Consecutive treatments of human lung epithelial cells with crocidolite asbestos results in resistance to apoptosis due to increased expression of Bcl-2

Abstract

2514

Exposure to asbestos is known to increase the incidence of lung cancer. However, the mechanism is not fully understood. Animal studies have shown that fibers that remain longer in the lung are the ones that are more carcinogenic. In this study, we investigated whether consecutive exposures of human lung epithelial (A549) cells to crocidolite, one of the most carcinogenic forms of asbestos, would result in increased cell survival, perhaps because of increased expression of anti-apoptotic proteins, such as Bcl-2. Exposure to crocidolite has been shown to cause cell cycle arrest and apoptosis. After a 6 μg/cm² treatment with crocidolite for 24 h we observed that only ∼ 30 % of the treated cells survived. In order to select the viable cells, treated cells were replated at low density (7 cells/cm²) and allowed to grow for 10 days. The surviving cells were treated again with crocidolite and this process was repeated for a total of 5 cycles. After 5 cycles of either 3 μg/cm² or 6 μg/cm² crocidolite treatment for 24 h, the percent survival of the consecutively treated surviving cells was compared to that of parental A549 cells after crocidolite treatment. The percent survival increased by ∼ 1.5 fold, from 60 % to 91 %, relative to parent cells, for 3 μg/cm² crocidolite treatment and by ∼ 2 fold, from 35 % to 68 %, relative to parent cells, for 6 μg/cm² crocidolite treatment. The cells surviving 5 cycles of treatment showed a decreased fragmentation of PARP protein after a subsequent treatment with crocidolite, indicating that apoptosis was inhibited. We examined whether expression of the anti-apoptotic protein, Bcl-2, or the pro-apoptotic protein Bax, was changed and whether modulation of its expression was related to increased cell survival in the consecutively treated A549 cells. Consecutive treatment caused an increased expression of Bcl-2 protein and decreased expression of Bax protein, resulting in a high ratio of Bcl-2 to Bax. Therefore, this study suggests that A549 cells with consecutive exposure to crocidolite acquired resistance to apoptosis and increased cell survival, due to an increased expression ratio of Bcl-2 to Bax. This may help to explain why fibers that persist in the lung are more carcinogenic, because repeated exposure of the cells leads to increased survival and perhaps increased mutations, leading to cancer. (This work was supported by NIH grant ES05814.)