Table 1.
PCR primers used in this study and the size of generated products.
Figure 1.
Effect of AN components on cytotoxicity, COX-2 expression and prostanoids production of GK and its modulation by catalase and PBL extract.
(A) Stimulation of PGF2α production of GK by ANE (50–1200 µg/ml) (n = 9). (B) Effect of arecoline on PGF2α production of GK (n = 3), (C) The regulation of ANE-induced PGE2 production of GK by catalase (500 and 1000 U/ml) (n = 11), (D) Catalase inhibited the ANE-induced COX-2 protein expression in GK as analyzed by western blotting. One representative western blotting picture was shown. (E) Catalase attenuated the ANE-induced cytotoxicity to GK (n = 7). (F) Effect of catechol on PGE2 production of GK (n = 4), (G) Effect of PBL extract on PGE2 production of GK (n = 3). Results were expressed as concentration (pg/ml) in the culture medium. (H) Cytotoxicity of ANE to GK (n = 6), (I) Cytotoxicity of arecoline to GK (n = 4), (J) Cytotoxicity of catechol to GK (n = 3). Results were expressed as MTT reduction (% of control, Mean ± SE). *denotes significant difference when compared with control. #denotes statistical significant difference when compared with ANE (800 µg/ml) solely-treated group (P<0.05).
Figure 2.
Role of cytochrome p450 and HO-1 on ANE-induced cytotoxicity and PGE2 production in GK.
(A) Expression of cytochrome p450 isoforms (1A1, 1A2, 2B6/B7, 2A6/A7, 2C8/19, 2E1, 2F1, 3A3, 3A4, 3A5, 3A7) in cultured GK. One representative RT-PCR picture was shown, (B) Stimulation of CYP1A1 mRNA expression in GK by ANE was noted as revealed by RT-PCR (C) α–naphthoflavone (10 and 25 µM) pretreatment and co-incubation prevented the ANE-induced PGE2 production in GK (n = 10). (D) Effect of α–naphthoflavone on ANE-induced cytotoxicity to GK (n = 7), (E) Effect of ANE on HO-1 gene expression of GK, (F) Effect of catalase on ANE-induced HO-1 gene expression of GK, (G) Effect of Zn-protoporphyrin on ANE-induced PGE2 production in GK. *denotes significant difference when compared with solvent control. #denotes statistical significant difference when compared with ANE (800 µg/ml) solely-treated group (P<0.05).
Figure 3.
Effect of ANE on EGFR activation and its role in ANE-induced COX-2 expression and PGE2 production of GK.
(A) AN extract (ANE) stimulated EGFR (Tyr845) phorphorylation within 60 min of exposure as analyzed by Pathscan p-EGFR ELISA. *denotes significant difference when compared with solvent control. (n = 10). (B) Pretreatment by PD153035 (1 and 5 µM, an EGFR antagonist) markedly attenuated the ANE-induced PGE2 production in GK. (C) Effect of PD153035 on ANE-induced COX-2 expression in GK. One representative PCR result was shown. *denotes significant difference when compared with solvent control. #denotes significant difference when compared with ANE (800 µg/ml)-treated group.
Figure 4.
Effect of ANE on Src and Ras activation and its role in ANE-induced COX-2 expression and PGE2 production of GK.
(A) GK were exposed to ANE (800 µg/ml) for different time points. Cell lysates were collected and used for analysis of Src phosphorylation (p-Src) by pathscan p-ELISA. Results were expressed as absorbance (OD450) (n = 9). *denotes significant difference when compared with control. (B) Inhibition of ANE-induced Src phosphorylation in GK by pp2, (C) pp2 inhibited the ANE-induced COX-2 mRNA and protein expression in GK. One representative PCR and western blotting result was shown. (D) pp2 prevented the ANE-induced PGE2 production in GK (n = 5). (E) ANE induced the activation of Ras in GK. GK were exposed to ANE (800 µg/ml) for different time points (30–150 secs). Cell lysates were collected and used for analysis of Ras activation using Ras activation assay kit (Pierce). GTPγS-activated was the positive control. (F) Manumycin A (1 and 5 µM) effectively prevented the ANE-induced COX-2 expression of GK, (G) Manumycin A also attenuated the ANE-induced PGE2 production of GK. *denotes significant difference when compared with solvent control. #denotes statistical significant difference when compared with ANE (800 µg/ml) solely-treated group (P<0.05)
Figure 5.
Effect of ANE on the cell cycle- and differentiation-related genes expression of GK and its modulation by various signal transduction inhibitors.
(A) Effect of different concentrations of ANE on cell cycle- and differentiation-related genes expression. Effect of (B), α-naphthoflavone, (C) pp2, (D) PD153035, (E) manumycin A and (F) aspirin on the ANE-induced changes of keratin 14, cyclin B1 and cdc25C gene expression of GK. One representative RT-PCR picture was shown.
Figure 6.
Effect of PBL and HC on the ANE-induced PGE2 production and Cox-2 expression of GK.
(A) Effect of PBL (250 and 500 µg/ml) on ANE-induced PGE2 production in GK, (B) Effect of HC (25 and 50 µM) on ANE-induced PGE2 production in GK, (C) Effect of PBL on ANE-induced Cox-2 expression in GK, (D) Effect of HC (25 and 50 µM) on ANE-induced Cox-2 expression in GK. One representative PCR picture was shown. The expression of β-actin was used as control. (E) Effect of curcumin on ANE-induced PGE2 production in GK (n = 7). (F) Effect of curcumin on ANE-induced cytotoxicity as analyzed by MTT assay. Results were expressed as % of control (as 100%), Mean ± SE (n = 4). (G) Effect of dicoumarol (10 and 20 µM) on ANE-induced PGE2 production in GK (n = 6). (H) Effect of dicoumarol (50 and 100 µM) on ANE-induced cytotoxicity as analyzed by MTT assay (n = 5). *denotes statistically significant difference when compared with solvent control group, #denotes significant difference when compared with ANE (800 µg/ml)-treated group.
Figure 7.
The proposed mechanism of ANE-induced molecular changes (CYP1A1, ROS, Src, EGFR, Ras, COX, PGE2, keratins and cyclin B1 etc.) in oral mucosal cells.
Signal transduction pathways responsible for the AN-induced alterations in cell proliferation, differentiation and inflammation of GK were shown.