Crebanine with purity above 97% was provided by Chengdu pureChem-standard Corp. (Chengdu, China); Fetal bovine serum (FBS) and dulbecco’s modified Eagle’s medium (DMEM) were obtained from Gibco (Thermo Fisher Scientific, Inc. United States); LY294002 (PI3K Inhibitor) was purchased from MedChemExpress (NJ, United States); N-acetylcysteine (NAC) was obtained from Sigma Chemical (St. Louis, MO, United States); Hoechst 32258 was obtained from Wuhan Servicebio Biotechnology Co., Ltd. (Wuhan, China); CCK-8 kit was purchased from Biosharp (Anhui, China); the ROS assay kit and JC-1 kit were obtained from Beyotime Biotechnology (Shanghai, China); Superoxide Dismutase (SOD) assay kit、Malondialdehyde (MDA) assay kit、Glutathione Peroxidase (GSH-PX) assay kit were purchased from Nanjing Jiancheng bioengineering research institute (Nanjing, China); Annexin V/PI apoptosis detection kit was obtained from BD Biosciences (NY, United States); the monoclonal antibody against Bax, caspase-3, Bcl-2, AKT, p-AKT, FoxO3a, β-actin and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Cell Signaling Technology (CST, United States); the monoclonal antibody against caspase-9, p-FoxO3a (Ser253) were obtained from Proteintech (Rosmont, IL, United States).

The Shanghai Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences, Shanghai, China) provided HepG2 cells. Growth of the cells was carried out in DMEM containing 10% FBS, 1% penicillin-streptomycin (100 U/mL penicillin and 100 U/mL streptomycin), and 37°C in humidified 5% CO₂ incubator (Thermo Forma Electron Co, Marietta, OH, United States). Cells were subcultured with fresh media at a frequency of 1:4 every two to 3 days.

Briefly, about 1 × 10⁴ cells/wells were plated into 96-well plates and exposed to crebanine (35, 70, 105, 140, 175, and 280 µM) for 24, 48, 72 h in a 37°C incubator. The cells were cultured in 10 µl of CCK-8 solution for 1 h. Optical density was determined using a microplate reader at a wavelength of 490 nm after incubation. The IC50 values were determined by plotting linear regression curves.

About 4 × 10³ cells/well were seeded in 6-well plate overnight. Cells were exposed to crebanine treatment (0, 35, 70, 105, 140, 175, and 280 µM) for 24 h, and then the cells were grown for 14 days with the media being switched out every 3 days. On the 14th day, cells were fixed with 4% paraformaldehyde for 15 min and then stained with crystal violet dye for 25 min. Then, phosphate-buffered saline (PBS) was used to gently wash each well before allowing it to air dry. Colony numbers were then calculated using Image J software (National Institutes of Health, United States).

HepG2 cells were seeded into 6-well plates at a density of 6 × 10⁵/well. After overnight incubation, cells were treated with crebanine for 24 h. Then, changes in cell morphology were imaged under an inverted phase-contrast microscope (Olympus, Hamburg, Germany).

Using 24-well transwell chambers with or without 50 µL of matrigel, cell migration and invasive ability were assessed based on the number of cells passing through the chambers. HepG2 cells were trypsinized after crebanine treatment, and about 5 × 10⁴ cells/well were placed in the upper chamber of the transwell and 0.5 mL of complete medium in the lower chamber before being placed in a 37°C incubator. After 24 h, to remove upper chamber cells, gently wash the chamber twice with PBS using a sterile cotton swab. After that, the cells were fixed with paraformaldehyde, stained with 0.1% crystal violet dye for 20 min, and examined with an inverted phase contrast microscope.

Application of hoechst 33258 staining to observe the morphology of apoptosis. Briefly, 1 × 10⁵ cells per well were cultured in 6-well plates and then treated with different concentrations of crebanine for 24 h. Subsequently, cells were washed three times with PBS at room temperature, and being fixed with 4% paraformaldehyde for 20 min and stained with 5 μg/mL hoechst 33258 for 30 min. Morphological changes of apoptotic cells were photographed using an inverted phase-contrast microscope.

