This study evaluated cell viability and cytokine release in immortalized human oral fibroblasts (hTERT-hNOFs) and keratinocytes (IHOK) exposed to a dental-impregnated gingival retraction cord. To prepare the extracts, dental gingival retraction cords impregnated with aluminum chloride hexahydrate were immersed in a cell culture medium for 24 h at 37 °C. hTERT-hNOFs and IHOK were cultured for 24 h. The cell culture medium was removed and extracts of the dental gingival retraction cords were added. After incubation with the extract solution, cell viability was evaluated using an MTT assay. The levels of the cytokines IL-1α and IL-8 were measured in the supernatants of each cell type. The cell viability after exposure to the extract solution for 10 min exceeded 70 % in both cell types. The ET50 values for hTERT-hNOF and IHOK were 35.75 and 28.98 min, respectively. For IHOK, the IL-1α level was (5.35 ± 5.22) pg/mL at 10 min, (3.58 ± 5.38) pg/mL at 20 min, and (2.85 ± 4.28) pg/mL at 60 min of exposure (p > 0.05). The IL-8 level in IHOK was (67.16 ± 18.70) pg/mL at 10 min, (78.36 ± 7.50) pg/mL at 20 min, and (111.9 ± 26.10) pg/mL at 60 min of exposure (p > 0.05). Cytokine release was not observed from hTERThNOFs. Based on these results, cell viability and cytokine release were confirmed in cells exposed to the impregnated gingival retraction cord. In addition, the application of the extracts to hTERT-hNOF and IHOK during the actual contact time and determination of ET50 may be beneficial for evaluating the biocompatibility of dental-impregnated gingival retraction cords.

Cellular microenvironment is an essential issue for regulating epithelial characteristics through the alteration of intricate signaling pathways and intercellular communications in different cell types. Thus, microenvironment influences tumor initiation, progression, and metastasis. This study aimed to investigate the relationship between microenvironment and epithelial property in HPV16 E6/E7-immortalized human oral keratinocytes (IHOKs). To investigate characteristics of IHOK cultured in different media, two media were used, which included keratinocyte growth media (KGM), F-medium composed of 3:1 ratio of DMEM and F-12 (P media) supplemented with 10% FBS and 1% penicillin/streptomycin. Proliferative property and invasive and migratory activity were observed. As results, proliferating activities of IHOK in different culture condition were changed. Likewise, migratory and invasive activities were also different depending on media types. These results suggest that cellular microenvironment can affect modification of biological properties of epithelial cells.

Considering the great potential of iron chelators at inhibiting the proliferation of tumor cells, in order to determine the molecular and biological basis for the effects of iron chelator in oral cancer, we investigated the effects of iron chelator, desferrioxamine (DFO), on the gene profiling analysis of immortalized human oral keratinocytes (IHOK), and oral cancer cells (HN12), using the cDNA microarray. We identified 46 clones cDNA exhibiting more than 2 fold overexpression in DFO treated IHOK and HN12 cells, and 94 cDNA reveal more than 2 fold down-regulated expression. Examination of gene expression that differs between DFO treated vs. control IHOK and HN12 cells apprear to be related to : cell cycle regulator, cell growth and apoptosis, signal transduction and stress. p21 for cell cell cycle factor was upregualted, and cyclin-cdk gene was decreased expression, so we observed cell cycle arrest in DFO treated IHOK and HN12 cells. In tumor growth, we have identified downregulation of hemidesmosomal protein (bullous pemphigoid antigen 1) and epiregulin expression in DFO treated IHOK and oral cancer cells. Signal transducers including mitogen-activated protein kinase-activated protein kinase 5, serine/thereonine kinase 6 were downregulated with DFO treated cells, suggesting the DFO regulates the p38 MAP kianse pathway in immortalized and maignant oral keratincytes. In conclusion, we have demonstrated the high-throughput utility of cDNA array hybridization in parallel to the gene expression analysis to identify genes that are expressed differentially in DFO treated with immortalized and malignant oral keratinocytes. The differentially expressed genes identified here should be informative in DFO-induced anti-cancer effects.

