Digoxigenin activates autophagy in hepatocellular carcinoma cells by regulating the PI3K/AKT/mTOR pathway

Abstract

Hepatocellular carcinoma (HCC) is recognized as a highly malignant tumor. Targeted combination immunotherapy, the initially approved regimen, is compromised by adverse side effects and low response rates during clinical treatment. Traditional Chinese medicine and its derived natural compounds, known for their anticancer effects, offer advantages of low toxicity and cost. In this study, we performed high-throughput phenotypic screening in vitro to identify promising anti-HCC drugs. Among 1,444 bioactive compounds, digoxigenin (DIG) was found to significantly impede HCC cell progression. We validated DIG’s therapeutic effects through assays such as cell counting by CCK8, lactate dehydrogenase, and colony formation. Analyses including transmission electron microscopy, western blotting, and immunofluorescence demonstrated that DIG inhibits HCC cell proliferation via autophagy. Network pharmacology and molecular docking studies suggest that DIG targets the PI3K/AKT/mTOR signaling pathway. Comparative treatments of Hep3B and Huh7 cells with DIG or mTOR inhibitors revealed similar inhibitory impacts, indicating that DIG induces autophagy by inhibiting the PI3K/AKT/mTOR pathway. In vivo studies confirmed that DIG halts the growth of subcutaneous xenograft tumors. In conclusion, DIG represents a potential HCC treatment by modulating the PI3K/AKT/mTOR pathway to induce autophagy. This research, via phenotypic screening, accelerates drug discovery and the development of novel therapies targeting the underlying mechanisms of liver cancer.

Introduction

In December 2020, the WHO/IARC released the latest global cancer statistics, revealing that primary liver cancer ranks as the sixth most common cancer globally and the third leading cause of cancer mortality [1]. Primary liver cancer, predominantly hepatocellular carcinoma (HCC), accounts for 85% of all cases [2]. Surgery is the primary treatment for early-stage HCC patients; however, the five-year survival rate for these patients, following curative surgical resection or interventional therapy, is approximately 40%, with recurrence and metastasis occurring in 50–70% of cases [2]. For advanced HCC, treatments such as chemotherapy are prevalent, but drugs like sorafenib often exhibit significant toxicities and resistance [3, 4]. Consequently, there is an urgent need for more effective treatments and alternative strategies.

In contrast to apoptosis and necrosis, autophagy involves the encapsulation of intracellular components by bilayer vesicles in cells, which are then transported to lysosomes for degradation [5]. LC3BI/II is recognized as an autophagy marker [6]. As a bridge between LC3 and polyubiquitinated proteins, p62 is selectively packaged into autophagosomes before being degraded by proteolytic enzymes in autophagosomes, which can also be used to evaluate autophagy levels [7]. Autophagy is bidirectional and has a crucial regulatory role in cancer [8]. It is significantly up-regulated under starvation, hypoxia, REDOX stress, and disease conditions, upon which it removes and degrades damaged cell structures, aging cell membranes, and unwanted biomacromolecules [9]. Therefore, autophagy can eliminate abnormal cells, including tumor cells, to maintain the stability of normal cell genomes [10]. Autophagy and NBR1 degradation are believed to prevent life-threatening metastatic recurrence because NBR1 regulates tumor cell heterogeneity and metastatic [11]. In addition, down-regulating SNE-induced autophagy-mediated epithelial-mesenchymal transition and inhibiting mTOR and SGK1 both improve prostate cancer autophagy and have synergistic anti-metastatic effects [12]. These outcomes mean that activating autophagy may present a novel therapeutic strategy for the treatment of cancer.

Traditional Chinese medicines contain many bioactive ingredients, some of which exert inhibitory effects on tumors [13]. About half of the anti-cancer drugs used in hospitals in the past 40 years have been natural small-molecule compounds derived from animals, plants, or microorganisms, mostly from medicines sold in ethnic pharmacies [14]. These drugs have benefits like being less harmful, targeting more therapeutic targets, and working better. Therefore, screening monomer compounds from traditional Chinese medicines may be a rapid and practical method of identifying new drugs for HCC. In this study, through screening a traditional Chinese medicine monomer drug library containing 1,444 compounds, we identify digoxigenin (DIG) as a good liver cancer curative candidate. Digoxigenin (DIG) is a naturally occurring compound that belongs to a family of cardiac glycosides, derived from digitalis plants, and has been shown to exert significant anti-cancer effects [15]. Previous research has demonstrated that strong cardiosides, like oleandum, can inhibit autophagy and down-regulate cell survival proteins [15, 16]. However, the poteatoside c and digoxin exhibit anti-liver cancer effects and can inhibit tumor progression by the main anti-cancer effects, and the mechanism of action of DIG remains unclear. Consequently, we examine the therapeutic effects of DIG on HCC and consider potential pathways that could underlie these results.

