Inhibitory effect of bufalin combined with Hedgehog signaling pathway inhibitors on proliferation and invasion and metastasis of liver cancer cells
Abstract
Bufalin, a topoisomerase II inhibitor, has garnered significant attention for its potential anticancer properties, particularly in hepatocellular carcinoma (HCC), a malignancy notorious for its late-stage diagnosis and limited treatment options. With only 30% of cases eligible for surgical resection and molecular targeted therapies benefiting a similarly small fraction of patients, alternative therapeutic strategies are urgently needed.
Recent investigations have demonstrated that bufalin effectively suppresses the proliferation, invasion, and metastatic potential of liver cancer cells through modulation of the Hedgehog (Hh) signaling pathway. In vitro studies utilizing the highly metastatic LM3 hepatoma cell line (HCC-LM3) revealed that treatment with bufalin, either alone or in combination with Hedgehog pathway inhibitors such as GANT61 and cyclopamine, significantly impeded tumor progression over a 72-hour period.
Mechanistically, bufalin was found to inhibit epithelial-mesenchymal transition (EMT), extracellular matrix (ECM) degradation, and angiogenesis by altering the expression of key proteins within the Hedgehog signaling cascade, including Ptch1, Gli1, and Gli3. Additionally, bufalin downregulated critical downstream targets such as MMP-2, MMP-9, β-catenin, and VEGF, which are known to facilitate tumor growth and metastasis. Conversely, it upregulated E-cadherin expression via Gli3 modulation, reinforcing cellular adhesion and reducing metastatic potential.
These findings suggest that bufalin, when combined with Hedgehog pathway inhibitors, can significantly diminish the aggressive biological behavior of liver cancer cells, offering a promising avenue for therapeutic intervention in HCC.
Introduction
Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide, with an annual incidence exceeding 600,000 cases. It ranks as the third leading cause of cancer-related mortality, accounting for 18.8% of deaths across all malignant tumors. In China, the burden of HCC is particularly severe, where it holds the second-highest cancer mortality rate and contributes to 53% of global deaths associated with liver cancer. Standard surgical treatments, such as liver resection and transplantation, are the primary therapeutic options, yet early diagnosis remains challenging due to the concealed pathogenesis and rapid progression of the disease, limiting the resection rate to just 30%.
Adding to the complexity of HCC treatment is its inherent resistance to conventional chemotherapy. Cytotoxic agents offer minimal survival benefits for patients with advanced disease and are further hindered by severe toxic effects on vital organs, including the heart, liver, and kidneys. While molecular targeted therapies have emerged as potential alternatives, they provide clinical benefits to only approximately 30% of patients. Additionally, HCC is characterized by early metastasis, with postoperative recurrence rates ranging from 40% to 70%, significantly impacting patient survival and quality of life. These factors highlight the urgent need for novel, low-toxicity drugs capable of effectively suppressing cellular proliferation, invasion, and metastasis.
Recently, increasing attention has been directed toward traditional Chinese medicines and their bioactive compounds as promising candidates for HCC therapy. Bufalin, a major digoxin-like immunoreactive component derived from Chan Su—a traditional Chinese medicine obtained from the skin and parotid venom glands of toads—has demonstrated significant pharmacological effects, including anticancer, anti-radiation, anti-clotting, analgesic, and cardiotonic properties. As a topoisomerase II inhibitor, bufalin has been extensively studied for its ability to hinder tumor progression. Research has shown that bufalin can inhibit proliferation and angiogenesis, promote cellular differentiation and apoptosis, reverse drug resistance, modulate gene expression in tumor cells, and regulate the immune system.
Previous investigations have indicated that bufalin markedly suppresses the proliferation, invasion, and metastasis of liver cancer cells by inducing cell cycle arrest at the S and G2 phases in BEL-7402 hepatoma cells, demonstrating a time- and dose-dependent inhibitory effect. However, the precise mechanisms underlying bufalin’s anti-tumor activity in hepatocellular carcinoma remain incompletely understood. The Hedgehog (Hh) signaling pathway, originally identified in Drosophila gene mutation research by Wieschaus and Nusslein-Volhard in 1980, is a highly conserved cellular signaling system crucial for regulating cell growth and survival. Studies have established the involvement of Hh signaling in the development of various malignancies, including gastric, pancreatic, and liver cancer.
