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Single Exposure of Human Oral Mucosa Fibroblasts to Ultraviolet B Radiation Reduces Proliferation and Induces COX-2 Expression and Activation
Research Work
Single Exposure of Human Oral Mucosa Fibroblasts to Ultraviolet B Radiation Reduces Proliferation and Induces COX-2 Expression and Activation Boza Y1,23H]-thymidine LQFRUSRUDWLRQDQG077DVVD\V S +20¿EUREODVWVKDGLQFUHDVHG&2;P51$H[SUHVVLRQDWDQGKUVDIWHULUUDGLDWLRQDQG3*(2 production ZDVHOHYDWHGDWDQGKUVSRVWLUUDGLDWLRQDVFRPSDUHGWRFRQWUROV S 7KHUHVXOWVVKRZHGDQLQKLELWRU\HIIHFWRIDVLQJOHGRVHRI89%LUUDGLDWLRQ RQ+20¿EUREODVWSUROLIHUDWLRQZLWKDQLQFUHDVHLQ&2;H[SUHVVLRQDQGDFWLYDWLRQ7KHUHIRUHSKRWRGDPDJHG¿EUREODVWVPD\SOD\DQGLPSRUWDQWUROHLQ the pathogenesis of UV-induced lesions of the lip. Rev. Clin. Periodoncia Implantol. Rehabil. Oral Vol. 3(3); 123-127, 2010. Key words: 2UDO¿EUREODVWV89%&2;SUROLIHUDWLRQ3*(2. INTRODUCTION The lip vermillion constitutes a transition tissue, between oral mucosa and skin, where oral mucosal cells (e.g., keratinocytes, ¿EUREODVWV PDVW FHOOV DQG HQGRWKHOLDO FHOOV DUH H[SRVHG WR GLIIHUHQW types of environmental insults including ultraviolet (UV) sunlight(1). Chronic exposure of the lip vermillion to sunlight results in several alterations at the epithelial and connective tissue compartments which are similar to those described in photodamaged skin(2-4). These alterations include hyperplasia and overexpression of p53 and cyclooxygenase COX-2 at WKH HSLWKHOLXP DQG HODVWRVLV DQG LQÀDPPDWRU\ LQ¿OWUDWLRQ DW WKH ODPLQD propria(2,5-7). UVB (290-320) light is an inherent component of sunlight. Although it affects predominantly the epidermis of the skin, UVB light also penetrates up to the reticular dermis, which is mainly composed of ¿EUREODVWVDQGH[WUDFHOOXODUPDWUL[ (&0 (8,9). Similar to skin, it is highly probable that the lamina propria of the lip is reached by UVB light, since oral mucosa has a higher capacity for UVB light absorption than other tissues(10) ,Q DGGLWLRQ GLIIHUHQFHV LQ (&0 SURGXFWLRQ DQG PDWUL[ metalloproteinase (MMP) gene expression have been described between VNLQ DQG RUDO PXFRVD ¿EUREODVWV VXJJHVWLQJ D GLIIHUHQW ¿EUREODVW phenotype depending upon the tissue of residence(11,12). Studies analyzing the effects of UVB light on cultured skin ¿EUREODVWVVKRZHGWKDWUHSHDWHGH[SRVXUHWRVXEF\WRWR[LFGRVHVRI89% induces premature senescence(13), resulting in a sharp decrease in cell proliferation(14) ,Q DGGLWLRQ 89% H[SRVXUH RI VNLQ ¿EUREODVWV VWLPXODWHV the expression of several cell cycle regulatory genes, such as p53, as ZHOO DV SURLQÀDPPDWRU\ JHQHV VXFK DV &2;(14,15). UVB-induced &2;H[SUHVVLRQLQFUHDVHVWKHSURGXFWLRQRISURVWDJODQGLQ(2 3*(2) by catalyzing the rate limiting step in the conversion of arachidonic acid into PGs(3) ,QFUHDVHG 3*(2 production has been implicated in skin photodamage and photocarcinogenesis, since it stimulates generation RIUHDFWLYHR[\JHQVSHFLHV 526 R[LGDWLYH'1$GDPDJHNHUDWLQRF\WH proliferation and angiogenesis(3,16,17).
Very few studies have analyzed the effects of UV radiation on RUDOPXFRVD¿EUREODVWIXQFWLRQ:LOOLDPVHWDO IRXQGWKDWKXPDQ JLQJLYDO ¿EUREODVWV LUUDGLDWHG ZLWK 89$ OLJKW KDG D VLJQL¿FDQW GHFUHDVH in proliferation(18), and Lim et al. (2008) found that UVC radiation caused JLQJLYDO ¿EUREODVW DSRSWRVLV(19). It is known that several factors induce &2; H[SUHVVLRQ LQ RUDO ¿EUREODVWV LQFOXGLQJ QLFRWLQH DUHFD QXW H[WUDFWV DUHFROLQH SURLQÀDPPDWRU\F\WRNLQHVDQG/36(20-23). However, the effects of UVB radiation on COX-2 expression and activation in oral PXFRVD ¿EUREODVWV DUH XQNQRZQ 7KHUHIRUH WKLV VWXG\ ZDV DLPHG DW characterizing the effects of a single exposure to subcytotoxic doses of 89% OLJKW RQ SUROLIHUDWLRQ &2; H[SUHVVLRQ DQG 3*(2 production by SULPDU\¿EUREODVWFXOWXUHVHVWDEOLVKHGIURPKXPDQRUDOPXFRVD +20 explants.
MATERIAL AND METHODS Isolation and Culture of Oral Mucosa Fibroblasts 3ULPDU\ ¿EUREODVW FXOWXUHV IURP KXPDQ RUDO PXFRVD +20 were isolated by the explant method as previously described(24)%ULHÀ\ using a 6 mm punch-biopsy (Acu-punch, Acuderm, Inc., Fort Launderdale, FL, USA) tissue explants were obtained from the oral mucosa lining the inner cheek of the oral cavity, in close proximity to the lip, from three healthy, non-smoking volunteers, with no history of drug intake in the past 6 months (2 females and 1 male, mean ages 22 ± 1 years). Informed consent was obtained from the subjects, and the surgical protocol was DSSURYHGE\WKH(WKLFV&RPPLWWHHRI7KH8QLYHUVLW\RI&RQFHSFLyQ&HOOV ZHUH FXOWXUHG LQ 'XOEHFFRV PRGL¿HG (DJOHV PHGLXP '0(0 *LEFR BRL, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, *LEFR%5/ ȝJPO SHQLFLOOLQVWUHSWRP\FLQ *LEFR%5/ ȝJPO IXQJL]RQH 86%LRORJLFDO6ZDPSVFRWW0$ DQGȝJPORIJHQWDP\FLQ 6LJPD6W/RXLV02 DW&LQD&22 atmosphere. All the assays were performed using fibroblasts between passages 4 and 10. HOM
1. Departamento de Estomatología Quirúrgica y Laboratorio de Biología y Patología Oral. Facultad de Odontología, Universidad de Concepción. Concepción, Chile. 2. Programa de Magíster en Ciencias Odontológicas, Mención Patología. Escuela de Graduados, Facultad de Odontología, Universidad de Chile. Santiago, Chile. 8QLGDG$FDGpPLFD2GRQWRORJtD)DFXOWDGGH0HGLFLQD3RQWL¿FLD8QLYHUVLGDG&DWyOLFDGH&KLOH6DQWLDJR&KLOH 4. Department of Pathology. College of Medicine, The Ohio State University. Columbus, Ohio, USA. Correspondence author: Dra. I. Gina Rojas. [email protected]. Depto. Estomatología Quirúrgica. Facultad de Odontología, Universidad de Concepción. Casilla 160-C. Concepción, Chile. Trabajo recibido el 05/11/2010. Aprobado para su publicación el 30/12/2010. 123
