Activation of AMP-activated protein kinase by temozolomide contributes to apoptosis in glioblastoma cells via p53 activation and mTORC1 inhibition

Abstract

Methylating drugs such as temozolomide (TMZ) are widely used in the treatment of brain tumors including malignant glioblastoma. The mechanism of TMZ-induced glioblastoma cell death and apoptosis, however, is not fully understood. Here, we tested the potential involvement of AMP-activated protein kinase (AMPK) in this process. We found that methylating agents TMZ and N-methyl-N'-nitro-N-nitrosoguanidine induce AMPK activation in primary cultured human glioblastoma and glioblastoma cell lines. TMZ-induced O(6)-methylguanine production is involved in AMPK activation. O(6)-benzylguanine, an O(6)-methylguanine-DNA methyltransferase inhibitor, enhances TMZ-induced O(6)-methylguanine production, leading to enhanced reactive oxygen species production, which serves as an upstream signal for AMPK activation. Activation of AMPK is involved in TMZ-induced glioblastoma cell death and apoptosis. AMPK inhibitor (Compound C) or AMPKα siRNA knockdown inhibits TMZ-induced glioblastoma cell death and apoptosis, whereas AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside enhances it. In further studies, we found that activation of AMPK is involved in TMZ-induced p53 activation and subsequent p21, Noxa, and Bax up-regulation. Activation of AMPK by TMZ also inhibits mTOR complex 1 (mTORC1) signaling and promotes anti-apoptosis protein Bcl-2 down-regulation, which together mediate TMZ-induced pro-cell apoptosis effects. Our study suggests that activation of AMPK by TMZ contributes to glioblastoma cell apoptosis, probably by promoting p53 activation and inhibiting mTORC1 signaling.

Figures

FIGURE 1.

TMZ induces AMPK activation in glioblastoma cells. Glioblastoma cell line U87MG (A and B) or U373MG (C and D) was treated with TMZ (250 μm) and cultured for different time points (0, 2, 4, 8, and 12 h) or treated with different doses of TMZ (0, 50, 250, and 500 μm) and cultured for 8 h. p-AMPKα (Thr-172), T-AMPKα, p-ACC (Ser-79), p-LKB1 (Ser-428), and β-actin were detected by Western blotting. AMPKα phosphorylation was quantified. U87MG cells were treated with different doses of TMZ (0, 50, 250, and 500 μm), and cell apoptosis was detected by Hoechst 33342 assay (E and quantified in F) and histone/DNA ELISA (G) 36 h later. Cell viability was detected by MTT assay (H) 48 h after TMZ treatment. Primary cultured human glioblastoma cells (I and J) were treated with TMZ (250 μm) and cultured for different time points (0, 2, 4, 8, and 12 h) or treated with different doses of TMZ (0, 50, 150, 250, and 500 μm) and cultured for 8 h. p-AMPKα (Thr-172), T-AMPKα, p-ACC (Ser-79), and p-LKB1 (Ser-428) were detected by Western blotting. Experiments in this figure were repeated at least three times, and similar results were obtained. *, p < 0.05 versus untreated group (Ctrl lane) (ANOVA). Scale bar, 25 μm.

FIGURE 2.

O6-MeG, ROS, and LKB1 are involved in TMZ-induced AMPK activation. A, glioblastoma U87MG cells were treated with 10 μm MNNG and cultured for 4, 8, and 12 h. p-AMPKα (Thr-172), p-ACC (Ser-79), p-LKB1 (Ser-428), T-AMPKα, and ERK1/2 were detected by Western blotting. B, U87MG cells were either left untreated or pretreated with O6-BG (10 μm) for 2 h, followed by TMZ (250 μm) or MNNG (10 μm) treatment for 8 h. AMPK activation was detected by Western blotting using the antibodies mentioned above. C, U87MG cells were pretreated with anti-oxidant NAc (400 μm) or O6-BG (10 μm) for the indicated time periods, followed by TMZ (250 μm) or MNNG (10 μm) treatment and cultured for the indicated times. ROS production was detected. D, U87MG cells were treated with 500 μm H2O2 or 1 mm AICAR and cultured for 2 and 4 h. p-AMPKα, p-ACC, and β-actin were detected by Western blotting. E, U87MG cells transfected with scramble (control) or LKB1 siRNAs were treated with 250 μm TMZ. LKB1/AMPK activation was detected by Western blotting. U87MG cells were pretreated with 10 μm Compound C (CC) or NAc (400 μm) for 2 h, followed by TMZ (250 μm) treatment. AMPK activation was detected by Western blotting 4 and 8 h later (F), cell viability was detected by MTT assay after 48 h (G), cell apoptosis was detected by histone/DNA ELISA (H), and Hoechst staining (I and quantified in J) after 36 h. U87MG cells transfected with scramble (Ctrl) or LKB1 siRNA were treated with 250 μm TMZ, and cell death and apoptosis were detected using the same methods described above (K–M). Experiments in this figure were repeated at least three times, and similar results were obtained. *, p < 0.05 versus TMZ-treated group in Ctrl cells (ANOVA). Scale bar, 25 μm.

