Upregulation of AMPK by 4‐O‐methylascochlorin promotes autophagy via the HIF‐1α expression

Abstract 4‐O‐methylascochlorin (MAC) is a derivative of ascochlorin, a prenyl‐phenol compound antibiotic isolated from the fungus Ascochyta viciae. MAC induces caspase/poly (ADP‐ribose) polymerase‐mediated apoptosis in leukemia cells. However, the effects of MAC on autophagy in cancer cells and the underlying molecular mechanisms remain unknown. Here, we show that MAC induces autophagy in lung cancer cells. MAC significantly induced the expression of autophagy marker proteins including LC3‐II, Beclin1, and ATG7. MAC promoted AMP‐activated protein kinase (AMPK) phosphorylation and inhibited the phosphorylation of mammalian target of rapamycin (mTOR) and its downstream signalling proteins P70S6K and 4EBP1. The AMPK activator AICAR upregulated LC3‐II expression through the AMPK/mTOR pathway similar to the effects of MAC. MAC‐induced LC3‐II protein expression was slightly reduced in AMPK siRNA transfected cells. MAC upregulated hypoxia‐inducible factor‐1α (HIF‐1α) and BNIP3, which are HIF‐1α‐dependent autophagic proteins. Treatment with CoCl2, which mimics hypoxia, induced autophagy similar to the effect of MAC. The HIF‐1α inhibitor YC‐1 and HIF‐1α siRNA inhibited the MAC‐induced upregulation of LC3‐II and BNIP3. These results suggest that MAC induces autophagy via the AMPK/mTOR signalling pathway and by upregulating HIF‐1α and BNIP3 protein expression in lung cancer cells.

growth alterations. 3 Autophagy involves the formation of autophagosomes through the extension of the phagophore, which generates vesicles that enclose damaged organelles within a double membrane before fusing with lysosomes. 4 It is a highly regulated process that depends on the interaction of approximately 30 autophagy-related genes (ATG) present from yeasts to mammals. In the ATG family, ATG8, also known as LC3, plays an essential role in the formation of the autophagosome. 5 It is localized in the autophagosomal membrane and is the main protein involved in the autophagy process. 6 The activation of LC3 is controlled by ATG4, which converts LC3 to LC3-I by removing its C-terminus. 5 LC3-I, which has an exposed glycine residue, is transformed into LC3-II by ATG3 through conjugation with phosphatidylethanolamine (PE), and LC3-II is recruited to the autophagosomal membrane through the action of the ATG12/ATG5/ ATG16 complex. [7][8][9] Autophagy is induced in response to stresses such as the hypoxic (low-oxygen) condition. During hypoxia stress, autophagy can be induced by hypoxia-inducible factor-1α (HIF-1α), an important transcription factor that functions in cell survival in response to hypoxia and according to the extent of exposure. 10 In addition to hypoxia stress, decreased energy and glucose supply induce autophagy via HIF-1α independent signalling pathways. These signalling mechanisms involve the AMP-activated protein kinase (AMPK) signal, which responds to nutritional stress. 11 A cascade of AMPK-triggered phosphorylation events eventually inhibits the ATP-consuming anabolic processes, stimulates the ATP-producing catabolic processes, and initiates autophagy. 12 Ascochlorin (ASC) is a prenyl-phenol compound isolated from the fungus Ascochyta viciae. ASC-related compounds were originally characterized as antiviral antibiotics. 13 ASC functions as an antibiotic, 14 promotion hypolipidemic activity, 15 anti-diabetic, 16 immunomodulation. 17 In addition to ASC, ASC-related derivatives like 4-O-carboxymethylascochlorin (AS-6) and 4-O-methylascochlorin (MAC) 16 have been reported to be therapeutic reagents for various physiological events. 18 Among the derivatives of ASC, MAC-induced apoptotic effects through caspase/poly (ADP-ribose) polymerasemediated apoptosis in leukemia cells. 13 However, the effects of MAC on autophagy in cancer cells and the underlying molecular mechanisms have not been investigated to date.
In this study, the effects of MAC on autophagy and the mechanism underlying MAC-mediated autophagy in lung cancer cells were investigated. Our results showed that MAC-induced autophagosome formation by upregulating LC3-II, Beclin1, and ATG7, and the effects of MAC on autophagy were related to the regulation of HIF-1α and the AMPK/mammalian target of rapamycin (mTOR) signalling pathway.

