Apoptotic Nuclei Research Articles

Dietary amino acid ergothioneine protects HT22 hippocampal neurons against H2O2-induced neurotoxicity via antioxidative mechanism

BackgroundErgothioneine (EGT) is a naturally occurring amino acid acquired through the dietary intake of foods, mainly mushrooms. This potent antioxidant is a cytoprotectant in several experimental models. However, its neuroprotective activities are scarcely available, especially in the hippocampal region associated with cognition. MethodsIn this study, the neuroprotective activity of EGT was investigated in hydrogen peroxide (H2O2)-induced neurotoxicity in HT22 mouse hippocampal neurons by focusing on antioxidation, mitochondrial functions, and anti-apoptotic activities. ResultsH2O2 drastically reduced the HT22 cells viability accompanied by a significant rise in reactive oxygen species (ROS), reduction of mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP), and increase in apoptotic nuclei. Co-incubation with 1 mM EGT significantly attenuated the H2O2-induced toxicity, reduced the ROS, and decreased the apoptotic nuclei, without affecting the MMP and ATP. EGT significantly increased expression levels of Nrf2, NQO1 and HO-1 antioxidant genes as well as mitochondrial CYC1 gene while Bcl-2 and Bax apoptotic genes were unaffected. ConclusionThis study suggests the potential of EGT as a neuroprotectant in hippocampal neurons mainly through its potent antioxidative activities. EGT could be further explored in vivo considering its effectiveness and safety profile.

Chronic retinal injury induced by white LED light with different correlated color temperatures as determined by microarray analyses of genome-wide expression patterns in mice

Widely used white light-emitting diodes (LEDs) currently deliver higher levels of blue light than conventional domestic light sources. The high intensity of the blue component is the main source of concern regarding possible health risks of LED to chronic light toxicity to the retina. Therefore, we analyzed retinal injury and genome-wide changes in gene expression induced by white LED light with different correlated color temperatures (CCTs) in a mouse model. Balb/c mice (10 weeks old) were exposed to LED light with CCTs of 2954, 5624, and 7378 K, at different illuminance levels (250, 500, 1000, and 3000 lx) and for different exposure times (7, 14, and 28 days). Hematoxylin and eosin staining revealed that exposure to 7378 K light at 250 lx for 28 days resulted in a significant reduction of outer nuclear layer (ONL) nuclei, whereas 2954 K light at <3000 lx led to only a mild reduction in the number of ONL nuclei. In addition, 5624 and 7378 K light at 3000 lx resulted in a significant increase in TUNEL-positive apoptotic nuclei, which was not found at an illuminance of 1000 lx. Genome-wide expression analyses showed that, compared to a control group, there were 121 upregulated differentially expressed genes (DEGs) and 458 downregulated DEGs found in the 7378 K group, and 59 upregulated and only 4 downregulated DEGs in the 2954 K group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the DEGs were involved in 341 GO terms and 16 related pathways for the 7378 K group and in 12 GO terms and 7 related pathways for the 2954 K group. Signal pathways related to ubiquitin potentially played an important role in light-induced retinal degeneration. Furthermore, retinal immunohistochemistry (IHC) indicated downregulation of ubiquitin and autophagy function caused by 7378 K light. Taken together, these results indicate that retinal injury in the mice induced by white LED light occurred in a CCT-dependent manner, and that light with a higher CCT was more likely to reduce ONL nuclei; however, the apoptosis pathway may not be the only mechanism involved. Based on genome-wide expression analyses and retinal IHC, the ubiquitin-mediated proteolysis signal pathway may have participated in the induction retinal degeneration.

Astrocytic palmitoylethanolamide pre-exposure exerts neuroprotective effects in astrocyte-neuron co-cultures from a triple transgenic mouse model of Alzheimer's disease

