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Arctigenin Triggers Apoptosis and Autophagy via PI3K/Akt/mTOR Inhibition in PC-3M Cells
2021 Volume 69 Issue 5 Pages 472-480
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2021 Volume 69 Issue 5 Pages 472-480
Arctigenin (ARG), a natural lignans compound isolated from Arctium lappa L. In this study, the anti-tumor effect of ARG on prostate cancer cell PC-3M and the mechanism of apoptosis and autophagy induced by phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway were discussed, and further confirmed by the joint treatment of ARG and PI3K inhibitor LY294002. Here, the effect of ARG on cell viability was evaluated in PC-3M cells by Cell Counting Kit-8 reagent (CCK-8) assay. After the treatment of ARG, colony formation assay was used to detect the anti-proliferation effect. Annexin V-fluoresceine isothiocyanate/propidium iodide (FITC/PI) kit and 4′,6-diamidino-2-phenylindole (DAPI) staining were used to detect the apoptosis level, and cell cycle changes were analyzed by flow cytometry. The expression of autophagy was detected by acridine orange staining. In addition, the expression levels of apoptosis and autophagy-related proteins were analyzed by Western blot. The result showed that different concentrations of ARG inhibited the proliferation of PC-3M cells. DAPI staining and flow cytometry showed that ARG induced PC-3M cell apoptosis and arrested cell in G0/G1 phase. Acridine orange staining showed that ARG induced autophagy in PC-3M cells. Western blot experiments showed that ARG inhibited the expression of Bcl-2, promoted the expression of Bax and cleaved caspase-3. At the same time, the expression of autophagy-related proteins LC3B-II and Beclin-1 increased after ARG treatment, but P62 decreased. In addition, further studies have shown that treatment with LY294002 enhanced the effects of ARG on the expression of proteins associated with apoptosis and autophagy, indicating that ARG may induce apoptosis and autophagy through PI3K/Akt/mTOR pathway.
Prostate cancer is one of the most common malignant tumors of the male urinary system and is a leading cause of death in male patients, and it is considered to be one of the most important problems to be solved in modern medicine.1) The data showed that prostate cancer was very prevalent in the European population, and it had serious impacts on male health. The number of patients has exceeded colorectal cancer and lung cancer.2–4) In China, the incidence and mortality of prostate cancer are below the world average, but in recent years, the number of illnesses and deaths has been increasing.5) At present, surgery, chemotherapy and radiation therapy have been the main methods of cancer treatment. For example, the development and research of drugs such as protein affinity reagents and steroid analogues are getting more and more attention.6,7) However, modern therapies have limitations, including resistance to cancer drugs, various types of side effects and damage to normal cells.8) Therefore, based on the current treatment of prostate cancer, the research and development of low-toxicity, high-efficiency drugs is still an important direction for the development of anticancer drugs.9)
Arctigenin (ARG) is the main active ingredient extracted from the dried and ripe fruits of Arctium lappa L.10) Modern pharmacological studies have found that it has anti-tumor,11) anti-viral,12) immune-regulation,13) hypoglycemic,14) anti-inflammatory15) and other effects. In our previous studies, it was confirmed that ARG had a significant tumor inhibition on H22 tumor-bearing mice. In addition, literature pointed out that ARG activated the p38 and c-Jun N-terminal kinase (JNK) pathways to induce HepG2 cell apoptosis by inducing excessive reactive oxygen species (ROS) generation.16) Besides, the literature reported that ARG caused autophagic death of estrogen receptor (ER)-positive human breast cancer cells and reduced ER expression by inhibiting the activation of the mammalian target of rapamycin (mTOR) signaling pathway.17) Previous studies have shown that ARG had inhibitory effects in a variety of prostate cancer cells. Hao et al.18) reported that the effect of ARG in prostate cancer cells LNCaP under obese conditions, and the results showed that ARG inhibited the growth of prostate cells LNCaP at a lower concentration. In addition, it has shown that ARG in combination with quercetin synergistically enhanced the antiproliferative effect in prostate cancer cells LAPC-4.19) However, the effect of ARG in PC-3M cells and its mechanism are still unclear, which is need to further explored.