Materials and methods

Reagents and antibodies

Traditional Chinese Medicine Library compounds (#L8300), digoxigenin (DIG, #S4396), Ac DEVD CHO (ADC, #S7901) and 3-methyladenine inhibitors (3MA, #S2767) were purchased from Selleck Chemicals (Houston, Texas, USA). Bafilomycin A1(Baf-A1, # MB5505-L) was purchased from Meilunbio (Dalian, Liaoning, China). Ferrostatin-1 inhibitor (Fer-1, #HY-100579) were purchased from MedChem Express. Rapamycin (Rapa, #AY-22989) was purchased from MedChem Express (San Jose, CA, USA). LC3I/II (#L7543) and p62 (#P0068) were purchased from Sigma-Aldrich (St. Louis, MO, USA). AKT (#YT0185), p-AKT (Ser473, #YP0006), and p-70S6K (Tr389, #YP1427) were purchased from ImmunoWay (Newark, USA). PI3K (#4249T), p-mTOR (Ser2448, #2971), mTOR (#2983T), LC3I/II (#2775)and GAPDH (#14C10) were obtained from Cell Signaling Technology (Danvers, MA, USA). p-PI3K (#bs-6417R) was purchased from Bioss (Beijing, China). Rac1 (#GB11621) was obtained from Servicebio (Wuhan, China). RHOA (#10749-1-AP) was obtained from Proteintech (Wuhan, China). The cyanine3 (A-10520) and goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibodies were bought by the Invitrogen brand (Thermo Fisher Scientific, Inc.).

Using a naked mouse model as a tumor and administering medication

The Animal Ethics Committee of China Science and Technology Industry Holdings (Shenzhen) Co., Ltd. (No. 202300185) approved all animal trials. Four-week-old male BALB/c nude mice were purchased from the Guangdong Medical Laboratory Animal Center. Mice were fed under special pathogen-free conditions, maintained at 25 ± 2 °C and 65% humidity, and supplied unrestricted access to food and water. Subcutaneous injections of Huh7 cells (1 × 107 in 0.1 ml phosphate buffered saline) were made into the right axilla of naked mice [17]. On day 12 after Huh7 injection, 24 mice with tumors ≥ 2 mm in diameter were randomly divided into 4 groups [18]: control group (0.9% NaCl/day), DIG group (2.8 mg/kg/day), DIG (2.8 mg/kg/day) + 3MA group (24 mg/kg/day), and DIG (2.8 mg/kg/day) + Rapa group (2 mg/kg/day) (n = 6). Mouse weight and tumor size were measured daily. After 10 days of continuous intraperitoneal treatment, the experiment was terminated, and the largest tumor size was less than 20 mm. Pentobarbital sodium (200 mg/kg) was intraperitoneally injected into the mice, the blood was removed from the orbital sinus, and the disappearance of a pain response was recorded (no response when the toe was pressed by hand or pliers), and cardiac arrest and apnea were observed to confirm animal death. The liver and subcutaneous tumors were then excised. The blood samples were taken to determine serum alanine aminotransferase (ALT, cat. no. C00921) and aspartate aminotransferase (AST, cat. no. C01021) levels (both from Jiancheng, Bioengineering Institute). The liver and tumor tissues were collected for Western blotting, immunohistochemistry, and hematoxylin and eosin (H&E) staining.

Cell culture

Shanghai Institutes for Biological Sciences (Shanghai, China) provided the human hepatoma cell line Hep3B, and the Huh7 cells were purchased from Suyan Biotechnology. Co., Ltd. The HCCLM3 cells were brought from the China Center for Type Culture Collection (CCTCC), and AML-12 and HepG2 cell lines were brought from Pricella Life Science & Technology Co., Ltd. (Wuhan, China). MIHA cell lines were brought from Fenghui Biotechnology Co., Ltd. (Hunan, China). Hep3B cells, HCCLM3 cells, HepG2 cells, and Huh7 cells were grown in DMEM/high-glucose medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37℃ and 5% CO₂. AML-12 cells were grown in special medium (Pricella Life Science & Technology, Wuhan, China) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37℃ and 5% CO₂. MIHA cells were grown in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37℃ and 5% CO₂.

Cell viability assay

Hep3B and Huh7 cells were seeded into 96-well plates at a density of 1 × 104 per well for the cell viability investigation. After 24 h of culture, the cells experienced the prescribed amount of DIG (1–10 µM) for 24–48 h at 37℃ and 5% CO₂. Pretreatment with 3MA (1 mM), Ac DEVD CHO (ADC, 50 µM), ferrostatin-1 inhibitor (Fer-1, 1 µM), and Rapa (1 µM) for 1 h was followed by treatment with DIG (1–2 µM) for 48 h. Finally, CCK8 reagent (#C0043, Beyotime, Shanghai, China) was used to detect absorbance at 450 nm enzyme-linked immunosorbent assay (Biotek Synergy H1, Burlington, Vermont, USA).

Lactate dehydrogenase assay

The lactate dehydrogenase (LDH) release rate was measured as follows: 1 × 10⁴ per well liver cancer cells were seeded into 96-well plates, which were then grown at 37 °C and 5% CO₂. After 24 h, tumor cells were subjected to the recommended dosage of small-molecule compound for 48 h (37 ℃, 5% CO₂). An LDH cytotoxicity assay kit (C0017; Beyotime, Shanghai, China) was used to conduct the LDH assay. Absorbance was measured at 490 nm and 680 nm via enzyme-linked immunosorbent assay (Biotek Synergy H1, Burlington, Vermont, USA).