Rev. Clin. Periodoncia Implantol. Rehabil. Oral Vol. 3(3); 123-127, 2010. ¿EUREODVWFXOWXUHVZHUHFKDUDFWHUL]HGE\LPPXQRF\WRFKHPLVWU\WRFRQ¿UP their phenotype with primary antibodies against vimentin (1:100) (Sigma, St Louis, MO, USA) and prolyl-4-hydroxylase (1:50) (Dako, Carpinteria, CA) followed by a secondary antibody coupled to an HRP detection system (Chemicon Int, Temecula, CA) and DAB as the chromogen (Vector Labs, Burlingame, CA). Growth Synchronization and UVB Light Exposure of HOM Fibroblasts 3UHYLRXVWR89%H[SRVXUH+20¿EUREODVWVZHUHSODWHGDQG maintained either in complete medium supplemented with 10% FBS for 12 hrs (unsynchronized cells) or synchronized according to the protocol by Gilroy et al. (2001)(25)%ULHÀ\FHOOVZHUHZDVKHGZLWK3%6SODWHGLQ 96-well or 100 mm plates, and then incubated in serum-free medium for 24 hrs, followed by medium containing 2.5% FBS for 20 hrs. Previous to LUUDGLDWLRQPHGLXPZDVUHSODFHGE\ȝORI3%6 *LEFR LQHDFKZHOORU PORI3%6LQWKHPPSODWHV7KHQ¿EUREODVWVZHUHLUUDGLDWHGZLWK 30 or 60 mJ/cm2RI89%HTXLYDOHQWWRDQGPLQLPDOHU\WKHPDOGRVHV 0('V UHVSHFWLYHO\ZLWK3KLOOLSV)689%ODPSV $PHULFDQ8OWUDYLROHW &RPSDQ\0XUUD\+LOO1- DVSUHYLRXVO\GHVFULEHG(26)/DPSVZHUH¿WWHG ZLWK.RGDFHO¿OWHUV (DVWPDQ.RGDN5RFKHVWHU1< WRHQVXUHHPLVVLRQ of UVB light only (290-320). The intensity of the UVB light at the bottom of each well or plate was measured with a UVX Digital radiometer (UVP, 8SODQG&$86$ &RQWURO¿EUREODVWVZHUHVKDPLUUDGLDWHG
Rojas IG y cols. Prostagandin E2 Assay +20¿EUREODVWVSODWHGLQPPSODWHVZHUHV\QFKURQL]HG and UVB irradiated. At 12 and 24 hrs after irradiation, supernatants were FROOHFWHGDQGWKHDPRXQWRI3*(2 secreted was determined by enzymelinked immunosorbent assay according to manufacturer´s instructions (Cayman Chemical, Ann Arbor, MI, USA). Results were expressed as PHDQ6'RIWKHSHUFHQWDJHRI3*(2 production (pg/ml) as compared to control. Statistical Analyses 6WDWLVWLFDO WHVWV ZHUH SHUIRUPHG XVLQJ -03,1 6$6 ,QVWLWXWH ,QF &DU\ 1& 86$ 'LIIHUHQFHV EHWZHHQ JURXSV ZHUH examined using Mann-Whitney or t-test, according to data distribution. 'LIIHUHQFHVZHUHFRQVLGHUHGVWDWLVWLFDOO\VLJQL¿FDQWZKHQS
RESULTS Effects of UVB on Proliferation of Fibroblasts Derived from Human Oral Mucosa 3ULPDU\ ¿EUREODVW FXOWXUHV ZHUH REWDLQHG IURP KXPDQ RUDO mucosa explants and characterized by immunocytochemistry. Greater than 95% of the cells stained positive for vimentin and prolyl-4-hydroxylase (Figure 1).
Cell Proliferation Assays 7RDVVHVV+20¿EUREODVWSUROLIHUDWLRQZLWKWKH>3H]-thymidine LQFRUSRUDWLRQ DVVD\ ¿EUREODVWV ZHUH FXOWXUHG LQ ZHOO SODWHV DW D density of 0.6-1 x 104 FHOOVPO &HOOV ZHUH 89%LUUDGLDWHG DQG ȝ&L [3+@WK\PLGLQH VSHFL¿F DFWLYLW\ &LPPRO 'XSRQW1(1 %RVWRQ 0$ was added to the culture medium for 3 to 48 hrs. After incubation, cells ZHUHWU\SVLQLVHGYDFXXPHGRQWRSDSHU¿OWHUV ȝPSRUHV DQGSODFHG LQPORIVFLQWLOODWLRQÀXLG (FRVFLQW/61DWLRQDO'LDJQRVWLFV86$ 7KH LQFRUSRUDWHG UDGLRDFWLYLW\ ZDV TXDQWL¿HG E\ D VFLQWLOODWLRQ FRXQWHU (Packard 1600 TR, Packard Instruments, Downers Grove, IL, USA). Results were expressed as cpm/well. )RU 077 SUROLIHUDWLRQ DVVD\ ¿EUREODVWV ZHUH FXOWXUHG LQ well plates, at a density of 1 x 104 cells/ml. At 6, 12 and 24 hrs post-UVB LUUDGLDWLRQȝORI077VROXWLRQZHUHDGGHGWRHDFKZHOODQGSURFHVVHG according to the manufacturer´s instructions. Absorbance was measured DWQPXVLQJDPLFURSODWHUHDGHU 0XOWLVNDQ(;7KHUPR&RUSRUDWLRQ USA). In addition, cell viability was determined at 24 and 48 hrs posttreatment by trypan blue exclusion assay as previously described(27). ([SHULPHQWVZHUHUHSHDWHGDWOHDVWWKUHHWLPHVZLWKZHOOVJURXS Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 7RWDO 51$ ZDV LVRODWHG IURP ¿EUREODVWV VHHGHG RQWR mm plates (0.6 x 106 cells/plate) with Trizol (Invitrogen, Rockville, MD), according to manufacturer´s instructions (n=6-7 plates/group at each WLPHSRLQW 51$ FRQFHQWUDWLRQ DQG QXFOHLF DFLG SXULW\ ZDV GHWHUPLQHG spectrophotometrically by UV absorbance. RT-PCR for COX-2 and GAPDH was performed as previously described(5) %ULHÀ\ ȝJ RI WRWDO 51$ ZDV UHYHUVHG WUDQVFULEHG XVLQJ ROLJRG7 SULPHUV DQG $09 UHYHUVH WUDQVFULSWDVH 3URPHJD 0DGLVRQ :, DQG ȝO RI F'1$ IURP HDFKVDPSOHZDVDPSOL¿HGE\3&5(DFK3&5ZDVFDUULHGRXWLQȝO of a reaction mix containing 10 mM Tris-HCl, 50 mM KCl, 3 mM MgCl2, P0RIHDFKG173ȝ0RIHDFKVHQVHDQGDQWLVHQVHSULPHUDQG RQHXQLWRI3ODQWLQXP7DT'1$SRO\PHUDVH ,QYLWURJHQ&DUOVEDG&$ $QHJDWLYHFRQWUROZKHUHWKHF'1$ZDVRPLWWHGIURPWKHUHDFWLRQPL[ ZDVUXQIRUHDFKDPSOL¿HGJHQH7KHIROORZLQJSULPHUVHTXHQFHVZHUH used: COX-2 sense 5´-GGT CTG GTG CCT GGT CTG ATG ATG-3´ and antisense 5´-GTC CTT TCA AGG AGA ATG GTG C-3´, product size 724 bp(28) and GAPDH sense 5´-CGG AGT CAA CGG ATT TGG TCG TAT-3´ and antisense 5´-GCC TTC TCC ATG GTT GGT GAA GAC-3´, product size 301 bp(29)7KHUHDFWLRQPL[WXUHZDVLQFXEDWHGIRUPLQXWHDW & DPSOL¿HG IRU 3&5 F\FOHV DQG HQGHG E\ LQFXEDWLRQ DW & IRU PLQXWHV(DFK3&5F\FOHFRQVLVWHGRIGHQDWXULQJIRUVHFRQGVDW & DQQHDOLQJIRUVHFRQGVDW&DQGH[WHQGLQJIRUPLQXWHVDW& $PSOL¿HGVDPSOHVZHUHYLVXDOL]HGDIWHUHOHFWURSKRUHVLVRQDJDURVH gels and staining with ethidium bromide, with a gel photodocumentation system (Vilber Lourmat, Marne La Vallee, France). Band densitometry was performed with Image J Program (Bethesda, MD). 124
Figure 1. 3KHQRW\SLF FKDUDFWHUL]DWLRQ RI SULPDU\ ¿EUREODVW FXOWXUHV LVRODWHG from human oral mucosa (HOM). Phase-contrast microscopy of plated HOM fibroblasts (A and B) and immunodetection of vimentin (C) and prolyl-4hydroxylase (D) expression in HOM fibroblasts.
