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In addition to its conventional role for cell proliferation and survival, the Raf/MEK/Extracellular signal-regulated kinase (ERK) pathway can also induce growth arrest and death responses, if aberrantly activated. Here, we determined a molecular basis of ERK1/2 signaling that underlies these growth inhibitory physiological outputs. We found that overexpression of ERK1 or ERK2 switches ΔRaf-1:ER-induced growth arrest responses to caspase-dependent apoptotic death responses in different cell types. These death responses, however, were reverted to growth arrest responses upon titration of cellular phospho-ERK1/2 levels by the MEK1/2 inhibitor AZD6244. These data suggest that a cellular threshold for active ERK1/2 levels exists and affects the cell fate between death and growth arrest. We also found that death-mediating ability of ERK2 is abolished by the catalytic site-disabling Lys52Arg replacement or significantly attenuated by the F-site recruitment site-disabling Tyr261Asn replacement, although unaffected by the mutations that disable the common docking groove or the dimerization interface. Therefore, ERK1/2 mediates death signaling dependently of kinase activity and specific physical interactions. Intriguingly, Tyr261Asn-replaced ERK2 could still mediate growth arrest signaling, further contrasting the molecular basis of ERK1/2-mediated growth arrest and death signaling. These data reveal a mechanism underlying the role of ERK1/2 as a focal point of Raf/MEK/ERK-mediated growth arrest and death signaling., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The objective of this study was to determine whether (5S)-5-(4-benzyloxy-3,5-dimethoxy-phenyl)-5,9-dihydro-8H-furo [3',4':6,7] naphtho [2,3-d] [1,3]dioxol-6-one (JNC-1043), which is a novel chemical derivative of β-apopicropodophyllin, acts as a novel potential anticancer reagent and radiosensitizer in colorectal cancer (CRC) cells. Firstly, we used MTT assays to assess whether JNC-1043 could inhibit the cell proliferation of HCT116 and DLD-1 cells. The IC50 values of these cell lines were calculated as 114.5 and 157 nM, respectively, at 72 h of treatment. Using doses approximating the IC50 values, we tested whether JNC-1043 had a radiosensitizing effect in the CRC cell lines. Clonogenic assays revealed that the dose-enhancement ratios (DER) of HCT116 and DLD-1 cells were 1.53 and 1.25, respectively. Cell-counting assays showed that the combination of JNC-1043 and γ-ionizing radiation (IR) enhanced cell death. Treatment with JNC-1043 or IR alone induced cell death by 50~60%, whereas the combination of JNC-1043 and IR increased this cell death by more than 20~30%. Annexin V-propidium iodide assays showed that the combination of JNC-1043 and IR increased apoptosis by more 30~40% compared to that induced by JNC-1043 or IR alone. DCFDA- and MitoSOX-based assays revealed that mitochondrial ROS production was enhanced by the combination of JNC-1043 and IR. Finally, we found that suppression of ROS by N-acetylcysteine (NAC) blocked the apoptotic cell death induced by the combination of JNC-1043 and IR. The xenograft model also indicated that the combination of JNC-1043 and IR increased apoptotic cell death in tumor mass. These results collectively suggest that JNC-1043 acts as a radiosensitizer and exerts anticancer effects against CRC cells by promoting apoptosis mediated by mitochondrial ROS. [ABSTRACT FROM AUTHOR]

The adipose tissue NLRP3 inflammasome has recently emerged as a contributor to obesity-related metabolic inflammation. Recent studies have demonstrated that the activation of the NLRP3 inflammasome cleaves gasdermin D (GSDMD) and induces pyroptosis, a proinflammatory programmed cell death. However, whether GSDMD is involved in the regulation of adipose tissue function and the development of obesity-induced metabolic disease remains unknown. The aim of the present study was to investigate the role of GSDMD in adipose tissue inflammation as well as whole-body metabolism using GSDMD-deficient mice fed a high-fat diet (HFD) for 30 weeks. The effects of GSDMD deficiency on adipose tissue, liver, and isolated macrophages from wild-type (WT) and GSDMD knockout (KO) mice were examined. In addition, 3T3-L1 cells were used to examine the expression of GSDMD during adipogenesis. The results demonstrate that although HFD-induced inflammation was partly ameliorated in isolated macrophages and liver, adipose tissue remained unaffected by GSDMD deficiency. Compared with the WT HFD mice, GSDMD KO HFD mice exhibited a mild increase in HFD-induced glucose intolerance with increased systemic and adipose tissue IL-1β levels. Interestingly, GSDMD deficiency caused accumulation of fat mass when challenged with HFD, partly by suppressing the expression of peroxisome proliferator-activated receptor gamma (PPARγ). The expression of GSDMD mRNA and protein was dramatically suppressed during adipocyte differentiation and was inversely correlated with PPARγ expression. Together, these findings indicate that GSDMD is not a prerequisite for HFD-induced adipose tissue inflammation and suggest a noncanonical function of GSDMD in regulation of fat mass through PPARγ. [ABSTRACT FROM AUTHOR]

