T-2 toxin-induced apoptosis involving Fas, p53, Bcl-xL, Bcl-2, Bax and caspase-3 signaling pathways in human chondrocytes

Abstract

Objective: To investigate the effects of T-2 toxin on expressions of Fas, p53, Bcl-xL, Bcl-2, Bax and caspase-3 on human chondrocytes.

Methods: Human chondrocytes were treated with T-2 toxin (1-20 ng/ml) for 5 d. Fas, p53 and other apoptosis-related proteins such as Bax, Bcl-2, Bcl-xL, caspase-3 were determined by Western blot analysis and their mRNA expressions were determined by reverse transcriptase-polymerase chain reaction (RT-PCR).

Results: Increases in Fas, p53 and the pro-apoptotic factor Bax protein and mRNA expressions and a decrease of the anti-apoptotic factor Bcl-xL were observed in a dose-dependent manner after exposures to 1-20 ng/ml T-2 toxin, while the expression of the anti-apoptotic factor Bcl-2 was unchanged. Meanwhile, T-2 toxin could also up-regulate the expressions of both pro-caspase-3 and caspase-3 in a dose-dependent manner.

Conclusion: These data suggest a possible underlying molecular mechanism for T-2 toxin to induce the apoptosis signaling pathway in human chondrocytes by regulation of apoptosis-related proteins.

Fig. 1

Effects of different concentrations of T-2 toxin on the cellular viability of chondrocytes estimated by MTT reduction. Cells were incubated in absence or presence of T-2 toxin at different concentrations for different time periods (1~5 d). MTT reduction was then calculated to indicate cellular proliferation. Results are expressed as mean±SD of six independent determinations. ^* P<0.05, significantly different from control values

Fig. 2

Western blotting analyses of T-2 toxin-induced expressions of Bcl-2, Bcl-xL, Bax, caspase-3, p53 and Fas proteins in human chondrocytes. The intensities of Bcl-2, Bcl-xL, Bax, caspase-3, p53 and Fas bands were normalized with respect to the intensities of β-actin bands detected on the same blots Lane 1: Untreated control; Lanes 2~4: Treatment with 1, 10 and 20 ng/ml of T-2 toxin for 5 d, respectively

Fig. 3

Ratios of (a) Bax/Bcl-2 and (b) Bax/Bcl-xL ^* P<0.05 vs control

Fig. 3

Ratios of (a) Bax/Bcl-2 and (b) Bax/Bcl-xL ^* P<0.05 vs control

Fig. 4

The levels of (a) caspase-3, (b) p53 and (c) Fas quantified by densitometric analysis of the three autoradiographs ^* P<0.05 vs control

Fig. 4

The levels of (a) caspase-3, (b) p53 and (c) Fas quantified by densitometric analysis of the three autoradiographs ^* P<0.05 vs control

Fig. 4

The levels of (a) caspase-3, (b) p53 and (c) Fas quantified by densitometric analysis of the three autoradiographs ^* P<0.05 vs control

Fig. 5

RT-PCR analysis of T-2 toxin-induced expressions of Bcl-2, Bcl-xL, Bax, caspase-3, p53 and Fas mRNA in human chondrocytes. Results were normalized according to GAPDH levels (n=3) Lane 1: Untreated control; Lanes 2~4: Treatment with 1, 10 and 20 ng/ml of T-2 toxin for 5 d, respectively

Fig. 6

Relative mRNA levels of (a) Bcl-xL, (b) Bax (c) Bcl-2, (d) caspase-3, (e) p53 and (f) Fas ^* P<0.05 vs control

Fig. 6

Relative mRNA levels of (a) Bcl-xL, (b) Bax (c) Bcl-2, (d) caspase-3, (e) p53 and (f) Fas ^* P<0.05 vs control

Fig. 6

Relative mRNA levels of (a) Bcl-xL, (b) Bax (c) Bcl-2, (d) caspase-3, (e) p53 and (f) Fas ^* P<0.05 vs control

Fig. 6

Relative mRNA levels of (a) Bcl-xL, (b) Bax (c) Bcl-2, (d) caspase-3, (e) p53 and (f) Fas ^* P<0.05 vs control

Fig. 6

Relative mRNA levels of (a) Bcl-xL, (b) Bax (c) Bcl-2, (d) caspase-3, (e) p53 and (f) Fas ^* P<0.05 vs control

Fig. 6

Relative mRNA levels of (a) Bcl-xL, (b) Bax (c) Bcl-2, (d) caspase-3, (e) p53 and (f) Fas ^* P<0.05 vs control