Abstract 3649: DAPK-dependent apoptosis induction after treatment of colorectal tumor cells with the histone deacetylase inhibitor panobinostat (LBH589)
Authors: Gandesiri Muktheshwar , Chakilam Saritha , Ocker Matthias , Christiansen Hans , Rave-Fraenk Margret , Rauchschwalbe Sonja , Ivanovska Jelena , Hartmann Arndt , Schneider-Stock Regine 
Published: February 2011

Objectives: Among Histone deacetylase inhibitors (HDACi), panobinostat is a relatively new compound showing potent antitumor activities. Although the signal transduction after panobinostat treatment has been studied in various tumor cell lines, the role of the death associated protein kinase (DAPK) in panobinostat-induced apoptosis has not been investigated to date. Dependent on the stimuli, a dual role for DAPK has been shown as apoptosis inducer or pro-survival factor. The aim of this study was to characterize the role of DAPK in panobinostat-induced apoptosis using HCT116-wildtype (DAPK-wt) and HCT116-DAPK knockdown (DAPK-ko) cells.

Methods: DAPK-ko stable cell line was generated using DAPK shRNA lentiviral particles (Santa Cruz, USA). DAPK-ko cells expressed only 30% of the original DAPK protein level. DAPK-wt and DAPK-ko cells were stimulated with 0.05µM panobinostat for 6, 24 or 48 h, respectively. Long term survival was studied by clonogenic assay. Anti-tumoral activity of the HDACi was assessed by immunoblotting for p21WAF-1, Ac-H3, and Ac-H4. Apoptosis induction was verified by PARP cleavage, BrdU proliferation assay, M30 cytodeath FACS, and caspase 3/7 ELISA. In vivo relevance of our findings was confirmed in a xenograft mouse model by Western Bloting and immunohistochemical analysis.

Results: As expected the acetylation of histones H3 and H4 was significantly induced in both cell types. Panobinostat significantly reduced the colony formation ability (long term survival) in DAPK-wt as well as in the DAPK-ko cells. Interestingly panobinostat exposure resulted in up-regulation of DAPK in both cell lines, whereas the protein level of the cell cycle regulator p21WAF1 was increased only in the DAPK-ko cells. DAPK-ko cells were more sensitive to panobinostat treatment than the DAPK-wt cells. They showed a higher PARP cleavage, an increase in caspase 3/7 activity, and a higher number of apoptotic cells in M30 cytodeath FACS. In a subcutaneous xenograft mouse model, DAPK-ko tumors were growing slower than DAPK-wt cells. Panobinostat treatment yielded a more effective size reduction in DAPK-ko tumors. In accordance with the in vitro data, DAPK-ko tumors showed a significantly higher level of p21WAF1 protein and a reduced Ki67-proliferation index in immunohistochemistry. DAPK protein expression and Ki-67 index were significantly correlated (p=0.001).

Conclusions: We have generated a suitable model for understanding DAPK-signaling as a new target for HDACi treatment in colorectal cancer cells. Our results show a clear involvement of DAPK in panobinostat-induced apoptosis. From these preliminary data we assume a pro-survival function of DAPK upon drug treatment. The association between DAPK, p21WAF1 and DNA damage response has to be further elucidated.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3649.