With the introduction of immunotherapy, significant improvement has been made in the treatment of head and neck squamous cell carcinoma (HNSCC). However, only a small subset of patients with HNSCC benefit from immunotherapy. The current biomarker, a programmed cell death protein ligand 1 (PD-L1) expression that is widely used in treatment decision making for advanced HNSCC, has only a moderate predictive value. Additionally, PD-L1-based assay has critical inherent limitations due to its highly dynamic nature and lack of standardization. With the advance in molecular techniques and our understanding of biology, more reliable, reproducible, and practical novel biomarkers are being developed. These include but are not limited to neoantigen/mutation characteristics, immune transcriptomes, tumor-infiltrating immune cell composition, cancer epigenomic, proteomics and metabolic characteristics, and plasma-based and organoid assays. [ABSTRACT FROM AUTHOR]

Dasatinib is an inhibitor of Src that has anti‐tumour effects on many haematological and solid cancers. However, the anti‐tumour effects of dasatinib on human oral cancers remain unclear. In this study, we investigated the effects of dasatinib on different types of human oral cancer cells: the non‐tumorigenic YD‐8 and YD‐38 and the tumorigenic YD‐10B and HSC‐3 cells. Strikingly, dasatinib at 10 µM strongly suppressed the growth and induced apoptosis of YD‐38 cells and inhibited the phosphorylation of Src, EGFR, STAT‐3, STAT‐5, PKB and ERK‐1/2. In contrast, knockdown of Src blocked the phosphorylation of EGFR, STAT‐5, PKB and ERK‐1/2, but not STAT‐3, in YD‐38 cells. Dasatinib induced activation of the intrinsic caspase pathway, which was inhibited by z‐VAD‐fmk, a pan‐caspase inhibitor. Dasatinib also decreased Mcl‐1 expression and S6 phosphorylation while increased GRP78 expression and eIF‐2α phosphorylation in YD‐38 cells. In addition, to its direct effects on YD‐38 cells, dasatinib also exhibited anti‐angiogenic properties. Dasatinib‐treated YD‐38 or HUVEC showed reduced HIF‐1α expression and stability. Dasatinib alone or conditioned media from dasatinib‐treated YD‐38 cells inhibited HUVEC tube formation on Matrigel without affecting HUVEC viability. Importantly, dasatinib's anti‐growth, anti‐angiogenic and pro‐apoptotic effects were additionally seen in tumorigenic HSC‐3 cells. Together, these results demonstrate that dasatinib has strong anti‐growth, anti‐angiogenic and pro‐apoptotic effects on human oral cancer cells, which are mediated through the regulation of multiple targets, including Src, EGFR, STAT‐3, STAT‐5, PKB, ERK‐1/2, S6, eIF‐2α, GRP78, caspase‐9/3, Mcl‐1 and HIF‐1α. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) are byproducts of cellular metabolism; they play a significant role as secondary messengers in cell signaling. In cells, high concentrations of ROS induce apoptosis, senescence, and contact inhibition, while low concentrations of ROS result in angiogenesis, proliferation, and cytoskeleton remodeling. Thus, controlling ROS generation is an important factor in cell biology. We designed a chlorin e6 (Ce6)-immobilized polyethylene terephthalate (PET) film (Ce6-PET) to produce extracellular ROS under red-light irradiation. The application of Ce6-PET films can regulate the generation of ROS by altering the intensity of light-emitting diode sources. We confirmed that the Ce6-PET film could effectively promote cell growth under irradiation at 500 μW/cm2 for 30 min in human umbilical vein endothelial cells. We also found that the Ce6-PET film is more efficient in generating ROS than a Ce6-incorporated polyurethane film under the same conditions. Ce6-PET fabrication shows promise for improving the localized delivery of extracellular ROS and regulating ROS formation through the optimization of irradiation intensity. [ABSTRACT FROM AUTHOR]

