Schisandrin B inhibits tumor progression of hepatocellular carcinoma by targeting the RhoA/ROCK1 pathway

Introduction

Hepatocellular carcinoma (HCC) has become one of the most frequently diagnosed forms of malignancy (1). HCC’s overall rate ranks sixth of all cancers and second in terms of mortality. Although the targeted therapy, the immunotherapy, and interventional therapy have become major strategies for treating cancer, treatment efficiency in HCC remains pessimistic due to rapid growth, metastasis, and drug resistance of HCC cells, and unfortunately these treatment options have limited success in HCC (2). One such agent is 5-fluorouracil (5-Fu), which inhibits thymidylate synthase and has shown a response rate of around 10% in HCC treatment (3). Accordingly, developing novel nontoxic and potent modulators to treat HCC is an urgent problem to be solved.

Schisandrin B (Sch. B), a potent dibenzocyclooctadiene derivative extracted from the fruit of the traditional Chinese medicinal plant Schisandra chinensis (Turcz.), has been reported to have significant inhibitory effects on a variety of solid malignancies, including colon, cervical, and breast cancers (4-6). Recently, He et al. reported that Sch. B inhibits the duplication of gastric cancer cells and promotes 5-fluorouracil (5-FU) drug sensitivity (7). Yan et al. found that Sch. B promotes docetacxel-induced inhibition of cervical cancer progression (5). Sch. B has also been shown to reduce neoplastic changes in osteosarcoma (8). However, there are fewer studies reporting on the suppression effect of Sch. B on HCC cells.

The RhoA/rho-associated protein kinase 1 (ROCK1) pathway has been found to participate in regulating duplication and metastasis in HCC cells (9). It has been suggested that targeting the RhoA/ROCK1 signaling pathway may be an effective strategy for inhibiting HCC and breast cancer progression (10,11). However, there is little reported in the existing literature about the role of Sch. B in regulating the RhoA/ROCK1 pathway.

Based on those researches, we propose a scientific hypothesis that Sch. B performs the therapeutic impact on HCC progression in vivo and in vitro in italic, which is related with the RhoA/ROCK signaling pathway. Our results have provided compelling experimental results for the development of Sch. B for HCC therapy. We present the following article in accordance with the ARRIVE reporting checklist (available at https://jgo.amegroups.com/article/view/10.21037/jgo-23-87/rc).

Methods

Animal grouping and Sch. B treatment

Firstly, we evaluated the inhibitory role of Sch. B on HCC using tumor-bearing mice model, which is well-established animal model of cancer. A total of thirty-two, half female and half male, 4–6-week-old, Balb/c nude mice were obtained from Liaoning Changsheng Biotechnology Co. Mice were bred at an airconditioned animal facility of China Medical University with 22±1 ℃ temperature and 45%±5% relative humidity. The entire animal protocol was approved by the Institutional Animal Care and Use Committee of China Medical University (No. CMU2021633), in compliance with institutional guideline for the care and use of animals. Once the tumor volume grew to 100 mm³, the mice were put into 4 groups of 8 mice each using a random number table: control (Con) group, low-dose Sch. B (Sch. B-L), medium-dose Sch. B (Sch. B-M), and high-dose Sch. B (Sch. B-H). Mice in the different groups were treated with either saline only, 100, 200, or 400 mg/kg/d of Sch. B solution respectively, by gavage daily for 21 days. Sch. B was purchased from Chengdu Pufei De Biotech Co., Ltd. Sch. B was dissolved in dimethyl sulfoxide (DMSO), then diluted in Phosphate Buffered Saline (PBS) at final concentration of 5 mg/mL, 10 mg/mL and 20 mg/mL. After the final drug treatment, the mice were euthanized, and tumors were collected, weighed, and stored appropriately for subsequent experiments. A protocol was measured and prepared before the study without registration.

Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) experiment

TUNEL assay was used to examine cell apoptosis. Formalin-fixed, paraffin-embedded tissues were sectioned at 4 µm thick using a microtome (Leica Biosystems RM2245 Microtome). Paraffin sections were prepared of the tumor tissues, and a TUNEL kit (Dalian Meilun Biotech, Dalian, China) was used to determine tumor tissue apoptosis. TUNEL procedures were carried out strictly according to the kit instructions. The tumor tissue was placed in an inverted fluorescent microscope (TE2000U, Nikon, Tokyo, Japan) to observe and count apoptotic cells.

Immunohistochemical staining (IHC)

Ki-67 and proliferating cell nuclear antigen (PCNA) was determined for evaluating cell proliferation. Paraffin sections were used for detection of Ki-67 and PCNA levels. After hydration, antigen repair, and permeabilization, Ki-67 antibody (#34330,1:300, CST Biological Reagents Co., Ltd., Shanghai, China) or PCNA antibody (#13110, 1:300, CST) was added dropwise on the tissue surface and incubated overnight at 4 ℃. The Ki-67 or PCNA positive cells were observed under a microscope (BX53M, Olympus, Tokyo, Japan) and the positive rate was calculated.

Cell culture

Human hepatocellular carcinoma cell line Huh-7 was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultivated in Roswell Park Memorial Institute (RPMI)-1640 with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a 37 ℃ incubator. The RhoA overexpression plasmid was synthesized by Genepharma (Shanghai, China). After Huh-7 cells were cultured, Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was used to perform cell transfection according to the instructions.

Drug concentration selection and grouping

Huh-7 cells were digested in 0.25% trypsin, then centrifuged at 1,500 rpm/min for 3 minutes, resuspended in fresh medium, adjusted to a concentration of 1.5×10⁵/mL, and inoculated in 96-well plates with 100 µL each. Next, 100 µL of medium containing different dosages of Sch. B at final concentrations of 40, 30, 20, 10, 5, 1, and 0 µM was added to each well. The cells were then incubated for 24 hours, after which Cell Counting Kit-8 (CCK-8) staining solution was added. The Huh-7 cells were then further cultured for 120 minutes. The absorbance was determined according to the protocol. Cell survival rate (%) = (A _{Sch. B}/A _Con) ×100%.

For the subsequent experiment, Huh-7 cells were put into the following groups: control, Sch. B, RhoA overexpression, and Sch. B + RhoA overexpression. The cells in the control group were transfected with vector solution. The cells in the RhoA overexpression group were transfected with RhoA overexpression virus solution for 48 hours. The Sch. B and Sch. B + RhoA groups were treated with 10 µM Sch. B for 24 hours.

Colony formation assay

Colony formation assay was performed to detect cell proliferation. Cells were digested and resuspended after 24 hours of Sch. B treatment, after which 500 cells/well were inoculated in 6-well plates and cultured for 14 days. The cells were then fixed in paraformaldehyde solution, followed by the addition of 1 mL of 0.1% crystalline violet staining solution and staining for 30 minutes. Cells were photographed using a digital camera, and the number of cell colonies was statistically analyzed.

Apoptosis examination

After digestion by ethylenediaminetetraacetic acid (EDTA)-free trypsin following 24 hours of drug treatment, Huh-7 cells were centrifuged at 1,500 rpm/min for 3 minutes. Apoptosis was assessed using an FITC Annexin V/PI Apoptosis Detection Kit, and the apoptosis rate was calculated.

Scratch wound healing assay

Scratch wound healing assay was used to examine cell migratory ability. HCC cells were cultivated and prepared as 1×10⁵/mL cell suspension and spread in 6-well plates at 2 mL/well. Once 80% of cells were fused, the cell surface was scored with a pipette tip. Cells on the plate were then photographed by a light microscope (Olympus) at 0 hours and 24 hours after drug administration, and the migration rate was determined. Wound healing rate (%) = (1−scratch area/original scratch area) × 100%.

