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Wogonin is considered to be an inhibitor of myeloid cell leukemia 1 and B-cell lymphoma 2, and a potential antitumor drug due to its ability to induce apoptosis in certain cancer cells; however, few previous studies have reported on wogonin-induced autophagy. The aim of the present study was to investigate the influence of wogonin on autophagy in human pancreatic cancer cells (HPCCs), elucidate its mechanism, and identify strategies to increase its effectiveness as an anti-cancer treatment. HPCCs were treated with wogonin and autophagy was detected in the cells. The mechanism of wogonin-related autophagy was investigated, and the antioxidant N-acetyl-L-cysteine (NAC) was used to assess the role of reactive oxygen species (ROS) in wogonin-related autophagy. The results demonstrated that wogonin may induce autophagy by activating the Beclin-1/phosphatidylinositol-3-kinase and ROS pathways in HPCCs, and may enhance ROS generation, followed by the activation of the AKT/ULK1/4E-BP1/CYLD pathway and inhibition of the mammalian target of rapamycin signaling pathway. The incubation of HPCCs with wogonin and the antioxidant NAC, revealed that the effects of wogonin-enhanced ROS generation on autophagy-related molecules were inhibited, contributing to the inhibition of autophagy and increasing the cell death ratio through apoptosis activation in HPCCs. These studies suggest that autophagy activation, via the ROS pathway, by the antitumor drug wogonin in HPCCs may partially reduce the antitumor effects of the drug, and that the antioxidant NAC may enhance the antitumor effectiveness of wogonin via the inhibition of ROS-enhanced autophagy and the subsequent promotion of apoptosis. Therefore, the present research suggests that wogonin combined with NAC may be a novel combination therapy for clinical pancreatic cancer therapy trials.
Pancreatic cancer has a mortality rate of >95%, which has not improved for 3 decades (
Wogonin (5,7-dihydroxy-8-methoxyflavone) is a potent inhibitor of myeloid cell leukemia 1 (Mcl-1) and B-cell lymphoma 2 (Bcl-2), which are anti-apoptotic proteins that are expressed in various tumors, including pancreatic cancer (
Autophagy has become a novel target for the treatment of certain types of cancer, and may prevent cell death (
Panc-1 and Colo-357 HPCCs were obtained from the American Type Culture Collection (Manassas, VA, USA). All cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C in an atmosphere containing 5% CO2. HPCCs were treated with 40 µM wogonin, 40 µM wogonin combined with 40 µM chloroquine (CQ), or 50 µM rapamycin for 24 h; in addition, HPCCs were pretreated with sterile water or 10 mM NAC for 2 h and then treated with 0.1% DMSO or 40 µM wogonin for 24 h. All cell culture reagents were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA).
The cells (5×106) were lysed for 30 min in radioimmunoprecipitation assay lysis buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS; Beyotime Institute of Biotechnology, Haimen, China] on ice then centrifuged at 12,000 ×
The degradation of autophagosomes is mediated by lysosomes; autolysis lowers the pH value and allows AO dye to penetrate into the acidic organelles and exhibit a red fluorescence; therefore, the intensity of red fluorescence following AO staining can be used to indicate the levels of autolysosomes (
When autophagy is induced, cytosolic LC3 is cleaved by hydrolysis to a shorter peptide (LC3 II) that is located on the membrane of the autophagosome; therefore, the expression levels of LC3 II may be used to estimate the level of autophagy. In order to estimate the location and processing of LC3-II, assessment of LC3-GFP puncta is an effective method (
Panc-1 cells (5×106) were plated onto 15-cm dishes and attached overnight. The cells were treated with DMSO or 40 µM wogonin for 12 h at 37°C then lysed for 30 min in hypotonic lysis buffer (Beyotime Institute of Biotechnology) on ice and centrifuged (1,000 ×
Cell Counting kit-8 (CCK8) was used to assess cell viability. Cells (1×104) were seeded into a 96-well plate and incubated overnight in the previously described conditions. The cells were pretreated with sterile water or 10 mM NAC for 2 h and then with 0.1% DMSO or 40 µM wogonin for 24 h. Following this, the medium was removed and the cells were washed three times with PBS. DMEM (90 µl) and CCK8 (10 µl) were subsequently added to each well and incubated for 1.5 h at 37°C; a microplate reader was used to measure the optical density (OD) at 450 nm.
