The human SMMC-7721, HepG2, and Huh-7 HCC cell lines were purchased from ATCC. The cells were cultured in Dulbecco’s Minimum Essential Medium (DMEM) (Gibco, USA) with 10% FBS (Foetal Bovine Serum, BI), 100 U/mL penicillin, and 100 U/mL streptomycin at 37°C in a humidified atmosphere with 5% CO₂. α-hederin was purchased from Chengdu Herbpurify CO., LTD (purity>98%). Dimethyl sulfoxide (DMSO) was used as solvent.

Cells were seeded at a density of 8×10³ cells per well in 96-well plate and treated with 0, 2.5, 5, 10, 20, 40, 80 uM of α-hederin for 12 h, 24 h or 48 h followed by incubating with MTT solution for 4 h. The absorbance of each well was measured at 490 nm using the plate reader (TECAN SPARK 10M). Cell proliferation was assessed using MTT according to the manufacturer’s protocol (Beyotime, China).

Antibodies: YAP (ab56701, abcam, 1:5000), Lats1 (ab70561, abcam,1:5000), p-YAP (ab62751, abcam,1:1000), Bax (ab32503, abcam,1:5000), Bcl-2 (ab196495, abcam,1:1000), TAZ (ab224239, abcam,1:5000), Caspase-3 (ab13847, abcam,1:500), GAPDH (ImmunoWay, YM3029,1:5000), Mst1 (CST,3682s,1:1000), TEAD1 (ab133533, abcam,1:5000), Cleaved-caspase3 (CST,9664s,1:1000), Phospho-Lats1/Lats2 (PA5-64591,Invitrogen,1:1000), Histone H3 (ImmunoWay, YM3038,1:5000).

Briefly, protein concentrations of tissues or cells were quantified by BCA protein assay kit (Thermo Scientific, Waltham, MA, USA). Sample of proteins (25 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to PVDF membrane, then blocked by 5% non-fat milk in PBST buffer (PBS buffer containing 0.05% Triton-100) for 1 h at room temperature. The membranes were incubated overnight with primary antibodies at 4°C, after 3 times washing in PBST buffer, the membranes were incubated with the secondary antibodies for 1 h at room temperature. Signals were detected by using an ECL substrate (Thermo Scientific) and exposure with the Tanon 5500 imaging system. The intensity of the bands was quantified by densitometry.

After drug treatment, cells were gently washed by PBS for three times and fixed with 4% fresh paraformaldehyde-phosphate (BL539A, Biosharp) for 15 min, then permeabilized with 0.5% Triton X-100 in PBS for 10 min at room temperature. After blocking in 10% goat serum and 5% BSA in PBS-T for 30 min, cells were incubated with primary antibody against YAP (1: 100) in 5% BSA overnight at 4°C. After washing three times with PBS-T, cells were incubated with secondary antibodies (1:200 dilution) for 1 h at room temperature. The coverslips were treated with DAPI for 5 min for nuclear staining. An inverted fluorescence microscope (Nikon Eclipse Ti, Nikon, Japan) was used for imaging.

Apoptosis assay was monitored by flow cytometry. Cells were seeded at a density of 3 ×10^5/ml in six-well plates. After attachment, cells were treated with α-hederin at different concentrations for 24 h. Supernatants of the cultures were collected and the attached cells were collected by digestion with pancreatin (S330JV, BasalMedia) followed by centrifugation (2000 rpm, 2min) after washing twice with PBS. Cells were re-suspended in 500 ul of 1×Binding Buffer and incubated with 5 ul of Annexin V-FITC and 5 ul of PI Staining Solution for 15 min. The next steps were followed according to the manufacturer’s instructions (Key GEN BioTECH).

For cell cycle analysis, cells were synchronized at the G0/G1 phase by serum starvation for 24 h and then released into cell cycle by re-addition of 10% FBS. After α-hederin treatment at different concentrations for 24 h, cells were collected and fixed in 75% ethanol at −20°C overnight, then re-suspended in precooled PBS for 3 times. Cell cycles were detected by the flow cytometer (Beckman Coulter) at 488 nm excitation wavelength and the data were analyzed with FlowJo/Modfit 5 software.

