Serum Deprivation-induced Apoptosis Research Articles

Genistein ameliorates starvation-induced porcine follicular granulosa cell apoptosis.

Genistein is an isoflavone which is beneficial for health, but little is known regarding its function on granulosa cell fate during follicular atresia. In the present study, we established an in-vitro model of porcine follicular granulosa cell apoptosis by serum-deprivation and showed that treatments with 1 μM and 10 μM genistein significantly reduced the apoptotic rate of granulosa cells compared to the blank control (p < 0.05). These results suggest that genistein at micromolar levels alleviates serum deprivation-induced granulosa cell apoptosis, and the ameliorative effect of genistein on granulosa cell apoptosis is likely to be able to inhibit nutrient depletion-induced follicular atresia. Further experimental results revealed that the expression of the autophagic marker protein LC3II in 100 nM - 10 μM genistein treatment increased in a dose-dependent manner and was higher than the control (p < 0.05). Genistein also dose-dependently promoted the phosphorylation of AMPK (adenosine 5'-monophosphate [AMP]-activated protein kinase) in granulosa cells. Poly(ADP-ribose) (pADPr) formation in genistein-treated groups was also notably higher than in the controls (p < 0.05). Collectively, genistein alleviates serum deprivation-induced granulosa cells in vitro through enhancing autophagy, which involving AMPK activation and PARylation signaling. However, further study should be taken to investigate the role of the above signaling on this process.

Molecular mechanism of EGFR signaling evoked by environmental pollutant 1,2-naphthoquinone

The epidermal growth factor receptor (EGFR) is the most extensively examined receptor tyrosine kinase. Several EGFR mutations and modifications have been shown to induce self-activation, which plays a central role in carcinogenesis. Recently, environmental chemicals such as PM2.5 can also activate EGFR and become risk factors for cancer. Although, the detailed mechanism remains unknown. In this study, we focused on 1,2-naphthoquinone (1,2-NQ) which is a secondary metabolite of naphthalene. Humans are exposed to 1,2-NQ through the combustion of fossil and diesel fuel and from tobacco smoke and PM2.5. Here, we demonstrate that 1,2-NQ is a novel EGFR-specific activator. We found that 1,2-NQ forms a covalent bond called N-arylation with EGFR Lys80 which is in the extracellular domain by LC-MS/MS. This modification activates the EGFR-Akt signaling pathway, which inhibits serum deprivation-induced apoptosis in A549 cells. Our study reveals an original mode of EGFR activation via covalent binding. We propose the correlation between EGFR activation without ligands and environmental pollutant-associated diseases such as cancer.

Long Non-Coding Transcript in LEF1 Locus Is a Novel Regulator of Cell Cycle and Apoptotic Pathways in Myeloid Leukemic Cells