FIGURE 3.

AMPK activation is involved in TMZ-induced cell death in vitro. A, glioblastoma U87MG cells were transfected with control (scrambled) or AMPKα1/2 siRNA for 48 h. AMPKα1/2, AMPKβ, ERK1/2, and β-actin expression levels were detected by Western blotting. Successfully AMPKα knocked down cells were used for further experiments. U87MG cells transfected with control or AMPKα siRNA were treated with 250 μm TMZ. B, cell viability was detected by MTT assay after 48 h. C–E, cell apoptosis was detected by histone/DNA ELISA (C) and Hoechst 33342 staining (D and quantified in E) after 36 h. F and G, U87MG cells were pretreated with AICAR (1 mm) followed by 250 μm TMZ treatment. Cell viability was detected by MTT assay after 48 h (F). Cell apoptosis was detected by histone/DNA ELISA after 36 h (G). H, primary cultured human glioblastoma cells were either left untreated or transfected with control (scramble) or AMPKα1/2 siRNA for 48 h. The expression level of AMPKα1/2, AMPKβ, and β-actin were detected by Western blotting. Successfully AMPKα knocked down cells were used for further experiments. DMSO, dimethyl sulfoxide. I, primary cultured human glioblastoma cells transfected with control or AMPKα siRNA were treated with 250 μm TMZ. Cell viability was detected by MTT assay after 48 h. J, cell apoptosis was detected by Hoechst 33342 staining after 36 h. Experiments in this figure were repeated at least three times, and similar results were obtained. *, p < 0.05 versus TMZ-treated group in control group (ANOVA). Scale bar, 25 μm.

FIGURE 4.

AMPK activation is involved in TMZ-induced p53 activation. A, glioblastoma U87MG cells were treated with 250 μm TMZ and cultured for the indicated times (0, 4, 8, 16, 24 and 48 h). AMPKα1, AMPKα2, ERK1/2, p-AMPKα (Thr-172), p-ACC (Ser-79), phosphorylation (Ser-15) and expression of p53 as well as p21, Noxa, and Bax were detected. B, p53 successfully knocked down U87MG cells (by tranfecting cell with a p53 shRNA plasmid and puromycin selection as described under “Experimental Procedures”) were treated with TMZ (250 μm) and cultured for 24 and 48 h. Phosphorylation (Ser-15) and expression of p53, p21, Noxa, Bax, p-AMPKα (Thr-172) and T-AMPK were detected. C, U87MG cells were pretreated with the AMPK inhibitor Compound C (CC, 10 μm) for 2 h, followed by 250 μm TMZ treatment for 24 h and 48 h. p-AMPKα (Thr-172), p-ACC (Ser-79), T-AMPKα, p-p53 (Ser-15), p21, and Bax were detected. D, U87MG cells transfected with control or AMPKα1/2 siRNA were treated with TMZ (250 μm) for 24 and 48 h. AMPKα1/2, AMPKβ, β-actin, p-AMPKα (Thr-172), p-ACC (Ser-79) as well as phosphorylation (Ser-15) and the expression level of p53, p21, Noxa, Bax, and p-ATM (Ser 1981) were detected. E–H, the association between AMPK and p53 after TMZ (250 μm, 12 and 24 h) (E and F) or AICAR (1 mm, 24 h) (G and H) was detected by co-immunoprecipitation (IP). I, IgG was used here as an internal control, whereas 30 μg of total cell lysate was used as input control. J, U87MG cells transfected with control or AMPKα1/2 siRNA were treated with AICAR (1 mm) or H2O2 (500 μm) for 24 h. The expression levels of AMPKα1/2, p-AMPKα (Thr-172), and AKT1, phosphorylation (Ser-15), and expression level of p53 were detected. IB, immunoblotting. K and L, p53 shRNA-transfected stable U87MG cells were treated with TMZ (250 μm). Cell viability was detected by MTT assay after 48 h (K). Cell apoptosis was detected by histone/DNA ELISA (L) after 36 h. Experiments in this figure were repeated at least three times and similar results were obtained. *, p < 0.05 versus same treatment in scramble siRNA-transfected group (ANOVA).

