Mechanisms of ceramide‐induced COX‐2‐dependent apoptosis in human ovarian cancer OVCAR‐3 cells partially overlapped with resveratrol

Ceramide is a member of the sphingolipid family of bioactive molecules demonstrated to have profound, diverse biological activities. Ceramide is a potential chemotherapeutic agent via the induction of apoptosis. Exposure to ceramide activates extracellular‐signal‐regulated kinases (ERK)1/2‐ and p38 kinase‐dependent apoptosis in human ovarian cancer OVCAR‐3 cells, concomitant with an increase in the expression of COX‐2 and p53 phosphorylation. Blockade of cyclooxygenase‐2 (COX‐2) activity by siRNA or NS398 correspondingly inhibited ceramide‐induced p53 Ser‐15 phosphorylation and apoptosis; thus COX‐2 appears at the apex of the p38 kinase‐mediated signaling cascade induced by ceramide. Induction of apoptosis by ceramide or resveratrol was inhibited by the endocytosis inhibitor, cytochalasin D (CytD); however, cells exposed to resveratrol showed greater sensitivity than ceramide‐treated cells. Ceramide‐treated cells underwent a dose‐dependent reduction in trans‐membrane potential. Although both ceramide and resveratrol induced the expressions of caspase‐3 and ‐7, the effect of inducible COX‐2 was different in caspase‐7 expression induced by ceramide compared to resveratrol. In summary, resveratrol and ceramide converge on an endocytosis‐requiring, ERK1/2‐dependent signal transduction pathway and induction of COX‐expression as an essential molecular antecedent for subsequent p53‐dependent apoptosis. In addition, expressions of caspase‐3 and ‐7 are observed. However, a p38 kinase‐dependent signal transduction pathway and change in mitochondrial potential are also involved in ceramide‐induced apoptosis. J. Cell. Biochem. 114: 1940–1954, 2013. © 2013 Wiley Periodicals, Inc.     

Co‐expression of NF‐κB‐p65 and phosphorylated NF‐κB‐p105 is associated with poor prognosis in surgically resectable non‐small cell lung cancer

Nuclear factor‐kappa B (NF‐κB) as a prognostic marker remains unclear in non‐small cell lung cancer (NSCLC). Here, we studied NF‐κB‐p65 (p65) expression and phosphorylated NF‐κB‐p105 (p‐p105) expression in NSCLC and correlated the finding with overall survival (OS) and clinicopathological features. A total of 186 archival samples from patients with surgically resectable NSCLC were probed with p65 and p‐p105 (Ser 932). The p65‐positive expression and p‐p105‐positive expression were defined as distinct nuclear p65 and cytoplasmic p‐p105 labelling in at least 1% of tumour cells, respectively. The positive staining of p65 alone, p‐p105 alone and co‐expression of p65 and p‐p105 were observed in 61 (32.8%), 90 (48.4%) and 35 (18.8%) patients, respectively. Co‐expression of p65 and p‐p105 but not of either p65 or p‐p105 alone was associated with a poor prognosis. Patients with co‐expression of p65 and p‐p105 had a shorter OS than others, median OS 26.5 months versus 64.1 months, HR 1.85 (95% CI: 1.18–2.91), P = 0.007. There was no statistically significant association between clinicopathological characteristics and either p65 or p‐p105 alone or co‐expression of p65 and p‐p105. This indicates that co‐expression of p65 and p‐p105 was a poor prognostic factor, and pathologic studies of NF‐κB expression could include multiple pathway components in NSCLC.     