Myelodysplastic syndrome and acute myeloid leukemia are hematologic disorders characterized by inefficient differentiation and aberrant control of cell growth and apoptosis. In early MDS, blasts, unable to differentiate, exhibit exacerbated proliferation and apoptosis. However, MDS progression and AML transformation are associated with a progressive resistance to apoptosis. The molecular mechanisms that control these cellular decisions are not fully understood. Long non-coding RNAs (lncRNA) have gained great attention in recent years as they have been implicated in a wide variety of biological functions. Here we focus on a long non-coding transcript encoded in the LEF1 locus as a novel regulator of apoptosis and proliferation signaling in hematologic disease.Our previous work using microarray technology identified a long non-coding RNA, antisense to LEF1 gene, which expression was reduced in myelodysplastic syndrome patients. We mapped the sequence to a previously characterized transcript, LEF1-AS1. This transcript is highly conserved and has been recently associated to B-cell differentiation. We observed a modulation of LEF1-AS1 during the first stages of in-vitro erythroid differentiation from CD34+ HSC to erythroblast (from collection day 6 to day 8 after addition of erythropoietin, n=6, 78% mean reduction, P<0.0001).Due to reported reduction on LEF1-AS1 expression in MDS and low endogenous expression of LEF1-AS1 in myeloid cells lines, we decided to overexpress the transcript in myeloid cell lines HL60 and K562 to study the function of the lncRNA. Stably transfected cells were obtained by DMRIE-C mediated transfection using pcDNA vector containing full length lef1-AS1 or empty pcDNA vector. Selection was carried out by addition of geneticin to the culture medium.Unexpectedly, after stable over-expression of LEF1-AS1, we could not detect any alteration of LEF1 expression in the mRNA or protein level in both myeloid cell lines (HL60 and K562). Despite of the lack of regulatory effect upon LEF1, we observed a significant inhibition in cell proliferation evaluated Carboxyfluorescein succinimidyl ester (CFSE) labelling (p<0.05).To investigate the antiproliferative effects of LEF1-AS1, we analyzed the cell cycle phase distribution of the stably transfected cell HL60 by flow cytometry using propidium iodide DNA staining. LEF1-AS1 over-expression resulted in a significant increase in the proportion of S-phase cells (p=0.01) and no difference in G2/M, suggesting intra S-phase imprisonment.Additionally, we evaluated the effect of LEF1-AS1 over-expression upon apoptosis in stably transfected HL60 using annexin/Propidium Iodide Apoptosis flow cytometry assay. We observed no difference in the apoptosis level between LEF1-AS1-overexpressing cells and control cells in basal culture conditions; however, LEF1-AS1 over-expression inhibited significantly serum deprivation-induced apoptosis (reduction of 50%, p<0.05).We screened several apoptosis and proliferation regulators by RT-PCR and western blotting and detected a strong increase in anti-apoptotic BCL2 and CDKN1A/p21 in HL60 cells over-expressing LEF1-AS1. This increase was observed in cells in exponential growth and starvation. Interestingly, we did not observe this antiapoptotic effect in K562, a cell line that do not express BCL2 endogenously.To confirm that the phenotypical effects of lef1-AS over -expression were not mediated by LEF1 function, we also over-expressed lef1-AS1 in Hela, a cell line lacking endogenous expression of LEF1. Empty vector and lef1-AS1-Hela stably-transfected-cells had no detectable expression of LEF1; however, a similar inhibition of cell proliferation and S-phase accumulation were observed.To further investigate the mechanism behind these phenotypical effects we carried out a RNA-pulldown experiment coupled with mass spectrometry to identify the proteins that bind to LEF1-AS1. Our preliminary results identified an apoptosis regulator protein in association to this transcript and we are currently working to unravel the implications of this interaction.Remarkably, BCL2 is simultaneously antiapoptotic and anti-proliferative and S-phase arrest has been associated to p21 up-regulation. Our results depict LEF1-AS1 as a fine-tuner of proliferation and survival decisions, acting, at least partially, through modulation of BCL2 and CDKN1A/p21. DisclosuresNo relevant conflicts of interest to declare.

PTEN mediates serum deprivation-induced cytotoxicity in H9c2 cells via the PI3K/AKT signaling pathway

The pathogenesis of acute myocardial infarction (AMI) is associated with cardiomyocyte necrosis and apoptosis. Numerous studies have determined the regulatory effects of Phosphatase and tensin homolog (PTEN) cell proliferation and apoptosis in other cell types. However, the potential role of PTEN in cardiomyocyte is unclear. In this study, we used H9c2 cells cultured under serum deprivation to simulate the apoptosis process of myocardial infarction. Small interference RNA (siRNA) of PTEN was used to knock down the expression of PTEN. Cell viability was determined by CCK-8. Cell proliferation was examined by Edu staining, and the protein expression was analyzed by Western blot. We also evaluated the generation of ROS, the degree of DNA damage, and cell apoptosis using immunofluorescence assay. As a result, we observed that serum deprivation in H9c2 cells increased PTEN expression. Functionally, the PTEN knockdown experiment using siRNA inhibited serum deprivation-induced cell apoptosis, ROS production, and DNA damage, whereas increased cell proliferation. All these effects could be reversed by phosphatidylinositol 3-kinase (PI3K) inhibitor, which indicated the PI3K/protein kinase B (AKT) might be the critical component of the PTEN effects during serum deficiency. In conclusion, our study indicated the role of the PTEN/PI3K/AKT pathway in serum deprivation-induced cytotoxicity in H9c2 cells.

