Background/Aims: Autophagy is an evolutionarily conserved catabolic mechanism to maintain energy homeostasis and to remove damaged cellular components, which plays an important role in the survival of various cells. Inhibiting autophagy is often applied as a new strategy to halt the growth of cancer cells. Methods: The effect of FOXO1 gene on cellular function and apoptosis and its underlying mechanisms were investigated in cultured QBC939 cells by the methylthiazoletetrazolium (MTT) assay, western blot, DCFDA mitochondrial membrane potential, and ATP content measurement. FOXO1 siRNA was applied to down-regulate FOXO1 expression in QBC939 cells. Results: Here we reported that FOXO1, acetylation of FOXO1 (Ac-FOXO1) and the following interaction between Ac-FOXO1 and Atg7 regulated the basal and serum starvation (SS)-induced autophagy as evidenced by light chain 3 (LC3) accumulation and p62 degration. Either treatment with FOXO1 siRNA or resveratrol, a sirt1 agonist, inhibited autophagic flux, resulting in oxidative stress, mitochondrial dysfunction (MtD) and apoptosis in QBC939 cells, which were attenuated by enhancing autophagy with rapamycin. On the contrary, inhibiting autophagic flux with 3-MA worsened all these effects in QBC939 cells. Conclusions: Taken together, our study for the first time identified FOXO1 as a potential therapeutic target to cure against human cholangiocarcinoma via regulation of autophagy, oxidative stress and MtD.

The incidence of cholangiocarcinoma is growing over the past decades [1, 2], and relative resistance of the tumors to chemotherapy leading to poor outcome is a thorny problem in patients with cholangiocarcinoma [3, 4]. Molecular insights into pathophysiology of cholangiocarcinoma cells and novel therapeutic strategies are urgent for the improved clinical management of patients with cholangiocarcinoma.

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved catabolic mechanism to maintain energy homeostasis and to remove damaged cellular components [5, 6]. FOXO1 is a member of the forkhead box O (FOXO) family, which a series of recent studies have demonstrated plays an important role in several intracellular functions, including autophagy [7], cell cycle [8], apoptosis [9], and tumor suppression [10]. Various experiments pointed out that post-translational modifications regulated FOXO1 transcriptional activities and modulated FOXO1 protein stability, DNA binding activity, protein-protein interactions, and subcellular localization, all of which are involved in regulating autophagic flux [7, 11].

Oxidative stress, characterized by increased ROS production, plays a key role in the regulation of cells survival [12]. Many drugs exert their negative effects on cancer cells are through increased the production of ROS [13, 14]. Mitochondrial dysfunction (MtD) represents a malfunction in biochemical processes, characterized by MMP collapse and decreased ATP production, also have an essential role in mediation of apoptosis [15, 16]. Impaired autophagic flux could result in oxidative stress and MtD, both of which represents a malfunction in biochemical processes [17, 18].

Therefore, the main aim of this study was to elucidate whether FOXO1 existed in autophagy regulation and whether impairing autophagic flux could lead to oxidative stress and MtD in QBC939 cells. Finally, we explored the possibility that FOXO1 served as the therapeutic target to cure human cholangiocarcinoma.

Materials

The established QBC939 was obtained from Cell Bank of Wuhan University (Wuhan, China). Resveratrol, Chloroquine (CQ), and rapamycin (RP) were from Sigma Chemical Co. (St. Louis, MO, USA). FOXO1, p-FOXO1, Ac-FOXO1, ub-FOXO1, LC3, p62, and GAPDH antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). All other chemicals were of analytical grade.

Cell Culture

The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum at 37°C in an atmosphere containing 5% CO2. When cells reached 80%-90% confluence, the cells were added into the DMEM (10% FCS in media) with different stimulation as indicated.

MTT asay

The methylthiazoletetrazolium (MTT) reduction assay was used as a qualitative index of cell viability. The effect of resveratrol on cell viability was assessed as percent cell viability compared to resveratrol non-treated control cells, which were arbitrarily assigned 100% viability.

Assessment of cell apoptosis

The cytosolic DNA-histone complexes generated during apoptotic DNA fragmentation in the treated QBC939 cells were evaluated using a cell death detection enzyme-linked immunosorbent assay kit (Cell Death Detection ELISA PLUS; Roche Applied Science, Indianapolis, IN, USA), according to the manufacturer’s protocol.

ROS

According to our previous study, we used 2’7’-dichlorofluorescin diacetate (DCFDA) to detect renal ROS production [19]. We incubated each 3-µm tissue cryosection with 10 µM DCFDA in the dark at 37°C for 30 min, before imaging using fluorescence microscopy.