To further investigate bufalin’s potential, this study examined whether it can impede the growth, invasion, and metastatic behavior of liver cancer cells by modulating the Hedgehog signaling pathway. Using in vitro culturing of human high-metastasis potential LM3 hepatoma cells (HCC-LM3), experimental findings revealed bufalin’s ability to influence key molecular pathways that drive tumor progression. These results suggest that bufalin may represent a promising therapeutic strategy for limiting HCC aggressiveness by targeting Hedgehog signaling, paving the way for further investigations into its clinical applicability.
Materials and methods
Reagents
Bufalin was obtained from Sigma Chemical Co. and prepared as a 10⁻¹ mol/L solution in anhydrous alcohol, stored at 4˚C to maintain stability. High-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS), essential for cell culture experiments, were sourced from Gibco. Various antibodies targeting key proteins involved in cellular signaling and tumor progression were acquired for experimental analysis. Specifically, β-actin, SHH, PTCH1, Gli1, MMP-2, E-cadherin, and β-catenin antibodies were procured from Cell Signaling Technology, while Gli3 antibody was obtained from Milipore. Additionally, MMP-9 and VEGF antibodies were purchased from Abcam.
To investigate the role of Hedgehog (Hh) signaling in cancer progression, selective pathway inhibitors were incorporated into the study. GANT61, an inhibitor specifically targeting Gli1 protein expression, and cyclopamine, which acts as a Smo protein inhibitor, were sourced from Selleck Chemicals LLC. These inhibitors enabled a precise modulation of the Hh pathway, facilitating an in-depth analysis of bufalin’s effects on liver cancer cells.
Cell lines
HCC-LM3 cells were obtained from the Liver Cancer Institute of Zhongshan Hospital Affiliated to Fudan University. The cells were cultured in high glucose DMEM supplemented with 10% FBS, 100 U/ml of penicillin and 100 µg/ml of streptomycin in a humidified atmosphere with 5% CO2 in air at 37˚C. Then, the cells in the logarithmic growth phase were collected for the following experiments.
Grouping
HCC-LM3 cells were cultured in vitro, they were divided into the control group, the bufalin group, the bufalin+GANT61 group, the bufalin+cyclopamine group and the bufalin+GANT61+cyclopamine group according to the experimental requirements.
Drug concentration
According to our previous studies (15), the 0.04 µg/ml of bufalin was chosen as an effective concentration. Following the manufacturer’s instructions and the related experiments, the 2.148 µg/ml of GANT61 and the 0.412 µg/ml of cyclopamine were chosen as effective concentrations, respectively.
Cell proliferation assay
The Cell Counting Kit-8 (CCK-8) assay, sourced from Dojindo Molecular Technologies, was utilized to assess cell proliferation. Cells in their logarithmic growth phase were seeded at a density of 100,000 cells per milliliter, with 100 µl distributed per well in 96-well plates. After an initial incubation period of 12 hours, the cells were exposed to different drug concentrations for analysis.
To establish a comparative framework, several experimental groups were prepared. The blank group received 200 µl of Dulbecco’s Modified Eagle’s Medium (DMEM) alone, serving as a baseline control. The control group was similarly treated with 200 µl of DMEM, ensuring that external variables did not interfere with the study. The bufalin group received 200 µl of bufalin solution, while the bufalin+GANT61 group was exposed to both 200 µl of bufalin and 200 µl of GANT61. Another set of cultures, designated as the bufalin+cyclopamine group, received a combination of 200 µl of bufalin and 200 µl of cyclopamine. Additionally, a bufalin+GANT61+cyclopamine group was prepared, in which cells were treated with 200 µl of bufalin, 200 µl of GANT61, and 200 µl of cyclopamine.
