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Plants serve as abundant sources of medicinal compounds and have been extensively utilized for disease treatment since ancient times [1]. Currently, several drugs used for treating human ailments originate from plants [2]. Some plant-derived compounds, such as taxanes and vinca alkaloids, have been employed in cancer therapy [3]. Plants, as factories for producing bioactive metabolites, warrant further exploration. It has been reported that approximately 70% of the global population relies to some extent on plants for basic primary healthcare [4]. Among these metabolites, steroids are one category, with some steroids purported to possess anticancer, antimicrobial, and anti-inflammatory activities [5,6]. Among them, stigmasterol is a compound with significant pharmacological potential. Many of its biological activities are attributed to stigmasterol, including anticancer effects [7]. However, the anticancer activity of stigmasterol against Hepatocellular Carcinoma (HCC) has not been adequately evaluated. HCC is a prevalent malignant tumor associated with a grim prognosis, particularly in advanced stages, significantly endangering human health [8]. Moreover, there is a notable upward trend in HCC incidence among females, surpassing a 2% increase [9]. The main treatment modalities for HCC include surgical resection and chemotherapy, but clinical outcomes remain unsatisfactory. Meanwhile, anticancer drugs used for treating HCC may lead to adverse reactions, negatively impacting the overall health of patients.

This study discovered that stigmasterol inhibits the proliferation of Huh7 and Hep3B cancer cells. The TLR4/MyD88/NF-κB pathway, known for its pivotal role in the proliferation and tumorigenesis of various cancers, has been validated in cancer cells. Here, we investigated stigmasterol's anticancer effect on hepatocellular carcinoma cell lines by evaluating the TLR4/MyD88/NF-κB signaling pathway.

Molecular docking

Using molecular docking techniques, we examined the interactions between core compounds and targets in the treatment of hepatocellular carcinoma (HCC). The structures of target proteins were downloaded from the PDB website (http://www.rcsb.org/), and the 3D structures of core compounds were obtained from the TCMSP database. Molecular docking and conformational scoring were performed using AutoDock software. Structural diagrams of the optimal docking results were generated using PyMOL.

Cell cultures, chemicals and reagents

Huh7 and Hep3B human hepatocellular carcinoma cell lines were purchased from HaiXing Biotechnology Co., Ltd. They were maintained in complete culture medium comprising 10% Fetal Bovine Serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were cultured in a CO2 incubator at 37°C with 98% humidity and 5% CO2 . Stigmasterol (98% purity by HPLC) and all other chemicals were purchased from KeBo Biotech Co., Ltd. (Lanzhou, Gansu, China).

CCK-8 assay

The impact of stigmasterol on Huh7 and Hep3B liver cancer cell proliferation was assessed using the CCK-8 assay. Initially, 5 × 103 cells were seeded into 96-well plates and cultured at 37°C with 5% CO2 . During incubation, cells were exposed to stigmasterol at concentrations ranging from 0 to 400 μM overnight. Following treatment, 10 μL of CCK-8 reagent was added to each well and incubated at 37°C for 50 minutes. Absorbance values were measured at 450 nm using a microplate reader. Cell proliferation rates were calculated relative to the optical density of the untreated control group.

Wound healing assay

Cells were cultured in DMEM or MEM complete medium supplemented with 10% FBS. Huh7 and Hep3B cells were seeded at a density of approximately 1 × 10⁶ cells per well in 6-well plates and allowed to reach 70-80% confluence after 24 hours of incubation. Subsequently, a scratch assay was conducted by creating vertical scratches in the center and on both sides of each well using a new 200 μL pipette tip. After aspirating the old medium, cells were washed with PBS and then treated with stigmasterol at concentrations of 0 μM, 100 μM, 200 μM, and 400 μM, with 4 mL per well. Following a further 48-hour incubation, the culture medium was aspirated, and images were captured. Data analysis was performed using ImageJ and GraphPad software.

