Human HCCLM3, SMMC7721, Hep3B and HepG2 cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences. HCCLM3, Hep3B and HepG2 cells were grown in DMEM (Gibco, United States) containing 10% heat-inactivated fetal bovine serum (FBS) (GIBCO, United States) and 1% penicillin/streptomycin (Sigma-Aldrich, United States). SMMC7721 were grown in 1640 (Gibco, USA) with 10% FBS and penicillin/streptomycin. The cells were maintained at 37°C in a humidified incubator containing 95% air and 5% CO2.

Dihydrotanshinone (DHTS) was purchased from Sigma-Aldrich (USA). DHTS was dissolved in DMSO to form a concentration of 10 μM stock solution. Anti- STAT3 (#9139), Phospho- Stat3 (Tyr705) (#9145), JAK2 (#3230), Phospho-Jak2 (Tyr1007/1008) (#3771), Bax (#5023), Bcl-2 (#), Cyt C (#4280), Survivin (#2808), cleaved caspase-3 (#9661), cleaved caspase-9 (#20750), cleaved caspase-7 (#8438) were from Cell Signaling Technology (United States). The secondary antibodies were purchased from LI-COR (United States).

The viability and inhibition of Cells was measured by the Cell Counting kit- 8 assay (CCK- 8 assay, Dojindo, Japan). Cells (5 × 10³ cells) were seeded into a 96- well microtiter plate per well. Then, HCCLM3, SMMC7721, Hep3B cells were treated with DHTS (1, 2, 4, 8, 16, or 32 μg/ml) for 24 h HepG2 cells were treated with DHTS (5, 10, 20, 40, 80, 160 μg/ml) for 24 h. Subsequently, 10 μL CCK-8 dye was added to each well and incubated for 2 h at 37°C. Then The absorbance was measured at a wavelength of 450 nm using an iMark microplate reader (Victor3 1420 Multilabel Counter, PerkinElmer).

Hoechst 33258 Assay was purchased from Beyotime Institute of Biotechnology (Shanghai, China). Hoechst 33258 is a blue fluorescent dye which is used for the detection of cells apoptosis. SMMC7721 were seeded into a sterile 6-well plate for 24 h. Subsequently, DHTS (1, 2, 4 μg/ml) was added for another 24 h. Then, the cells were fixed in 4% paraformaldehyde solution for 30 min and incubated with Hoechst 33258 dye for 10 min. After washing three times with PBS, apoptotic morphological features were examined and photographed under a fluorescence microscope (OLYMPUS, Japan).

Cells were cultured in 6-well plates for 24 h, Then, the cells were treated with DHTS (1, 2, 4 μg/ml) for 24 h. After treatment, cells were collected and washed twice with cold PBS. Then, cells were resuspended in binding buffer at a concentration of 1 × 10⁶ cells/ml. Next, 5 μL Annexin V-PE and 10 μL 7-AAD or 50 μL PI staining (for cell cycle detection) were added in the cell suspension and incubated in the dark for 15 min at room temperature. The percentage of apoptotic cells was evaluated by flow cytometric analysis (FACSCalibur, Becton-Dickinson).

The harvested cells were lyzed with cold RIPA buffer (Beyotime, China) containing protease and phosphatase inhibitors and PMSF (Beyotime, China) and total proteins were extracted. The protein samples (30 µg) were separated using 12% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore, Unites States). Membranes were blocking with 5% milk at room temperature for 1 h. After washing three times with PBS, the bands were incubated at 4°C overnight with the different primary antibodies at the recommended concentrations. After three washes with PBST, the bands were incubated with the secondary fluorescent antibody at 37°C in the dark for 1 h. After washing three times, the bands were analyzed using the Odyssey Infrared Imaging system (LI-COR, Unites States).

After treatment, cells were fixed with 4% paraformaldehyde for 30 min at room temperature. Cells were then permeabilized with 0.2% Triton X-100 for 15 min. After three washes, the permeabilized cells were blocked with 5% bovine serum albumin (BSA) for 30 min. Then, cells were incubated with 1:100 dilution of Stat3 (mouse antibody) overnight at 4°C. The next day, cells were incubated in a 1:100 dilution of goat anti-mouse Ig G antibody (Invitrogen) for 1 h at room temperature. The nuclei were counterstained using DAPI (Beyotime, China). Images of cells were visualized under fluorescence microscope (Olympus, Japan).