The final concentrations of bufalin, GANT61, and cyclopamine in the culture medium were precisely controlled at 0.04, 2.148, and 0.412 µg/ml, respectively. After a 72-hour incubation period, cell viability was assessed according to the manufacturer’s instructions using the CCK-8 assay. To ensure the reliability of the data, each experiment was performed in triplicate.
The cell inhibition ratio was calculated using the following formula:
Cell inhibition ratio (%) = [1 – (average absorbance of the treated group – average absorbance of the blank group) / (average absorbance of the control group – average absorbance of the blank group)] × 100%.
These measurements provided a quantitative evaluation of drug efficacy in inhibiting cell proliferation, offering insight into potential therapeutic applications.
Flow cytometry for cell cycle phase
Cell cycle analysis was conducted using flow cytometry in conjunction with a DNA content quantitation assay kit. Cells in the logarithmic growth phase were seeded at a density of 100,000 cells per milliliter, with 100 µl allocated per well in 6-well plates. After a 12-hour initial incubation, cells were subjected to various drug treatments designed to assess their response to bufalin and Hedgehog pathway inhibitors.
To ensure consistency across experimental conditions, multiple groups were established. The control group received 2 ml of Dulbecco’s Modified Eagle’s Medium (DMEM), serving as a baseline comparison. The bufalin group was treated with 2 ml of bufalin solution, while another set of cultures received both 2 ml of bufalin and 2 ml of GANT61 (bufalin+GANT61 group). Similarly, a separate group was exposed to 2 ml of bufalin and 2 ml of cyclopamine (bufalin+cyclopamine group). The final experimental condition involved a combined treatment with 2 ml of bufalin, 2 ml of GANT61, and 2 ml of cyclopamine (bufalin+GANT61+cyclopamine group). The respective concentrations of bufalin, GANT61, and cyclopamine in the culture medium were precisely maintained at 0.04, 2.148, and 0.412 µg/ml.
Following a 72-hour drug incubation period, cells were harvested and processed through a series of preparatory steps for flow cytometry analysis. This involved centrifugation at 2000 rpm for 5 minutes, followed by two washes in cold phosphate-buffered saline (PBS) to remove residual media. Cells were then fixed in 70% ethanol and stored at 4˚C for two hours. After additional washing with PBS, fixed cells underwent incubation with RNase at 37˚C for 30 minutes to degrade any residual RNA before being stained with 400 µl of propidium iodide (PI) at 4˚C for 30 minutes in a dark environment to visualize DNA content.
Each sample was subsequently analyzed using CellQuest software to determine the percentage of cells in the G1, S, and G2 phases of the cell cycle. To ensure reliability, all experiments were performed in triplicate, providing robust and reproducible insights into the effects of bufalin and Hedgehog pathway inhibitors on cellular progression.
Analysis of cell apoptosis
Cells in the logarithmic growth phase were plated at a density of 10×104 cells/ml, then, 100 µl/well in 6-well plates. Twelve hours later, the cells were treated with various concentrations of all drugs. A total of 2 ml DMEM was injected into culture plate in the control group, 2 ml bufalin was injected into culture plate in the bufalin group, 2 ml bufalin and 2 ml GANT61 were injected into culture plate in the bufalin+GANT61 group, 2 ml bufalin and 2 ml cyclopamine were injected into culture plate in the bufalin+cyclopamine group, 2 ml bufalin, 2 ml GANT61 and 2 ml cyclopamine were injected into culture plate in the bufalin+GANT61+cyclopamine group.
The final concentrations of buffalin, GANT61 and cyclopamine added to the cell culture medium for each group were 0.04, 2.148 and 0.412 µg/ml, respectively. After 72 h of treatment with different agents, cells were harvested, centrifuged and washed twice with cold 0.1 M PBS at 2000 rpm for 5 min, then suspended in 100 µl 1X binding buffer. Next, cells were incubated with 5 µl Annexin V and 5 µl PI for 15 min at 25˚C in dark room. Finally, each sample was injected with 400 µl 1X binding buffer, phase distributions of the cell cycle and hypodiploid DNA were determined by flow cytometry and Annexin V-FITC apoptosis detection kit I (BD Biosciences, San Jose, CA, USA). Then assessed for cell apoptosis by CellQuest software (Becton-Dickinson). Each experiment was performed in triplicate.