Liraglutide regulates proliferation, differentiation, and apoptosis of preosteoblasts through a signaling network of Notch/Wnt/Hedgehog signaling pathways.

This study aims to investigate whether liraglutide can affect proliferation, osteogenic differentiation and serum deprivation-induced apoptosis of preosteoblast cell line MC3T3-E1 through the Notch, Wnt/β-catenin, and Hedgehog (Hh) signaling pathways. MC3T3-E1 cells were exposed to different treatments (via Notch inhibitor DAPT, an Hh inhibitor cyclopamine, or serum deprivation) or transfections of different siRNAs (targeting glucagon-like peptide-1 receptor (GLP-1R), β-catenin, or Gli1) in the presence or absence of 100 nM liraglutide. Cell proliferation, mRNA levels of osteogenic differentiation-related genes, mRNA and protein levels of the Notch and Hh signaling pathway proteins, and apoptosis-related proteins were assessed. Liraglutide significantly increased proliferation of MC3T3-E1 cells, expression levels of the Notch and Hh signaling pathway proteins and β-catenin, and mRNA levels of osteogenic differentiation-related genes and TCF7L2. Moreover, liraglutide promoted a translocation of β-catenin, increased a ratio of Bcl-2/Bax proteins, reduced serum deprivation-induced apoptosis of MC3T3-E1 cells, and a ratio of caspase-3/procaspase-3. However, a cotreatment with liraglutide and DAPT reversed the alterations. A cyclopamine treatment and knockdowns of GLP-1R, Gli1, and β-catenin significantly reduced the expression of Notch proteins. Furthermore, the knockdown of GLP-1R, β-catenin, or Gli1 significantly increased apoptosis, which could be inhibited by liraglutide. In summary, liraglutide can promote proliferation and differentiation of MC3T3-E1 cells, and inhibit their serum deprivation-induced apoptosis by activating both the Notch and Hh signaling pathways involving β-catenin and Gli1. These results provide a therapeutic foundation that patients with diabetes and osteoporosis may be cured with treatments of liraglutide.

Corrigendum] Nicorandil protects mesenchymal stem cells against hypoxia and serum deprivation-induced apoptosis

After the publication of the above article, the authors have realized that Figs.2 and 4 in their paper were published with incorrect images; regarding Fig.2, the data featured in Fig.2A (for the H/SD+Nico 1000µM panel) were repeated with those featured in Fig.1C (the 6h H/SD panel), and the data shown for Bcl-2 in Fig.4C were selected incorrectly. These errors arose inadvertently as a consequence of misassembling the figures. The revised versions of Figs.2 and 4, featuring the corrected data panels for the above‑mentioned experiments, are shown on the next page. Note that the revised data shown for these Figures do not affect the overall conclusions reported in the paper. The authors express their gratitude to the Editor of International Journal of Molecular Medicine for allowing them the opportunity to publish this corrigendum, and apologize to the readership for any inconvenience caused. [the original article was published in International Journal of Molecular Medicine 36: 415-423, 2015; DOI: 10.3892/ijmm.2015.2229].

Protective effects of acetyl-l-carnitine against serum and glucose deprivation-induced apoptosis in rat adipose-derived mesenchymal stem cells

Low survival rate of grafted mesenchymal stem cells (MSC) in injured tissue is one of the major limitations of stem cell therapy. One of the most important factors that limits the MSCs survival rate and retention is ischemic stress, which can lead to damage to all components of the cell. In particular, it can damage mitochondria, that play an important role in apoptosis with releasing apoptotic factors. Therefore, we investigated the protective effects of Acetyl-L-carnitine (ALCAR) against serum and glucose deprivation (SGD) in adipose-derived mesenchymal stem cells (AD-MSCs). We measured cell viability, proliferation, and apoptosis in cells experiencing SGD stress for 8 h with exposure to varying concentrations of ALCAR. Results showed that ALCAR protects cells against SGD stress by reducing apoptosis. Its protective effects are associated with reductions in cleaved caspase-3 and attenuation of apoptosis. Result showed that ALCAR exhibits protective effects against SGD-induced damage to AD-MSCs by enhancing the expression of survival signals and by decreasing the expression of death signals.

