| Purpose: Oxidative stimulation induced by hydrogen peroxide (H2O2) on human epithelial cells (HLECs) was performed to observe the effects on cell proliferation, caveolin expression, and cholesterol depletion in HLECs caused by methyl-β-cyclodextrin (MβCD) was also studied.
Methords:SRA01/04 HLECs were exposed to H2O2 or MβCD of various concentrations and durations. We used a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrozolium bromide (MTT) assay to measure the effect of H2O2 on the proliferation of SRA01/04 HLECs. The distributions of caveolins after oxidative stimulation were probed by fluorescence microscopy and laser scanning confocal microscopy. Immunoblotting was performed to analyze alterations of caveolins expression.
Results:We observed that the proliferation of SRA01/04 HLECs under 0.1 mM H2O2 for 10 min or longer was significantly reduced (* p<0.05, F=11.63). Laser scanning microscopy showed immunofluorescent caveolins in SRA01/04 HLECs under 0.1 mM H2O2 for 10 min or longer, caveolins were largely confined to intracellular domains. Western blots showed both membrane and total caveolin protein (22 kDa) levels in SRA01/04 HLECs treated with 0.1, 0.2, 0.5 or 1.0 mM H2O2 for 30 min were significantly reduced, compared with the untreated (* p<0.05, F=6.149, or *p<0.05, F=14.489, respectively). In addition, the membrane and total caveolin protein level after treated with 0.1mM (* p<0.05, F=6.843, or * p<0.05, F=7.944, respectively) H2O2 for different durations also down regulated. Fluorescence microscopy also showed that phosphorylated caveolin-1 was distributed near the focal adhesions of the cells.
Conclusion:This study concludes that the responses of HLECs to oxidative stress may include down regulation of caveolin and phosphorylation of caveolin-1 on tyrosine 14, and that MβCD also down regulates caveolin while depleting cholesterol in HLECs.
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