Cellular Preoxygenation Partially Attenuates the Antitumoral Effect of Cisplatin despite Highly Protective Effects on Renal Epithelial Cells

Abstract

Our previous in vitro studies demonstrated that oxygen pretreatment significantly protects human embryonic renal tubular cell against acute cisplatin- (CP-) induced cytotoxicity. The present study was designed to investigate whether this protective effect is associated with decreasing therapeutic effects of cisplatin on malignant cells. For this purpose, cultured human embryonic kidney epithelial-like (AD293), cervical carcinoma epithelial-like (Hela), and ovarian adenocarcinoma epithelial-like (OVCAR-3) cells were subjected to either 2-hour pretreatment with oxygen (≥90%) or normal air and then to a previously determined 50% lethal dose of cisplatin for 24 hours. Cellular viability was evaluated via MTT and Neutral Red assays. Also, activated caspase-3 and Bax/Bcl-2 ratio, as the biochemical markers of cell apoptosis, were determined using immunoblotting. The hyperoxic preexposure protocol significantly protects renal AD293 cells against cisplatin-induced toxicity. Oxygen pretreatment also partially attenuated the cisplatin-induced cytotoxic effects on Hela and OVCAR-3 cells. However, it did not completely protect these cells against the therapeutic cytotoxic effects of cisplatin. In summary, the protective methods for reducing cisplatin nephrotoxic side effects like oxygen pretreatment might be associated with concurrent reduction of the therapeutic cytotoxic effects of cisplatin on malignant cells like cervical carcinoma (Hela) and ovarian adenocarcinoma (OVCAR-3) cells.

Figure 1

Effect of cisplatin on cell viability. Different cells types (AD293-(a), Hela-(b), and OVCAR-3-(c)) were incubated with variant doses of cisplatin for 24 h. Cell viability was determined by MTT and Neutral Red assays. Data are mean ± SD; n = 6–8 wells for each group/cell type; ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 versus control.

Figure 2

Hyperoxic preconditioning prevents cisplatin-induced cells death. AD293 (a), Hela (b), and OVCAR-3 (c) cells were treated with nearly pure oxygen (≥90%) preconditioning (2 h) and then cisplatin (50, 35, and 30 μM for AD293, Hela, and OVCAR-3 cells, resp.) was added for an additional 24 h. Cell viability was then determined using the MTT and Neutral Red assays. Data are mean ± SEM; n = 6–8 wells for each group; ^∗∗∗p < 0.001 versus both “Air + Veh” and “O₂ + Veh” groups (in (a), (b), and (c) parts); ^&p < 0.05, ^&&p < 0.01, and ^&&&p < 0.001 versus “Air + CP” group (in (a), (b), and (c) parts).

Figure 3

Western blot analysis of the caspase-3 protein activation, Bax, Bcl-2, and Bax : Bcl-2 ratio of AD293 cells. Cells were exposed to cisplatin (50, 35, and 30 μM for AD293, Hela, and OVCAR-3 cells, resp.) and cisplatin plus hyperoxic preconditioning (2 h) for 24 h. Each value in the graph represents the mean ± SEM band density ratio for each group. Beta-actin was used as an internal control (n = 4).

Figure 4

Western blot analysis of the caspase-3 protein activation, Bax, Bcl-2, and Bax : Bcl-2 ratio of Hela cells. Cells were exposed to cisplatin (50, 35, and 30 μM for AD293, Hela, and OVCAR-3 cells, resp.) and cisplatin plus hyperoxic preconditioning (2 h) for 24 h. Each value in the graph represents the mean ± SEM band density ratio for each group. Beta-actin was used as an internal control (n = 4).

Figure 5

Western blot analysis of the caspase-3 protein activation, Bax, Bcl-2, and Bax : Bcl-2 ratio of OVCAR-3 cells. Cells were exposed to cisplatin (50, 35, and 30 μM for AD293, Hela, and OVCAR-3 cells, resp.) and cisplatin plus hyperoxic preconditioning (2 h) for 24 h. Each value in the graph represents the mean ± SEM band density ratio for each group. Beta-actin was used as an internal control (n = 4).