A novel role of vitronectin in promoting survival of mesenchymal stem cells under serum deprivation stress

BackgroundDue to their immunomodulatory and trophic support functions, mesenchymal stem cells (MSCs) are promising in the field of cell-based regenerative medicine. However, MSC survival post-transplantation is challenged by various microenvironment stress factors. Here, we investigated the role of vitronectin (VTN) in the survival strategy of MSCs under serum deprivation stress condition.MethodsProliferation kinetics and cell adhesion of MSCs under serum deprivation were determined from population doublings and cell-matrix de-adhesion studies, respectively. mRNA and protein expression levels of VTN were confirmed by qRT-PCR and Western blotting, respectively. Immunofluorescence technique revealed distribution of VTN under serum deprivation stress. siRNA and inhibitor-based studies were performed to confirm the role and regulation of VTN. Apoptosis and cell cycle status of MSCs were assessed using flow cytometric analysis.ResultsSubjecting MSCs to serum deprivation led to significant increase in cell spread area and cell-matrix adhesion. An upregulation of VTN expression was noted with an arrest in G0/G1 phase of cell cycle and no appreciable apoptotic change. Pro-survival PI3kinase pathway inhibition led to further increase in VTN expression with no apoptotic change. siRNA-mediated inhibition of VTN resulted in reversal in G0/G1 cell cycle arrest and a marked increase in apoptosis, suggesting a role of VTN in preventing serum deprivation-induced apoptotic cell death. In addition, p65 knockdown resulted in downregulation of VTN establishing an association between NF-κβ pathway and VTN.ConclusionsVTN was identified as a survival factor in providing protection from serum deprivation-induced apoptosis in MSCs.

Histone demethylase Jmjd3 modulates osteoblast apoptosis induced by tumor necrosis factor-alpha through directly targeting RASSF5

ABSTRACT Purpose: Regulation of gene expression is fine-tuned by a dynamic equilibrium between repressive modifications and transcriptional activation of histone tails. Jumonji domain-containing 3 (Jmjd3), also known as KDM6B, is a specific histone demethylase for trimethylation on histone H3 lysine 27 (H3K27me3) that specifically removes the methylation of H3K27me3 and promotes gene expression. Our previous study showed that Jmjd3 inhibits serum deprivation-induced osteoblast apoptosis. In this study, we clarified the role of Jmjd3 in tumor necrosis factor-alpha (TNF-α)-induced osteoblast apoptosis. Materials and Methods: Jmjd3 activity was inhibited by GSK-J4. Transfection of osteoblastic murine MC3T3-E1 cells with short hairpin RNA (shRNA) was used to establish stable Jmjd3 knockdown cells. Osteoblast apoptosis was detected using Annexin V-APC/PI staining, cysteinyl aspartate specific protease-3 (caspase-3) activity assays, and Western blot. Real-time polymerase chain reaction (PCR) and chromatin immunoprecipitation (ChIP) assays were performed to clarify the mechanism responsible for Jmjd3-regulated osteoblast apoptosis induced by TNF-α. Results: Based on Annexin V-APC/PI staining, caspase-3 activation, and poly ADP-ribose polymerase (PARP) cleavage, pretreatment with GSK-J4 and knockdown of Jmjd3 by shRNA transfection each inhibited osteoblast apoptosis. Furthermore, knockdown of Jmjd3 decreased the expression of Ras association domain family 5 (RASSF5), which is a pro-apoptotic gene of the Ras associated domain family. H3K27me3 levels in the promoter region of RASSF5 were up-regulated in the Jmjd3 knockdown cells. Conclusions: Jmjd3 regulated TNF-α-induced osteoblast apoptosis by targeting RASSF5.

