Protective effects of antioxidants against smokeless tobacco-induced oxidative stress and modulation of Bcl-2 and p53 genes in human oral keratinocytes

The oral use of chewing tobacco has greatly increased in recent years, and this usage is associated with cancers of the mouth, lip, nasal cavities, es ophagus and gut. Oral cancer accounts for 3% of all cancers in U.S.A. and i s the seventh most common cancer. Previous studies in our laboratory have d emonstrated the protective abilities of a novel IH636 grape seed proanthocy anidin extract (GSPE) against reactive oxygen species both in vitro and in vivo models, and provided significantly better protection as compared to vi tamins C, E and beta -carotene. In the recent past, we have demonstrated sm okeless tobacco (STE)-induced oxidative stress, apoptotic cell death in a p rimary culture of normal human oral keratinocytes (NHOK), and have compared the protective abilities of vitamins C and E, singly and in combination, a nd GSPE in this pathobiology [Free Rad. Biol. Med., 26, 992-1000 (1999)]. I n the present study, we have assessed the protective role of vitamins C and E, and GSPE against STE-induced modulation of intracellular oxidized state s in NHOK cells as demonstrated by laser scanning confocal microscopy. Appr oximately 11%, 26%, 28% and 50% protection were observed following incubati on with vitamin C, vitamin E, a combination of vitamins C plus E, and GSPE, respectively. DNA fragmentation was assessed as an index of oxidative DNA damage and similar results were observed. Furthermore, the cellular viabili ty and functional roles of Bcl-2, p53 and c-myc genes were assessed in STE- induced oxidative stress in NHOK cells. NHOK cells were treated with STE (0 -200 mug/ml) for 24h and changes in the expression of Bcl-2, p53 and c-myc genes were measured by reverse transcriptase-polymerase chain reaction (RT- PCR), and the protective effect of GSPE was assessed. Approximately a 2.0-f old increase in p53 gene expression was observed following incubation of th e oral keratinocytes with 100 mug/ml of STE, beyond which the expression of p53 decreased, confirming increased apoptotic cell death with a higher con centration of STE as reported earlier. GSPE significantly modulated STE-ind uced changes in p53. The expression of antiapoptotic Bcl-2 gene decreased w ith STE treatment and the expression of Bcl-2 gene increased significantly following preincubation with GSPE. No significant change in the expression of transcription factor c-myc gene responsible for cell cycle growth was ob served following incubation with STE and/or GSPE. Thus, c-myc may not be in volved in STE-induced cytotoxicity towards NHOK cells. These results sugges t that antioxidant protection of STE-induced cellular injury is associated with alterations in Bcl-2 and p53 expression.