Effects of butein on human osteosarcoma cell proliferation,apoptosis,and autophagy through oxidative stress

Cell and cell culture

The human OS cell line 143B was obtained from Zhongqiaoxinzhou Biotech (Shanghai, China). The cells were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM; Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) 100 U/mL penicillin and 100μg/mL streptomycin at 37°C with 5% CO₂.

Reagents and antibodies

Butein (purity of > 98%) was purchased from Shanghai Yuanye Biotechnology, Ltd. (Shanghai, China). Stock solution at 100 mM was made in DMSO (Sigma, St. Louis, MO, USA) and stored in the dark at −20°C. 3-Methyladenine (3-MA), broad-spectrum caspase inhibitor z-VAD-fmk-VAD(VAD), SP600125(SP), MK2206(MK), and N-acetyl-L-cysteine (NAC) were purchased from Target Mol. Hoechst staining kit, terminal deoxynucleotidyl transferase -mediated dUTP Nick- End Labeling (TUNEL) kit and JC-1 kit were bought from Beyotime Institute of Biotechnology (Jiangsu, China). The antibodies to GPX4, LC3B, phospho-AKT, β-actin and GAPDH were purchased from Proteintech (Chicago, IL, USA). The antibody to p-mTOR and p-JNK were bought from Cell Signaling Technology.

Cell proliferation assay

The cells were incubated overnight in a 96-well plate and then treated with various concentrations of 0,6.25,12.5, 25,50, and 75μM for 24 and 48 h. Cell viability was assessed using cell counting kit-8 (CCK8) (Dojindo, Kumamoto, Japan) according to the protocol. In brief, the cells were washed with phosphate buffer saline (PBS) and incubated with CCK8 working solution at 37°C for 1–2 h. To determine cell viability, the optical density (OD) at 450 nm was measured using a microplate reader (Tecan infinite M1000 Pro).

Colony formation assay

To examine the capacity of single cells to form a colony, the 143B cells were seeded in 6-well plates at a density of 500 cells/well. After 24 h, the cells were treated with various concentration of 5,7.5, and10 μM for about 14 days. Then, the cells were washed twice with PBS, fixed with 4% paraformaldehyde, and then stained with 0.2% crystal violet solution for 15 min. The clones with>50 cells were counted under a light microscope (ZEISS, Germany), and the images were captured using a camera.

Cell cycle analysis by flow cytometry

The role of butein in cell cycle distribution was analyzed using the propidium iodide (PI)/RNase staining buffer. Briefly, the cells were incubated in 6-well plates and treated with butein or DMSO for 24 h. Then, the cells were harvested, washed twice with ice-cold PBS, and fixed overnight with precooled 75% ethanol at 4°C. Next, the cells were incubated with 500 μL of PI/RNase A staining buffer (PI/RNase A at 9:1) in the dark for 30 min at RT. After staining, the samples were evaluated using the flow cytometer (FACSCalibur, BD, San Jose, CA, USA) and FlowJo™ software.

Transwell invasion assay

The cells that were either pretreated or not treated with butein and IL-6 for 24h were collected and re-suspended in culture medium. Afterward, the upper chambers coated with Matrigel matrix (BD, USA) were filled with 200 μL of cell suspension. Then, the upper chambers were placed into a 24-well plate that containing 12% FBS-supplemented DMEM (600 μl). After incubation for 24h, the cells were fixed with 4% polyformaldehyde, and stained with 0.1% crystal violet for 30 min. The cells at the upper surface were softly wiped off using a cotton swab. Images were captured under an inverted microscope (ZEISS, Germany) and then the cell number was calculated.

Morphological apoptosis

Hoechst 33342 staining was conducted to observe apoptosis. The 143B cells were inoculated on the coverslips in 6-well plates overnight and treated with butein for 24 h. After treatment, the Hoechst 33342 solution was used to analyze morphological changes in the nucleus according to the manufacturer's instructions (Beyotime, China). Finally, the cells were observed under a fluorescence microscope (ZEISS, Germany).

TUNEL assay

The 143B cells were plated on coverslips in 24-well plates and treated with butein for 24 h. After 24 h, apoptosis in the 143B cells was examined using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay kit (Beyotime, China) according to the manufacturer’s instructions. In brief, cells were fixed with 4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.3% Triton X-100 for 5 min at RT. Then, the cells were incubated with 50 μL of TUNEL test solution in the dark at 37 ° C for 1 hour. The nuclei were stained with Hoechst staining solution (Beyotime, China). Finally, the TUNEL-positive cells were examined under a fluorescence microscopy (ZEISS, Germany).