Mitochondrial membrane potential

The MMP of QBC939 was monitored using JC-1, a MMP-sensitive fluorescent dye, as described previously [19]. Briefly, the dissociated QBC939 were washed twice with Hank’s balanced salt solution (Sigma, St Louis, MO, USA), and incubated in the dark with JC-1 (7.5 mmol/l; 30 min at 37°C, Sigma, St Louis, MO, USA), then cells were washed with JC-1 washing buffer, and fluorescence was detected by fluorescence-assisted cell sorting for QBC939 cells. The relative MMP was calculated using the ratio of J-aggregate/monomer (590/520 nm).

ATP content measurement

ATP levels were determined in the QBC939 cells with a luciferase-based bioluminescence assay kit (Sigma-Aldrich, St Louis, MO, USA) in a FLUO star Optima reader according to the manufacturer’s instructions. Total ATP levels were the results of the luminescences normalized by protein concentrations.

Western Blotting

Total cellular proteins from cells of respective group were extracted by lysing cells with buffer containing 150mM NaCl, 0.1% Triton X-100, 0.5% Deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50mM Tris–HCl (pH 7.0), and 1mM ethylenediaminetetraacetic acid (EDTA). Detection of protein expression by Western blot was carried out according to established protocols. The primary antibodies used were as follows: LC3 (1: 1000), p62 (1: 2000), and GAPDH (1: 1000). The relative intensity of each band, respectively, was normalized to GAPDH.

Statistical analysis

Quantitative data are presented as mean±SD. Statistical significance was determined using one-way analysis of variance (ANOVA); p < 0.05 was considered statistically significant.

Endogenous FOXO1 is required for basal and SS-induced autophagy in human cholangiocarcinoma QBC939 cells

To determine whether FOXO1 is involved in the autophagic process, we first compared the levels of FOXO1 expression and those of two biological markers of autophagy, namely LC3-II accumulation and p62 degradation under normal conditions and serum starvation (SS) in human cholangiocarcinoma QBC939 cells. Compared to the normal group, FOXO1 levels in SS group cells was elevated and seemed to be related to the autophagic process, as evidenced by increased p62 degradation and LC3-II accumulation (Fig. 1A through C). The autophagy-associated p62 degradation was blocked and LC3-II was further accumulated in the presence of CQ (lysosomal protease inhibitors) in response to SS stimuli (Fig. 1A through C).

Fig. 1.

Knocking down FOXO1 impaired basal and SS-induced autophagy in human cholangiocarcinoma QBC939 cells. (A) Immunofluorescence staining for LC3 in QBC939 cells after various treatments, as indicated. (B) Western blot analysis revealed the expression of LC3II/LC3I, p62, FOXO1, and GAPDH proteins in QBC939 cells after various treatments, as indicated. (C) Graphical presentation indicates the relative abundance levels of LC3II/LC3I, p62, and FOXO1 after normalization with GAPDH. #P<0.05 vs. normal control, *P<0.05 vs. SS or SS+siRNA Con.

Fig. 1.

Knocking down FOXO1 impaired basal and SS-induced autophagy in human cholangiocarcinoma QBC939 cells. (A) Immunofluorescence staining for LC3 in QBC939 cells after various treatments, as indicated. (B) Western blot analysis revealed the expression of LC3II/LC3I, p62, FOXO1, and GAPDH proteins in QBC939 cells after various treatments, as indicated. (C) Graphical presentation indicates the relative abundance levels of LC3II/LC3I, p62, and FOXO1 after normalization with GAPDH. #P<0.05 vs. normal control, *P<0.05 vs. SS or SS+siRNA Con.

Close modal

To further clarify the role of endogenous FOXO1 in inducing autophagy, a FOXO1 RNA interference (RNAi) or a non-specific control RNAi was transfected into human cholangiocarcinoma QBC939 cells. SS treatment induced p62 degradation and LC3-II accumulation in non-specific RNAi-treated human cholangiocarcinoma QBC939 cells but not in the FOXO1 knockdown QBC939 cells (Fig. 1A through C).

