡、坏死、自噬性死亡以及最近提出的凋亡性坏死都是已经阐明的细胞死亡方式。对caspase活性的深入研究结果表明畜禽宰后肌肉细胞死亡可能涉及了凋亡过程。坏死作为一种突发性的非程序化死亡过程,由于具有胞浆中Ca2+升高、ATP快速消耗、线粒体膜通透性增加等特征,可能更适于解释宰后肌肉细胞变化过程。最近凋亡性坏死学说的提出又增加了宰后肌肉细胞死亡机制的复杂性。对畜禽宰后肌肉细胞死亡机制的研究任重而道远。     

Selenomethionine activates selenoprotein S,suppresses Fas/FasL and the mitochondrial pathway,and reduces Escherichia coli-induced apoptosis of bovine mammary epithelial cells

Escherichia coli is a major environmental pathogen causing bovine mastitis, characterized by cell death and mammary tissue damage. Apoptosis, a form of cell death, has an important role in the pathogenesis of mastitis. Selenium, an essential trace element, protects against mastitis by acting through several biochemical pathways, potentially including prevention of apoptosis. Our objective was to investigate whether selenomethionine (SeMet) attenuated E. coli-induced apoptosis in bovine mammary epithelial cells (bMEC). These cells were cultured in vitro and treated with 0, 5, 10, 20, and 40 μM SeMet for 12 h, with or without E. coli (multiplicity of infection of 5) for 8 h. Treatment with SeMet/Z-IE(OMe)TD(OMe)-FMK (ZIK)/Z-LE(OMe)HD(OMe)-FMK (ZLK, specific inhibitors of caspase-8 and -9, respectively) significantly counteracted effects of E. coli on bMEC. Specifically, SeMet upregulated selenoprotein S (SeS) and increased mitochondrial membrane potential and the ratio of Bcl-2 and Bax. Furthermore, it decreased protein expressions of Fas, FasL, FADD, cleaved caspase-8, cytochrome c, cleaved caspase-9, and cleaved caspase-3, namely, decreasing protein expression of the Fas/FasL and mitochondrial pathways. Furthermore, it downregulated total apoptosis indexes in E. coli-infected bMEC. Although ZIK and ZLK (specific inhibitors of caspases 8 and 9, respectively) significantly inhibited Fas/FasL and the mitochondrial apoptotic pathway and apoptosis indexes, respectively, substantial apoptosis still occurred. In conclusion, SeMet attenuated E. coli-induced apoptosis in bMEC by activating SeS, associated with Fas/FasL and mitochondrial pathways.     

Apoptosis of bovine ovarian surface epithelial cells by Fas antigen/Fas ligand signaling

Ovarian surface epithelial cells (OSEs), a single layer of cells that cover the surface of the ovary, undergo turnover at the site of follicular rupture at ovulation. Greater than 90% of ovarian cancers arise from the OSEs. The objective of this study was to determine whether OSEs have the capacity to regulate their own demise through expression of Fas antigen (Fas) and Fas ligand (FasL) and activation of Fas-mediated apoptosis. In initial experiments, primary cultures of bovine OSEs responded to treatment with recombinant FasL by undergoing apoptosis. The percentage of cell death was not affected by the presence or absence of serum in the media or by co-treatment with interferon-gamma, a treatment shown to potentiate Fas-mediated apoptosis in a number of cell types. Subsequent experiments tested the ability of stress-inducing drugs, anisomycin and daunorubicin, to promote apoptosis by stimulating an endogenous Fas-FasL pathway in OSEs. Treatment with FasL, anisomycin or daunorubicin induced cell death and this was suppressed by co-treatment with a peptide inhibitor of caspases, ZVAD. Treatment with anisomycin or daunorubicin in the presence of ZVAD increased expression of FasL mRNA and protein but did not alter expression of Fas mRNA or protein. Treatment of OSEs with a recombinant protein that blocks interaction of FasL with Fas (Fas:Fc) reduced apoptosis in response to anisomycin and daunorubicin, indicating that drug-induced apoptosis was mediated at least partially through endogenous Fas-FasL interactions. In summary, OSEs undergo apoptosis in response to stress-inducing drugs through activation of an endogenous Fas pathway.     

