Mitochondrial programmed cell death pathways in yeast

Whether or not yeast cell death is altruistic, apoptotic, or otherwise analogous to programmed cell death in mammals is controversial. However, growing attention to cell death mechanisms in yeast has produced several new papers that make a case for ancient origins of programmed death involving mitochondrial pathways conserved between yeast and mammals.     

Airborne fine particulate matter induces multiple cell death pathways in human lung epithelial cells

Our group was the first one reporting that autophagy could be triggered by airborne fine particulate matter (PM) with a mean diameter of less than 2.5 μm (PM_2.5) in human lung epithelial A549 cells, which could potentially lead to cell death. In the present study, we further explored the potential interactions between autophagy and apoptosis because it was well documented that PM_2.5 could induce apoptosis in A549 cells. Much to our surprise, we found that PM_2.5-exposure caused oxidative stress, resulting in activation of multiple cell death pathways in A549 cells, that is, the tumor necrosis factor-alpha (TNF-α)-induced pathway as evidenced by TNF-α secretion and activation of caspase-8 and -3, the intrinsic apoptosis pathway as evidenced by increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic protein Bcl-2, disruption of mitochondrial membrane potential, and activation of caspase-9 and -3, and autophagy as evidenced by an increased number of double-membrane vesicles, accompanied by increases of conversion and punctuation of microtubule-associated proteins light chain 3 (LC3) and expression of Beclin 1. It appears that reactive oxygen species (ROS) function as signaling molecules for all the three pathways because pretreatment with N-acetylcysteine, a scavenger of ROS, almost completely abolished TNF-α secretion and significantly reduced the number of apoptotic and autophagic cells. In another aspect, inhibiting autophagy with 3-methyladenine, a specific autophagy inhibitor, enhanced PM_2.5-induced apoptosis and cytotoxicity. Intriguingly, neutralization of TNF-α with an anti-TNF-α special antibody not only abolished activation of caspase-8, but also drastically reduced LC3-II conversion. Thus, the present study has provided novel insights into the mechanism of cytotoxicity and even pathogenesis of diseases associated with PM_2.5 exposure.     

Induction of programmed cell death in human retinoblastoma Y79 cells by C2-ceramide

C₂-ceramide, a cell-permeable analogue of ceramide, induced significant, dose- and time-dependent death in human retinoblastoma Y79 cells. Dying cells strongly displayed the morphology of apoptosis as characterized by microscopic evidence of cell shrinkage, membrane blebbing, nuclear and chromatin condensation and degeneration of the nucleus into membrane-bound apoptotic bodies. Upon induction of apoptosis Y79 cells evidence early phosphatidylserine externalization, as shown by annexin V-FITC. Apoptosis was also assessed by monitoring changes in cell granularity by staining with the combined fluorescent dyes acridine orange and ethidium bromide. C₂-ceramide induced these morphological changes without a concomitant production of oligonucleosomal fragments responsible for the DNA ladder and without changes in p53 protein level. Apoptosis was accompanied by accumulation of a modified Bcl-2 protein with a slower-mobility form, and by proteolytic cleavage of PARP. The effect seemed to be specific for C₂-ceramide, as C₂-dihydroceramide, or other amphiphilic lipid analogues, or products of ceramide hydrolysis were ineffective. The effect also depended on mRNA and protein synthesis as it was markedly inhibited by actinomycin D and cycloheximide. Sphingomyelinase and interleukin-l, which are known to activate the sphingomyelin turnover leading to ceramide generation, also induced apoptosis mimicking the effects of ceramide. These findings propose ceramide as an activator of the suicidal program in Y79 cells.     

Mitochondrial apoptotic pathways induced by Drosophila programmed cell death regulators

Multicellular organisms eliminate unwanted or damaged cells by cell death, a process essential to the maintenance of tissue homeostasis. Cell death is a tightly regulated event, whose alteration by excess or defect is involved in the pathogenesis of many diseases such as cancer, autoimmune syndromes, and neurodegenerative processes. Studies in model organisms, especially in the nematode Caenorhabditis elegans, have been crucial in identifying the key molecules implicated in the regulation and execution of programmed cell death. In contrast, the study of cell death in Drosophila melanogaster, often an excellent model organism, has identified regulators and mechanisms not obviously conserved in other metazoans. Recent molecular and cellular analyses suggest, however, that the mechanisms of action of the main programmed cell death regulators in Drosophila include a canonical mitochondrial pathway.     