The levels of intracellular ROS generation were detected through measuring the conversion of cell-permeable 2,7-dichlorofluorescein diacetate (DCFH-DA; Beyotime Institute of Biotechnology) to fluorescent dichlorofluorescein (DCF). Cells (1×104) were seeded into 96-well plates and incubated overnight in the standard conditions. The cells were pretreated with sterile water or 10 mM NAC for 2 h and then treated with 0.1% DMSO or 40 µM wogonin for 24 h. The medium was removed and the cells were washed three times with PBS prior to incubation with DCFH-DA at 37°C for 20 min. A fluorescence microplate reader, with 488 nm excitation wavelength and 525 nm emission wavelength, was used to determine the levels of ROS in the cells.
The intracellular levels of GSH were measured by GSH Assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. The OD value of GSH was detected with a fluorescence microplate reader (Tecan, Männedorf, Switzerland) at 420 nm. In addition, the intracellular levels of O2− were detected by Superoxide Assay kit (Beyotime Institute of Biotechnology), and the fluorescence value of O2− was measured at a wavelength of 550 nm.
Following pretreatment with sterile water or 10 mM NAC for 2 h and treatment with 0.1% DMSO or 40 µM wogonin for 24 h, the cells were suspended in 0.25% Trypsin-EDTA (Thermo Fisher Scientific, Inc.), and 0.4% (w/v) trypan blue solution was added to each 2-µl suspension; the ratio of the cell suspension to the trypan blue solution was 9:1. The cells were counted under a light microscope. As the dead cells fail to exclude the dye, the cell death rate was calculated using the following equation: Total cell death rate = (no. dyed cells / total no. cells) × 100%.
TEM is the gold standard for the assessment of autophagosomes (
Control siRNA and siRNA specifically targeted to Beclin-1 (GAG AUC UUA GAG CAA AUG ATT) were purchased from RiboBio Co., Ltd. (Guangzhou, China). Cells were plated in 6-well plates and grown to ~80% confluence before transient transfections with siRNAs (100 pmol per well) were performed using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific), according to the manufacturer's instructions. After a 36-h transfection, 0.1% DMSO or 40 µM wogonin were added for an additional 24 h prior to collection of the cells for western blotting.
The cells were pretreated with sterile water or 10 mM NAC for 2 h and then treated with 0.1% DMSO or 40 µM wogonin for 24 h, prior to collection and washing three times in PBS. The cells were then resuspended in 100 µl 1X binding buffer (10 mM HEPES/NaOH pH 7.4, 140 mM NaCl and 2.5 mM CaCl2), and 5 µl of Annexin V-fluorescein isothiocyanate (FITC) (Beyotime Institute of Biotechnology) and 5 µl of propidium iodide (PI; Beyotime Institute of Biotechnology) were added into the suspension, which was gently vortexed and incubated at room temperature for 15 min in the dark. The samples were assessed using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), and the data were analyzed by CellQuest Pro software (version 3.3; BD Biosciences) to determine the ratios of apoptotic cells.
Data are presented as the mean ± standard deviation from triplicated experiments and SPSS 19.0 software (IBM SPSS, Armonk, NY, USA) was used to analyze the data. Two-way analysis of variance was used to analyze the differences between the groups. P<0.05 was considered to indicate a statistically significant result.
