Mitochondrial Condensation Research Articles

An update on the role of ferroptosis in the pathogenesis of osteoporosis.

Ferroptosis is a novel form of programmed cell death, distinguished from apoptosis, autophagy, and programmed necrosis and has received much attention since it was defined in 2012. Ferroptotic cells physiologically exhibit iron metabolism dysregulation, oxidative stress, and lipid peroxidation. Morphologically, they show plasma membrane disruption, cytoplasmic swelling, and mitochondrial condensation. Osteoporosis is taken more and more seriously as the proportion of the aging population continues to increase globally. Interestingly, ferroptosis has been demonstrated to be involved in the development and progression of osteoporosis in many extant studies. The review summarizes iron metabolism, lipid peroxidation, and the different regulatory signals in ferroptosis. Changes in signaling mechanisms within osteoblasts, osteoclasts, and osteocytes after ferroptosis occur are explained here. Studies showed ferroptosis play an important role in different osteoporosis models (diabetes osteoporosis, postmenopausal osteoporosis, glucocorticoid-induced osteoporosis). Inhibitors and EC (Exos) targeting ferroptosis could ameliorate bone loss in osteoporotic mice by protecting cells against lipid peroxidation. Shortly, we hope that more effective and appropriate clinical therapy means will be utilized in the treatment of osteoporosis.

Exposure to bisphenol A induces neurotoxicity associated with synaptic and cytoskeletal dysfunction in neuro-2a cells.

Bisphenol A (BPA) has been reported to injure the developing and adult brain. However, the underlying mechanism still remains elusive. This study used neuro-2a cells as a cellular model to investigate the neurotoxic effects of BPA. Microtubule-associated protein 2 (MAP2) and tau protein maintain microtubule normal function and promote the normal development of the nervous system. Synaptophysin (SYP) and drebrin (Dbn) proteins are involved in regulating synaptic plasticity. Cells were exposed to the minimum essential medium (MEM), 0.01% (v/v) DMSO, and 150μM BPA for 12, 24, or 36h. Morphological analysis revealed that the cells in the BPA-treated groups shrank and collapsed compared with those in the control groups. CCK-8 and lactate dehydrogenase assay (LDH) assays showed that the mortality of neuro-2a cells increased as the BPA treatment time was prolonged. Ultrastructural analysis further revealed that cells demonstrated nucleolar swelling, dissolution of nuclear and mitochondrial membranes, and partial mitochondrial condensation following exposure to BPA. BPA also decreased the relative protein expression levels of MAP2, tau, and Dbn. Interestingly, the relative protein expression levels of SYP increased. These results indicated that BPA inhibited the proliferation and disrupted cytoskeleton and synaptic integrity of neuro-2a cells.

Wogonoside attenuates liver fibrosis by triggering hepatic stellate cell ferroptosis through SOCS1/P53/SLC7A11 pathway.

Wogonoside (WG) is a flavonoid chemical component extracted from Scutellaria baicalensis, which exerts therapeutic effects on liver diseases. Ferroptosis, a novel form of programmed cell death, regulates diverse physiological/pathological processes. In this study, we attempted to investigate a novel mechanism by which WG mitigates liver fibrosis by inducing ferroptosis in hepatic stellate cells (HSCs). A CCl4 -induced mouse liver fibrosis model and a rat HSC line were employed for in vivo and in vitro experiments, both treated with WG. Firstly, the levels of the fibrotic markers α-smooth muscle actin (α-SMA) and α1(I)collagen (COL1α1) were effectively decreased by WG in CCl4 -induced mice and HSC-T6 cells. Additionally, mitochondrial condensation and mitochondrial ridge breakage were observed in WG-treated HSC-T6 cells. Furthermore, ferroptotic events including depletion of SLC7A11, GPX4 and GSH, and accumulation of iron, ROS and MDA were discovered in WG-treated HSC-T6 cells. Intriguingly, these ferroptotic events did not appear in hepatocytes or macrophages. WG-elicited HSC ferroptosis and ECM reduction were dramatically abrogated by ferrostatin-1 (Fer-1), a ferroptosis inhibitor. Importantly, our results confirm that SOCS1/P53/SLC7A11 is a signaling pathway which promotes WG attenuation of liver fibrosis. On the contrary, WG mitigated liver fibrosis and inducted HSC-T6 cell ferroptosis were hindered by SOCS1 siRNA and pifithrin-α (PFT-α). These findings demonstrate that SOCS1/P53/SLC7A11-mediated HSC ferroptosis is associated with WG alleviating liver fibrosis, which provides a new clue for the treatment of liver fibrosis.