Lovastatin‐mediated MCF‐7 cancer cell death involves LKB1‐AMPK‐p38MAPK‐p53‐survivin signalling cascade

There is increasing evidence that statins, which are widely used in lowering serum cholesterol and the incidence of cardiovascular diseases, also exhibits anti‐tumour properties. The underlying mechanisms by which statins‐induced cancer cell death, however, remain incompletely understood. In this study, we explored the anti‐tumour mechanisms of a lipophilic statin, lovastatin, in MCF‐7 breast cancer cells. Lovastatin inhibited cell proliferation and induced cell apoptosis. Lovastatin caused p21 elevation while reduced cyclin D1 and survivin levels. Lovastatin also increased p53 phosphorylation, acetylation and its reporter activities. Results from chromatin immunoprecipitation analysis showed that p53 binding to the survivin promoter region was increased, while Sp1 binding to the region was decreased, in MCF‐7 cells after lovastatin exposure. These actions were associated with liver kinase B1 (LKB1), AMP‐activated protein kinase (AMPK) and p38 mitogen‐activated protein kinase (p38MAPK) activation. Lovastatin's enhancing effects on p53 activation, p21 elevation and survivin reduction were significantly reduced in the presence of p38MAPK signalling inhibitor. Furthermore, LKB1‐AMPK signalling blockade abrogated lovastatin‐induced p38MAPK and p53 phosphorylation. Together these results suggest that lovastatin may activate LKB1‐AMPK‐p38MAPK‐p53‐survivin cascade to cause MCF‐7 cell death. The present study establishes, at least in part, the signalling cascade by which lovastatin induces breast cancer cell death.     

Nanostructured p‐n Junctions for Kinetic‐to‐Electrical Energy Conversion

Piezoelectric ZnO nanorods grown on a flexible substrate are combined with the p‐type semiconducting polymer PEDOT:PSS to produce a p‐n junction device that successfully demonstrates kinetic‐to‐electrical energy conversion. Both the voltage and current output of the devices are measured to be in the range of 10 mV and 10 μA cm^?2. Combining these figures for the best device gives a maximum possible power density of 0.4 mW cm^?3. Systematic testing of the devices is performed showing that the voltage output increases linearly with applied stress, and is reduced significantly by illumination with super‐band gap light. This provides strong evidence that the voltage output results from piezoelectric effects in the ZnO. The behavior of the devices is explained by considering the time‐dependent changes in band structure resulting from the straining of a piezoelectric material within a p‐n junction. It is shown that the rate of screening of the depolarisation field determines the power output of a piezoelectric energy harvesting device. This model is consistent with the behavior of a number of previous devices utilising the piezoelectric effect in ZnO.     

A trs20 Mutation That Mimics an SEDT‐Causing Mutation Blocks Selective and Non‐Selective Autophagy: A Model for TRAPP III Organization

TRAPP is a multisubunit complex that functions in membrane traffic. Mutations in the mammalian TRAPP protein C2 are linked to the skeletal disorder spondyloepiphyseal dysplasia tarda (SEDT) that is thought to arise from an inability to secrete procollagen from the endoplasmic reticulum. Here, we show that C2 binds to the SNARE protein Syntaxin 5 and this interaction is weakened by an SEDT‐causing missense mutation (D47Y). Interestingly, the equivalent mutation (D46Y) in the yeast C2 homolog Trs20p does not block anterograde traffic but did affect endocytosis. The trs20D46Y mutation interfered with the interaction between Trs20p and Trs85p (TRAPP III‐specific subunit), Trs120p and Trs130p (TRAPP II‐specific subunits). Size exclusion chromatography suggested that this yeast mutation destabilized the TRAPP III complex that is involved in autophagy. We further show that this mutation blocks both the selective cytosol‐to‐vacuole (cvt) pathway as well as non‐selective autophagy. We demonstrate that the apparent molecular size of the TRAPP III complex is dependent upon membranes, and that the presence of TRAPP III is dependent upon Atg9p. Finally, we demonstrate that lipidated Bet3p is enriched in TRAPP III and that lipidation increases the efficiency of autophagy. Our study suggests that Trs20p acts as an adaptor for Trs85p and Trs120p and reveals complexities in TRAPP III assembly and function. The implications of C2D47Y in SEDT are discussed .     

p53 and p21(Waf1) Are Recruited to Distinct PML‐Containing Nuclear Foci in Irradiated and Nutlin‐3a‐Treated U2OS Cells