Palmitoylethanolamide (PEA) is an endogenous lipid mediator that, also by blunting astrocyte activation, demonstrated beneficial properties in several in vitro and in vivo models of Alzheimer's disease (AD).In the present study, we used astrocyte-neuron co-cultures from 3xTg-AD mouse (i.e. an animal model of AD) cerebral cortex to further investigate on the role of astrocytes in PEA-induced neuroprotection. To this aim, we evaluated the number of viable cells, apoptotic nuclei, microtubule-associated protein-2 (MAP2) positive cells and morphological parameters in cortical neurons co-cultured with cortical astrocytes pre-exposed, or not, to Aβ42 (0.5 μM; 24 h) or PEA (0.1 μM; 24 h).Pre-exposure of astrocytes to Aβ42 failed to affect the viability, the number of neuronal apoptotic nuclei, MAP2 positive cell number, neuritic aggregations/100 μm, dendritic branches per neuron, the neuron body area, the length of the longest dendrite and number of neurites/neuron in 3xTg-AD mouse astrocyte-neuron co-cultures. Compared to neurons from wild-type (non-Tg) mouse co-cultures, 3xTg-AD mouse neurons co-cultured with astrocytes from this mutant mice displayed higher number of apoptotic nuclei, lower MAP2 immunoreactivity and several morphological changes. These signs of neuronal suffering were significantly counteracted when the 3xTg-AD mouse cortical neurons were co-cultured with 3xTg-AD mouse astrocytes pre-exposed to PEA.The present data suggest that in astrocyte-neuron co-cultures from 3xTg-AD mice, astrocytes contribute to neuronal damage and PEA, by possibly counteracting reactive astrogliosis, improved neuronal survival. These findings further support the role of PEA as a possible new therapeutic opportunity in AD treatment.

Acetyl-11-keto-\u03b2-boswellic acid enhances the cisplatin sensitivity of non-small cell lung cancer cells through cell cycle arrest, apoptosis induction, and autophagy suppression via p21-dependent signaling pathway

Cisplatin-based therapy is a widely used chemotherapeutic regimen for non-small cell lung cancer (NSCLC); however, drug resistance limits its efficacy. Acetyl-11-keto-β-boswellic acid (AKBA), a bioactive compound from frankincense, has been shown to exert anti-cancer effects. The aim of this study is to explore the potential of AKBA in combination with cisplatin as a new regimen for NSCLC. CCK8 assay and clone formation assay were used to determine the effects of AKBA in combination with cisplatin on cell viability of NSCLC cell lines. A three-dimensional spherification assay was used to simulate in vivo tumor formation. Flow cytometry was performed to examine cell cycle distribution and the percentages of apoptotic cells. The associated proteins and mRNA of cell cycle, apoptosis, and autophagy were measured by western blotting and real-time fluorescence quantitative PCR. Immunofluorescence assay was used to test apoptotic nuclei and autolysosome. Small interfering RNA experiments were used to silence the expression of p21. Combination treatment of AKBA and cisplatin inhibited cell viability, clone formation, and three-dimensional spherification, enhanced G0/G1 phase arrest, increased the percentages of apoptotic cells, and decreased the ratio of positive autolysosomes, compared with cisplatin alone. AKBA in combination with cisplatin suppressed the protein expressions of cyclin A2, cyclin E1, p-cdc2, CDK4, Bcl-xl, Atg5, and LC3A/B, and upregulated p27 and p21 mRNA levels in A549 cells. Downregulation of p21 decreased G0/G1 phase arrest and the percentages of apoptotic cells, and promoted autophagy in NSCLC A549 cells. Our study demonstrates that AKBA enhances the cisplatin sensitivity of NSCLC cells and that the mechanisms involve G0/G1 phase arrest, apoptosis induction, and autophagy suppression via targeting p21-dependent signaling pathway.

Methylmercury exposure during the vulnerable window of the cerebrum in postnatal developing rats

The developing brain is known to be sensitive to the toxic effects of methylmercury (MeHg). The effects of toxic levels of MeHg exposure during the most seemingly vulnerable window of the cerebrum are not well studied. In this study, we aimed to examine the specific effects of toxic levels of MeHg on neurobehavior, neurodegeneration, and selenoenzyme activity in the cerebrum of infant rats. Male Wistar rats (n = 8/group) were orally treated with MeHg at an acute toxic dose (8 mg Hg/kg/day) for 10 consecutive days starting on postnatal day 14 (P14). The MeHg-exposed rats showed a significant reduction in body weight after day 8 and severe neurological symptoms similar to dystonia on day 12 (P25). Motor coordination deficits determined using the rotarod performance test and short-term memory impairment determined using the Y-maze task were observed in the MeHg-exposed rats on day 11 (P24). The MeHg-exposed rats sacrificed on day 12 showed severe cerebral neuronal degeneration, reactive astrocytosis, and TUNEL-positive apoptotic nuclei, with the cerebral Hg concentration of 15.0 ± 1.6 μg/g. Furthermore, the activities of glutathione peroxidase and thioredoxin reductase in the cerebrum in MeHg-exposed rats were lower than those in control. These results indicate that MeHg exposure to infant rats will be useful to predict the effects of MeHg at the cerebral growth spurt in humans.