Ultrastructural and hormonal changes related to harmaline-induced treatment in Arabidopsis thaliana (L.) Heynh. root meristem

Harmaline is an indole alkaloid with demonstrated phytotoxicity and recognized pharmacological applications. However, no information is available concerning its mode of action on plant metabolism. Therefore, the present work evaluated bioherbicide mode of action of harmaline on plant metabolism of Arabidopsis thaliana (L.) Heynh. Harmaline induced a strong inhibitory activity on root growth of treated seedlings, reaching IC50 and IC80 values of 14 and 29 μM, respectively. Treated roots were shorter and thicker than control and were characterized by a shorter root meristem size and an increase of root hairs production. Harmaline induced ultrastructural changes such as increment of cell wall thickness, higher density and condensation of mitochondria and vacuolization, appearance of cell wall deposits, increment of Golgi secretory activity and higher percentage of aberrant nuclei. The ethylene inhibitor AgNO3 reversed high root hair appearance and increment of root thickness, and pTCSn::GFP transgenic line showed fluorescence cytokinin signal in stele zone after harmaline treatment that was absent in control, whereas the auxin signal in the transgenic line DR5 was significantly reduced by the treatment. All these results suggest that the mode of action of harmaline could be involving auxin, ethylene and cytokinin synergic/antagonistic action.

PO-242 Myoferlin controls mitochondrial structure and metabolism in pancreatic ductal adenocarcinoma, and affects tumor aggressiveness

IntroductionPancreatic ductal adenocarcinoma (pdac) is the most common type of pancreatic cancer, and the third leading cause of cancer related death. therapeutic options remain very limited and are still based on classical chemotherapies. cell fraction can survive to the chemotherapy and is responsible for tumor relapse. it appears that these cells rely on oxydative phosphorylation (oxphos) for survival.Myoferlin, a membrane protein involved in cell fusion was recently shown by our laboratory to be overexpressed in pancreatic cancer.Material and methodsWe used pancreatic cancer cell lines depleted in myoferlin to assess mitochondrial function with an extracellular flux analyser. pancreas cancer samples from the institutional biobank with matched pet scan data were used to correlate myoferlin abundance and glycolysis.results and discussionsin the present study, we discovered that myoferlin was more expressed in cell lines undergoing (oxphos) than in glycolytic cell lines. in the former cell lines, we showed that myoferlin silencing reduced oxphos activity and forced cells to switch to glycolysis. the decrease in oxphos activity is associated with mitochondrial condensation and network disorganization. an increase of dynamin-related protein (drp)-1 phosphorylation in myoferlin-depleted cells led us to suggest mitochondrial fission, reducing cell proliferation, atp production and inducing autophagy and ros accumulation. electron microscopy observation revealed mitophagy, suggesting mitochondrial alterations.To confirm the clinical importance of myoferlin in pdac, we showed that low myoferlin expression was significantly correlated to high overall survival. myoferlin staining of pdac sections was negatively correlated with several 18fdg pet indices indicating that glycolytic lesions had less myoferlin. these observations are fully in accordance with our in vitro data.conclusionas the mitochondrial function was associated with cell chemoresistance, the metabolic switch induced by myoferlin silencing could open up a new perspective in the development of therapeutic strategies. among them, targeting functional domains (c2, dysf, …) of myoferlin should be a priority.

3',5-dihydroxy-3,4',7-trimethoxyflavone-induces ER-stress-associated HCT-116 programmed cell death via redox signaling.