After 12 hrs of cell plating in 96-well plates at a density of 0.6 x 104 cells/well, growth medium was replaced by a thin layer of PBS DQG+20¿EUREODVWVZHUHVXEMHFWHGWRRUP-FP2 of UVB light or sham-irradiation. Proliferation was assessed from 3 to 48 hrs after UVB irradiation with the [3H]-thymidine incorporation assay. Analysis of the results showed that UVB irradiation, at both 30 and 60 mJ/cm2, caused D VLJQL¿FDQW GHFUHDVH LQ +20 ¿EUREODVW SUROLIHUDWLRQ IURP WR KUV DIWHU 89% LUUDGLDWLRQ S $129$ 7XNH\.UDPHU WHVWV )LJXUH 2). Analysis of cell viability by trypan blue exclusion assay showed no increase in cell death (more than 95% cell viability) at 24 and 48 hrs after UVB exposure with 30 and 60 mJ/cm2 (data not shown). Therefore, the 60 mJ/cm2GRVHRI89%LUUDGLDWLRQZDVXVHGLQWKHVXEVHTXHQWH[SHULPHQWV
6LQJOH([SRVXUHRI+XPDQ2UDO0XFRVD)LEUREODVWVWR8OWUDYLROHW%5DGLDWLRQ5HGXFHV3UROLIHUDWLRQDQG,QGXFHV&2;([SUHVVLRQDQG$FWLYDWLRQ Effects of UVB Irradiation on COX-2 mRNA Expression by Synchronized HOM Fibroblasts 7R DVVHVV &2; DW WKH P51$ OHYHO +20 ¿EUREODVWV ZHUH synchronized in their cell cycle in order to avoid changes in COX2 expression that were not related to UVB radiation, as previously described(25)7RWDO51$ZDVVXEVHTXHQWO\H[WUDFWHGDWDQG hrs after UVB irradiation (60 mJ/cm2) and subjected to RT-PCR. Samples were visualized in agarose gels stained with ethidium bromide (Figure 4A) and analyzed by densitometry. Analysis of densitometric units of &2; EDQGV QRUPDOL]HG WR *$3'+ VKRZHG D VLJQL¿FDQW LQFUHDVH LQ WKHSHUFHQWDJHRIUHODWLYH&2;P51$H[SUHVVLRQDWPLQDQGKUV DIWHU 89% LUUDGLDWLRQ DV FRPSDUHG WR VKDPLUUDGLDWHG FRQWUROV S t-test) (Figure 4B).
Figure 2. (IIHFWRIDVLQJOHGRVHRI89%UDGLDWLRQRQSUROLIHUDWLRQRI+20¿EUREODVWV Cells grown at a density of 0.6 x 104 cells/well for 12 hrs, were exposed to one single dose of 30 or 60 mJ/cm2 of UVB, and proliferation was assessed from 3 to 48 hrs after UVB -or sham-irradiation (control) with [3H]-thymidine incorporation assay. Results are expressed as mean ± SD of one representative experiment (n=8 wells/ group) of at least 5 independent experiments.
S $129$ DQG7XNH\ .UDPHU WHVW IRU VKDPLUUDGLDWHG +20 ¿EUREODVWV DV FRPSDUHGWR89%LUUDGLDWHG¿EUREODVWV DQGP-FP2).
The effects of UVB on HOM proliferation was also assessed RQ SUHYLRXVO\ V\QFKURQL]HG ¿EUREODVWV XVLQJ ERWK >3H]-thymidine incorporation and MTT assays, at 6, 12 and 24 hrs after UVB irradiation with 60 mJ/cm26LPLODUWRQRQV\QFKURQL]HGFHOOVDVLJQL¿FDQWGHFUHDVH LQSUROLIHUDWLRQRI89%LUUDGLDWHG+20¿EUREODVWVZDVREVHUYHGDWDOOWKH WLPH SRLQWV DQDO\]HG DV FRPSDUHG WR VKDPLUUDGLDWHG ¿EUREODVWV ZLWK ERWKDVVD\V S WWHVWDQG0DQQ:KLWQH\ )LJXUH
Figure 4. (IIHFW RI D VLQJOH GRVH RI 89% UDGLDWLRQ RQ &2; P51$ H[SUHVVLRQ E\V\QFKURQL]HG+20¿EUREODVWV7RWDO51$ZDVH[WUDFWHGIURP89%DQGVKDP LUUDGLDWHG FRQWURO +20¿EUREODVWV [5 cells/plate) and subjected to RT-PCR WRDVVHVV&2; ES DQG*$3'+ ES P51$H[SUHVVLRQ3URGXFWVZHUH visualized in 1.5% agarose gels stained with ethidium bromide. Representative gels of one of the experiments for 0.5 and 12 hrs after UVB irradiation are shown in (A). Relative densitometric units of COX-2 bands (normalized to GAPDH) were calculated. Results are expressed in percentage of corresponding control as mean 6(0RIWZRSRROHGH[SHULPHQWV Q SODWHVJURXSDWDQGKUVQ SODWHV group at 6 and 24 hrs) (B).
S WWHVW IRU89%LUUDGLDWHG¿EUREODVWVDVFRPSDUHGWRFRQWUROVDWDQG hrs after UVB exposure.
Figure 3. (IIHFWRIDVLQJOHGRVHRI89%UDGLDWLRQRQSUROLIHUDWLRQRIV\QFKURQL]HG +20¿EUREODVWV Cells grown at a density of 1 x 104 cells/well, were synchronized in their cell cycle and exposed to one single dose of 60 mJ/cm2 of UVB. Proliferation was assessed at 6, 12 and 24 hrs after UVB exposure or sham-irradiation (control) with [3H]-thymidine incorporation (A) and MTT (B) assays. Results are expressed as mean ± SD of one representative experiment repeated at least three times with similar results (n=8 wells/group at each time point). A)
S 0DQQ:KLWQH\ IRU89%LUUDGLDWHG¿EUREODVWVDVFRPSDUHGWRFRQWUROV DWKUV
S WWHVW IRU89%LUUDGLDWHG¿EUREODVWVDVFRPSDUHGWRFRQWUROV at 12 and 24 hrs. B)
S WWHVWDQG0DQQ:KLWQH\ IRU89%LUUDGLDWHG¿EUREODVWVDVFRPSDUHG to controls at 6, 12 and 24 hrs.
Effects of UVB on PGE2 Production by Synchronized HOM Fibroblasts 3*(2 SURGXFWLRQ ZDV PHDVXUHG E\ (,$ IURP WKH supernatants of synchronized HOM fibroblasts at 12 and 24 hrs after UVB irradiation. The results showed a significant increase in 3*( 2 production by HOM fibroblasts at both 12 and 24 hrs after UVB LUUDGLDWLRQDVFRPSDUHGWRFRQWUROV S DQGS UHVSHFWLYHO\ Mann Whitney) (Figure 5).
125
Rojas IG y cols.
Rev. Clin. Periodoncia Implantol. Rehabil. Oral Vol. 3(3); 123-127, 2010.
Figure 5. (IIHFW RI D VLQJOH GRVH RI 89% UDGLDWLRQ RQ 3*(2 production by V\QFKURQL]HG+20¿EUREODVWV3*(2OHYHOV SJPO ZHUHDVVHVVHGE\(,$DWDQG KUVDIWHU89%LUUDGLDWLRQIURPVXSHUQDWDQWVRIV\QFKURQL]HG+20¿EUREODVWV x 105 cells/plate). Results are expressed in percentage of corresponding control as PHDQ6(0RIWZRSRROHGH[SHULPHQWV Q SODWHVJURXSDWKUVDQGQ SODWHV group at 24 hrs).
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S 0DQQ:KLWQH\ IRU89%LUUDGLDWHG¿EUREODVWVDVFRPSDUHG to sham-irradiated at 12 and 24 hrs after UVB exposure, respectively.