Autophagy and apoptosis are the main ways of tumor cell death and the main pathways that antitumor drugs exert their efficacy.20) Autophagy is a catabolic process which carried out in eukaryotic cells. Autophagy can inhibit normal cells from mutating in the early stage of tumorsand protect cells, and and it also can inhibit tumor genesis and development by eliminating damaged organelles, such as mitochondria and Golgi apparatus.21) Apoptosis belongs to programmed cell death, which has the characteristics of cell atrophy, nuclear fragmentation and the formation of apoptotic bodies.22) Although apoptosis and autophagy have different physiological processes and morphological characteristics, there is a complex and close relationship between them.23,24) Apoptosis and autophagy are regulated by different signaling transduction pathways, but both are regulated by the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling pathway, which regulates cell cycle progression, cell survival and differentiation and is a classic signaling pathway which regulated apoptosis and autophagy.25,26) In recent years, many studies have confirmed that many active ingredients derived from medicinal plants could induce tumor cell apoptosis and autophagy through the PI3K/Akt/mTOR signaling pathway.27,28) Changes in the expression of PI3K/Akt/mTOR signaling pathway may lead to the occurrence of tumors. Therefore, an in-depth understanding of the PI3K/Akt/mTOR signaling pathway will contribute to the development of anticancer drugs.29,30) Therefore, this study explored the effect of ARG and its mechanism on prostate cancer cells PC-3M. Our data suggested that ARG inhibited PC-3M cells proliferation, triggered apoptosis and induced PC-3M cells autophagy via the PI3K/AKT/mTOR signaling pathway. These results provide an important basis for the future research and development of ARG.
PC-3M cells were treated with different concentrations of ARG (0, 6.25, 12.5, 25, 50, 100 and 200 µM). After 48 h, the cell viability was measured by Cell Counting Kit-8 reagent (CCK-8) assay. We found that ARG exhibited inhibitory effect on the growth of PC-3M cells significantly, and its inhibitory effect continued to increase with increasing doses (Fig. 1c). The IC50 of ARG was 13.49 ± 1.65 µM at 48 h. Therefore, we chose 6.25, 12.5 and 25 µM ARG in subsequent experiments. In addition, colony formation experiments showed that ARG reduced the number of colony formation of PC-3M cells significantly. And its inhibitory effect increased in a dose-dependent manner (Fig. 1d).
ARG inhibited proliferation in PC-3M cells. (a) The chemical structure of ARG. (b) HPLC chromatogram of ARG. (c) PC-3M cells were treated with different concentrations of ARG (0, 6.25, 12.5, 25, 50, 100 and 200 µM) for 48 h, and the cell viability was identified by CCK-8. (d) PC-3M cells were treated with ARG (0, 6.25, 12.5 and 25 µM) for one week, and the image of clone formation was performed. The value is the average of three repeated experiments. * p < 0.05, ** p < 0.01 vs. the control group.
As shown in Figs. 2a, and b, PC-3M cells were treated with different concentrations of ARG (0, 6.25, 12.5 and 25 µM) for 48 h. With the increasing concentration of ARG, the distribution of cells in the G0/G1 phase was increased significantly, the change of cells in G2/M phase was not obvious, and the distribution of cells in S phase was reduced significantly. Therefore, cell cycle testing data showed that ARG could induce G0/G1 phase arrest in PC-3M cells.
ARG arrested cell cycle at G0/G1 phase. (a) PC-3M cells were treated with various concentrations of ARG (0, 6.25, 12.5 and 25 µM) for 48 h, and the cell cycle was analysised by flow cytometry. (b) The percentages of G0/G1, S and G2/M phase. The value is the average of three repeated experiments. * p < 0.05, ** p < 0.01 vs. the control group.
The results of Annexin V-fluoresceine isothiocyanate/propidium iodide (FITC/PI) double staining method were shown in Figs. 3a, and b, compared with the control group, the apoptosis rate of PC-3M cells increased significantly after 6.25, 12.5 and 25 µM ARG treament. The apoptotic rates were 7.61, 14.32 and 24.5%, which were all higher than the control group 4.18%. In addition, the results of 4′,6-diamidino-2-phenylindole (DAPI) staining (Fig. 3c) indicated that ARG induced apoptosis. We further detected apoptosis-related proteins by Western blotting. The results were shown in Figs. 3d, and e, compared with the control group, the protein expression levels of Bax and cleaved caspase-3 in PC-3M cells gradually increased as the ARG concentration increased, while the protein expression level of Bcl-2 gradually reduced.