Quercetin, a well cognized bioactive flavone possessing great medicinal value, has limited usage. The rapid gastrointestinal digestion of quercetin is also a major obstacle for its clinical implementation due to low bioavailability and poor aqueous solubility. 3',5-dihydroxy-3,4',7-trimethoxyflavone (DTMF), a novel semi-synthetic derivative of quercetin, is known to modulate several biological activities. Therefore, in the present study we examined the cytotoxic mechanism of DTMF in concentration-dependent manner (25, 50, and 100μM; 24h) against HCT-116 human colon carcinoma cells. The cytotoxic potential of DTMF was characterized based on deformed cell morphology, increased ROS accumulation, loss of mitochondrial membrane potential (ΔѰm), increased mitochondrial mass, chromatin condensation, and typical DNA-fragmentation in HCT-116 cells. The results showed that DTMF-induced enhanced ROS production at higher concentration (100μM) as evidenced by upregulated expression of ER stress and apoptotic proteins with concomitant increase in PERK, CHOP, and JNK levels, when compared to N-acetyl cysteine (NAC, ROS inhibitor) treated HCT-116 cells, which depicts that DTMF might act as a crucial mediator of apoptosis signaling. Collectively, our results suggest that DTMF stimulates ROS-mediated oxidative stress, which in turn induces PERK-CHOP and JNK pathway of apoptosis to promote HCT-116 cell death.

RNA Distribution During Flock House Virus Infection

The mechanisms involved in the specific packaging of multipartite genomes by (+) RNA viruses remain poorly understood. In order to gain insight into the location of genomic RNA encapsidation, we employed fluorescence in situ hybridization (FISH) to visualize the sub‐cellular distribution of (+) vRNA in cells infected with the model nodavirus, Flock House virus (FHV). During FHV assembly, genomic RNAs 1 and 2 are encapsidated into a T = 3 icosahedral capsid. Previously it has been shown that while vRNA replication and CP synthesis are initially targeted to different cellular locations (mitochondria and ER, respectively), these regions converge into perinuclear clusters late in infection. Our analysis of the cellular distribution of FHV RNA during infection, however, suggests that the vast majority of (+) RNA is trafficked towards the periphery of the cell relatively early during the infectious cycle (within 3 hours post infection) and does not relocate to the perinuclear region during mitochondrial condensation (15 hours post infection). In addition, the (+) RNA fits well within the region occupied by CP at the time points we tested. Our findings show that RNA transport precedes genome packaging and virus assembly. This suggests that genomic RNA is likely released from vRNA replication sites fairly soon after synthesis and trafficked to peripheral sites prior to encapsidation. Our analysis in mammalian cells also revealed that in the absence of B2, a small virus‐encoded protein known to bind dsRNAs, RNA2 is stalled in cytoplasmic aggregates while RNA1 trafficking is largely unaffected.

Ultrastructure and metabolic activity of pea mitochondria under clinorotation

Experimental data on the mitochondrial ultrastructure and tissue respiration in root apex as well as metabolic activity of the organelles isolated from pea seedling roots after 5-day of clinorotation are presented. It was shown that mitochondrial condensation in the distal elongation zone correlated with an increased rate of oxygen uptake on 7%. We also observed increase in rate of malate oxidation and respiratory control ratio increased simultaneously with a decreased in efficiency of oxidative phosphorylation. Such character of mitochondrial rearrangements in simulated microgravity is assumed to be a consequence of adaptation to these conditions.

The natural compound trans‐chalcone induces programmed cell death in Arabidopsis thaliana roots

Chalcone (1,3-diphenyl-2-propen-1-one) is an aromatic ketone precursor of important molecules in plants such as flavonoids or anthocyanins. Its phytotoxicity has been demonstrated on different plant species, but to date little is known about the mechanisms of action of this secondary metabolite at plant cellular level. Detailed analysis by light and transmission electron microscopy (TEM) was conducted to examine the root meristems' ultrastructure of control and chalcone-treated Arabidopsis seedlings. Mitochondrial dysfunction was analysed by measuring mitochondrial membrane potential with JC-1 fluorochrome. Finally, acridine orange/ethidium bromide staining was used for the detection of programmed cell death. Microscopy revealed tissue alterations, inhibition of root hair formation and important changes after 7 and 14 d at the chalcone IC(50) value. Chalcone-treated cells showed signs of programmed cell death such as mitochondrial condensation, disruption of organelles and chromatin fragmentation. Acridine orange/ethidium bromide staining confirmed the programmed cell death, which could be induced by the reduction of mitochondrial transmembrane potential (ΔΨ(m)) that was detected after chalcone treatment. These results confirm the phytotoxic activity of chalcone on Arabidopsis seedlings, the alteration of mitochondrial membrane potential and the induction of programmed cell death.

