Warangkana Chunglok

Association of gp91phox homolog Nox1 with anchorage-independent growth and MAP kinase-activation of transformed human keratinocytes.

Among five members of the NADPH oxidase (Nox) family, Nox1 confers mitogenic properties and is implicated to participate in the process of cell transformation. We have established two phenotypes of carcinogenesis model by ethanol treatment of human gingival keratinocytes immortalized with E6/E7 oncogenes of human papillomavirus type16: immortalized (EPI) nontransformed cells with epithelium-like morphology and more advanced transformed (FIB) cells with spindle fibroblastic-shape morphology. FIB membranes possessed a 63-kDa Nox1 protein at higher levels and exhibited 2.8-fold higher capability for superoxide and hydroxyl radical generation, compared with EPI membranes. Both EPI and FIB cells expressed more abundant Nox1 protein at a proliferating stage than that at a quiescent confluent phase. Immunofluorescence staining with an anti-Nox1 antibody showed that immunoreactive materials were distributed in the whole interior of both types of cells, while they were preferentially localized in the nuclei of FIB cells. Nuclei isolated from EPI and FIB cells contained a 63 kDa-Nox1 protein. Compared with EPI cells, FIB cells expressed elevated levels of Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase proteins. Furthermore, JNK2 was constitutively phosphorylated in FIB cells. Together, our data strongly implicate Nox1 in redox-mediated signaling related to cellular activation of human keratinocytes at a more advanced stage of transformation.

Synthesis, cytotoxicity against human oral cancer KB cells and structure–activity relationship studies of trienone analogues of curcuminoids

A general method for the synthesis of substituted (1E,4E,6E)-1,7-diphenylhepta-1,4,6-trien-3-ones, based on the aldol condensations of substituted 4-phenylbut-3-en-2-ones and substituted 3-phenylacrylaldehydes, was achieved. The natural trienones 4 and 5 have been synthesized by this method, together with the trienone analogues 9-20. These analogues were evaluated for their cytotoxic activity against human oral cancer KB cell line. The structure-activity relationship study has indicated that the analogues with the 1,4,6-trien-3-one function are more potent than the curcuminoid-type function. Analogues with meta-oxygen function on the aromatic rings are more potent than those in the ortho- and para-positions. Free phenolic hydroxy group is more potent than the corresponding methyl ether analogues. Among the potent trienones, compounds 11, 18 and 20 were more active than the anticancer drug ellipticine. All compounds were also evaluated against the non-cancerous Vero cells and it was found that compounds 11, 12 and 17 were much less toxic than curcumin (1); they showed high selectivity indices of 35.46, 33.46 and 31.68, respectively. These analogues are regarded as the potent trienones for anti-oral cancer study.

Demethoxycurcumin from Curcuma longa Rhizome Suppresses iNOS Induction in an in vitro Inflamed Human Intestinal Mucosa Model

BACKGROUND It is known that inducible nitric oxide synthase (iNOS)/nitric oxide (NO) plays an integral role during intestinal inflammation, an important factor for colon cancer development. Natural compounds from Curcuma longa L. (Zingiberaceae) have long been a potential source of bioactive materials with various beneficial biological functions. Among them, a major active curcuminoid, demethoxycurcumin (DMC) has been shown to possess anti-inflammatory properties in lipopolysaccharide (LPS)-activated macrophages or microglia cells. However, the role of DMC on iNOS expression and NO production in an in vitro inflamed human intestinal mucosa model has not yet been elucidated. This study concerned inhibitory effects on iNOS expression and NO production of DMC in inflamed human intestinal Caco-2 cells. An in vitro model was generated and inhibitory effects on NO production of DMC at 65 μM for 24-96 h were assessed by monitoring nitrite levels. Expression of iNOS mRNA and protein was also investigated. DMC significantly decreased NO secretion by 35-41% in our inflamed cell model. Decrease in NO production by DMC was concomitant with down-regulation of iNOS at mRNA and protein levels compared to proinflammatory cytokine cocktail and LPS-treated controls. Mechanism of action of DMC may be partly due to its potent inhibition of the iNOS pathway. Our findings suggest that DMC may have potential as a therapeutic agent against inflammation-related diseases, especially in the gut.

Ursodeoxycholyl Lysophosphatidylethanolamide Protects Against CD95/Fas-Induced Fulminant Hepatitis.

