Abstract 3220: Doxorubicin-induced senescence in hepatocellular carcinoma cells

Abstract

Abstract Introduction: Doxorubicin (DOX, adriamycin) is an anti-cancer drug commonly used in treatment of several human solid cancers including unresectable hepatocellular carcinoma (HCC). Therapeutic effects of DOX have been linked to DNA intercalation and topoisomerase inhibition as well as oxidative stress induction. As DNA damage and oxidative stress have been identified as potential mediators of cellular senescence, we tested whether DOX is able to trigger senescence arrest in HCC cells. Methods: HepG2 (wt p53) and Huh7 (p53-Y220C) cell lines were exposed to different doses of DOX and their responses were studied over a period of 6 days. Senescence was tested by morphological changes and senescence-associated beta-galactosidase (SABG) activity. DNA damage response was studied at the levels phospho-H2AX, phospho-ATM, phospho-ATR, phospho-CHK1, phospho-CHK2 and 53BP1. Cell cycle analysis was performed by flow cytometry and cell cycle protein expression analysis. We also analyzed histone methylation changes in response to DOX. Results: High dose DOX (500ng/ml) treatment caused p53 accumulation, induced p21 levels and triggered apoptosis, as shown by flow cytometry. Low dose (LD) DOX treatment (25ng/ml and 50ng/ml) did not induce apoptosis, but triggered a senescence response at day 3 in both HepG2 and Huh-7 HCC cell lines. Senescence phenotype was characterised with flattened and enlarged shape, and strong positivity of SABG activity (> 80% in three days, >90% in six days). LD DOX treatment caused a slight increase in p53 and p21 protein levels in correlation with SABG positivity in HepG2 cells. After treating cells 3 days with LD 50ng/ml of DOX, we found that 53BP1 and p-H2AX localized to DNA damage foci. ATM-Chk2-Cdc25-A signal transduction pathway but not the ATR-Chk1-Cdc25 pathway was activated to arrest the cell cycle following DNA damage in G2 phase both in HepG2 and Huh-7 cells. We obtained G2/M arrest reaching as high as 90% for both cell lines. In correlation with this observation, we also showed downregulation of cdc2, and cyclin B1 proteins, key modulators of G2/M cell cycle transition, using immunoblotting while cyclin A1 protein levels remained unchanged. Using this model, we also explored the global epigenetic histone methylation marks in LD DOX treated cells using both immunofluorescence and immunoblotting technique. Upon treatment with LD DOX, we observed increase in H3K27Me3, H3K36Me3 and H4K20Me3 levels and a corresponding decrease in monomethyl forms of these histones, H3K27Me1, H3K36Me1 and H4K20Me1. We did not detect any change on H3K4Me3, H4K36Me2 and H4K20Me2 methylation levels. Taken together, our observations indicate that LD DOX treatment of HCC cells induces p53-independent senescence arrest at phase G2/M. This was associated with a DNA damage response associated with global changes in histone methylation. 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 3220.