The involvement of regulated in development and DNA damage response 1 (REDD1) in the pathogenesis of intervertebral disc degeneration

Regulated in development and DNA damage response 1 (REDD1) is an evolutionarily conserved, ubiquitous protein that responds to various cell stresses. Studies have proved REDD1 is involved in many diseases, such as osteoarthritis and cancer. The present study aimed to investigate the potential role of REDD1 in the pathogenesis of intervertebral disc degeneration (IDD). Analysis of clinical tissue samples showed REDD1 expression was up-regulated during IDD and was correlated with the grade of disc degeneration. Overexpression of REDD1 in normal human nucleus pulposus (NP) cells resulted in extracellular matrix (ECM) degeneration. Further, we investigated the function of REDD1 using a serum deprivation-induced IDD vitro model and found that REDD1 was up-regulated in a temporal manner. However, hypoxia abolished this increase through down-regulation of NF-κB. Knockdown of REDD1 or NF-κB by si-RNA significantly rescued ECM from degeneration both in normoxia and hypoxia. In addition, NF-κB/REDD1 mediated the protection of hypoxia from serum deprivation-induced apoptosis and autophagy in NP cells. These results suggest that REDD1 might play a pivotal role in IDD pathogenesis, thereby potentially providing a new therapeutic target for IDD treatment.

NGAL promotes recruitment of tumor infiltrating leukocytes.

We have previously shown that Neutrophil Gelatinase-Associated Lipocalin (NGAL) is strongly expressed in thyroid carcinomas, especially of anaplastic type, where it protects neoplastic cells from serum deprivation-induced apoptosis and enhances tumor invasivity by regulating MMP-9 activity. Here we demonstrate that NGAL-containing conditioned medium from human anaplastic thyroid carcinoma (ATC) cells is able to induce monocyte migration via up-regulation of a number of different chemokines. The enhanced chemokines transcription is due to the NGAL-mediated intracellular iron uptake. Very importantly, mice tumor allografts raised from subcutaneous injection of syngeneic colon carcinoma cell lines, expressing high levels of NGAL, show a dense leukocyte infiltrate which strongly decreases in tumor allografts from NGAL-depleted cell injected mice.Our results indicate that the NGAL promotes leukocytes recruitment in tumor microenvironment through iron-mediated chemokines production.

17β-estradiol protects nucleus pulposus cells from serum deprivation-induced apoptosis and regulates expression of MMP-3 and MMP-13 through promotion of autophagy

Serum deprivation is a likely contributor to intervertebral disc (IVD) degeneration (IVDD).17β-estradiol (E2) have been noted to protect nucleus pulposus cells (NPCs) against apoptosis. Autophagy and apoptosis play a paramount role in maintaining the homeostasis of IVD. So far, little research has been published on whether autophagy plays a role for the E2 mediated protection of NPCs. The aim of this study is to understand whether autophagy is involved in the protective effect of E2 against serum deprivation-induced cell apoptosis and expression of matrix metalloproteinase (MMP)-3 and MMP-13. mCherry-GFP-LC3-adenovirus transfection is used to monitor autophagy detection. The expression levels of autophagy-related proteins were measured by Western blotting, Apoptosis and MMPs were detected by flow cytometry and Western blotting. Accordingly, Autophagy and apoptosis was detected in NP cells under serum deprivation conditions, the autophagy incidence began to reached a peak value at 48 h, the apoptosis and MMPs incidence began reached a minimum value treat with E2 (10−7 M). Whereas the combined use of E2 and 3-MA led to a dramatic decrease in autophagy, while aberrantly elevated expression levels of apoptotic and MMPs. These data suggest that serum deprivation-induced apoptosis and MMP-3, MMP-13, which was efficiently suppressed by the E2 through promoting autophagy in rat NPCs.

Human Urine Extract Cell Differentiation Agent 2 Protects PC12 Cells from Serum Deprivation-Induced Apoptosis Accompanied with Priming of Extracellular Signal-Regulated Kinase Activation and Differentiation Induction.