DISCUSSION The results of this in vitro study showed that exposure of +20 ¿EUREODVWV WR D VLQJOH VXEF\WRWR[LF GRVH RI 89% OLJKW P-FP2) VLJQL¿FDQWO\ UHGXFHG FHOO SUROLIHUDWLRQ GXULQJ WKH ¿UVW KUV DIWHU 89% exposure as assessed by [3H]-thymidine incorporation and MTT assays. 89%LUUDGLDWHG +20 ¿EUREODVWV DOVR KDG LQFUHDVHG &2; P51$ expression at 0.5 and 12 hrs after UVB exposure and enhanced COX-2 DFWLYLW\DVUHÀHFWHGE\DQLQFUHDVHLQ3*(2 production at 12 and 24 hrs after irradiation. Very few studies have assessed the effects of UV light on SUROLIHUDWLRQ RI ¿EUREODVWV IURP WKH RUDO FDYLW\(18). Williams et al. (1992) showed that UVA light, with exposure times ranging from 15 seconds to PLQXWHVVLJQL¿FDQWO\GHFUHDVHG>3H]-thymidine incorporation by gingival ¿EUREODVWV(18). On the other hand, the effects of UVB light on proliferation of ERWKOXQJPXFRVDDQGVNLQ¿EUREODVWVDUHZHOONQRZQ(30-32). Straface et al. IRXQGWKDWHDUO\H[SRVXUHRIOXQJPXFRVD¿EUREODVWV SDVVDJHV to a single subcytotoxic dose of UVB (200 mJ/cm2 UHVXOWHGLQDVLJQL¿FDQW GHFUHDVHLQFHOOSUROLIHUDWLRQVLPLODUWRQRQLUUDGLDWHGOXQJ¿EUREODVWVIURP later passages (greater than 11 passages)(30). Chainiaux et al. (2002) using KXPDQ GLSORLG VNLQ ¿EUREODVWV H[SRVHG WR UHSHDWHG VXEF\WRWR[LF 89% doses (62.5 mJ/cm2) also found decreased proliferation, as assessed by [3H]-thymidine incorporation(31). Therefore, UVB irradiation had a similar LQKLELWRU\HIIHFWRQRUDOPXFRVD¿EUREODVWSUROLIHUDWLRQDVSUHYLRXVO\VKRZQ IRUVNLQDQGOXQJ¿EUREODVWV Since the MTT assay allows determination of both, cell proliferation and cytotoxicity(33), the results may also suggest that the SUROLIHUDWLRQLQKLELWLRQLQ+20¿EUREODVWVLUUDGLDWHGZLWKP-FP2 was due to reduced cell viability. Previously, mild to moderate cell cytotoxicity ZDV GHVFULEHG IRU VNLQ ¿EUREODVWV DW KUV DIWHU LUUDGLDWLRQ ZLWK 89% doses of 40 and 60 mJ/cm2(32). However, in this study, no increase in cell death was found, as assessed by trypan blue exclusion assay, at 24 and 48 hrs after UVB irradiation (more than 95% viability), suggesting higher UHVLVWDQFHRIRUDOPXFRVD¿EUREODVWVWR89%H[SRVXUHDVFRPSDUHGWR VNLQ¿EUREODVWV
Increased COX-2 expression has been described for UVBLUUDGLDWHGVNLQ¿EUREODVWV(15). It has been shown that transcriptional control RI&2;LVDVVRFLDWHGZLWK WKHSUROLIHUDWLRQVWDWXVRI¿EUREODVWV&2; XSUHJXODWLRQ ZDV REVHUYHG LQ TXLHVFHQW FHOOV ZKHUHDV &2; GRZQ UHJXODWLRQ ZDV IRXQG LQ SUROLIHUDWLQJ ¿EUREODVWV(25,34). In a similar way, 89%LUUDGLDWHGRUDOPXFRVD¿EUREODVWVZKLFKSUHVHQWHGDQLQKLELWLRQRI their proliferative response, also had and increase in COX-2 expression DQGDFWLYDWLRQ7KHUHIRUHWKHLQKLELWLRQRI¿EUREODVWSUROLIHUDWLRQE\89% irradiation may have both, a protective effect by reducing expansion of SKRWRGDPDJHG ¿EUREODVWV DQG RQ WKH RWKHU KDQG D SURFDUFLQRJHQLF effect by increasing COX-2 expression and activation. ,QWKHODVWGHFDGH¿EUREODVWVKDYHHPHUJHGDVFUXFLDOUHJXODWRUV RIHSLWKHOLDODQGHQGRWKHOLDOFHOOIXQFWLRQDVZHOODV(&0FRPSRVLWLRQ(35). 'XULQJFDUFLQRJHQHVLVVWURPDO¿EUREODVWVXQGHUJRVHYHUDOFKDQJHVWKDW UDQJH IURP DQ DFWLYDWHG WR D P\R¿EUREODVW SKHQRW\SH7KHVH FKDQJHV are strongly correlated with tumor progression(35). Increased COX-2 H[SUHVVLRQE\VWURPDO¿EUREODVWVKDVEHHQGHVFULEHGLQODU\QJHDOOXQJ and colon carcinogenesis with a direct association with prognosis(36-38). Several carcinogens increase COX-2 expression in oral mucosa ¿EUREODVWV LQFOXGLQJ QLFRWLQH DQG DUHFD QXW H[WUDFW(20,22). The present study showed that UVB radiation, which is a complete carcinogen, also LQGXFHV &2; H[SUHVVLRQ DQG DFWLYDWLRQ LQ RUDO PXFRVD ¿EUREODVWV Future work should focus on elucidating the signal transcription pathways that participate in UVB-induced COX-2 upregulation in oral mucosa ¿EUREODVWV ZKLFK PD\ LQYROYH 1)κB activation as has already been GHVFULEHG IRU 89%LUUDGLDWHG VNLQ ¿EUREODVWV DQG QLFRWLQHWUHDWHG JLQJLYDO¿EUREODVWV(15,21). Previous studies describing COX-2 overexpression at the epithelium of photodamaged lips(5), as well as, the present results VKRZLQJLQFUHDVHG&2;H[SUHVVLRQLQ89%LUUDGLDWHG+20¿EUREODVWV VWURQJO\VXJJHVWWKDW&2;PRGXODWLRQPD\EHRIFOLQLFDOVLJQL¿FDQFH for prevention and treatment of lip carcinogenesis. It has been already shown that selective COX-2 inhibitors, such as Celecoxib, are effective LQ UHGXFLQJ 89%LQGXFHG FKURQLF LQÀDPPDWLRQ DQG WXPRU IRUPDWLRQ LQ the skin(26,39)(SLGHPLRORJLFDOVWXGLHVKDYHVKRZQWKDWOLSFDQFHUKDVD higher metastasis occurrence than skin cancer(40), which may be due to phenotypic differences between epithelial and stromal cells from skin and lip tissues(11,12). Future studies should continue to investigate the role of ¿EUREODVWVDQGWKHLULQWHUDFWLRQVZLWKVWURPDOFHOOVGXULQJOLSSKRWRGDPDJH DQG FDUFLQRJHQHVLV DV ZHOO DV WKH WKHUDSHXWLF EHQH¿W RI PRGXODWLQJ ¿EUREODVW IXQFWLRQ DQG SKHQRW\SH LQ WKH WUHDWPHQW RI SUHPDOLJQDQW DQG malignant lesions of the lip.
CONFLICT OF INTEREST STATEMENT
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ACKNOWLEDGMENTS This research was supported by the Chilean Council for Science DQG7HFKQRORJ\&21,&<7JUDQWV)21'(&<7 DQG 7050210 (International collaboration grant). Dr. Yadira Boza wishes to WKDQNWKHJRYHUQPHQWRI&RVWD5LFDIRU¿QDQFLQJKHUJUDGXDWHVWXGLHV
REFERENCES 1. Lundeen RC, Langlais RP, Terezhalmy GT. Sunscreen protection for lip mucosa: a review and update. J Am Dent Assoc, 1985; 111: 617-621. .DXJDUV *( 3LOOLRQ 7 6YLUVN\ -$ 3DJH '* %XUQV -&$EEH\ /0 Actinic cheilitis: a review of 152 cases. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 1999; 88: 181-186. 5XQGKDXJ-()LVFKHU60&\FORR[\JHQDVHSOD\VDFULWLFDOUROHLQ UV-induced skin carcinogenesis. Photochem Photobiol, 2008; 84: 322329. 4. Yaar M, Gilchrest BA. Photoageing: mechanism, prevention and therapy. Br J Dermatol, 2007; 157: 874-887. 5. Rojas IG, Martínez A, Brethauer U et al. Actinic cheilitis: epithelial 126
expression of COX-2 and its association with mast cell tryptase and PAR2. Oral Oncol, 2009; 45: 284-290. 0DUWtQH]$%UHWKDXHU8%RUODQGR-6SHQFHU0/5RMDV,*(SLWKHOLDO expression of p53, mdm-2 and p21 in normal lip and actinic cheilitis. Oral Oncol, 2008; 44: 878-883. 1HWR 3LPHQWHO '5 0LFKDODQ\ 1 $OFKRUQH 0 $EUHX 0 %RUUD 5& Weckx L. Actinic cheilitis: histopathology and p53. J Cutan Pathol, 2006; 33: 539-544. 8. Rosette C, Karin M. Ultraviolet light and osmotic stress: activation of WKH-1.FDVFDGHWKURXJKPXOWLSOHJURZWKIDFWRUDQGF\WRNLQHUHFHSWRUV Science, 1996; 274: 1194-1197.