ABSTRACT Increased activation of CD95/Fas by Fas ligand in viral hepatitis and autoimmunity is involved in pathogenesis of fulminant hepatitis and liver failure. We designed a bile-acid phospholipid conjugate ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE with LPE containing oleate at the sn-1) as a hepatoprotectant that was shown to protect against fulminant hepatitis induced by endotoxin. We herein further assessed the ability of UDCA-LPE to prevent death receptor CD95/Fas-induced fulminant hepatitis. C57BL/6 mice were intravenously administered with CD95/Fas agonistic monoclonal antibody (Jo-2) with or without 1 h pretreatment with 50 mg/kg UDCA-LPE. Jo-2 administration caused massive hepatocyte damage as seen by histology, and this was associated with a significant decrease in hepatic phosphatidylcholine (PC), lysoPC, and lysophosphatidylethanolamine levels. By histology, UDCA-LPE pretreatment improved hepatocyte damage and restored the loss of these phospholipids in part by a mechanism involving an inhibition of cytosolic phospholipaseA2 expression. Accordingly, Jo-2 treatment increased hepatic expression of cleaved caspase 8, caspase 3, and poly (ADP-Ribose) polymerase-1, and on the other hand decreased that of anti-apoptotic cellular FLICE-inhibitory protein. UDCA-LPE pretreatment was able to reverse all these changes. Moreover, UDCA-LPE attenuated inflammatory response by lowering the levels of Jo-2-induced proinflammatory cytokines TNF-&agr;, IL-6, and IL-1&bgr; in liver and serum. UDCA-LPE was also able to decrease the levels of stimulated Th1/Th17 cytokines in Jo-2-primed isolated splenocytes. Taken together, UDCA-LPE exhibited potent anti-inflammatory effects against CD95/Fas-induced fulminant hepatitis.

2′,4-Dihydroxy-3′,4′,6′-trimethoxychalcone from Chromolaena odorata possesses anti-inflammatory effects via inhibition of NF-κB and p38 MAPK in lipopolysaccharide-activated RAW 264.7 macrophages

Abstract Context: Immune dysregulation has been implicated in the pathogenesis of many diseases. Macrophages play a crucial role contributing to the onset, progression, and resolution of inflammation. Macrophage inflammatory mediators are of considerable interest as potential targets to treat inflammatory diseases. Objective: The present study was conducted to elucidate the anti-inflammatory mechanism of 2′,4-dihydroxy-3′,4′,6′-trimethoxychalcone (1), the major chalcone isolated from Chromolaena odorata (L.) R.M.King & H.Rob, against lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophages. Materials and methods: Cell viability, nitric oxide (NO), and proinflammatory cytokines of LPS-activated RAW 264.7 cells were measured by MTT, Griess, and ELISA assays, respectively. Cell lysates were subjected to Western blotting for investigation of protein expression. Results and discussion: Treatment with the major chalcone 1 significantly attenuated the production of NO and proinflammatory cytokines, tumor necrosis factor-α, interleukin-1β, and interleukin-6 in a dose-dependent manner. The chalcone suppressed nuclear factor-κB (NF-κB) stimulation by preventing activation of inhibitor κB kinase (IKK) α/β, degradation of inhibitor κB (IκB) α, and translocation of p65 NF-κB into the nucleus. Additionally, the chalcone markedly repressed the phosphorylation of p38 mitogen-activated protein kinase (MAPK), but no further inhibition was detected for c-Jun N-terminal activated kinases or extracellular regulated kinases. Thus, suppression of NF-κB and p38 MAPK activation may be the core mechanism underlying the anti-inflammatory activity of 2′,4-dihydroxy-3′,4′,6′-trimethoxychalcone (1). Conclusion: These findings provide evidence that 2′,4-dihydroxy-3′,4′,6′-trimethoxychalcone (1) possesses anti-inflammatory activity via targeting proinflammatory macrophages. This anti-inflammatory chalcone is a promising compound for reducing inflammation.

