Introduction

Gastric cancer is the third leading cause of cancer death worldwide, and approximately 700,000 cases of stomach cancer are diagnosed each year [1]. Chemotherapy is the major clinical treatment for advanced gastric cancer, but the development of effective chemotherapeutic drug is rather slow. Drug resistance and the toxicity of chemotherapy are great limitations to this kind of treatment [2]. Therefore, the study of new treatments that overcome the aforementioned drawbacks is essential.

Vitamin E succinate (VES; α-tocopherol succinate), a vitamin E derivative, has potent anticancer effects on a variety of cancers. Previous research has shown that VES can induce human gastric cancer SGC-7901 cell apoptosis through many signaling pathways, such as extrinsic Fas, endoplasmic reticulum stress, and mitogen-activated protein kinase (MAPK) pathways [3–5]. VES, which has anticancer effects, shows no toxicity to normal cells and tissues in vitro and in vivo [6–12]. However, the relationship between VES and autophagy and the underlying molecular mechanisms is poorly defined.

Autophagy is a dynamic and highly regulated process that plays a critical role in degradation of long-lived proteins, bulk cytoplasm, and organelles [13–16]. This process begins with the formation of double-membrane autophagosomes initiated by PI3 kinase (PI3K) type III-Atg6/Beclin-1 cascade [17]. After the formation of autophagosomes, two ubiquitin-like systems exist: the Atg8/LC3-phosphatidylethanolamine conjugate system and the Atg12–Atg5 conjugate system. Both systems are involved in the expansion of the autophagosomes [18]. Autophagy is regulated by numerous cross talking signaling pathways, which function in response to many forms of cellular stress, such as hypoxia, starvation, and radiation [19]. The classic PI3K/Akt-mTOR signaling pathway is considered as a crucial negative regulator for the formation of autophagosomes [20]. mTOR activity is controlled by PI3K/Akt signaling pathway. Activated Akt phosphorylates tuberous sclerosis complex 2 (TSC2) prevents formation of the inhibitory TSC1/TSC2 heterodimer and then activates TOR complex 1 (TORC1), which is a functionally distinct complex of mTOR. When mTOR is activated, it stimulates protein synthesis by phosphorylating key translation regulators, i.e., ribosomal protein S6 kinase (P70S6K) and eukaryotic initiation factor 4E binding protein 1 (4EBP-1) [21, 22]. Recent studies have shown that AMPK activation may lead to autophagy through the negative regulation of mTOR [23–26]. Activated AMPK inhibits TORC1 activity by phosphorylation and activation of the negative regulator TSC2 [27]. AMPK can directly phosphorylate ULK1 (Unc-51-like kinase 1) to induce autophagy [28]. In the present study, we investigated the cellular responses of autophagy process induced by VES in human gastric cancer cells, and whether the accumulation of AMPK was involved in these processes. We also probed whether crosstalk is present between AMPK and Akt/mTOR signaling pathways. Our studies would provide a better understanding for the implication of autophagy in VES-mediated anticancer activities.

Materials and methods

Cells and cell culture

Human gastric cancer SGC-7901 cells were obtained from Chinese Academy of Sciences Cell Resource Center. Cells were maintained in RPMI1640 medium (Hyclone, SH30809.01B) supplemented with 10% fetal bovine serum (FBS) (SiJiQing, 13011-8611), 10,000 IU penicillin, and 10,000 μg/mL streptomycin. The cells were cultured in a humidified 5% CO2 atmosphere at 37 °C.

Chemical treatment and antibodies

VES (Sigma, T3126) dissolved in sterile absolute ethanol; 3-methyladenine (3-MA) (Sigma, M9281), chloroquine (CQ) (Sigma, C6628), and IGF-1 (Abcam, ab123776) dissolved in phosphate-buffered saline (PBS); AICAR (Sigma, A9978), LY294002 (Sigma, L9908), Compound C (Sigma, P5499), and Rapamycin (RAPA) (Sigma, 37094) dissolved in dimethyl sulfoxide (DMSO) were added to the medium to final concentrations, as described in each experiment. Cells grown to 60% confluency were switched to 2% FBS medium, and the culture was allowed to expand for 24 h before any treatment was performed. For 3-MA, CQ, RAPA, AICAR, LY294002, Compound C, and IGF-1 plus VES treatment, cells were first incubated with 5 mM 3-MA, 20 μM CQ, 100 nM RAPA, 1 mM AICAR, 10 μM LY294002, and 10 μM Compound C for 2 h, respectively. Then, VES was added, and the cells were incubated for 24 h. For IGF-1 plus VES treatment, cells were pretreated with 100 ng/mL IGF-1 for 1 h and then incubated with VES for 24 h.