6LQJOH([SRVXUHRI+XPDQ2UDO0XFRVD)LEUREODVWVWR8OWUDYLROHW%5DGLDWLRQ5HGXFHV3UROLIHUDWLRQDQG,QGXFHV&2;([SUHVVLRQDQG$FWLYDWLRQ 9. Clydesdale GJ, Dandie GW, Muller HK. Ultraviolet light induced injury: LPPXQRORJLFDO DQG LQÀDPPDWRU\ HIIHFWV Immunol Cell Biol, 2001; 79: 547-568. 10. Yokoo S, Yamagami S, Mimura T et al. UV absorption in human oral mucosal epithelial sheets for ocular surface reconstruction. Ophthalmic Res, 2006; 38: 350-354. 11. McKeown ST, Barnes JJ, Hyland PL, Lundy FT, Fray MJ, Irwin CR. 0DWUL[PHWDOORSURWHLQDVHGLIIHUHQFHVLQRUDODQGVNLQ¿EUREODVWVJ Dent Res, 2007; 86: 457-462. 6WHSKHQV3'DYLHV.-2FFOHVWRQ1HWDO6NLQDQGRUDO¿EUREODVWV exhibit phenotypic differences in extracellular matrix reorganization and matrix metalloproteinase activity. Br J Dermatol, 2001; 144: 229-237. 13. Chen W, Kang J, Xia J et al. p53-related apoptosis resistance and tumor suppression activity in UVB-induced premature senescent human VNLQ¿EUREODVWVInt J Mol Med, 2008; 21: 645-653. 'HEDFT&KDLQLDX[)%RUORQ&3DVFDO7HWDO5HSHDWHGH[SRVXUHRI KXPDQVNLQ¿EUREODVWVWR89%DWVXEF\WRWR[LFOHYHOWULJJHUVSUHPDWXUH senescence through the TGF-beta1 signaling pathway. J Cell Sci, 2005; 118: 743-758. 0DUZDKD 9 &KHQ<+ +HOPV ( HW DO7ROLJR WUHDWPHQW GHFUHDVHV FRQVWLWXWLYHDQG89%LQGXFHG&2;OHYHOVWKURXJKSDQG1)NDSSD% dependent repression of the COX-2 promoter. J Biol Chem, 2005; 280: 32379-32388. 16. Tober KL, Wilgus TA, Kusewitt DF, Thomas-Ahner JM, Maruyama 72EHU\V]\Q70,PSRUWDQFHRIWKH(3 UHFHSWRULQFXWDQHRXV89% LQGXFHGLQÀDPPDWLRQDQGWXPRUGHYHORSPHQWJ Invest Dermatol, 2006; 126: 205-211. 2EHU\V]\Q701RQPHODQRPDVNLQFDQFHU LPSRUWDQFHRIJHQGHU immunosuppressive status and vitamin D. Cancer Lett, 2008; 261: 127136. 18. Williams JK, Davidson SF, Johnson SG, Hsu HS, Das SK. An in vitro VWXG\RQWKHHIIHFWRI89$UDGLDWLRQRQKXPDQJLQJLYDO¿EUREODVWVBr J Plast Surg, 1992; 45: 349-353. 19. Lim W, Ko M, Lee S et al. Ultraviolet-C-induced apoptosis protected E\ QP ODVHU LUUDGLDWLRQ LQ KXPDQ JLQJLYDO ¿EUREODVWV Photomed Laser Surg, 2008; 26: 215-220. 20. Chang YC, Tsai CH, Yang SH, Liu CM, Chou MY. Induction of F\FORR[\JHQDVH P51$ DQG SURWHLQ H[SUHVVLRQ LQ KXPDQ JLQJLYDO ¿EUREODVWV VWLPXODWHG ZLWK QLFRWLQH J Periodontal Res, 2003; 38: 496501. 1DNDR62JDWD<6XJL\D+1LFRWLQHVWLPXODWHVWKHH[SUHVVLRQRI F\FORR[\JHQDVH P51$ YLD 1)NDSSD% DFWLYDWLRQ LQ KXPDQ JLQJLYDO ¿EUREODVWVArch Oral Biol, 2009; 54: 251-257. 22. Tsai CH, Chou MY, Chang YC. The up-regulation of cyclooxygenase-2 H[SUHVVLRQLQKXPDQEXFFDOPXFRVDO¿EUREODVWVE\DUHFROLQH DSRVVLEOH UROHLQWKHSDWKRJHQHVLVRIRUDOVXEPXFRXV¿EURVLVJ Oral Pathol Med, 2003; 32: 146-153.
:LOJXV 7$ .RNL $7 =ZHLIHO %6 5XEDO 3$ 2EHU\V]\Q 70 &KHPRWKHUDSHXWLF HI¿FDF\ RI WRSLFDO FHOHFR[LE LQ D PXULQH PRGHO RI ultraviolet light B-induced skin cancer. Mol Carcinog, 2003; 38: 33-39. 5XGROSK0,%R]D<
127
Single Exposure of Human Oral Mucosa Fibroblasts to Ultraviolet B Radiation Reduces Proliferation and Induces COX-2 Expression and Activation Boza Y1,2
Very few studies have analyzed the effects of UV radiation on RUDOPXFRVD¿EUREODVWIXQFWLRQ:LOOLDPVHWDO IRXQGWKDWKXPDQ JLQJLYDO ¿EUREODVWV LUUDGLDWHG ZLWK 89$ OLJKW KDG D VLJQL¿FDQW GHFUHDVH in proliferation(18), and Lim et al. (2008) found that UVC radiation caused JLQJLYDO ¿EUREODVW DSRSWRVLV(19). It is known that several factors induce &2; H[SUHVVLRQ LQ RUDO ¿EUREODVWV LQFOXGLQJ QLFRWLQH DUHFD QXW H[WUDFWV DUHFROLQH SURLQÀDPPDWRU\F\WRNLQHVDQG/36(20-23). However, the effects of UVB radiation on COX-2 expression and activation in oral PXFRVD ¿EUREODVWV DUH XQNQRZQ 7KHUHIRUH WKLV VWXG\ ZDV DLPHG DW characterizing the effects of a single exposure to subcytotoxic doses of 89% OLJKW RQ SUROLIHUDWLRQ &2; H[SUHVVLRQ DQG 3*(2 production by SULPDU\¿EUREODVWFXOWXUHVHVWDEOLVKHGIURPKXPDQRUDOPXFRVD +20 explants.