Colony formation assay

Cell proliferation was assessed using a colony formation assay. Hep3B and Huh7 cells were seeded in six-well plates at a density of 500 cells per well and cultured under conditions of 37 °C and 5% CO₂ until most single colonies contained over 50 cells. Every three days, the medium was replaced, and cell conditions were evaluated. After cloning, the cells were treated with DIG at concentrations of 1–2 µM for 48 h. Subsequently, the medium was discarded, and the cells were washed twice with PBS. Each well was then fixed with 1 ml of 4% paraformaldehyde for 30–60 min, followed by another wash with PBS. Next, 1 mL of crystal violet dye was added to each well; the cells were stained for two hours, rinsed three times with PBS, air-dried, and photographed using a digital camera.

Cell scratch test

A scratch wound-healing test was performed to evaluate the inhibitory effects of the treatment on cell migration. Tumor cells (4 × 10⁵ cells per well) were seeded into six-well plates. At 0 and 48 h, or before and after treatment, photos of the same scratch were obtained using an optical microscope (Ts2R-FL, Nikon Corporation).

Flow cytometric analysis

Annexin V-FITC Apoptosis Detection Kit (Elabscience, E-CK-A211) was used for cell apoptosis analysis according to the protocol.

Transmission electron microscopy

The prescribed concentration of DIG (2 µM) was administered to Hep3B and Huh7 cells for 48 h following their incubation for 24 h at 37 °C and 5% CO₂. Cells were first pretreated with 3MA (1 mM) for 1 h, then subjected to DIG (2 µM) for 48 h under the same conditions. Subsequently, cells were digested with 0.5% pancreatic enzyme, centrifuged at 1,000 rpm for 5 min, and fixed at 4 °C using electron microscope fixative. After 2–4 h, acetone and ethanol were employed to dehydrate the cells, which were then embedded in epoxy resin. Ultrathin sections of 70 nm were stained with uranyl acetate and lead citrate, and examined under a transmission electron microscope (model HT7800, Hitachi Ltd.).

Immunofluorescence

For immunofluorescence analysis, 0.8 × 10⁵ cell species per well were treated with DIG (2 µM) with or without 3MA (1 mM) in 24-well plates. After incubation for 48 h, PBS was used to wash the cells three times. After that, each well received 0.5 ml of 4% paraformaldehyde for 15 min, followed by 15 min of permeabilization with 1% TritonX-100 and 1 h of incubation with goat serum. Subsequently, the cells were incubated overnight with LC3 antibody (1:200 dilution) at 4 °C, washed with TBST, and incubated in the dark with goat anti-rabbit fluorescent secondary antibody (A-10520, Cy3-labeled Goat Anti-Rabbit IgG; 1:200; Invitrogen, Carlsbad, CA, USA) for 1 h. DAPI ready-made solution (#C0065, Sorabio, Beijing, China; 1:500 dilution) was employed for nuclear staining for 5 min with an immunofluorescence anti-quenching agent seal. Lastly, cells were examined and taken pictures with a fluorescent microscope (Ts2R-FL; Nikon Corporation, Tokyo, Japan).

Western blot analysis

Proteins were collected as follows split with precooled RIPA cleavage buffer (#89900 Thermo Scientific, Wilmington, DE, USA). A BCA protein detection kit (#23277, Thermo Scientific, Wilmington, DE, USA) was used to determine protein concentrations. Approximately 20–30 µg of protein was diffused in a 5% concentration gel and a 10% separation gel. The SDS polyacrylamide gel was then used to transfer the proteins to a PVDF membrane. Following transfer, 5% milk was used to seal the PVDF membrane for 1 h at room temperature. The primary antibody was incubated overnight on a shaker at 4 °C. Goat anti-rabbit IgG antibody labeled with HRP (1:200) was incubated at room temperature for 1 h. The identified proteins were then photographed using a ChemiDoc MP imaging system (BIO-RAD, Hercules, CA, USA) and viewed using a supersensitive electrogenerated chemiluminescence solution (WBKLS0500, Millipore Sigma).

RNA isolation and reverse transcriptionquantitative PCR (RT-qPCR)

Total RNA was isolated from the HCC cells using AGRNAex RNA extraction reagent (Accurate Biotechnology Co., Ltd.). Following the manufacturer’s instructions, cDNA was created using a backward transcription kit (cat. no. AG11728, Jiangsu Accuracy Biotechnology Co., Ltd.). A SYBR premixed kit (cat. no. AG11718, Jiangsu Accuracy Biotechnology Co., Ltd.) was used for the qPCR procedure. All RT-qPCR analyses were run at least three times. The primer sequences were designed by Servicebio (GM2041, Wuhan, China), and those sequences for PCR were as follows: PIK3CA forward, 5’-TGCTGTTCGGTGCTTGGA-3’ and reverse, 5’-ATACATCCCACATGCACGACA-3’; AKT1 forward, 5’-TACTCTTTCCAGACCCACGACC-3’ and reverse, 5’-CCCGGTACACCACGTTCT-3’;mTOR forward, 5’-CCGAGAGATGAGTCAAGAGGAGTC-3’ and reverse,5’-GGGAGGAGGTTCCGAAGATAGTT-3’;GAPDH forward, 5’-GGAAGCTTGTCATCAATGGAAATC-3’ and reverse, 5’-TGATGACCCTTTTGGCTCCC-3’.

