Abstract 3875: Persimmon leaf extract inhibit gamma ray and heavy ion induced DNA damage checkpoint activity in A549 lung adenocarcinoma cells

Abstract

Abstract Persimmon leaves have been commonly used as traditional medicines in Asia. They have variety kinds of health benefits in such prevention of cancer, hypertension, and inflammation. However, a few of scientific evidences are reported to date. Recent study reported that the cell cycle checkpoint system is playing a pivotal role in DNA damage response, and it is expected that the discovery of checkpoint inhibitor will be an effective assistance of anti-cancer therapy. Checkpoint signaling cascades are critically modulated by ATM (Ataxia telangiectasia-mutated) and ATR (ATM-Rad3-related) protein kinases. Generally, ATM primarily responds to ionizing radiation-induced DNA double-strand breaks, whereas ATR is activated by stalled replication resulting from UV and some genotoxic drugs. In the previous study, we demonstrated that a flavonoid-enriched persimmon leaf extract (FEPLE) promoted cytotoxic effect of cancer cells by chemotherapeutic agents inhibiting the checkpoint activity, especially in ATM dependent pathway. Here, we investigated whether FEPLE inhibits checkpoint activity during DNA damage response induced by radiation such as gamma irrradiation (γIR) and heavy ion (HI) treatment. Dried persimmon leaves were prepared by a decoction for 30 min. A soluble extract was subsequently partitioned into ethyl acetate layer which is flavonoid-enriched persimmon leaves extract (FEPLE). FEPLE included eight flavonol components, non-galloylated flavonoids (kaempferol 3-O-glucoside, kaempferol 3-O-galactoside, quercetin 3-O-glucoside, quercetin 3-O-galactoside) and galloylated flavonoids (their 2″-gallated flavonol glucoside). γIR (2.5 to 20 Gy) and HI (2.5 to 20 Gy) were performed in National Institute of Radiological Sciences (NIRS) and Heavy Ion Medical Accelerator in Chiba (HIMAC), respectively. Human lung adenocarcinoma A549 cells were incubated with FEPLE (0, 1, 10, 30 μg/mL) at one hour prior to γIR or HI treatment. Intracellular levels of phosphorylation in each checkpoint molecule such as p53, SMC1 and Chk1 were determined by western blot using phospho-specific antibodies of Ser15, Ser966 and Ser345, respectively. Phosphorylation on Ser10 of histone H3 was measured by flow-cytometry as a marker of cells in mitosis. The phosphorylation of p53, SMC1 and Chk1 were increased by the treatment of γIR and HI. FEPLE reduced the phosphorylation of checkpoint proteins in both γIR and HI. Furthermore, ATM activity, estimated by the phosphorylation of Ser1981, was inhibited by the treatment of FEPLE. The pre-treatment of FEPLE significantly prevented the decrease of mitotic cells in HI exposed cells. These results indicated that DNA damage checkpoint system in γIR or HI exposed cells were abrogated by FEPLE treatment through the inhibition of ATM-dependent signaling pathway. FEPLE might be useful for clinical application by combining γIR or HI cancer therapy. 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 3875.