Acetylation of FOXO1 and its interaction with Atg7 is also associated with serum starvation induced autophagy in QBC939 cells

Because a post-translational modification of cytosolic FOXO1 were reported to be involved in triggering autophagy [7], cytosolic proteins of QBC939 cells were immunoprecipitated with anti-ubiquitylation, anti-phosphorylation or anti-acetylation antibodies. No detectable changes in ubiquitylation or phosphorylation of cytosolic FOXO1 were observed in QBC939 cells before or after the treatments (Fig. 2A and 2B). By contrast, a significant increase in acetylated FOXO1 was detected in serum-starved QBC939 cells (Fig. 2A and 2B). Because a previous report that have proven that FOXO1 acetylation and its interaction with Atg7 is involved in regulating the autophagic process. Subsequently, we examined whether acetylated FOXO1 interacts with Atg7 is associated with enhanced autophay under SS condition. Expectedly, SS enhanced the interaction between endogenous Atg7 and Ac-FOXO1 (Fig. 2C through 2F).

Fig. 2.

Acetylation of FOXO1 is also responsible for SS-induced autophagy in QBC939 cells. (A) Western blot analysis revealed the expression of Ac-FOXO1, p-FOXO1, ub-FOXO1, and GAPDH proteins in QBC939 cells under control or SS condition. (B) Graphical presentation indicates the relative abundance levels of Ac-FOXO1, p-FOXO1, and ub-FOXO1 after normalization with GAPDH. (C, E) Co-immunoprecipitation of Atg and Ac-FOXO1 in QBC939 cells. Cells were treated as indicated, and the cell lysate was then extracted for co-immunoprecipitation with anti-Ac-FOXO1or Atg7 followed by probing with anti-acetylated Atg7 or Ac-FOXO1 (n=3). (D, F) Graphical presentation shows the relative quality of Atg7 and Ac-FOXO1 after normalization with Ac-FOXO1 or Atg7 respectively. #P<0.05 vs. normal control.

Fig. 2.

Acetylation of FOXO1 is also responsible for SS-induced autophagy in QBC939 cells. (A) Western blot analysis revealed the expression of Ac-FOXO1, p-FOXO1, ub-FOXO1, and GAPDH proteins in QBC939 cells under control or SS condition. (B) Graphical presentation indicates the relative abundance levels of Ac-FOXO1, p-FOXO1, and ub-FOXO1 after normalization with GAPDH. (C, E) Co-immunoprecipitation of Atg and Ac-FOXO1 in QBC939 cells. Cells were treated as indicated, and the cell lysate was then extracted for co-immunoprecipitation with anti-Ac-FOXO1or Atg7 followed by probing with anti-acetylated Atg7 or Ac-FOXO1 (n=3). (D, F) Graphical presentation shows the relative quality of Atg7 and Ac-FOXO1 after normalization with Ac-FOXO1 or Atg7 respectively. #P<0.05 vs. normal control.

Close modal

Inhibition of acetylation of FOXO1 by Resveratrol imparied autophagic flux in QBC939 cells

Resveratrol is a sirt1 agonist and sirt1 has been confirmed to be responsible for the deacetylation of FOXO1[20]. Hence, we explored the possibility that resveratrol could interrupt the autophay process in QBC939 cells. Expectedly, after incubation with 20 µM of resveratrol for 48 h, FOXO1 acetylation (Fig. 3A and 3B) and its interaction with Atg7 were attenuated (Fig. 3D and 3E), accompanied by impaired autophagic flux as evidenced by increased LC3 and p62 accumulation (Fig. 3A through 3C). Similar results could be found under SS condition (Data not shown).

Fig. 3.

Resveratrol imparied autophagic flux in QBC939 cells . (A) Western blot analysis revealed the expression of LC3II/LC3I, p62, Ac-FOXO1, and GAPDH proteins in QBC939 cells in the presence or absence of Res for 48h. (B) Graphical presentation indicates the relative abundance levels of LC3II/LC3I, p62, and Ac-FOXO1 after normalization with GAPDH. (C) Immunofluorescence staining for LC3 in QBC939 cells after various treatments, as indicated. (D) Co-immunoprecipitation of Atg7 and Ac-FOXO1 in QBC939 cells. Cells were treated as indicated, and the cell lysate was then extracted for co-immunoprecipitation with anti-Atg7 followed by probing with anti-Ac-FOXO1 (n=3). (E) Graphical presentation shows the relative quality of Ac-FOXO1 after normalization with Atg7 respectively. #P<0.05 vs. normal control.

Fig. 3.

Resveratrol imparied autophagic flux in QBC939 cells . (A) Western blot analysis revealed the expression of LC3II/LC3I, p62, Ac-FOXO1, and GAPDH proteins in QBC939 cells in the presence or absence of Res for 48h. (B) Graphical presentation indicates the relative abundance levels of LC3II/LC3I, p62, and Ac-FOXO1 after normalization with GAPDH. (C) Immunofluorescence staining for LC3 in QBC939 cells after various treatments, as indicated. (D) Co-immunoprecipitation of Atg7 and Ac-FOXO1 in QBC939 cells. Cells were treated as indicated, and the cell lysate was then extracted for co-immunoprecipitation with anti-Atg7 followed by probing with anti-Ac-FOXO1 (n=3). (E) Graphical presentation shows the relative quality of Ac-FOXO1 after normalization with Atg7 respectively. #P<0.05 vs. normal control.