Macrophage inflammatory prote in -3α (M1P-3a 01' CCL20) is an intr땅uing molecule in CanCel‘ irrununotherapy‘ but M1P-3 a expr ession and signaling are not well under s tood in oral cancer cell s. We investigated CCL20 expression a nd signal trans duction by treating immortal ized hllman oral keratinocyte (IHO찌 and oral ca ncer (뻐 4) cells with defe roxa llline (DFO) and examined the mRNA express ion 01' CCL20 using RT- PCR and ELI8A. lHOK and HN4 cells treated with DFO sbowed increased mRNA and protein expression 01' CCL20. and the upregulation 01' DFO-induced CCL20 expression was higher in IHOK cells than in HN4 cells 8elective inhibitors of p38 and ERKl/2 abol ished DF'O- induced CCL20 expression in both lHOK and HN12 cells. and p38 and ERKl/2 inhibitors prevented DFO- induceddegradati on 01' 1 -κ B and NF'-K B activation. Activation 01' c-fos and c-jun also occmred fo l lowing DFO treatment in IHOK and HN4 cells Collectively, these results suggest that DFO-indllced M1 P- 3a. which is involved in the MAP kinase‘ c-fos, c-jun, and NF-K B pathways, may be an important mediator of the a ntitumor immune response in oral keratinocytes ancl warrants con sideration as a target molecule for oral cancer t reatment

Heme oxygenase-l (HO-l) exhibits cyt oprotective effects in many different cell types and is induced by nicotine exposure in human gingival fibroblasts‘ However‘ therole of HO- l in cancer cells exposed to nicotine has not previously been descnbed We investigated the effects of nicotine on HO-l protein expression and cell viability in immortalized (IHOK) and malignant (HN12) human ora l keratinocyte cells using the MTT assay and Western blotting. We al so examined the involvement of t he phosphoinosit ide-3-0H- kinase (PI3K), mitogen-acti vated protein kinase (MAPK) , and nucJear factor-κ B (NF-κ B) signaling pathways in nicotine-induced cytotoxicity and HO- l levels in IHOK and HN12 cell s‘ Nicotine induced HO- l pro ducti on and had cytotoxic effects on cells in both a concentration- and time-dependent manner. Nicotine-induced cytotox icity and accumulation of HO- l were greater in JJ-IOK cells than in HN12 cells Molecular inhibitors of the ERK, p38 MAP kinase, PI3K, and NF-κ B signaling pathways blocked the cytotoxic effects and induction of J-IO-l expression by nicotine. Treatmen t with an t ioxida nts (bil irubin, N-acetyl cysteine) protected cells against nicotine-induced cytotoxicity and blocked the upregula tion of J-IO- l, the effects of which were more pronounced in II-IOK cells than in HN12 cells Collecti vely, these results suggest that J-IO- l plays a principal role in the protective response to nicotine in oral cancel and immortalized keratinocytes. Moreover, the addition of exogenous antioxidants may help to protect oral epithelial cells as chemopreventive agents against nicotine-induced oxidative stress.

Sulfur is commonly used in Asia as a n herba l medicine to treat infl ammation and cancel‘. and potent chemopreventive effects have been demonstrated in various in vivo and in vitromodels for sulfur-containing compounds found in naturally occun‘ ing products. Here, we report the growth inhibitory and apoptosis-related effects of a newly developedhigh- purity eclible sulfur (ES) on immortali zecl human oral keratinocytes (IHOKs) and on oral cancer cells representing two stages of oral can cer (HN4‘ HN12) basecl on an 3-(4. 5-Dimethylt hiazol-2-yl)-2.5-cliphenyl tetrazolium bromide (MTT) a ssay, Western blotting, cell cycle analysis, ancl nuclear staining. The puri ty of the ES used in th is s tucly was verified by high performance liquid chromat ography (HPLC) , amino acid analysis and energy di spersive spectroscopy (EDS). ES inhibitecl the proliferation of immor talized and malignant oral kerati nocytes in a dose- and time-dependent manner FITC-Annexin V staining. DNA fragmentation testing. and Hoechst 33258 staining revealed that ES inhibits cell growth via apoptosis . ES blocked cell-cycle progression at the sub- Gl phase, with decreased expression 0 1' cyclins Dl, D2, and E, and t heir activating partners cdk2, cdk4, and cdk6‘ and a concomitant induction of p53 and p21/WAF1. Furthermore, ES treatment increased the cytosolic level of cytochrome c a nd resulted in caspase-3 activation‘ and thi s effect was correlated wi th Bax up- regulation and Bcl-2 down- regulation Taken together, these clata suggest that ES is a potential chemopreventive and chemotherapeutic agent for oral cancel