Tanswell cell invasion assay

The Matrigel was dissolved at room temperature, and Transwell chambers were positioned at the center of a 24-well culture plate. Fifty microliters of dissolved Matrigel were applied to the upper chamber of each Transwell, followed by incubation in a 37鈩� cell culture incubator for 15 minutes. Huh7 and Hep3B cells in logarithmic phase were harvested and adjusted to a concentration of approximately 5 × 105 cells/mL using serum-free DMEM or MEM medium. Two hundred microliters of cell suspension containing varying concentrations (0, 100, 200, 400 μM) of diosgenin were added to the upper chamber, while the lower chamber received 600 μL of DMEM medium supplemented with 10% fetal bovine serum. Each experimental group was replicated three times and then placed in a 37鈩�, 5% CO₂ cell culture incubator for 48 hours.

Following incubation, the supernatant was removed, and cells on the upper side of the membrane were gently wiped off with a cotton swab. Subsequent to PBS washing (2-3 times), cells were fixed with polyformaldehyde for 30 minutes and stained with crystal violet for an additional 30 minutes. Three random fields were selected for photography, and cell counting was performed using Image J software.

Colony formation assay

Huh7 and Hep3B cells were initially seeded at a density of 200 cells per well. Following a 24-hour incubation period to promote attachment, the cells were subjected to different concentrations (0, 100, 200, and 400 µM) of stigmasterol. Subsequently, after 15 days of cell culture, the cells underwent two washes with phosphate-buffered saline (PBS) and were fixed with paraformaldehyde to facilitate colony formation. The resulting colonies were then stained with crystal violet for around 30 minutes and quantified using an optical microscope.

Apoptosis assay

Following a 48-hour treatment with various concentrations of stigmasterol (0, 100, 200, 400 µM) in Huh7 and Hep3B cell cultures, the culture medium was aspirated, and cells underwent trypsinization without EDTA. Subsequently, the cells were harvested into centrifuge tubes. Afterward, they underwent two washes with pre-chilled PBS (centrifuged at 12,000 rpm for 5 minutes to eliminate the supernatant), followed by the gradual addition of 500 μL of binding buffer into each centrifuge tube to generate cell suspensions. Then, Annexin V-FITC (5 μL) and Propidium Iodide (PI) (5 μL) were sequentially introduced into each centrifuge tube, gently mixed by pipetting, and left to incubate in darkness at room temperature for 5 to 15 minutes. Flow cytometry analysis was conducted within 1 hour.

Cell cycle analysis

Following a 48-hour treatment of Huh7 and Hep3B cell cultures with varying stigmasterol concentrations (0, 100, 200, 400 µM), the subsequent procedures were executed: Initially, cells underwent two rounds of pre-chilled PBS washing and subsequent centrifugation for cell counting. Subsequently, cells were adjusted to a density of approximately 1 × 10₆ cells/mL, suspended in 75% pre-chilled ethanol, and incubated overnight at -20°C for fixation. The fixed cells were then retrieved, subjected to centrifugation to eliminate the supernatant, washed twice with PBS, and resuspended in PBS. Following this, 50 μL of PI staining solution was applied for staining for 30 minutes, after which the staining solution was washed off, and the cells were resuspended once more in PBS. Ultimately, flow cytometry was utilized to analyze cell cycle distribution.

Western blotting

Proteins were extracted using RIPA buffer supplemented with protease inhibitors, and their concentrations were determined using the BCA assay kit. Afterward, equal amounts of protein were loaded onto SDS-PAGE gel and transferred onto PVDF membranes. To prevent nonspecific binding, the membrane-bound proteins were blocked with 5% non-fat milk in TBST buffer. Primary antibodies, tagged with various markers specific to their antigens, were then applied and incubated for 2 hours at 37°C. After three washes, secondary antibodies were added and incubated for 1 hour at 37°C. Protein immunoblotting was subsequently conducted using an enhanced chemiluminescence detection kit (Thermo Scientific, Rockford, IL, USA), with β-actin serving as an internal reference for normalization.

Statistics

Statistical analysis was carried out utilizing GraphPad software (version 9.0), and findings are depicted as mean ± standard deviation. Group comparisons were conducted via one-way Analysis of Variance (ANOVA) for multiple groups, while paired comparisons between two groups were evaluated utilizing the LSD method. A significance threshold of α=0.05 was established, and discrepancies were deemed statistically significant when the p-value was below 0.01.