Analysis of cell migration and invasion
Cells in the logarithmic growth phase were plated at a concentration of 100,000 cells per milliliter, with 100 µl added to each well in 6-well plates. After 12 hours of incubation, different drug treatments were applied to assess their effects on cellular behavior. Experimental groups were carefully structured to compare responses to various conditions. The control group received 2 ml of Dulbecco’s Modified Eagle’s Medium (DMEM), while the bufalin group was exposed to 2 ml of bufalin solution. Other groups included bufalin combined with GANT61, bufalin with cyclopamine, and a combined treatment with bufalin, GANT61, and cyclopamine, each receiving 2 ml of their respective solutions. The final concentrations of bufalin, GANT61, and cyclopamine were maintained at 0.04, 2.148, and 0.412 µg/ml, respectively.
To evaluate cell migration, a Transwell permeable support system was employed, utilizing 24-well Transwell filters composed of polyvinylidene fluoride with an 8 µm pore size. After 72 hours of drug exposure, cells were harvested, resuspended in fresh DMEM at a density of 1,000,000 cells per milliliter, and transferred into the upper chamber at 100 µl per well. A serum-containing medium of 500 µl was simultaneously added to the lower chamber to facilitate migration. Following a 48-hour incubation period, non-migratory cells on the upper surface of the filter were removed using cotton swabs, while those that successfully traversed the Matrigel to the lower membrane were stained with Giemsa for 10 minutes. The number of migrated cells was quantified across five fields per triplicate filter using an inverted microscope. The cell migration ratio was calculated as follows:
Cell migration ratio (%) = (number of migrated cells in the treated group / number of migrated cells in the control group) × 100%.
A similar approach was used for the cell invasion assay, with the key distinction being the inclusion of Matrigel in the wells. Matrigel was diluted at a 1:4 ratio in DMEM and allowed to set for 24 hours prior to cell seeding, ensuring an additional barrier for assessing invasive potential. The number of invaded cells was quantified using the same microscopy method, with the cell invasion ratio determined through:
Cell invasion ratio (%) = (number of invaded cells in the treated group / number of invaded cells in the control group) × 100%.
These assays provided insights into the effects of bufalin and Hedgehog pathway inhibitors on cellular migration and invasion, offering a valuable perspective on their potential applications in cancer research.
Cell adhesion assay
The 96-well flat-bottomed plates were precoated with 50 µl/well of 1:4 DMEM-diluted Matrigel at 4˚C overnight. Cells in the logarithmic growth phase were plated at a density of 10×104 cells/ml, then 100 µl/well in 6-well plates. Twelve hours later, the cells were treated with various concentrations of the drugs. A total of 2 ml DMEM was injected into culture plate in the control group, 2 ml bufalin was injected into culture plate in the cufalin group, 2 ml bufalin and 2 ml GANT61 were injected into culture plate in the bufalin+GANT61 group, 2 ml bufalin and 2 ml cyclopamine were injected into culture plate in the bufalin+cyclopamine group, 2 ml bufalin, 2 ml GANT61 and 2 ml cyclopamine were injected into culture plate in the bufalin+GANT61+cyclopamine group.
The final concentrations of buffalin, GANT61 and cyclopamine added to the cell culture medium for each group were 0.04, 2.148 and 0.412 µg/ml, respectively. After 72 h of treatment with different agents, cells were harvested and suspended in new DMEM with 10% FBS at a density of 1×106 cells/ml. Then, the collected cells were seeded into a 96-well plate at 100 µl/ well, blocking at 37˚C for 2 h. Only 100 µl of DMEM without cells was injected in each well as a blank group. The average numbers of adhered cells were counted using the CCK-8. The 96-well plate was put into enzyme-labeled instrument, and OD value was obtained by 450 nm wavelength detection. Each experiment was performed in triplicate. The cell adhesion ratio was calculated by the following formula: Cell adhesion ratio (%) = (average OD value of treated group/average OD value of control group) x 100%.