To assess in vitro proliferation, the cells were supplemented with fresh medium containing 10 μM EdU and incubated for 2 h at room temperature after α-hederin treatment at different concentrations. Cell nuclei were counterstained with DAPI at room temperature for 10 min. EdU-positive cells were quantified using fluorescence microscopy (Nikon Eclipse Ti, Nikon, Japan).

Cells were seeded in six-well plates and treated with different concentrations of α-hederin for 24 h. Then, the medium was replaced with α-hederin free medium and continued to incubate for about 14 days. The cells were fixed with 4% paraformaldehyde and stained with Giemsa stain at room temperature. After a final series of rinses and air dry, photographs were taken for colony quantification.

Total RNA was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer instructions. 1 μg of total RNA was reversed transcription to cDNA by using the Evo M-MLV RT Mix Kit with gDNA Clean for qPCR (AG11728, ACCURATE BIOTECHNOLOGY, HUNAN, Co.,Ltd). The qPCR was performed using the SYBR Green Premix Pro Taq HS qPCR Kit (AG11701, ACCURATE BIOTECHNOLOGY, HUNAN, Co., Ltd). The expression level of individual genes was analyzed by the comparative Ct method (2^-ΔΔCt method) and normalized according to the expression of the housekeeping gene.

All animal experiments were approved by our Animal Ethics Committee of Nanjing University of Chinese Medicine (Nanjing, China), and all experiments were performed in accordance with relevant guidelines and regulations. For each mouse, 0.2 ml (2.5×10⁷/ml) HepG2 cell suspension was injected subcutaneously into the right axillary region of female BALB/c nude mice (6 weeks). All animals were maintained in a pathogen-free and temperature-controlled environment with 12 h light/dark cycle and standard laboratory diet. The animals were randomized into three groups (n=8 per group): control group, α-hederin group (5 mg/kg), and positive group DDP (Cisplatin, 5 mg/kg). Tumor growth was calculated with calipers every three days, and the volume was calculated according to the formula: volume = (width)² × length/2. The body weight of the mice was recorded every three days. At the end of experiments, xenotransplant tumors (three mice per group were chosen randomly to be identified by western blot), livers, mouse blood and kidney were harvested for additional analysis.

For fluorescence imaging in vivo, mice were imaged using an IVIS Spectrum In Vivo Imaging System (Caliper Life Sciences). HepG2 cells were transfected with a green fluorescent protein (GFP) vector [Zhongqiao Xinzhou Science and Technology Co. (Shanghai, China)]. Prior to in-vivo imaging, the mice were anesthetized with isoflurane. Excitation of fluorophore were performed at 488 nm for GFP. The signals were analyzed with Living Image Software (PerkinElmer).

The xenograft tumor tissues from different groups of mice were fixed in 4% paraformaldehyde for over 12 h and were embedded in paraffin. Then, the tissues were cut into 4 μm thick sections and stained with hematoxylin and eosin (HE).

For Immunohistochemistry, paraffin sections were incubated with a blocking solution and then incubated with anti-YAP antibody (Abcam, ab52771) at 4°C overnight. After washing, sections were incubated with biotinylated secondary antibodies at room temperature for 30 min. Sections were visualized with 3, 3’-diaminobenzidine and then counterstained and dehydrated for microscopic observation (×100, ×200, Nikon, Japan).

The One Step TUNEL Apoptosis Assay Kit (Beyotime) was used for TUNEL assay. The 4um-thick paraffin‐embedded tissue sections were dewaxed with xylene and rehydrated in an ethanol gradient. Then, the slices were treated with 20 ug/ml proteinase K for 30min at 37°C. After washed three times with PBS for 5min, the slices were incubated with TUNEL reaction mixture for 1 h at 37°C away from light, washed three times in PBS, then incubated with DAPI-containing anti-fluorescence quenching tablets and observed under a fluorescence microscope.

