β-Lapachone-induced reactive oxygen species (ROS) generation mediates autophagic cell death in glioma U87 MG cells

Autophagy is mainly responsible for the degradation of long-lived proteins and subcellular organelles. Autophagy is responsible for the non-apoptotic cell death, and plays a crucial role in regulating cellular functions. β-Lapachone is a quinone-containing compound originally obtained from the lapacho tree in South America. Here, we show that β-lapachone induces death in U87 MG cells, which is not inhibited by blockers of pan-caspase or necrosis. β-Lapachone-induced cell death gradually increased in a time-dependent manner in U87 MG cells, which were partly prevented by pretreatment of a specific inhibitor of NQO1 (dicoumarol). These results suggested that β-lapachone-induced cell death was mediated by NQO1-independent as well as NQO1-dependent cell death pathways. During progression of β-lapachone-induced cell death, translocation and processing of LC3 as well as an increase in acidic vesicular organelles, as assessed by acridine orange staining, were observed. Furthermore, β-lapachone-induced cell death was inhibited by either a knockdown of beclin-1/Atg-6 or Atg-7 gene expression or by autophagy inhibitors (3-methyl adenine or bafilomycin A1). Reactive oxygen species (ROS) were involved in β-lapachone-induced autophagic cell death of U87 MG glioma cells, because β-lapachone induced ROS production and antioxidant N-acetylcysteine (NAC) decreased autophagic cell death. Our results collectively demonstrate that ROS mediate β-lapachone-induced autophagic cell death in U87 MG glioma cells.     

A functionalized single-walled carbon nanotube-induced autophagic cell death in human lung cells through Akt�CTSC2-mTOR signaling

Nanoparticles are now emerging as a novel class of autophagy activators. Functionalized single-walled carbon nanotubes (f-SWCNTs) are valuable nanomaterials in many industries. This article is designed to assess the autophagic response for f-SWCNTs exposure in vitro and in vivo. A few types of f-SWCNTs were screened in human lung adenocarcinoma A549 cells for the autophagic response and related pathways in vitro. Formation of autophagosomes and LC3-II upregulation were confirmed on the basis of electron microscopy and LC3 western blotting for COOH-CNT, but not for PABS-CNT and PEG-CNT. MTT assay showed marked increase in cell viability, when COOH-CNT was added to cells in the presence of autophagy inhibitor 3MA, ATG6 or TSC2 siRNA. Consistent with the involvement of the Akt–TSC1/2–mTOR pathway, the phosphorylation levels of mTOR, mTOR''s substrate S6 and Akt were shown significantly decreased in A549 cells on treatment with COOH-CNT using western blotting. What''s more, autophagy inhibitor 3MA significantly reduced the lung edema in vivo. In a word, COOH-CNT induced autophagic cell death in A549 cells through the AKT–TSC2–mTOR pathway and caused acute lung injury in vivo. Inhibition of autophagy significantly reduced COOH-CNT-induced autophagic cell death and ameliorated acute lung injury in mice, suggesting a potential remedy to address the growing concerns on the safety of nanomaterials.     

WIN induces apoptotic cell death in human colon cancer cells through a block of autophagic flux dependent on PPARγ down-regulation

Cannabinoids have been reported to possess anti-tumorigenic activity in cancer models although their mechanism of action is not well understood. Here, we show that the synthetic cannabinoid WIN55,212-2 (WIN)-induced apoptosis in colon cancer cell lines is accompanied by endoplasmic reticulum stress induction. The formation of acidic vacuoles and the increase in LC3-II protein indicated the involvement of autophagic process which seemed to play a pro-survival role against the cytotoxic effects of the drug. However, the enhanced lysosomal membrane permeabilization (LMP) blocked the autophagic flux after the formation of autophagosomes as demonstrated by the accumulation of p62 and LC3, two markers of autophagic degradation. Data also provided evidence for a role for nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) in cannabinoid signalling. PPARγ expression, at both protein and mRNA levels, was significantly down-regulated after WIN treatment and its inhibition, either by specific antagonists or by down-regulation via gene silencing, induced effects on cell viability as well as on ER stress and autophagic markers similar to those obtained in the presence of WIN. Moreover, the observation that the increase in p62 level and the induction of LMP were also modified by PPARγ antagonists seemed to indicate that PPARγ down-regulation was crucial to determinate the block of autophagic flux, thus confirming the critical role of PPARγ in WIN action. In conclusion, at our knowledge, our results are the first to show that the reduction of PPARγ levels contributes to WIN-induced colon carcinoma cell death by blocking the pro-survival autophagic response of cells.     