Antibodies were obtained from the following sources: AMPKα (Cell signaling, 5831S), p-AMPKα (Thr172) (Cell signaling, 2535S), ACC (Cell signaling, 3662), p-ACC (Ser97) (Cell signaling, 11818), Akt (Cell signaling, 9272), p-Akt (S473) (Cell signaling, 9271S), mTOR (Cell signaling, 2972S), p-mTOR (Ser2448) (Cell signaling, 5536S), P70S6K (Cell signaling, 5707S), p-P70S6K (T389) (Cell signaling, 9205S), 4EBP-1 (Cell signaling, 8594S), p-4EBP-1 (T37/46) (Cell signaling, 9459S), LC3 (Sigma, L7543), Beclin-1 (Cell signaling, 3495), β-actin (Santa Cruz, sc-1616), Alkaline phosphatase-conjugated second antibody anti-rabbit IgG (Promega, S373B), and Fluorescein-Conjugated AffiniPure Goat anti-Rabbit IgG (H+L) (ZSGB-BIO, ZF-0311).

Western blot analyses

The cells were lysed in the RIPA buffer mixed with Phosphatase Inhibitors Cocktail (Roche, 04906845001) or Protease Inhibitors (Roche, 11206893001). After incubation for 30 min at 4 °C, the sample was centrifuged at 15,000×g for 8 min at 4 °C, and the supernatant was collected as whole cell lysate. Equivalent amounts of proteins were separated on 10% SDS–PAGE gels and transferred to a polyvinylidene difluoride membrane (Merck Millipore, IPFL00010). The blots were blocked in 5% (w/v) bovine serum albumin (BSA) and incubated with a 1:1000 dilution of primary antibodies overnight at 4 °C, followed by incubation with goat anti-rabbit alkaline phosphatase-conjugated IgG secondary antibody. The bolts were then detected by using the Western Blue Stabilized Substrate for alkaline phosphatase (Promega, S3841). The blots were analyzed by using Chemilmager 4000 (Alpha Innotech, USA).

Quantitative real-time PCR

Total RNA was isolated with Trizol reagent (Invitrongen, 10296010) according to the manufacturer’s instructions. Total RNA at 1 μg was reverse-transcribed into cDNA with High-Capacity cDNA Reverse Transcription Kit (Invitrongen, 4368813). For Real-time PCR, 1 μL of the cDNA was performed in triplicate with SYBR® Select Master Mix (Invitrongen, 4472980) for 10 min at 95 °C for initial denaturation, followed by 40 cycles of segments of 95 °C for 15 s and 60 °C for 60 s in the 7500 Fast real-time PCR system (Applied Biosystems). Primers were designed using Primer Premier 5 software. The primer sequences and the expected length of PCR products are shown in Table 1. The expression levels of the LC3, Beclin-1, and AMPK genes were normalized against the expression levels of the housekeeping gene β-actin.

Table 1 Sequence of primers
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RNA interference

Small interfering AMPK RNA sequences (5′-UGG CAA ACA GGU UGA AUU Gtt-3′) and negative control siRNA were purchased in Ambion. When the cells were grown to 40–50% confluency, Opti-MEM I Reduced Serum Medium was used to suspend the cells. The siRNA at 100 pmol was added to 250 μL Opti-MEM I Reduced Serum Medium. Two copies of the 5 μL Lipofectamine 2000 reagent (Invitrogen, 11668027) were added to 250 μL Opti-MEM I Reduced Serum Medium. The above-mentioned siRNA-containing solution and the Lipofectamine 2000 solution were mixed and added to cells. After incubation for 6 h, the conventional culture solution was changed with 10% RPMI-1640 medium. Then, cells were subjected to 20 μg/mL VES for another 24 h.

Immunocytochemistry

After treatment, human gastric cancer SGC-7901 cells were fixed in 4% paraformaldehyde (Thermo Fisher, NC9245948), perforated with 0.5% Triton X-100 (BIOSHARP, 0694), and then blocked with 1% bovine serum albumin (Roche, 735094) in PBS. Cells were stained with rabbit anti-LC3 (1:200) primary antibody, visualized using Fluorescein-Conjugated AffiniPure Goat anti-Rabbit IgG (H+L), and counterstained with DAPI dihydrochloride (Sigma, 30670). These fluorescent-stained cells were observed under a confocal microscope (Nikon, Japan). Final images were a non-contrast-adjusted merge of two channels. The number of LC3-positive dots per cell was quantified by Image-Pro Plus. At least ten cells in two independent experiments were analyzed randomly.