Inhibition effects and induction of apoptosis of flavonoids on the prostate cancer cell line PC-3 in vitro

Myricetin and myricitrin are naturally occurring flavonoids have been suggested to play a role in inhibition of proliferation and transformation of carcinogenic cell. However, the underlying molecular mechanisms of their activity have not yet to be revealed. The aim of the present study was to clarify the molecular mechanisms of apoptosis cell on the prostate cancer induced by myricetin, myricitrin, quercetin and quercitrin. The MTT assay confirmed that myricetin had the strongest inhibitory effect on human prostate cancer cell line PC-3, myricitrin was second, and quercitrin was the weakest. A noticeable synergistic effect was observed with the inhibition of cell proliferation when myricetin was used in combination with myricitrin. In the concentration range of 37.5–300 μmol/L, the inhibitory effects of these flavonoids were enhanced with increasing dose and treatment time. The acridine orange analysis and annexin V–FITC/PI double-staining results confirmed that myricetin and myricitrin were effective in inducing PC-3 cell apoptosis. The results showed that myricetin was more effective than myricitrin in inducing cell apoptosis. The apoptosis rate increased with increasing flavonoid concentration in a dose dependent manner. A synergistic effect was observed on the apoptosis rate when myricetin was used in combination with myricitrin.     

Thioredoxin system inhibitors as mediators of apoptosis for cancer therapy

The thioredoxin (Trx) system is a major antioxidant system integral to maintaining the intracellular redox state. It contains Trx, a redox active protein, which regulates the activity of various enzymes including those that function to counteract oxidative stress within the cell. Trx can also scavenge reactive oxygen species (ROS) and directly inhibits proapoptotic proteins such as apoptosis signal-regulating kinase 1 (ASK1). The oxidized form of Trx is reduced by thioredoxin reductase (TrxR). The cytoplasm and mitochondria contain equivalent Trx systems and inhibition of either system can lead to activation of apoptotic signaling pathways. There are a number of inhibitors with chemotherapy applications that target either Trx or TrxR to induce apoptosis in cancer cells. Suberoylanilide hydroxamic acid (SAHA) is effective against many cancer cells and functions by up-regulating an endogenous inhibitor of Trx. Other compounds target the selenocysteine-containing active site of TrxR. These include gold compounds, platinum compounds, arsenic trioxide, motexafin gadolinium, nitrous compounds, and various flavonoids. Inhibition of TrxR leads to an accumulation of oxidized Trx resulting in cellular conditions that promote apoptosis. In addition, some compounds also convert TrxR to a ROS generating enzyme. The role of Trx system inhibitors in cancer therapy is discussed in this review.     

Serine Protease Inhibitors as Good Predictors of Meat Tenderness: Which Are They and What Are Their Functions?

Since years, serine proteases and their inhibitors were an enigma to meat scientists. They were indeed considered to be extracellular and to play no role in postmortem muscle proteolysis. In the 1990's, we observed that protease inhibitors levels in muscles are a better predictor of meat tenderness than their target enzymes. From a practical point of view, we therefore choose to look for serine protease inhibitors rather than their target enzymes, i.e. serine proteases and the purpose of this report was to overview the findings obtained. Fractionation of a muscle crude extract by gel filtration revealed three major trypsin inhibitory fractions designed as F1 (Mr:50–70 kDa), F2 (Mr:40–60 kDa) and F3 (Mr:10–15kD) which were analyzed separately. Besides antithrombin III, an heparin dependent thrombin inhibitor, F1 and F2 comprised a large set of closely related trypsin inhibitors encoded by at least 8 genes bovSERPINA3-1 to A3-8 and able to inhibit also strongly initiator and effector caspases. They all belong to the serpin superfamily, known to form covalent complexes with their target enzymes, were located within muscle cells and found in all tissues and fluids examined irrespective of the animal species. Potential biological functions in living and postmortem muscle were proposed for all of them. In contrast to F1 and F2 which have been more extensively investigated only preliminary findings were provided for F3. Taken together, these results tend to ascertain the onset of apoptosis in postmortem muscle. However, the exact mechanisms driving the cell towards apoptosis and how apoptosis, an energy dependent process, can be completed postmortem remain still unclear.     

Biomarkers of meat tenderness: Present knowledge and perspectives in regards to our current understanding of the mechanisms involved

Biomarkers of the meat quality are of prime importance for meat industry, which has to satisfy consumers' expectations and, for them, meat tenderness is and will remain the primary and most important quality attribute. The tenderization of meat starts immediately after animal death with the onset of apoptosis followed by a cooperative action of endogenous proteolytic systems. Before consideration of the biomarkers identified so far, we present here some new features on the apoptotic process. Among them, the most important is the recent discovery of a complex family of serpins capable to inhibit, in a pseudo-irreversible manner, caspases, the major enzymes responsible of cell dismantling during apoptosis. The biomarkers so far identified have been then sorted and grouped according to their common biological functions. All of them refer to a series of biological pathways including glycolytic and oxidative energy production, cell detoxification, protease inhibition and production of Heat Shock Proteins. Some unusual biomarkers are also presented: annexins, galectins and peroxiredoxins. On this basis, a detailed analysis of these metabolic pathways allowed us to identify some domains of interest for future investigations. It was thus emphasized that mitochondria, an important organelle in the production of energy from carbohydrates, lipids and proteins are a central element in the initiation and development of apoptosis. It was therefore stressed forward that, in fact, very little is known about the postmortem fate of these organelles and their multiple associated activities. Other topics discussed here would provide avenues for the future in the context of identifying reliable predictors of the ultimate meat tenderness.     