MATERIAL AND METHODS Isolation and Culture of Oral Mucosa Fibroblasts 3ULPDU\ ¿EUREODVW FXOWXUHV IURP KXPDQ RUDO PXFRVD +20 were isolated by the explant method as previously described(24)%ULHÀ\ using a 6 mm punch-biopsy (Acu-punch, Acuderm, Inc., Fort Launderdale, FL, USA) tissue explants were obtained from the oral mucosa lining the inner cheek of the oral cavity, in close proximity to the lip, from three healthy, non-smoking volunteers, with no history of drug intake in the past 6 months (2 females and 1 male, mean ages 22 ± 1 years). Informed consent was obtained from the subjects, and the surgical protocol was DSSURYHGE\WKH(WKLFV&RPPLWWHHRI7KH8QLYHUVLW\RI&RQFHSFLyQ&HOOV ZHUH FXOWXUHG LQ 'XOEHFFRV PRGL¿HG (DJOHV PHGLXP '0(0 *LEFR BRL, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, *LEFR%5/ ȝJPO SHQLFLOOLQVWUHSWRP\FLQ *LEFR%5/ ȝJPO IXQJL]RQH 86%LRORJLFDO6ZDPSVFRWW0$ DQGȝJPORIJHQWDP\FLQ 6LJPD6W/RXLV02 DW&LQD&22 atmosphere. All the assays were performed using fibroblasts between passages 4 and 10. HOM
1. Departamento de Estomatología Quirúrgica y Laboratorio de Biología y Patología Oral. Facultad de Odontología, Universidad de Concepción. Concepción, Chile. 2. Programa de Magíster en Ciencias Odontológicas, Mención Patología. Escuela de Graduados, Facultad de Odontología, Universidad de Chile. Santiago, Chile. 8QLGDG$FDGpPLFD2GRQWRORJtD)DFXOWDGGH0HGLFLQD3RQWL¿FLD8QLYHUVLGDG&DWyOLFDGH&KLOH6DQWLDJR&KLOH 4. Department of Pathology. College of Medicine, The Ohio State University. Columbus, Ohio, USA. Correspondence author: Dra. I. Gina Rojas. [email protected]. Depto. Estomatología Quirúrgica. Facultad de Odontología, Universidad de Concepción. Casilla 160-C. Concepción, Chile. Trabajo recibido el 05/11/2010. Aprobado para su publicación el 30/12/2010. 123
Rev. Clin. Periodoncia Implantol. Rehabil. Oral Vol. 3(3); 123-127, 2010. ¿EUREODVWFXOWXUHVZHUHFKDUDFWHUL]HGE\LPPXQRF\WRFKHPLVWU\WRFRQ¿UP their phenotype with primary antibodies against vimentin (1:100) (Sigma, St Louis, MO, USA) and prolyl-4-hydroxylase (1:50) (Dako, Carpinteria, CA) followed by a secondary antibody coupled to an HRP detection system (Chemicon Int, Temecula, CA) and DAB as the chromogen (Vector Labs, Burlingame, CA). Growth Synchronization and UVB Light Exposure of HOM Fibroblasts 3UHYLRXVWR89%H[SRVXUH+20¿EUREODVWVZHUHSODWHGDQG maintained either in complete medium supplemented with 10% FBS for 12 hrs (unsynchronized cells) or synchronized according to the protocol by Gilroy et al. (2001)(25)%ULHÀ\FHOOVZHUHZDVKHGZLWK3%6SODWHGLQ 96-well or 100 mm plates, and then incubated in serum-free medium for 24 hrs, followed by medium containing 2.5% FBS for 20 hrs. Previous to LUUDGLDWLRQPHGLXPZDVUHSODFHGE\ȝORI3%6 *LEFR LQHDFKZHOORU PORI3%6LQWKHPPSODWHV7KHQ¿EUREODVWVZHUHLUUDGLDWHGZLWK 30 or 60 mJ/cm2RI89%HTXLYDOHQWWRDQGPLQLPDOHU\WKHPDOGRVHV 0('V UHVSHFWLYHO\ZLWK3KLOOLSV)689%ODPSV $PHULFDQ8OWUDYLROHW &RPSDQ\0XUUD\+LOO1- DVSUHYLRXVO\GHVFULEHG(26)/DPSVZHUH¿WWHG ZLWK.RGDFHO¿OWHUV (DVWPDQ.RGDN5RFKHVWHU1< WRHQVXUHHPLVVLRQ of UVB light only (290-320). The intensity of the UVB light at the bottom of each well or plate was measured with a UVX Digital radiometer (UVP, 8SODQG&$86$ &RQWURO¿EUREODVWVZHUHVKDPLUUDGLDWHG
Rojas IG y cols. Prostagandin E2 Assay +20¿EUREODVWVSODWHGLQPPSODWHVZHUHV\QFKURQL]HG and UVB irradiated. At 12 and 24 hrs after irradiation, supernatants were FROOHFWHGDQGWKHDPRXQWRI3*(2 secreted was determined by enzymelinked immunosorbent assay according to manufacturer´s instructions (Cayman Chemical, Ann Arbor, MI, USA). Results were expressed as PHDQ6'RIWKHSHUFHQWDJHRI3*(2 production (pg/ml) as compared to control. Statistical Analyses 6WDWLVWLFDO WHVWV ZHUH SHUIRUPHG XVLQJ -03,1 6$6 ,QVWLWXWH ,QF &DU\ 1& 86$ 'LIIHUHQFHV EHWZHHQ JURXSV ZHUH examined using Mann-Whitney or t-test, according to data distribution. 'LIIHUHQFHVZHUHFRQVLGHUHGVWDWLVWLFDOO\VLJQL¿FDQWZKHQS
RESULTS Effects of UVB on Proliferation of Fibroblasts Derived from Human Oral Mucosa 3ULPDU\ ¿EUREODVW FXOWXUHV ZHUH REWDLQHG IURP KXPDQ RUDO mucosa explants and characterized by immunocytochemistry. Greater than 95% of the cells stained positive for vimentin and prolyl-4-hydroxylase (Figure 1).
Cell Proliferation Assays 7RDVVHVV+20¿EUREODVWSUROLIHUDWLRQZLWKWKH>3H]-thymidine LQFRUSRUDWLRQ DVVD\ ¿EUREODVWV ZHUH FXOWXUHG LQ ZHOO SODWHV DW D density of 0.6-1 x 104 FHOOVPO &HOOV ZHUH 89%LUUDGLDWHG DQG ȝ&L [3+@WK\PLGLQH VSHFL¿F DFWLYLW\ &LPPRO 'XSRQW1(1 %RVWRQ 0$ was added to the culture medium for 3 to 48 hrs. After incubation, cells ZHUHWU\SVLQLVHGYDFXXPHGRQWRSDSHU¿OWHUV ȝPSRUHV DQGSODFHG LQPORIVFLQWLOODWLRQÀXLG (FRVFLQW/61DWLRQDO'LDJQRVWLFV86$ 7KH LQFRUSRUDWHG UDGLRDFWLYLW\ ZDV TXDQWL¿HG E\ D VFLQWLOODWLRQ FRXQWHU (Packard 1600 TR, Packard Instruments, Downers Grove, IL, USA). Results were expressed as cpm/well. )RU 077 SUROLIHUDWLRQ DVVD\ ¿EUREODVWV ZHUH FXOWXUHG LQ well plates, at a density of 1 x 104 cells/ml. At 6, 12 and 24 hrs post-UVB LUUDGLDWLRQȝORI077VROXWLRQZHUHDGGHGWRHDFKZHOODQGSURFHVVHG according to the manufacturer´s instructions. Absorbance was measured DWQPXVLQJDPLFURSODWHUHDGHU 0XOWLVNDQ(;7KHUPR&RUSRUDWLRQ USA). In addition, cell viability was determined at 24 and 48 hrs posttreatment by trypan blue exclusion assay as previously described(27). ([SHULPHQWVZHUHUHSHDWHGDWOHDVWWKUHHWLPHVZLWKZHOOVJURXS Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 7RWDO 51$ ZDV LVRODWHG IURP ¿EUREODVWV VHHGHG RQWR mm plates (0.6 x 106 cells/plate) with Trizol (Invitrogen, Rockville, MD), according to manufacturer´s instructions (n=6-7 plates/group at each WLPHSRLQW 51$ FRQFHQWUDWLRQ DQG QXFOHLF DFLG SXULW\ ZDV GHWHUPLQHG spectrophotometrically by UV absorbance. RT-PCR for COX-2 and GAPDH was performed as previously described(5) %ULHÀ\ ȝJ RI WRWDO 51$ ZDV UHYHUVHG WUDQVFULEHG XVLQJ ROLJRG7 SULPHUV DQG $09 UHYHUVH WUDQVFULSWDVH 3URPHJD 0DGLVRQ :, DQG ȝO RI F'1$ IURP HDFKVDPSOHZDVDPSOL¿HGE\3&5(DFK3&5ZDVFDUULHGRXWLQȝO of a reaction mix containing 10 mM Tris-HCl, 50 mM KCl, 3 mM MgCl2, P0RIHDFKG173ȝ0RIHDFKVHQVHDQGDQWLVHQVHSULPHUDQG RQHXQLWRI3ODQWLQXP7DT'1$SRO\PHUDVH ,QYLWURJHQ&DUOVEDG&$ $QHJDWLYHFRQWUROZKHUHWKHF'1$ZDVRPLWWHGIURPWKHUHDFWLRQPL[ ZDVUXQIRUHDFKDPSOL¿HGJHQH7KHIROORZLQJSULPHUVHTXHQFHVZHUH used: COX-2 sense 5´-GGT CTG GTG CCT GGT CTG ATG ATG-3´ and antisense 5´-GTC CTT TCA AGG AGA ATG GTG C-3´, product size 724 bp(28) and GAPDH sense 5´-CGG AGT CAA CGG ATT TGG TCG TAT-3´ and antisense 5´-GCC TTC TCC ATG GTT GGT GAA GAC-3´, product size 301 bp(29)7KHUHDFWLRQPL[WXUHZDVLQFXEDWHGIRUPLQXWHDW & DPSOL¿HG IRU 3&5 F\FOHV DQG HQGHG E\ LQFXEDWLRQ DW & IRU PLQXWHV(DFK3&5F\FOHFRQVLVWHGRIGHQDWXULQJIRUVHFRQGVDW & DQQHDOLQJIRUVHFRQGVDW&DQGH[WHQGLQJIRUPLQXWHVDW& $PSOL¿HGVDPSOHVZHUHYLVXDOL]HGDIWHUHOHFWURSKRUHVLVRQDJDURVH gels and staining with ethidium bromide, with a gel photodocumentation system (Vilber Lourmat, Marne La Vallee, France). Band densitometry was performed with Image J Program (Bethesda, MD). 124
Figure 1. 3KHQRW\SLF FKDUDFWHUL]DWLRQ RI SULPDU\ ¿EUREODVW FXOWXUHV LVRODWHG from human oral mucosa (HOM). Phase-contrast microscopy of plated HOM fibroblasts (A and B) and immunodetection of vimentin (C) and prolyl-4hydroxylase (D) expression in HOM fibroblasts.