During autophagy, LC3-I is converted to LC3-II by lipidation through a ubiquitin-like system which involves Atg3 and Atg7; therefore, LC3 is associated with autophagic vesicles, and the presence of LC3 in autophagosomes and the presence LC3-II are the indicators of autophagy (
Wogonin-induced autophagy was revealed to be time- and dose-dependent (
AO staining demonstrated that wogonin markedly enhanced the expression of autolysosomes, and the red fluorescence intensity was greater than for DMSO (Panc-1, P=0.003; Colo-357, P=0.007;
A co-immunoprecipitation pull-down assay was used to investigate the mechanisms underlying the effects of wogonin in HPCCs. Immunoprecipitation of Beclin-1 was able to pull down PI3K, and immunoprecipitation of PI3K was able to pull down Beclin-1, indicating that Beclin-1 and PI3K are bound to each other in HPCC (
Beclin-1 and PI3K expression may be induced by wogonin; when Beclin-1 was knocked down with a specific siRNA, the LC3-II conversion was lower as compared with the control, although it was not absent (
ROS was observed to mediate the survival and proliferation of cancer cells; therefore, the current study investigated the variations in ROS levels induced by wogonin in HPCCs. The results demonstrated that wogonin decreases the levels of glutathione (GSH;
As an antioxidant, NAC is considered as inhibitor of ROS. In order to negate the role of ROS in wogonin-induced-autophagy, HPCCs were co-treated with 10 mM NAC and 40 µM wogonin. The results demonstrated that the quantity of LC3-GFP puncta in cells co-treated with NAC and wogonin was lower, as compared with the cells treated with wogonin alone (Panc-1, P=0.007; Colo-357, P=0.006;
Similarly, LC3 expression was inhibited by NAC, as demonstrated when HPCCs were co-treated with NAC and wogonin (
Additional pro-autophagy pathways were detected to confirm whether they are involved in ROS-mediated autophagy following wogonin treatment. Previous reports (
To investigate the role of ROS-mediated autophagy in wogonin-induced-apoptotic cell death, HPCCs were treated with wogonin or co-treated with NAC and wogonin. The results demonstrated that co-treatment with NAC and wogonin was able to significantly enhance the wogonin-induced cell death ratio (Panc-1, P=0.024; Colo-357, P=0.037;
To the best of our knowledge, the present study is the first to demonstrate that wogonin-induced autophagy is regulated by the Beclin-1/PI3K and ROS signaling pathways. Wogonin is able to activate Beclin-1 and PI3K and promote ROS generation, inducing ROS-mediated autophagy, through activating ULK1, AKT, 4E-BP1 and CYLD, and inhibiting the mTOR signaling pathway (
Autophagy contributes to the survival of cells under various stresses, including starvation, drug stimulation and radiation, and is therefore a protective mechanism (
In mammalian cells, PI3K can bind to Beclin-1 through special ECD and CCD domains to form the Beclin-1/PI3K core complex, and then participate in the mediation of phosphorylation and ubiquitination, eventually inducing autophagy (
ROS can aggravate cell injury by oxidative stress. As the cell reacts to the injury, autophagy is activated to promote cell survival (
In summary, the findings of the present study indicate that wogonin can induce autophagy through activation of the Beclin-1/PI3K complex and triggering the ROS-mediated autophagy pathway. The pro-autophagy molecules mTOR, ULK1, AKT, 4E-BP1 and CYLD are involved in the ROS-mediated autophagy following wogonin treatment. Furthermore, the antioxidant NAC can inhibit wogonin-induced autophagy through inhibition of ROS generation, and enhance the ratio of wogonin-induced apoptotic cell death. In the future, wogonin combined with NAC may be a novel combination therapy for clinical pancreatic cancer therapy trials.