Progestin inhibition of cell death in human breast cancer cell lines

Previously, we have shown that progestins both stimulate proliferation of the progesterone receptor (PR)-rich human breast cancer cell line T47D and protect from cell death, in charcoal-stripped serum-containing medium. To lessen the variability inherent in different preparations of serum, we decided to further characterize progestin inhibition of cell death using serum starvation to kill the cells, and find that progestins protect from serum-starvation-induced apoptosis in T47D cells. This effect exhibits specificity for progestins and is inhibited by the antiprogestin RU486. While progestin inhibits cell death in a dose–responsive manner at physiological concentrations, estradiol-17β surprisingly does not inhibit cell death at any concentration from 0.001 nM to 1 μM. Progestin inhibition of cell death also occurs in at least two other human breast cancer cell lines, one with an intermediate level of PR, MCF-7 cells, and, surprisingly, one with no detectable level of PR, MDA-MB-231 cells. Further, we have found progestin inhibition of cell death caused by the breast cancer chemotherapeutic agents doxorubicin and 5-fluorouracil. These data are consistent with the building body of evidence that progestins are not the benign hormones for breast cancer they have been so long thought to be, but may be harmful both for undiagnosed cases and those undergoing treatment.     

Induction of programmed cell death in Arabidopsis and rice by single-wall carbon nanotubes

? Premise of the study: Single-walled carbon nanotubes (SWCNTs) have many unique structural and mechanical properties. Their potential applications, especially in biomedical engineering and medical chemistry, have been increasing in recent years, but the toxicological impact of nanoparticles has rarely been studied in plants. ? Methods: We exposed Arabidopsis and rice leaf protoplasts to SWCNTs and examined cell viability, DNA damage, reactive oxygen species generation, and related gene expression. We also tested the effects of nanoparticles on Arabidopsis leaves after injecting a SWCNT solution. EM-TUNEL (electron-microscopic terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) and a cerium chloride staining method were used. ? Key results: SWCNTs caused adverse cellular responses including cell aggregation, chromatin condensation along with a TUNEL-positive reaction, plasma membrane deposition, and H(2)O(2) accumulation. The effect of SWCNTs on the survival of cells was dose dependent, with 25 μg/mL inducing 25% cell death in 6 h. In contrast, activated carbon, which is not a nano-sized carbon particle, did not induce cell death even 24 h after treatments. The data indicated that the nano-size of the particle is a critical factor for toxicity. Moreover, endocytosis-like structures with cerium chloride deposits formed after SWCNT treatment, suggesting a possible pathway for nanoparticles to traverse the cell membrane. ? Conclusions: Consequently, SWCNTs have an adverse effect on protoplasts and leaves through oxidative stress, leading to a certain amount of programmed cell death. Although nanomaterials have great advantages in many respects, the benefits and side effects still need to be assessed carefully.     

VK3诱导悬浮培养的胡萝卜细胞凋亡

Menadione (VK3), a quinone that undergoes redox cycles leading to the formation of superoxide radicals, was found to induce cell death in suspension culture of carrot cells. The effect of menadione was in a dose-dependent manner. 100-800 mumol/L menadione caused 10-33 percent cell death. When concentration of menadione reached 1 mmol/L, 100 percent of cell death was observed. DNA cleavage, a hallmark of apoptosis was further studied. DNA ladders were observed in cells treated with 600 and 800 mumol/L menadione but not with lower concentration treatments where only very low percentage of cell death was found. There was no DNA ladders in the cells treated with 1 mmol/L menadion indicating that necrosis may occur. In situ detection of nuclear DNA fragmentation by TUNEL reaction revealed fragmented nuclear DNA in cells treated with 100-800 mumol/L menadion but not in cells treated with 1 mmol/L menadione.     