Measurement of mitochondrial membrane potential (MMP)

The MMP was measured using the JC-1 Assay Kit (Beyotime, China) according to the manufacturer's instructions. Briefly, 143B cells were seeded in 6-well plates and treated with butein for 24h. After treatment, the cells were incubated with 1mL of JC-1 solution for 20 min at 37°C. The cells were observed under a fluorescence microscope (ZEISS, Germany), and mitochondrial depolarization was indicated by a decrease in the red/green fluorescence intensity ratio.

Flow cytometric analyses of apoptosis

The 143B cells were sub-cultured in 6-well plates and treated with butein at indicated concentration for 24 h. Then, the cells were collected, washed twice with chilled PBS, and then resuspended in the 1X binding buffer. Aliquots of 105 cells were stained with 5 μL of APC Annexin V and 5 μL of PI for 15 min at RT in the dark. After staining, the samples were evaluated using the flow cytometer (FACSCalibur, BD, San Jose, CA, USA) and FlowJo™ software.

Measuring apoptotic activity

The 143B cells were treated with butein with or without NAC. Afterward, the cells were collected and lysed on ice with lysis buffer (Beyotime, China) and centrifuged at 20000×g for 15 min at 4°C. The supernatants were collected to detect the activities of caspase-8, -9, and -3 by using colorimetric assay kits according to the manufacturer’s instructions. The caspase activity was evaluated by measuring the absorbance at 405 nm. The results were then standardized to the protein concentrations.

Oxidative stress detection

To determine whether butein induced oxidative stress in the 143B cells, we measured the levels of ROS, MDA, GSH/GSSG ratio, and GPX4 protein expression level. The production of ROS was detected by using the ROS Assay Kit (Beyotime, China). In short, the cells were seeded in 6-well plates and treated with butein for 24 h. The cells were stained with 10 μM DCFH-DA at 37°C in the dark for 30 min. Then, the cells were washed thrice with serum-free DMEM and the ROS level was measured using a fluorescence microscopy (ZEISS, Germany). The lipid peroxidation assay was conducted using the MDA assay kit. Briefly, the 143B cells were treated with butein. Then, the cells were collected and lysed with lysis buffer and then centrifuged at 12000×g for 10 min. Subsequently, the cell supernatants were collected and added with the test solution. Then, the EP tubes containing the samples and test solution were kept in boiling water for 15 min and cooled to RT. Finally, the MDA level was evaluated by measuring the absorbance at 532 nm and standardized to the protein concentrations. The GSH/GSSG assay kit was used to detect the levels of total glutathione and oxidized glutathione. The 143B cells were treated with butein for 24h. Next, the cells were washed with PBS. Then, the cell samples were subjected to rapid freezing and thawing twice with liquid nitrogen and 37°C water bath. After centrifugation at 10000×g for 10 min, the cell supernatants were harvested and processed with the test solution. Finally, the GSH level was evaluated by measuring the absorbance at 412 nm. Total glutathione minus GSSG represents the GSH level.

Western blot assay

The cells were seeded in 100-mm dishes and treated with butein and SP or MK for 24 h. The 143B cells samples were lysed in cooled RIPA buffer containing protease inhibitor mixture (Beyotime, China) for 30 min. Then, the lysates were centrifuged at 12,000g and4 °C for 15 min and the supernatant was collected. The protein concentration was quantified using the BCA protein assay kit (Beyotime biotechnologies, Jiangsu, China) according to the manufacturer’s instruction. Equal amounts of protein were separated by SDS-PAGE and transferred to the polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk for 1 h at room temperature (RT) and then incubated with primary antibodies overnight at 4°C, subsequently incubated with species-specific peroxidase-conjugated secondary antibodies for 1 h at RT. Each band was detected by using a chemiluminescence reagent and visualized using a Bio-Rad imaging system.

Transmission electron microscopy

The 143B cells were treated with butein for 24 h. The treated cells were fixed with 2.5% glutaraldehyde for 1 h at RT and post-fixed with 1% osmium tetroxide for another 1 h. The cell pellets were dehydrated in graded ethanol series, and then embedded in epon. Representative areas were chosen for ultrathin sectioning. After making ultrathin sections, the sections were stained with uranyl acetate and lead citrate. The morphology of the autophagosomes was examined under a transmission electron microscope. (H-7650, Hitachi, Tokyo, Japan).