ARG induced apoptosis in PC-3M cells. (a, b) PC-3M cells were treated with different concentrations of ARG (0, 6.25, 12.5 and 25 µM) for 48 h, and the cells were analysised by flow cytometry after Annexin V-FITC/PI staining. (c) DAPI staining was performed after ARG treatment in PC-3M cells. (d, e) The expression levels of apoptosis-related proteins Bcl-2, Bax and cleaved caspase-3 were analyzed by Western blotting. The value is the average of three repeated experiments. * p < 0.05, ** p < 0.01 vs. the control group.
The influence that the number of lysosomes in PC-3M cells treated with different concentrations of ARG was detected by acridine orange staining. As shown in Fig. 4a, compared with the control group, the number of lysosomes in PC-3M after ARG treatment increased significantly, and the number of lysosomes increased with the rise of ARG concentration. On this basis, the changes in autophagy-related protein expression were detected by Western blotting. Beclin-1, LC3B and P62 are the key proteins that induce autophagy. LC3B-II is formed by the transformation of LC3B-I.31,32) As shown in Figs. 4b, and c, the expression of Beclin 1 and LC3B-II in ARG groups cells increased in a concentration-dependent manner compared to the control cells, while the expression of P62 decreased in a concentration-dependent manner compared to the control cells, further confirmed that ARG could induce autophagy in PC-3M cells.
ARG induced autophagy in PC-3M cells. (a) PC-3M cells were treated with various concentrations of ARG (0, 6.25, 12.5, and 25 µM) for 48 h, stained them with acridine orange and recorded the images with a microscope. (b, c) Thethe expression levels of autophagy-related proteins Beclin-1, LC3B and P62 were analyzed by Western blotting. The value is the average of three repeated experiments. * p < 0.05, ** p < 0.01 vs. the control group.
PI3K/Akt/mTOR signaling pathway is the upstream pathway that regulates apoptosis and autophagy. In order to further understand the potential mechanism that ARG promoted PC-3M cell death, this study conducted a detailed detection of PI3K/Akt/mTOR signaling pathway and related proteins by Western blot. The results were shown in Figs. 5a, b, after treated PC-3M cells with 6.25, 12.5 and 25 µM ARG for 48 h, compared with the control group, the expression of phosphorylated (p)-PI3K, p-Akt, and p-mTOR decreased with the rise of ARG concentration, the expression of PI3K, Akt, and mTOR did not change significantly. The expression of p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR gradually decreased. To further explore the role of PI3K/Akt/mTOR pathway in the regulation of apoptosis and autophagy, the PI3K inhibitor LY294002 and ARG were co-treated with PC-3M cells. Compared with the ARG alone administration group, after treatment with ARG (25 µM) and LY294002 (10 µM) for 48 h in PC-3M cells, the expression of p-PI3K/PI3K, p-Akt/Akt and p-mTOR/mTOR were significantly reduced (Figs. 5c, d). In addition, after treatment with LY294002, apoptosis-and autophagy-related proteins were also tested. Compared with the ARG alone group, after treatment with ARG (25 µM) and LY294002 (10 µM) in PC-3M cells, the expression of Bax and cleaved caspase-3 increased, and the expression of Bcl-2 gradually decreased (Figs. 5e, f). The expression level of Beclin-1 and LC3B-II increased, while the expression level of P62 decreased (Figs. 5g, h). Therefore, these data proved that ARG could induce apoptosis and autophagy through PI3K/Akt/mTOR signaling pathway.