The STAT3 plasmid, STAT3 shRNA plasmid, and control plasmid were purchased from Miaoling Bioscience and Technology (Wuhan, China). All plasmids contain enhanced green fluorescent protein (EGFP) and anti-puromycin gene site. The STAT3 plasmid, STAT3 shRNA plasmid, or NC plasmid was co-transfected with packaging plasmids pSPAX2 (#12260, addgene) and pMD2. G (#12259, addgene) into HEK293T cells by using transfection reagent Lipofectamine 2000 (Invitrogen) to produce lentiviruses. After transfection, the supernatants containing the lentiviruses were collected and used to infect SMMC7721. The expression of EGFP of cells was used to evaluate the efficiency of viral infection by using fluorescence microscope. 5 mg/ml puromycin was added into medium to select the successfully transduced cells. Western blotting was performed to evaluate efficiency of transfection.

Male BALB/c nude mice, 5–6 weeks of age, were purchased from Beijing HFK Experimental Animal Center (Beijing, China). All procedures of the mice were approved by the Committee on the Ethics of Animal Experiments and performed in accordance with National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. All mice were kept in specific pathogen-free (SPF) animal house and fed with autoclaved food, sterile water, and filtered air. Nude mice were randomly divided into four groups, and each group contained 6 mice. Mice were subcutaneously inoculated SMMC7721 cells on the lower right flank. The xenograft tumors were allowed to grow until 100 mm³ in size before giving the treatment of DHTS (5, 10, or 15 mg/kg, via intraperitoneal injection) thrice a week. The tumor volume (TV) was calculated according to the following formula: TV (mm3) = d2 × D/2 (d and D are the shortest and longest diameters, respectively). TV was measured every alternate day.

The cell death of the xenograft tumors was detected by TUNEL kits (Roche, United States). Briefly, harvested tumors were fixed in 4% paraformaldehyde, and sliced into 4 μm thickness. Subsequently, the sections were deparaffinized and rehydrated. Then, the sections were incubated with TUNEL according to the manufacturer’s instructions. The TUNEL-positive cells were observed and analyzed under a fluorescence microscope (Olympus, Japan).

Experimental results were represented as mean ± standard deviation and analyzed using the IBM Statistical Product and Service Solutions (SPSS) software. Statistical differences of the results between groups were performed with one-way ANOVA test and Student’s test. Results were considered statistically when p value was less than 0.05.

In order to verify the ability of DHTS in exhibiting inhibitory effects on cell viability. HCC cancer cells HCCLM3, SMMC7721, Hep3B were treated with 1, 2, 4, 8, 16, and 32 μM DHTS. HepG2 cell was cultured with 5, 10, 20, 40, 80,160 μM DHTS. The antitumor effects of the DHTS were detected by CCK-8 assay at different time points (12, 24 and 36 hours). As shown in the cell survival curves, the proliferation of four HCC cell lines were significantly inhibited by DHTS in a concentration- and time-dependent manner (Figures 1B–E). The inhibitory difference of different cell lines maybe the different genetic backgrounds. It is worth noting that the IC50 of SMMC7721 for 24 h is approximately 2 μM, which is lower than the values of the other cell lines. Among the four cell lines, SMMC7721 exerted the best inhibitory effect on the cell viability at 24 h (Figure 1D). Thus, we choose the SMMC7721 by the best inhibitory effect.

Decreased viability of HCC cells after DHTS exposure prompted us to detect the effect of DHTS on apoptosis of cells. As the previous results showed that SMMC7721 cells were more sensitive to DHTS, we selected 24 h as the functional timepoint of apoptosis for SMMC7721 cells treated with DHTS. We performed a Hoechst assay to investigate the morphological characteristics of apoptosis of cells after DHTS exposure. The apoptosis of SMMC7721 cells was induced by the presence of DHTS compared with control (p < 0.05) (Figure 2A,B). The apoptotic cells of 4 μM DHTS showed much more condensed nuclei and bright-blue fluorescent than the control medium (p < 0.05) (Figure 2A,B). Overall, these results suggest that DHTS may promote the apoptosis of HCC cells.

Staining of SMMC7721 cells with Annexin PE/7-AAD was used to detect the apoptosis of SMMC7721 cells after DHTS exposure 24 h (p < 0.05) (Figure 2C,D). The results demonstrated that DHTS could significantly promote apoptosis of cells in a dose-dependent.

To further verify the results above, we used western blot to detect the expression of apoptosis-related proteins (Bax, bcl-2, Cyt C, Survivin, and caspase-3,7,9) in SMMC7721 cells treated by DHTS (0, 1, 2, and 4 μM) for 24 h. The results showed that after DHTS treatment for 24 h, the proapoptotic protein Bax gradually increased, and the increase was most significant when DHTS concentration was 4 μM (p < 0.05) (Figure 3A,B). At the same time, inhibition of apoptotic protein Bcl-2 was gradually down-regulated with the increase of DHTS concentration (p < 0.05) (Figure 3A,C). Cyt C, which can activate caspase-mediated apoptosis, was up-regulated with an increase of dose of DHTS treatment (p < 0.05) (Figure 3A,D). The expression of apoptotic inhibitor protein survivin decreased, and the expression level was the lowest when DHTS concentration was 4 μM (p < 0.05) (Figure 3A,E). Mitochondrial apoptosis-related protein cleaved caspase-3,7,9 increased significantly with the increase of DHTS concentration (Figures 3F–I). The results indicated that DHTS could promote apoptosis in HCC cell line SMMC7721.