Statistical analysis

The quantitative data are presented as the mean ± SD. Fisher’s Exact test, unpaired student's t-test, one-way ANOVA and two-way ANOVA were used to compare differences between the groups. All statistical analyses were performed using GraphPad Prism 7.0 (San Diego, CA, USA). p-values < 0.05 were considered as statistically significant.

Butein inhibited 143B cells viability

The CCK8 assay and colony formation test were used to examine the anti-proliferative effect of butein on the 143B cells. As shown in (Figure 1(a)), cell proliferation activity was significantly decreased in the dose- and time-dependent manner. The IC50 for 143B cells at 24h and 48h were 23.9 μM and 11.7 μM, respectively. Additionally, the colony formation test demonstrated that butein treatment significantly inhibited colony formation (Figure 1(b)). These results suggested that butein inhibited 143B cells proliferation.OPEN IN VIEWER

Butein induced cell cycle arrest at G2/M phase in 143B cells

To determine the relationship between growth inhibition and cell cycle arrest, we examined the effect of butein on cell cycle progression. As shown in Figure 1(c), butein treatment led to the accumulation of cells in the G2/M phase, accompanied by G0/G1 phase and S phase reduction. These results indicated butein inhibited 143B cell growth by inducing cell cycle arrest at the G2/M phase.

Butein inhibits the invasiveness of 143B cells stimulated by IL-6

Because 143B cells are highly aggressive OS cells, and inflammatory factors in cancer often promotes tumor cell invasion. Next, we investigated the effect of butein on the invasiveness of 143B cells stimulated by IL-6. Transwell invasion test showed that butein significantly inhibited the invasion of 143B cells upon IL-6 treatment (Figure 2(a)). We further investigated the effect of butein on E-cadherin expression, which indicated that butein upregulated E-cadherin expression in 143B cells upon IL-6 treatment. In addition, butein decreased MMP-9 and MMP2 protein expression in the presence of IL-6. IL-6 can increase the phosphorylation of STAT3, and the increase of STAT3 phosphorylation level can promote cancer cell metastasis. Thus, we investigated the effect of butein on the STAT3 signal in the IL-6-stimulated 143B cells. Inevitably, butein inhibited STAT3 phosphorylation (Figure 2(b)). These results indicated that butein can effectively inhibit the invasion of 143B cells.OPEN IN VIEWER

Butein induced apoptosis of 143B cells

Butein-induced apoptosis was investigated through Hoechst 33258 staining, TUNEL assay, MMP assay, and Annexin V/PI assay. As shown in Figure 3(a), the 143B cells treated with butein for 24 h showed some apoptotic characteristics as indicated by the red arrows, such as chromatin condensation, and nuclear fragmentation. TUNEL staining results further revealed that butein treatment led to an increase in DNA fragmentation in the 143B cells (Figure 3(b)). MMP (ΔΨm) loss is a crucial step in the process of apoptosis. Next, we analyzed it by using JC-1 probe and found a decrease in the red/green fluorescence intensity ratio following butein treatment (Figure 3(c)). Additionally, we quantified apoptosis through Annexin V-PE/APC double staining. The results demonstrated that the exposure to 12.5µM–50μM butein lead to a dose-dependent increase in the number of apoptotic cells (Figure 3(d)) To further verify that butein induced apoptosis of 143B cell, we determined the activities of caspsase-3, -8, and -9. As shown in Figure 3(e), the caspase-3 activity was significantly increased in the 143B cells treated with butein (p < 0.05). The activity of caspase 8 (an extrinsic apoptotic pathway initiator caspase) and caspase 9 (an intrinsic apoptotic pathway initiator caspase) was also significantly increased in a dose-dependent manner (p < 0.05), which indicated that butein triggered both extrinsic and intrinsic apoptosis in the 143B cells. Moreover, VAD (a caspase inhibitor) could reverse a certain proportion of cell death (Figure 3(f)).OPEN IN VIEWER