ARG induced apoptosis and autophagy through PI3K/Akt/mTOR signaling pathway in PC-3M cells. (a, b) PC-3M cells were treated with different concentrations of ARG (0, 6.25, 12.5 and 25 µM) for 48 h, the expression levels of PI3K, Akt, mTOR and their phosphorylation forms were analyzed by Western blotting. (c, d) PC-3M cells were treated with 10 µM LY294002 and 25 µM ARG for 48 h, the expression levels of PI3K, Akt, mTOR and their phosphorylation forms were analyzed by Western blotting. (e, f) PC-3M cells were treated with 25 µM ARG, LY294002, 25 µM ARG + 10 µM LY294002 for 48 h, the expression levels of apoptosis-related proteins Bcl-2, Bax and cleaved caspase-3 were analyzed by Western blotting. (g, h) PC-3M cells were treated with 25 µM ARG, LY294002, 25 µM ARG + 10 µM LY294002 for 48 h, and then the changes in the expression levels of autophagy-related proteins Beclin-1, LC3B and P62 were analyzed by Western blotting. The value is the average of three repeated experiments. * p < 0.05, ** p < 0.01 vs. the control group.
Lignans compounds are widely present in Chinese herbal medicines and have rich pharmacological effects. Recent studies have shown that many lignans compounds exhibited potential anticancer activity. Mainly mechanisms were inhibiting cell proliferation, inducing cell apoptosis and autophagy, blocking cell cycle and inhibiting cell migration.33,34) ARG is an important lignans compound with anticancer effect. In recent years, the anti-tumor activity of ARG has attracted widespread attention. Studies showed that ARG may be a promising alternative anti-cancer drug for the treatment of prostate cancer.35,36) Prostate cancer is one of the most common malignancies in male reproductive system.37) Prostate cancer is caused by many factors such as excessive cell proliferation and abnormal differentiation. In this study, androgen-independent prostate cancer cell PC-3M was studied. PC-3M cells were derived from human prostate cancer epithelium, belonging to the subline of PC-3 cells, which have strong metastasis and invasion.38) Abnormal cell differentiation and uncontrolled proliferation are two distinct characteristics of malignant cells and normal cells. Many published literatures have shown that ARG could inhibit tumor cells proliferation.39,40) In this study, the results of CCK-8 and colony formation assay showed that the inhibition rate of PC-3M cells proliferation at different concentrations of ARG was significantly increased compared with the control group, and the inhibition effect on cell colony formation was significant, and the inhibition rate was increased in a dose-dependent manner. The cell cycle is closely related to a series of pathophysiological processes such as cell growth, proliferation, differentiation, apoptosis and carcinogenesis. The cell cycle is a process closely regulated by genes. If cell cycle regulation is abnormal, it will cause uncontrolled cell growth and proliferation, then leadto tumor development and even metastasis. Studies have shown that many drugs could cause cell arrest in G0/G1, S or G2/M phase.41) Literature reported that the compound apigetrin which found from many natural products had antitumor effects in vitro. It caused cell arrest in G2/M phase and further inhibited the proliferation of AGS cells.42) In this study, the analysis of ARG on PC-3M cell cycle was detected by flow cytometry. The results showed that ARG could block PC-3M cells in G0/G1 phase.
Apoptosis is a process of genetically programmed cell death, which plays a vital role in the development of tumor cells.43) Apoptosis includes endogenous and exogenous modes. Although endogenous and exogenous apoptotic pathways have different causes and activation processes, they eventually converge on the caspase family44) caspase-3 is one of the main performers of apoptosis. It can specifically cut a variety of substrates, result in DNA fragmentation and finally lead to cell apoptosis.45) Bcl-2 and Bax are both key regulators of apoptosis, but their roles are opposite. Bcl-2 has the effect of inhibiting cell apoptosis, and Bax has the effect of promoting cell apoptosis.46) To explore whether ARG can induce PC-3M cell apoptosis and play an anti-tumor effect. In this experiment, DAPI staining and flow cytometry were performed to analyze the apoptosis rate. At the same time, the expression level of apoptosis-related protein was detected by Western blotting. It was found that the number of apoptotic cells increased significantly after trement with different concentrations of ARG for 48 h, and it was dose-dependent. In addition, the expression of apoptosis-related proteins Bax and cleaved caspase-3 increased, and the expression of Bcl-2 decreased.