Heme oxygenase-l (HO-l) exhibits cyt oprotective effects in many different cell types and is induced by nicotine exposure in human gingival fibroblasts‘ However‘ therole of HO- l in cancer cells exposed to nicotine has not previously been descnbed We investigated the effects of nicotine on HO-l protein expression and cell viability in immortalized (IHOK) and malignant (HN12) human ora l keratinocyte cells using the MTT assay and Western blotting. We al so examined the involvement of t he phosphoinosit ide-3-0H- kinase (PI3K), mitogen-acti vated protein kinase (MAPK) , and nucJear factor-κ B (NF-κ B) signaling pathways in nicotine-induced cytotoxicity and HO- l levels in IHOK and HN12 cell s‘ Nicotine induced HO- l pro ducti on and had cytotoxic effects on cells in both a concentration- and time-dependent manner. Nicotine-induced cytotox icity and accumulation of HO- l were greater in JJ-IOK cells than in HN12 cells Molecular inhibitors of the ERK, p38 MAP kinase, PI3K, and NF-κ B signaling pathways blocked the cytotoxic effects and induction of J-IO-l expression by nicotine. Treatmen t with an t ioxida nts (bil irubin, N-acetyl cysteine) protected cells against nicotine-induced cytotoxicity and blocked the upregula tion of J-IO- l, the effects of which were more pronounced in II-IOK cells than in HN12 cells Collecti vely, these results suggest that J-IO- l plays a principal role in the protective response to nicotine in oral cancel and immortalized keratinocytes. Moreover, the addition of exogenous antioxidants may help to protect oral epithelial cells as chemopreventive agents against nicotine-induced oxidative stress.

Cell behavior of the transformed cells is known to affect by interaction with extracellular matrix (ECM) proteins and integrin. To investigate the alterations of both integrin expression and cell-matrix interaction during neoplastic conversion of human oral kerationcytes, we studied expression levels of integrin subunits by flow cytometry and cellular responses to the ECM proteins in normal human oral keratinocytes (NHOKs), HPV-immortalized HOK-16B line, and three oral cancer cell lines established from HOK-16B line, CTHOK-16B-BaP, CTHOK-16B-DMBA, and CTHOK-16B-Dexa lines. The expression levels of α and β integrin subunits were shown decreased tendency in human oral keratinocytes undergoing immortalization and tumorigenic transformation except CTHOK-16B-DMBA line tested. Although αuβ6 integrin is known to be highly expressed in squamous cell carcinomas, and the altered integrin expression is suspected to be associated with cellular carcinogenesis, αu integrin subunit and αuβ6 egrin did not express in oral cancer cell lines tested. Cell behavior to the ECM proteins in HOK-16B line was generally similar to that of exponentially proliferating NHOKs. The adhesion activity profiles of type I collagen were very similar to that of its laminin counterparts, but fibronectin showed minimal adhesion activity under our conditions compared to the BSA control. The ability of the CTHOK-16B-BaP line to spread upon type I collagen and laminin markedly decreased, but migration was notably increased on type I collagen. In contrast, CTHOK-16B-DMBA and CTHOK-16B-Dexa lines spread less but migrated more upon type I collagen than immortalized HOK-16B line. These data indicate that downregulation of integrin subunits causes the changes of cellular responses to the ECM proteins during neoplastic conversion of human oral keratinocytes, and that cellular responses to the ECM proteins in oral cancer cell lines established by exposing different carcinogens are variable according to chemical carcinogens treatment.

