Human gingival mucosal keratinocytes exhibiting anchorage-independent growth express increased inducible nitric oxide synthase: regulation by MAP kinases

Chunglok, Warangkana; Ittarat, Wanida; Tomakidi, Pascal; Schmidt, Rainer; Stremmel, Wolfgang; Chamulitrat, Waleé

doi:10.1016/j.niox.2004.09.004

Cited by 9 publications

(9 citation statements)

References 39 publications

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“…Concerning K14, this parallels the above-described situation for the FIB monolayers but points to post-transcriptional K14 regulation. This also holds true for mesenchymal vimentin, which appears less abundant in EPI when compared with FIB (Chunglok et al 2004). In EEs of FIB cells, the clear detection of vimentin can be seen at both the gene expression and protein levels, whereas EPI and IHGK show sporadic protein presence concomitant with strong gene expression.…”

Section: Discussionmentioning

confidence: 73%

“…By contrast, the FIB phenotype is completely devoid of this keratin. An almost vice versa situation has been found for vimentin, which can be seen in EPI cells in only a scattered distribution, whereas it is homogeneously distributed in monolayer cultures of FIB cells (Chunglok et al 2004). These findings point to the acquisition of EMT features by FIB cells.…”

Section: Introductionmentioning

confidence: 86%

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Discrimination of epithelium-like and fibroblast-like phenotypes derived from ethanol-treated immortalised human gingival keratinocytes in epithelial equivalents

et al. 2008

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Ethanol treatment of immortalised human gingival keratinocytes (IHGK) yields in an epithelium-like (EPI) and fibroblast-like (FIB) phenotype. With respect to the stratified gingival epithelium, putative structural and molecular differences assigning cells to these phenotypes have not, to date, been analysed in a three-dimensional tissue/epithelial context. Therefore, we generated epithelial equivalents (EEs) in organotypic co-cultures of IHGK, EPI and FIB cells for 1 and 2 weeks and conducted protein and gene expression studies on the EEs for epithelial biomarkers including keratin K14, integrin subunits alpha6 and beta1, E-cadherin, and mesenchymal vimentin. As in the EEs of IHGK and EPI, indirect immunofluorescence revealed continuous expression of beta1 integrin in EEs of FIB cells. However, FIB cells exhibited a significant down-regulation in K14 and integrin alpha6 protein and a loss of E-cadherin at week 2, whereas vimentin was increased. FIB EEs were devoid of transcripts for E-cadherin at both time points, although transcription of the other genes remained constant in all phenotypes. Thus, the FIB phenotype exhibited a poor epithelial structure coinciding with disturbances in the expression of epithelial biomarkers and the persistence of mesenchymal vimentin. Transcription analysis revealed post-transcriptional regulation of vimentin in IHGK and EPI and of K14 and alpha6 in FIB cells. Our findings indicate that differences in the epithelial integrity and expression of molecules in EEs allow for the discrimination of EPI and FIB cells. This suggests that FIB cells share features of epithelial-mesenchymal transition and reflect a more progressive stage in epithelial cell transformation.

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Section: Discussionmentioning

confidence: 73%

Section: Introductionmentioning

confidence: 86%

Discrimination of epithelium-like and fibroblast-like phenotypes derived from ethanol-treated immortalised human gingival keratinocytes in epithelial equivalents

et al. 2008

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“…Furthermore, we showed that FIB cells expressed increased levels of redox-sensitive inducible nitric oxide synthase (iNOS) [37]. iNOS is shown to be associated with tumor invasiveness consistent with increased iNOS at mRNA and protein levels in FIB cells.…”

Section: Ethanol Increases Neoplastic Progression Of Gm16 To Cells Exmentioning

confidence: 74%

“…The MEK1/2 inhibitor U0126 could reduce detectable iNOS mRNA levels suggesting that MAP kinases were upstream regulators of iNOS transcription. U0126 caused both morphological and functional reversion of FIB cells indicating involvement of MAP kinases in these functions [37].…”

Section: Ethanol Increases Neoplastic Progression Of Gm16 To Cells Exmentioning

confidence: 98%

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

Sattayakhom¹,

Chunglok²,

Ittarat³

et al. 2014

Redox Biology

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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.

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“…Total cDNA of 500 ng/ml was applied to Thermal cycler PCR machine to amplify DNA template. The sequences of the PCR primers were as follows: 5'-CGG TGC TGT ATT TCC TTA CGA GGC GAA GAA-3'(forward) and 5'-GGT GCT GCT TGT TAG GAG GTC AAG TAA AGG-3'(reverse) for iNOS (Chunglok et al, 2004) and 5-TGG ATA TTG TTG CCA TCA ATG ACC-3'(forward) and 5'-GAT GGC ATG GAC TGT GGT CAT G-3'(reverse) for GAPDH (Chunglok et al, 2004). Finally, PCR products were analyzed by electrophoresis in 1.5% agarose gel and visualized under ultraviolet light.…”

Section: Rt-pcr Analysis Of Inos Mrnamentioning

confidence: 99%

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

Somchit

Changtam

Kimseng³

et al. 2014

Asian Pacific Journal of Cancer Prevention

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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.

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Human gingival mucosal keratinocytes exhibiting anchorage-independent growth express increased inducible nitric oxide synthase: regulation by MAP kinases

Cited by 9 publications

References 39 publications

Discrimination of epithelium-like and fibroblast-like phenotypes derived from ethanol-treated immortalised human gingival keratinocytes in epithelial equivalents

Discrimination of epithelium-like and fibroblast-like phenotypes derived from ethanol-treated immortalised human gingival keratinocytes in epithelial equivalents

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

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

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