Substance P (SP) is known to be expressed in the nerve fibers of dental pulp and periodontal tissues. It was recently reported that SP expression increased in response to orthodontic force. In the present study, we investigated the effect of SP on expression of mineralization markers and heme oxygenase-1 (HO-1) in human immortalized periodontal ligament (IPDL) cells. Cell viability was measured using a 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) assay. The expression of mineralization markers, including alkaline phosphatase (ALP), osteonectin (ON) and bone sialoprotein (BSP), and heme oxygenase-1 (HO-1) was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. SP did not significantly change human IPDL cell viability, with the exception of the 24 hour treatment group. Treatment of human IPDL cells with 10-10 to 10-⁴M SP upregulated mineralization marker and HO-1 expression in a time- and concentration-dependent manner. Our results suggest that SP may modulate osteoblastic cell differentiation of human IPDL cells through a mechanism involving HO-1 expression.

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.

Previous in vi tro studies demonstrated that H202 or carbamide peroxide cou ld penetrate i nto pul p chambers through enamel and dentin (Benetti et a l., 2004; G okay et a l. , 2004‘ Suli eman et al .. 2005) ‘ Recently. Lee et al.(2006) demonstrated that H20Z enhanced the diffe rentiation of odontoblast like cell line, whereas it inhibited osteogenic diffe rentiation in pre 。steobl astic cell line, as seen by its efl"ecLs on an early difï"erentiation marker. ALP activity. I-lowever. the effects of HZ02 have not been well elucidated in primary cultured human pulp cells ln th is study‘ we investigated whether HO- 1 is involved in H20 2-induced cytotoxicity and examined the production 0 1" dent in sia lophosphoprotein (DSPP) and other minera li zation markers, in human pulp cells H20Z dec1'eased cell viabili ty. but increased HO-l and DSPP expression in a concentra t ion and time dependent manner. Inhibitors of guanylate cyclase, PI3K. ERK, and p38 MAP kinase blocked J-!?,0 2- induced cytot oxicity and the expression of HO-1 and DSPP mRNAs in pulp cells. These data suggest that t he induction of HO-l by H202 in pu lp cells plays a protective role against the cytotoxic effects of H202 and stimulates DSPP expression. resulting in prematu re oclontoblast differentiation th rough pathways t hat involve cGMP. p38. ancl ERK

The aim of t his study was to investigate the cytotoxic and ni t ric oxide (NO)-inducing effects of bismuth oxide (Bi203)-containi ng Portland cement (BPC) on human dental pulp cells. We also assessed whether heme oxygenase-l (HO-l) is involved in BPC-induced cytotox.icity in dental pulp cells Cytotoxicity and NO production induced by BPC were higher than those induced by Portland cement (PC) at 12 and 24 hours, and the former grad ua lly decreased to the level observed for PC. HO- l and inducible nitric oxide synthase (iNOS) mRNA expressions in the BPC group showed maximal increase at 24 hours. and it gradually decreased with increasing cultivation tlme Hemin treatment reversed the BPC-induced cytotoxicity ‘ whereas zinc protoporphyrin IX treatment increased the cytotoxicity. These results suggested that NO production by BPC correlates with HO-l exp1'ession in dental pulp cells Moreover ‘ BPC- induced HO-l expression in dental pulp cells plays a protective 1'ole against the cytotox.ic effects of BPC.