Relationship between autophagy and apoptotic cell death in human neuroblastoma cells treated with paraquat: could autophagy be a "brake" in paraquat-induced apoptotic death?

Paraquat (PQ) (1, 1'-dimethyl-4, 4'-bipyridinium dichloride), a widely used herbicide, has been suggested as a potential etiologic factor for the development of Parkinson's disease (PD). In neurons from patients with PD display characteristics of autophagy, a degradative mechanism involved in the recycling and turnover of cytoplasmic constituents from eukaryotic cells. Low concentrations of paraquat have been recently found to induce autophagy in human neuroblastoma cells, and ultimately the neurons succumb to apoptotic death. Whereas caspase inhibition retarded cell death, autophagy inhibition accelerated the apoptotic cell death induced by paraquat. These findings suggest a relationship between autophagy and apoptotic cell death in human neuroblastoma cells treated with paraquat and open a new line of investigation to advance our knowledge regarding the origin of PD.     

Visualization of co-localization in Aβ42-administered neuroblastoma cells reveals lysosome damage and autophagosome accumulation related to cell death

Aβ42 [amyloid-β peptide-(1-42)] plays a central role in Alzheimer's disease and is known to have a detrimental effect on neuronal cell function and survival when assembled into an oligomeric form. In the present study we show that administration of freshly prepared Aβ42 oligomers to a neuroblastoma (SH-SY5Y) cell line results in a reduction in survival, and that Aβ42 enters the cells prior to cell death. Immunoconfocal and immunogold electron microscopy reveal the path of the Aβ42 with time through the endosomal system and shows that it accumulates in lysosomes. A 24?h incubation with Aβ results in cells that have damaged lysosomes showing signs of enzyme leakage, accumulate autophagic vacuoles and exhibit severely disrupted nuclei. Endogenous Aβ is evident in the cells and the results of the present study suggest that the addition of Aβ oligomers disrupts a crucial balance in Aβ conformation and concentration inside neuronal cells, resulting in catastrophic effects on cellular function and, ultimately, in cell death.     

Status of topoisomerase-2β protein in all-trans retinoic acid–treated human neuroblastoma (SK-N-SH) cells

Of the mammalian topoisomerase (Topo)-2 isozymes (α and β), Topo-2β protein has been reported to regulate neuronal development and differentiation. However, the status of Topo-2β in all-trans retinoic acid (ATRA)-treated human neuroblastoma (SK-N-SH) cells is not understood. More information about the effects of ATRA on SK-N-SH cells is needed to reveal the role of ATRA in the regulation of Topo-2β levels and spontaneous regression of SK-N-SH cells to predict the clinical activity. This study was proposed to investigate the status and role of Topo-2β protein in ATRA-induced survival and neuronal differentiation of SK-N-SH cells. Microscopic, sodium dodecyl sulfate polyacrylamide gel electrophoresis after immunoprecipitations and Western blot analysis were used to study and compare Topo-2β protein among 10 µM ATRA-treated SK-N-SH cells and controls at different time points. The level of Topo-2β protein increased in the initial days of treatment but markedly decreased upon induction of differentiation by ATRA in later stages. Upon ATRA treatment, SK-N-SH cells stretched, exhibited neurite extensions, and acquired a neuronal phenotype. Both treated and untreated SK-N-SH cells were able to migrate, occupy the scratched area, and completely recolonized 24 hours later. These results suggest an indirect role of Topo-2β protein in regulation of genes involved in cell migration and differentiation of ATRA-treated SK-N-SH cells. This study suggests that Topo-2β may be part of activation/repression of protein complexes activated by epigenetic modifying agents, differentiating signals, and inducible locus. However, detailed studies are needed to explore the ATRA-downstream genes leading to Topo-2β regulation and regulatory proteins of neuronal differentiation.     