Annexin V- FITC/PI staining kit was used to measure apoptotic cells in this experiment. In 6-well plates, about 6 × 10⁵ cells/well were planted and incubated overnight, followed by a 24 h crebanine treatment. Subsequently, the cells were collected by centrifugation, gently washed twice with PBS, and stained with annexin V-FITC/PI for 25 min. Subsequently, the frequency of apoptotic cells was determined using a flow cytometer (CytoFLEX, Backman Counter, CA, United States) under the manufacturer’s instructions.

A fluorescent probe DCFH-DA was used in fluorescence microscopy and flow cytometry to measure changes in intracellular ROS level. About 6 × 10⁵ cells/well were seeded in 6-well plate overnight and then treated with crebanine for 24 h. Subsequently, the cells were collected by centrifugation, suspended in PBS, and loaded with 20 µM DCFH-DA at 37°C for 30 min. Following fluorochrome incubation, cells were washed twice with PBS and analyzed immediately with a flow cytometer using a FL-1 filter with an excitation wavelength of 480 nm.

Superoxide dismutase (SOD), glutathione (GSH-PX) and malondialdehyde (MDA) assay kits were used to detect intracellular levels of SOD, GSH-PX and MDA, respectively. In 6-well plates, about 6 × 10⁵ cells/well were planted and incubated overnight, followed by a 24 h crebanine treatment, then washed twice with ice-cold PBS and the supernatant was collected by centrifugation for 4 min, then the kit instructions were followed and finally detected on a microplate reader.

JC-1 assay kit was used to detect variation in mitochondrial membrane potential (MMP). A 6-well plate was seeded with about 5 × 10⁵ cells/well, and it was left incubating overnight. The cells were then exposed to crebanine at various doses for 24 h. Following that, cells were obtained by centrifugation, suspended in PBS, and dyed with JC-1 for 20 min at 37°C in accordance with the manufacturer’s instructions. The cells were then collected and given two rinses in JC-1 staining buffer (1×). A flow cytometer was used to immediately analyze stained cells.

Cells in 6-well plate were washed 3 times with PBS and fixed with 4% paraformaldehyde for 25 min; then, samples were treated in 0.2% Triton X-100 for 15 min and closed with PBS containing 2% BSA for 30 min at room temperature. After that, cells were incubated with primary antibody overnight at 4°C, followed by incubation with FITC-coupled secondary antibody for 2 h at room temperature. Cells were washed three times with PBS and fixed with PBS containing DAPI for 15 min. Cells were visualized under a fluorescent microscope.

In 6-well plates, about 6 × 10⁶ cells were cultivated per well, and they were incubated at 37°C overnight. Crebanine was applied to the cells for 24 h. The cells were then homogenized and dissolved in RIPA buffer in the presence of a protease inhibitor to extract cell proteins. Protein concentrations were measured using the BCA protein determination kit. Samples were separated on 10%–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electro-transfer onto PVDF membranes. The primary antibodies of caspase-3 (1:1000), caspase-9 (1:1000), PARP (1:1000), AKT (1:2000), p-AKT (1:1500), FoxO3a (1:1000), p-FoxO3a (Ser253) (1:1000), and β-actin (1:4000)—were then incubated on the PVDF membranes for at least an overnight period at 4°C. Following three 10-min TBST washes, the membranes were incubated for 2 h at room temperature with diluted horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000). The membranes were detected using the Odyssey Dual Color Infrared Laser Imaging System (LI-COR, United States) after three TBST washes lasting 10 min each. The outcomes were quantified with Image J software.

Data from at least three independent experiments are presented as the mean ± SD. All data were analyzed by the SPSS 17.0 software, Changes over time and differences between treatment and control groups were analyzed using a one-way analysis of variance (ANOVA) followed by the Tukey’s multiple comparison post-hoc test. If the p-value was less than 0.05, the difference was considered statistically significant.