Induction of a ricinosomal-protease and programmed cell death in tomato endosperm by gibberellic acid

Several examples of programmed cell death (PCD) in plants utilize ricinosomes, organelles that appear prior to cell death and store inactive KDEL-tailed cysteine proteinases. Upon cell death, the contents of ricinosomes are released into the cell corpse where the proteinases are activated and proceed to degrade any remaining protein for use in adjacent cells or, in the case of nutritive seed tissues, by the growing seedling. Ricinosomes containing pro-SlCysEP have been observed in anther tissues prior to PCD and ricinosome-like structures have been observed in imbibed seeds within endosperm cells of tomato. The present study confirms that the structures in tomato endosperm cells contain pro-SlCysEP making them bona fide ricinosomes. The relative abundance of pro- versus mature SlCysEP is suggested to be a useful indicator of the degree of PCD that has occurred in tomato endosperm, and is supported by biochemical and structural data. This diagnostic tool is used to demonstrate that a sub-region of the micropylar endosperm surrounding the emerged radical is relatively long-lived and may serve to prevent loss of mobilized reserves from the lateral endosperm. We also demonstrate that GA-induced reserve mobilization, SlCysEP accumulation and processing, and PCD in tomato endosperm are antagonized by ABA.     

Achievements and perspectives in yeast acetic acid-induced programmed cell death pathways

The use of non-mammalian model organisms, including yeast Saccharomyces cerevisiae, can provide new insights into eukaryotic PCD (programmed cell death) pathways. In the present paper, we report recent achievements in the elucidation of the events leading to PCD that occur as a response to yeast treatment with AA (acetic acid). In particular, ROS (reactive oxygen species) generation, cyt c (cytochrome c) release and mitochondrial function and proteolytic activity will be dealt with as they vary along the AA-PCD time course by using both wild-type and mutant yeast cells. Two AA-PCD pathways are described sharing common features, but distinct from one another with respect to the role of ROS and mitochondria, the former in which YCA1 acts upstream of cyt c release and caspase-like activation in a ROS-dependent manner and the latter in which cyt c release does not occur, but caspase-like activity increases, in a ROS-independent manner.     

Estrogen receptor signaling pathways in human non-small cell lung cancer

Lung cancer is the most common cause of cancer mortality in male and female patients in the US. The etiology of non-small cell lung cancer (NSCLC) is not fully defined, but new data suggest that estrogens and growth factors promote tumor progression. In this work, we confirm that estrogen receptors (ER), both ERalpha and ERbeta, occur in significant proportions of archival NSCLC specimens from the clinic, with receptor expression in tumor cell nuclei and in extranuclear sites. Further, ERalpha in tumor nuclei was present in activated forms as assessed by detection of ER phosphorylation at serines-118 and -167, residues commonly modulated by growth factor receptor as well as steroid signaling. In experiments using small interfering RNA (siRNA) constructs, we find that suppressing expression of either ERalpha or ERbeta elicits a significant reduction in NSCLC cell proliferation in vitro. Estrogen signaling in NSCLC cells may also include steroid receptor coactivators (SRC), as SRC-3 and MNAR/PELP1 are both expressed in several lung cell lines, and both EGF and estradiol elicit serine phosphorylation of SRC-3 in vitro. EGFR and ER also cooperate in promoting early activation of p42/p44 MAP kinase in NSCLC cells. To assess new strategies to block NSCLC growth, we used Faslodex alone and with erlotinib, an EGFR kinase inhibitor. The drug tandem elicited enhanced blockade of the growth of NSCLC xenografts in vivo, and antitumor activity exceeded that of either agent given alone. The potential for use of antiestrogens alone and with growth factor receptor antagonists is now being pursued further in clinical trials.     