Immunohistochemistry

For immunohistochemistry staining, 3 mm thick paraffin slices were employed. The sections were then baked at 60 °C for 60 min, dewaxed with xylene, dehydrated with gradient alcohol, and the antigen was extracted with citric acid solution at 100 °C (cat. no. P0086, Beyotime Institute of Biotechnology) for 20 min. The antigen was followed by PBS for 5 min, three times. Following a 60 min period of closure with goat serum and a soak in 3% hydrogen peroxide, the sections were incubated with p-mTOR, mTOR, or LC3B (1:200 dilution) in 5% BSA overnight at 4 °C. The sections were then washed with 0.25% PBS and treated at 37 °C for 30 min with a secondary antibody (cat. no. KIT5020; MXB Biotechnologies).The slices were then stained with DAB for 3 min at room temperature (MXB Biotechnology Co., Ltd., catalog number DAB-0031), and hematoxylin was used to stain the nuclei. Then, using an Axio Imager M2 microscope from Zeiss AG, the cells were examined.

Network pharmacology and molecular docking analysis

First, targets linked to DIG and liver cancer were found using the GeneCards database (http://www.genecards.org/) and SwissTargetPrediction data (http://www.swisstargetprediction.ch/).R language software (version 4.1.1) was used to calculate an intersection map of drug and disease targets. The analysis of KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment was conducted using the David database (http://david.ncifcrf.gov/). Using R language software (version 4.1.1), the KEGG signal path results were displayed to show the function of junctions in linked signal paths. The 2D structure of the active components is available thanks to Chem3D software’s optimization of the PubChem database (http://pubchem.ncbi.nlm.nih.gov). Protein structures were found in the PDB database (http://www.rcsb.org). Protein and small-molecule ligands were processed using AutoDockTools software, molecular docking was performed using AutoDock-Vina to calculate the binding energies, and data visualization was performed using Pymol.

Statistical analysis

In this study, we used the mean ± variance of at least three independent experimental replicates. Differences between two or more groups were subjected to either-tests or one-way analysis of variance. Statistical significance was set at P < 0.05 when the analysis was conducted using Prism8 (GraphPad Software, La Jolla, CA, USA).

Results

Screening of drugs that can inhibit tumor proliferation

To identify compounds with anti-cancer proliferative properties, we screened 1,444 small-molecule compounds from the Selleckchem compound library in Hep3B cells. Various methods were employed to assess the ability of these compounds to inhibit cell proliferation at a concentration of 10 µM [19] (Fig. 1A). An initial screening condition was set where the cell survival rate had to be below 70%. Results from the CCK8 assay indicated that 88 compounds fulfilled this criterion (Fig. 1B). After excluding those already researched in hepatocellular carcinoma, 50 compounds remained. The concentration was then lowered further, and compounds demonstrating a cell survival rate below 80% were selected (Fig. 1C). Secondary screening revealed that six compounds inhibited Hep3B cell growth by over 20% at concentrations under 10 µM. For further details, refer to the annotations in Fig. 1C. These six compounds’ growth-inhibitory effects were subsequently confirmed through an LDH release assay (Fig. 1D). Integration of CCK8 and LDH assays showed that DIG (C23H34O5) exhibited the most significant inhibitory impact on liver cancer cell proliferation (Fig. 1E). Additionally, DIG demonstrated a comparable cytotoxic effect on other liver cancer cell lines, such as HepG2 and HCCLM3 (Supplementary Fig. 1A and B). The efficacy of DIG in curbing cancer cell proliferation was further validated using the CCK8 assay across various time points (24–48 h) and concentrations (1–10 µM) (Fig. 1F and G). In contrast, AML12 and MIHA cells displayed no notable differences compared to the control group following treatment with DIG (1–10 µM) (Supplementary Fig. 1C and D). Moreover, the half-maximal inhibitory concentration (IC50) of DIG was determined (Fig. 1H), and alterations in cell morphology were observed using an optical microscope (Fig. 1J). The impact of varying doses of DIG on the proliferation and motility of HCC cells was assessed using cloning and wound healing assays (Fig. 1I, K-L; Supplementary Fig. 1E-H). The experiments showed a significant inhibition of both proliferation and migration in the DIG-treated groups relative to controls. Furthermore, to delineate the effect of cell proliferation on migration in scratch assays, the expression levels of migration-associated proteins, RHOA and RAC1, were analyzed via Western blot. The results indicated that DIG reduced the protein expression levels of RHOA and RAC1 in Hep3B and Huh7 cells (Supplementary Fig. 2A-D). These findings suggest that DIG substantially diminishes the proliferation capacity of liver cancer cells.

Fig. 1

Screening of small-molecule drugs with the ability to inhibit liver cancer cell proliferation. (A) Multiple screenings and identification of small-molecule compounds. (B) Survival rate of Hep3B cells treated with candidate drugs, with the red box indicating a cell viability of 70% post-treatment with 10 µM of the drug. (C) Each dot within the screening dots represents the proportion of cell viability after treatment with less than 10µM of a medicine not previously investigated in liver cancer. Dots in various colored boxes denote compounds with cell viability less than 80%. (D) Lactate dehydrogenase release. (E) Chemical composition of Digoxigenin (DIG). (F and G) Activities of Hep3B and Huh7 cells following exposure to varying concentrations of DIG at different times. (H) 48-hour 50% inhibitory concentration (IC50) in Hep3B and Huh7 cells. (I) Colony formation of hepatocellular carcinoma (HCC) cells treated with DIG (1–2 µM). (J) HCC cells treated with 1–2 µM DIG for 48 h (scale bar = 100 μm). (K and L) Assessment of the migration ability of HCC cells treated with 1–2 µM DIG for 48 h using a scratch test; representative images are displayed (scale bar = 200 μm). Compared to the control group, statistical significance was indicated as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. NS indicates Not Significant; TCM denotes Traditional Chinese Medicine, while DIG represents digoxigenin

