Role of autophagy in histone deacetylase inhibitor-induced apoptotic and nonapoptotic cell death

Abstract

Autophagy is a cellular catabolic pathway by which long-lived proteins and damaged organelles are targeted for degradation. Activation of autophagy enhances cellular tolerance to various stresses. Recent studies indicate that a class of anticancer agents, histone deacetylase (HDAC) inhibitors, can induce autophagy. One of the HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA), is currently being used for treating cutaneous T-cell lymphoma and under clinical trials for multiple other cancer types, including glioblastoma. Here, we show that SAHA increases the expression of the autophagic factor LC3, and inhibits the nutrient-sensing kinase mammalian target of rapamycin (mTOR). The inactivation of mTOR results in the dephosphorylation, and thus activation, of the autophagic protein kinase ULK1, which is essential for autophagy activation during SAHA treatment. Furthermore, we show that the inhibition of autophagy by RNAi in glioblastoma cells results in an increase in SAHA-induced apoptosis. Importantly, when apoptosis is pharmacologically blocked, SAHA-induced nonapoptotic cell death can also be potentiated by autophagy inhibition. Overall, our findings indicate that SAHA activates autophagy via inhibiting mTOR and up-regulating LC3 expression; autophagy functions as a prosurvival mechanism to mitigate SAHA-induced apoptotic and nonapoptotic cell death, suggesting that targeting autophagy might improve the therapeutic effects of SAHA.

Fig. 1.

SAHA induces autophagy and LC3 up-regulation in MEF cells. (A) MEF cells seeded in six-well dishes were treated with the indicated concentration of SAHA for 8 h. Where indicated, 20 nM Baf A1 was added 2 h before harvesting cells. Cell extracts were analyzed by Western blot using antibodies against the indicated proteins. The accumulation of LC3-II (faster migrating form) relative to LC3-I (slower migrating form) is indicative of the induction of autophagy. (B) MEF cells stably expressing GFP-LC3 grown on glass cover-slips were either left untreated or treated with 5 μM SAHA for 24 h. Cells were then fixed with 3.7% PFA, processed for imaging, and visualized under the confocal microscope using the 60× magnification objective. (C) MEF cells were seeded in six-well dishes and treated with 20 μM of SAHA for 20 h followed by 20 nM Baf A1 for a further 4 h. Cell extracts were analyzed by Western blot analysis using the indicated antibodies. (D) Wild-type or ATG3^−/− MEF cells were treated with the indicated concentrations of SAHA for 24 h. (E) A semiquantitative RT-PCR detecting LC3 expression was performed using RNA extracted from wt MEFs either treated with 10 μM SAHA or left untreated. GAPDH RT-PCR was used as a loading control.

Fig. 2.

SAHA triggers autophagy by suppressing mTOR and activating ULK1. (A) ULK1/2 DKO or MEFs with intact ULK1/2 expression (wild-type, wt MEFs) were treated with the indicated concentrations of SAHA for 24 h. Baf A1 (20 nM) was included 2 h before harvesting and cell lysates were analyzed by Western blot using the indicated antibodies. (B) MEF cells were either left untreated, amino acid-starved for 2 h, or treated with 5 μM of SAHA for 16 h. Cell extracts were then analyzed by Western blot analysis using antibodies against the proteins indicated. A change in migration of ULK1, 4EBP, and p70S6K to faster migrating forms is indicative of protein dephosphorylation and thus mTOR inactivation.

Fig. 3.

SAHA induces autophagy in T98G glioblastoma cells. (A) T98G glioblastoma cell lines expressing either control shRNA or shRNA targeting ATG7 sequences were treated with SAHA using the indicated concentrations and times. Cell lysates were then subjected to Western blot analysis using the following antibodies: anti-ATG7 to confirm knockdown efficiency, anti-LC3 to analyze autophagy induction and anti-actin as a loading control. (B) T98G stably expressing GFP-LC3 were grown on glass cover-slips and treated with 10 μM SAHA for the indicated times. Slides were then fixed with PFA and analyzed by confocal microscopy using a 20× magnification objective.

Fig. 4.

ATG7 knockdown in T98G cells increases SAHA-induced apoptotic cell death. (A) An in vitro caspase-3 assay performed on cell lysates from T98G cells expressing control or ATG7 shRNA. Cells were treated with various concentrations of SAHA in the presence or absence of the pan-caspase inhibitor, zVAD, at 10 μM. The y axis values correspond to RFU as a measure of caspase activity using a fluorescence-based caspase-3 substrate. Error bars correspond to SEM values of at least three independent assays. (B) Western blot analysis to detect expression of the active cleaved caspase-3 levels. Extracts are taken from T98G cells expressing control or ATG7 shRNA left untreated or treated with SAHA for 24 h. (C) Cell survival assay using Resazurin dye. T98G cells expressing control or ATG7 shRNA were seeded in a 96-well plate at 500 cells per well and treated with 10 μM SAHA for the indicated times. Resazurin dye was then added to each well and plate and was further incubated for 3 h before measurement of the Resazurin fluorescence. Cell numbers were then deduced from a standard curve and survival was measured relative to untreated samples, which corresponded to 100% survival. Error bars correspond to SEM values of at least three independent assays. (D) T98G cells expressing control or ATG7 shRNA were seeded in a six-well plate, left untreated, or treated with SAHA for 48 h. Phase-contrast images of cells were then captured under Nikon fluorescent scope using a 20× magnification objective.

Fig. 5.

ATG7 knockdown decreases cell survival in the absence of apoptosis. (A) Western blot analysis of T98G cells expressing control or ATG7 shRNA. Cells were left untreated or treated as indicated with a combination of SAHA and zVAD at 10 μM each for 48 h. (B) Cell survival assay using Resazurin dye as in Fig. 4C except that SAHA concentration was used at 20 μM and zVAD treatment at 10 μM was included in all samples. Error bars correspond to SEM values of at least three independent assays. (C) Cell detachment was analyzed as in Fig. 4D (20× magnification), except that 10 μM zVAD was included along with SAHA treatment. (D) Colony formation assay of T98G cells treated with 20 μM SAHA and 10 μM zVAD for 48 h. Cells were further grown for 2 wk in fresh media before fixation, staining and counting of colonies.