Diterpenoid trigonoreidon B isolated from Trigonostemon reidioides alleviates inflammation in models of LPS-stimulated murine macrophages and inflammatory liver injury in mice

The roots of Trigonostemon reidioides, Thai medicinal plant, have long been used as an antidote, laxative, and antiasthmatic, and also used as folk remedy for relieving inflammatory symptoms from poisonous insect and snake bites as well as abscesses and sprains. Here, we studied anti-inflammatory effects of a major diterpenoid named trigonoreidon B (TR-B) isolated from T. reidioides roots in lipopolysaccharide (LPS)-activated RAW264.7 macrophages and D-galactosamine (D-GalN)/LPS-induced inflammatory liver injury in mice. RAW264.7 cells were treated with TR-B or other available minor diterpenoids, and cell viability was determined by AlamarBlue. The levels of inflammatory mediators were determined by nitrite assay, ELISA, and luminescence. NF-κB nuclear translocation was investigated by indirect immunofluorescence. Expression levels were determined by real-time PCR and Western blotting. Transaminases and caspase activities were determined by using assay kits. Our results showed that TR-B was able to suppress PI3K/Akt activation and inflammatory induction in LPS-activated macrophages. These events were concomitant with TR-Bs ability to hamper activated generation of reactive oxygen species, nitric oxide, prostaglandin E2, and cytokines as well as NF-κB p65 nuclear translocation. In an in vivo model of inflammatory liver injury, an administration of TR-B protected mice from D-GalN/LPS-induced liver injury by suppressing the elevation of serum TNF-α, transaminase activities, and hepatocyte apoptosis as well as an improvement of liver histopathology. During protection against liver damage, TR-B also prevented the loss of Akt phosphorylation. Collectively, the results of this present study suggested that TR-B exerted an anti-inflammatory effect via attenuating macrophage-mediated inflammation and inflammatory liver injury in vivo. TR-B may represent a promising lead compound for anti-inflammatory drug development.

Study designs to investigate Nox1 acceleration of neoplastic progression in immortalized human epithelial cells by selection of differentiation resistant cells

To investigate the role of NADPH oxidase homolog Nox1 at an early step of cell transformation, we utilized human gingival mucosal keratinocytes immortalized by E6/E7 of human papillomavirus (HPV) type 16 (GM16) to generate progenitor cell lines either by chronic ethanol exposure or overexpression with Nox1. Among several cobblestone epithelial cell lines obtained, two distinctive spindle cell lines – FIB and NuB1 cells were more progressively transformed exhibiting tubulogenesis and anchorage-independent growth associated with increased invasiveness. These spindle cells acquired molecular markers of epithelial mesenchymal transition (EMT) including mesenchymal vimentin and simple cytokeratins (CK) 8 and 18 as well as myogenic alpha-smooth muscle actin and caldesmon. By overexpression and knockdown experiments, we showed that Nox1 on a post-translational level regulated the stability of CK18 in an ROS-, phosphorylation- and PKCepilon-dependent manner. PKCepilon may thus be used as a therapeutic target for EMT inhibition. Taken together, Nox1 accelerates neoplastic progression by regulating structural intermediate filaments leading to EMT of immortalized human gingival epithelial cells.

Cytotoxic alkaloids against human colon adenocarcinoma cell line (HT-29) from the seed embryos of Nelumbo nucifera

Phytochemical investigation of the CHCl3 extract of the seed embryos of Nelumbo nucifera Gaertn resulted in the isolation of a new naturally occurring bisbenzylisoquinoline alkaloid, O-methylneferine (1), together with five known alkaloids, neferine (2), armepavine (3), (–)-(1R)-N-methylcoclaurine (4), nuciferine (5), and pronuciferine (6). The structures of these compounds were characterized by spectroscopic methods and comparison of physical properties with those reported in the literature. Among them, compounds 1 and 2 exhibited significant activity against human colon adenocarcinoma cell line (HT-29), with IC50 values of 0.70 and 1.61 µM, respectively, which were 8- and 3.5-fold higher than that of the reference anticancer drug, doxorubicin (IC50 5.63 µM). Moreover, compounds 1 and 2 displayed less cytotoxic activity against the non-cancerous HEK 239 cells with the IC50 values of 42.48 and 12.19 μM, respectively, whereas the cytotoxicity of doxorubicin against this cell line was 0.22 μM. The very potent cytotoxicity against HT-29 cell line and very high selectivity index (60.6-fold) of the alkaloid 1 is of particular significant; it could be considered as a promising structure lead for anti-colon cancer drug development.