DIG induces autophagy in hepatocellular carcinoma cells

Morphological changes and subcellular structures were observed in cells using electron microscopy. Subcellular monolayer or bilayer vacuole-like structures were more prevalent in DIG-treated cells compared to the untreated group, suggesting the formation of autophagosomes (Fig. 2A and B, supplementary Fig. 2E). To ascertain whether the anti-tumor activity of DIG is mediated by autophagy, Hep3B and Huh7 cells were treated with DIG both with and without various cell death inhibitors, as assessed by the CCK8 assay. The addition of Fer-1 (an iron death inhibitor) and ADC (a pyroptosis inhibitor) failed to inhibit DIG-induced cell death, whereas 3MA (an autophagy inhibitor) significantly reversed this effect (Fig. 2C and D). Furthermore, Annexin V-FITC/PI staining and flow cytometry analysis showed that apoptosis rates did not significantly increase after DIG treatment (Supplementary Fig. 2F-I), ruling out apoptosis as a pathway for DIG-induced liver cancer cell death. However, the potential for DIG to induce apoptosis in other tumor cells remains to be determined and necessitates further experimental investigation. Western blot analysis revealed concentration-dependent increases and decreases in the expression levels of autophagy-associated proteins LC3BII/I and p62, respectively, in cells treated with various concentrations of DIG compared to untreated controls (Fig. 2E-G). Additionally, immunofluorescence detection revealed a marked increase in bright green fluorescence in cells treated with DIG, indicative of LC3B autophagosome formation in liver cancer cells (Fig. 2H and I). These findings suggest that DIG promotes autophagy in liver cancer cells.

Fig. 2

DIG induced autophagy in liver cancer cells. (A and B) Transmission electron microscopy was used to detect autophagy in Hep3B and Huh7 cells that had been exposed to DIG at the recommended concentration for 48 h, scale bar = 5 μm. (C and D) Hep3B and Huh7 cells were pretreated with 3MA, AcDEVD, CHO, and Fer-1 inhibitors for 1 h, then treated with DIG for 48 h for CCK8 detection. (E-G) Expression of autophagy-related proteins in hepatoma cells treated with DIG, as detected by western blotting. (H and I) Immunofluorescence detection of LC3 autophagosome expression in cancer cells treated with DIG (×20). LC3B expression (green) was significantly increased. The nucleus is defined by DAPI staining (blue )( scale bar = 50 μm). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with the control group. NS, not significant; Con, control; Fer-1, ferrostatin-1; 3MA, 3-methyladenine; DIG, digoxigenin

Autophagy inhibitor downregulates DIG-induced cancer cell death

However, an increase in LC3-II levels may be associated with an increase in autophagy flux and a decrease in autophagosome degradation. To distinguish between these two possibilities, we verified the involvement of DIG in autophagy using 3MA and Baf-A1, which were the inhibitor of autophagy. HCC cells treated with DIG + 3MA showed that 3MA pretreatment restored tumor cells viability compared to the DIG group, suggesting that 3MA can alleviate DIG-induced cell death (Fig. 3A and B). Moreover, DIG-induced suppression of wound-healing ability was dramatically restored by 3MA, according to scratch test results, which demonstrated that DIG greatly hindered the ability of HCC cells to heal wounds (Fig. 3C and D, supplementary Fig. 3A and B). Subsequently, DIG induced a significant reduction in single- or double-layer vacuole-like intracellular subcellular structures in 3MA-pretreated cells (Fig. 3E and F, supplementary Fig. 3C). Immunofluorescence also confirmed that 3MA pretreatment reversed DIG-induced autophagosome formation (Fig. 3G and H). At the same time, we assessed intracellular DIG-induced autophagic protein levels after pretreatment with 3MA by western blotting and found that 3MA reversed DIG-generated LC3B-II levels and increased p62 levels (Fig. 3I and J). Additionally, we used another autophagy inhibitor (Bafilomycin A1, Baf-A1). Compared to cells cultured with Baf-A1 alone, the LC3B-II autophagosome spots increased in cells co-incubated with DIG and Baf-A1(supplementary Fig. 3D and E), and the Western blot results showed that the expression levels of LC3B-II and p62 proteins were higher (suplementary Fig. 3F and G), indicating that DIG can promote the formation of autophagosomes. In summary, DIG can enhance autophagy by promoting the autophagy flux, thereby inducing hepatocellular carcinoma cell death.