Similar to apoptosis, autophagy is another form of programmed cell death. Normal level of autophagy is a process to maintain cell homeostasis, while excessive upregulation of autophagy can lead to autophagic death. In recent years, it has been shown that ARG could induce autophagy in various cancers. For examples, Jiang et al.47) reported that ARG induced apoptosis and autophagy of glioma cells in a dose-dependent manner, and confirmed that ARG induced apoptosis and autophagy through Akt/mTOR signaling pathway by detecting apoptotic- and autophagy-related proteins. Wang et al.48) showed that ARG could enhance the sensitivity of colorectal cancer cells by activating autophagy, thereby induced apoptosis and inhibited cell growth. Although there were obvious differences between autophagy and apoptosis in terms of biochemical metabolic pathways and morphology, more and more data showed that there were also complex connections between autophagy and apoptosis. It has been found that autophagy and apoptosis could occur simultaneously and assist each other to promote cell death.49) Qian et al.50) reported when arsenic trioxide was used to treat human T-lymphocytic leukemia, autophagy and apoptosis were simultaneously activated, and they acted synergistically to promote cell death. In addition, it has been shown when VK2 acted on leukemia cells HL-60, VK2 could induce autophagy and apoptosis at the same time.51) Fluorescence microscope observation of autophagy vesicles with double-layer membrane structure is the most direct and classic method to observe autophagy. After autophagosomes bind to lysosomes, acid phosphatase remains in the body. In this study, compared with the control group, the aggregation of orange-red fluorescent spots increased in the administration group, indicated an increase in the number of autophagic lysosomes. It is preliminarily proved from the morphology that different concentrations of ARG induced autophagy in PC-3M cells. LC3 plays an important role in the formation and expansion of autophagosome membranes. LC3 is transformed into free form LC3-I under the action of proteolytic enzymes.52) The conversion of free LC3-I to membrane-bound LC3-II is one of the key factors to induce autophagy. LC3-II is closely related to autophagosomes, increases with the increase of autophagosome membrane, which is a sign of autophagy.53) In mammals, LC3 mainly includes three types: LC3A, LC3B and LC3C. Among them, LC3B is one of the types of LC3and is generally considered to be an important molecular marker for autophagy. Beclin-1 is a kind of autophagy related gene (Atg) expressed protein and is closely related to the formation of autophagosomes.54) P62 is another marker of autophagy, it is a key autophagy receptor, it interacts with LC3 and binds to autophagosomes.55) With the rise of ARG concentration, the expression level of Beclin-1 and LC3B-II gradually increased, while the expression level of p62 gradually decreased. Obviously, ARG could induce autophagy in PC-3M cells.
Finally, in order to further explore the potential mechanism of ARG-induced apoptosis and autophagy in PC-3M cells, we studied the PI3K/Akt/mTOR signaling pathway that is closely related to these two death pathways. PI3K/Akt/mTOR is a classical signaling pathway that participates in the regulation of cell growth, survival, proliferation, death, energy metabolism and other important biological processes.56) Akt is a critical factor in the signaling pathway, which mediates autophagy through multiple pathways. Phosphorylated Akt is an important mediator of carcinogenesis. mTOR is the main negative regulator of autophagy and plays an important role in tumorigenesis. PI3K can activate its downstream substrate Akt and cause its phosphorylation. p-Akt can further activate its downstream target protein mTOR. If p-Akt and p-mTOR are highly active, it will cause the disorder of many downstream signaling pathways regulated by them, which plays an important role in tumor cells.57) In this experiment, Western blot was used to detect the expression of PI3K, Akt, mTOR and their phosphorylated forms in the PI3K/Akt/mTOR signaling pathway in PC-3M cells treated with ARG. The results showed that the expression levels of p-PI3K, p-Akt, and p-mTOR decreased with the rise of drug concentration, while the expression of PI3K, Akt, and mTOR did not change significantly. In addition, in order to further determine the apoptosis and autophagy induced by ARG through the PI3K/Akt/mTOR signaling pathway, we used PI3K inhibitor LY294002 and ARG to co-treat PC-3M cells. The results showed that compared with the ARG alone administration group, the co-treatment of LY294002 and ARG significantly reduced the phosphorylation expression of PI3K, Akt and mTOR. At the same time, the expression of apoptosis-related protein Bax and cleaved caspase-3 increased significantly, while the expression of Bcl-2 decreased. The expression of autophagy-related proteins (LC3B-II and Beclin-1) increased significantly, while the expression of P62 decreased. In summary, all results indicated that ARG-induced apoptosis and autophagy may be achieved through PI3K/Akt/mTOR signaling pathway.