Mountain ginseng has been used generally as a pharmacopuncture for cancer therapy in clinical practice in Northeast Asia. Nonetheless, there have been few scientific reports for the anticancer action of mountain ginseng. In this study, we investigated whether mountain ginseng extract (MGE) could inhibit the growth of breast cancer in in vitro and in vivo models. MGE showed stronger cytotoxicity than farm-cultivated ginseng extract (FGE) through promoting ROS generation. Also MGE dose-dependently brought about mitochondrial dysfunction in MCF-7 cells. In addition, MGE induced apoptosis through enhancing the activities of caspase-3/7 by regulation of expression of Bcl-2, Bax, cytochrome c, and cleaved caspase-3 in the MCF-7 cells. Consistent with the in vitro results, MGE significantly reduced tumor weights compared with FGE in mice transplanted with MCF-7 cells, and it regulated the expression of apoptosis-related proteins, such as Bcl-2, Bax, cytochrome c, cleaved caspase-3, and cleaved PARP, in the tumor tissues. Additionally, MGE included higher total ginsenoside contents than FGE. In conclusion, MGE, which is richer in ginsenosides, exerts a stronger anticancer action than FGE in breast cancer. The anticancer action of MGE may be closely correlated with caspase-mediated apoptosis through upregulating ROS generation. Therefore, these findings may be helpful for a clinical understanding of the anticancer mechanism of MGE for breast cancer patients. [ABSTRACT FROM AUTHOR]

We investigated the anti-angiogenic and pro-apoptotic effects of robustaflavone (RF), a naturally occurring biflavonoid, on human umbilical vein endothelial cells (HUVECs). RF inhibited HUVEC proliferation and showed cytotoxicity that inhibited HUVEC viability. RF-induced apoptosis was characterized by flow cytometry and caspase 3 analysis. We found that RF increased the number of sub-G1 cells and terminal deoxynucleotidyl transferase dUTP nick end-labeled cells. Additionally, RF induced caspase 3 and poly (ADP-ribose) polymerase activation. Potential molecular targets were identified using a human apoptosis antibody array. RF upregulated Bax, Bad, cleaved caspase 3, p21, and phosphorylated p53 levels. RF induced mitochondrial membrane potential loss and the release of cytochrome c and apoptosis-inducing factor. Cell cycle arrest at G0/G1 phase and the downregulation of Cdk4, Cdk6, and cyclin D1 expression were induced by RF. In vivo anti-angiogenic effects were investigated using a tumor allograft animal model and a Matrigel plug assay. RF reduced the volumes and weights of CT-26 cell-derived tumors. The blood vessel density was significantly decreased in RF-treated tumors. RF also inhibited VEGF-A-stimulated blood vessel formation in vivo in Matrigel plugs. These results suggest that RF can potentially inhibit angiogenesis-dependent tumor growth and metastasis. [ABSTRACT FROM AUTHOR]

Age and acute hyperglycemia are known risk factors of myocardial ischemia-reperfusion injury. We investigated age-related difference in the effect of acute hyperglycemia on myocardial ischemia-reperfusion injury in Sprague-Dawley rats (young, 3 months; middle-aged, 10-12 months; and old, 22-24 months). The rats received 1.2 g/kg dextrose or normal saline and were subjected to coronary artery occlusion for 45 minutes followed by reperfusion for 240 minutes. Infarct size and ejection fraction were measured. The levels of apoptosis-related proteins (C-PARP, Bcl-2, Bax, and cytochrome c) and autophagy-related proteins (Bnip3, Beclin-1, Atg5, and LC3B-II) were evaluated. Infarct size increased with acute hyperglycemia in young and middle-aged rats but not in old rats, whereas the reduction of ejection fraction after ischemia-reperfusion was aggravated by acute hyperglycemia in all age groups. Acute hyperglycemia increased Bnip3 and Beclin-1 expressions after ischemia-reperfusion in young and middle-aged rats but not in old rats, whereas it increased the expression of Bax, cytochrome c, Atg5, and LC3B-II only in young or middle-aged rats. Conclusively, acute hyperglycemia does not aggravate myocardial ischemia-reperfusion injury in old rats, unlike in young and middle-aged rats. This heterogeneity may be due to attenuated changes in protein signaling after ischemia-reperfusion injury under acute hyperglycemia in old rats. [ABSTRACT FROM AUTHOR]