Antiproliferative activity of fucoidan was associated with the induction of apoptosis and autophagy in AGS human gastric cancer cells

Fucoidan, a sulfated polysaccharide purified from brown algae, possesses a variety of pharmacologic effects, including antiinflammatory, antioxidant, and anticancer properties; however, the underlying action mechanisms are not completely understood. This study investigated the possible mechanisms through which fucoidan exerts its antiproliferative action in cultured AGS human gastric adenocarcinoma cells. We found that fucoidan effectively inhibits the growth of AGS cells by inducing autophagy, as well as apoptosis. Apoptosis by fucoidan treatment was associated with the downregulation of antiapoptotic Bcl-2 and Bcl-xL expression, loss of mitochondrial membrane potential, activation of caspases, and concomitant degradation of poly-(ADP-ribose) polymerase protein. In addition, the morphological study indicated a characteristic finding of autophagy, such as the formation of autophagosomes in fucoidan-treated AGS cells. Furthermore, markers of autophagy, namely, the conversion of microtubule-associated protein light chain 3 (LC3)-I to LC3-II and increased beclin-1 accumulation, were observed. Overall, the present data suggest that fucoidan induces apoptotic and autophagic cell death, and both apoptotic and autophagic mechanisms contribute to the fucoidan-induced AGS cell death.     

Caspase activity and expression of cell death genes during development of human preimplantation embryos

It has been observed that apoptosis occurs in human blastocysts. In other types of cell, the characteristic morphological changes seen in apoptotic cells are executed by caspases, which are regulated by the BCL-2 family of proteins. This study investigated whether these components of the apoptotic cascade are present throughout human preimplantation development. Developing and arrested two pronucleate embryos at all stages were incubated with a fluorescently tagged caspase inhibitor that binds only to active caspases, fixed, counterstained with 4,6-diamidino-2-phenylindole (DAPI) to assess nuclear morphology and examined using confocal microscopy. Active caspases were detected only after compaction, at the morula and blastocyst stages, and were frequently associated with apoptotic nuclei. Occasional labelling was seen in arrested embryos. Expression of proapoptotic BAX and BAD and anti-apoptotic BCL-2 was examined in single embryos using RT-PCR and immunohistochemistry. BAX and BCL-2 mRNAs were expressed throughout development, whereas BAD mRNA was expressed mainly after compaction. Simultaneous expression of BAX and BCL-2 proteins within individual embryos was confirmed using immunohistochemistry. The onset of caspase activity and BAD expression after compaction correlates with the previously reported appearance of apoptotic nuclei. As in other types of cell, human embryos express common molecular components of the apoptotic cascade, although apoptosis appears to be suppressed before compaction and differentiation.     

Cytotoxic activity to acute myeloid leukemia cells by Antp-TPR hybrid peptide targeting Hsp90

We previously reported that Antp-TPR hybrid peptide inhibited the interaction of Hsp90 with TPR2A and had selective cytotoxic activity discriminating between normal and cancer cells to induce cancer cell death. In this study, we investigated the cytotoxic activity of Antp-TPR peptide toward acute myeloid leukemia (AML) cells. It was demonstrated that Antp-TPR peptide induced AML cell death in cell lines such as U937, K562, THP-1, and HL-60 via activation of caspases 3 and 7, and disruption of mitochondrial membrane potential. Conversely, Antp-TPR peptide did not reduce the viability of normal cells including peripheral blood mononuclear cells (PBMCs), although both geldanamycin and 17-AAG, small-molecule inhibitors of Hsp90, mediated cytotoxicity to these normal cells at low concentrations. In addition, mutation analysis of TPR peptide demonstrated that the highly conserved amino acids Lys and Arg were critical to the cytotoxic activity. These results indicated that Antp-TPR hybrid peptide would provide potent and selective therapeutic options in the treatment of AML.     