Fig. 3

The autophagy inhibitor 3MA downregulates DIG-induced cancer cell death. (A and B) After 3MA pretreatment in hepatoma cells, DIG-induced cell death was significantly decreased. (C and D) Scratch test results (scale bar = 100 μm). (E and F) Autophagy of HCC cells pretreated with 1 mM 3MA in the presence of DIG, as observed by transmission electron microscopy. (G and H) LC3B expression detected by immunofluorescence staining (scale bar = 50 μm). (I and J) Pretreated with 1 mM 3MA, then cancer cells were treated with 2 µM DIG. Western blot was used to find the expression level of proteins connected to autophagy. Compared to the control group, Con, control; 3MA, 3-methyladenine; DIG, digoxigenin; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with the control group; # P < 0.01, ##P < 0.01, ### P < 0.001and #### P < 0.0001 compared to DIG treated group

DIG inhibits the PI3K/AKT/mTOR pathway in HCC cells

To explore potential therapeutic targets and associated signaling pathways of DIG in hepatocellular carcinoma (HCC), we employed network pharmacology to predict DIG’s mechanism of action. We identified 89 common potential target genes for DIG in the treatment of liver cancer by selecting the intersection of gene targets associated with liver cancer and DIG (Fig. 4A). To elucidate the biological mechanisms underlying these potential DIG targets, we conducted KEGG pathway enrichment analysis, identifying 145 pathways. The enrichment analysis, ordered by P-value, indicated significant enrichment in several pathways, including the cancer signaling pathway, PI3K-AKT signaling pathway, cancer proteoglycan signaling pathway, MAPK signaling pathway, and Ras signaling pathway, all of which involve the PI3K-AKT signaling pathway (Fig. 4B). Western blot analysis was performed for PI3K, AKT, and mTOR, as the PI3K/AKT/mTOR signaling pathway is closely associated with autophagy. Additionally, DIG reduced the phosphorylation of AKT, PI3K, and mTOR (Fig. 4C and F). RT-qPCR verification of these three targets revealed no interaction between DIG and PIK3CA, AKT1, or mTOR at the transcriptional level, reinforcing the notion that DIG influences these targets at the protein level (Fig. 4G and H). Subsequently, we performed molecular docking analysis of DIG with PIK3CA, AKT1, and mTOR. An affinity of less than − 5.0 kJ/mol was considered indicative of significant binding activity [20]. The binding energy results demonstrated that DIG strongly binds with PIK3CA, AKT1, and mTOR, with mTOR showing the highest affinity (Fig. 4I and J), potentially exerting a considerable regulatory effect on the activity of these target proteins.

Fig. 4

In HCC cells, DIG blocked the PI3K/AKT/mTOR pathway. (A) Venn diagram of the overlapping genes of DIG and HCC. (B) KEGG enrichment analysis for common targets by the DAVID database. (C-F) Western blot analysis was used to determine the amounts of expression of PI3K, AKT, and mTOR in HCC cells after a 48-h treatment with 2 µM DIG. (G and H) mRNA expression levels of PI3K, AKT, and mTOR detected by RT-qPCR. (I) Visualization of molecular docking between DIG, PIK3CA, and mTOR. (J) Molecular docking scores represented as heat maps

Digoxigenin may regulate hepatocellular carcinoma cell autophagy through the PI3K/AKT/mTOR signaling pathway

According to electron microscopy and western blotting analysis, DIG induced autophagy and inhibited the proliferation of HCC cells. Network pharmacological prediction revealed that DIG can bind to PIK3CA, AKT1, and mTOR, with mTOR exhibiting the highest binding energy. Furthermore, this interaction evidently enriched the PI3K-AKT signaling pathway, which is closely related to autophagy. Additionally, western blot analysis of MIHA cells post-DIG treatment showed that DIG minimally affects the PI3K/AKT/mTOR signaling pathway in normal liver cells at a specific dose, potentially indicating specificity for liver cancer cells (Supplementary Fig. 4A and B). The interactions between DIG and mTOR were further explored using rapamycin, an mTOR inhibitor. CCK8 assays of HCC cells treated with DIG + Rapamycin indicated that, compared to treatment with DIG alone, rapamycin pre-treatment notably inhibited cell proliferation (Fig. 5A and B). Moreover, LC3B immunofluorescence assays showed significant increases in autophagosome formation in the presence of DIG following rapamycin pre-treatment (Fig. 5C and D). Lastly, rapamycin treatment markedly decreased the phosphorylation of AKT, PI3K, mTOR, and p70s6k, and enhanced the expression of the autophagy-associated protein LC3B induced by DIG (Fig. 5E and J).

Fig. 5

The mTOR signaling pathway regulates DIG-induced autophagy. (A and B) Cell viability was detected by the CCK8 method. (C and D) After pretreatment with rapamycin for 1 h and DIG was used for 48 h, and the immunofluorescence method was used to detect the changes in LC3B fluorescence. (scale bar = 50 μm). (E-J) HCC cells pretreated with 1µM of rapamycin for 1 h were cultured with 2µM of DIG for 48 h, and the levels of PI3K/AKT/mTOR pathway-related proteins were evaluated by western blotting. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with the control group; # p < 0.01, ##P < 0.01, and ### p < 0.001 compared to DIG treated group. NS, not significant; Con, control; Rapa, rapamycin; DIG, digoxigenin