Abstract Rhus verniciflua stokes (RVS) has been used as a functional food to cure inflammatory diseases in Korea. In the present study, we carry out an investigation of the cellular mechanism of a 36 kDa glycoprotein isolated from RVS fruit (RVS glycoprotein) during the apoptosis of human gastrointestinal epithelial HCT116 cells induced by the hemolytic toxin (VvhA) produced by V. vulnificus. Recombinant protein (r) VvhA produced by V. vulnificus stimulated apoptosis by activating the phosphorylation of protein kinase C (PKC) through the production of intracellular reactive oxygen species (ROS). However, RVS glycoprotein significantly inhibited the level of ROS production and PKC activation in rVvhA-stimulated HCT116 cells. Interestingly, we found that RVS glycoprotein has inhibitory effects on the phosphorylation of c-Jun N-terminal kinase (JNK) and nuclear factor-kappa B (NF-κB), which are responsible for the expression of Bax and cleaved caspase-3 in HCT116 cells treated with rVvhA, respectively. On the basis of these results, we suggest that RVS glycoprotein blocks mitochondrial apoptotic cell death induced by rVvhA via the inhibition of ROS-mediated signaling events in HCT116 cells. Highlights • RVS glycoprotein blocks cell death induced by V. vulnificus , VvhA. • RVS glycoprotein reduces ROS production to recover cell death induced by rVvhA. • RVS glycoprotein suppresses PKC/JNK/NF-κB pathway activated by rVvhA. • RVS glycoprotein inhibits expression of Bax and cleaved-caspase 3 induced by rVvhA. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the effect of tacrolimus ointment on corneal epithelium. Methods: Eight-week-old male BALB/c mice were divided into 3 groups. In group 1, no injury was made. In group 2, the central 2-mm corneal epithelium was scraped off. In group 3, the whole corneal and limbal epithelium was removed after absolute ethanol application. In each group, the corneas were observed and treated daily with 0.03% or 0.1% tacrolimus ointment (Protopic®). The viability of cultivated human corneal epithelial cells was also examined after 24-h incubation with various concentrations of tacrolimus. Results: Tacrolimus ointment significantly delayed the epithelial healing in corneas with epithelial injuries (groups 2 and 3) in dose- and frequency-dependent manners, whereas it did not have any effects on uninjured corneas (group 1). The proportion of terminal deoxynucleotidyl transferase-mediated nick end labeling-positive cells and mRNA levels of inflammatory cytokines were higher in tacrolimus-treated corneas, compared with controls. Tacrolimus did not directly reduce the viability of corneal epithelial cells in culture. Conclusions: Topical application of tacrolimus ointment onto the ocular surface induced toxicity in the cornea with epithelial defects through the impairment of epithelial healing and induction of apoptosis. [ABSTRACT FROM AUTHOR]

ABSTRACT: Host factors such as nutritional status and immune cell state are important for vaccine efficacy. Inflammasome activation may be important for triggering vaccine‐induced humoral and cell‐mediated immune responses. Formulations with alum as a typical adjuvant to overcome the effects of host factors have recently been shown to induce inflammasome activation, which augments vaccine efficacy. Apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC) is one of the main components of inflammasomes, but it is not clear whether ASC affects the vaccine‐induced immune response. Herein, we used two types of vaccines: inactivated influenza vaccine not formulated with alum, and HPV vaccine formulated with alum. We gave the vaccines to ASC knockout (ASC−/−) mice to investigate the role of ASC in vaccine efficacy. Influenza vaccine‐immunized ASC−/− mice did not show antibody titers in week 2 after the first vaccination. After boosting, the antibody titer in ASC−/− mice was about half that in wild type (WT) mice. Furthermore, a cytotoxic T‐lymphocyte response against influenza vaccine was not induced in ASC−/− mice. Therefore, vaccinated ASC−/− mice did not show effective protection against viral challenge. ASC−/− mice immunized with alum‐formulated HPV vaccine showed similar antibody titers and T‐cell proliferation compared with immunized WT mice. However, the HPV vaccine without alum induced up to threefold lower titers of HPV‐specific antibody titers in ASC−/− mice compared with those in WT mice. These findings suggest that alum in vaccine can overcome the ASC‐deficient condition. [ABSTRACT FROM AUTHOR]