Protein expression assay with western blot technique. Cells in the logarithmic growth phase were plated at a density of 10×104 cells/ml, then 5 ml/flask in cell culture flask. Twelve hours later, the cells were treated with various concentrations of the drugs. A total of 2 ml DMEM was injected into culture flask in the control group, 2 ml bufalin was injected into culture flask in the bufalin group, 2 ml bufalin and 2 ml GANT61 were injected into culture flask in the bufalin+GANT61 group, 2 ml bufalin and 2 ml cyclopamine were injected into culture flask in the bufalin+cyclopamine group, 2 ml bufalin, 2 ml GANT61 and 2 ml cyclopamine were injected into culture flask in the bufalin+GANT61+cyclopamine group.
The final concentrations of buffalin, GANT61 and cyclopamine added to the cell culture medium for each group were 0.04, 2.148 and 0.412 µg/ml, respectively. After cultured for 72 h, cells in various groups were, respectively, washed with ice-cold 0.01 M PBS and extracted in protein lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Protein concentrations were assayed with the BCA protein assay kit (Beyotime Institute of Biotechnology). Protein samples of cell lysates were mixed with 5X sodium dodecyl sulfate (SDS) loading buffer (dilution 1:4), boiled for 5 min, then separated on 10% SDS polyacryl- amide gels. After electrophoresis, proteins were transferred onto polyvinylidene fluoride membranes, blocked in 5% non-fat dry milk in phosphate-buffered saline with Tween-20 (PBST) for 1 h, and incubated with corresponding antibodies against β-actin (dilution 1:2,000), SHH (dilution 1:1,000), PTCH1 (dilution 1:1,000), Gli1 (dilution 1:1,000), Gli3 (dilution 1:1,000), E-cadherin (dilution 1:1,000), β-catenin (dilution 1:1,000), MMP-2 (dilution 1:1,000), MMP-9 (dilution 1:5,000) and VEGF (dilution 1:2,000) overnight at 4˚C.
The membranes were washed three times with PBST and incubated for 1 h at room temperature with a horseradish peroxidase-conjugated secondary antibody (dilution 1:1,000). After washing again three times with PBST, blots were incubated with ECL plus chemiluminescence substrate (Millipore, Billerica, MA, USA) and digital images were acquired using a ChemiDoc system employing Quantity One software (Shanghai Peiqing, Shanghai, China). Three independent blots were analyzed for each protein.
Statistical analysis
Data were analyzed using analysis of variance (SPSS 18.0 and Graphpad Prism 5; SAS Institute, Inc., Cary, NC, USA). Data are expressed as the mean values ± standard of the mean. P-values of <0.05 were consid- ered statistically significant. Results Bufalin demonstrates a potent inhibitory effect on the proliferation of HCC-LM3 cells. The CCK-8 assay results indicate that bufalin alone reduces cell proliferation by 33.68%, whereas its combination with Hedgehog (Hh) signaling pathway inhibitors further enhances this effect. The bufalin+GANT61 group shows a 38.28% inhibition rate, bufalin+cyclopamine leads to a 58.30% inhibition rate, and the bufalin+GANT61+cyclopamine group exhibits the strongest suppression at 72.71%. Statistical analysis confirms that inhibiting key proteins in the Hh pathway, such as Gli1 and Smo, significantly strengthens bufalin’s antiproliferative effect, with simultaneous inhibition of both proteins yielding the most pronounced result. Bufalin also influences the cell cycle of HCC-LM3 cells, specifically by inducing a blockade in the S and G2 phases, limiting cell cycle progression. Previous studies have shown that bufalin progressively decreases the percentage of G1 phase cells in a time- and dose-dependent manner. In this study, the bufalin+GANT61+cyclopamine group displayed the lowest proportion of G1 phase cells, while the percentage of G2+S phase cells increased correspondingly across different experimental conditions. The G2+S phase percentage rose from 38.05% in the control group to 69.76% in the bufalin+GANT61+cyclopamine group, with all bufalin-combined treatments showing significantly higher G2+S phase accumulation compared to bufalin alone. These findings reinforce bufalin's