Our study investigated the cytotoxicity of crebanine on HepG2 cells by treating them with varying concentrations (0, 35, 70, 105, 140, 175, and 280 µM) over 24–72 h. We analyzed the cytotoxicity of crebanine using CCK-8 assays. As shown in Figure 1B, HepG2 cells treated with crebanine showed a sharp decline in viability in a dose- and time-dependent manner compared with the control group. In addition, the IC50 value at 24, 48, and 72 h for cytotoxicity on HepG2 cells was 111.77 ± 5.40 µM, 65.07 ± 0.35 µM, 23.68 ± 2.04 µM respectively. These values showed that HepG2 cells were sensitive to crebanine treatment. Therefore, we chose 35, 105, and 175 µM of crebanine as the experimental group in our subsequent experiments. To determine population dependence and long-term proliferation capacity, cells were incubated with crebanine at concentrations of 35, 105, and 175 µM for 14 days before colony formation. In Figures 1C,D, crebanine significantly reduced HepG2 cells’ ability to form clones, indicating that it inhibited cancer cell proliferation over time and dose-dependently. Subsequently, the impact of crebanine on cell growth was then assessed by examining morphological alterations in HepG2 cells with an inverted microscope. As shown in Figure 1E, crebanine induced significant morphological changes in cancer cells. In the treatment groups, cell bodies generally became smaller and rounder than those of the control group; additionally, significant cellular debris appeared in the culture media, and cell count dropped. From these results, we can konw that crebanine inhibited both proliferation and death of cells.

We used transwell assays to examine crebanine’s impact on HepG2 cells’ capacity for invasion and migration. The findings of treating the cells for 24 h with various doses of crebanine showed that it considerably reduced the ability of HepG2 cells to invade and migrate, and higher crebanine concentrations had stronger inhibitory effects (Figures 2A,B). This finding suggested that crebanine suppressed the migration and invasion of HepG2 cells.

To determine whether crebanine’s cytotoxicity is due to apoptotic events, we first used Hoechst 33258 to observe nuclear morphological changes during apoptosis. As shown in Figure 2C, cancer cells in the control group were in a good condition-only slightly stained, whereas HepG2 cells in the crebanine-treated group showed a rapid decrease in viability, obvious cell shrinkage, chromatin concentration, and appearance of both fluorescent nuclear and apoptotic fragments. In other words, crebanine causes apoptosis in a dose-dependent way. In addition, we used the flow cytometry to identify a specific apoptosis rate. As can be seen in Figure 2D, crebanine treatment considerably increased the apoptosis rate of cancer cells compared to the control group, with the maximum apoptosis rate of 43.5% occurring in cells that received the highest dose of crebanine. This result further demonstrated the pro-apoptotic effect of crebanine.

In addition, to understand whether crebanine-induced apoptosis was due to excessive production of ROS in HepG2 cells, we detected intracellular ROS production using 2′,7′-dichlorodiacetate fluorescein (DCFH-DA) staining after drug administration. Based on the experimental results in Figures 3A,B, crebanine caused an increase in the level of ROS in hepatocellular carcinoma cells. In addition, SOD, GSH, and MDA changed with the level of oxidative stress, therefore, detecting their fluctuations was beneficial for us to better determine the effect of kebanine on cancer cells. According to the experimental results as shown in Figure 3C, we found that as the concentration of crebanine administration increased, while stimulating the production of ROS, the production of MDA also gradually increased, while SOD and GSH decreased accordingly, and they, as important compounds for maintaining the intracellular redox state, also proved the burst of oxidative stress caused by crebanine from the side.

Additionally, using the ROS inhibitor NAC substantially reduced the impact of increasing ROS production (4 mM) (Figures 3D,E), Also, the results of Figure 4A suggest that NAC as an anti-oxidative stress compound can modify the effects of SOD, MDA, and GSH on redox status to some extent. Also, the ability of crebanine to induce apoptosis in cancer cells was significantly reduced when we pretreated hepatocellular carcinoma cells with NAC (Figure 4B), confirming that crebanine could induce apoptosis by activating oxidative stress.