We have examined the effect of NO donor, S-nitl‘ oso-N-acetyl-DL-penicillamine(SNAP) on heme oxygenase-1 (HQ-l) ex pression in human oral immortalized & malignant keratinocytes, and investigated in the control of keratinocyte proliferation evidence tha t HO-1 cou ld be involved in a low dose of NO, NO inhibitor, HOinducer, and HO inhibitor medi ated cytoprotect ion against cytotoxi city induced by a high dose of NO Oral keratinocyte growth inhibitory or anti-proliferative effects were exerted by with SNAP and hemin in a dose- and cul tivation time dependent manner The level of HQ-1 protein was increased in all cell types after exposure hernin dose, and the hemin induced HQ-1 protein achieved at higher maximum level by 12 hrs in all kind of cells , The pretreatment of cells with 0, 2 μ M SNAP reduced 1 mM SNAP-induced death in IHOK and HN4 cells , These cytoprotective effects on high dose of NO induced HQ-1 expresion and cell ular toxicity were blocked by low dose of SNAP, HCB, and ZnPP IX supporting the involvement of HQ-1 in high dose NO induced growth arrest or cell death, But these cytoprotection pattern is different from immortalized and malignant keratinocytes , These results indirectly demonstrate that HQ-1 could be involved in cytoprotection by NO priming against high dose NO induced cytotoxicity in immortalized and maigla nt oral keratinocytes, Thus, HQ-1 might be an important cellular target of NO donor, with clinical implications for the pre vention of inJlammatory di seases and anti-tumor immunity

Sulfur is commonly used in Asia as an herbal medicine to treat inflammation and cancer , a nd potent chemo preventi ve effects have been demons tra ted in various in vivo and in vitromodels for s ul fur-containing compounds found in natura l1y occurring product s. Here, we 1'eport the growth inhibitory and apoptosis-related effects of a n ewly developedhigh-puri ty edible sulfur(ES) on immo1'tali zed human o1'al ke1'atinocytes(IHOKs) and on oral cancer cells representing two stages of oral cancer (HN4‘ HN12) based on an 3-(4,5-Dimethylthiazol-2-yl) -2.5- diphe n yltetrazolium bromide(MTI) assay, Western blotting, cell cycle analysis, and nuclear staining. The puri ty of the ES used in thi s study was ve1'ified by high performance liquid chromatog1'aphy (HPLC) , ami no acid analysis and energy dispersive spectroscopy(EDS). ES inhibited the prolife1'ation of imrnortalized and ma lig nant o1'al kerati nocytes in a dose- and time-dependent manne1' FITC-Annex.in V staining, DNA fragmentation t esting. and Hoechst 33258 s taining revealed that ES inhibits cell growth via apoptosis. ES bl ocked cell-cycle prog1'ession at t he sub-Gl phase‘ wi th decreased expression of cyclins Dl, D2‘ and E, and their activating partn ers cdk2‘ cdk4‘ and cdkfì, and a concomitant induction of p53 and p21/WAF1. Furthe1'more, ES treatment in creased the cytosolic level of cytochrome c and resulted in caspase- 3 activation‘ and thi s effect was co1'1'elated with Bax up-regulation and Bcl-2 down-1'egulation Taken together‘ these data suggest that ES is a potential chemopreventive and chemotherapeut ic agent fo r oral ca ncer

Interlellkin • 8(IL-8) is an important cytokine involved in tllmor growth and angiogenesis in a variety of malig nancies. bllt the regll lation of IL-8 in 01 외 cancer cells are llnderstood . We invesLigated whether mi togen-activated protein kinases pathway is involved in iron chelator-mediated lL-8 produdion in inunortalized and malignant oral keratinocytes. In this study we examined the role of p38 and extracellular signal- reglllated kinase• 1/2 in the expression of [L-8 by DFO. Incllbation of IHOK and HN12 cel ls with DF'O increased the expression of 11-8 mRNA. as well as the release of IL-8 protein. The signal transdllction study revealed that both p38 and ERK1/2 were significantly activated in response to DFO. Accord ingly. the selective inhibitors for both kinases‘ eit her a lone or combination. abolished DFO- induced lL-8 secretion. indicating an importance of MAP kinase pathway. Interestingly. however‘ inhibition of the p38 and ERK pathway more attenuated IL-8 secretion in IHOK than in HN12 cells. DFO induced NF-kB activation , suggesting a NF-kB- dependent mechanism in DFO- induced IL-8 production. In addition, p38 and ERK inhi bition resulted in the accelerated degradation of lL-8 mRNA, suggesting that in IHOK and HN12 cells, p38 and ERK cunLr iullLe Lo DFO imluced IL-8 secretion by IHOK and HN12 cells via a posttranscriptional mechanism that involves stabilization 01' the IL-8 transcript. Finally. we investigatecl llsing specific inhibitors : 8NP and G8NO for NO c1onor. PDTC for potent inhibitor of NF-kB. Cycloheximide for inhibition of de novo protein synthesis. We observecl 8NP ancl PD1'C clepenclent IL-8 gene incluction in IHOK cell s. but not in HN12 cells used specific inhibitors‘ Collectively. these results demonstrate that‘ targeting MAP kinase ancl NF-kB pathway may be a potentiaI approacb to controlling the angiogenes is ancl growth 이 human oral cancers