reactive oxygen species
N-acetyl-L-cysteine
human pancreatic cancer cells
Wogonin induces autophagy in HPCC. (A) HPCCs were treated with 40 µM wogonin, wogonin combined with 40 µM CQ or 50 µM rapamycin for 24 h; LC3 expression was induced by wogonin, indicating its ability to induce autophagy. (B) HPCCs were treated with various doses of wogonin for 24 h, and wogonin-induced autophagy was found to be dose-dependent, with an optimal dose of 40 µM. (C) HPCCs were treated with 40 µM wogonin and incubated for various times; wogonin-induced autophagy was time-dependent (optimal time, 24 h). (D) HPCCs were treated with 40 µM wogonin and processed for fluorescent microscopy for 24 h; the level of autolysosomes markedly increased following treatment with wogonin. (E) Following GFP-LC3 transfection, cells were treated with 0.1% DMSO or 40 µM wogonin for 24 h, and the LC3-GFP puncta-positive cells were counted (≥5 puncta was considered positive); the number of LC3-GFP puncta-positive cells was increased with wogonin treatment (***P<0.005). Data are presented as the mean ± standard deviation from triplicated experiments. (F) TEM was used to detect autophagic vacuoles (arrows), indicating that wogonin treatment induced autophagy. HPCC, human pancreatic cancer cell; CQ, chloroquine, LC3, microtubule-associated protein 1A/1B-light chain 3; GFP, green fluorescent protein; TEM, transmission electron microscopy; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; DMSO, dimethyl sulfoxide.
Wogonin activates the Beclin-1/PI3K signaling pathway. (A and B) HPCCs were treated with 40 µm wogonin or 0.1% DMSO for 12 h. Antibodies targeting (A) Beclin-1 or (B) PI3K were added to immunoprecipitate the Beclin-1- or PI3K-containing complexes and then IB for PI3K or Beclin-1 was performed. The results revealed that Beclin-1 and PI3K were pulled down after PI3K and Beclin-1 were downregulated, respectively, and wogonin could upregulate Beclin-1 and PI3K, which indicated that Beclin-1 and PI3K were bound to one another, and wogonin could promote integration to form the Beclin-1/PI3K complex. (C) HPCCs were transfected with control siRNA or siRNA-Beclin-1; at 36 h post-transfection, HPCCs were treated with 0.1% DMSO or 40 µM wogonin for 24 h and then immunoblotted for LC3 and Beclin-1, revealing that LC3 expression was decreased following Beclin-1 knowckdown. (D) HPCCs expressing LC3-GFP were transfected with control siRNA or siRNA-Beclin-1. At 36 h post-transfection, HPCCs were treated with 0.1% DMSO or 40 µM wogonin for 24 h, and the LC3-GFP puncta-positive cells were counted (≥5 puncta was considered positive). After Beclin-1 was knocked down, the number of positive cells was significantly decreased, but not reduced to zero, which indicated that the Beclin-1/PI3K complex was involved in, but not solely responsible for, wogonin-induced autophagy. Data are presented as the mean ± standard deviation from triplicated experiments. **P<0.01; ***P<0.005. HPCC, human pancreatic cancer cell; CQ, chloroquine, LC3, microtubule-associated protein 1A/1B-light chain 3; GFP, green fluorescent protein; TEM, transmission electron microscopy; DMSO, dimethyl sulfoxide; siRNA, small interfering RNA; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Con, control; IgG, immunoglobulin G; IB, immunoblotting; IP, immunoprecipitation.
Wogonin may promote ROS generation in HPCCs. HPCCs were pretreated with sterile water or NAC for 2 h and then treated with 0.1% DMSO or 40 µM wogonin for 24 h. (A) The levels of GSH were determined using an ELISA kit at 420 nm. Wogonin reduced the intracellular levels of GSH, and wogonin combined with NAC could significantly increase the intracellular levels of GSH. (B) The levels of O2− were determined using an ELISA kit at 550 nm. The intracellular levels of O2− were promoted by wogonin treatment and significantly inhibited by wogonin and NAC co-treatment. The levels of ROS were evaluated by DCFH-DA using (C) a fluorescence microscope or (D) a fluorescence microplate reader. ROS generation was promoted by wogonin treatment alone, but was inhibited by wogonin and NAC co-treatment. These results indicate that wogonin promotes ROS generation in HPCCs, and the antioxidant NAC can attenuate the effect of wogonin on ROS generation. Data are presented as the mean ± standard deviation from triplicated experiments. *P<0.05; ***P<0.005. HPCC, human pancreatic cancer cell; GSH, glutathione; DMSO, dimethyl sulfoxide; ROS, reactive oxygen species; NAC, N-acetyl-cysteine; DCFH-DA, 2,7-dichlorofluorescein diacetate; OD, optical density; DCF, 2,7-dichlorofluorescein.