Xylodiol from Xylopia langsdorfiana induces apoptosis in HL60 cells

An atisane diterpene was isolated from Xylopia langsdorfiana St. Hilaire & Tulasne, Annonaceae, leaves, ent-atisane-7α,16α-diol (xylodiol). Preliminary study showed that xylodiol was cytotoxic and induced differentiation on human leukemia cell lines. However, the molecular mechanisms of xylodiol-mediated cytotoxicity have not been fully defined. Thus, we investigated the anti-tumor effect of xylodiol in human leukemia HL60 cell line. Xylodiol induced apoptosis and necrosis. HL60 cells treated with xylodiol showed biochemical changes characteristic of apoptosis, including caspases-8, -9 and -3 activation and loss of mitochondrial transmembrane potential (∆ Ψm). However, there was a condensation rather than swelling of mitochondria. Moreover, the formation of condensed mitochondria and the loss of ∆ Ψm occurred downstream of caspase activation. Cyclosporine A did not protect HL60 cells from the cytotoxic effects of xylodiol, suggesting that the loss of ∆ Ψm is a late event in xylodiol-induced apoptosis. Oxidative stress was involved in xylodiol-induced apoptosis. Thus, we conclude that activated caspases cleave cellular proteins resulting in mitochondrial damage leading to mitochondrial condensation, loss of ∆ Ψm and ROS release from the mitochondria. ROS can further induce and maintain a collapse of ∆ Ψm leading to cellular damage through oxidation of lipids and proteins resulting in apoptotic cell death.

Biomedical Imaging and Visualization Model of Mitochondrial Dysfunction and Oxidative Stress in Alzheimer Disease

Mitochondrial dysfunction and oxidative stress are among the earliest events linked to Alzheimer Disease and might play a causative role in disease onset and progression. In this paper, a biomedical imaging and visualization model was set to investigate the mitochondrial dysfunction and oxidative stress in Aβ/Cu2+-treated cells, which contribute in a significant manner to bioenergetic failure and mitochondrial dysfunction, by measuring mitochondrial membrane potential, Ca2+, chromatin condensation and ROS. This evaluating method opens a window for analyzing the protective effect of a certain substance in improving mitochondrial function and preventing oxidative stress in AD development.

Abstract 2154: Simultaneous analysis of cell death mechanisms and oxidative stress using live cell fluorescence microscopy

Abstract Reactive oxygen species (ROS) can directly damage DNA, proteins and lipids, while increased oxidative stress has been reported to be associated with apoptotic and autophagic cell death. Importantly, increased oxidative stress and dysregulation of cell death mechanisms have been linked with cancer development. To better understand the connection between increased oxidative stress and cell death pathways, it is important to be able to monitor both of these events simultaneously in the same cell and amongst mixed populations of cells. The goal of this study was to utilize multi-parametric fluorescence microscopy to examine the temporal progression of oxidative stress relative to cell death. By using a fluorogenic caspase substrate in combination with a near infrared fluorescent probe for ROS, we observed that increased oxidative stress was followed by activation of caspase-3/7 after induction by several agonists. We examined the effect of various agonists in both primary cells as well as cancer cell lines. Loss of mitochondrial membrane potential, nuclear condensation, and cell proliferation were also measured to establish a more comprehensive assessment of the cellular response to stress in these models. By simultaneously examining multiple parameters over time, we were able to define the temporal progression of apoptosis relative to the onset of oxidative stress. Furthermore, we were able to characterize the mechanism of cell death by discriminating between cells which were apoptotic (active caspase-3/7), autophagic (LC3B-positive autophagosomes), or both. This multi-parametric approach provided detailed spatial information at the cellular level so that correlations and temporal resolution could be determined between oxidative stress and cell death mechanisms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2154. doi:10.1158/1538-7445.AM2011-2154

Free Rhodium (II) citrate and rhodium (II) citrate magnetic carriers as potential strategies for breast cancer therapy