Promyelocytic leukemia nuclear bodies (PML‐NBs) are multiprotein complexes that include PML protein and localize in nuclear foci. PML‐NBs are implicated in multiple stress responses, including apoptosis, DNA repair, and p53‐dependent growth inhibition. ALT‐associated PML bodies (APBs) are specialized PML‐NBs that include telomere‐repeat binding‐factor TRF1 and are exclusively in telomerase‐negative tumors where telomere length is maintained through alternative (ALT) recombination mechanisms. We compared cell‐cycle and p53 responses in ALT‐positive cancer cells (U2OS) exposed to ionizing radiation (IR) or the p53 stabilizer Nutlin‐3a. Both IR and Nutlin‐3a caused growth arrest and comparable induction of p53. However, p21, whose gene p53 activates, displayed biphasic induction following IR and monophasic induction following Nutlin‐3a. p53 was recruited to PML‐NBs 3–4 days after IR, approximately coincident with the secondary p21 increase. These p53/PML‐NBs marked sites of apparently unrepaired DNA double‐strand breaks (DSBs), identified by colocalization with phosphorylated histone H2AX. Both Nutlin‐3a and IR caused a large increase in APBs that was dependent on p53 and p21 expression. Moreover, p21, and to a lesser extent p53, was recruited to APBs in a fraction of Nutlin‐3a‐treated cells. These data indicate (1) p53 is recruited to PML‐NBs after IR that likely mark unrepaired DSBs, suggesting p53 may either be further activated at these sites and/or function in their repair; (2) p53–p21 pathway activation increases the percentage of APB‐positive cells, (3) p21 and p53 are recruited to ALT‐associated PML‐NBs after Nutlin‐3a treatment, suggesting that they may play a previously unrecognized role in telomere maintenance. J. Cell. Biochem. 111: 1280–1290, 2010. © 2010 Wiley‐Liss, Inc.     

Determination of p‐Aminobenzenesulfonic acid based on the electrochemiluminescence quenching of tris (2,2’‐bipyridine)‐ ruthenium (II)

This study describes the quenching effects of p‐aminobenzenesulfonic acid (p‐ABSA) based on electrochemiluminescence (ECL) of the tris (2,2^’‐bipyridyl)‐ruthenium(II)(Ru(bpy)₃²⁺)/tri‐n‐propylamine (TPrA) system in aqueous solution. Quenching behaviours were observed with a 200‐fold excess of p‐ABSA over Ru(bpy)₃²⁺. In the presence of 0.1 M TPrA, the Stern‐Volmer constant (K_SV) of ECL quenching was as high as 1.39 × 10⁴ M^‐1 for p‐ABSA. The logarithmic plot of inhibited ECL versus concentration of p‐ABSA was linear over the range of 6.0 × 10^‐6 ‐3.0 × 10^‐4 mol/L. The corresponding limit of detection was 1.2 × 10^‐6 mol/L for p‐ABSA (S/N = 3). The mechanism of quenching is believed to involve an energy transfer from the excited‐state luminophore to a dimer of p‐ABSA and the adsorption of free radicals of p‐ABSA at the electrode surface that impeded the oxidation of the Ru(bpy)₃²⁺/TPrA system. Copyright © 2012 John Wiley & Sons, Ltd.     

BBR induces apoptosis in HepG2 cell through an Akt‐ASK1‐ROS‐p38MAPKs‐linked cascade