Wogonin induces autophagy through the ROS signaling pathway in HPCC. HPCC were pretreated with sterile water or NAC for 2 h, then treated with 0.1% DMSO or 40 µM wogonin for 24 h. (A) LC3-GFP puncta-positive cells were counted (≥5 puncta was considered positive) and the results indicated that wogonin could increase the number of LC3-GFP puncta-positive cells, whereas co-treatment with NAC and wogonin attenuated this increase. Data are presented as the mean ± standard deviation from triplicated experiments. **P<0.01; ***P<0.005. (B) AO staining for autolysosomes indicated that autolysosomes were activated by wogonin treatment, but inhibited by NAC and wogonin co-treatment. (C) Immunoblotting for LC3 revealed that the expression levels of LC3 were reduced by NAC and wogonin co-treatment compared with wogonin single treatment. (D) The levels of p-mTOR, t-mTOR, ULK1, AKT, 4E-BP1 and CYLD were evaluated by immunoblot. Wogonin could downregulate the expression of mTOR, and upregulate the expression levels of ULK1, AKT, 4E-BP1 and CYLD; these effects were significantly inhibited by NAC and wogonin co-treatment. AO, acridine orange; HPCC, human pancreatic cancer cell; DMSO, dimethyl sulfoxide; ROS, reactive oxygen species; NAC, N-acetyl-cysteine; LC3, microtubule-associated protein 1A/1B-light chain 3; GFP, green fluorescent protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mTOR, mammalian target of rapamycin; P-, phosphorylated; T-, total; ULK1, Unc-51 like autophagy activating kinase 1; AKT, protein kinase B; 4E-BP1, 4E-binding protein-1; CYLD, cylindromatosis.
NAC enhances wogonin-induced apoptotic cell death. HPCCs were pre-treated with sterile water or NAC for 2 h, then treated with 0.1% DMSO or 40 µM wogonin for 24 h. (A) Analysis of the cell death ratio by trypan blue revealed that wogonin could enhance the cell death ratio, and this effect could be further promoted by co-treatment with NAC. (B) Cell viability was determined by CCK8; cell viability was reduced by wogonin and was further inhibited by co-treatment with NAC. (C) Caspase-3 levels were assessed by immunoblotting, revaling that the expression level of caspase-3 could be upregulated by wogonin, and was further promoted by co-treatment with NAC. (D) Flow cytometry revealed that wogonin could increase the cell apoptosis ratio, and the increased effect of co-treatment with NAC was significant. Data are presented as the mean ± standard deviation from triplicated experiments. *P<0.05; **P<0.01. HPCC, human pancreatic cancer cell; DMSO, dimethyl sulfoxide; NAC, N-acetyl-cysteine; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; CCK-8, cell counting kit-8.
The signaling pathways involved in wogonin-induced autophagy. Wogonin activates Beclin-1 and PI3K. Wogonin also promotes ROS generation, inducing ROS-mediated autophagy by activating the ULK1, AKT, 4E-BP1 and CYLD signaling pathways, and inhibiting the mTOR signaling pathway. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mTOR, mammalian target of rapamycin; P-, phosphorylated; T-, total; ULK1, Unc-51 like autophagy activating kinase 1; AKT, protein kinase B; 4E-BP1, 4E-binding protein-1; PI3K, phosphatidylinositol-3-kinase; ROS, reactive oxygen species; CYLD, cylindromatosis.