ability to disrupt cell cycle progression by targeting Hh signaling components. Bufalin also effectively induces apoptosis in HCC-LM3 cells. The apoptotic rate observed in untreated control cells is 17.72%, whereas treatment with bufalin alone increases apoptosis to 47.26%. The addition of GANT61 and cyclopamine further amplifies the apoptotic response, reaching 59.91% and 66.51%, respectively. The bufalin+GANT61+cyclopamine group produces the highest apoptotic rate at 76.77%, demonstrating that blocking both Gli1 and Smo proteins significantly enhances bufalin-induced apoptosis. Furthermore, bufalin suppresses the migration of HCC-LM3 cells, with its inhibitory effect magnified when combined with Hh pathway inhibitors. Bufalin alone reduces migration to 65.08%, but co-treatment with GANT61 lowers the migration rate to 42.75%, and the bufalin+GANT61+cyclopamine group produces the most substantial reduction at 15.13%. The bufalin+cyclopamine treatment exhibits a slightly stronger inhibitory effect than bufalin+GANT61, confirming the importance of blocking different sites within the Hh pathway. The synergistic effect of bufalin and Hh inhibitors provides compelling evidence that targeting Gli1 and Smo enhances its suppressive activity against cancer cell migration and metastasis. Overall, the findings suggest that bufalin exerts strong antiproliferative, apoptotic, and anti-migratory effects on HCC-LM3 cells, which are significantly enhanced when combined with Hedgehog signaling inhibitors. These results highlight bufalin’s potential as a promising therapeutic agent for liver cancer treatment, particularly in strategies aimed at disrupting cell cycle progression, inhibiting metastasis, and promoting apoptosis. Bufalin effectively inhibits the invasion of HCC-LM3 cells, with its effects significantly enhanced when combined with Hedgehog (Hh) signaling pathway inhibitors. Following 72 hours of treatment, the invasion rates were observed to be 49.77% in the bufalin group, 33.48% in the bufalin+GANT61 group, 27.73% in the bufalin+cyclopamine group, and the lowest at 16.96% in the bufalin+GANT61+cyclopamine group. Statistical analysis confirms that bufalin alone reduces invasion, but the combined use of Hh pathway inhibitors further amplifies this effect, with cyclopamine demonstrating a stronger synergistic inhibition than GANT61. Bufalin also suppresses adhesion of HCC-LM3 cells, with adhesion rates decreasing progressively across treatment groups. Cells treated with bufalin alone had an adhesion rate of 73.92%, which dropped to 47.02% with GANT61, 33.86% with cyclopamine, and 20.91% when both inhibitors were combined. The lowest adhesion rate observed in the bufalin+GANT61+cyclopamine group underscores the powerful inhibitory effect achieved through dual pathway inhibition, with cyclopamine demonstrating a stronger impact on adhesion suppression than GANT61. Additionally, bufalin influences key protein expressions in the Hh signaling pathway. While SHH protein expression remained unchanged across all treatment conditions, Ptch1 expression was notably inhibited, particularly when bufalin was combined with cyclopamine. Gli1 protein expression showed a significant reduction in bufalin-treated groups, especially when combined with either GANT61 or cyclopamine, with cyclopamine exerting a stronger inhibitory effect. Similarly, Gli3 expression was downregulated by bufalin, but the most pronounced reduction was observed when combined with cyclopamine, highlighting the role of Smo protein inhibition in strengthening bufalin’s effects. Bufalin further modulates the expression of downstream target proteins in the Hh signaling pathway, including β-catenin, E-cadherin, MMP-2, MMP-9, and VEGF. Bufalin alone downregulated β-catenin expression, with its inhibitory effect enhanced through combination treatments, particularly with GANT61+cyclopamine. E-cadherin expression was upregulated, reinforcing cellular adhesion, with bufalin+GANT61+cyclopamine inducing the strongest effect. MMP-2 and MMP-9, key regulators of extracellular matrix degradation, were