Suppression of cell proliferation and programmed cell death by dexamethasone during postnatal lung development

Prematurely born babies are often treated with glucocorticoids. We studied the consequences of an early postnatal and short dexamethasone treatment (0.1-0.01 microg/g, days 1-4) on lung development in rats, focusing on its influence on peaks of cell proliferation around day 4 and of programmed cell death at days 19-21. By morphological criteria, we observed a dexamethasone-induced premature maturation of the septa (day 4), followed by a transient septal immatureness and delayed alveolarization leading to complete rescue of the structural changes. The numbers of proliferating (anti-Ki67) and dying cells (TdT-mediated dUTP nick end labeling) were determined and compared with controls. In dexamethasone-treated animals, both the peak of cell proliferation and the peak of programmed cell death were reduced to baseline, whereas the expression of tissue transglutaminase (transglutaminase-C), another marker for postnatal lung maturation, was not significantly altered. We hypothesize that a short neonatal course of dexamethasone leads to severe but transient structural changes of the lung parenchyma and influences the balance between cell proliferation and cell death even in later stages of lung maturation.     

Transgenic human programmed cell death 5 expression in mice suppresses skin cancer development by enhancing apoptosis

AimsWe sought to probe the role of human programmed cell death 5 (PDCD5) in vivo and to understand its mechanisms.Main methodsA transgenic mouse model of human PDCD5 was generated by pronuclear microinjection. Apoptosis in tissues of three independent transgenic mouse lines was quantified by terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling (TUNEL) and compared to wild type littermates. Their lifespan was compared. 8-Week PDCD5 mice and wild type mice (at a group of 5) were treated with carcinogen 3-methylcholanthrene (3-MC) at 5 μg per week to induce skin cancer. Cancer development was measured by examining hematoxylin and eosin (H&E) stained skin sections after 5 weeks and 10 weeks treatment. Protein expression was determined by Western blot and apoptosis of skin cells was quantified by TUNEL.Key findingsStarting from 5 months after birth, significant autonomous apoptosis was observed in multiple tissues of transgenic mice including skin, liver, spleen, adrenal gland and thyroid gland comparing to their wild type littermates. The average lifespan of PDCD5 mice was reduced to 9.75 months (normally 24–30 months). Moreover, carcinogen 3-MC induced skin cancer development was attenuated in the lesion of PDCD5 transgenic mice by enhancing apoptosis. Pro-apoptotic protein Bax expression was up-regulated in the 3-MC treated skin of transgenic mice.SignificanceThese results suggest PDCD5 plays an antitumor role by enhancing apoptosis in animal physiological settings. Therefore, PDCD5 is a potential target for cancer therapy.     

The expression of "tissue" transglutaminase in two human cancer cell lines is related with the programmed cell death (apoptosis).

The expression of "tissue" transglutaminase (tTG) in two human tumor cell lines (the cervix adenocarcinoma line HeLa-TV and the neuroblastoma cells SK-N-BE-2) was found to be in correlation with the rate of physiological cell death (apoptosis) in culture. We investigated the effect of retinoic acid (RA) and alpha-difluoromethylornithine (DFMO) in order to elucidate the relationship between tTG expression and apoptosis. RA led to a 6-fold increase of tTG activity in HeLa-TV cells and to a 12-fold increase in SK-N-BE(2) cells, which was paralleled in both cell lines by a proportional increase in the number of apoptotic bodies recovered from the cultures. On the contrary, DFMO determined a dramatic reduction of tTG expression and of the apoptotic index. Immunohistochemical analysis using an anti-tTG antibody showed that the enzyme was accumulated in both cell lines within typical apoptotic bodies. Immunocytochemistry and cell cloning of SK-N-BE(2) line demonstrated that tTG was absent in cells showing neurite outgrowth, indicating that the enzyme expression is not associated with neural differentiation, even though both phenomena are elicited by retinoic acid. On the whole, these data indicate that also in tumors tTG activation takes place in cells undergoing apoptosis. The enzyme is activated in apoptotic cells to form cross-linked protein envelopes which are insoluble in detergents and chaotropic agents. The number of insoluble protein envelopes as well as the N,N-bis(gamma-glutamyl)polyamine cross-links is related with both tTG expression and apoptotic index, strongly suggesting the participation of the enzyme in the apoptotic program.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular effectors of multiple cell death pathways initiated by photodynamic therapy