Kynostatin and 17β-estradiol prevent the apoptotic death of human neuroblastoma cells exposed to HIV-1 protease

A significant number of adult male patients with acquired immunodeficiency syndrome develop cerebral atrophy and progressive brain disorders such as dementia complex and neuropsychiatric problems. Upon entering the brain via activated macrophages or microglias, the human immunodeficiency type 1 virus (HIV-1) may produce cytotoxic factors such as HIV-1 envelope protein (gp 120) and protease. Owing to significant proteolysis of nonviral proteins, the protease derived from HIV-1 may be detrimental to brain cells and neurons. Our results revealed that HIV-1 protease, at nanomolar concentrations, was as potent as gp 120 in causing neurotoxicity in human neuroblastoma neurotypic SH-SY5Y cells. As shown by the Oncor ApopTag staining procedure, HIV-1 protease significantly increased the number of apoptotic cells over the serum-free controls. Moreover, HIV-1 protease-induced neurotoxicity was blocked by a selective protease inhibitor, kynostatin (KNI-272). Antioxidants such as 17-estradiol, melatonin, andS-nitrosoglutathione also prevented protease-induced neurotoxicity. These findings indicate that oxidative proteolysis may mediate HIV-1 protease-induced apoptosis and the degeneration of neurons and other brain cells. Centrally active protease inhibitors and antioxidants may play an important role in preventing cerebral atrophy and associated dementia complex caused by HIV-1.     

IL-1β induces ER stress in a JNK dependent manner that determines cell death in human pancreatic epithelial MIA PaCa-2 cells

The proinflammatory cytokine, IL-1β (Interleukin-1β) is a significant determinant of pancreatic apoptosis and cell death that are common characteristics during diabetes. Using human derived pancreatic MIA PaCa-2 cells, we describe one of the underlying molecular mechanisms behind this observation. Incubation of these cells with IL-1β at doses from 0.5 to 3.0 ng/ml caused significant cell death at 36 h. This was accompanied with marked increases in JNK and p38 phosphorylation together with increased levels of the endoplasmic reticulum (ER) stress markers, namely BiP, CHOP, GADD34, ATF4 and sXBP1. IL-1β also led to increased phosphorylation of eIF2α and all these events could be prevented by pretreatment with the JNK inhibitor, SP600125. A time course study indicated that while IL-1β mediated JNK phosphorylation was induced as early as 2 h of IL-1β treatment, induction of the ER stress markers was evident at later time points. IL-1β stimulated JNK phosphorylation was observed even in the presence of the ER stress inhibitor, 4-phenyl butyrate and the decrease in cell viability was significantly prevented in the presence of the JNK inhibitor. All these suggest that JNK activation is a pre-requisite for ER stress induction and cell death. Reports till date have consistently demonstrated JNK activation as a consequence of ER stress induction by IL-1β in the pancreas. We show here for the first time that the activation of JNK by IL-1β is a prelude to the subsequent induction of ER stress and cell death. These therefore suggest that the JNK-ER stress axis is critical in deciding the decreased survival status by IL-1β in MIA PaCa-2 cells.     

Synthetic menthyl α/β-(1→6)-diglucopyranosides-induced cell death in human leukemia cells is dependent on caspases

A series of alkyl α/β-(1→6)-diglucopyranosides 1-12 were synthesized and assessed for cytotoxicity against HL-60, U937, Molt-3 and MCF-7 cancer cell lines. The menthyl derivatives displayed strong cytotoxic properties showing IC(50) values between 6 and 16 μM. Furthermore, we demonstrated that the selected synthetic (+)-menthyl β-(1→6)-diglucopyranoside 5 induces apoptotic cell death in human leukemia cells through a mechanism that involves activation of multiple caspases. Cell death was completely prevented by the non-specific caspase inhibitor z-VAD-fmk and found to be associated with the release of cytochrome c, an increase in the expression of Bax levels and a decrease in the generation of reactive oxygen species.     