BackgroundRhodium (II) citrate (Rh2(H2cit)4) has significant antitumor, cytotoxic, and cytostatic activity on Ehrlich ascite tumor. Although toxic to normal cells, its lower toxicity when compared to carboxylate analogues of rhodium (II) indicates Rh2(H2cit)4 as a promising agent for chemotherapy. Nevertheless, few studies have been performed to explore this potential. Superparamagnetic particles of iron oxide (SPIOs) represent an attractive platform as carriers in drug delivery systems (DDS) because they can present greater specificity to tumor cells than normal cells. Thus, the association between Rh2(H2cit)4 and SPIOs can represent a strategy to enhance the former's therapeutic action. In this work, we report the cytotoxicity of free rhodium (II) citrate (Rh2(H2cit)4) and rhodium (II) citrate-loaded maghemite nanoparticles or magnetoliposomes, used as drug delivery systems, on both normal and carcinoma breast cell cultures.ResultsTreatment with free Rh2(H2cit)4 induced cytotoxicity that was dependent on dose, time, and cell line. The IC50 values showed that this effect was more intense on breast normal cells (MCF-10A) than on breast carcinoma cells (MCF-7 and 4T1). However, the treatment with 50 μM Rh2(H2cit)4-loaded maghemite nanoparticles (Magh-Rh2(H2cit)4) and Rh2(H2cit)4-loaded magnetoliposomes (Lip-Magh-Rh2(H2cit)4) induced a higher cytotoxicity on MCF-7 and 4T1 than on MCF-10A (p < 0.05). These treatments enhanced cytotoxicity up to 4.6 times. These cytotoxic effects, induced by free Rh2(H2cit)4, were evidenced by morphological alterations such as nuclear fragmentation, membrane blebbing and phosphatidylserine exposure, reduction of actin filaments, mitochondrial condensation and an increase in number of vacuoles, suggesting that Rh2(H2cit)4 induces cell death by apoptosis.ConclusionsThe treatment with rhodium (II) citrate-loaded maghemite nanoparticles and magnetoliposomes induced more specific cytotoxicity on breast carcinoma cells than on breast normal cells, which is the opposite of the results observed with free Rh2(H2cit)4 treatment. Thus, magnetic nanoparticles represent an attractive platform as carriers in Rh2(H2cit)4 delivery systems, since they can act preferentially in tumor cells. Therefore, these nanopaticulate systems may be explored as a potential tool for chemotherapy drug development.

Rat kidney mitochondrial damage induced by acute cadmium administration

To evaluate effect of acute cadmium administration on mitochondria from rat kidney. 24 male Sprague-Dawley (SD) rats were randomly divided into four groups. Four groups of rats were injected with saline, 0.6, 1.2, and 1.8 mg/kg weight subcutaneously, once daily, for 5 days, respectively. Ultrastructural change of rat kidney mitochondria was observed, and respiration function, membrane potential, mitochondria swelling, and superoxide level were determined. Ultrastructural changes included matrix vacuolation, swelling and condensation of mitochondria. In group of 1.8 mg/kg body weight, the oxygen consumption rate during state 3 respiration [(6.25 +/- 0.61) nmol/L O2 x min(-1) x mg(-1)] and RCR value (2.45 +/- 0.23) were significantly lower than those of control group [(9.66 +/- 1.16) nmol/L O2 x min(-1) x mg(-1)] (P < 0.05), indicating respiration inhibition. The membrane potential and superoxide level of the same group were 85.89% +/- 3.82% and 116.33% +/- 3.06% of control values (P < 0.05), respectively. Acute cadmium administration can cause rat kidney mitochondrial damage in a dose-effect manner, including inhibition of respiration, dissipation of membrane potential, swelling of mitochondria matrix. Such damage might be related to the increase of mitochondrial free radical.