Photodynamic therapy (PDT) is a recently developed anticancer modality utilizing the generation of singlet oxygen and other reactive oxygen species, through visible light irradiation of a photosensitive dye accumulated in the cancerous tissue. Multiple signaling cascades are concomitantly activated in cancer cells exposed to the photodynamic stress and depending on the subcellular localization of the damaging ROS, these signals are transduced into adaptive or cell death responses. Recent evidence indicates that PDT can kill cancer cells directly by the efficient induction of apoptotic as well as non-apoptotic cell death pathways. The identification of the molecular effectors regulating the cross-talk between apoptosis and other major cell death subroutines (e.g. necrosis, autophagic cell death) is an area of intense research in cancer therapy. Signaling molecules modulating the induction of different cell death pathways can become useful targets to induce or increase photokilling in cancer cells harboring defects in apoptotic pathways, which is a crucial step in carcinogenesis and therapy resistance. This review highlights recent developments aimed at deciphering the molecular interplay between cell death pathways as well as their possible therapeutic exploitation in photosensitized cells.     

Plant lectins: targeting programmed cell death pathways as antitumor agents

Lectins, a group of highly diverse, carbohydrate-binding proteins of non-immune origin that are ubiquitously distributed in plants, animals and fungi, are well-characterized to have numerous links a wide range of pathological processes, most notably cancer. In this review, we present a brief outline of the representative plant lectins including Ricin-B family, proteins with legume lectin domains and GNA family that can induce cancer cell death via targeting programmed cell death pathways. Amongst these above-mentioned lectins, we demonstrate that mistletoe lectins (MLs), Ricin, Concanavalin A (ConA) and Polygonatum cyrtonema lectin (PCL) can lead to cancer cell programmed death via targeting apoptotic pathways. In addition, we show that ConA and PCL can also result in cancer cell programmed death by targeting autophagic pathways. Moreover, we summarize the possible anti-cancer therapeutic implications of plant lectins such as ConA, Phaseolus vulgaris lectin (PHA) and MLs that have been utilized at different stages of preclinical and clinical trials. Together, these findings can provide a comprehensive perspective for further elucidating the roles of plant lectins that may target programmed cell death pathways in cancer pathogenesis and therapeutics. And, this research may, in turn, ultimately help cancer biologists and clinicians to exploit lectins as potential novel antitumor drugs in the future.     

Localization of cathepsin B in two human lung cancer cell lines

We demonstrated the cysteine proteinase cathepsin B in two human lung tumor cell lines by cytochemical and immunocytochemical methods. The cell lines were derived from a squamous cell carcinoma of the lung (HS-24) and a metastasis to the adrenal gland from an adenocarcinoma of the lung (SB-3). For comparison and control, normal human lung fibroblasts cells (Wi-38) were also investigated. Intracellular cathepsin B activity was detected in all three cell lines. SB-3 and the normal fibroblast cells showed almost equal cathepsin B activity, which was considerably stronger than that in the HS-24 cells. Specific inhibitors for cathepsin B (E64, leupeptin, antipain) suppressed its activity completely. Stefin A, the physiological cathepsin B inhibitor, was less effective; this might depend on its limited penetrability into living cells. Localization of the cathepsin B was performed by conventional immunofluorescence microscopy and laser scanning microscopy. With specific anti-cathepsin B antibodies, the enzyme was localized in HS-24, SB-3, and Wi-38 fibroblast cells within perinuclear granules representing the lysosomal compartment. In the SB-3 cells, we additionally localized a minor fraction of the enzyme bound to the plasma membrane in a speckled distribution, accessible to the antibodies from the outside. This direct demonstration of cathepsin B distribution supports biochemical data about the dual localization of the enzyme in tumor cells. It also supports the possibility of a direct involvement of cathepsin B in the degradation of the extracellular matrix, and thus a contribution of the enzyme in invasion and metastasis.