Shear stress modulation of IL-1β-induced E-selectin expression in human endothelial cells

Background

Endothelial cells (ECs) are continuously exposed to hemodynamic forces imparted by blood flow. While it is known that endothelial behavior can be influenced by cytokine activation or fluid shear, the combined effects of these two independent agonists have yet to be fully elucidated.

Methodology

We investigated EC response to long-term inflammatory cues under physiologically relevant shear conditions via E-selectin expression where monolayers of human umbilical vein ECs were simultaneously exposed to laminar fluid shear and interleukin-1ß (shear-cytokine activation) in a parallel plate flow chamber.

Results and Conclusion

Naïve ECs exposed to shear-cytokine activation display significantly higher E-selectin expression for up to 24 hr relative to ECs activated in static (static-cytokine). Peak E-selectin expression occurred after 8–12 hr of continuous shear-cytokine activation contrary to the commonly observed 4–6 hr peak expression in ECs exposed to static-cytokine activation. Cells with some history of high shear conditioning exhibited either high or muted E-selectin expression depending on the durations of the shear pre-conditioning and the ensuing shear-cytokine activation. Overall, the presented data suggest that a high laminar shear enhances acute EC response to interleukin-1ß in naïve or shear-conditioned ECs as may be found in the pathological setting of ischemia/reperfusion injury while conferring rapid E-selectin downregulation to protect against chronic inflammation.     

Sulindac induces apoptotic cell death in susceptible human breast cancer cells through, at least in part, inhibition of IKKβ

Sulindac is a non-steroidal anti-inflammatory agent with anti-tumor activities that include the induction of apoptosis in various cancer cells and the inhibition malignant transformation. However, the molecular mechanisms underlying these effects are unclear. Recently, it has been shown that sulindac can inhibit NF-κB activation. Here, we demonstrate that sulindac induces apoptotic cell death in susceptible human breast cancer cells through, at least in part, inhibition of IKKβ activity. More specifically, when we compared two different human breast cancer cell lines, Hs578T, which has relatively low basal IKKβ activity, and MDA-MB231, which has relatively high basal IKKβ activity, we found that MDA-MB231 was markedly more sensitive to sulindac-induced apoptosis than Hs578T. This was associated with greater caspase-3 and -9 activity in sulindac-treated MDA-MB231 cells. Using a combination of chemical kinase inhibitors and siRNA-mediated knockdown of specific kinases, we found that sulindac inhibits IKKβ, which, in turn, leads to the p38 MAPK-dependent activation of JNK1. Together, these findings suggest that sulindac induces apoptosis in susceptible human breast cancer cells through, at least in part, the inhibition of IKKβ and the subsequent p38 MAPK-dependent activation of JNK1. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. A-Mi Seo and Seoug-Woo Hong have contributed equally.     

Biotin attenuation of oxidative stress,mitochondrial dysfunction,lipid metabolism alteration and 7β-hydroxycholesterol-induced cell death in 158N murine oligodendrocytes

