Apoptotic and autophagic cell death induced by histone deacetylase inhibitors

Abstract

Histone deacetylase (HDAC) inhibitors can induce programmed cell death in cancer cells, although the underlying mechanism is obscure. In this study, we show that two distinct HDAC inhibitors, butyrate and suberoylanilide hydroxamic acid (SAHA), induced caspase-3 activation and cell death in multiple human cancer cell lines. The activation of caspase-3 was via the mitochondria/cytochrome c-mediated apoptotic pathway because it was abrogated in mouse embryonic fibroblasts with knockout of Apaf-1, the essential mediator of the pathway. Overexpression of Bcl-XL in HeLa cells also blocked caspase activation by the HDAC inhibitors. Nevertheless, Apaf-1 knockout, overexpression of Bcl-XL, and pharmacological inhibition of caspase activity did not prevent SAHA and butyrate-induced cell death. The cells undergoing such caspase-independent death had unambiguous morphological features of autophagic cell death. Therefore, HDAC inhibitors can induce both mitochondria-mediated apoptosis and caspase-independent autophagic cell death. Induction of autophagic cell death by HDAC inhibitors has clear clinical implications in treating cancers with apoptotic defects.

Fig. 1.

Butyrate and SAHA induced apoptosis, caspase-3 activation, and cytochrome c release in HeLa cells. HeLa cells were harvested after 2 days of treatment with indicated concentrations of butyrate or SAHA. (A) Cell death was measured by Hoechst dye 33342 staining followed by flow cytometry as described. BuT, butyrate; Con, control. (B) Cytosolic proteins of each sample were assayed for caspase-3 activity as described. (C) Cytosolic proteins (40 μg) were subjected to immunoblotting against cytochrome c (Cyto). Immunoblotting against β-tubulin was performed as the loading control.

Fig. 2.

Apaf-1 is required for butyrate and SAHA-induced caspase-3 activation but not cell death. Apaf-1 wild-type (WT, filled bars) and knockout (KO, open bars) MEFs were harvested after the treatment with butyrate (A) or SAHA (B) for 2 days or 3 days, and caspase-3 activation was measured as described. Cell death of the KO cells was documented by light microscopy after treatment of butyrate (C) or SAHA (D) with indicated concentrations.

Fig. 3.

Butyrate and SAHA induced caspase-independent cell death. (A) Apaf-1 wild-type (WT, filled bars) and knockout MEFs (KO, open bars) were treated with 5 μM SAHA or 50 mM butyrate in the presence or absence of 40 μM Z-VAD-FMK (Z-VAD), as indicated. After treatment (2 days for the WT cells and 3 days for the KO cells), the cells were harvested for measurement of caspase-3 activity. (B) Microscopic pictures of the treated cells showing cell death.

Fig. 4.

Overexpression of Bcl-XL blocked butyrate- and SAHA-induced cytochrome c release and caspase-3 activation, but not cell death. HeLa cell lines stably transfected with Bcl-XL (HeLa-Bcl-XL) or vector alone (HeLa) were treated for 2 days with various concentrations of butyrate or SAHA as indicated. (A) Cells were harvested, and cytosolic proteins (40 μg) were subjected to immunoblotting to detect Bcl-XL, cytochrome c, and β-tubulin. (B) Caspase-3 activity was measured after treatment (filled bars, control HeLa cells; open bars, Bcl-XL-overexpressing cells). (C) Two days after treatment with 10 mM butyrate or 2.5 μM SAHA, death of the Bcl-XL-overexpressing HeLa cells was documented by light microscopy.

Fig. 5.

HDAC inhibitors induced caspase-independent, autophagic cell death. (A–D) Apaf-1-knockout MEFs were treated without (A) or with (B–D) 5 μM SAHA for 36 h, and transmission electron microscopic study was conducted as described. Nuclei were labeled N. Autophagic structures, autophagosome (arrowheads) and autophagic vacuoles (arrows), were detected in SAHA-treated cells. C and D are pictures with higher magnification showing detailed autophagosome structure. (E and F) Bcl-XL-overexpressing HeLa cells, without (E) or with (F) treatment with 5 μM SAHA for 2 days. The arrowhead in F shows an autophagosome fusing with an autophagic vacuole. (G) Arrowheads show double membrane structures approaching mitochondria (from a SAHA-treated Apaf-1-knockout MEF cell). (H) A SAHA-treated, Bcl-XL-overexpressing HeLa cell, containing a single large autophagic vacuole and very few mitochondria.