After 12 hrs of cell plating in 96-well plates at a density of 0.6 x 104 cells/well, growth medium was replaced by a thin layer of PBS DQG+20¿EUREODVWVZHUHVXEMHFWHGWRRUP-FP2 of UVB light or sham-irradiation. Proliferation was assessed from 3 to 48 hrs after UVB irradiation with the [3H]-thymidine incorporation assay. Analysis of the results showed that UVB irradiation, at both 30 and 60 mJ/cm2, caused D VLJQL¿FDQW GHFUHDVH LQ +20 ¿EUREODVW SUROLIHUDWLRQ IURP WR KUV DIWHU 89% LUUDGLDWLRQ S $129$ 7XNH\.UDPHU WHVWV )LJXUH 2). Analysis of cell viability by trypan blue exclusion assay showed no increase in cell death (more than 95% cell viability) at 24 and 48 hrs after UVB exposure with 30 and 60 mJ/cm2 (data not shown). Therefore, the 60 mJ/cm2GRVHRI89%LUUDGLDWLRQZDVXVHGLQWKHVXEVHTXHQWH[SHULPHQWV
6LQJOH([SRVXUHRI+XPDQ2UDO0XFRVD)LEUREODVWVWR8OWUDYLROHW%5DGLDWLRQ5HGXFHV3UROLIHUDWLRQDQG,QGXFHV&2;([SUHVVLRQDQG$FWLYDWLRQ Effects of UVB Irradiation on COX-2 mRNA Expression by Synchronized HOM Fibroblasts 7R DVVHVV &2; DW WKH P51$ OHYHO +20 ¿EUREODVWV ZHUH synchronized in their cell cycle in order to avoid changes in COX2 expression that were not related to UVB radiation, as previously described(25)7RWDO51$ZDVVXEVHTXHQWO\H[WUDFWHGDWDQG hrs after UVB irradiation (60 mJ/cm2) and subjected to RT-PCR. Samples were visualized in agarose gels stained with ethidium bromide (Figure 4A) and analyzed by densitometry. Analysis of densitometric units of &2; EDQGV QRUPDOL]HG WR *$3'+ VKRZHG D VLJQL¿FDQW LQFUHDVH LQ WKHSHUFHQWDJHRIUHODWLYH&2;P51$H[SUHVVLRQDWPLQDQGKUV DIWHU 89% LUUDGLDWLRQ DV FRPSDUHG WR VKDPLUUDGLDWHG FRQWUROV S t-test) (Figure 4B).
Figure 2. (IIHFWRIDVLQJOHGRVHRI89%UDGLDWLRQRQSUROLIHUDWLRQRI+20¿EUREODVWV Cells grown at a density of 0.6 x 104 cells/well for 12 hrs, were exposed to one single dose of 30 or 60 mJ/cm2 of UVB, and proliferation was assessed from 3 to 48 hrs after UVB -or sham-irradiation (control) with [3H]-thymidine incorporation assay. Results are expressed as mean ± SD of one representative experiment (n=8 wells/ group) of at least 5 independent experiments.
S $129$ DQG7XNH\ .UDPHU WHVW IRU VKDPLUUDGLDWHG +20 ¿EUREODVWV DV FRPSDUHGWR89%LUUDGLDWHG¿EUREODVWV DQGP-FP2).
The effects of UVB on HOM proliferation was also assessed RQ SUHYLRXVO\ V\QFKURQL]HG ¿EUREODVWV XVLQJ ERWK >3H]-thymidine incorporation and MTT assays, at 6, 12 and 24 hrs after UVB irradiation with 60 mJ/cm26LPLODUWRQRQV\QFKURQL]HGFHOOVDVLJQL¿FDQWGHFUHDVH LQSUROLIHUDWLRQRI89%LUUDGLDWHG+20¿EUREODVWVZDVREVHUYHGDWDOOWKH WLPH SRLQWV DQDO\]HG DV FRPSDUHG WR VKDPLUUDGLDWHG ¿EUREODVWV ZLWK ERWKDVVD\V S WWHVWDQG0DQQ:KLWQH\ )LJXUH
Figure 4. (IIHFW RI D VLQJOH GRVH RI 89% UDGLDWLRQ RQ &2; P51$ H[SUHVVLRQ E\V\QFKURQL]HG+20¿EUREODVWV7RWDO51$ZDVH[WUDFWHGIURP89%DQGVKDP LUUDGLDWHG FRQWURO +20¿EUREODVWV [5 cells/plate) and subjected to RT-PCR WRDVVHVV&2; ES DQG*$3'+ ES P51$H[SUHVVLRQ3URGXFWVZHUH visualized in 1.5% agarose gels stained with ethidium bromide. Representative gels of one of the experiments for 0.5 and 12 hrs after UVB irradiation are shown in (A). Relative densitometric units of COX-2 bands (normalized to GAPDH) were calculated. Results are expressed in percentage of corresponding control as mean 6(0RIWZRSRROHGH[SHULPHQWV Q SODWHVJURXSDWDQGKUVQ SODWHV group at 6 and 24 hrs) (B).
S WWHVW IRU89%LUUDGLDWHG¿EUREODVWVDVFRPSDUHGWRFRQWUROVDWDQG hrs after UVB exposure.
Figure 3. (IIHFWRIDVLQJOHGRVHRI89%UDGLDWLRQRQSUROLIHUDWLRQRIV\QFKURQL]HG +20¿EUREODVWV Cells grown at a density of 1 x 104 cells/well, were synchronized in their cell cycle and exposed to one single dose of 60 mJ/cm2 of UVB. Proliferation was assessed at 6, 12 and 24 hrs after UVB exposure or sham-irradiation (control) with [3H]-thymidine incorporation (A) and MTT (B) assays. Results are expressed as mean ± SD of one representative experiment repeated at least three times with similar results (n=8 wells/group at each time point). A)
S 0DQQ:KLWQH\ IRU89%LUUDGLDWHG¿EUREODVWVDVFRPSDUHGWRFRQWUROV DWKUV
S WWHVW IRU89%LUUDGLDWHG¿EUREODVWVDVFRPSDUHGWRFRQWUROV at 12 and 24 hrs. B)
S WWHVWDQG0DQQ:KLWQH\ IRU89%LUUDGLDWHG¿EUREODVWVDVFRPSDUHG to controls at 6, 12 and 24 hrs.
Effects of UVB on PGE2 Production by Synchronized HOM Fibroblasts 3*(2 SURGXFWLRQ ZDV PHDVXUHG E\ (,$ IURP WKH supernatants of synchronized HOM fibroblasts at 12 and 24 hrs after UVB irradiation. The results showed a significant increase in 3*( 2 production by HOM fibroblasts at both 12 and 24 hrs after UVB LUUDGLDWLRQDVFRPSDUHGWRFRQWUROV S DQGS UHVSHFWLYHO\ Mann Whitney) (Figure 5).
125
Rojas IG y cols.
Rev. Clin. Periodoncia Implantol. Rehabil. Oral Vol. 3(3); 123-127, 2010.