Although substance P (SP). a potent pro-inflammatory peptide, is involved in inflammation and immune responses, the effect of SP 011 the expression of macl'ophage inJlammatol'Y protein 3a (MIP-3a. CCL20) in periodontal ligament (PDL) cells a l'e unknown Equally as enigmatic is the link between SP. the stress protein heme oxygenase-l (HO-l) , and CCL20 product ion. We investigated whether SP induces the release of chemokine CCL20 from irrunortalized POL (IPDL) cells. and further claif’y SP mediated pathways . We also exarnined the relationship between HO-l and CCL20 by treating POL cells with SP Incubating IPOL cells with SP incl'eased ex pl'ession of CCL20 mRNA and CCL20 protein in a dose-time dependent manner. Highly selective p38 and ERKl/2 inhibitors abl'ogated SP-induced expression of CCL20 lD IPOL cells SP is also responsible fo l' ini tiating phosphorylation of I/( B‘ degl'adation of IK B. and activation of NF-/( B. SP induced expression of HO-l in both a concentration- and time-dependent manner. and CCL20 refl ected similal' patterns. The inductive effects of SP on HO-l and CCL20 were enhanced by HO- l inducer hemin and the membrane-permea ble cGMP analog 8-bromo-cGMP Conversely, this pathway was inhi bited by the HO-l inhibitor zinc Pl'otoporphyrin IX (ZnPP IX) and the selective inhibitor of guanylate cyclase‘ 1H- [1. 2. 4]uxad iazole[4, 3-alquinoxal i n- 1-one (ODQ) We report hel'ein the pathway that connects SP a long with other modulators 0 1' neuroimmunoregulationto the induction of HO-1 and the inflanunatol'y mediatol' MIP- 3a /CCL20 in IPDL cel ls. which play an impol'tant role in the development 0 1' pe- I'iodontitis or inflammation during ol'thodontic tooth movement

Epithelial-mesenchymal interaction is well known to have an importance during the organ development as well as cell growth and differentiation. However, in vitro experimental model is not well developed to reproduce in vivo cellular micro-environment which provide a epithelial-mesenchymal interaction. The aims of this study were to develop and evaluate the in vitro experimental model that maintains epithelial-mesenchymal interaction by organotypic raft culture, and to characterize biologic properties of three-dimensionally reconstituted human normal oral kertinocyte(NHOK) and immortalized human oral keratinocytes(IHOK) by histological and immunohistochemical analysis. The results were as follows; 1. Best condition of three dimensionally reconstituted IHOK & HaCaT cells are 14 days air-exposure cultivation, 3 days of submerged state, and dermal equivalent consisting type I collagen and IGF cells. 2. In comparison to IHOK, there was better preservation of the overall epidermal strucutures in oral cracioma cells (HN30) & HaCaT cells by organotypic cultures. But orgnaotypic co-culture of the normal keratinocyte showed the thinnest epithelial layer formation. 3. PCNA was detected primarily in the basal layer of normal mucosa and NHOK, whereas was shown throught the epithellium except surface layer of IHOK cells, and it's expression was similar to that of CIS of biopsied patient's tissue 4. Involucrin is expressed in the upper layer of oral mucosa and NHOK raft, but staining for involucrin was induced in the IHOK rafts indicating differentiation is incomplete, and the staining pattern in the IHOK raft was not uniform. 5. Normal oral keratinocyte raft showed weak immunostaining for p53, and p53 expression of IHOK raft increased rahter than in NHOK. In organotypic cultures of normal cells and IHOK, p53 expression was restricted to the proliferative part of epithelium. This is consistent with expression pattern in biopsy specimens of the normal and CIS tissue. 6. In artifically reconstructed NHOK, the pattern of keratin staining showed both similarity and differences from that of intact normal mucosa. An obvious difference was increased expression of CK10 & CK19, and decreased expression of CK6 in a reconstructed NHOK rather than in normal mucosa, and similar expression was in CK4 and CK16. 7. CK19 & CK16 were strongly positive in HPV immortlaized keratinocyte rafts rahter than in NHOK, an indicator of premalignant or malignant changes, while CK10 & CK6 were decreased, and organotypic cultures of IHOK express was similar keratin expression as epithelial dysplasia or CIS tissue. These results suggest that three-dimensional organotypic co-culture of normal oral & immortalized keratinocytes with dermal equivalent consisting type I collagen and fibroblasts results in similar morphologic and immunohistochemical characteristics to in vivo patient specimens. Thus this organotypic system can be used for studing mechanism of epitehlial-mesenchymal interaction in particular regulating epidermal diffenentiation and morphogenes