It has been reported that elevated ROS levels can induce apoptosis through a series of downstream pathways. Mitochondrial dysfunction is one of the major effects of ROS production, which first leads to a decrease in membrane potential, and then induces abnormal expression of apoptosis-related proteins such as PARP, Caspase-3, Caspase-9, Bax, Bcl-2 (Plackova et al., 2016). Therefore, to assess whether crebanine caused mitochondrial alterations in hepatocellular carcinoma cells, we examined the mitochondrial membrane potential. After administration of crebanine at different concentrations, flow cytometry results for this detection are shown in Figures 5A,B, the mitochondrial membrane potential of HepG2 cells treated with crebanine decreased in a concentration-dependent manner, suggesting the involvement of mitochondrial apoptosis pathway in crebanine-induced apoptosis. Western blot results in Figure 5C and the protein relative expression in Figure 5D showed that the levels of Bax, cleaved-PARP, cleaved-caspase-3 and cleaved-caspase-9 were significantly increased after crebanine treatment, whereas the expression level of Bcl-2 was decreased.

Furthermore, to verify whether mitochondrial disorders were due to oxidative stress, we examined mitochondrial membrane potential and the corresponding changes in the expression of these mitochondrial apoptosis-related protein markers after pretreatment with NAC. The results in Figures 6A,B show that NAC delayed the decay of mitochondrial membrane potential and also down-regulated cleaved PARP, cleaved caspase-9, cleaved caspase-3 and Bax, and up-regulated Bcl-2; as seen in Figures 6C,D. These findings provide strong evidence that while inducing ROS, crebanine also triggers mitochondrial dysfunction, further causing cell damage and death.

The PI3K/AKT signaling pathway plays a crucial role in the biological functions of growth, proliferation, apoptosis, autophagy, drug resistance, and angiogenesis in various diseases. AKT is a core regulator in the PI3K/AKT signaling pathway, when activated, AKT up-regulates various effector molecules, particularly its downstream target FoxO3a, to promote tumor development. Therefore, in this study, to mechanistically explore the anti-tumor toxicity of crebanine, the effect of crebanine on PI3K/AKT/FoxO3a pathway was examined.

The PI3K/AKT signaling pathway plays a crucial role in the biological functions of growth, proliferation, apoptosis, autophagy, drug resistance, and angiogenesis in various diseases. AKT is a core regulator in the PI3K/AKT signaling pathway, when activated, AKT up-regulates various effector molecules, particularly its downstream target FoxO3a, to promote tumor development. Therefore, in this study, to mechanistically explore the anti-tumor toxicity of crebanine, the effect of crebanine on PI3K/AKT/FoxO3a pathway was examined. As shown in Figure 6E, the expression level of AKT remained unchanged after crebanine treatment, whereas that of p-AKT decreased in a dose-dependent way. Although its downstream target FoxO3a was similarly unchanged, AKT inactivation directly down-regulated phosphorylated FoxO3a (Figure 7F).

We also pretreated HepG2 cells with NAC for 1 h to examine the impact of ROS generation on the AKT/FoxO3a signaling pathway. The results in Figures 7A,B showed that NAC partially reversed the previous trend of crebanine-induced downregulation of p-AKT and p-FoxO3a (Ser253), whereas AKT and FoxO3a were not significantly changed. This led us to hypothesize that the inhibition of the PI3K/AKT/FoxO3a pathway by crebanine was at least in part caused by the buildup of ROS. In addition, we used immunofluorescence experiments to localize p-FoxO3a (Ser253), and the experimental results shown in Figure 8A showed that p-FoxO3a (Ser253) was indeed more concentrated in the cytoplasm in the control group without the administration of crebanine, and the gradual decrease of p-FoxO3a (Ser253) was indeed inhibited under the conditions of NAC administration, which fully supported our above conclusion.

Besides, LY294002 is a common PI3K/AKT signaling inhibitor. A significant inhibition of AKT and FoxO3a phosphorylation after treatment with a combination of 35 mM LY294002 and crebanine was observed (Figures 6C,D), Similarly, the results of immunofluorescence experiments shown in Figure 8B also suggest that our LY294002 has a significant role in promoting the inhibition of p-FoxO3a (Ser253) cytoplasmic transfer by crebanine, further verifying the relationship between PI3K/AKT/FoxO3a pathway and crebanine-induced cytotoxicity. In conclusion, these results suggested that crebanine-induced cytotoxicity on HepG2 cells could occur through the PI3k/AKT/FoxO3a signaling pathway.