| Cells culture and materials
Human non-small lung cancer cell lines A549, H1793, and H23 cells were obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were cultured in RPMI 1640 (Thermo Scientific, Logan, UT, USA), containing 10% of fetal bovine serum and 1% of antibiotic mixture. These were incubated at 5% of CO 2 , 37°C. MAC was supplied Chugai Pharmaceutical (Tokyo, Japan).

| Cell viability assay
Human

| Morphology
Cells were cultured in 60 mm dish and incubated until they reached 60% confluence. Then, media were changed fresh RPMI 1640 media and treat MAC for 12 hours, and cells were photographed.

| Immunofluorescence microscopy
Cells were cultured and treated on poly-L-lysine-coated coverslips before being fixed in 100% ethanol for 1 minute at room tempera-

| Statistical analysis
All in vitro results are representative of at least three independent experiments performed in triplicate. *P < 0.05, statistically significant between experimental and control values. Significance of differences between experimental and control values was calculated using ANOVA with Newman-Keuls multicomparison test.

| Effects of ASC and ASC derivatives on autophagosome formation in A549 lung cancer cells
The typical morphological feature of autophagy is the formation of autophagosomes, which fuse with lysosomes and expose cargo to lysosomal enzymes, causing degradation of the contents. 19 The effect of ASC and its derivatives AS-6 and MAC on autophagosome formation ( Figure 1A) was investigated by examining morphological changes of A549 lung cancer cells. Incubation of A549 cells with 20 μmol/L ASC, AS-6, or MAC for 12 hours induced vesicle formation ( Figure 1B). LC3 and p62 (also known as SQSTM1) are the key factors for the autophagy. 20 Accumulation of LC3-II allows assessment of autophagy initiation, whereas p62 is degraded by autophagy. The effect of the three chemicals on autophagosome formation was investigated by western blot analysis of LC3-I/II and p62 expression. As shown in Figure 1C, AS-6 and MAC but not ASC upregulated LC3-II. AS-6 and MAC increased the LC3-II/LC3-I ratio by approximately 3-and 10-fold, respectively, whereas ASC had no effect on the LC3-II/LC3-I ratio. MAC also completely degraded p62.
These data suggest that MAC induces autophagy more effectively than the other ASC derivatives. Therefore, the mechanism of MACinduced autophagy was analysed in this study.

| MAC triggered autophagosome formation in various lung cancer cell lines
The effects of MAC on autophagy were examined in three lung can-

| MAC-induced autophagy-related gene expression
To determine whether autophagy was induced by MAC in lung cancer cells, the expression of LC3-I/II, a key autophagy marker protein, was measured by western blotting. As shown in Figure 3A  have been studied for their roles in autophagosome formation and maturation. 22 We showed that MAC upregulated Beclin1 and ATG7 in a time-dependent manner ( Figure 3D). These results suggest that MAC promotes autophagy by upregulating LC3-II, Beclin1, and ATG7 protein expression in lung cancer cells. We next evaluated changes in p62 and TFEB expression. p62 is selectively bind to LC3 and degraded by lysosome in the final stages of the autophagy process. 23 The expression of p62 was increased at 6 hours after treatment with MAC. However, it began to decline at 12 hours compared with those in untreated ( Figure 3E). TFEB, which is a regulator of autophagy to lysosomal biogenesis, 24 also began to increase at 12 hours after MAC treatment. These results suggest that MAC promotes autolysosome by regulating p62 and TFEB protein expression in lung cancer cells.