Statistical analysis

Multiple comparisons were analyzed using one-way analysis of variance (ANOVA), and individual differences were tested by the Fisher’s protected least-significant difference test after demonstrating the existence of significant intergroup difference by one-way ANOVA. Two-group comparisons were performed using Student’s t test. Results are shown as mean ± SD values, with a minimum of three separate experiments for each issue addressed. p values of less than 0.05 were considered significant.

Result

VES-induced autophagy in SGC-7901 cells

We investigated whether VES could elicit autophagy in SGC-7901 cells by Western blot analysis. As shown in Fig. 1a, significantly increased protein expressions of LC3 (LC3-II) and Beclin-1 were observed in SGC-7901 cells treated with VES in both dose- and time-dependent manners. VES-induced cleavage of LC3 and the increased expression of Beclin-1 were attenuated by the treatment of 5 mM 3-MA. Then, the mRNA levels of LC3 and Beclin-1 were also assessed with quantitative Real-time PCR. As shown in Fig. 1b, both the levels of mRNA of LC3 and Beclin-1 significantly increased after VES treatment at 20 μg/mL when compared with the control group.

Fig. 1
figure 1

Autophagy is induced in SGC-7901 cells after VES treatment. a Protein levels of LC3 and Beclin-1 were measured using immunoblot analysis. SGC-7901 cells were treated with various concentrations of VES for 24 h and 20 μg/mL VES treated for different times. In 3-MA plus VES treatment, cells were first incubated with 5 mM 3-MA for 2 h. RAPA (100 nM) as a positive control. b LC3 and Beclin-1 mRNA expressions in response to VES were examined by quantitative Real-time PCR analysis. c Representative images of LC3 fluorescent staining in SGC-7901 cells with or without VES treatment. Cells were treated with different doses of VES for 24 h. DMOS (<0.1%) as a negative control. (a) DMSO; (b) 5 μg/mL VES; (c) 10 μg/mL VES; (d) 20 μg/mL VES; (e) 3-MA+VES. d Immunoblot analysis of LC3 expression from lysates of SGC-7901 cells treated with various concentrations of VES for 24 h in the absence or presence of lysosomal inhibitors (20 μM CQ), *compared with control p < 0.05, #compared with the group of 20 μg/mL VES, p < 0.05

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Furthermore, the immunofluorescence technique was used to observe the LC3-positive fluorescence spots and distribution of LC3 in the SGC-7901 cells after the VES treatment. Negative control failed to show LC3 fluorescent spots, whereas the LC3 fluorescent spots aggregation gradually increased with increasing VES concentration; 3-MA can significantly weaken the VES-induced LC3-positive spots (Fig. 1c).

The role of VES is related to initiation of autophagy instead of the inhibition of lysosome degradation. SGC-7901 cells were co-treated with CQ and VES to further prove that autophagy could be triggered by VES. LC3-II accumulated with increasing VES dose. When cells were co-treated with CQ and VES, the expression of LC3-II was higher than when CQ was absent (Fig. 1d).

VES treatment increased the phosphorylation of AMPK and its substrate

The AMPK subunits were examined to define their roles on VES-treated SGC-7901 cells; AICAR, as an adenosine analog to activate AMPK, was used as a positive control in this set-up. As shown in Fig. 2, both phosphorylations of AMPKα at Thr172 and the AMPK substrate acetyl-CoA carboxylase (ACC) increased upon VES treatment in both dose- and time-dependent manners. AMPK was involved in VES-induced autophagy.