Dapsone prolong delayed excitotoxic neuronal cell death by interacting with proapoptotic/survival signaling proteins

BackgroundDapsone prevents ischemic injury, inhibits apoptosis and shows functional improvement post-ischemia. However, its effect on proapoptotic or survival proteins in delayed ischemia remains unclear. MethodsMale adult Wistar rats were subjected to middle cerebral artery occlusion (MCAO) for 90 min followed by 24 h of ischemic reperfusion (I/R). Dapsone [9.375 or 12.5 mg/kg, intraperitoneally (IP)] was administered at 3, 6 and 12 h of I/R followed by behavioural assessment, brain infarction, histological alteration and cell viability study. Further, dapsone (25 and 50 µM) was added at 3, 6 and 12 h after L-glutamate (100 µM) in primary cortical culture (DIV 14) and cell viability, cytotoxicity, apoptosis was observed. Proteins expression were observed using immunocytochemistry. All experiments were performed after 24 h of I/R (In-Vivo) and 24 h of recovery post glutamate insult (In-Vitro). ResultsReduced brain infarction, improved neurobehavioural functions in addition to reduction in abnormal morphological structures of ischemic brain and improvement in cell viability was observed with treatment of dapsone (12.5 mg/kg) administered upto 6 h. Similarly, dapsone (25, 50 µM) increased cell survival post glutamate insult in cortical culture (In-vitro). Further, dapsone treatment at delayed hours (6 h) reduced apoptotic nuclei and proapoptotic proteins JNK, PTEN, Calpain, Caspase 3 expression along with activation of prosurvival protein BDNF expression post-glutamate insult. ConclusionOur results suggest that dapsone has the potential to limit the neuronal damage post-glutamate insult in delayed hours (6 h) through repressing proapoptotic proteins JNK, PTEN, Calpain, Caspase-3 of cerebral ischemia along with activation of pro-survival protein BDNF.

P1387IRON CITRATE BLOCKS THE PROGRESSION OF CALCIUM DEPOSITION BY REVERTING APOPTOSIS AND AUTOPHAGY IN VSMCS

Abstract Background and Aims Cardiovascular disease is the first cause of mortality in chronic kidney disease (CKD) induced by vascular calcification. Since, in advanced CKD, iron based phosphate binders have been proposed to treat hyperphosphatemia, we studied, in high-phosphate (Pi) calcified VSMC, the effect of iron citrate (iron) on the progression of calcium deposition. Method We treated VSMCs with 5 mM Pi and iron citrate to evaluate Ca deposition by Alizarin Red destaining, DNA fragmentation by ELISA, gene expression by RT-PCR and protein expression by Western blot. Results High-Pi calcification was blocked when iron was added from day 7 to 15 (1.30±0.03vs0.61±0.02; OD/mg protein; day 15; PivsPi+Fe, p&amp;lt;0.01). Since iron added prophylactically prevented apoptosis and potentiated autophagy we studied the role of these two processes in the therapeutic protocol. Adding iron from day 7 to day 11 decreased apoptotic cell number (17.3±2.6vs11.6±1.6; Annexin V; % positive cells; day 11; PivsPi+Fe; p&amp;lt;0.05, figure). To confirm the effect of iron on apoptosis, we demonstrated its action in blocking the H2O2-induced increase in calcification when added from day 7 to day 14 (1.37±0.03 vs. 0.96±0.05; H2O2+Pi day 7-14 vs Fe+H2O2+Pi day 7-14; day 14; OD/mg protein; p&amp;lt;0.01). We then investigated the mechanism of the anti-apoptotic effect by studying the pro-survival axis GAS6/AXL, finding that iron treatment from day 9 to day 14 counteracted the protein down-regulation, and induced their de-novo synthesis. Supporting the effect of therapeutic added iron on apoptosis we found a decrease in apoptotic nuclei both in VSMCs and aortic rings after iron treatment from day 7 to 15 (3.8±0.2vs2.3±0.3 and 4.0±0.3vs2.2±0.2; apoptotic nuclei; arbitrary score; day 15; PivsPi+Fe; VSMCs and aortic rings; p&amp;lt;0.05). Moreover, iron added from day 9 to day 15 potentiated autophagic flux and increased the number of autophagosomes with damaged mitochondria. Conclusion Iron exerts an anti-apoptotic and pro-autophagic effect on established calcified VSMC when simil-osteoblastic transformation was already occurred.