| MAC activated autophagy through the AMPK signalling pathway
AMPK induces autophagy by stimulating the ULK1 and Beclin1 proteins mTOR, a downstream regulator of AMPK, stimulates protein synthesis and inhibits autophagy by disrupting the interaction between ULK1 and AMPK. 25 We therefore examined the effect of

| MAC-induced autophagy was related to hypoxia in lung cancer cells
As HIF-1 signalling responds to oxygen deficiency, AMPK, an energy deficiency sensor that responds to stressful environments, may play a fundamental role in cancer control and longevity. 26 Therefore, we  Figure 5D). However, EBSScontaining media weakly induced LC3-II expression and had no effect on the other factors. MAC and CoCl 2 upregulated HIF-1α expression, whereas EBSS treatment had no effect on HIF-1α expression ( Figure 5E). These results indicated that MAC induces HIF-1α expression and autophagy similar to the hypoxia environment.

| MAC promotes autophagy by regulating AMPK/HIF-1α expression in lung cancer cells
To

| DISCUSSION
Autophagy is a lysosome-mediated degradation mechanism under various stress conditions, such as the high temperatures, starvation conditions, and hypoxic condition. 30,31 The autophagy process starts with the formation of autophagosomes, which fuse with lysosomes to become autolysosomes and then remove damaged protein and organelles through material recycling, to promote long-life. [32][33][34] It was recently reported that autophagy plays an important role in the processes of immunity, infection, inflammation, and tumour formation. [35][36][37][38][39] In the present study, the autophagic effects of ASC, AS-6, and MAC in lung cancer cells were investigated. First, the three chemicals were assessed for their induction of autophagy with LC3 expression. ASC did not affect LC3-II expression, but AS-6 and MAC significantly increased LC3-II expression in A549 cells (Figure 1). In the previous study, AS-6 induced autophagy through ER stress by increasing the essential autophagic proteins Beclin1, ATG5, and LC3-II in liver carcinoma cells. 40 However, MAC increased the LC3-II expression more than the AS-6. As MAC has a 4-O-methyl group to be substituted for the 4-O-hydroxyl group of ASC, the methyl group of MAC is important for the activation of autophagy. 41 Autophagy was related to many members of the ATG family in various stages of autophagosome formation. We investigated the expression of LC3 and Beclin1, which used as autophagy markers, for assessing the autophagy induction by MAC in lung cancer cells.
MAC downregulated LC3-II and Beclin1 (Figure 3). ATG gene family is involved in vesicle formation, processing, and maturation. The E1 ligase ATG7 plays a central role in the elongation of the autophagosomal vesicles, an essential process for damaged-protein loading, but it also functions in ubiquitin-like reactions involving the lipidation of LC3 and the conjugation of the ATG5 band ATG12, which is essential for lysosomal fusion. 42 We found that MAC increased ATG7 expression in a time-dependent manner. These results suggest that MAC induces autophagy through the ATG family, including LC3, Beclin1, and ATG7.
According to the previous study, AMPK activation is a cellular sensor for the energy balance status, and suppresses cell growth and proliferation in the tumour survival pathway. Considering that AMPK is inactive in the high-glucose condition, mTOR may be one of the major targets of AMPK signalling. The mTOR signalling plays an essential role in carcinogenesis, which is attributable to its functions in the regulation of growth and metabolism. 43 45 It was speculated that the hypoxia-specific proteins BNIP3 and HIF-1α were related to the increase of the autophagy-associated proteins. BNIP3 is stimulated by hypoxia and induces autophagy by weakening the binding of the Bcl-2/Beclin1 complex. 10 Hypoxia also induced autophagy by the upregulation of ATG5 and ATG7. 46 MAC increased BNIP3, Beclin1 and ATG7 expression, and HIF-1α nuclear translocation ( Figure 5). The low levels of oxygen and nutrients enhance autophagy signalling in the tumour microenvironment. 47 Therefore, the autophagic activities under the treatment of MAC, CoCl 2 (mimic hypoxia), or EBSS media (amino acid deprivation) were compared.
In Figure 5, all the conditions induced autophagosome formation, but EBSS selectively increased LC3-II. According to a previous study, EBSS increased the phosphorylation of Beclin1 without the regulation of Beclin1 expression. 48 Our results also showed that the EBSS medium did not increase the Beclin1 expression. As

CONFLI CT OF INTEREST
The authors confirm that there are no conflicts of interest.