Abstract. Casticin, a natural polymethoxyflavone isolated from A. annua, V. trifolia, and V. agnus‐castus induces apoptosis in cancer cells by activating FoxO3a. However, whether casticin inhibits in vitro carcinogenesis and cancer stem cell (CSC) characteristics, and whether casticin activates FoxO

Casticin, a natural polymethoxyflavone isolated from A. annua, V. trifolia, and V. agnus‐castus induces apoptosis in cancer cells by activating FoxO3a. However, whether casticin inhibits in vitro carcinogenesis and cancer stem cell (CSC) characteristics, and whether casticin activates FoxO3a in small cell lung cancer (SCLC) cells remain unclear. We here demonstrated that casticin decreased sphereand colony-formation capabilities, and downregulated uPAR and CD133 in second-generation spheres, which were considered as lung cancer stem-like cells (LCSLCs), from SCLC H446 cells, in a concentration-dependent manner. In addition, casticin dose-dependently elevated the phosphorylation levels of AMPK and ACC, and reduced p-FoxO3a expression. The above effects were attenuated by AMPK knockdown with small interfering RNAs (siRNAs). FoxO3a silencing resulted in decreased protein expression of FoxO3a, increased in vitro carcinogenesis and CSC characteristics, with no appreciable effects on AMPK and ACC phosphorylation, and displayed similar activities to those neutralizing the effects of casticin on in vitro carcinogenesis and CSC characteristics. These findings reveal a novel mechanism for regulating AMPK/FoxO3a signaling in response to casticin, suggesting a new strategy for SCLC therapy by targeting cancer stem-like cells. Introduction Small cell lung cancer (SCLC), a poorly differentiated and highly aggressive tumor, constitutes approximately 15% of all lung cancers (1). SCLC patients often present with metastasis at diagnosis, ruling out surgery as a treatment option (2). There is currently no approved targeted drugs for SCLC, and no effective method for early diagnosis is available; meanwhile, most patients treated with conventional therapies, including chemotherapy and radiotherapy, show recurrence after a short period of time, which results in poor patient prognosis (3). To improve the survival of patients with SCLC, developing new and efficacious candidate agents is urgently required. Emerging evidence suggests that cancer stem cells (CSCs), a subpopulation of tumor cells, have the properties of self-renewal, heterogeneous progeny, drug-resistance, and carcinogenesis in vitro and in vivo (4,5). Multiple SCLC characteristics, such as aggressiveness and high metastatic potential, suggest that this cancer could be enriched in CSCs (6). Furthermore, drug resistance may be attributable to the occurrence of a CSC subpopulation in SCLC (6). Therefore, a therapeutic strategy targeting CSCs may help cure malignant tumors, including SCLC (7). FOXO3a is considered an evolutionarily conserved transcription factor involved in various cellular processes, including cell cycle arrest, DNA repair and tumor suppression (8). In cancer progression, FOXO3a inhibition stimulates cell transformation and angiogenesis (9). Conversely, FOXO3a overexpression suppresses cancer cell growth, induces apoptosis, and reduces tumor size by regulating downstream effectors (10). These findings suggest a tumor suppressor role for FOXO3a, which could constitute a potential target for cancer treatment. Our previous findings demonstrated that casticin, a natural polymethoxyflavone isolated from A. annua, V. trifolia, and V. agnus‐castus that has been widely used in traditional Chinese medicine as an anti‐inflammatory drug for thousands of years, not only induces growth suppression, apoptosis and cell cycle arrest in hepatocellular carcinoma (11) and breast cancer cells (12), but also promotes apoptosis in ovarian cancer SKOV3 cells (13) through FOXO3a activation. However, whether casticin inhibits in vitro carcinogenesis and Casticin suppresses the carcinogenesis of small cell lung cancer H446 cells through activation of AMPK/FoxO3a signaling QIAN GONG1*, XIAOZHENG CAO2*, JIANGUO CAO3,4, XIAOHONG YANG3,4 and WENBIN ZENG2 1Pharmacy Department, Hunan Cancer Hospital/The Affiliated Cancer Hospital of Xiangya School of Medicine; 2School of Pharmaceutical Sciences, and Molecular Imaging Research Center, Central South University; 3Department of Pharmaceutical Science, Medical College, Hunan Normal University; 4Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Changsha, Hunan 410013, P.R. China Received January 10, 2018; Accepted June 12, 2018 DOI: 10.3892/or.2018. 6547 Correspondence to: Dr Jianguo Cao, Department of Pharmaceu tical Science, Medical College, Hunan Normal University, 371 Tongzipo Road, Changsha, Hunan 410013, P.R. China E-mail: caojianguo2005@126.com *Contributed equally