To investigate the potential neuroprotective effect of human urine extract cell differentiation agent 2 (CDA-2) by the model of serum deprivation-induced apoptosis of PC12 cells and study the underlying molecular mechanisms. Apoptosis of PC12 cells was induced by serum deprivation. CDA-2 at doses of 0.5-4 mg/mL was used to treat the serum-deprived PC12 cells. The cellular viability was measured by sulforhodamine B binding assay and the cell apoptosis was determined by flow cytometer. Western blot was used to analyze the expression of differentiation markers and activity of extracellular signal-regulated kinase (ERK). The cellular morphology was examined under an inverted microscope. CDA-2 inhibited apoptotic cell death of serum-deprived PC12 cells in a dose-dependent manner. Expression of low- and mid-sized neurofilaments was observed in serum-deprived PC12 cells treated with CDA-2 or nerve growth factor (NGF). However, CDA-2 did not induce proliferation of these cells like NGF. The morphology of CDA-2 treated cells was changed from rounded to neuron-like flat polygonal shape in contrast to the extensive neurite outgrowth induced by NGF. CDA-2 transiently induced the phosphorylation of ERK in serum deprived-PC12 cells and the expression of neurofilaments induced by CDA-2 was attenuated by mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) inhibitor PD98059. Human urine extract CDA-2 showed a potential neuroprotective activity which may correlate with ERK activation and differentiation induction.

Ginsenoside Rg1 inhibits apoptosis by increasing autophagy via the AMPK/mTOR signaling in serum deprivation macrophages.

Ginsenoside Rg1 (Rg1) has been widely used in a broad range of cardiovascular and cerebral-vascular diseases because of its unique therapeutic properties. However, the underlying mechanisms of Rg1 in the treatment of atherosclerosis have not been fully explored. This study sought to determine the precise molecular mechanisms on how Rg1 might be involved in regulating apoptosis in serum deprivation-induced Raw264.7 macrophages and primary bone marrow-derived macrophages. Results demonstrated that Rg1 treatment effectively suppressed apoptosis and the expression of phosphorylation level of mTOR induced by serum deprivation in Raw264.7 macrophages; the expressions of autophagic flux-related proteins including Atg5, Beclin1, microtubule-associated protein 1 light chain 3 (LC3), p62/SQSMT1, and the phosphorylation level of AMPK were concomitantly up-regulated. 3-Methyl-adennine (3-MA), the most widely used autophagy inhibitor, strongly up-regulated the expression of cleaved caspase-3, and blocked the anti-apoptosis function of Rg1 in macrophages. Importantly, autophagic flux was activated by Rg1, while Beclin1 knockdown partially abolished the anti-apoptosis of Rg1. Moreover, compound C, an AMPK inhibitor, partially decreased the expressions of phosphorylation of mTOR, Atg5, Beclin1, LC3, and p62/SQSMT1, which were increased by Rg1. AICAR, an AMPK inducer, promoted the protein expressions of phosphorylation of mTOR, Atg5, Beclin1, LC3, and p62/SQSMT1. In conclusion, Rg1 significantly suppressed apoptosis induced by serum deprivation in macrophages. Furthermore, Rg1 could effectively induce the autophagic flux by attenuating serum deprivation-induced apoptosis in Raw264.7 macrophages through activating the AMPK/mTOR signaling pathway.

Inhibition of retinal ganglion cell apoptosis: regulation of mitochondrial function by PACAP.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an endogenous peptide with neuroprotective effects on retinal neurons, but the precise mechanism underlying these effects remains unknown. Considering the abundance of mitochondria in retinal ganglion cells (RGCs), we postulate that the protective effect of PACAP is associated with the regulation of mitochondrial function. RGC-5 cells were subjected to serum deprivation for 48 hours to induce apoptosis in the presence or absence of 100 nM PACAP. As revealed with the Cell Counting Kit-8 assay, PACAP at different concentrations significantly increased the viability of RGC-5 cells. PACAP also inhibited the excessive generation of reactive oxygen species in RGC-5 cells subjected to serum deprivation. We also showed by flow cytometry that PACAP inhibited serum deprivation-induced apoptosis in RGC-5 cells. The proportions of apoptotic cells and cells with mitochondria depolarization were significantly decreased with PACAP treatment. Western blot assays demonstrated that PACAP increased the levels of Bcl-2 and inhibited the compensatory increase of PAC1. Together, these data indicate protective effects of PACAP against serum deprivation-induced apoptosis in RGCs, and that the mechanism of this action is associated with maintaining mitochondrial function.