Mitochondrial dysfunction and oxidative stress are involved in neurodegenerative diseases associated with an enhancement of lipid peroxidation products such as 7β-hydroxycholesterol (7β-OHC). It is, therefore, important to study the ability of 7β-OHC to trigger mitochondrial defects, oxidative stress, metabolic dysfunctions and cell death, which are hallmarks of neurodegeneration, and to identify cytoprotective molecules. The effects of biotin were evaluated on 158N murine oligodendrocytes, which are myelin synthesizing cells, exposed to 7β-OHC (50?µM) with or without biotin (10 and 100?nM) or α-tocopherol (positive control of cytoprotection). The effects of biotin on 7β-OHC activities were determined using different criteria: cell adhesion; plasma membrane integrity; redox status. The impact on mitochondria was characterized by the measurement of transmembrane mitochondrial potential (ΔΨm), reactive oxygen species (ROS) overproduction, mitochondrial mass, quantification of cardiolipins and organic acids. Sterols and fatty acids were also quantified. Cell death (apoptosis, autophagy) was characterized by the enumeration of apoptotic cells, caspase-3 activation, identification of autophagic vesicles, and activation of LC3-I into LC3-II. Biotin attenuates 7β-OHC-induced cytotoxicity: loss of cell adhesion was reduced; antioxidant activities were normalized. ROS overproduction, protein and lipid oxidation products were decreased. Biotin partially restores mitochondrial functions: attenuation of the loss of ΔΨm; reduced levels of mitochondrial O₂^?? overproduction; normalization of cardiolipins and organic acid levels. Biotin also normalizes cholesterol and fatty acid synthesis, and prevents apoptosis and autophagy (oxiapoptophagy). Our data support that biotin, which prevents oligodendrocytes damages, could be useful in the treatment of neurodegeneration and demyelination.     

Porphyromonas gingivalis promotes malignancy and chemo-resistance via GSK3β-mediated mitochondrial oxidative phosphorylation in human esophageal squamous cell carcinoma

Our prior studies have confirmed that long-term colonization of Porphyromonas gingivalis (Pg) and overexpression of the inflammatory factor glycogen synthase kinase 3β (GSK3β) promote the malignant evolution of esophageal squamous cell carcinoma (ESCC). We aimed to investigate the functional mechanism by which Pg could promote ESCC malignancy and chemo-resistance through GSK3β-mediated mitochondrial oxidative phosphorylation (mtOXPHOS), and the clinical implications. The effects of Pg and GSK3β on mtOXPHOS, malignant behaviors and response to paclitaxel and cisplatin treatment of ESCC cells were evaluated by in vitro and in vivo studies. The results showed that Pg induced high expression of the GSK3β protein in ESCC cells and promoted the progression and chemo-resistance via GSK3β-mediated mtOXPHOS in human ESCC. Then, Pg infection and the expression of GSK3β, SIRT1 and MRPS5 in ESCC tissues were detected, and the correlations between each index and postoperative survival of ESCC patients were analysed. The results showed that Pg-positive ESCC patients with high-expression of GSK3β, SIRT1 and MRPS5 have significant short postoperative survival. In conclusion, we demonstrated that the effective removal of Pg and inhibition of its promotion of GSK3β-mediated mtOXPHOS may provide a new strategy for ESCC treatment and new insights into the aetiology of ESCC.     

Expression of human β-defensin-2 gene induced by CpG-DNA in human B cells

Defensins have a broad range of antimicrobial activity against bacteria, fungi, and viruses. The expression of human β-defensin-2 (hBD-2) is prevalently observed in epithelial cells and is induced by bacterial infection. Here, we have shown that the expression of the hBD-2 gene and release of hBD-2 protein into the medium is up-regulated in response to CpG-DNA in human B cell line RPMI 8226. The induction of hBD-2 was dependent on CG sequence and phosphorothioate backbone-modification. This was also confirmed in primary human lymphocytes. To shed light on the molecular mechanism involved in hBD-2 induction by CpG-DNA, we examined the contribution of the NF-κB signaling pathway in RPMI 8226 cells. Suppression of MyD88 function and inhibition of NF-κB nuclear localization blocked hBD-2 induction. The NF-κB pathway inhibitors also abolished hBD-2 induction. These results may contribute to a better understanding on the therapeutic effects of CpG-DNA against infectious diseases.     