Involvement of protein S-nitrosylation in regulating beef apoptosis during postmortem aging

This study was the first attempt to explore the effect of protein S-nitrosylation on the progress of apoptosis in postmortem beef semimembranosus muscle (SM). Five beef SM were incubated with S-nitrosoglutathione (GSNO, nitric oxide donor), control, or Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME, nitric oxide synthase inhibitor). Results suggest that compared to the control, more chromatin condensation, nucleusfragmentation, apoptoticbody formation, and mitochondrialswelling were observed in the l-NAME group while these apoptosis-related morphological changes were retarded in the GSNO group. Notably, there were fewer apoptotic nuclei in the GSNO group and more apoptotic nuclei in the l-NAME group compared to the control (P < 0.05). Additionally, caspase-3 and -9 activities and caspase-3 activation were greatly decreased by GSNO treatment and increased by l-NAME treatment (P < 0.05). The morphological and biochemical results indicate that protein S-nitrosylation could play a negative regulatory role in beef apoptosis during postmortem aging.

Effects of eEF1A2 knockdown on autophagy in an MPP+-induced cellular model of Parkinson’s disease

1-Methyl-4-phenylpyridinium ion (MPP+) is widely used to induce a cellular model of Parkinson’s disease (PD) in dopaminergic cell lines. Downregulation of the protein translation elongation factor 1 alpha (eEF1A) has been reported in the brain tissue of PD patients. eEF1A2, an isoform of eEF1A, is associated with lysosome biogenesis that involves the autophagy process. However, the role of eEF1A2 on autophagic activity in PD has not been elucidated. In this work, we investigated the role of eEF1A2 on autophagy using eEF1A2 siRNA knockdown in differentiated SH-SY5Y neuronal cells treated with MPP+. We found that eEF1A2 was upregulated in differentiated cells, which could be silenced by eEF1A2 siRNA. Significantly, cells treated with MPP+ after eEF1A2 knockdown showed a decreased number of LC3 puncta, decreased LC3-II/LC3-I ratio, and decreased phospho-Beclin-1, compared to the MPP+ alone group. These cells showed extensive areas of mitochondria damage, with a reduction of mitochondrial membrane potential, but reduced mitophagy as indicated by the reduced colocalization of LC3 puncta with damaged mitochondria. Cells with eEF1A2 siRNA plus MPP+ treatment aggravated α-synuclein accumulation but reduced colocalization with LC3. As a result, eEF1A2 knockdown decreased viability, increased apoptotic nuclei, increased caspase-3/7 activation and increased cleaved caspase-3 when cells were treated with MPP+. These results suggest that eEF1A2 is essential for dopaminergic neuron survival against MPP+, in part through autophagy regulation.

Anti-steatotic linagliptin pleiotropic effects encompasses suppression of de novo lipogenesis and ER stress in high-fat-fed mice

AimTo investigate the effects of linagliptin treatment on hepatic energy metabolism and ER stress in high-fat-fed C57BL/6 mice. MethodsForty male C57BL/6 mice, three months of age, received a control diet (C, 10% of lipids as energy, n = 20) or high-fat diet (HF, 50% of lipids as energy, n = 20) for 10 weeks. The groups were randomly subdivided into four groups to receive linagliptin, for five weeks, at a dose of 30 mg/kg/day added to the diets: C, C-L, HF, and HF-L groups. ResultsThe HF group showed higher body mass, total and hepatic cholesterol levels and total and hepatic triacylglycerol levels than the C group, all of which were significantly diminished by linagliptin in the HF-L group. The HF group had higher hepatic steatosis than the C group, whereas linagliptin markedly reduced the hepatic steatosis (less 52%, P < 0.001). The expression of Sirt1 and Pgc1a was more significant in the HF-L group than in the HF group. Linagliptin also elicited enhanced GLP-1 concentrations and a reduction in the expression of the lipogenic genes Fas and Srebp1c. Besides, HF-L showed a reduction in the genes related to endoplasmic reticulum stress Chop, Atf4, and Gadd45 coupled with reduced apoptotic nuclei immunostaining. ConclusionLinagliptin caused a marked reduction in hepatic steatosis as a secondary effect of its glucose-lowering property. NAFLD countering involved reduced lipogenesis, increased beta-oxidation, and relief in endoplasmic reticulum stress, leading to reduced apoptosis and better preservation of the hepatic structure. Therefore, linagliptin may be used, preferably in diabetic patients, to avoid the progression of hepatic steatosis.

