Quercetin induces mitochondria mediated apoptosis and protective autophagy in human glioblastoma U373MG.

Abstract

Flavonoid의 일종인 quercetin은 다양한 암세포 주에 대하여 항암효능이 있는 것으로 알려져 있다. 본 연구는 교모세포종인 U373MG에 대한 quercetin의 세포사멸효능과 그 기작에 대해 연구하였다. Quercetin 처리 시간과 농도 의존적으로 세포죽음이 일어났다. Hoechst 33342 염색을 통한 apoptotic body 형성, flow cytometic analysis를 통한 Sub-G1기의 축적과 mitochondrial membrane potential 감소, apoptosis 관련 단백질의 발현증가와 caspase-3, 9 의 활성 증가로 인한 apoptosis에 의한 세포죽음을 확인하였다. 또한 acridine orange 염색과 immunoblot analysis를 통하여 autophagy 현상도 일어나고 있음을 확인하였다. Autophagy inhibitor인 chloroquine 전처리를 통하여 autophagy 현상이 세포를 보호하기 위한 protective autophagy임을 확인하였으며, 이러한 autophagy를 억제하였을 때 apoptosis가 증진되어 세포죽음을 더욱 유도하는 것을 확인할 수 있었다.

Quercetin is one of the dietary flavonoids known to have antitumor effect against several types of cancer by promoting apoptotic cell death and inducing cell cycle arrest. In this study, anticancer effect of quercetin has been evaluated against p53-mutant human glioblastoma cells U373MG. Quercetin exhibited time- and concentration-dependent cytotoxicity against U373MG. Induction of apoptotic cell death by quercetin was observed by fragmented nuclei, increased sub-G1 population and decreased mitochondrial membrane potential. Further, proteolytic activation of caspase-3, -7, and cleavage of PARP (Poly (ADP-Ribose) Polymerase) substantiated the occurrence of apoptotic cell death. Also, translocation of p53 from cytosol to mitochondria lead to release of cytochrome C from mitochondria into cytosol. Concomitantly, quercetin induced formation of acidic vesicular organelles, conversion of LC3II, indicating that quercetin also induced autophagy. Importantly, pre-treatment with chloroquine, an autophagy inhibitor, enhanced quercetin-mediated apoptotic cell death. These results show that quercetin caused protective autophagy in U373MG and the ability of quercetin in association of autophagy inhibitor may improve the ultimate outcome of glioblastoma therapy.