Is testosterone important for women as they age?

In women, is testosterone an obligatory precursor for extra-gonadal estradiol production after menopause, and has important direct androgenic genomic and nongenomic effects. Testosterone levels in women decline with age from the 4th decade, reaching a nadir in the 7th decade of life. Testosterone levels are positively associated with sexual function in women, and multiple randomised placebo-controlled trials have shown that testosterone therapy can be effective for the treatment of female sexual dysfunction in postmenopausal women. However, low testosterone in ageing women has multiple potential undesirable consequences that have been somewhat ignored. In the brain, testosterone exhibits neuroprotection against oxidative stress, serum deprivation-induced apoptosis and soluble amyloid beta toxicity, and clinical trials suggest exogenous testosterone enhances cognitive performance in postmenopausal women. Lower free testosterone in older women is associated with a decline in bone mineral density and a greater risk of hip fracture. Observational studies implicate testosterone as having favourable cardiovascular effects measured by surrogate outcomes, however associations between endogenous testosterone and CVD risk, and total mortality, particularly in older women are yet to be established.

A miRNA-101-3p/Bim axis as a determinant of serum deprivation-induced endothelial cell apoptosis

Serum deprivation or withdrawal induces apoptosis in endothelial cells, resulting in endothelial cell dysfunction that is associated with cardiovascular disease. However, there is still limited information on the role of miRNA in serum deprivation-induced apoptosis. Here we found that serum deprivation increased caspase-dependent apoptosis through miRNA-101-3p downregulation, without altering expression of its host gene RNA 3′-terminal phosphate cyclase-like 1, which was highly correlated with suppressed expression levels of Dicer and Argonaute 2 (Ago2), indicating that miR-101-3p is post-transcriptionally elevated in serum-deprived conditions. The decreased miR-101-3p caused elevated Bim expression by targeting its 3′-untranslated region (3′-UTR). This resulted in activation of the intrinsic pathway of apoptosis via interaction with Bcl-2, decreased mitochondrial membrane potential, cytochrome c release, mitochondrial reactive oxygen species (ROS) production, and caspase activation. These events were abrogated by miR-101-3p mimic and the proapoptotic Bim siRNA, which suggest a determinant role of the miR-101-3p/Bim axis in serum deprivation-induced apoptosis. The apoptosis induced by miR-101-3p-mediated Bim expression is mediated by both caspase-3 and -1, which are activated by two distinct intrinsic mechanisms, cytochrome c release and ROS-induced inflammasome activation, respectively. In other words, the antioxidant inhibited endothelial cell death mediated by caspase-1 that activated caspase-7, but not caspase-3. These findings provide mechanistic insight into a novel function of miR-101-3p in serum withdrawal-induced apoptosis triggered by activating two different intrinsic or mitochondrial apoptosis pathways, implicating miR-101-3p as a therapeutic target that limits endothelial cell death associated with vascular disorders.

BNN27, a 17-Spiroepoxy Steroid Derivative, Interacts With and Activates p75 Neurotrophin Receptor, Rescuing Cerebellar Granule Neurons from Apoptosis.

Neurotrophin receptors mediate a plethora of signals affecting neuronal survival. The p75 pan-neurotrophin receptor controls neuronal cell fate after its selective activation by immature and mature isoforms of all neurotrophins. It also exerts pleiotropic effects interacting with a variety of ligands in different neuronal or non-neuronal cells. In the present study, we explored the biophysical and functional interactions of a blood-brain-barrier (BBB) permeable, C17-spiroepoxy steroid derivative, BNN27, with p75NTR receptor. BNN27 was recently shown to bind to NGF high-affinity receptor, TrkA. We now tested the p75NTR-mediated effects of BNN27 in mouse Cerebellar Granule Neurons (CGNs), expressing p75NTR, but not TrkA receptors. Our findings show that BNN27 physically interacts with p75NTR receptors in specific amino-residues of its extracellular domain, inducing the recruitment of p75NTR receptor to its effector protein RIP2 and the simultaneous release of RhoGDI in primary neuronal cells. Activation of the p75NTR receptor by BNN27 reverses serum deprivation-induced apoptosis of CGNs resulting in the decrease of the phosphorylation of pro-apoptotic JNK kinase and of the cleavage of Caspase-3, effects completely abolished in CGNs, isolated from p75NTR null mice. In conclusion, BNN27 represents a lead molecule for the development of novel p75NTR ligands, controlling specific p75NTR-mediated signaling of neuronal cell fate, with potential applications in therapeutics of neurodegenerative diseases and brain trauma.