FIGURE 5.

AMPK activation mediates TMZ-induced mTORC1 inhibition in vitro. A, glioblastoma U87MG cells were treated with TMZ (250 μm) for the indicated times. p-S6K (Thr-389), p-4E-BP1 (Ser-65), p-S6 (Ser-235/236), T-AMPKα, and AKT1 were detected. B, U87MG cells transfected with scramble (Ctrl) or AMPKα1/2 siRNA were treated with TMZ (250 μm) for 24 h. p-4E-BP1 (Ser-65), p-S6 (Ser-235/236), and relative loading controls were detected. C, U87MG cells were pretreated with Compound C (CC, 10 μm) for 2 h followed by 250 μm TMZ treatment for 24 h. p-S6K (Thr-389), AKT1, and T-AMPKα were detected. D, U87MG cells were transfected with TSC2/TSC1 shRNA for 48 h. Successfully transfected cells were selected by puromycin. After selection, cells were treated with TMZ (250 μm) for 24 h. p-4E-BP1 (Ser-65), p-S6 (Ser-235/236), TSC1, TSC2, and AKT1 were detected. E, glioblastoma U87MG cells were treated with TMZ (250 μm) for the indicated times. Bcl-2 expression was detected by Western blotting. F, U87MG cells were treated with rapamycin (100 nm) for 12 h. mTOR, p-S6K (Thr-389), p-S6 (Ser-235/236), p-AKT (Ser-473), AKT1, and Bcl-2 were detected. G, U87MG cells were transfected with scramble (Ctrl) or mTOR siRNA for 48 h. mTOR, p-mTOR (Ser-2448), p-S6K (Thr-389), p-AKT (Ser-473), AKT1, and Bcl-2 were detected. H, U87MG cells transfected with control (scramble) or AMPKα1/2 siRNA were treated with TMZ (250 μm) for 48 h. The expression levels of AMPKα1/2, Bcl-2, and β-actin were detected. I, U87MG cells were pretreated with Compound C (CC, 10 μm) for 2 h, followed by 250 μm TMZ treatment for 48 h. Bcl-2 and β-actin were detected. The effects of AICAR (1 mm) and H2O2 (250 μm, 24 h) on Bcl-2 expression were also detected. J, primary cultured human glioblastoma cells transfected with control (scramble) or AMPKα1/2 siRNA were treated with TMZ (250 μm) for 48 h. The expression levels of AMPKα1/2, AMPKβ, p53, p21, Bcl-2, and p-S6K as well as loading control β-actin were detected. K and L, U87MG cells were pretreated with rapamycin (100 nm) for 2 h, followed by 250 μm TMZ treatment. Cell viability (48 h later) was detected by MTT assay. Cell apoptosis (36 h later) was detected by histone/DNA ELISA. M, U87MG cells were transfected with scramble (Ctrl), mTOR, or Bcl-2 siRNA for 48 h followed by TMZ (250 μm) treatment for another 48 h. Cell viability was detected by MTT assay. N, proposed signal pathway involved in this study is shown. TMZ induces AMPK activation in glioblastoma cells, DNA damage caused by TMZ plays an important role in ROS production and following LKB1/AMPK activation. Activation of AMPK contributes TMZ-induced glioblastoma cell apoptosis, probably by regulating p53 (positively) and mTORC1 (negatively) pathways as well as altering the expression of apoptosis-associated proteins, including p21, Bax, Noxa, and Bcl-2. In addition to AMPK, ATM and possible other DNA repair proteins may also be involved in p53 activation by TMZ. Experiments in this figure were repeated at least three times, and similar results were obtained. *, p < 0.05 versus the TMZ-treated group (ANOVA).