DIG inhibits tumor growth in vivo through the PI3K/AKT/mTOR pathway

Subsequently, we investigated DIG-induced autophagy through the PI3K/AKT/mTOR pathway in vivo. Nude mice’s right axilla received a subcutaneous injection of Huh7 cells. The mice were intraperitoneally injected with DIG, DIG + 3MA, DIG + Rapa, or the equivalent volume of sterile saline once modeling was declared effective (after the tumor was achieved) for 10 consecutive days (Fig. 6A and C). As shown in Fig. 6D, the weight loss of animals in the normal saline group gradually decreased due to tumor consumption. In the later stage, due to the heavier tumor burden, the weight of the saline group and DIG + 3MA group was higher than that of the other two groups (Fig. 6D). Thus, DIG inhibited tumor growth, whereas 3MA reversed this phenomenon (Fig. 6E). Subsequently, to assess any changes in liver function, the mice’s serum ALT and AST levels were examined, but the results showed no statistically significant differences between DIG, DIG + 3MA, DIG + Rapa, and normal saline groups (Fig. 6F and G). In addition, H&E staining and immunohistochemical analysis of tumor tissues showed that 3MA reversed the expression of p-mTOR and LC3B in mouse tissues compared to DIG group (Fig. 6H and I). At the same time, Western immunoblot detection of tumor tissue also proved this (Supplementary Fig. 4C and D). These results indicate that DIG is effective in treating liver cancer in vivo and that its mechanism of action involves the PI3K/AKT/mTOR pathway.

Fig. 6

DIG inhibits tumor growth in vivo through the PI3K/AKT/mTOR pathway. (A) Diagrammatic representation of the in vivo tests. (B) Pictures of mice having successfully transplanted subcutaneous tumors (n = 4). (C) Pictures of mouse tumors implanted beneath the skin. (D) Mouse body weight (n = 6). (E) Tumor volume changes while taking medicine (n = 6). (F and G) Measured ALT and AST values, which are indicators of liver function (n = 6). (H) Using hematoxylin and eosin staining, a mouse tumor’s morphology is displayed, scale bar = 100 μm. (I) LC3B, mTOR, and p-mTOR protein expression in tumor tissues were found using immunohistochemical techniques, scale bar = 100 μm

Discussion

A prevalent cancer that accounts for a significant portion of cancer-related mortality is HCC [21]. Owing to improvements in diagnostic methods and treatment strategies, including the implementation of targeted therapy and immunotherapy, the life expectancy of patients with HCC has improved [22,23,24]. However, because the early symptoms of HCC are not immediately apparent, discovery and diagnosis are frequently put off, which leads to advanced disease stages and subpar treatment outcomes [25]. Comprehensive treatment is provided in the late stages of HCC; however, the survival benefits for patients are limited. Consequently, a top objective continues to be the creation of more potent HCC medications and the discovery of novel therapeutic targets. Anti-cancer medications are being used to treat various malignancies by activating various cancer cell death mechanisms, such as autophagy, apoptosis, iron death, necrosis, and other regulated death pathways [26]. Traditional Chinese medicines and natural drugs have exhibited substantial potential for the development of anti-cancer drugs. Indeed, many small-molecule compounds exhibit powerful anti-cancer activities by regulating autophagy [27]. The dual mTOR inhibitor AZD2014 reduces cell growth and promotes autophagy, making undifferentiated thyroid cancer cells susceptible to paclitaxel [28]. In this study, we employed a library of monomer small-molecule compounds from traditional Chinese medicine to find a small-molecule drug (DIG) that can efficiently suppress the growth of HCC cells and cause cancer cell death by focusing on the PI3K/AKT/mTOR pathway.

DIG is a natural compound that occurs as a derivative of the digitalis plant; however, research on its role in cancer is limited. To the best of our knowledge, this is the first study to report the mechanism underlying DIG-induced death. In this study, the Hep3B cell line integrates the complete hepatitis B virus genome and has the ability to synthesize a variety of human plasma proteins. It is an important cell line model for studying primary liver cancer [29]. In addition, the Huh7 cell line was isolated from the tissue of a patient with well-differentiated hepatocellular carcinoma. The cells are AFP-positive, characterized by being HBV-negative and susceptible to hepatitis C virus (HCV) [30]. Therefore, given the close relationship between HCC development and hepatitis virus infection [31], this study focused specifically on autophagy-mediated cell death in two tumor cell lines. According to our findings, DIG treatment dramatically decreased the quantity of HCC cells as well as their capacity to migrate and form colonies, as well as the viability of HCC cells. Moreover, after DIG interfered with normal liver cells, such as AML-12, MIHA, etc., no significant inhibitory effect on autophagy or cell proliferation was observed compared with the control group, indicating that DIG has certain specificity for tumor cells.Transmission electron microscopy was also used to detect the usual morphological characteristics of DIG-induced autophagy in HCC cells, including a marked increase in vacuole-like intracellular and subcellular structures with a single or double layer. Hep3B and Huh7 cells were treated with DIG in the absence or presence of a number of cell death inhibitors to further investigate if this type of cell death was connected to autophagy. These studies showed that 3MA stopped DIG-induced cancer cells from dying. This suggests that autophagic death may play a big role in the death of DIG-induced cancer cells.

