autophagy-inducing effects in HCT116 colon cancer cells. Autophagy plays a cytoprotective role in apigenin-induced apoptosis, and the combination of apigenin and an autophagy

Apigenin (4',5,7-trihydroxyflavone) is a natural flavonoid, shown to have chemopreventive and/or anticancer properties in a variety of human cancer cells. The involvement of autophagy in apigenin-induced apoptotic cell death of HCT116 human colon cancer cells was investigated. Apigenin induced suppression of cell growth in a concentrationdependent manner in HCT116 cells. Flow cytometric analyses indicated that apigenin resulted in G2/M phase arrest. This flavone also suppressed the expression of both cyclin B1 and its activating partners, Cdc2 and Cdc25c, whereas the expression of cell cycle inhibitors, such as p53 and p53-dependent p21CIP1/WAF1, was increased after apigenin treatment. Apigenin induced poly (ADP-ribose) polymerase (PARP) cleavage and decreased the levels of procaspase-8, -9 and -3. In addition, the apigenin-treated cells exhibited autophagy, as characterized by the appearance of autophagosomes under fluorescence microscopy and the accumulation of acidic vesicular organelles by flow cytometry. Furthermore, the results of the western blot analysis revealed that the levels of LC3-II, the processed form of LC3-I, was increased by apigenin. Treatment with the autophagy inhibitor 3-methyladenine (3-MA) significantly enhanced the apoptosis induced by apigenin, which was accompanied by an increase in the levels of PARP cleavage. These results indicate that apigenin has apoptosisand autophagy-inducing effects in HCT116 colon cancer cells. Autophagy plays a cytoprotective role in apigenin-induced apoptosis, and the combination of apigenin and an autophagy inhibitor may be a promising strategy for colon cancer control. Introduction Apigenin (4',5,7-trihydroxyflavone; Fig. 1A), a naturally occurring flavone, it is widely distributed in many fruits and vegetables such as parsley, onions, apples, tea and chamomile. In recent years, apigenin has been increasingly recognized as a cancer chemopreventive agent. The chemopreventive aspects of apigenin have been evaluated both in vitro and in vivo. Apigenin has been shown to be growth inhibitory in a variety of human cancer cell lines including colon, pancreatic, oral squamous, lung and leukemia cells (1-5). An important effect of apigenin is to increase the stability of the tumor suppressor p53 gene in normal cells. It has been shown that apigenin induced G2/M cell cycle arrest in colon cancer cells (1), and in vivo it is involved in p21CIP1/WAF1-independent pathway for inhibitory phosphorylation of p34 (Cdc2) and concomitant G2/M arrest in mouse keratinocytes (6). Apigenin was shown to induce apoptosis in a variety of cancer cells (3,4,7,8). Apigenin has shown to inhibit tumor cell invasion and metastases by regulating the hypoxia-inducible factor 1-α protein level and to inhibit transforming growth factor β 1-induced vascular endothelial growth factor expression in human prostate cancer cells (9). Moreover, apigenin has been reported to potentiate the effect of tumor necrosis factor-related apoptosis-inducing ligand, paclitaxel, ABT-263, 5-fluorouracil (5-FU) and cisplatin against various human cancers (10-13). Autophagy, an evolutionarily conserved process, sequesters and degrades long-lived cellular proteins and organelles through the lysosomal machinery (14,15). The purpose of autophagy is the recycling of cellular components to sustain metabolism under stress conditions such as nutrient deprivation and to prevent accumulation of damaged proteins and organelles (16). The first evidence for a role of autophagy in cancer was found by Liang et al (17). The autophagy-promoting activity of beclin-1 in human breast cancer cells is associated with inhibition of MCF7 cellular proliferation (17). It is reported that beclin-1, a phylogenetically conserved protein that is essential for autophagy, can inhibit tumorigenesis and is expressed at decreased levels in human breast carcinoma. Recent evidence indicates that the phosphoinositide 3-kinase (PI3K), Akt and mammalian target of rapamycin (mTOR) pathway, which is activated in many types of cancer (18) is important in autophagy regulation, especially through activating mTOR kinase, leading to suppression Apigenin-induced apoptosis is enhanced by inhibition of autophagy formation in HCT116 human colon cancer cells YUJIN LEE, BOKYUNG SUNG, YONG JUNG KANG, DONG HWAN KIM, JUNG-YOON JANG, SEONG YEON HWANG, MINJUNG KIM, HYUN SOOK LIM, JEONG-HYUN YOON, HAE YOUNG CHUNG and NAM DEUK KIM Division of Pharmacy, College of Pharmacy, Molecular Inflammation Research Center for Aging Intervention (MRCA), Pusan National University, Busan 609-735, Republic of Korea Received December 30, 2013; Accepted February 24, 2014 DOI: 10.3892/ijo.2014.2339 Correspondence to: Dr Nam Deuk Kim, Division of Pharmacy, College of Pharmacy, Pusan National University, Busan 609-735,