Is hypothermia a stress condition in HepG2 cells? Expression and localization of Hsp70 in human hepatoma cell line

To understand hypothermia as a stress condition we determined the expression and localization of Hsp70 under hyperthermic and hypothermic stress in human hepatoma HepG2 cells. Western blot analysis indicates that there was a statistically significant increase of Hsp70 expression under thermal stresses. Immunohistochemically, the distribution of inducible Hsp70 in stressed cells showed a granular pattern mostly in the cytoplasm. At subcellular level, Hsp70 was localized in the nucleus, vacuoles, cytoskeletal components and dispersed throughout the cytoplasm. Accumulation of Hsp70 in cells under hypothermia could be related to restitution of cell equilibrium modified by this thermal stress condition. The protective effect of hypothermia could be associated with promotion of Hsp expression. We suggest that hypothermia is a stress capable of inducing Hsp70 expression in human HepG2 cells.     

Protein kinase C-α activation promotes recovery of mitochondrial function and cell survival following oxidant injury in renal cells

We demonstrated that nonselective PKC activation promotes mitochondrial function in renal proximal tubular cells (RPTC) following toxicant injury. However, the specific PKC isozyme mediating this effect is unknown. This study investigated the role of PKC-α in the recovery of mitochondrial functions in oxidant-injured RPTC. Wild-type PKC-α (wtPKC-α) and inactive PKC-α mutants were overexpressed in RPTC to selectively increase or block PKC-α activation. Oxidant (tert-butyl hydroperoxidel; TBHP) exposure activated PKC-α in RPTC but decreased PKC-α levels in mitochondria following treatment. Uncoupled and state 3 respirations and activities of complexes I and IV in TBHP-injured cells decreased to 55, 44, 49, and 65% of controls, respectively. F(0)F(1)-ATPase activity and ATP content in injured RPTC decreased to 59 and 60% of controls, respectively. Oxidant exposure increased reactive oxygen species (ROS) production by 210% and induced mitochondrial fragmentation and 52% RPTC lysis. Overexpressing wtPKC-α did not block TBHP-induced ROS production but improved respiration and complex I activity, restored complex IV and F(0)F(1)-ATPase activities, promoted recovery of ATP content, blocked mitochondrial fragmentation, and reduced RPTC lysis to 14%. In contrast, inhibiting PKC-α 1) induced mitochondrial hyperpolarization and fragmentation; 2) blocked increases in ROS production; 3) prevented recovery of respiratory complexes and F(0)F(1)-ATPase activities, respiration, and ATP content; and 4) exacerbated TBHP-induced RPTC lysis. We conclude that 1) activation of PKC-α prevents mitochondrial hyperpolarization and fragmentation, decreases cell death, and promotes recovery of mitochondrial respiration and ATP content following oxidant injury in RPTC; and 2) respiratory complexes I and IV and F(0)F(1)-ATPase are targets of active PKC-α.     

18β-Glycyrrhetinic acid potentiates Hsp90 inhibition-induced apoptosis in human epithelial ovarian carcinoma cells via activation of death receptor and mitochondrial pathway

The Hsp90 inhibition has been shown to induce apoptosis in various cancer cells. The licorice compounds may enhance the anti-cancer drug effect. However, effect of the licorice compounds on the Hsp90 inhibition-induced apoptosis in ovarian cancer cells has not been studied. To assess the ability of 18β-glycyrrhetinic acid to promote apoptosis, we examined whether 18β-glycyrrhetinic acid potentiated the Hsp90 inhibitor-induced apoptosis in the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. Radicicol and geldanamycin induced a decrease in Bid, Bcl-2, Bcl-xL and survivin protein levels, an increase in Bax levels, the mitochondrial transmembrane potential loss, cytochrome c release, activation of caspases (-8, -9, and -3), cleavage of PARP-1, and an increase in the tumor suppressor p53 levels. 18β-Glycyrrhetinic acid enhanced Hsp90 inhibitor-induced apoptosis-related protein activation, nuclear damage, and cell death. The results suggest that 18β-glycyrrhetinic acid may potentiate the Hsp90 inhibition-induced apoptosis in ovarian carcinoma cell lines via the activation of the caspase-8- and Bid-dependent pathways and the mitochondria-mediated cell death pathway, leading to activation of caspases. Combination of Hsp90 inhibitors and 18β-glycyrrhetinic acid may confer a benefit in the treatment of epithelial ovarian adenocarcinoma.