In contrast to death forms such as apoptosis and necrosis, autophagy involves the encapsulation of intracellular components by bilayer vesicles in cells, which are then transported to lysosomes for degradation [5]. Disruption in the regulation of autophagy leads to a series of diseases, such as cancer [32]. Consequently, autophagy is crucial for the emergence and progression of cancer [33]. A significant reduction in the level of autophagy leads to the accumulation of dysfunctional mitochondria, increased production of reactive oxygen species, and DNA mutagenesis, which facilitates tumor transformation [34]. Additionally, LC3B is active during autophagy. LC3B-I turns during autophagy into LC3B-II, which subsequently attaches to the membrane of the autophagosome. As a result, LC3B is frequently utilized as a marker for the development of self-degrading, and the LC3-II/I ratio can be used to determine the degree of autophagy [35]. Furthermore, when autophagy activity is weakened or the autophagy system is damaged, p62 accumulates continuously in the cytoplasm, which is negatively correlated with autophagic activity [7]. Therefore, using immunofluorescence and western blotting, we assessed the levels of expression of proteins related to autophagy. The results showed that DIG significantly changed the expression levels of autophagy-associated proteins such as LC3B, whereas 3MA reversed this phenomenon, which may be related to the stage of autophagosome formation. These findings strongly imply autophagy was involved in the inhibitory effect of DIG on HCC cell development and demonstrate a favorable correlation between LC3B expression in HCC cells and DIG concentration. These findings offer fresh proof of DIG’s anti-cancer actions on HCC, specifically that it can induce autophagy. However, the autophagy inhibitory effect of 3-MA is achieved by inhibiting class III PI3K, but this effect is transient. Therefore, 3-MA weakens the regulation of DIG on two autophagy-related proteins, which may have other potential biological effects in addition to inhibiting autophagy, and its potential impact on the PI3K/AKT/mTOR signaling pathway may need to be further investigated. in-depth research to clarify.

To clarify the mechanism of DIG-induced autophagy in HCC, we applied a comprehensive network pharmacology and in vivo experimental validation strategy based on a drug target database and a liver cancer target database and screened 89 core target proteins according to intersections of the Venn diagram. The DAVID database’s KEGG enrichment analysis of these 89 core targets revealed a close connection to the PI3K/AKT signaling pathway and its downstream pathways. Autophagy and the PI3K/AKT/mTOR signaling pathway are tightly related [36]. Then, we used western blotting to assess the expression levels of PI3K, AKT, and mTOR and discovered that DIG decreased their phosphorylation. RT-qPCR was used to demonstrate that DIG interacted with PIK3CA, AKT1, and mTOR at the protein level rather than at the transcriptional level. Moreover, molecular docking showed that DIG exhibited good binding ability with PIK3CA, AKT1, and mTOR and was most strongly bound to mTOR, which provided insights into the potential mechanism of action of DIG in HCC. A key regulator of rapamycin metabolism is the mammalian target of the rapamycin (mTOR) pathway. In fact, when autophagy was activated in PDAC cells, the mTOR/p70S6K signaling pathway was blocked, leading to autophagic death [37]. In another study, the inhibition of mTOR promoted autophagy, thereby inhibiting osteosarcoma growth [38]. Therefore, we performed western blotting and immunofluorescence to evaluate the effects of rapamycin on liver cancer cells. The results showed a similar significant anti-cancer effect for DIG and the mTOR inhibitor Rapa. This is consistent with other studies showing that rapamycin blocks autophagy by inhibiting mTOR phosphorylation, thereby inhibiting pancreatic cancer progression [39]. Last but not least, in vivo research demonstrated that DIG has reasonably bio-safe and anti-cancer effects by significantly inhibiting mTOR, which has an impact on mTOR’s positive feedback control of the PI3K/AKT signaling pathway [40], thereby inhibiting the PI3K/AKT/mTOR signaling pathway and promoting autophagy (Fig. 7). In addition, the activity of the PI3K/AKT/mTOR pathway was not significantly inhibited in normal cells. Taken together, the data indicate that DIG has a small effect on normal liver cells at a certain dose and may have certain specificity, thus providing support for its potential as an anti-tumor treatment.

In conclusion, we suggest that DIG, a naturally occurring substance, is a novel inducer of autophagy with the capacity to impede HCC cell proliferation. We also provide a fresh mechanism for DIG’s ability to block the PI3K/AKT/mTOR signaling pathway, which causes autophagy in cancer cells and has anti-cancer effects in HCC. These findings indicate a fresh approach to the treatment of HCC and highlight DIG as a potential new medication. This study, however, had certain drawbacks. First, it is unclear whether other molecular targets are implicated because the pharmacological mechanism of DIG in HCC is complicated and cannot be fully understood. Second, while network pharmacological analysis and animal tests were used to examine the mechanism of action of DIG on HCC, PI3K/AKT pathway inhibitors were not used in this study to further highlight the inhibitory effect of DIG on HCC. Such as AKT inhibitors, we will pay more attention to the rigor of experimental design in the future to guide future research directions. In the future, we will further explore the mechanism of DIG inducing autophagy in tumor cells. Finally, the limited HCC cell lines used in this study and future research can explore broader applicability across different cell lines.

Fig. 7

The proposed model of Digoxigenin-mediated autophagic cell death in HCC cells via the PI3K / AKT / mTOR pathway. Digoxigenin Blockade of the PI3K / AKT / mTOR signaling pathway mainly by binding to mTOR further led to autophagy in HCC cells (of course, Digoxigenin also has a certain ability to bind to PI3K and AKT). Specifically, the up-regulation of autophagy protein LC3B, but the downregulation of P62, increasing autophagic flux, and autophagosomes fuse with lysosomes, leading to HCC cell death