1'0 determine Lhe ll1echanism of cell c1eath incluced by iron chelators. we explored the pathways of the three structurally relatecl ll1 itogen-activatecl protein(MAP) kinase subfami li esduring iron cbelator- inclucecl apoptosis ancl differentiation of oral precancerous ancl cancel‘ cells. The iron chelator c1 eferoxamine(DFO) exertecl potent timeancl c1ose-c1epenclent inhi bitory effects on the growth of IHOK and HN4 cells The major mechanism of growth inhibition following DFO treatment was fOllncl to be apoptosis incluction. as assessecl by annexin V-FITC staining. cell cycle analysis‘ DNA lacldering, a ncl Hoechst staining. We report that DF'O s trongly activates the p38 MAP kinase and extracell ular signal- regu lated kinase(ERK). but c10es not activate the c-Jun N-terminal kin ase/ stl않s-activaLecl protein kinase(JNK/8APK) . Of the three MAP kinase blockers usecl‘ the selective p38 MAP kinase inhibitor 8ß203580 ancl ERK inhibitor PD98059 protected oral premaIignant ancl malignant cells againsL iron chelator- nclllced cell death. which incl icates that the p38 MAP kinase serves as a major mecliator 01' apoptos is induced by this iron chelator DFO also evoked the release of cytochrome c from mitochondria, and incluced the activation of caspase-3 ancl caspase-8 in oral cancer cells, which suggests that apoptosis occurs via the mi tochoncl ri on - mecl iaLed pathway. DFO enhanced the expression of Bax in IHOK ancl HN4 cells. consistent witll thei r p53 status Moreover. DFO downregulatecl the expression 01' Bcl-2 in oral cancer cells. which suggests that DFO- incluced apoptos is 01' oraJ cancer and precancerous cells may be mediatecl by an increase in the ratio of pro-apoptotic to anti-apoptotic proteins. ln terestingJy, trcatmcnt 01' IHOK ancl HN4 cel ls with 8B203580 abolishecl cytochrome c release‘ as wel l as the activation of caspase-3 and caspase-8. DFO suppressecl the expression of epithelial di ffe rentiation markers, such as involucrin, t ransglutaminase II. CK6. and CK19. ancl this suppression was blockecl by p38 ancl ERK MAP kinase ll1hlbltors The oral premalignant(IHOK) ancl malignant cell s(I-lN4) showed differential responses to DFO with respect to the expression of cel l cycle regulatory proteins. cell growth. ancl apoptosis. Coll ectively. the current study reveals that p38 MAP kinase plays an ill1 portant role in iron chela tor-mecliatecl cel l cleath and in the suppression of differentiation of oral premalignantandmalignanLcell s.by activating a c10wnstream apoptotic cascade that executes the ceIl c1eath pathway

뻐ny studìes have shown the anti-proli ferative effects of irondeprivation on cancer cell s‘ but the effects 01' iron-chelators on oral cancer have not been clearly elucidated , To investigate the effects of an iron chelato r, desferrioxamine( 01"O).on the growth of ilIllTIortali zed human o1'al ke ratinocytes(IHOK), primary oraJ cancer cel ls(HN4)‘ metastatic oral cancer cell s(HN12) , and human skin keratìnocytes(HaCaT) in the MTr assay, three-dimensional(3D) raft cul tmes, Western blott ing, cell cycJ e analysis‘ nuclear staining‘ and cytochrome c expression for apoptosis s ig naling pathway were used OFO inhibited the growth of immortalized IHOK and HaCaT and mal ignant HN4 and HN12 keratinocytes in a time- and dose-dependent manner according to the MTT assay, The 3D organotypic cu l tu re also revealed that OF'O-treated cells showed less epithelial maturation, less surface keratinizati on‘ and de creased epithelia l thickness, The major mechanìsm of growth inhìbition with the micromolar 0 1"0 treatment was by the induction of apoptosis‘ which was supported by nuclear OAPI staining, ONA fragmentation analysis, and J10w cytometric analysis for sub-Gl phase ar rest and Annexin V-1"ITC stainìng, Furthermore‘ Bax expression in creased together with p53 and p21WAF1!CIPl, whìle the Bcl-2 expression decreased in the immortalized and malig nant keratinocytes treated with 01"0 , Time-dependent cytochrome c from mitochondria was observed in D1"O-treated [l-IOK and 0 1'머 cancel‘ ceJJ s, and was accompanied by the activation of caspase-3 in IHOK cells. These resu lts demonstrate that 0 1"0 has growth inhibitory effects on immortalized and malignant oral keratinocyLes Lhrough the induction of apoptosis and suggest that further evaluation of OFO as a potcntial thcrapcutic agent for human oral precancerous and cancerous lesions is warranted