Protein phosphatase 2A Cα regulates proliferation, migration, and metastasis of osteosarcoma cells

Osteosarcoma is the most frequent primary bone tumor. Serine/threonine protein phosphatase 2A (PP2A) participates in regulating many important physiological processes, such as cell cycle, growth, apoptosis, and signal transduction. In this study, we examined the expression and function of PP2A Cα in osteosarcoma cells. PP2A Cα expression was expected to be higher in malignant osteosarcoma tissues. PP2A Cα expression level and PP2A activity was higher in malignant osteosarcoma LM8 cells compared with that in primary osteoblasts and in the osteoblast-like cell line MC3T3-E1. Okadaic acid, an inhibitor of PP2A, reduced cell viability and induced apoptosis in LM8 cells. PP2A Cα-knockdown LM8 cells (shPP2A) exhibited less striking filopodial and lamellipodial structures than that in original LM8 cells. Focal adhesion kinase phosphorylation and NF-κB activity decreased in shPP2A-treated cells. Sensitivity to serum deprivation-induced apoptosis increased in shPP2A-treated cells, accompanied by a lower expression level of anti-apoptotic BCL-2 in these cells. Reduction of PP2A Cα resulted in a decrease in the migration ability of LM8 cells in vitro. Reduction in PP2A Cα levels in vivo suppressed proliferation and metastasis in LM8 cells. PP2A Cα expression was also higher in human osteosarcoma MG63 and SaOS-2 cells than that in primary osteoblasts and MC3T3-E1 cells, and reduction in PP2A Cα levels suppressed the cell proliferation rate and migration ability of MG63 cells. These results indicate that PP2A Cα has a critical role in the proliferation and metastasis of osteosarcoma cells; therefore, its inhibition could potentially suppress the malignancy of osteosarcoma cells.

CD317 Promotes the survival of cancer cells through apoptosis-inducing factor.

BackgroundLow nutrient environment is a major obstacle to solid tumor growth. However, many tumors have developed adaptive mechanisms to circumvent the requirement for exogenous growth factors.MethodsHere we used siRNA interference or plasmid transfection techniques to knockdown or enhance CD317 expression respectively, in mammalian cancer cells, and subjected these CD317-manipulated cells to serum deprivation to study the role of CD317 on stress-induced apoptosis and the underlying mechanism.ResultsWe report that CD317, an innate immune gene overexpressed in human cancers, protected cancer cells against serum deprivation-induced apoptosis. In tumor cells, loss of CD317 markedly enhanced their susceptibility to serum deprivation-induced apoptosis with no effect on autophagy or caspase activation, indicating an autophagy- and caspase-independent mechanism of CD317 function. Importantly, CD317 knockdown in serum-deprived tumor cells impaired mitochondria function and subsequently promoted apoptosis-inducing factor (AIF) release and nuclear translocation but had little effect on mitochondrial and cytoplasmic distributions of cytochrome C, a pro-apoptotic factor released from mitochondria that initiates caspase processing in response to death stimuli. Furthermore, overexpression of CD317 in HEK293T cells inhibits serum deprivation-induced apoptosis as well as the release and nuclear accumulation of AIF.ConclusionOur data suggest that CD317 functions as an anti-apoptotic factor through the mitochondria-AIF axis in malnourished condition and may serve as a potential drug target for cancer therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0391-2) contains supplementary material, which is available to authorized users.