Berberine (BBR) has indicated significant antimicrobial activity against a variety of organisms including bacteria, viruses, and fungi. The mechanism by which BBR initiates apoptosis remains poorly understood. In the present study, we demonstrated that BBR exhibited significant cytotoxicity in human hepatoma HepG2 cells. Herein, we investigated cytotoxicity mechanism of BBR in HepG2 cells. The results showed that the induction of apoptosis in HepG2 cells by BBR was characterized by DNA fragmentation, an increased percentage of annexin V, and the activation of caspase‐3. The expressions of Bcl‐2 protein and pro‐caspase‐3 were reduced by BBR in HepG2 cells. However, Bax protein was increased in the cells. BBR‐induced apoptosis was preceded by increased generation of reactive oxygen species (ROS). NAC treatment, a scavenger of ROS, reversed BBR‐induced apoptosis effects via inhibition of Bax activation and Bcl‐2 inactivation. BBR‐induced, dose‐dependent induction of apoptosis was accompanied by sustained phosphorylation of MAP Kinases (JNK and p38 MAPK), ASK1, Akt, and p53. Furthermore, SB203580, p38 inhibitor, reduced the apoptotic effect of BBR, and blocks the generation of ROS and NO as well as activation of Bax. We found that the treatment of HepG2 cells with BBR triggers generation of ROS through Akt phosphorylation, resulting in dissociation of the ASK1‐mediated activation of JNK and p38 pathways. J. Cell. Biochem. 109: 329–338, 2010. © 2009 Wiley‐Liss, Inc.     

Role of mitogen‐activated protein kinase in Zn‐BC‐AM PDT‐induced apoptosis in nasopharyngeal carcinoma cells

Photodynamic therapy (PDT) with a recently developed photosensitizer Zn‐BC‐AM was found to effectively induce apoptosis in a well‐differentiated nasopharyngeal carcinoma (NPC) HK‐1 cell line. Sustained activation of p38 mitogen‐activated protein kinase (MAPK) and c‐jun N‐terminal kinase (JNK) as well as a transient increase in activation of extracellular signal‐regulated kinase (ERK) were observed immediately after Zn‐BC‐AM PDT. A commonly used p38 MAPK/JNK pharmacological inhibitor PD169316 was found to reduce PDT‐induced apoptosis of HK‐1 cells. PD169316 also prevented the loss of Bcl‐2 and Bcl‐xL in PDT‐treated HK‐1 cells. However, inhibition of JNK with SP600125 had no effect on Zn‐BC‐AM PDT‐induced apoptosis while inhibition of ERK with PD98059 or p38 MAPK with SB203580 significantly increased Zn‐BC‐AM PDT‐induced apoptosis. Further study showed that knockdown of the p38β isoform with siRNA also increased Zn‐BC‐AM PDT‐induced apoptosis, indicating that the anti‐apoptotic effect of PD169316 in PDT‐treated HK‐1 cells was probably independent of p38 MAPK or JNK activation. Taken together, the results suggest that inhibition of p38β and ERK may enhance the therapeutic efficacy of Zn‐BC‐AM PDT on NPC cells. It should be noted that data only based on the use of PD169316 should be interpreted in caution. Copyright © 2010 John Wiley & Sons, Ltd.     

Sodium 5,6‐benzylidene‐L‐ascorbate induces oxidative stress,autophagy, and growth arrest in human colon cancer HT‐29 cells

Our previous studies have demonstrated the oxidative stress properties of sodium ascorbate (SAA) and its benzaldehyde derivative (SBA) on cancer cell lines, but the molecular mechanisms mediating their cytotoxicity remain unclear. In this study, we treated human colon cancer HT‐29 cells with SAA and SBA, and found a significant exposure time‐dependent increase of cytotoxicity in both treatments, with a higher cytotoxicity for 24 h with SAA (IC₅₀ = 5 mM) than SBA (IC₅₀ = 10 mM). A short‐term treatment of cells with 10 mM SAA for 2 h revealed a destabilization of the lysosomes and subsequent induction of cell death, whereas 10 mM SBA triggered a remarkable production of reactive oxidative species, phosphorylation of survival kinase AKT, expression of cyclin kinase‐dependent inhibitor p21, and induction of transient growth arrest. The crucial role of p21 mediating this cytotoxicity was confirmed by isogenic derivatives of the human colon carcinoma HCT116 cell lines (p21^+/+ and p21^?/?), and immunoprecipitation studies with p21 antibody. The SAA cytotoxicity was blocked by co‐incubation with catalase, whereas the SBA cytotoxicity and its subsequent growth arrest were abolished by N‐acetyl‐L‐cysteine (NAC), but was not affected by PI3K phosphorylation inhibitor LY294002, or catalase, suggesting two separated oxidative stress pathways were mediated by these two ascorbates. In addition, neither active caspase 3 nor apoptotic bodies but autophagic vacuoles associated with increased LC3‐II were found in SBA‐treated HT‐29 cells; implicating that SBA induced AKT phosphorylation‐autophagy and p21‐growth arrest in colon cancer HT‐29 cells through an NAC‐inhibitable oxidative stress pathway. J. Cell. Biochem. 111: 412–424, 2010. © 2010 Wiley‐Liss, Inc.     