We used three-dimensional Matrigel culture system to examine the morphognesis of normal and malignant salivary glands cell in vitro including acinar cells(AC), myoepithelial cell(MC), salivary gland adenocarcinoma cells(SGT), mucoepidermoid carcinoma cells(MEC), and immortalized human salivary gland cells(HSG). For this purpose, normal and salivary gland tumor cells cultured in 3-D Matrigel, and characterized histologically and immunohistochemically, compared with same cells grown on monolayer culture and patient tissue from biopsy. 1. In three-dimensional Matrigel culture, HSG cells form acinar structure, SGT cells shows duct like structure, and other AC, MC and MEC cells dont' form any structure , and their morphology was different from that of monolayer cells. 2. Matrigel involved cell proliferation at a similar pattern to cells on plastic monolayer cell cultures, and monolayer cell revealed higher cell viability than that of Matrigel cultured cells. 3. All salivary glands cells on Matrigel or monolayer showed strong PCNA expression, and there is no expression difference in these cells. But some cells including myoepithelial cells in normal and salivary gland tumor tissue showing PCNA lavelling, so there is PCNA expression difference among normal and tumor tissue cells. 4. Actin expression was noted in AC cells on Matrigel, were rare expressed in the other cells except in MEC cells, and was present in myoepithelial cell and ductal cells of normal gland tissue. There is actin expression difference between tissue and cultured cells . 5. S-100 immunoreaction was moderateively positive in MC cells of monolayer culture, myoepithelial cells of normal tissue and pleomorphic adenoma, all cancer cells of mucoepidermoid carcinoma tissue, but significantly decreased in all salivary cells on Matrigel. 6. TGase 2 expression was prominent in MC cells of monolayer and Matrigel cultured, in myoepithelial cells of normal gland and pleomorphic adenoma, epidermoid cells of mucoepidermopid carcioma, and strong reaction in MEC and AC cells of monolayer and Matrigel cultured. 7. Expression of CK in monolayer culture showed strong reaction to CK6 in all sailvary gland cells, and mild reaction to CK10 and CK16 for all salivary cells, CK16 and CK19 expression in monolayer culture was similar to that of Matrigel culture. 8. CK6 and CK10 expression was strongest in AC and MC cells on Matrigel, and CK 4 was negative reaction in AC, SGT, MEC cells, strong reaction in MC cells but mild in SGT cells on Matrigel. Expression of CK was rare in HSG cells compared with other salivary gland cells, CK16 was prominent in SGT cells, CK10 and CK16 showed strongest expression in MEC cells of Matrigel. 9. Monolayer culture of HSG cell shwoing strong reaction to CK6, moderate to CK19 and mild to the others CK, but 3D cultured HSG cells reveal mild expression to CK16, and rare to others CK, intercallated duct in normal gland tissue showing strong to CK19, and mild to the others Ck, so there are CK expression difference in tissue, monolayer and 3-D cultured cells. 10. Monolayer culture of MEC cells represent strong reaction to CK6, mild to other CK, 3-D cells showing increased CK expression including CK6, epidermoid cells and intermediate cells in mucoepidermoid carcinoma tissue reveal positive to CK6 and CK16, mucous cell positive to CK10 and CK19, so Matrigel showed similar CK pattern compared to mucoepidermoid carcinoma tissue rather than monolThese data indicate that the interaction of salivary gland cells with basement membrane is an important factor in salivary gland development and cytodifferentiation, so this model system will be useful to study acinar or ductal differentiation in vitro.ayer cultred.

In this study, anaerobic digestion tests for municipal solid wastes were performed in order to estimate their methane production rates. To simulate methane production rate, the first derivatives of the sigmoidal equations were calculated. The sigmoidal equations used were Gompertz and Logistic equations. Also, diauxic growth was employed to simulate methane production rate from municipal solid wastes. The equations were fitted to simulate methane production rate by nonlinear regression with a Marquardt-Levenberg algorithm. This is a statistical analysing methodology to minimize the sum of the squares of the differences between the predicted and measured values. Data fittings obtained by using the equations were compared statistically by using the RMSE (Root mean square error) and AIC (Akaike's information criterion). The equations derived from sigmoidal equations successfully described methane production rate for municipal solid wastes. RMSE and AIC were decreased significantly for the equations considering diauxic growth, compared to the equations without diauxic growth. These results showed that the equations derived from sigmoidal equations with diauxic growth can be a useful tool to simulate methane production rate for municipal solid wastes.