Fig. 2
figure 2

Increased expression of AMPK in response to VES. a Dose-dependent analysis of p-AMPK, AMPK, p-ACC, and ACC in SGC-7901 cells. Cells were exposed to VES at the indicated concentrations for 24 h. AICAR at 1 mM was used as a positive control. b Time-dependent analysis of p-AMPK, AMPK, p-ACC, and ACC in SGC-7901 cells. Cells were treated with 20 μg/mL VES for the indicated time periods, * and # p < 0.05 compared to control

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Silencing AMPK attenuates VES-induced autophagy

To further define the role of AMPK in VES-induced autophagy, AMPK was silenced by siRNA method. The transfection efficiency is shown in Fig. 3a. The reduction of LC3 and Beclin-1 expressions demonstrated that VES-mediated autophagy was inhibited (Fig. 3b). Notably, AMPK silencing by siRNA also reduced the mRNA expression of LC3 (Fig. 3c). In addition, we also used immunofluorescence to observe the LC3-positive fluorescence dots and distribution of LC3, and the result showed that the LC3-positive dots induced by VES was significantly weakened when AMPK was silenced (Fig. 3d). Collectively, these results suggest that VES signals autophagy in SGC-7901 cells via the activation of AMPK.

Fig. 3
figure 3

AMPK-mediated VES-induced autophagy. a Transfection efficiency. Cells were transfected with negative control or AMPK-siRNA for 48 h. Then, the mRNA expression of AMPK was detected by quantitative Real-time PCR and protein level of AMPK was measured by immunoblot analysis. b Cells were transfected with AMPK-siRNA for 48 h and were treated with 20 μg/mL VES for 24 h. Western blot was used to measure the protein expressions of LC3 and Beclin-1. c Cells were treated with the same method as B. Quantitative Real-time PCR was used to measure the mRNA expression of LC3. d Cells were treated with the same method as B. Immunofluorescence was used to observe the fluorescence dots of LC3. 1 Negative control. 2 AMPK-siRNA. 3 20 μg/mL VES. 4 AMPK-siRNA plus 20 μg/mL VES. *Compared with control p < 0.05, #compared with the group of VES alone, p < 0.05

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Akt-mTOR signaling pathway was inhibited by VES

We examined whether Akt and mTOR phosphorylations were involved in VES-induced autophagy in SGC-7901 cells. As shown in Fig. 4a, both Akt (S473) and mTOR (S2448) phosphorylations were inhibited by VES treatment in SGC-7901 cells. In addition, the dephosphorylations of Akt and mTOR mediated by VES relied on the increased doses of VES. The phosphorylation status of both P70S6K (T389) and 4EBP-1 (T37/46) were also examined with immunoblot analysis. As shown in Fig. 4b, the VES-treated dephosphorylations of both P70S6K (T389) and 4EBP-1 (T37/46) were in a dose-dependent manner.

Fig. 4
figure 4

VES suppressed the Akt/mTOR signaling pathway. Immunoblot analysis of both total and phosphorylation levels of Akt (S473), mTOR (S2448), P70S6K (T389), and 4EBP-1 (T37/46) from lysates of SGC-7901 cells treated with various concentrations of VES for 24 h, * and # p < 0.05 compared to respective treatment group in control medium

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mTOR signaling was inhibited by VES through AMPK activation

To define whether mTOR phosphorylation was regulated by VES-activated AMPK, the cells were treated with AMPK activator AICAR or inhibitor compound C before VES treatment. The p-AMPK was significantly activated when cells were treated with 1 mM AICAR or AICAR plus VES. However, the phosphorylations of mTOR, P70S6K, and 4EBP-1 decreased (Fig. 5a). AMPK was inhibited when the cells were administered with 10 μM Compound C. The p-AMPK expression was significantly inhibited. The phosphorylations of mTOR, P70S6K, and 4EBP-1 were clearly increased. The p-AMPK was weakened when co-treated with Compound C and VES, compared with VES treatment. Meanwhile, the p-mTOR, p-P70S6K, and p-4EBP-1 were enhanced (Fig. 5b). When the AMPK was silenced with transfecting siRNA before VES treatment, the results showed that both p-AMPK and total AMPK were significantly inhibited, but the phosphorylations of mTOR, p70S6K, and 4EBP-1 increased (Fig. 5c).

Fig. 5
figure 5

VES inhibited mTOR signaling through AMPK activation. a AMPK activation inhibited mTOR-related signaling. SGC-7901 cells were incubated in the presence or absence of 1 mM AICAR for 2 h, and then were treated with 20 μg/mL VES for 24 h. After cell lysis, both the total and phosphorylation levels of AMPK, mTOR, P70S6K, and 4EBP-1 were analyzed by protein gel blotting. b AMPK inhibition activated mTOR-related signaling. Cells were incubated in the presence or absence of 10 μM Compound C for 2 h and then were treated with 20 μg/mL VES for 24 h. Protein gel blotting was used to measure both the total and phosphorylation levels of AMPK, mTOR, P70S6K, and 4EBP-1. c AMPK silencing activated mTOR-related signaling. Cells were transfected with control siRNA or AMPK-siRNA for 48 h and then treated with VES for 24 h. Protein expressions of total and phosphorylation levels of AMPK, mTOR, P70S6K, and 4EBP-1 were analyzed by protein gel blotting