significantly suppressed, with bufalin+GANT61+cyclopamine showing the highest reduction rates. VEGF, essential for angiogenesis, was markedly decreased following bufalin treatment, with GANT61 exhibiting a stronger suppressive effect on VEGF expression than cyclopamine. These findings demonstrate that bufalin exerts potent anticancer effects on HCC-LM3 cells, inhibiting invasion, adhesion, and metastatic potential. Its efficacy is significantly enhanced when combined with Hedgehog pathway inhibitors, suggesting a promising therapeutic strategy for targeting liver cancer progression through synergistic molecular inhibition. Discussion The invasion and metastasis of tumors are complex pathological processes involving multiple genes and stages. Metastasis typically begins after extensive cancer cell proliferation, followed by a sequence of pathological events such as matrix degradation, interstitial infiltration, tissue-specific chemotaxis, vascular invasion, cancer cell escape, selection of target organs, and secondary tumor formation at distant sites. Various signaling pathways—including MAPK, PI3K/AKT, Wnt, Hedgehog, Hippo, JAK/STAT, TGF-β, and Notch—play pivotal roles in tumor progression by influencing cell proliferation, apoptosis, differentiation, and angiogenesis. The Hedgehog (Hh) signaling pathway is a highly conserved intercellular communication system composed of key proteins such as Hedgehog (Hh), Patched-Smoothened (Ptch-Smo), Glioma (Gli), Costal-2 (Cos2), Fuse (Fu), Suppressor of Fuse (SuFu), and protein kinase A (PKA). While Hh, Smo, Gli, and Fu act as positive regulators of signaling, PTCH, Cos2, and PKA serve as inhibitory factors. Upon activation of the Gli family transcription factors, Gli translocates into the nucleus, where it modulates the expression of target genes involved in crucial biological functions such as embryonic development, cellular renewal, and angiogenesis. These genes include CCND1, CCND2, VEGF, Snail, Wnts, Igf2, Bcl-2, and Bcl-XL. GANT61, a specific inhibitor of Gli1, directly suppresses Gli1 transcription. Previous research has demonstrated that GANT61 effectively disrupts the Hedgehog signaling pathway in bile duct cancer cells, induces apoptosis, and exhibits potent anticancer properties. Cyclopamine, a non-steroidal alkaloid extracted from certain lily plants, inhibits Smo protein activation, thereby preventing downstream signaling. Unlike the canonical Hedgehog pathway, certain non-canonical mechanisms of activation do not depend on Smo. Consequently, cyclopamine selectively blocks the canonical pathway, whereas GANT61, by inhibiting Gli1, effectively suppresses both canonical and non-canonical pathways. Studies involving the neutralizing antibody for SHH in liver cancer cell lines such as Hep3B, Huh7, and PLC/PRF/5 have demonstrated decreased endogenous expression of Hedgehog pathway target genes, leading to enhanced apoptosis. Experimental results suggest that bufalin does not influence SHH protein expression nor alter its levels upon blocking Gli1 or Smo. Bufalin, however, effectively downregulates Ptch1 protein expression, with cyclopamine further enhancing this inhibitory effect. Unlike cyclopamine, GANT61 does not significantly affect Ptch1 suppression, indicating that bufalin primarily inhibits Ptch1 expression via Smo regulation. Further analysis shows that blocking Gli1 or Smo significantly amplifies bufalin’s suppressive effect on Gli1 expression. Additionally, while bufalin alone downregulates Gli3 protein levels, inhibition of Smo—rather than Gli1—enhances this effect, suggesting that bufalin modulates Gli3 expression through Smo regulation. Loss of balance between tumor cell proliferation and apoptosis leads to excessive cellular growth and tumorigenesis. Previous studies have confirmed that bufalin induces cell cycle arrest in SK-Hep-1 liver cancer cells at the G2/M phase, accompanied by decreased expression of cyclin A and cyclin B. In vivo studies utilizing orthotopic liver cancer transplantation models have demonstrated bufalin’s ability to trigger apoptosis, evidenced