Next, we measured the effect of DHTS on the growth of SMMC7721 cells. SMMC7721 cells were treated with 0, 1, 2, 4 μM DHTS, respectively. After 24 h, cell cycle phase distribution was evaluated by flow cytometry. The results showed that DHTS induced G2/M cell cycle arrest in SMMC7721 cells (p < 0.05) (Figures 4A–C). We observed the accumulation of G2/M phase in SMMC7721 cells when the DHTS concentration was 2 μM. Greatest accumulation in G2/M phase was noted with DHTS 4 μM G2/M DNA damage is an important check point. The checkpoint insures that cells don’t initiate mitosis before they have repaired the damaged DNA after replication. Cells with a defective G2/M checkpoint enter mitosis without repairing their DNA, leading to death after cell division (Lee et al., 2020). DHTS induces G2/M cell cycle arrest in SMMC7721 cells. G2/M is an important DNA damage check-point and numbers of proteins are involved in the regulation of the check-point. Key molecules among them are cyclin B1, CDC 2. We also assessed the levels of these proteins in SMMC7721 cells. Western Blot results showed that DHTS could lead to a decrease in Cyclin B1 and CDC2 levels (p < 0.05) (Figures 4D–F). In summary, our data suggests that DHTS-induced reduction in SMMC7721 survival is associated with G2/M-phase arrest and apoptotic cell death.

STAT3 can be involved in the regulation of apoptosis, invasion, migration and angiogenesis. We further performed western blot experiments to verify the effect of DHTS on STAT3. After DHTS treatment, the protein expression of p-STAT3 decreased in a concentration-dependent manner (p < 0.05) (Figure 5D,E).

JAK2 is known to stimulate STAT3 phosphorylation on tyrosine Tyr705 in many cancer cells. We speculated that whether DHTS is able to inhibits the phosphorylation of STAT3 by inhibiting the phosphorylation of JAK2. Indeed, DHTS inhibits phosphorylated-JAK2 in a dose-dependent manner (p < 0.05) (Figure 5D,F).

We further used immunofluorescence experiment to verify the effect of DHTS on STAT3 nuclear translocation, considering that nuclear translocation is the core of transcription factor function. In the DHTS treatment, STAT3 was inhibited from shifting to the nucleus of the HCC cells (p < 0.05) (Figure 5A). In the western blot, SMMC7721 cells were exposed to DHTS for 24 h. Nuclear and Cytoplasmic extracts of DHTS-treated or untreated cells were extracted. After DHTS treatment, the nuclear expression of p-STAT3 decreased (p < 0.05) (Figure 5B,C).

The activation of JAK2/STAT3 pathway in response to DHTS was evaluated. The protein expression of phosphorylated-JAK2 and phosphorylated-STAT3, were decreased in DHTS group (Figure 5D). To confirm the effect of DHTS on JAK2/STAT3 signaling in SMMC7721 cells, the STAT3 knockdown and overexpression in SMMC7721 cells were detected. Western-blot showed that protein levels of STAT3 and phosphorylated-STAT3 were higher in STAT3 overexpression SMMC7721 cells (p < 0.05) (Figure 5G,H) and lower in STAT3 knockdown SMMC7721 cells (p < 0.05) (Figure 5G,H). After DHTS treatment, p-STAT3 was decreased both in STAT3 overexpression and knockdown SMMC7721 cells (p < 0.05) (Figures 5G–J).

All results support that DHTS may regulate the proliferation and apoptosis via inhibiting the activation of JAK2/STAT3.

Our final objective was to determine whether DHTS inhibits the growth of DHTS cells in vivo. The SMMC7721 cells were seeded into BALB/C nude mice. Tumor weight and size were significantly reduced in the DHTS groups (at 5, 10 and 15 mg/kg) (p < 0.05) (Figures 6A–C). It’s noted that no animal death in all groups. TUNEL staining of tumor tissues of each group showed that efficient apoptosis occurred in the tumor mass from treatment groups, whereas the degree of apoptosis differed in each cluster. Also, 15 mg/kg DHTS group exhibited a distinct apoptosis compared with control (p < 0.05) (Figure 6D). Furthermore, we evaluated the level of STAT3 in tumor specimens by Western blot. The results revealed that DHTS decreased the expression levels of p-STAT3 (p < 0.05) (Figure 6E). These results indicate that DHTS has significant antitumor effect in vivo.