Different pathways lead to mitochondrial fragmentation during apoptotic and excitotoxic cell death in primary neurons

Mitochondrial fragmentation is recognized to be an important event during the onset of apoptosis. In this current study, we have used single cell imaging to investigate the role of the mitochondrial fission protein DRP-1 on mitochondrial morphology and mitochondrial fragmentation in primary hippocampal neurons undergoing necrotic or apoptotic cell death. Treatment of neurons with 500 nM staurosporine (apoptosis) or 30 μM glutamate (l-Glu; excitotoxic necrosis) produced a fragmentation and condensation of mitochondria, which although occurred over markedly different time frames appeared broadly similar in appearance. In neurons exposed to an apoptotic stimuli, inhibiting DRP-1 activity using overexpression of the dominant negative DRP-1(K38A) slowed the rate of mitochondrial fragmentation and decreased total cell death when compared to overexpression of wild-type DRP-1. In contrast, responses to l-Glu appeared DRP-1 independent. Similarly, alterations in the fission/fusion state of the mitochondrial network did not alter mitochondrial Ca(2+) uptake or the ability of l-Glu to stimulate excitotoxic Ca(2+) overload. Finally, apoptosis-induced mitochondrial fragmentation was observed concurrent with recruitment of Bax to the mitochondrial membrane. In contrast, during glutamate excitotoxicity, Bax remained in the cytosolic compartment. We conclude that different pathways lead to the appearance of fragmented mitochondria during necrotic and apoptotic neuronal cell death.

Effect of Ram Age on Structural and Functional Competence of Frozen-Thawed Spermatozoa in Dairy Sheep

The objectives of this study were to investigate the influence of ram age on structural and functional competence of frozen-thawed spermatozoa and to test the hypothesis that increasing number of sperm bound to the zona pellucida in vitro was associated with decreasing in vivo fertility of frozen semen. Rams were allocated into two groups. Each group consisted of five rams aged either 1-2 years (young) or 4-5 years (mature). Three successive ejaculates were collected from each ram using an artificial vagina. Only ejaculates of >or= 2.5 x 10(9) sperm/ml and 80% sperm progressive motility were pooled per ram, diluted with Bioxcell medium and frozen in 0.25 ml straws. The end points of post-thawing semen evaluation were computer-assisted cell motility analysis, sperm capacitation (chlortetracycline assay), simultaneous assessment of plasma membrane integrity, mitochondrial membrane potential and condensation status of nucleus, per-cell analysis of lipid peroxidation using C11-BODIPY(581/591), sperm-hemizona binding (HZB) ability and sperm fertility after laparoscopic insemination of ewes (n = 114) in the progestagen-synchronized oestrus. The results showed that mature rams had significantly lower values of sperm hyperactivated motility and peroxidized sperm, higher percentages of live non-capacitated sperm and sperm cells with intact plasma membrane, functional mitochondria and condensed chromatin, as well as, greater lambing rate and ewe prolificacy. Sperm HZB binding ability was higher (p < 0.05) for young than for mature rams. Significant correlations were found between number of spermatozoa bound to the zona pellucida and semen fertility (r = -0.63 to -0.71). In conclusion, mature rams have better semen quality and in vivo fertility than young rams. Cryocapacitation can be involved in decreasing ram semen fertility as evidenced by the high number of spermatozoa bound to the zona pellucida in vitro.

Ultrastructural alterations in midgut and Malpighian tubules of Boettcherisca peregrina exposure to cadmium and copper

The effects of Cu and Cd at their at their low concentrations (80 μg/g diet) on the morphology and ultrastructure of the midgut and Malpighian tubules of Boettcherisca peregrina larvae were observed by light and transmission electron microscopy. After exposure to both metals, the midgut got darker, shorter, and thicker than in control, and many strumae occurred on the surface of the midgut. Similarly, Malpighian tubules got shorter and thinner. Ultrastructural alterations in the midgut included mitochondrial condensation, swelling, and lysis. The rough endoplasmic reticulum (rER) showed dilation and vesiculation. The microvilli were shortened and disorganized. The stored glycogens increased and many mineral spherites appeared along with lipid droplets decreased. Ultrastructural alterations observed in the Malpighian tubules included rER vesiculation and mitochondria swelling with loss of cristae. Shortened and disordered microvilli, increased numbers of large hydropic vacuoles, and mineral spherites were also observed.