Validation results of the interaction between stigmasterol and the TLR4/MyD88/NF-κB signaling pathway-associated protein molecules

According to reference, smaller binding energies indicate better docking results. The heatmap results demonstrate that stigmasterol exhibits favorable binding affinity with the target proteins in the TLR4/MyD88/NF-κB signaling pathway. Visualization using PyMOL software reveals hydrogen bonding interactions between the receptor proteins and the ligand small molecules. Refer to Figure 1 and Table 1 for more details.

 

Figure 1: Illustrates the visualization of the docking between stigmasterol and core proteins of the TLR4/MyD88/NF-κB signaling pathway.

Table 1: Presents the binding energies of stigmasterol with target proteins.

Stigmasterol inhibits the proliferation of liver cancer Huh7 and Hep3B cells

The results of treating Huh7 and Hep3B cells with different concentrations of stigmasterol, as detected by the CCK-8 assay, show a gradual decrease in cell proliferation activity with increasing stigmasterol concentrations (0, 100, 200, and 400 µM). Compared to the normal control group, the differences in each stigmasterol concentration group were statistically significant (P<0.01), as detailed in Figure 2. After 48 hours of treatment with stigmasterol, 400 µM exhibited the strongest inhibitory effect on both Huh7 and Hep3B cells. Therefore, we selected concentrations of 100, 200, and 400 µM as experimental concentrations for stigmasterol.

 

Figure 2: A) Survival dose-effect of Hep3b; B) Aging values of Hep3B survival rate; C) survival dose-effect of Huh7; D) Aging values of Huh7 survival rate.

Note: The survival rate of the control group was set to 1. The survival rate of the experimental group was calculated as the ratio of the number of cells in the experimental group to the number of cells in the control group. Statistical significance was indicated as follows: **for P<0.01, ***for P<0.001, and ****for P<0.0001.

The inhibitory effect of stigmasterol on the migratory capability of Huh7 and Hep3B cells

After treating Huh7 and Hep3B cell cultures with different concentrations of stigmasterol for 48 hours, the migratory capability of the cells was evaluated using the cell scratch and Transwell chamber assays. The results demonstrate that compared to the normal control group, the migratory ability of Huh7 and Hep3B cells is significantly inhibited following stigmasterol treatment (Figure 3), with differences being statistically significant (P<0.01).

 

Figure 3: A) Effect of stigmasterol on migration of Hep3B and Huh7; B) Wound healing rate of Hep3B; C) Wound healing rate of Huh7.

Note: L: 100 μM, M: 200 μM, H: 400 μM, *** : P<0.001, **** : P<0.0001.

The inhibitory effect of stigmasterol on the clonogenic ability of Huh7 and Hep3B cells

Following treatment with different concentrations of stigmasterol for 48 hours, the clonogenic ability of Huh7 and Hep3B cells was assessed using a colony formation assay. The results indicate that compared to the normal control group, the clonogenic capacity of Huh7 and Hep3B cells is suppressed after stigmasterol treatment (Figure 4), with statistically significant differences (P<0.01).

 

Figure 4: A) Effect of Stigmasterol on clonal formation of Hep3B and Huh7; B) Number of Hep3B clones formed; C) Number of Huh7 clones formed.

Note: L: 100 μM, M: 200 μM, H: 400 μM, **:P<0.01 锛� ***:P<0.001, ****:P<0.0001.

Effect of stigmasterol on invasion of Huh7 and Hep3B cells

Compared with the control group, the inhibitory effect of stigmasterol on the invasion of Hep3B and Huh7 cells was enhanced in the L, M, and H groups after 48 h of stigmasterol intervention. Particularly, the concentration of 400 μM of stigmasterol showed the most significant enhancement of inhibitory effect after 48 h of intervention, which was statistically significant (p<0.0001) (Figure 5).

 

Figure 5: A) Influence of stigmasterol on invasion of Hep3B and Huh7; B) Relative invasion count of Hep3B; C) Relative invasion counts of Huh7.

Note: L: 100 μM, M: 200 μM, H: 400 μM, **: P<0.01锛�***: P<0.001, ****: P<0.0001.

The effect of stigmasterol on apoptosis and cell cycle of Huh7 and Hep3B cells

Following treatment with different concentrations of stigmasterol for 48 hours, the apoptosis rate and cell cycle of Huh7 and Hep3B cells were analyzed using annexin V-FITC/PI flow cytometry. The results demonstrate a significant increase in apoptosis rate compared to the normal control group across all stigmasterol concentration groups (P<0.01, Figure 4), while the cell cycle is arrested compared to the normal control group (P<0.01, Figure 6A-F).