Mapping the relationship between proliferation and morphology in the developing mouse face

There is a long‐standing prediction that small changes in proliferation and apoptosis during the time frame of facial morphogenesis act to shape the face. Further, many studies show genetic alterations that cause structural birth defects affect local proliferation or apoptosis. Yet, it is unclear how much of local change in regional proliferation would be necessary to cause a defect. Here, we set out to understand the relationship between growth, morphology and proliferation and test that prediction that targeted proliferation shapes the developing face by quantifying proliferation and apoptosis in 3D and relating it to the growth of the face. We use whole mount staining for proliferation and apoptosis markers, whole tissue clearing methods, lightsheet microscopy and atlas and machine learning based quantification methods to identify individual proliferating or apoptotic nuclei within a 3D tissue structure at a set time point. We also employee geometric morphometric analysis of the same tissue structure to quantify overall morphology. By collecting data at various time points across facial development (E9.5–E11.5) and quantifying the age of each embryo, we are able to relate cell biological level growth to tissue level growth and morphological change and relate these two parameters in a way not performed previously.Support or Funding InformationNIH NIDCR R01‐DE019638 to RM and BH, NSERC Discovery to BH, and CIHR Foundation grant to BH and RM, CIHR postdoctoral fellowship to RMG.Atlas based quantification of proliferation: A) Maximum Projection of the embryo highlighting the external morphology ‐ lateral view. B) Proliferation staining (phospho‐Histone H3) ‐ lateral view. C–D) Surface morphology of the atlas (n=5) C ‐ anterior view, D ‐ Lateral view. E–F) Heat map of proliferating cells (no density correction) E ‐ anterior view, F ‐ Lateral view.Figure 1

Effect of bisphenol Z and its analogs on reproductive health of C. elegans

Bisphenol A (BPA) is an organic synthetic molecule used in thousands of different plastic‐based products throughout the world. Over the years BPA has been repeatedly linked to neurobehavioral, cardiovascular and reproductive disorders. Because of growing human health concerns, BPA has been increasingly replaced by structurally similar chemicals such as bisphenol S (BPS) and bisphenol Z (BPZ). In recent years, some of these analogs have been under scrutiny by scientists whose research uncovered adverse health effects in model organisms. Whereas numerous studies suggest that BPS may be as toxic as BPA, the impact of BPZ is less understood. To assess its toxicity, we exposed the nematode C. elegans to BPZ at a final concentration of 1 mM throughout their development (from eggs to adulthood). Following exposure, we observed a marked reduction in the brood size compared to the wild type. Moreover, the BPZ‐exposed gonads showed a significant increase in the number of apoptotic nuclei. In the search for potentially safer alternatives, we synthesized three novel analogs of BPZ. Interestingly, these compounds exhibited even stronger effect on the reproductive ability of C. elegans. Our findings suggest that, similar to BPA and BPS, exposure to BPZ and its analogs results in reproductive toxicity.Support or Funding InformationDepartment of Natural Sciences, Lawrence Technological University.