Abstract: Preclinical Research & Development Angelicin is a furocoumarin derived from Psoralea corylifolia L. fruit that has anti‐inflammatory and anti‐tumor activity. In the present study, the effect of angelicin in enhancing tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL)‐induced apoptotic cell death was studied in Caki (renal carcinoma) cells. Angelicin alone and TRAIL alone had no effect on apoptosis, but in combination these compounds markedly induced apoptosis in the cancer cell lines while not inducing apoptosis in normal cells. The combination treatment induced accumulation of the sub‐G1 population, DNA fragmentation, and activated caspase 3 activity in Caki cells, induced down‐regulation of c‐FLIP expression post‐translationally, and over‐expression of c‐FLIP markedly blocked apoptosis induced by combined treatment with angelicin plus TRAIL. This study provides evidence that angelicin might be a TRAIL sensitizer. [ABSTRACT FROM AUTHOR]

TNF-related apoptosis-inducing ligand (TRAIL) is a death ligand that can induce apoptosis in cells expressing its cognate death receptors (DRs). Previously, we demonstrated the therapeutic potential of recombinant human TRAIL in experimental rheumatoid arthritis (RA) models. However, the mechanisms of how DR-mediated apoptosis elicits these actions is not known. Here, we show that systemically administering a potent, long-acting PEGylated TRAIL (TRAIL PEG ) is profoundly anti-rheumatic against two complementary experimental RA mouse models, collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA), via targeting IL-17 secreting Th17 cells and regulatory T cells (Treg). Systemic administration of TRAIL PEG after disease onset ameliorated the severity of inflammatory arthritis including arthritis indices, paw thickness, cartilage damage and neutrophil infiltration in both CIA and CAIA models. Additionally, the levels of inflammatory molecules (p-p65, ICAM-1, Cox-2, MMP3, and iNOS), pro-inflammatory cytokines (TNF-α, IL-1β, IFN-γ, IL-6, IL-17) and accumulation of activated macrophages were significantly reduced after the TRAIL PEG treatment. Importantly, TRAIL PEG decreased the number of pro-inflammatory Th17 cells in inflamed arthritic joints through TRAIL-induced apoptosis while increasing anti-inflammatory Treg population in vivo . These results suggest that TRAIL PEG ameliorates autoimmunity by targeting the Th 17-Tregs axis, making it a promising candidate drug for the treatment of RA. [ABSTRACT FROM AUTHOR]

Nivolumab is a fully human IgG4 monoclonal antibody targeting the programmed death-1 (PD-1) receptor that blocks interactions between PD-1 and its ligands on tumor cells to prevent T-cell exhaustion in patients with cancer. It has demonstrated efficacy in multiple tumor types, including melanoma, non-small-cell lung cancer, and renal cell carcinoma. This analysis assessed the immunogenicity of nivolumab and its impact on pharmacokinetics, safety, and efficacy in patients with solid tumors enrolled in 6 clinical studies. The incidence and prevalence of antidrug antibodies (ADAs) were determined by validated electrochemiluminescence assays in samples collected during nivolumab treatment and up to 100 days after the last dose. Confirmed positive samples from the 6 studies were also tested for presence of neutralizing antibodies (NAbs). Among 1086 nivolumab-treated patients, 138 patients (12.7%) were ADA positive (relative to baseline), only 3 (0.3%) of whom were persistently positive for ADA, and 9 (0.8%) were NAb positive at 1 time point. The presence of ADAs was not associated with hypersensitivity, infusion reactions, or loss of efficacy and had minimal impact on nivolumab clearance. Additionally, the presence of NAbs was not associated with loss of efficacy. In conclusion, immunogenicity of nivolumab is not clinically meaningful. [ABSTRACT FROM AUTHOR]

The anticancer properties and mechanism of action of omega-3 polyunsaturated fatty acids (ω3-PUFAs) have been demonstrated in several cancers; however, the mechanism in lung cancer remains unclear. Here, we show that docosahexaenoic acid (DHA), a ω3-PUFA, induced apoptosis and autophagy in non-small cell lung cancer (NSCLC) cells. DHA-induced cell death was accompanied by AMP-activated protein kinase (AMPK) activation and inactivated phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling. Knocking down AMPK and overexpressing Akt increased mTOR activity and attenuated DHA-induced cell death, suggesting that DHA induces cell death via AMPK- and Akt-regulated mTOR inactivation. This was confirmed in Fat-1 transgenic mice, which produce ω3-PUFAs. Lewis lung cancer (LLC) tumor cells implanted into Fat-1 mice showed slower growth, lower phospho-Akt levels, and higher levels of apoptosis and autophagy than cells implanted into wild-type mice. Taken together, these data suggest that DHA-induced apoptosis and autophagy in NSCLC cells are associated with AMPK activation and PI3K/Akt inhibition, which in turn lead to suppression of mTOR; thus ω3-PUFAs may be utilized as potential therapeutic agents for NSCLC treatment. [ABSTRACT FROM AUTHOR]

