Comparison of Glioblastoma Cell Culture Platforms Based on Transcriptional Similarity with Paired Tissue.

No standardized in vitro cell culture models for glioblastoma (GBM) have yet been established, excluding the traditional two-dimensional culture. GBM tumorspheres (TSs) have been highlighted as a good model platform for testing drug effects and characterizing specific features of GBM, but a detailed evaluation of their suitability and comparative performance is lacking. Here, we isolated GBM TSs and extracellular matrices (ECM) from tissues obtained from newly diagnosed IDH1 wild-type GBM patients and cultured GBM TSs on five different culture platforms: (1) ordinary TS culture liquid media (LM), (2) collagen-based three-dimensional (3D) matrix, (3) patient typical ECM-based 3D matrix, (4) patient tumor ECM-based 3D matrix, and (5) mouse brain. For evaluation, we obtained transcriptome data from all cultured GBM TSs using microarrays. The LM platform exhibited the most similar transcriptional program to paired tissues based on GBM genes, stemness- and invasiveness-related genes, transcription factor activity, and canonical signaling pathways. GBM TSs can be cultured via an easy-to-handle and cost- and time-efficient LM platform while preserving the transcriptional program of the originating tissues without supplementing the ECM or embedding it into the mouse brain. In addition to applications in basic cancer research, GBM TSs cultured in LM may also serve as patient avatars in drug screening and pre-clinical evaluation of targeted therapy and as standardized and clinically relevant models for precision medicine.

A lignan from Alnus japonica inhibits glioblastoma tumorspheres by suppression of FOXM1.

Forkhead Box M1 (FOXM1) is known to regulate cell proliferation, apoptosis and tumorigenesis. The lignan, (-)-(2R,3R)-1,4-O-diferuloylsecoisolariciresinol (DFS), from Alnus japonica has shown anti-cancer effects against colon cancer cells by suppressing FOXM1. The present study hypothesized that DFS can have anti-cancer effects against glioblastoma (GBM) tumorspheres (TSs). Immunoprecipitation and luciferase reporter assays were performed to evaluate the ability of DFS to suppress nuclear translocation of ß-catenin through ß-catenin/FOXM1 binding. DFS-pretreated GBM TSs were evaluated to assess the ability of DFS to inhibit GBM TSs and their transcriptional profiles. The in vivo efficacy was examined in orthotopic xenograft models of GBM. Expression of FOXM1 was higher in GBM than in normal tissues. DFS-induced FOXM1 protein degradation blocked ß-catenin translocation into the nucleus and consequently suppressed downstream target genes of FOXM1 pathways. DFS inhibited cell viability and ATP levels, while increasing apoptosis, and it reduced tumorsphere formation and the invasiveness of GBM TSs. And DFS reduced the activities of transcription factors related to tumorigenesis, stemness, and invasiveness. DFS significantly inhibited tumor growth and prolonged the survival rate of mice in orthotopic xenograft models of GBM. It suggests that DFS inhibits the proliferation of GBM TSs by suppressing FOXM1. DFS may be a potential therapeutic agent to treat GBM.

Self-Weighing Behaviors of Diverse Community-Dwelling Adults Motivated for a Lifestyle Change.

We aimed to understand adults' self-weighing behaviors and explore significant predictors of body mass index (BMI) accuracy based on self-reported height and weight in a diverse sample of community-dwelling adults. Methods: In this cross-sectional study, 531 adults participating in a physical activity program or a weight loss program were analyzed. Participants' self-reported and objectively measured weight, height, weight scale ownership, self-weighing behaviors, and medical history were collected. Results: The mean age (standard deviation) was 50.0 (12.0) years with a range of 24 to 78 years. Out of 531 participants, 455 (85.7%) were women. The study population was diverse (58.9% non-White). In total, 409 (77.0%) participants had a weight scale at home, but only 222 (41.8%) weighed themselves at least once a week. The weight and BMI underestimation became much more significant as the participant's weight increased (p ≤ 0.001). Employment status, high cholesterol, and low objectively measured weight were significant predictors of self-reported BMI accuracy after controlling for potential confounding factors (p < 0.05). Interestingly, ownership of a home weight scale and the frequency of self-weighing behavior were not significantly associated with the accuracy of self-reported BMI (p > 0.05). Conclusion: The accuracy of the participants' BMI, based on self-reported height and weight, was significantly associated with employment status, high cholesterol, and low objectively measured weight, suggesting that BMI accuracy depends on multi factors.

[Corrigendum] Neurotoxin ß­N­methylamino­L­alanine induces endoplasmic reticulum stress­mediated neuronal apoptosis.

Following the publication of the above article and a Corrigendum published in July 2018, the authors have noted an additional error, associated with the presentation of Fig. 1C. Fig 1C showed that ß­N­methylamino­L­alanine induces neuronal apoptotic cell death; however, an error was made in the compilation of this figure and an incorrect band image was selected for α­actinin, the loading control panel for Fig. 1C. A corrected version of Fig. 1 is shown opposite, incorporating the correct α­actinin protein bands in Fig. 1C. This change affects neither the interpretation of the data nor conclusions of this work. We regret that this further error went unnoticed at the time, and thank the Editor for allowing us the opportunity to publish this additional Corrigendum. [the original article was published in the Molecular Medicine Reports 14: 4873­4880, 2016; DOI: 10.3892/mmr.2016.5802].