To sum up, ARG could inhibit the proliferation and induce apoptosis and autophagy of PC-3M cells through the PI3K/Akt/mTOR pathway, ultimately play an anti-tumor effect. Therefore, the data of this study indicated that ARG induced autophagy and apoptosis through the PI3K/Akt/mTOR signaling pathway may be a new direction for target prostate cancer therapy in future drug development and clinical practice.
ARG was obtained from the ethanol extracts of the dried and mature fruits of Arctium lappa L. The papers published by our research group clarified the specific extraction and separation process of ARG.58) The chemical structure of ARG was shown in Fig. 1a. The purity of the obtained ARG was detected by HPLC, and the gradient was CH3OH/H2O (55 : 45, 1 mL/min, 25 min) elution (Fig. 1b) (98% purity). The obtained ARG was dissolved in quantitative dimethyl sulfoxide (DMSO) and diluted with 1640 to different concentrations before using.
Chemicals Reagents and AntibodiesRPMI-1640 medium, Phosphate Buffered Saline (PBS), Trypsin, Tris-Buffered Saline with Tween 20 (TBST, 10X), Fetal bovine serum (FBS) and antibiotics (penicillin/streptomycin) were purchased from Thermo Fisher Scientific (Beijing, China). DMSO was purchased from Shanghai Aladdin Co., Ltd., (Shanghai, China). Cell Counting Kit-8 reagent (CCK-8) was obtained from Good Laboratery Practice biosclence (California Montclair, CA, U.S.A.). DAPI staining, Crystal Violet Staining Solution, PI/RNase staining, Annexin V fluorescein isothiocyanate (FITC) and Propidium iodide (PI) assay kit were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Anti-Bcl2; Bax; cleavedcaspase-3; PI3K; p-PI3K; AKT; p-AKT; mTOR; p-mTOR; Beclin-1; LC3B; P62; β-actin and Horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit and anti-mouse, respectively) were purchased from Abcam (Cambridge, U.K.). RIPA lysate and Pierce™ BCA protein detection kit were purchased from Beyotime (Nanjing, China). 5× protein loading buffer was purchased from Biosntech (Beijing, China). PVDF membrane was purchased from Thermo Fisher (Shanghai, China). Hypersensitive enhanced chemiluminescence (ECL) Chemiluminescence Kit was purchased from Beyotime (Shanghai, China). The PI3K inhibitor LY294002 was purchased from Good Laboratery Practice bioscience.
Cell Culture and TreatmentThe human prostate cancer cell line PC-3M was purchased from the Cell Bank of Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI-1640 medium with 10% FBS and 1% penicillin and streptomycin, and placed in a 37 °C and 5% CO2 cell incubator. When the cells exceed 80% of the culture flask, passed them once, and took the cells in the logarithmic growth phase for experiment.
Cell Viability AssayInoculate PC-3M cells into 96-well plates at a density of 5 × 103/well, 100 µL per well, and cultured in a 37 °C, 5% CO2 incubator. After the cells adhered, different concentrations of ARG (0, 6.25, 12.5, 25, 50, 100 and 200 µM) were added as the experimental group, and the same concentration of DMSO was used as the control group . After continuing to culture 48 h in the incubator, added 10 µL CCK-8 to each well and incubated for 2 h in the incubator. Measure the absorbance value (optical density (OD)) at A450 nm with a microplate reader. Calculation: cell viability rate (%) = (experimental group OD-zero adjustment group OD)/(control group OD-zero adjustment group OD) × 100%.