Close modal

Imparied autophagy either by FOXO1 siRNA or resveratrol treatment contributes to decreased cell ability and increased cell apoptosis in QBC939 cells

To further characterize the role of autophagic activity in resveratrol-induced QBC939 cells injury, we also blocked autophagic activity and then assessed apoptosis. Our results showed that addition of 20uM resverratrol for 48 h inhibited human cholangiocarcinoma cell viability and increase in cell apoptosis compared with the normal group. Treating QBC939 cells with the autophagy inhibitors 3-Methyladenine (3-MA) resulted in an enhancement of QBC939 damage induced by resveratrol as indicated by the increased apoptosis and decreased cell ability. On the contrary, treatment with Rap, the most frequent strategy used to increase autophagic activity, reduced QBC939 cells damage induced by resverratrol. FOXO1 siRNA treatment also decreased cell viability and increased apoptosis in QBC939 cells compared with the group (Fig. 4A through 4D).

Fig. 4.

Inhibiting autophagic flux by targeting FOXO1 inhibits cell viability and induces cell death in QBC939 cells. (A) Western blot analysis revealed the expression of p62 and GAPDH proteins in QBC939 cells after various treatments, as indicated. (B) Graphical presentation indicates the relative abundance levels of p62 after normalization with GAPDH. (C) Cell viability and (D) Apoptotic of QBC939 cells after various treatments, as indicated. Values are presented as the mean ± standard deviation from six independent experiments. #P<0.05, vs. Control, *P<0.05 vs. Res or Res+siRNA Con.

Fig. 4.

Inhibiting autophagic flux by targeting FOXO1 inhibits cell viability and induces cell death in QBC939 cells. (A) Western blot analysis revealed the expression of p62 and GAPDH proteins in QBC939 cells after various treatments, as indicated. (B) Graphical presentation indicates the relative abundance levels of p62 after normalization with GAPDH. (C) Cell viability and (D) Apoptotic of QBC939 cells after various treatments, as indicated. Values are presented as the mean ± standard deviation from six independent experiments. #P<0.05, vs. Control, *P<0.05 vs. Res or Res+siRNA Con.

Close modal

Impared autophagy caused either by FOXO1 siRNA or resveratrol treatmentleads to oxidative stress and MtD in QBC939 cells

Since autophagy inhibition could result in oxidative stress [21] and mitochondrial dysfunction [22], both of which have been approved to participate in cell death. We firstly evaluate the ROS production in QBC939 cells after the RES stimuli. Expectedly, ROS level was significantly increased compared with the normal QBC939 cells (Fig. 5A). To evaluate MtD, we used two independent parameters: mitochondrial membrane potential (MMP) and adenosine-50-triphosphate (ATP) levels. MMP collapse has been shown to play an important role in mediation of apoptosis, in which it allows the release of cytochrome c, activation of caspase-9, and subsequently lead to the apoptosis of cells [23]. Second, mitochondria are the energy powerhouses of cells and make the most use of the cell’s ATP, tumor cells consumes a large amount of energy supplied as ATP, so any disruption in this supply is likely to cause cell death [15]. Expectedly, resveratrol reduced MMP as indicated by the reduction of JC-1 fluorescence at 590/520 nm (Fig. 5B). Similarly, resveratrol reduced ATP production (Fig. 5C). Interestedly, RP treatment attenuated the ROS levels and improved the MtD in QBC939 cells, whereas CQ further increased the level of ROS and impaired the MtD in QBC939 cells. FOXO1 siRNA treatment also decreased cell viability and increased apoptosis in QBC939 cells compared with the group (Fig. 5A trough 5C).

Fig. 5.

Inhibiting autophagic flux by targeting FOXO1 worsens oxidative stress and MtD in QBC939 cells . (A) Quantification of 2’,7’-dichlorofluorescein (DCF) fluorescence in QBC939 cells after various treatments, as indicated (B) Mito-chondrial membrane potential and (C) ATP content were determined. Values are presented the mean ± standard deviation (n=6). # P<0.05, vs. Control, *P<0.05 vs. Res or Res+siRNA Con.

Fig. 5.

