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ABSTRACT Smoking increases the risk of cardiovascular disorders and leads to damage caused by inflammation and oxidative stress. The actin cytoskeleton is a key player in the response to inflammatory stimuli and is an early target of cellular oxidative stress. The purpose of this study was to investigate the changes in actin cytoskeleton dynamics in human endothelial EA.hy926 cells exposed to cigarette smoke extract (CSE). Immunostaining revealed that CSE exposure resulted in modification of the actin cytoskeleton and led to cell rounding in a dose- and time-dependent manner. In addition, the intracellular calcium concentration was increased by treatment with CSE. Pr etreatment with antioxidants (lipoic acid, glutathione, N-acetyl cysteine, aminoguanidine, α-tocopherol, and vitamin C) significantly attenuated the CSE-induced actin cytoskeleton reorganization and cell rounding. Calcium ion chelators (EGTA, BAPTA-AM AM) and a potent store-operated calcium channel inhibitor (MRS 1845) also reduced CSE-induced intracellular calcium changes and attenuated actin cytoskeleton reorganization and cell morphology change. Moreover, the CSE-induced intracellular calcium increase was suppressed by pretreatment with the inositol trisphosphate receptor (IP3R) inhibitor xestospongin C, the phospholipase C ( PLC) inhibitor U-73122, and the protein kinase C (PKC) inhibitor GF109203X. These results suggest that reactive oxygen species production and intracellular calcium increase play an essential role in CSE-induced actin disorganization and cell rounding through a PLC-IP3-PKC signaling pathway. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1293-1306, 2016. [ABSTRACT FROM AUTHOR]

Sterile alpha motif (SAM)- and leucine-zipper-containing kinase (ZAK) plays a role in the regulation of cell cycle progression and oncogenic transformation. The ZAK gene generates two transcript variants, ZAKα and ZAKβ, through alternative splicing. In this study, we identified that ZAKα proteins were upregulated in tumor tissues, whereas ZAKβ proteins were mostly expressed in corresponding normal tissues. The ectopically expressed ZAKβ proteins in cancer cells inhibited cancer cell proliferation as well as anchorage-independent growth. The ZAKβ:ZAKα protein ratio played a role in the regulation of the cyclic adenosine monophosphate (cAMP) signaling pathway, whereas high ZAKβ protein levels led to the activation of cAMP response element binding protein 1 (CREB1) and exerted antitumor properties. Overexpression of ZAKβ or CREB1 cDNAs in cancer cells inhibited anchorage-independent growth and also reduced the levels of cyclooxygenase 2 (Cox2) and β-catenin proteins. Cancer cells treated with doxorubicin (Doxo) resulted in the switching from the expression of ZAKα to ZAKβ and also inhibited cancer cell growth in soft agar, demonstrating that pharmacological drugs could be used to manipulate endogenous reprogramming splicing events and resulting in the activation of endogenous antitumorigenic properties. We showed that the two ZAK transcript variants, ZAKα and ZAKβ, had opposite biological functions in the regulation of tumor cell proliferation in that ZAKβ had powerful antitumor properties and that ZAKα could promote tumor growth., Competing Interests: The authors declare that they have no competing interests.

Curcumin, a popular yellow pigment of the dietary spice turmeric, has been reported to inhibit cell growth and to induce apoptosis in a wide variety of cancer cells. Although numerous studies have investigated anticancer effects of curcumin, the precise molecular mechanism of action remains unidentified. Whereas curcumin mediates cell survival and apoptosis through mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling cascades, its impact on the upstream regulation of MAPK is unclear. The leucine-zipper and sterile-α motif kinase alpha (ZAKα), a mitogen-activated protein kinase kinase kinase (MAP3K), activates the c-Jun N-terminal kinase (JNK) and NF-κB pathway. This paper investigated the prospective involvement of ZAKα in curcumin-induced effects on cancer cells. Our results suggest that the antitumor activity of curcumin is mediated via a mechanism involving inhibition of ZAKα activity., Competing Interests: The authors declare that there are no conflicts of interests.

