Doxorubicin induces the persistent activation of intracellular transglutaminase 2 that protects from cell death

Abstract

The activation of transglutaminase 2 (TG2), an enzyme that catalyzes post-translational modifications of proteins, has been implicated in apoptosis, cell adhesion and inflammatory responses. We previously reported that intracellular TG2 is activated under oxidative stress conditions, such as ultraviolet irradiation, ischemia-reperfusion, and hypoxia. In this study, we examined the effect of genotoxic stress on the intracellular activity of TG2 using doxorubicin which generates reactive oxygen species that lead to double-strand breakage of DNA. We demonstrated that doxorubicin elicits the persistent activation of TG2. Doxorubicin-induced TG2 activity was suppressed by treatment with caffeine at the early phase, N-acetylcysteine at the mid-phase, and EGTA at the late phase. However, treatment with a blocking antibody against TGFβ or toll-like receptor 2 showed no effect on TG2 activity, indicating that at least three different signaling pathways may be involved in the process of TG2 activation. In addition, using MEF cells defective for TG2 and cells overexpressing an activesite mutant of TG2, we revealed that doxorubicin-induced cell death is inversely correlated with TG2 activity. Our findings indicate that the persistent activation of TG2 by doxorubicin contributes to cell survival, suggesting that the mechanism-based inhibition of TG2 may be a novel strategy to prevent drug-resistance in doxorubicin treatment.

Fig. 1.

Doxorubicin induces the persistent activation of intracellular TG2 in HeLa cells. (A) The intracellular TG activity of HeLa cells treated with doxorubicin (1 μg/ml) was monitored at 0, 6, 12, 24 and 48 h. (B) Western blot analysis for TG2 expression and incorporation of 5-biotinamidopentylamine to substrates (BP-incorporation) in doxorubicin (1 μg/ml) treated cells. (C, D) The intracellular TG activity was measured in HeLa cells after the exposure to doxorubicin (1 μg/ml) for the indicated times in the presence of N-acetylcysteine (NAC; 1 mM) or TGFβ-neutralizing antibody (30 μg/ml). The data represent the mean values ± standard deviations based on 3 independent experiments. ^*, p < 0.05; ^**, p < 0.01.

Fig. 2.

Doxorubicin-induced TG2 activation is inhibited by calcium chelators and caffeine. (A, B) The effect of BAPTA-AM (20 μM), EGTA (2 mM), or TLR2-neutralizing antibody (10 μg/ml) on TG activity after exposure to doxorubicin (1 μg/ml) was monitored in HeLa cells. (C) Dose-dependent effect of caffeine. The TG activity was measured in HeLa cells after 6 h of doxorubicin treatment (1 μg/ml). (D) Time-dependent effect of caffeine. TG activity in the presence of caffeine (10 mM) was monitored in doxorubicin (1 μg/ml)-treated HeLa cells. The data represent the mean values ± standard deviations based on 3 independent experiments. ^**, p < 0.01.

Fig. 3.

TG2 expression promotes the survival of doxorubicin-treated MEFs. (A) Wild type (TG2^+/+) and TG2-null (TG2^−/−) MEFs were treated with 0.1 and 0.5 μg/ml of doxorubicin for 4 days. (B) MEFs were treated with 0.5 μg/ml doxorubicin for 2 and 4 days. The extent of cell death was evaluated by trypan blue exclusion staining. Cell death was expressed as the percentage of dead cells out of the total number of cells. (C) MEFs were treated with 0.1 and 0.5 μg/ml of doxorubicin for 24 h. Each sample was analyzed for the intracellular activity of TG2. The data represent the mean values ± standard deviations based on 3 independent experiments. ^*, p < 0.05; ^**, p < 0.01.

Fig. 4.

TG2 activity is required for the survival of doxorubicin-treated cells. (A) HEK293 cells and HEK293 cells stably transfected with wild-type TG2 (293-TG2) or an active-site mutant of TG2 (293-C277S) were treated with doxorubicin (1 μg/ml) for 24 h. Each sample was analyzed for intracellular TG2 activity. (B) The HEK293, 293-TG2 and 293-C277S cells were treated with doxorubicin (1 μg/ml) for 48 h, and the extent of cell death was evaluated by trypan blue exclusion staining. The data represent the mean values ± standard deviations based on 3 independent experiments. (C) Cells were treated with 1 μg/ml doxorubicin for 48 h, and the TG2 expression and cleavage of caspase-3 were subsequently determined by Western blot analysis. ^*, p < 0.05; ^**, p < 0.01.