Abstract: Recent findings suggest that Notch-1 signaling contributes to neuronal death in ischemic stroke, but the underlying mechanisms are unknown. Hypoxia inducible factor-1α (HIF-1α), a global regulator of cellular responses to hypoxia, can interact with Notch and modulate its signaling during hypoxic stress. Here we show that Notch signaling interacts with the HIF-1α pathway in the process of ischemic neuronal death. We found that a chemical inhibitor of the Notch-activating enzyme, γ-secretase, and a HIF-1α inhibitor, protect cultured cortical neurons against ischemic stress, and combined inhibition of Notch-1 and HIF-1α further decreased neuronal death. HIF-1α and Notch intracellular domain (NICD) are co-expressed in the neuronal nucleus, and co-immunoprecipitated in cultured neurons and in brain tissue from mice subjected to focal ischemic stroke. Overexpression of NICD and HIF-1α in cultured human neural cells enhanced cell death under ischemia-like conditions, and a HIF-1α inhibitor rescued the cells. RNA interference-mediated depletion of endogenous NICD and HIF-1α also decreased cell death under ischemia-like conditions. Finally, mice treated with inhibitors of γ-secretase and HIF-1α exhibited improved outcome after focal ischemic stroke, with combined treatment being superior to individual treatments. Additional findings suggest that the NICD and HIF-1α collaborate to engage pro-inflammatory and apoptotic signaling pathways in stroke. [Copyright &y& Elsevier]

Calsenilin is a calcium sensor protein that interacts with presenilin and increases calcium-triggered neuronal apoptosis, and γ-secretase activity. Notch is a cell surface receptor that regulates cell-fate decisions and synaptic plasticity in brain. The aim of the present study was to characterize the role of calsenilin as a regulator of the γ-secretase cleavage of Notch in ischemic stroke. Here, we determined the modulation of expression level and cellular distribution of calsenilin in neurons subjected to ischemic-like conditions. The levels of calsenilin and presenilin were increased in primary neurons after oxygen and glucose deprivation. Furthermore, calsenilin was found to enhance the γ-secretase cleavage of Notch and to contribute to cell death under ischemia-like conditions. The inhibition of γ-secretase activity and a presenilin deficiency were both found to protect against calsenilin-mediated ischemic neuronal death. The expression of calsenilin was found to be increased in brain following experimental ischemic stroke. These findings establish a specific molecular mechanism by which the induction of calsenilin enhances Notch activation in ischemic stroke, and identify calsenilin as an upstream of the γ-secretase cleavage of Notch. [ABSTRACT FROM AUTHOR]

Purpose: Diabetes mellitus is an important aetiological factor in intervertebral disc degeneration. The disappearance of notochordal cells in the nucleus pulposus is thought to be the starting point for intervertebral disc degeneration. A cellular effect of diabetes mellitus on apoptosis of notochordal cells and intervertebral disc degeneration has been recently reported. However, how the duration and severity of diabetes mellitus affects viability of notochordal cells and intervertebral disc degeneration is still unknown . Methods: Rat notochordal cells were isolated, cultured, and placed in either 10 % foetal bovine serum (FBS) (normal control) or 10 % FBS plus three different high glucose concentrations (0.1 M, 0.2 M, and 0.4 M) (experimental conditions) for one, three, five and seven days, respectively. We identified and quantified the degree of proliferation and apoptosis, caspase activities, and cleavages of Bid and cytochrome-c. In addition, we examined the cells for expression of matrix metalloproteinases (MMPs) and their tissue inhibitors of metalloproteinases (TIMPs). Results: Each three high glucose concentrations significantly decreased proliferation and increased apoptosis of notochordal cells from culture days one to seven in a dose-dependent manner. Compared with those of 10 % FBS, caspase-9 and -3 activities and cleavage of Bid and cytochrome-c were significantly increased in each three high glucose concentrations, accompanied by increased expression of MMP-1, -2, -3, -7, -9, and -13 and TIMP-1 and -2. Conclusions: High glucose concentration significantly decreased proliferation and increased apoptosis of notochordal cells via the intrinsic pathway with dose- and time-dependent effects. We also found that expression of MMPs and TIMPs was increased with dose- and time-dependent effects. Therefore, these results suggest that aggressive glucose control from an early stage of diabetes mellitus should be recommended to prevent or limit intervertebral disc degeneration. [ABSTRACT FROM AUTHOR]