 

Figure 6: A) Effects of stigmasterol on apoptosis of Hep3B and Huh7; B) Apoptosis rate of Hep3B; C) Apoptosis rate of Huh7; D) Distribution of stigmastero's influence on Hep3B and Huh7 cycles; E) Cell cycle distribution of Hep3B; F) Cell cycle distribution of Huh7.

Note: L: 100 μM, M: 200 μM, H: 400 μM, * : P<0.05, **: P<0.01锛� ***: P<0.001, ****: P<0.0001.

The impact of stigmasterol on the expression of TLR4/ MyD88/NF-κB signaling pathway-associated proteins in Huh7 and Hep3B cells

Following treatment with different experimental concentrations (0, 100, 200, 400 µM) of stigmasterol for 48 hours, the expression levels of TLR4, MyD88, and NF-κB proteins in Huh7 and Hep3B cells were examined using Western blot analysis. The results indicate that compared to the control group, the expression of TLR4, MyD88, and NF-κB is downregulated in all experimental concentration groups (P<0.01) (Figure 7).

 

Figure 7: A) Protein expression in Hep3B group; B) Protein expression in Huh7 group; C) Relative expression of TLR4 in Hep3B; D) Relative expression of TLR4 protein in Huh7; E) Relative expression of MyD88 in Hep3B; F) Relative expression of protein MyD88 in Huh7; G) Relative expression of NF-κB in Hep3B; H) Relative expression of NF-κB in Huh7.

Note: L: 100 μM, M: 200 μM, H: 400 μM, * : P<0.05锛�**: P<0.01锛�***: P<0.001, ****: P<0.0001.

Natural products have long been used for the treatment of various diseases and conditions. Different plant extracts and decoctions have been employed to treat various pathological conditions in different medical systems, including traditional medical systems in China, as well as Ayurvedic and Unani medicine. In most cases, treatments based on these traditional remedies have produced very effective results. Indeed, researchers worldwide have leveraged the knowledge from these traditional systems to isolate compounds effective for treating various pathological conditions. Several compounds currently used as drugs have been isolated from plants. For instance, compounds such as podophyllotoxin, taxane drugs, and several others have been isolated from plants and have shown significant therapeutic potential.

In this study, the anticancer effects of stigmasterol on liver cancer Huh7 and Hep3B cells were examined, demonstrating the ability of this compound to inhibit the proliferation and colony-forming potential of these cells. These results align with other studies in which plant-derived sterols have been reported to possess anticancer properties. For example, stigmasterol has been shown to inhibit the proliferation of breast cancer cells.

Apoptosis and cell cycle arrest are two important mechanisms through which anticancer drugs exert their antiproliferative effects on cancer cells. While apoptosis completely eliminates cancer cells from the body, cell cycle arrest prevents the division of these cells, leading to lethal outcomes. In this study, we observed that stigmasterol triggered apoptosis and G2/M cell cycle arrest in Huh7 and Hep3B cancer cells.

Our findings are also supported by previous studies that demonstrated the ability of sterols to induce apoptosis and cell cycle arrest in cancer cells. For example, a plant-derived sterol, γ-sitosterol, has been shown to induce cell cycle arrest and apoptosis in cancer cells.

In this study, we found that stigmasterol can inhibit the metastatic potential of liver cancer cells. The TLR4/MyD88/NF-κB signaling pathway is highly activated in cancer cells and plays a role in tumor progression. Interestingly, in this study, we observed that stigmasterol can inhibit the TLR4/MyD88/NF-κB signaling pathway, suggesting that stigmasterol could be a potential candidate for the treatment of liver cancer.

The images in this article are processed by Photoshop software

Chun Yu carried out experiments and wrote the manuscript. Yanhua Ma provides technical guidance. All authors have seen and approved the fnal version of the manuscript being submitted.

The paper was co-funded by the National Natural Science Foundation of China (81860821) and the Gansu Joint Research Foundation (23JRRA1523).

In the manuscript paper.

Not applicable.

Not applicable.

The authors declare that they have no competing interests.

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