[Corrigendum] Neurotoxin ß­N­methylamino­L­alanine induces endoplasmic reticulum stress­mediated neuronal apoptosis.

Following the publication of the article, the authors noted an error associated with the presentation of Fig. 4A. Fig 4 showed that overexpression of HSP70 suppresses ER stress-mediated neuronal death induced by ß­N­methylamino­L­alanine (BMAA). An error was made in the compilation of this Figure, and the band images shown in the HA panel for Fig. 1A were selected incorrectly. A corrected version of Fig. 4 is shown below. This change affects neither the interpretation of the data nor conclusions of this work. We regret that this error occurred, and thank the Editor for allowing us the opportunity to publish this Corrigendum. [the original article was published in the Molecular Medicine Reports 14: 4873-4880, 2016; DOI: 10.3892/mmr.2016.5802].

Neurotoxin ß­N­methylamino­L­alanine induces endoplasmic reticulum stress­mediated neuronal apoptosis.

ß-N-methylamino-L-alanine (BMAA) is a neurotoxin that is closely associated with the incidence of amyotrophic lateral sclerosis, Parkinson's disease and Alzheimer's disease. In cultured neuronal cells, BMAA notably induces the upregulation of endoplasmic reticulum (ER) chaperons and activates the unfolded protein response (UPR) receptor pathways of protein kinase RNA­like endoplasmic reticulum kinase, inositol­requiring kinase 1 and transcription factor 6. The ER stress­specific protein CCAAT/­enhancer­binding protein homologous protein (CHOP) affords pro­apoptotic responses that cause mitochondrial damage and caspase activation. BMAA also induces the activation of mitogen­activated protein kinase member c­JUN N­terminal kinase, p38 and extracellular signal­regulated kinase, which have been suggested to be involved in the signaling pathway of UPR­mediated apoptosis. Inhibition of ER stress using ER stress antagonist, salubrinal, attenuated the expression of CHOP and alleviated neuronal death. Overexpression of heat shock protein 70 suppressed the activation of UPR receptors and UPR­evoked apoptotic signaling. The present findings demonstrated that ER stress induced by BMAA is the important mediator of neuronal injury and apoptotic death, and suggests development in novel therapeutic strategies for treatment.

Cyclophilin B protects SH-SY5Y human neuroblastoma cells against MPP(+)-induced neurotoxicity via JNK pathway.

Parkinson's disease (PD) is the second most common neurodegenerative disorder of aging. PD involves a progressive loss of dopaminergic neurons in the substantia nigra pars compacta. 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyidine (MPTP) and its toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) inhibit the complex I of the mitochondrial electron transport chain, and have been widely used to construct PD models. Cyclophilin B (CypB) is an endoplasmic reticulum protein that binds to cyclosporine A as a cyclophilin family member. CypB has peptidyl-prolyl cis-trans isomerase (PPIase) activity. We investigated the protective effects of overexpressed CypB on MPP+-induced neurocytotoxicity in SH-SY5Y human neuroblastoma cells. Overexpressed CypB decreased MPP(+)-induced oxidative stress through the modulation of antioxidant enzymes including manganese superoxide dismutase and catalase, and prevented neurocytotoxicity via mitogen-activated protein kinase, especially the c-Jun N-terminal kinase pathway. In addition, CypB inhibited the activation of MPP(+)-induced the pro-apoptotic molecules poly (ADP-ribose) polymerase, Bax, and Bcl-2, and attenuated MPP(+)-induced mitochondrial dysfunction. The data suggest that overexpressed CypB protects neuronal cells from MPP+-induced dopaminergic neuronal cell death.

Hypoxia induces cyclophilin B through the activation of transcription factor 6 in gastric adenocarcinoma cells.

Hypoxia is an important form of physiological stress that induces cell death, due to the resulting endoplasmic reticulum (ER) stress, particularly in solid tumors. Although previous studies have indicated that cyclophilin B (CypB) plays a role in ER stress, there is currently no direct information supporting the mechanism of CypB involvement under hypoxic conditions. However, it has previously been demonstrated that ER stress positively regulates the expression of CypB. In the present study, it was demonstrated that CypB is transcriptionally regulated by hypoxia-mediated activation of transcription factor 6 (ATF6), an ER stress transcription factor. Subsequently, the effects of ATF6 on CypB promoter activity were investigated and an ATF6-responsive region in the promoter was identified. Hypoxia and ATF6 expression each increased CypB promoter activity. Collectively, these results demonstrate that ATF6 positively regulates the expression of CypB by binding to an ATF6-responsive region in the promoter, which may play an important role in the attenuation of apoptosis in the adaption to hypoxia. These results suggest that CypB may be a key molecule in the adaptation of cells to hypoxic conditions.

Cyclophilin A regulates JNK/p38-MAPK signaling through its physical interaction with ASK1.