Colony Formation AssayPC-3M cells in cell culture flask were seeded into 6-well plates at a density of 1000 cells/well. After the cells adhered, the cells were treated with different concentrations of ARG (0, 6.25, 12.5 and 25 µM). After culturing for 1 week, taken out the 6-well plate, discarded the medium, then washed the cells 3 times with PBS at 37 °C. Then added 1 mL 4% paraformaldehyde to each well for 20 min. Discarded the paraformaldehyde and washed the cells 3 times with PBS. Then added 1 mL of 1% crystal violet staining solution and stained for 15 min at room temperature. Finally, washed the staining solution with tap water, and then dried the 6-well culture plate at room temperature. A camera was used to photograph the 6-well culture plate.
Cell Cycle AssayThe logarithmic growth phase cells were seeded in 6-well plates (2 mL/well) at a density of 5 × 105 per well. After overnight in a 37 °C, 5% CO2 incubator, different concentrations of ARG (0, 6.25, 12.5 and 25 µM) continued to process for 48 h. Then 6-well plates were taken out, washed them twice with 4 °C pre-cooled PBS, added 1 mL pre-cooled 70% ethanol, mixed gently, and fixed at 4 °C overnight. Then collected the cells by centrifugation, washed them twice with PBS, and added 500 µL PI/RNase staining buffer to resuspend the cells. Then, the cells were incubated at 37 °C for 30 min in the dark, detected by flow cytometry and analyzed by FlowJo software (FACSVerse, BD Biosciences, U.S.A.).
Annexin V-FITC/PI Assay for ApoptosisPC-3M cells were seeded in 6-well plates at a density of 5 × 105/well. After the cells adhered to the wall at the next day, discarded the supernatant culture medium, and then added different concentrations of ARG (0, 6.25, 12.5 and 25 µM). After 48 h, the cells were collected and washed twice with 4 °C pre-cooled PBS. Added 400 µL Annexin V-FITC binding buffer to each group, then added5 µL Annexin V-FITC and 10 µL PI solution and mixed well. Finally, stained at room temperature for 30 min in the dark, and used flow cytometry to detect the apoptosis rate (FACSVerse, BD Biosciences).
DAPI StainingInoculated PC-3M cells in 6-well plates, discarded the supernatant culture medium after the cells adhered to the wall, and add edmedia containing different concentrations of ARG. After treatment for 48 h, washed twice with PBS. Fixed with 4% paraformaldehyde at room temperature for 15 min, and washed twice with PBS. Added 500 µL DAPI staining solution and stained for 20 min at room temperature in the dark. Finally, used a fluorescence microscope to observe the staining and took pictures (LEICA DMi8, Germany).
Acridine Orange (AO) StainingPC-3M cells were seeded in 6-well plates and allowed to settle overnight at 37 °C. Then discarded the supernatant culture medium, and the cells were treated with ARG (0, 6.25, 12.5 and 25 µM) for 48 h. After that, the cells were washed twice with PBS, fixed with chilled 70% ethanol at room temperature for 15 min, and washed twice with PBS. Five hundred microliters acridine orange staining solution was added in the 6-well plate and stained for 15 min at room temperature in the dark. Finally, used a fluorescence microscope to observe the staining (LEICA DMi8).
Western BlotThe cells were treated as indicated, harvested and then washed twice with ice-cold PBS for total protein extraction. The protein concentration was determined with an Enhanced BCA Protein Assay Kit. Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skim milk in Tris-Buffered Saline with Tween 20 (TBST, 1X) buffer. Then, the membranes were incubated with primary antibodies at 4 °C overnight. After that, the membranes were washed 3 times with TBST buffer and incubated with the secondary antibodies at room temperature for 2 h. After the membranes were washed with TBST buffer 3 times. Blots were visualized with an ECL detection system. Protein quantification was performed using ImageJ. The densitometry readings of the bands were normalized according to the expression of β-actin.
Statistical AnalysesGraphPad Prism 5.0 statistical software was used for statistical analysis. The results were expressed as mean ± standard deviation. One-way ANOVA was used for the analysis between groups, and then t test was used. p < 0.05 indicated that the difference was statistically significant. (p < 0.05*, p < 0.01**)
This work was supported by Grants from the Science and Technology Research Projects in “the 12th Five-Year Plan” of Jilin Provincial Education Department (201459).
The authors declare no conflict of interest.