The cytotoxic effect of 3- O-acetyloleanolic acid, an oleanolic acid derivative isolated from the seeds of Vigna sinensis K., was investigated in human colon carcinoma HCT-116 cells. 3- O-acetyloleanolic acid dose-dependently inhibited the viability of HCT-116 cells. Apoptosis was characterized by detection of cell surface annexin V and sub-G1 apoptotic cell populations. The number of immunostained cells with annexin V-FITC was increased after treatment with 3- O-acetyloleanolic acid. The sub-G1 cell population was also increased. Expression of TRAIL-mediated apoptosis signaling-related death receptor DR5 was increased in 3- O-acetyloleanolic acid-treated HCT-116 cells. Activation of caspase-8 and caspase-3, critical mediators of extrinsic apoptosis signaling, was also increased by 3- O-acetyloleanolic acid. The results indicate that 3- O-acetyloleanolic acid induces apoptosis in HCT-116 cells mediated by an extrinsic apoptosis signaling cascade via up-regulation of DR5. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Purpose: The disappearance of notochordal cells by apoptosis is thought to be the starting point of intervertebral disc degeneration. The aim of this study was to determine the apoptotic pathway of notochordal cells as well as the anti-apoptotic potential of caspase inhibitors. Methods: Rat notochordal cells were isolated, cultured, and placed in either 0 % (apoptosis-promoting condition) or 10 % (normal control) foetal bovine serum (FBS). We identified and quantified apoptotic cell deaths and caspase activities. In addition, we examined the cells for expression of nerve growth factor (NGF) and its two receptors-TrkA (survival signal) and p75 (apoptotic signal)-and downstream pathways. Finally, we analysed the degree of anti-apoptotic effects of caspase inhibitors on the cells. Results: The apoptotic rate and expressions of caspase-8 (extrinsic pathway), -9 (intrinsic pathway), and -3 (common executioner) of notochordal cells were increased in 0 % FBS compared with those in 10 % FBS. Expressions of NGF, p75 receptor and JNK downstream pathways were also increased in 0 % FBS. In contrast, expressions of the TrkA receptor and Akt and MAPK downstream pathways were decreased in 0 % FBS. Pancaspase, capase-9 and capase-8 inhibitors significantly reduced apoptotic cell death. Conclusions: Our results suggest that notochordal cells undergo apoptosis through both the intrinsic and extrinsic pathways by activation of NGF, p75 receptor, and the JNK downstream pathway. We also found that apoptosis of notochordal cells can be attenuated by caspase inhibitors. Caspase inhibitors may play a therapeutic role in delaying the starting point of disc degeneration that is due to inappropriate or premature excessive apoptosis of notochordal cells. [ABSTRACT FROM AUTHOR]

J Oral Pathol Med (2012) 41: 540-546 Background: Toll-like receptor (TLR) agonists have anticancer effect by inducing apoptosis or activating immune cells. In this study, we investigated whether imiquimod, TLR7 agonist, inhibits the proliferation of oral cancer cells. Methods: Toll-like receptor 7 expression and IL-6/8 production by imiquimod were examined using RT-PCR and Enzyme-linked immunosorbent assay, respectively. Cell viability was examined by MTT assay. To examine apoptotic cell death, Annexin V/PI staining for flow cytometry and Western blot analysis were performed. Necrotic cell death was determined by leakage of lactate dehydrogenase (LDH), HMGB1, and PI staining in imiquimod-treated oral squamous cell carcinoma (OSCC) cells. Results: Toll-like receptor7 mRNA was expressed in OSCC cells. Imiquimod induced IL-6 and IL-8 production in OSCC cells, suggesting the functional expression of TLR7. Imiquimod inhibited cells proliferation in a dose-dependent manner. The ratio of annexin V-positive cells and cleaved caspase-3/7 was increased by imiquimod treatment in OSCC cells, suggesting that imiquimod-induced cell death in OSCC cells may be owing to apoptosis. In addition, LDH secretion and PI staining were detected in OSCC cells treated with imiquimod, showing that imiquimod also induced necrotic cell death in the OSCC cells. Conclusions: Imiquimod inhibited effectively the growth of OSCC cells by inducing apoptosis and necrosis. [ABSTRACT FROM AUTHOR]