All data are presented as the mean ± standard deviation (SD) of at least three independent experiments. Student’s t-test determined statistical differences between samples and the Bonferroni post hoc procedure was performed for a one-way analysis of variance (ANOVA) of statistical comparisons between more than two samples. GraphPad Prism 7.0 (GraphPad, La Jolla, CA, USA) was used to create the resulting data charts. Statistical significance was considered to be p < 0.05 or p < 0.01.

To investigate the effects of α-hederin on HCC cells proliferation, we treated HCC cell lines with indicated concentrations of α-hederin for 12 h, 24 h, and 48 h. Notably, α-hederin inhibited the growth of HCC cell lines in a time and dose-dependent manner ( Figure 1A ). The IC50 values of HepG2, SMMC-7721, Huh-7 cells at 24 h were 18.5 µM, 17.72 µM, 21.89 µM, respectively. Accordingly, the treatment of α-hederin at 10 µM (low concentration) and 20 µM (high concentration)for 24 h was selected for subsequent experiments. By Flow cytometry, we found thatα-hederin significantly promoted the accumulation of the G2/M phase compared with the control group ( Figure 1B ). In addition, the EdU assay revealed a marked reduction of the proliferative ability of HepG2 and SMMC-7721 cells after being treated with low and high concentrations of α-hederin ( Figure 1C ).

To further confirm the inhibitory effect of α-hederin on hepatoma cell proliferation, cell clone formation experiment was performed ( Figure 1D ). Our results clearly displayed that α-hederin inhibited the colony-formation at the indicated concentration. The results described above indicate that α-hederin exerted an inhibitory effect on the proliferation of HCC cells.

Apoptosis of HCC cells induced by α-hederin was analyzed by flow cytometry and morphological observation. We found that α-hederin increased the proportion of apoptotic cells ( Figure 2A ). Compared with that of control cells, the proportion of apoptosis in HepG2 cells treated with 10 µM or 20 µM α-hederin increased from 3.95% to 26.13% and 69.58% respectively. Similar results were found in SMMC-7721 cells, the proportion of apoptotic cells increased from 4.85% to 18.78% and 54.7% respectively by 10 µM or 20 µM α-hederin treatment. The effect of apoptosis induced by α-hederin was verified at the level of protein expression. The apoptosis-related proteins were determined by Western blot. The results revealed that the expression of Bax and cleaved-caspase3 were obviously increased by α-hederin treatment, while the expression of Bcl-2 and caspase-3 in HepG2 and SMMC-7721 cells were significantly decreased in a dose-dependent manner ( Figure 2B ). Furthermore, the expression of genes associated with apoptosis and proliferation were measured. As shown in Figure 2C , the proliferation-associated genes including CTGF, BIRC2, AREG, and Cyclin D1 in HCC cells were significantly decreased by α-hederin treatment compared with the control cells.

YAP is a downstream protein of Hippo signaling pathway, and plays a key role in hepatic carcinogenesis. We firstly investigated the native expression of YAP at gene and protein level in Huh-7, HepG2, and SMMC-7721 cells. As shown in Figure 3A , both YAP gene and protein highly expressed in HepG2 and SMMC-7721 cells compared with Huh-7 cells. Therefore, in the present study, HepG2 cells and SMMC-7721 cells were used. Crucial molecules of Hippo signaling pathway in HepG2 and SMMC-7721 cells were further examined. We found that α-hederin treatment suppressed YAP/TAZ protein expression, but elevated the expression of Mst1, Lats1, P-Lats, P-YAP in a dose dependent manner. Accordingly, the level of TEAD1 transcription factor was downregulated ( Figure 3B ). RT-qPCR results revealed that α-hederin treatment effectively enhanced Mst1 and Lats1 gene expression while downregulated YAP gene expression in HepG2 and SMMC-7721 cells ( Figure 3C ).

YAP is inactivated by phosphorylation followed by degradation in the cytoplasm (7, 18, 19). To verify whether α-hederin could inhibit YAP signaling activity, fluorescence staining followed by confocal microscopy was used. Our results showed that in both HepG2 and SMMC-7721 cells, α-hederin treatment cells revealed a significantly decreased distribution of green fluorescence in the nucleus and an increased cytoplasmic staining in comparing with that in control cells ( Figure 3D ), and the reduced level of nuclear YAP was confirmed by immunoblotting ( Figure 3E ). These results suggested that the inhibitory effect of α-hederin on HCC cell proliferation may be due to the suppression of YAP activation caused by the upregulated Mst1 activation.