Histological structure of skeletal muscle in disuse muscle atrophy and role of exercise

Disuse muscle atrophy is one of the most important research topics in fields of rehabilitation and clinical medicine. It refers to morphological and functional alteration of skeletal muscle under the states of hypokinesia, immobilization and weightlessness. This review analyzes a number of structural and functional changes in the skeletal muscle tissue in case of muscle disuse which appeared in loss in the muscle mass, reduction in the transverse diameter of most of muscle fibers, wide spaces between muscle fibers, increase collagen fiber deposition and appearance of apoptotic nuclei in muscle fibers besides sensorial dysfunction such as hypo mechanical responsiveness and diminished contractile function. In addition to the ultrastructural alteration, which include loss of sarcomere organization, indistinguishable A and I bands, irregular distorted Z-line with disruption of myofilaments and fused giant accumulated mitochondria. The review summarizes also the molecular mechanisms of skeletal muscle disuse atrophy which could be resulted from an alteration in the activity of oxidative stress enzymes and lead to increase in protein degradation and/or a reduction in protein synthesis. Moreover, the review discusses the efficiency of potential countermeasures of stretching exercise as a very powerful stimulant of muscle growth and muscle protein synthesis to counteract the structural and functional alterations in muscle tissue caused by disuse. The review showed that stretching exercise can increase the transverse sectional area of muscle fibers, produce a rise in the number of sarcomeres in series and prevent muscle atrophy. Passive stretching also completely prevented the decrease in myonuclear number induced by inactivity. Moreover, stretching accelerated the amelioration of the range of motion limitation and prevent the deposition of connective tissue resulted from disuse muscle atrophy.

Effects of human recombinant granulocyte-colony stimulating factor treatment during in vitro culture on porcine pre-implantation embryos.

Granulocyte-colony stimulating factor (G-CSF), a pleiotropic cytokine, belongs to the hematopoietic growth factor family. Recent studies have reported that G-CSF is a predictive biomarker of oocyte and embryo developmental competence in humans. The aim of our study was to determine whether CSF3 and its receptor (CSF3R) were expressed in porcine maternal reproductive tissues (oviduct and uterus), cumulus cells, and embryos and to investigate the effects of human recombinant G-CSF (hrG-CSF) supplementation during in vitro culture (IVC) on the developmental competence of pre-implantation embryos. To do this, we first performed reverse-transcription polymerase chain reaction (RT-PCR). Second, we performed parthenogenetic activation (PA), in vitro fertilization (IVF), and somatic cell nuclear transfer (SCNT) to evaluate the embryonic developmental potential after hrG-CSF supplementation based on various concentrations (0 ng/mL, 10 ng/mL, 50 ng/mL, and 100 ng/mL) and durations (Un-treated, Days 0–3, Days 4–7, and Days 0–7) of IVC. Finally, we examined transcriptional levels of several marker genes in blastocysts. The results of our study showed that CSF3 transcript was present in all samples we assessed. CSF3-R was also detected, except in cumulus cells and blastocysts from PA. Furthermore, 10 ng/mL and Days 0–7 were the optimal concentration and duration for the viability of in vitro embryonic development, especially for SCNT-derived embryos. The rate of blastocyst formation and the total cell number of blastocysts were significantly enhanced, while the number and index of apoptotic nuclei were significantly decreased in optimal condition groups compared to others. Moreover, the transcriptional levels of anti-apoptotis- (BCL2), proliferation- (PCNA), and pluripotency- (POU5F1) related genes were dramatically upregulated. In conclusion, for the first time, we demonstrated that CSF3 and CSF3R were expressed in porcine reproductive organs, cells, and embryos. Additionally, we determined that hrG-CSF treatment improved porcine embryonic development capacity in vitro.

Interleukin-6 Response of C2C12 Myotubes Stimulated with Lipopolysaccharide and Lipoic Acid.