Analysis of programmed cell death in mouse fetal oocytes

We report a short-term culture system that allows to define novel characteristic of programmed cell death (PCD) in fetal oocytes and to underscore new aspects of this process. Mouse fetal oocytes cultured in conditions allowing meiotic prophase I progression underwent apoptotic degeneration waves as revealed by TUNEL staining. TEM observations revealed recurrent atypical apoptotic morphologies characterized by the absence of chromatin margination and nuclear fragmentation; oocytes with autophagic and necrotic features were also observed. Further characterization of oocyte death evidenced DNA ladder, Annexin V binding, PARP cleavage, and usually caspase activation (namely caspase-2). In the aim to modulate the oocyte death process, we found that the addition to the culture medium of the pan-caspase inhibitors Z-VAD or caspase-2-specific inhibitor Z-VDVAD resulted in a partial and transient prevention of this process. Oocyte death was significantly reduced by the antioxidant agent NAC and partly prevented by KL and IGF-I growth factors. Finally, oocyte apoptosis was reduced by calpain inhibitor I and increased by rapamycin after prolonged culture. These results support the notion that fetal oocytes undergo degeneration mostly by apoptosis. This process is, however, often morphologically atypical and encompasses other forms of cell death including caspase-independent apoptosis and autophagia. The observation that oocyte death occurs mainly at certain stages of meiosis and can only be attenuated by typical anti-apoptotic treatments favors the notion that it is controlled at least in part by stage-specific oocyte-autonomous meiotic checkpoints and when activated is little amenable to inhibition being the oocyte able to switch back and forth among different death pathways.     

Revisiting the conversion of muscle into meat and the underlying mechanisms

The conversion of muscle into meat is a complex process in which all mechanisms responsible for the development of meat qualities are very likely interdependent. Colour and flavour are thus both dependent on oxidative mechanisms. Oxidation and proteolysis are probably two processes involved in the development of meat tenderness. This paper reviewed the consequences of programmed cell death or apoptosis on muscle cells structure and biochemistry and on meat qualities as well. We therefore look at different new hypothesis susceptible to highlight the meat science field and provide new supports for a more dynamic meat research. One of them which would have appeared evident for our purpose since a decade, deals with the fact that, after animal bleeding, muscle cells have no other alternative to only enter the programmed cell death procedure or apoptosis. If we introduce an early phase corresponding to apoptosis, taking place before the rigor onset and overlapping it, we will see that the known consequences of that process bring forward possible answers to still unexplained observations. After an overview of the actual state-of-the-art in meat science, we will introduce the programmed cell death and its underlying mechanisms. We then described the strong analogies between the known consequences of apoptosis and the postmortem changes affecting a set of different muscle characteristics.     

Mitochondria-targeted disruptors and inhibitors of cytochrome c/cardiolipin peroxidase complexes: a new strategy in anti-apoptotic drug discovery

The critical role of mitochondria in programmed cell death leads to the design of mitochondriotropic agents as a strategy in regulating apoptosis. For anticancer therapy, stimulation of proapoptotic mitochondrial events in tumor cells and their suppression in surrounding normal cells represents a promising paradigm for new therapies. Different approaches targeting regulation of components of mitochondrial antioxidant system such as Mn-SOD demonstrated significant antitumor efficiency, particularly in combination therapy. This review is focused on a newly discovered early stage of mitochondria-dependent apoptosis - oxidative lipid signaling involving a mitochondria-specific phospholipid cardiolipin (CL). Cytochrome c (cyt c) acts as a CL-specific peroxidase very early in apoptosis. At this stage, the hostile events are still secluded within the mitochondria and do not reach the cytosolic targets. CL oxidation process is required for the release of pro-apoptotic factors into the cytosol. Manipulation of cyt c interactions with CL, inhibition of peroxidase activity, and prevention of CL peroxidation are prime targets for the discovery of anti-apoptotic drugs acting before the "point-of-no-return" in the fulfillment of the cell death program. Therefore, mitochondria-targeted disruptors and inhibitors of cyt c/CL peroxidase complexes and suppression of CL peroxidation represent new strategies in anti-apoptotic drug discovery.     

A selection system for human apoptosis inhibitors using yeast.

Apoptosis is a regulated series of events which leads to the death and elimination of mammalian cells during development or in disease control. It is regulated in part by members of the Bcl-2 family of genes. Some of these stimulate cell death, while others prevent it. Expression of one of these death inducers, Bax-alpha (Bax), in the yeast Saccharomyces cerevisiae induces growth arrest and subsequently can cause cell death. Proteins of the Bcl-2 family that are known to inhibit apoptosis in mammalian cells overcome Bax-induced growth arrest in yeast. We describe here a system for isolation of human genes that are able to overcome Bax sensitivity in yeast. Two novel proteins, identified with this system, have been named 'Bax antagonists selected in saccharomyces' (BASS). These proteins not only overcome toxicity of Bax in yeast but also protect mammalian cells from apoptosis that is induced by staurosporine or Bax overexpression. We find that BASS2 is the more effective of the two genes.