Figure 5. (IIHFW RI D VLQJOH GRVH RI 89% UDGLDWLRQ RQ 3*(2 production by V\QFKURQL]HG+20¿EUREODVWV3*(2OHYHOV SJPO ZHUHDVVHVVHGE\(,$DWDQG KUVDIWHU89%LUUDGLDWLRQIURPVXSHUQDWDQWVRIV\QFKURQL]HG+20¿EUREODVWV x 105 cells/plate). Results are expressed in percentage of corresponding control as PHDQ6(0RIWZRSRROHGH[SHULPHQWV Q SODWHVJURXSDWKUVDQGQ SODWHV group at 24 hrs).
S DQG
S 0DQQ:KLWQH\ IRU89%LUUDGLDWHG¿EUREODVWVDVFRPSDUHG to sham-irradiated at 12 and 24 hrs after UVB exposure, respectively.
DISCUSSION The results of this in vitro study showed that exposure of +20 ¿EUREODVWV WR D VLQJOH VXEF\WRWR[LF GRVH RI 89% OLJKW P-FP2) VLJQL¿FDQWO\ UHGXFHG FHOO SUROLIHUDWLRQ GXULQJ WKH ¿UVW KUV DIWHU 89% exposure as assessed by [3H]-thymidine incorporation and MTT assays. 89%LUUDGLDWHG +20 ¿EUREODVWV DOVR KDG LQFUHDVHG &2; P51$ expression at 0.5 and 12 hrs after UVB exposure and enhanced COX-2 DFWLYLW\DVUHÀHFWHGE\DQLQFUHDVHLQ3*(2 production at 12 and 24 hrs after irradiation. Very few studies have assessed the effects of UV light on SUROLIHUDWLRQ RI ¿EUREODVWV IURP WKH RUDO FDYLW\(18). Williams et al. (1992) showed that UVA light, with exposure times ranging from 15 seconds to PLQXWHVVLJQL¿FDQWO\GHFUHDVHG>3H]-thymidine incorporation by gingival ¿EUREODVWV(18). On the other hand, the effects of UVB light on proliferation of ERWKOXQJPXFRVDDQGVNLQ¿EUREODVWVDUHZHOONQRZQ(30-32). Straface et al. IRXQGWKDWHDUO\H[SRVXUHRIOXQJPXFRVD¿EUREODVWV SDVVDJHV to a single subcytotoxic dose of UVB (200 mJ/cm2 UHVXOWHGLQDVLJQL¿FDQW GHFUHDVHLQFHOOSUROLIHUDWLRQVLPLODUWRQRQLUUDGLDWHGOXQJ¿EUREODVWVIURP later passages (greater than 11 passages)(30). Chainiaux et al. (2002) using KXPDQ GLSORLG VNLQ ¿EUREODVWV H[SRVHG WR UHSHDWHG VXEF\WRWR[LF 89% doses (62.5 mJ/cm2) also found decreased proliferation, as assessed by [3H]-thymidine incorporation(31). Therefore, UVB irradiation had a similar LQKLELWRU\HIIHFWRQRUDOPXFRVD¿EUREODVWSUROLIHUDWLRQDVSUHYLRXVO\VKRZQ IRUVNLQDQGOXQJ¿EUREODVWV Since the MTT assay allows determination of both, cell proliferation and cytotoxicity(33), the results may also suggest that the SUROLIHUDWLRQLQKLELWLRQLQ+20¿EUREODVWVLUUDGLDWHGZLWKP-FP2 was due to reduced cell viability. Previously, mild to moderate cell cytotoxicity ZDV GHVFULEHG IRU VNLQ ¿EUREODVWV DW KUV DIWHU LUUDGLDWLRQ ZLWK 89% doses of 40 and 60 mJ/cm2(32). However, in this study, no increase in cell death was found, as assessed by trypan blue exclusion assay, at 24 and 48 hrs after UVB irradiation (more than 95% viability), suggesting higher UHVLVWDQFHRIRUDOPXFRVD¿EUREODVWVWR89%H[SRVXUHDVFRPSDUHGWR VNLQ¿EUREODVWV
Increased COX-2 expression has been described for UVBLUUDGLDWHGVNLQ¿EUREODVWV(15). It has been shown that transcriptional control RI&2;LVDVVRFLDWHGZLWK WKHSUROLIHUDWLRQVWDWXVRI¿EUREODVWV&2; XSUHJXODWLRQ ZDV REVHUYHG LQ TXLHVFHQW FHOOV ZKHUHDV &2; GRZQ UHJXODWLRQ ZDV IRXQG LQ SUROLIHUDWLQJ ¿EUREODVWV(25,34). In a similar way, 89%LUUDGLDWHGRUDOPXFRVD¿EUREODVWVZKLFKSUHVHQWHGDQLQKLELWLRQRI their proliferative response, also had and increase in COX-2 expression DQGDFWLYDWLRQ7KHUHIRUHWKHLQKLELWLRQRI¿EUREODVWSUROLIHUDWLRQE\89% irradiation may have both, a protective effect by reducing expansion of SKRWRGDPDJHG ¿EUREODVWV DQG RQ WKH RWKHU KDQG D SURFDUFLQRJHQLF effect by increasing COX-2 expression and activation. ,QWKHODVWGHFDGH¿EUREODVWVKDYHHPHUJHGDVFUXFLDOUHJXODWRUV RIHSLWKHOLDODQGHQGRWKHOLDOFHOOIXQFWLRQDVZHOODV(&0FRPSRVLWLRQ(35). 'XULQJFDUFLQRJHQHVLVVWURPDO¿EUREODVWVXQGHUJRVHYHUDOFKDQJHVWKDW UDQJH IURP DQ DFWLYDWHG WR D P\R¿EUREODVW SKHQRW\SH7KHVH FKDQJHV are strongly correlated with tumor progression(35). Increased COX-2 H[SUHVVLRQE\VWURPDO¿EUREODVWVKDVEHHQGHVFULEHGLQODU\QJHDOOXQJ and colon carcinogenesis with a direct association with prognosis(36-38). Several carcinogens increase COX-2 expression in oral mucosa ¿EUREODVWV LQFOXGLQJ QLFRWLQH DQG DUHFD QXW H[WUDFW(20,22). The present study showed that UVB radiation, which is a complete carcinogen, also LQGXFHV &2; H[SUHVVLRQ DQG DFWLYDWLRQ LQ RUDO PXFRVD ¿EUREODVWV Future work should focus on elucidating the signal transcription pathways that participate in UVB-induced COX-2 upregulation in oral mucosa ¿EUREODVWV ZKLFK PD\ LQYROYH 1)κB activation as has already been GHVFULEHG IRU 89%LUUDGLDWHG VNLQ ¿EUREODVWV DQG QLFRWLQHWUHDWHG JLQJLYDO¿EUREODVWV(15,21). Previous studies describing COX-2 overexpression at the epithelium of photodamaged lips(5), as well as, the present results VKRZLQJLQFUHDVHG&2;H[SUHVVLRQLQ89%LUUDGLDWHG+20¿EUREODVWV VWURQJO\VXJJHVWWKDW&2;PRGXODWLRQPD\EHRIFOLQLFDOVLJQL¿FDQFH for prevention and treatment of lip carcinogenesis. It has been already shown that selective COX-2 inhibitors, such as Celecoxib, are effective LQ UHGXFLQJ 89%LQGXFHG FKURQLF LQÀDPPDWLRQ DQG WXPRU IRUPDWLRQ LQ the skin(26,39)(SLGHPLRORJLFDOVWXGLHVKDYHVKRZQWKDWOLSFDQFHUKDVD higher metastasis occurrence than skin cancer(40), which may be due to phenotypic differences between epithelial and stromal cells from skin and lip tissues(11,12). Future studies should continue to investigate the role of ¿EUREODVWVDQGWKHLULQWHUDFWLRQVZLWKVWURPDOFHOOVGXULQJOLSSKRWRGDPDJH DQG FDUFLQRJHQHVLV DV ZHOO DV WKH WKHUDSHXWLF EHQH¿W RI PRGXODWLQJ ¿EUREODVW IXQFWLRQ DQG SKHQRW\SH LQ WKH WUHDWPHQW RI SUHPDOLJQDQW DQG malignant lesions of the lip.
CONFLICT OF INTEREST STATEMENT
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ACKNOWLEDGMENTS This research was supported by the Chilean Council for Science DQG7HFKQRORJ\&21,&<7JUDQWV)21'(&<7 DQG 7050210 (International collaboration grant). Dr. Yadira Boza wishes to WKDQNWKHJRYHUQPHQWRI&RVWD5LFDIRU¿QDQFLQJKHUJUDGXDWHVWXGLHV
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