Our previous study explored the dual effect of lipoic acid on the regulation of IL-6 expression in C2C12 myotubes. However, the specific mechanism remains unclear. To evaluate IL-6 signaling in skeletal muscle, pCMV6-IL-6 was overexpressed in C2C12 myotubes. The real-time quantitative polymerase chain reaction was used to detect mRNA expression. Immunohistochemistry and a DeadEnd colorimetric TUNEL system were used to detect IL-6 localization and analyze the apoptosis in IL-6-overexpressing cells, respectively. A caspase-3/CPP32 colorimetric assay and Western blotting were used to analyze caspase-3 activity and protein expression, respectively. Our results showed the overexpressed IL-6 was not only located in the cytosol but also on the intracellular side of the cell membrane. Moreover, the nucleus did not demonstrate IL-6 overexpression. The DeadEnd colorimetric apoptosis detection assay results demonstrated that apoptotic nuclei were present in IL-6-overexpressing cells. However, the overexpressed IL-6 failed to promote caspase-3 activity. Notably, the exogenous pyrogen lipopolysaccharide (LPS) significantly promoted IL-6 mRNA expression and caspase-3 activity but did not induce apoptotic cell formation. Moreover, lipoic acid significantly upregulated IL-6, IL-6Ra, and gp130 mRNA expression and significantly increased caspase-3 activity but did not induce apoptotic cell formation. Lipoic acid significantly increased the p-Akt level in untreated cells but not in LY294002-treated cells. Taken together, our results suggesting that the overexpressed IL-6-induced apoptosis may not be mediated by caspase-3. LPS-induced IL-6 mRNA expression may not be involved in IL-6 classical signaling or trans-signaling in C2C12 myotubes. Lipoic acid-induced IL-6 mRNA expression may be mediated by IL-6 classical signaling in C2C12 myotubes. [Figure: see text].

Quinacrine causes apoptosis in human cancer cell lines through caspase-mediated pathway and regulation of small-GTPase

Quinacrine (QC), an FDA-approved anti-malarial drug, has shown to have anticancer activities. Due to its 'shotgun' nature, QC has become an inevitable candidate for combination chemotherapy. There is lack of study of the molecular interplay between colorectal cancer (CRC) microenvironment and its metastasis. In this study, we focused on the differential anti-cancerous effect of QC on two different human cancer cell lines, HCT 116 and INT 407. Results suggest that cytotoxicity increased in both the cell lines with an increase in QC concentration. The expression patterns of small-GTPases and caspases were altered significantly in QC-treated cells compared to non-treated cells. HSP70 and p53 showed comparable differences in the expression pattern. The wound-healing assay showed an increase in the denuded zone, with an increase in the concentration of QC. The formation of apoptotic nuclei increased with a rise in the concentration of QC in both the cell lines. The decrease and increase in caspase 9 and caspase 3 expression respectively were studied, confirming apoptosis by the extrinsic pathway.

Andrographolide mitigates cardiac apoptosis to provide cardio-protection in high-fat-diet-induced obese mice.

Excessive intake of high fat diet (HFD) and associated obese conditions are critical contributors of cardiac diseases. In this study, an active metabolite andrographolide from Andrographis paniculata was found to ameliorate HFD-induced cardiac apoptosis. C57/BL6 mouse were grouped as control (n = 9), obese (n = 8), low dose (25 mg/kg/d) andrographolide treatment (n = 9), and high dose (50 mg/kg/d) andrographolide treatment (n = 9). The control group was provided with standard laboratory chow and the other groups were fed with HFD. Andrographolide was administered through oral gavage for 1 week. Histopathological analysis showed increase in apoptotic nuclei and considerable cardiac-damages in the obese group signifying cardiac remodeling effects. Further, Western blot results showed increase in pro-apoptotic proteins and decrease in the proteins of IGF-1R-survival signaling. However, feeding of andrographolide significantly reduced the cardiac effects of HFD. The results strongly suggest that andrographolide supplementation can be used for prevention and treatment of cardiovascular disease in obese patients.

Treatment of Brain Edema by Wogonoside Is Associated with Inhibition of Neuronal Apoptosis and SIRT1 Activation in Rats.

BackgroundBrain edema and neuronal apoptosis are closely associated with loss of neurological function and death in rats with subarachnoid hemorrhage (SAH). The present study investigated the effect of wogonoside on brain edema induced by SAH in rats and studied the mechanism involved.Material/MethodsThe rats were intra-gastrically administered 10, 20, 50, 100, 150 and 200 mg/kg doses of wogonoside 24 h prior to SAH induction. Western blotting was used to assess levels of pro-apoptotic protein, SIRT1, ZO-1, and p53 protein expression. Apoptotic nuclei were detected using immunofluorescence and TUNEL staining.ResultsWogonoside treatment significantly suppressed edema formation in SAH-induced rats. Pre-treatment with wogonoside exhibited an inhibitory effect on SAH-induced extravascular Evans blue staining in rats. The expression of ZO-1, Occludin, and Claudin-5 proteins was increased by wogonoside in the SAH-induced rats. The inhibitory effect of SAH was completely reversed in the rats treated with the 200 mg/kg dose of wogonoside. The expression of SIRT1 protein was upregulated, and p53 and AC-p53 were downregulated by wogonoside in SAH rats. Wogonoside treatment significantly reduced SAH-mediated promotion of Bax, Puma, Noxa, Bid, and cleaved Caspase-3 expression. In the SAH-induced rats, pre-treatment with wogonoside reduced the TUNEL-positive cell count.ConclusionsThe present study demonstrated that wogonoside prevents brain edema development and apoptosis of neurons in rats by promoting SIRT1 expression and suppression of p53 activation. Therefore, wogonoside has therapeutic potential for the treatment of edema and needs to be investigated further to completely define the mechanism involved.