by nuclear pyknosis, chromatin condensation, cytoplasmic vacuolation, and apoptotic body formation. Experimental data from the current study reinforce these findings, showing that bufalin’s antiproliferative effect on liver cancer cells is significantly enhanced when Gli1 or Smo proteins are inhibited. The strongest suppression of proliferation occurs when both Gli1 and Smo are blocked simultaneously. Additionally, bufalin treatment results in a marked increase in the percentage of HCC-LM3 cells arrested in the S and G2 phases when combined with Gli1 or Smo inhibitors. This suggests that bufalin induces apoptosis in HCC-LM3 cells and that Hedgehog signaling pathway inhibitors synergistically enhance bufalin’s apoptogenic potential. Bufalin plays a crucial role in inhibiting epithelial-mesenchymal transition (EMT) in HCC-LM3 cells, a key process that enhances the invasive and metastatic potential of malignant tumors. EMT leads to significant changes in cellular properties, including cytoskeletal rearrangement, altered phenotypes, and reduced adhesive strength, allowing tumor cells to acquire mobility and invasiveness. This transition is marked by the downregulation of epithelial markers such as E-cadherin and the upregulation of mesenchymal markers like β-catenin. E-cadherin, a Ca²⁺-dependent transmembrane glycoprotein, promotes cellular adhesion, whereas β-catenin interacts with various proteins to facilitate intercellular connectivity. The present study demonstrates that bufalin not only inhibits migration, invasion, and adhesion in HCC-LM3 cells but also significantly upregulates E-cadherin expression while downregulating β-catenin. Hedgehog (Hh) signaling pathway inhibitors, GANT61 and cyclopamine, enhance bufalin’s regulatory effect on EMT markers, with the strongest inhibition observed when both inhibitors are applied together. Extracellular matrix (ECM) degradation is another critical factor in tumor invasion and metastasis. ECM consists of structural proteins, adhesion molecules, glycosaminoglycans, and proteoglycans, forming a complex microenvironment for cell interactions. Tumor cells produce protein degradation enzymes, such as matrix metalloproteinases (MMPs), to degrade ECM components and create pathways for invasion. MMPs, including MMP-2 and MMP-9, require metal cofactors such as Ca²⁺ and Zn²⁺ for activation. Bufalin inhibits ECM degradation by reducing the expression of MMP-2 and MMP-9, an effect further enhanced by the inhibition of Gli1 or Smo proteins in the Hh pathway. The strongest suppression occurs when both inhibitors are used in combination with bufalin, highlighting the synergistic effect. Angiogenesis is fundamental to tumor growth and metastasis, as it provides essential nutrients and oxygen to proliferating cancer cells. Bufalin effectively inhibits the proliferation of vascular endothelial cells, as demonstrated in human umbilical vascular endothelial cell studies. Additionally, downregulation of VEGF, a major driver of angiogenesis, was observed in hepatocellular carcinoma following bufalin treatment. GANT61 and cyclopamine further amplify the VEGF-suppressive effect of bufalin, with GANT61 exhibiting stronger inhibition compared to cyclopamine. Bufalin combined with both inhibitors achieves maximum VEGF suppression.
The findings in this study collectively indicate that bufalin’s therapeutic potential is significantly enhanced when used in conjunction with Hedgehog pathway inhibitors. Blocking key proteins such as Gli1 and Smo strengthens bufalin’s ability to inhibit proliferation, invasion, and metastasis of liver cancer cells. Mechanistically, bufalin suppresses EMT, ECM degradation, and angiogenesis by regulating the expression of Ptch1, Gli1, and Gli3. Additionally, bufalin modulates key downstream molecules, reducing MMP-2, MMP-9, and β-catenin levels while promoting E-cadherin expression. The combination of bufalin and Hedgehog signaling inhibitors provides a promising approach to mitigating malignant behaviors in liver cancer cells, offering valuable insights into future therapeutic strategies.