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AMPK was activated by VES through the inhibition of Akt

To test whether Akt signaling was involved in AMPK activation, the expression of p-AMPK, AMPK, p-Akt, and Akt were determined in SGC-7901 cells treated with 100 ng/mL Akt activator IGF-1 or 10 μM Akt inhibitor LY294002. The increased p-Akt activation and simultaneously decreased p-AMPK activation were observed when the cells were treated with IGF-1. Meanwhile, the VES-attenuated p-Akt activation was increased and VES-induced p-AMPK activation was decreased (Fig. 6a). On the contrary, when cells were treated with LY294002, lower p-Akt activation and higher p-AMPK activation compared with control were revealed. In addition, more obvious decrease of p-Akt activation and increase of p-AMPK activation were observed under combined treatment of VES and LY294002 (Fig. 6b).

Fig. 6
figure 6

VES-activated AMPK through Akt inhibition. a Akt activation inhibited AMPK signaling. SGC-7901 cells were incubated in the presence or absence of 100 ng/mL IGF-1 for 2 h and then were treated with 20 μg/mL VES for 24 h. After cell lysis, both the total and phosphorylation levels of Akt and AMPK were analyzed by protein gel blotting. b Akt inhibition activated AMPK signaling. Cells were incubated in the presence or absence of 10 μM LY294002 for 2 h, and then treated with 20 μg/mL VES for 24 h. Protein gel blotting was used to measure the expression of Akt, p-Akt, AMPK, and p-AMPK, * and # p < 0.05 compared to control

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Discussion

VES, a vitamin E analog, is a promising chemoprevention agent used in a variety of cancer models [29–32]. VES exhibits potent anti-proliferative effects on human tumor cells and selectively induces apoptosis of tumor cells in vitro and in vivo, but not of normal cells [31, 33]. A recent study also showed that VES induced autophagy in human gastric cancer cells [34]. Consistent with previous reports, our study showed that VES induced autophagy in gastric cancer cells, and AMPK played an important role in VES-induced autophagy. (Fig. 7).

Fig. 7
figure 7

Schematic model illustrating the potential pathway associated with VES-induced autophagy in SGC-7901 cells. VES-induced autophagic activation is mediated by the Akt/AMPK/mTOR pathway. VES causes the phosphorylation of AMPK at Thr172 and activates AMPK by decreasing the phosphorylation of Akt. This leads to inhibition of mTOR signaling and its downstream tartrates P70S6 K and 4EBP-1, thereby negatively regulating autophagy. Silencing of AMPK by using siRNA could markedly attenuate VES-induced autophagy

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Autophagy is a highly regulated process of sequestrating cytoplasmic proteins into lytic compartments and is characterized by the formation of the autophagosome, which then fuses with lysosomes where the contents are degraded [35, 36]. The formation of autophagosome was initiated by PI3 kinase type III-Atg6/Beclin-1 cascade and was expanded by Atg8/LC3-phosphatidylethanolamine conjugate system. Accumulating anticancer agents have been documented to trigger the cellular autophagic process, but the signaling pathway involved in autophagy is still controversial. In this study, we detect the regulatory effect of VES on autophagy in human gastric cancer SGC-7901 cells. The results indicated that VES is a strong inducer of autophagy, as evidenced by the significant increase of LC3 and Beclin-1 expressions. LC3 is a homolog of yeast Atg8 and is generally used as a marker protein for tracing the autophagic process. LC3 is post-translationally modified by an ubiquitination-like reaction and cleaved off to assume a soluble form, LC3-I, thereby exposing its carboxyl terminal glycine. Upon autophagy induction, LC3-I is modified with phosphatidylethanolamine at the glycine residue and becomes LC3-II, which is bound to both the outer and inner membranes of the autophagosome. Therefore, the formation of LC3-II is a good marker to monitor the occurrence of autophagosome formation [37]. Beclin-1, as a key molecule, is involved in the formation of autophagosome by PI3 kinase type III-Atg6/Beclin-1 cascade. Our study demonstrated that VES treatment increased both LC3 and Beclin-1 expressions in dose- and time-dependent manners in SGC-7901 cells. In addition, the mRNA of LC3 and Beclin-1 also increased after VES treatment. The immunofluorescence study also supports this conclusion.