To further verify the role of Mst1/2 in the suppression of hepatoma cell proliferation caused by α-hederin, HepG2 and SMMC-7721 cells were treated with Mst1/2 inhibitor XMU-MP-1 (3 μmol) for 3 h or 6 h before exposure to α-hederin. Immunoblot results revealed that XMU-MP-1 effectively inhibited the expression of Mst1 and downstream kinase Lats1 ( Figure 4A ). Additionally, XMU-MP-1 suppressed the α-hederin-induced reductions in cell density and the irregular morphology of HepG2 and SMMC-7721 cells. Moreover, the results of flow cytometric analysis showed XMU-MP-1 treatment not only ameliorated apoptosis, but also prevented cell cycle arrest induced by α-hederin ( Figures 4B, C ). Data from the EdU assay showed that XMU-MP-1 treatment reversed the inhibition of proliferation caused by α-hederin in HepG2 and SMMC-7721 cells ( Figure 4D ).

To further validate whether the decreased YAP activation induced by α-hederin was due to the increased Mst 1/2 activity. HepG2 and SMMC-7721 cells were treated with α-hederin or a combination of α-hederin/XMU-MP-1 for 24 h. Immunoblot revealed that XMU-MP-1 treatment reversed the α-hederin-induced expression changes of Mst1, Lats1, P-Lats1/2, YAP, P-YAP, TAZ in HCC cells ( Figure 5A ). These results were also supported by RT-qPCR analysis ( Figure 5B ). Moreover, XMU-MP-1 could partially reverse the α-hederin-induced decrease of the mRNA expression of YAP target genes involved in proliferation and apoptosis( Figure 5C ). Furthermore, the immunofluorescence analysis results showed that XMU-MP-1 treatment prevented the reduction of nuclear YAP induced by α-hederin ( Figure 5D ). Our results demonstrated that the Hippo signaling pathway played a critical role in α-hederin-medicated inhibition of cell proliferation and promotion of apoptosis in HCC cell lines.

Although we have been already verified that α-hederin could inhibit the proliferation of human HCC cells and induce its apoptosis in vitro, but whether α-hederin could display the same effects in vivo should be investigated. HepG2 cells which are stably transformed with green fluorescent protein were injected into BALB/c nude mice, then treated with α-hederin or DDP as a positive control. In-vivo fluorescence imaging confirmed that the fluorescence intensity of HepG2 cells was significantly attenuated and the areas of fluorescence were restricted by α-hederin treatment ( Figure 6A ). Compared with the control mice, the α-hederin-treated mice exhibited a smaller tumor size and lower tumor weight ( Figures 6B, C, E ). Besides, the kidney index and bodyweight of mice in the α-hederin group were higher than in the DDP group, this means α-hederin has low side effects ( Figures 6D, F, G ). The sections of the HepG2 xenograft tumor were analyzed by HE staining ( Figure 6H ). Compared with the control group, neoplastic cells in α-hederin or DDP treatment group underwent cell death as evidenced by nuclear pyknosis and loss disintegration of cellular architecture, this result was consistent with restricted xenograft tumor size ( Figures 6B, C ) and induced apoptosis in Figure 2 . The expression of YAP was analyzed in tumor sections ( Figure 6I ) and total proteins were extracted from tumor tissue ( Figure 6J ). After treatment with α-hederin and DDP, the expression of the apoptosis‐promoting protein (Bax) was upregulated, whereas the apoptosis‐inhibiting protein (Bcl-2) was downregulated. Besides, the main upstream Hippo pathway kinases (MST1, LATS1) were upregulated. More importantly, the expression of P-YAP was increased, whereas that of YAP was decreased after treatment with α-hederin and DDP. Besides, TUNEL staining assay showed more apoptosis cells in α-hederin and DDP group ( Figure 6K ).