Autophagic Cell Death of Human Pancreatic Tumor Cells Mediated by Oleandrin, a Lipid-Soluble Cardiac Glycoside

Lipid-soluble cardiac glycosides such as bufalin, oleandrin, and digitoxin have been suggested as potent agents that might be useful as anticancer agents. Past research with oleandrin, a principle cardiac glycoside in Nerium oleander L. (Apocynaceae), has been shown to induce cell death through induction of apoptosis. In PANC-1 cells, a human pancreatic cancer cell line, cell death occurs not through apoptosis but rather through autophagy. Oleandrin at low nanomolar concentrations potently inhibited cell proliferation associated with induction of a profound G(2)/M cell cycle arrest. Inhibition of cell cycle was not accompanied by any significant sub G1 accumulation of cells, suggesting a nonapoptotic mechanism. Oleandrin-treated cells exhibited time- and concentration-dependent staining with acridine orange, a lysosomal stain. Subcellular changes within PANC-1 cells included mitochondrial condensation and translocation to a perinuclear position accompanied by vacuoles. Use of a fluorescent oleandrin analog (BODIPY-oleandrin) revealed co-localization of the drug within cell mitochondria. Damaged mitochondria were found within autophagosome structures. Formation of autophagosomes was confirmed through electron microscopy and detection of green fluorescent protein-labeled light chain 3 association with autophagosome membranes. Also observed was a drug-mediated inhibition of pAkt formation and up-regulation of pERK. Transfection of Akt into PANC-1 cells or inhibition of pERK activation by MAPK inhibitor abrogated oleandrin-mediated inhibition of cell growth, suggesting that the reduction of pAkt and increased pERK are important to oleandrin's ability to inhibit tumor cell proliferation. The data provide insight into the mechanisms and role of a potent, lipid-soluble cardiac glycoside (oleandrin) in control of human pancreatic cancer proliferation.

Mitochondria play an important role in 17β-estradiol attenuation of H2O2-induced rat endothelial cell apoptosis

Studies have shown salutary effects of 17beta-estradiol following trauma-hemorrhage on different cell types. 17beta-Estradiol also induces improved circulation via relaxation of the aorta and has an anti-apoptotic effect on endothelial cells. Because mitochondria play a pivotal role in apoptosis, we hypothesized that 17beta-estradiol will maintain mitochondrial function and will have protective effects against H(2)O(2)-induced apoptosis in endothelial cells. Endothelial cells were isolated from rats' aorta and cultured in the presence or absence of H(2)O(2), a potent inducer of apoptosis. In additional studies, endothelial cells were pretreated with 17beta-estradiol. Flow cytometry analysis revealed H(2)O(2)-induced apoptosis in 80.9% of endothelial cells; however, prior treatment of endothelial cells with 17beta-estradiol resulted in an approximately 40% reduction in apoptosis. This protective effect of 17beta-estradiol was abrogated when endothelial cells were cultured in the presence ICI-182780, indicating the involvement of estrogen receptor (ER). Fluorescence microscopy revealed a 17beta-estradiol-mediated attenuation of H(2)O(2)-induced mitochondrial condensation. Western blot analysis demonstrated that H(2)O(2)-induced cytochrome c release from mitochondrion to cytosol and the activation of caspase-9 and -3 were decreased by 17beta-estradiol. These findings suggest that 17beta-estradiol attenuated H(2)O(2)-induced apoptosis via ER-dependent activation of caspase-9 and -3 in rat endothelial cells through mitochondria.

Structures related to the germ plasm in mouse

This report presents data from ultrastructural and morphometric studies on the germinal-body-like structures, nuage, nuage-mitochondrial clusters and chromatoid bodies in 4.5-day embryo cells and spermatogenic cells of the laboratory mouse Mus musculus. In the 4.5-day embryo cells the germinal-body-like structures that, according to previous data, arise by condensation of mitochondria in Graafian oocytes, were found not to undergo any ultrastructural alterations. In spermatogonia the germinal-body-like structures presumably were transformed into nuage that functioned as 'intermitochondrial cement' binding the mitochondrial clusters. In primary spermatocytes mitochondria aggregated by nuage were found with large vacuoles containing membraneous conglomerates that were obviously excreted by organelles into the cytoplasm. The chromatoid bodies that arose in spermatocytes and finally disintegrated in the posterior part of late spermatids seemed not to be implicated in the pathway of the germinal-body-like structure. The dispersion of chromatoid bodies was noted to be accompanied by excretion of membraneous conglomerates by late spermatid mitochondria. The spermatozoa were not found to contain either the germinal-body-like structures or any other germ-plasm-related structures.