The role of LINC00094/miR‐224‐5p (miR‐497‐5p)/Endophilin‐1 axis in Memantine mediated protective effects on blood‐brain barrier in AD microenvironment

The dysfunction of the blood‐brain barrier (BBB) is one of the main pathological features of Alzheimer's disease (AD). Memantine (MEM), an N‐methyl‐d ‐aspartate (NMDA) receptor antagonist, has been reported that been used widely for AD therapy. This study was performed to demonstrate the role of the MEM in regulating BBB permeability in AD microenvironment as well as its possible mechanisms. The present study showed that LINC00094 was dramatically increased in Abeta_1‐42‐incubated microvascular endothelial cells (ECs) of BBB model in vitro. Besides, it was decreased in MEM‐incubated ECs. Silencing LINC00094 significantly decreased BBB permeability, meanwhile up‐regulating the expression of ZO‐1, occludin and claudin‐5. Furthermore, silencing LINC00094 enhance the effect of MEM on decreasing BBB permeability in AD microenvironment. The analysis of the mechanism demonstrated that reduction of LINC00094 inhibited Endophilin‐1 expression by up‐regulating miR‐224‐4p/miR‐497‐5p, promoted the expression of ZO‐1, occludin and claudin‐5, and ultimately alleviated BBB permeability in AD microenvironment. Taken together, the present study suggests that the MEM/LINC00094/miR‐224‐5p (miR‐497‐5p)/Endophilin‐1 axis plays a crucial role in the regulation of BBB permeability in AD microenvironment. Silencing LINC00094 combined with MEM provides a novel target for the therapy of AD.     

Down‐regulation of miR‐10a‐5p in synoviocytes contributes to TBX5‐controlled joint inflammation

MicroRNAs are considered to play critical roles in the pathogenesis of human inflammatory arthritis, including rheumatoid arthritis (RA). The purpose of this study was to determine the relationship between miR‐10a‐5p and TBX5 in synoviocytes and evaluate their contribution to joint inflammation. The expression of miR‐10a‐5p and TBX5 in the synovium of RA and human synovial sarcoma cell line SW982 stimulated by IL‐1β was determined by RT‐qPCR and Western blotting. The direct interaction between miR‐10a‐5p and TBX5 3′UTR was determined by dual‐luciferase reporter assay in HeLa cells. Mimics and inhibitors of miR‐10a‐5p were transfected into SW982 cells. TBX5 was overexpressed by plasmid transfection or knocked down by RNAi. Proinflammatory cytokines and TLR3 and MMP13 expressions were determined by RT‐qPCR and Western blotting. Down‐regulated expression of miR‐10a‐5p and up‐regulation of TBX5 in human patients with RA were found compared to patients with OA. IL‐1β could reduce miR‐10a‐5p and increase TBX5 expression in SW982 cells in vitro. The direct target relationship between miR‐10a‐5p and 3′UTR of TBX5 was confirmed by luciferase reporter assay. Alterations of miR‐10‐5p after transfection with its mimic and inhibitor caused the related depression and re‐expression of TBX5 and inflammatory factors in SW982 cells. Overexpression of TBX5 after pCMV3‐TBX5 plasmid transfection significantly promoted the production of TLR3, MMP13 and various inflammatory cytokines, while this effect was rescued after knocking down of TBX5 with its specific siRNA. We conclude that miR‐10a‐5p in a relation with TBX5 regulates joint inflammation in arthritis, which would serve as a diagnostic and therapeutic target for RA treatment.