Objective. Toll-like receptors (TLR) signaling has dual effect of promoting tumor progression and anti-cancer property. This study was designed to determine the effect of polyinosinic-polycytidilic acid (poly I:C), a TLR3 agonist, on the proliferation of oral cancer cells. Materials and methods: Human oral squamous cell carcinoma cell lines, YD-10B and YD-8, were used. TLRs expression was examined by RT-PCR and IL-8 production by poly I:C was examined by ELISA. Cell proliferation was determined by MTT assay. Flow cytometry and Western blot analysis were performed to determine the molecular mechanism of poly I:C-induced cell death. Results. TLR3 was functionally expressed in YD-10B and YD-8 cells. Treatment of poly I:C inhibited the cell growth in a dose-dependent manner. Flow cytometry and Western blot analysis revealed that poly I:C induced apoptosis via a mitochondria-dependent pathway. In addition, combination treatment with poly I:C and paclitaxel more significantly inhibited cell proliferation compared with poly I:C or paclitaxel alone. Conclusions. Poly I:C effectively inhibits oral cancer cell proliferation and can be considered as a candidate to improve the inhibitory effect of anti-cancer drugs. [ABSTRACT FROM AUTHOR]

Background/Aims: ω3-polyunsaturated fatty acids (ω3- PUFAs) are known to possess anticancer properties. However, the relationship between ω3-PUFAs and β-catenin, one of the key components of the Wnt signaling pathway, in human pancreatic cancer remains poorly characterized. Methods: Human pancreatic cancer cells (SW1990 and PANC-1) were exposed to two ω3-PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), to investigate the relationship between ω3-PUFAs and the Wnt/β-catenin signaling pathway in vitro. Mouse pancreatic cancer (PANC02) cells were implanted into fat-1 transgenic mice, which express ω3 desaturases and result in elevated levels of ω3-PUFAs endogenously. The tumor size, levels of Wnt/β-catenin signaling molecules and apoptosis levels were analyzed to examine the influence of ω3-PUFAs in vivo. Results: DHA and EPA significantly inhibited cell growth and increased cell death in pancreatic cancer cells. DHA also reduced β-catenin expression, T cell factor/lymphoid-enhancing factor reporter activity and induced β-catenin/Axin/GSK-3β complex formation, a known precursor to β-catenin degradation. Furthermore, Wnt3a, a natural canonical Wnt pathway ligand, reversed DHA-induced growth inhibition in PANC-1 cells. Immunohistochemical analysis showed aberrant upregulation and increased nuclear staining of β-catenin in tumor tissues from pancreatic cancer patients. However, β-catenin levels in tumor tissues from fat-1 transgenic mice were reduced with a significant increase in apoptosis compared with those from control mice. Conclusion: ω3-PUFAs may be an effective therapy for the chemoprevention and treatment of human pancreatic cancer. Copyright © 2011 S. Karger AG, Basel and IAP [ABSTRACT FROM AUTHOR]

Aberrant miR-21 expression is closely associated with cell proliferation, anti-apoptosis, migration, invasion, and metastasis in various cancers. However, the regulatory mechanism of miR-21 biogenesis is largely unknown. Here, we demonstrated that the tumor suppressor PTEN negatively regulates the expression of oncogenic miR-21 at the post-transcriptional level. Moreover, our results suggest that PTEN plays such a role through the indirect interaction with the Drosha complex. To elucidate how PTEN regulates pri- to pre-miR-21 processing, we attempted to find PTEN-interacting proteins and identified an RNA-regulatory protein, RNH1. Using the sensor to monitor pri-miR-21 processing, we demonstrated that RNH1 is necessary and sufficient for pri-miR-21 processing. Moreover, our results propose that the nuclear localization of RNH1 is important for this function. Further analysis showed that RNH1 directly interacts with the Drosha complex and that PTEN blocks this interaction. Taken together, these results suggest that the PTEN-mediated miR-21 regulation is achieved by inhibiting the interaction between the Drosha complex and RNH1, revealing previously unidentified role of PTEN in the oncogenic miR-21 biogenesis. [ABSTRACT FROM AUTHOR]