Inhibiting autophagic flux by targeting FOXO1 worsens oxidative stress and MtD in QBC939 cells . (A) Quantification of 2’,7’-dichlorofluorescein (DCF) fluorescence in QBC939 cells after various treatments, as indicated (B) Mito-chondrial membrane potential and (C) ATP content were determined. Values are presented the mean ± standard deviation (n=6). # P<0.05, vs. Control, *P<0.05 vs. Res or Res+siRNA Con.

Close modal

Cholangiocarcinoma remains one of the most difficult tumors to treat in clinical practice and new therapeutic modalities are currently needed. The induction of cholangiocarcinoma apoptosis is considered to be one promising therapeutic strategy.

It has been recognized that control of autophagy progression in cancer cells is an effective strategy to halt tumor growth [24, 25], as the molecular analyses of human cancers have revealed that autophagy regulators are frequently deregulated in most of the common malignancies [26, 27]. For this reason, autophagy inhibition has been regarded as a promising anticancer strategy to inhibit the multiple cellular processes. Impaired autophagy could influencing the energy metabolism, protein balance, oxidatice stress, and MtD [17, 28, 29]. Hence, knowing the molecular mechanism by which the autophagy is regulated could help us to find a potential therapeutic target to solve this urgent problem.

Regulation of the expression and transcriptional activity of FOXO1 is involved in promoting cellular autophagy [7, 30]. In our study, SS condition could upregulate the expression of FOXO1,which regulate both basal and SS-induced autophagy in QBC939 cells. However agents could directly influencing the expression of FOXO1 is far from clear. Posttranslational modifications including phosphorylation and acetylation of FOXO1 were reported to participate in the regulation of autophagy [31, 32], According to our findings, acetylation of the FOXO1 level and interaction between Ac-FOXO1 and Atg7, which has also been confirmed to promote autophagy, were also found in QBC939 cells and enhanced by SS stimulation. Hence, we speculate that blocking the acetylation of the FOXO1 may be a new strategy to inhibit the autophagy process in QBC939 cells.

Resveratrol, a SIRT1 agonist, has recently been acknowledged to activate a mitochondrial apoptotic pathway in human cervical cancer cell [33, 34]. SIRT1 is a NAd+ dependent deacetylation enzyme. Frampton G et al. have demonstrated that treatment with progranulin, a secreted growth factor, decreased Sirt1 expression and increased the acetylation of FOXO1, resulting in the cytoplasmic accumulation of FOXO1 and enhanced proliferation in cholangiocytes [35]. However the effect of resveratrol on cholangiocarcinoma is far from clear. In our present investigation, we showed that resveratrol significantly inhibited the acetylation of FOXO1 and interaction between Ac-FOXO1 and Atg7, leading to a blocking autophagy in QBC939 cells, subsequently inducing apoptosis and lower viability of QBC939 cells (Fig. 1 through 3). These results suggested that resveratrol may be examined as an effective chemotherapeutic agent against cholangiocarcinoma.

ROS are known to function as second messengers for signal transduction in many cells and excessive production of ROS can lead to oxidative stress, loss of cell function and apoptosis or necrosis [36, 37]. MtD represents a malfunction in biochemical processes, characterized by MMP collapse, which has been shown to have an essential role in mediation of apoptosis [38]. Furthermore, mitochondria are the energy powerhouses of cells and make the most use of the cell’s ATP. Cancer cells consumed a large amount of energy supplied as ATP, so any disruption in this supply is likely to cause apoptosis of cell [39]. Our in vitro data demonstrate that treatment of QBC939 cells with resveratrol inhibited autophagy progression and increased the oxidative stress and MtD (Fig. 2). Additionally, pharmacological inhibition of autophagy worsens resveratrol -induced oxidative stress, MtD, and apoptosis whereas promotion autophagy with RP attenuates these effects, indicating that oxidative stress and MtD results from inhibition of autophagy is one of the mechanisms by which resveratrol induces the apoptosis of QBC939 cells (Fig. 4 and 5).

Our study provides first evidence that FOXO1 exists in autophagy regulation and impairing autophagic flux could lead to oxidative stress and MtD in human cholangiocarcinoma QBC939 cells, which indicates that FOXO1 could served as a potential therapeutic target to cure against human cholangiocarcinoma.

This study was supported by the National Natural Science Foundation of China (81172266), the Life Health Technology Foundation of Jiangsu province (BL2012031), and the “333 engineering” Foundation of Jiangsu province (BRA2015472).

The author(s) declare that they have no competing interests.

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W. He and A. Zhang contributed equally to this work.

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