Butein triggered autophagy, which protects cell survival

To determine whether butein could induce autophagy in the OS cells. We first used LysoTracker Red to label the cellular acidic compartments, such as lysosomes and autolysosomes. Figure 4(a) shows that butein-treated 143B cells exhibited more acidic vesicular organelles (AVOs) in the cytoplasm. Furthermore, we determined LC3B expression by western blotting. As shown in Figure 4(b), butein increased LC3B-II level in the 143B cell in a dose-dependent manner. Finally, we observed autophagosomes formation directly through TEM. The results showed that the number of autophagic vacuoles was significantly increased in the cytoplasm of butein-treated cells compared with that in the control cells (Figure 4(c)).OPEN IN VIEWER

Autophagy plays a dual role in cancer treatment; its role is to protect cell survival or lead to cell death. To determine the effect of butein-induced autophagy, we used 3-MA to block butein-induced autophagy in the OS cells. As shown in Figure 4(d), cell viability was decreased compared with that under butein treatment alone. As shown in Figure 4(e), 3-MA treatment increased the extent of butein -induced cell death. These data suggested that butein mediated autophagy promoted 143B cells survival.

Butein induced oxidative stress, and activated the JNK signaling pathway in human 143B cells

Our result revealed that the MMP decreased sharply in the butein-treated 143B cells. Mitochondria are the main source of ROS inside cells. This led us to assume that butein may trigger the release of ROS from mitochondria. Therefore, the fluorescent probe DCFH-DA was used to investigate whether butein treatment could enhance ROS production. As shown in Figure 5(a), the fluorescence signal was significantly increased in butein-treated 143B cells. Additionally, butein increased the level of lipid peroxidation, as evidenced by an increase in the MDA level (Figure 5(b)). The cellular redox balance is critical to cell fate. GSH is the main cell redox buffer, and the GSH/GSSG ratio is an important indicator of cell redox status. After butein treatment, the ROS level was significant increased. Thus, we believed that the antioxidant system exhibited defects, and thus lacked the ability to scavenge ROS. The results revealed that butein significantly reduced the GSH/GSSG ratio (Figure 5(c)). Additionally, the expression level of GPX4, an enzyme that can effectively reduce lipid peroxide, was decreased (Figure 5(d)). Therefore, we considered that butein induced oxidative stress in human 143B cells by damaging the GSH antioxidant defense of the143B cells. However, the ROS level was significantly reduced when the ROS scavenger NAC was used (Figure 5(e)).OPEN IN VIEWER

Next, we investigated the effect of butein on the JNK signaling pathway. Our results showed that butein increased the phosphorylation level of JNK in concentration-dependent manner (Figure 5(f)).

To further investigated the roles of the JNK signaling pathway, we used NAC and SP were used. Notably, the CCK8 analysis indicated that NAC and SP significantly attenuated butein-induced cell viability inhibition (Figure 6(a)). Flow cytometric analysis demonstrated that NAC had an obvious inhibitory effect on butein-induced G2/M phase arrest (Figure 6(b)) and cell apoptosis, and SP also had an inhibitory effect on butein-induced apoptosis (Figure 6(c)). NAC is more effective in preventing cell death than SP. The activities of caspsase-3 were also obviously reversed by NAC treatment (Figure 6(d)). Interestingly, NAC pretreatment reduced butein-induced autophagy, which was determined by western blot analysis (Figure 6(e)). Furthermore, NAC blocked JNK phosphorylation (Figure 6(e)), whereas SP did not suppress ROS generation (Figure 5(e)). These results suggested that JNK activation following the enhanced ROS production contributed to butein-induced autophagy and apoptosis.OPEN IN VIEWER

Butein induced apoptosis and autophagy by blocking the Akt/mTOR signaling pathway in human 143B cells

To further understand the molecular mechanism of the antitumor effect of butein, we investigated the effect of butein on the Akt/mTOR signaling pathway. MK (a AKT inhibitor) was used. As shown in Figure 7(a), butein decreased the phosphorylation level of AKT and mTOR. The CCK8 assay exhibited that MK could strengthen the cell inhibitory effect (Figure 7(b)). Flow cytometric analysis demonstrated that MK had led to an obvious increase in cell apoptosis (Figure 7(c)). Additionally, pretreatment of OS cells with MK increased butein induced autophagy, which was determined by the western blot analysis. (Figure 7(d)) Furthermore, NAC increased the phosphorylation levels of Akt and mTOR (Figure 7(e)). These results suggest that butein could induce apoptosis and autophagy through the ROS-Akt/mTOR signaling pathway in human 143B cells.OPEN IN VIEWER