Cyclophilin A (CypA), a member of the immunophilin family, is predominantly localized in the cytoplasm. The peptidylprolyl isomerase (PPIase) activity of CypA has been demonstrated to be involved in diverse cellular processes, including intracellular protein trafficking, mitochondrial function, pre-mRNA processing, and maintenance of multiprotein complex stability. In this study, we have demonstrated that CypA regulates apoptosis signaling-regulating kinase 1 (ASK1) through its direct binding. ASK1 is a member of MAPK kinase kinase (MAP3K) family, and selectively activates both JNK and p38 MAPK pathways. Here, we also report that CypA negatively regulates phosphorylation of ASK1 at Ser966, and that CypA reduces ASK1 and its downstream kinases of the JNK and p38 signaling. ASK1 is known to induce caspase-3 activation and apoptosis, and CypA inhibited ASK1-mediated apoptosis by decrease in caspase-3 activity under cellular stress conditions. Overall, we conclude that CypA negatively regulates ASK1 functions by its physical interaction with ASK1.

p53 negatively regulates Pin1 expression under ER stress.

Accumulating evidence suggests that endoplasmic reticulum (ER) stress plays a major role in the development of many diseases. A previous study indicated that the apoptotic regulator p53 is significantly increased in response to ER stress and participates in ER stress-induced apoptosis. However, the regulators of p53 expression during ER stress are still not fully understood. Here, we investigated whether p53 contributes to the impairment of Pin1 signaling under ER stress. We found that treatment with thapsigargin, a stimulator of p53 expression and an inducer of ER stress, decreased Pin1 expression in HCT116 cells. Also, we identified functional p53 response elements (p53REs) in the Pin1 promoter. Overexpression of p53 significantly decreased Pin1 expression in HCT116 cells while abolition of p53 gene expression induced Pin1 expression. Pin1 expression was significantly increased by treatment with the p53 inhibitor pifithrin-α or down-regulation of p53 expression. Taken together, ER stress decreased Pin1 expression through p53 activation, and this mechanism may be associated with ER stress-induced cell death. These data reported here support the importance of Pin1 as a potential target molecule mediating tumor development.

Apelin is transcriptionally regulated by ER stress-induced ATF4 expression via a p38 MAPK-dependent pathway.

Apelin, which is an endogenous ligand for the orphan G-protein-coupled receptor APJ, was reported to be up-regulated by hypoxia-inducible factor 1-α (HIF1-α) in hypoxia- and insulin-treated cell systems. However, a negative transcriptional regulator of apelin has not yet been identified. In this study, we showed that apelin is down-regulated by ATF4 via the pro-apoptotic p38 MAPK pathway under endoplasmic reticulum (ER) stress. First, we analyzed the human apelin promoter to characterize the effects of ER stress on apelin expression in hepatocytes. Treatment with thapsigargin, an inducer of ER stress, and over-expression of ATF4 decreased apelin expression in hepatocytes. This work identified an ATF4-responsive region within the apelin promoter. Interestingly, ATF4-mediated repression of apelin was dependent upon the N-terminal domain of ATF4. C/EBP-ß knockdown experiments suggest that C/EBP-ß, which acts as an ATF4 binding partner, is critical for the ER stress-induced down-regulation of apelin. We also demonstrated that ATF4 regulates apelin gene expression via p38 pathways. Ectopic expression of constitutively active MKK6, an upstream kinase of p38, suggested that activation of the p38 pathway is sufficient to induce ATF4-mediated repression of apelin. Moreover, apelin enhanced cell migration in a wound healing assay in a p38 MAPK-dependent manner. Furthermore, analysis of caspase-3 activation indicated that ATF4 knockdown up-regulated apelin expression, leading to the inability of MKK6 (CA) to exert pro-apoptotic effects. Taken together, our results suggest that ATF4-mediated repression of apelin contributes substantially to the pro-apoptotic effects of p38.

Neuroprotective effects of overexpressed cyclophilin B against Aß-induced neurotoxicity in PC12 cells.

Accumulated amyloid-ß (Aß) is a well-known cause of neuronal apoptosis in Alzheimer disease and functions in part by generating oxidative stress. Our previous work suggested that cyclophilin B (CypB) protects against endoplasmic reticulum (ER) stress. Therefore, in this study we examined the ability of CypB to protect against Aß toxicity. CypB is present in the neurons of rat and mouse brains, and treating neural cells with Aß(25-35) mediates apoptotic cell death. Aß(25-35)-induced neuronal toxicity was inhibited by the overexpression of CypB as measured by cell viability, apoptotic morphology, sub-G1 cell population, intracellular reactive oxygen species accumulation, activated caspase-3, PARP cleavage, Bcl-2 proteins, mitogen-activated protein kinase (MAPK) activation, and phosphoinositide 3-kinase (PI-3-K) activation. CypB/R95A PPIase mutants did not reduce Aß(25-35) toxicity. We showed that Aß(25-35)-induced apoptosis is more severe in a CypB knockdown model, confirming that CypB protects against Aß(25-35)-induced toxicity. Consequently, these findings suggest that CypB may protect against Aß toxicity by its antioxidant properties, by regulating MAPK and PI-3-K signaling, and through the ER stress pathway.