Anti-Migratory and Pro-Apoptotic Properties of Parvifloron D on Triple-Negative Breast Cancer Cells.

Medicinal plants are important sources of new bioactive compounds with potential anticancer activity. Parvifloron D (ParvD) is an abietane diterpenoid, isolated in high amounts from Plectranthus ecklonii Benth. Previous reports have suggested potential therapeutic properties for ParvD. ParvD has shown pro-apoptotic and cytotoxic effects in leukemia and melanoma cell lines. However, to the best of our knowledge, there are no studies in triple-negative breast cancer (TNBC) models. TNBC is a breast cancer subtype characterized by an aggressive behavior with poor clinical outcomes and weak overall therapeutic responses to the current treatment options. This work aimed at evaluating the anticancer effect of ParvD in MDA-MB-231 cells, a model of human TNBC. To obtain sufficient amounts of purified ParvD the efficiency of several extraction methods was compared. ParvD (0.1–10 µM) decreased cell viability in a concentration-dependent manner. Treatment with ParvD (5 µM) significantly increased the percentage of apoptotic nuclei and exposure to 3 µM ParvD increased the sub-G1 population. Since altered cell adherence, migration, and invasion are determinant processes for the formation of metastases, the effect of ParvD on these cellular processes was tested. Although treatment with ParvD (1 µM) had no effect on cell-substrate attachment, ParvD (1 µM) significantly reduced cell chemotaxis and invasion. This is the first report describing the proapoptotic effect of ParvD in TNBC cells. Moreover, for the first time we have shown that ParvD reduces cell motility, unraveling potential anti-metastatic properties.

Primary Intraosseous Xanthomas of the Jaws: A Series of Six Cases Including an Example with Formation of Apoptosis-Related Hyaline Globules, So-Called "Thanatosomes".

Primary intraosseous xanthomas of the jaws (PIXJ) are rare and predominantly affect the posterior mandible (86%) of normolipemic patients, with a mean age of 30years and no gender predilection. Clinically, PIXJ exhibit indolent biologic behavior; curettage is considered treatment of choice. Only 36 PIXJ have been reported. Apoptosis-related hyaline globules (HGs), also known as "thanatosomes", have not been previously reported in PIXJ. Cases diagnosed as xanthoma of bone were retrieved. Six cases fulfilling currently accepted criteria were identified and their clinicopathologic and immunohistochemical properties are presented herein. Mean age for PIXJ was 21.8years (range = 12-33) and F:M ratio = 2:1. All cases presented as well-demarcated, unilocular or multilocular radiolucencies. Microscopically, PIXJ featured sheets of lipid-laden macrophages with eosinophilic or foamy cytoplasm. A secondary fibroblastic population lacking storiform pattern was evident in two cases. Adipocytes (3/6), peripheral neurovascular bundles (1/6), bone fragments (3/6) and dystrophic calcifications (3/6) were observed enclosed by the xanthoma cells. Notably, one case exhibited numerous, spherical, eosinophilic HGs containing apoptotic nuclei. PIXJ were consistently CD68(+) and negative for CD1α and S100. CD45 decorated lymphocytes and the membrane of foamy histiocytes. Xanthoma cells stained for lysozyme and plasma proteins including alpha-1 antitrypsin (AAT), IgG and IgA in one probed case. HGs were lysozyme(+), AAT(+), IgG(+), IgA(+), PAS(+) and diastase-resistant, and fuchsinophilic with Masson's trichrome. PIXJ represent infrequent, solitary, mandibular lesions with a predilection for the second and third decade of life. Thanatosomes associated with cell injury and death can be present in PIXJ.