The involved mechanisms in autophagy regulation are multifactorial. Among these, AMPK integrates input information from multiple upstream signal transduction pathways and has been well identified as a vital positive regulator in autophagy. AMPK also serves as a key regulator of cell survival or death in response to various pathological stresses, including oxidative stress, endoplasmic reticulum stress, hypoxia, and osmotic stress [38, 39]. AMPK activation can regulate the tumor suppressor gene P53, mTOR, ULK1, and other downstream signal molecular activities involved in autophagy [28, 40, 41]. As an elementary mechanism, autophagy promotes cell survival under stress situations. Emerging evidence has presented the critical role of AMPK axis on autophagy [42]. Therefore, we hypothesize that AMPK would be involved in autophagy activation induced by VES in human gastric cancer cells. In the present study, we found that VES could activate AMPK in cultured SGC-7901 cells in a dose- and time-dependent manners. Increased AMPK activity upon VES treatment was further proven by analysis of the phosphorylation of the AMPK substrate acetyl-CoA carboxylase. Additionally, AMPK inhibition by siRNA significantly reduced the protein and mRNA expressions of LC3 and Beclin-1. Immunofluorescence study also showed that silencing AMPK by siRNA significantly weakened the VES-induced fluorescence dots of LC3, which suggested that VES-mediated autophagy was inhibited. All of the above-mentioned results demonstrated that the AMPK signaling pathway is involved in VES-induced autophagy.

The Akt-mTOR signaling pathway is the major negative signaling pathway against autophagy [43]. The serine/threonine protein kinase Akt mediates mTOR activity [44] and positively regulates mTOR, which is a major component of autophagy regulation. mTOR phosphorylation increases the levels of downstream targets, such as P70S6K and 4EBP-1, to regulate many different cellular processes [45, 46]. The P70S6K phosphorylation level is important in the initiation of the translation of proteins associated with cell growth and proliferation [47]. Our data demonstrated that VES dephosphorylated Akt and mTOR as well as P70S6K and 4EBP-1 (the two best-characterized targets of mTOR1 complex) in gastric cancer cells, thereby emphasizing the functional importance of the involved Akt-mTOR signaling in VES-mediated autophagy in gastric cancer cells.

The signaling by which VES induces autophagy has been suggested to be a direct action of mTOR through AMPK activation [34]. As a central signal integrator, mTOR receives signals arising from nutrients, growth factors, and many cellular kinases including AMPK [40, 48]. Phosphorylation of AMPK activates downstream signaling that leads to mTOR inhibition and triggers autophagy [49], which is consistent with the AMPK function of initiating catabolic processes [25]. Our data showed that the activity of mTOR and its downstream targets, including P70S6 K and 4EBP-1, was inhibited after AMPK activation because of AICAR treatment. On the contrary, pre-treatment with AMPK inhibitor Compound C could attenuate the VES-induced inhibition of mTOR and its downstream targets. Then, AMPK silencing also weakened VES-induced inhibition of mTOR. Therefore, VES-induced AMPK activation prohibits mTOR signaling, and then triggers the initiation of autophagy.

Akt has been shown to be an effective cellular antagonist of AMPK [50–52]. It has been reported that high glucose suppresses AMPK T172 phosphorylation through Akt-dependent phosphorylation of AMPK S485, which is a function to inhibit AMPK activation by multiple stimulations [53]. Our study also highlights the importance of the crosstalk between AMPK and Akt. When cells were treated with Akt activator IGF-1, the VES-induced AMPK activation was inhibited. On the contrary, for cells treated with Akt inhibitor LY294002, the VES-induced AMPK activation strengthened.

This study had some limitations. The relationship between VES-induced autophagy and the effect of VES to suppress tumor growth have not been explored. Therefore, the significance of this autophagy is unknown in terms of the VES anticancer effect. Moreover, autophagy flux should be detected when AMPK was inhibited or activated by Compound C or AICAR. However, autophagy may be influenced by Compound C or AICAR through other signaling pathways besides AMPK axis.

Collectively, this study highlights the significance of AMPK involved in VES-induced autophagy in SGC-7901 cells. VES initially triggers an AMPK-mediated autophagic process via Akt/AMPK/mTOR signaling. Information provided in this paper would be valuable for further studies. This study also contributes to explore the underlying mechanisms of VES-induced autophagy in human gastric cancer cells.