Histone deacetylase inhibitors (HDACi) are promising anti-cancer agents, however, their mechanisms of action remain unclear. In acute myeloid leukemia (AML) cells, HDACi have been reported to arrest growth and induce apoptosis. In this study, we elucidate details of the DNA damage induced by the HDACi vorinostat in AML cells. At clinically relevant concentrations, vorinostat induces double-strand breaks and oxidative DNA damage in AML cell lines. Additionally, AML patient blasts treated with vorinostat display increased DNA damage, followed by an increase in caspase-3/7 activity and a reduction in cell viability. Vorinostat-induced DNA damage is followed by a G2-M arrest and eventually apoptosis. We found that pre-treatment with the antioxidant N-acetyl cysteine (NAC) reduces vorinostat-induced DNA double strand breaks, G2-M arrest and apoptosis. These data implicate DNA damage as an important mechanism in vorinostat-induced growth arrest and apoptosis in both AML cell lines and patient-derived blasts. This supports the continued study and development of vorinostat in AMLs that may be sensitive to DNA-damaging agents and as a combination therapy with ionizing radiation and/or other DNA damaging agents.
Histone deacetylase inhibitors (HDACi) have recently emerged as promising anticancer agents. However, the mechanisms by which HDAC inhibitors arrest proliferation and induce apoptosis in tumor cells is far from clear. Activation of the stress MAP kinases and induction of DNA damage by different HDACi have been reported, however, the potential role of the MAP kinase p38 in the antitumor activity of these drugs has not been described. P38 is known to be activated independently or downstream of DNA damage. The purpose of this study was to elucidate the mechanisms by which the HDACi vorinostat (Zolinza®) triggers apoptosis in haematological malignant cells. We show that DNA damage as well as activation of p38 occurs relatively early in acute myeloid leukemia (AML) cell lines after treatment with vorinostat. Using comet assays, we detected direct evidence of early DNA damage and western blotting revealed induction of the DNA damage response proteins ATM and Chk2. We performed cell cycle analysis, and observed within the vorinostat-treated AML cell population, cells exiting G1 and accumulating in the G2-M phase of the cell cycle, where they subsequently underwent apoptosis. Notably, downregulation of p38 by shRNA or inhibition of p38 and β activity by the inhibitor SB203580 significantly decreased both G2-M accumulation and apoptosis induced by vorinostat, indicating a pro-apoptotic p38 function. Interestingly, several other HDACi tested all induced p38 activation but, depending on the HDACi, this activation was found to be either pro or antiapoptotic. The short-chain fatty acid sodium butyrate (NaB) requires p38 for induction of apoptosis, like vorinostat. On the other hand, LBH589, from the structural class encompassing vorinostat, does not depend on p38 for induction of apoptosis. Furthermore, p38 serves as a pro-survival signal when induced by the benzimide MGCD0103. In conclusion, we have shown that vorinostat-induced apoptosis in AML cells is preceded by generation of DNA damage and accumulation of cells in the G2-M phase of the cell cycle. Further, G2-M arrest and apoptosis induction (but not DNA damage) by vorinostat requires activation of the p38 MAP kinase, which is not the case for all HDACi. Therefore, a better understanding of the role of p38 MAPK in the action of specific HDACi may help in the development of rational combination regimes including these targeted agents. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A188.
Histone deacetylase inhibitors (HDACi) have recently emerged as promising anticancer agents. Some HDACi have advanced into Phase II and III clinical trials, both as a single agent and in combination with cytotoxics for a variety of malignancies. Encouraging results have been obtained in acute myeloid leukemia (AML). However, HDACi target many pathways and the full mechanism responsible for their anti-neoplastic activity is still far from clear. An understanding of the molecular mechanisms underlying resistance to HDACi may help to elucidate their mechanism of action and may be of relevance in an attempt to design more effective combination strategies. The purpose of this study is to understand the molecular alterations associated with resistance to vorinostat (Zolinza®). A vorinostat resistant clone (U937-VR) was derived from the AML cell line U937 using a dose escalation protocol. The vorinostat-resistant cells (U937-VR) are able to grow in 2 µM vorinostat without induction of apoptosis. U937-VR cells are cross-resistant to the cytotoxic effects of other HDACi, as measured by propidium iodide staining and caspase 3/7 activation. Using western blot, we observed that vorinostat exposure increases tubulin acetylation in U937-VR cells while being incapable of inducing histone acetylation. These data demonstrate effective drug import into the cells, an event that is otherwise commonly altered in drug resistant cells. Furthermore, western blotting reveals a drastic difference in the protein level of the histone acetyltransferase CBP (CREB binding protein) between VR-U937 and parental U937 cells, whereas no alterations in the protein levels of the main HDACs targeted by vorinostat is seen. This indicates that the balance between HDAC and HAT activity might be perturbed. Finally, the resistance index (the ratio between the LD50 of resistant and sensitive cells) of different drugs was evaluated by measuring apoptosis using propidium iodide staining. U937 parental and U937-VR cells have equivalent LD50 for the DNA damaging agent cisplatin and for the microtubule stabilizing agent taxol. In contrast, we observed that U937-VR cells exhibit increased sensitivity toward the proteasome inhibitors MG132 and Bortezomib. This increased sensitivity correlates with a higher level of ubiquitinated protein accumulation in U937-VR. Cumulatively, our data suggest that the U937-VR cells are not multidrug resistant and that the apoptotic cascade remains functional. The importance of the alteration in HAT level and the acquired sensitivity to proteasome inhibitor by chronic exposure to HDACi need further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 722. doi:10.1158/1538-7445.AM2011-722
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