The present study aimed to verify the effects of DFO on PDL cells, with particular emphasis on focusing on osteoblastic differentiation. Its mechanisms related to heme oxygenase-1 (HO-1) pathway were also analyzed. DFO increased the expression of HO-1 and early osteoblastic differentiation markers, such as alkaline phosphatase (ALP) and bone sialoprotein (BSP). DFO upregulated heme oxygenase-1. Treatment with HO-1 siRNA blocked the DFO-stimulated osteoblastic differentiation and HO-1 expression. The NF-kB inhibitor pyrrolidine dithiocarbamate, phosphatidylinositol 3-kinase inhibitor Wortmannin, and p38 MAPK inhibitor U0126 blocked the effects of DFO on HO-1 expression and osteoblastic differentiation in PDL cells. Collectively, these data suggest that DFO promotes osteoblastic differentiation and induces the expression of defense protein HO-1 probably via PI3K, p38 MAPK, and NF-kB signalling pathways in PDL 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

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