Date Published: February 3, 2017
Publisher: Public Library of Science
Author(s): Sophia P. M. Sok, Norhafiza M. Arshad, Mohamad Nurul Azmi, Khalijah Awang, Bulent Ozpolat, Noor Hasima Nagoor, Sujit Kumar Bhutia.
Autophagy plays a role in deciding the fate of cells by inducing either survival or death. 1’S-1-acetoxychavicol acetate (ACA) is a phenylpropanoid isolated from rhizomes of Alpinia conchigera and has been reported previously on its apoptotic effects on various cancers. However, the effect of ACA on autophagy remains ambiguous. The aims of this study were to investigate the autophagy-inducing ability of ACA in human non-small cell lung cancer (NSCLC), and to determine its role as pro-survival or pro-death mechanism. Cell viability assay was conducted using MTT. The effect of autophagy was assessed by acridine orange staining, GFP-LC3 punctate formation assay, and protein level were analysed using western blot. Annexin V-FITC/PI staining was performed to detect percentage of cells undergoing apoptosis by using flow cytometry. ACA inhibits the cell viability and induced formation of cytoplasmic vacuoles in NSCLC cells. Acidic vesicular organelles and GFP-LC3 punctate formation were increased in response to ACA exposure in A549 and SK-LU-1 cell lines; implying occurrence of autophagy. In western blot, accumulation of LC3-II accompanied by degradation of p62 was observed, which further confirmed the full flux of autophagy induction by ACA. The reduction of Beclin-1 upon ACA treatment indicated the Beclin-1-independent autophagy pathway. An early autophagy inhibitor, 3-methyaldenine (3-MA), failed to suppress the autophagy triggered by ACA; validating the existence of Beclin-1-independent autophagy. Silencing of LC3-II using short interfering RNA (siRNA) abolished the autophagy effects, enhancing the cytotoxicity of ACA through apoptosis. This proposed ACA triggered a pro-survival autophagy in NSCLC cells. Consistently, co-treatment with lysosomal inhibitor, chloroquine (CQ), exerted a synergistic effect resulting in apoptosis. Our findings suggested ACA induced pro-survival autophagy through Beclin-1-independent pathway in NSCLC. Hence, targeting autophagy pathway using autophagy inhibitor such as CQ represented a novel promising approach to potentiate the cytotoxicity of ACA through apoptosis in NSCLC.
Lung cancer is the most common cancer worldwide; accounting for 1.82 million new cases and 1.6 million deaths in 2012 . Among the lung cancer cases, non-small cell lung cancer (NSCLC) contributes to approximately 85% and has a low 5-year survival rate . Conventional cancer therapies such as surgery, chemotherapy and radiotherapy were found to have limitation in maintaining its effectiveness during the course of therapy which lead to recurrence and acquired apoptosis resistance in long term treatment . Hence, it is crucial to elucidate the underlying reason to improve the efficiency of the available therapeutic agents. Emerging evidences proposed that identifying the role played by autophagy in cancer could be a strategy to overcome resistance towards chemotherapy due to the fact its potential in eliciting a pro-survival or pro-death effect in response to metabolic and therapeutic stresses [4, 5].
The naturally occurring ginger compound, ACA, has been extensively studied which revealed its potential as an anti-cancer agent through activation of apoptosis [12–14]. However, the ability of ACA to induce autophagy in NSCLC remains unclear. Our current study revealed that ACA induced autophagy in NSCLC as evidenced by the accumulation of AVO and GFP-LC3-II along with elevated LC3-II protein level indicating the recruitment LC3-II on autophagosomes. Furthermore, degradation of p62 observed further implied that ACA induced autophagy flux. This is the first study reporting on autophagy inducing ability of ACA in NSCLC. Natural occurring compounds such as curcumin and resveratrol were also found to activate autophagy and/or apoptosis via reactive oxygen species (ROS) pathway in oral and colon carcinoma respectively [22–24]. Previous studies reported that ACA can stimulate the production of ROS in myeloid leukemia and hepatocellular carcinoma [25, 26]. This might be the possible mechanism of ACA regulating autophagy in both A549 and SK-LU-1 cells.
Collectively, our current study demonstrated for the first time that ACA induced autophagy is through Beclin-1-independent/non-canonical pathway in NSCLC. In addition, we showed autophagy inhibitor CQ or inhibition of autophagy gentically by knockdown of LC3 using siRNA promoted ACA-induced cell death through the apoptosis mechanism. These knowledge suggested targeting autophagy mechanism in cancer therapy may be a promising approach to sensitized NSCLC to ACA treatment.