Iron has a I"ole not on ly in the sy nthesis of hemoglobin but also in cell growth, including tumor development and progression. Excess iron aids tumor development by catalyzin g t he production of oxygen radicals that may be proximate carcinogens and by being a limiting nutrient to the growth and repli cation of cancer cells . Iron chelators have been shown to inhibit the growth and/or induce thc apoptosis of malignant cell lines from leukemia‘ neuroblastoma, melanoma. hepatoma, Kaposi's sarcoma, and cervical cancer. To ow' knowledge, iron chelating agents man ifesting anti-oral cancer effects has not been reported so fa r, and there a re no comparative studies on the elTects of c1esferrioxamine(DFO) on skin keratinocytes vs oral keratinocytes and on imm o J떠li zed cells vs oral cancer cell s. We have found that the iron chelators, deferoxamine(DFO) exert potent time- and dose-dependent inhibitory effec ts on the growth of IHOK and HN4 cells. The major mechanism of growth inhibition after DFO t reatment was by induction of apoptosis, which is supported by AnnexinV-FITC staining. cell cycle analysi s‘ DNA laddering and Hochest staining. We reported that the ch elator st rongly activates p38 MAP kinase and ex tracellul ar signal - regul ated kinase(ERK) , but not activates c-Jun N-terminal kinase/stress-activated protein kin ase(JNK/8APK) . Interl eukin -8(IL-8) is an important cytokine involved in tumor growth and angiogenesis in a va ri ety of rnalignancies‘ bu t the regulation of IL-8 in oral can cer cells are understood. We investigated whether mi togen-activatecl protein kinases pathway is involved in iron chelator-mediated IL-8 proclllction in immorta li zecl a ncl ma lignant ora l keratinocytes. In this study we examined the role of p38 ancl extracellular s ignal- reglll atecl kinase-l/2 in t he expression of IL-8 by DFO.

Studies to evaluate distribution of markers in normal keratinocyte and their immortalized keratinocyte are appropriate to evaluate the normal and preneoplastic lesion of oral cancers as biochemical and cytochemical changes associate with tumorigenesis being not completely understood. Complementary DNA microarray containing 6000 sequence -verified cDNA elements was used to systematically characterize the variation in gene expression patterns of NHOK cells vs. immortalized keratinocyte by HPV16 E6-E7(IHOK). Examination of gene expression that is 85 clones cDNAs exhibits greater than 2 fold overexpression in NHOK probes relative to IHOK probe, 147 cDNAs reveal greater 2 fold overexpression in IHOK relative to NHOK probe.The high similarity in gene expression (96.5%) between IHOK and NHOK cells suggests that only an additional 232/6720 (3.5%) of the genome is differentially gene activated during HPV16 immoratlized keratinocyte growth and differentiation. Examination of gene expression that differs between NHOK and IHOK cellsapprear to be related to : cell adhesion & recognition, cell cycle regulator, apoptosis, transciption factors, growth factors and therir receptors, cytoskeletal and extracellular matrix proteins, signal transduction modulators and effectors, and miscellaneous. The gene expression of cell recognition factor such as endothelin 1, collagen IV, fibronectin, and SPR1 in IHOK were upregulated. Distinct or duplicated cDNA clones representing the same gene were typically clustered in adjacent rows in the clustered gene map. Therefore the differentially expressed and identified genes should be informative in studying oral epithelial cell carcinogenesis and such studies should foster the research of molecular markers allowing to assess the phenotypeof malignant epithelial tumor.