Andrographolide is the most abundant diterpene lactone in Andrographis paniculata, which is widely used as a traditional medicine in Southeast Asia. Heme oxygenase 1 (HO-1) is an antioxidant enzyme encoded by a stress-responsive gene. HO-1 has been reported to inhibit the expression of adhesion molecules in vascular endothelial cells (EC). Intercellular adhesion molecule (ICAM-1) is an inflammatory biomarker that is involved in the adhesion of monocytes to EC. In this study, we investigated the effect of andrographolide on the expression of ICAM-1 induced by tumor necrosis factor alpha (TNF-alpha) in EA.hy926 cells and the possible mechanisms involved. Andrographolide (2.5-7.5 microM) inhibited the TNF-alpha-induced expression of ICAM-1 in a dose-dependent manner and resulted in a decrease in HL-60 cell adhesion to EA.hy926 cells (p < 0.05). In parallel, andrographolide significantly induced the expression of HO-1 in a concentration-dependent fashion (p < 0.05). Andrographolide increased the rate of nuclear translocation of nuclear factor erythroid 2-related 2 (Nrf2) and induced antioxidant response element-luciferase reporter activity. Transfection with HO-1-specific small interfering RNA knocked down HO-1 expression, and the inhibition of expression of ICAM-1 by andrographolide was significantly reversed. These results suggest that stimulation of Nrf2-dependent HO-1 expression is involved in the suppression of TNF-alpha-induced ICAM-1 expression exerted by andrographolide.

Monascus sp. fermented products are known for their antihypercholesterolemic effects; however, their antioxidant and anti-inflammatory activities are different from those of many plant-derived foods. To evaluate the effect of turmeric addition into the medium on the antioxidant and anti-inflammatory activities of Monascus pilosus fermented products, we cultured uninoculated PDB medium (PDB), inoculated PDB medium (MP), uninoculated turmeric-containing PDB medium (PDBT), and inoculated turmeric-containing PDB medium (MPT). The broth and mycelia were collected, freeze-dried, and extracted to evaluate their free radical scavenging and iron-chelating activities, inhibition of peroxidation, phenolic and curcuminoid contents, and cellular antioxidant activity. The effects of the extracts on cell viability, cytokines and nitric oxide (NO) production, and expression of enzymes that regulate antioxidation and inflammation were also evaluated. The results showed that MPT had a significantly higher antioxidant activity than PDB, MP, and PDBT at all fermentation time points; moreover, the fermentation process significantly increased the phenolic and curcuminoid contents of MPT. As compared with MP, MPT had a more significant effect on down-regulating the production of NO and TNF-alpha as well as the expression of inducible nitric oxide synthase, cyclooxygenase-2, glutathione peroxidase, superoxidase dismutase, and catalase. After the inherent levels of antioxidant and anti-inflammatory capacities were increased, the modified M. pilosus fermented product demonstrated a higher antiatherosclerotic value than the unmodified product.

Epidemiologic studies have shown a strong association between cigarette smoking and cardiovascular diseases. Various oxidative species and free radicals are produced during cigarette smoking and these lead to endothelial dysfunction and inflammation. Expression of adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1, and adhesion of leukocytes are present in atherosclerosis. We showed previously that a nonfractionated cigarette smoke extract (CSE) induces surface expression of ICAM-1 and E-selectin in human umbilical vein endothelial cells (HUVEC). We then investigated the role of the MAPKs (ERK1/2, JNK, and p38) and AP-1 and the role of actin cytoskeleton reorganization in the CSE-induced expression of ICAM-1 and E-selectin. Western blot analysis showed that CSE treatment rapidly and significantly caused phosphorylation of JNK and ERK1/2 but not of p38. Cytochalasin D (an actin filament disruptor) partially inhibited CSE-induced ICAM-1 and E-selectin surface expression. However, inhibitors of ERK1/2 (PD98059) and JNK (SP600125) did not attenuate the CSE-induced ICAM-1 and E-selectin surface expression. The results of electrophoretic mobility shift assay showed that CSE enhanced AP-1 binding activity. Therefore, CSE activated AP-1 and upregulated ICAM-1 and E-selectin surface expression in HUVEC seem to be via an MAPK-independent pathway. Moreover, the dynamic reorganization of the actin cytoskeleton seems to be required for the CSE-induced surface expression of ICAM-1 and E-selectin., ((c) 2008 Wiley-Liss, Inc.)