Transwell assay

Transwell assay was used to examine cell invasion rate. Matrigel matrix gel and RPMI 1640-free medium were diluted 1:8 on ice and 50 µL Matrigel matrix gel was aliquoted to the upper chamber of the Transwell insert. Huh-7 cells were aliquoted to the upper layer at 4×10⁴/well, followed by the addition of medium with FBS into the lower layer. After 48 hours cultivation, the Transwell chamber was removed, 4% paraformaldehyde was used for incubation for 15 minutes, and crystal violet solution was used to stain the cells for 30–40 minutes. Cells were then imaged by a light microscope (Olympus), and the cell invasion rate was calculated.

Western blot assay

RhoA and ROCK1 levels were examined by western blot.Tumor tissues or Huh-7 cells were used for western blot. Whole proteins were purified using a protein extraction kit (Promega, Beijing, China). After quantified, 30 µg whole protein was loaded on sodium dodecyl by BCA method sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, transferred onto a polyvinylidene difluoride (PVDF) membrane, and then blocked at skim milk solution. Anti-RhoA (ab187027, Abcam) and anti-ROCK1 (ab134181, Abcam) antibodies were added and incubated overnight at 4 ℃. Diluted secondary antibodies (1:1,000, Abcam) were aliquoted and incubated for 2 hours. After detection by enhanced chemiluminescence (ECL) luminescence kit (Thermo Fisher Scientific, Madison, WI, USA), the protein blots were imaged in a gel imager and photographed. The grey scale values were calculated by Image J software [National Institutes of Health (NIH) image software].

Statistical analysis

Measurements are showed as mean ± standard deviation (mean ± SD). Statistical analysis was performed by SPSS 21.0. Students t test was used for two-group comparisons. One-way analysis of variance (ANOVA) was performed for multiple comparison, and the least significant difference (LSD) test was used for two-way comparisons. P<0.05 was considered statistically significant.

Results

Sch. B inhibits tumor weight and volume in HCC tumor-bearing mice

We firstly explored the effect of Sch. B on HCC. HCC tumor-bearing mice were prepared and treated with different doses of Sch. B. As demonstrated in Figure 1A,1B, tumor volume in the Sch. B-L, Sch. B-M and Sch. B-H group were statistically reduced compared with that of control group (P<0.05). Moreover, as showed in Figure 1C, tumor weight in the Sch. B-L, Sch. B-M and Sch. B-H group were also remarkably decreased compared with that of control group (P<0.05).

Figure 1 The weight and volume of tumor in each group. (A) Representative pictures of tumors. (B) Tumor volume data (n=8). (C) Tumor weight results (n=8). *P<0.05 vs. Con group.

Sch. B promotes cell apoptosis of HCC

Cell apoptosis of tumor tissue under Sch. B treatment was detected by TUNEL assay (Figure 2A). There was no significant difference in apoptotic cells between the Sch. B-L and Con groups (P>0.05). Interestingly, there were markedly more apoptotic cells in the Sch. B-M group and Sch. B-H group than that in the Con group (P<0.05).

Figure 2 Apoptotic cells in tumor tissue and the percentage of Ki-67 or PCNA positive cells in tumor tissues of tumor-bearing mice. (A) Apoptotic cells in tumor-bearing mice stained by DAPI marked by blue staining and TUNEL marked by green staining (n=3). Magnification, ×400. (B) Ki-67 or PCNA positive cells stained by IHC (%) (n=3). Magnification, ×400. *, P<0.05 vs. Con group. ns, P>0.05 vs. Con group. ^#, P<0.05 vs. Sch. B-L group. PCNA, proliferating cell nuclear antigen; IHC, immunohistochemical staining; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick-end labeling; DAPI, 4',6-diamidino-2-phenylindole.

Sch. B inhibits HCC cell duplication in mice tumor models

To examine the regulatory role of Sch. B on HCC duplication, Ki-67 or PCNA expression in tumor tissue of Huh-7 xenograft mice were examined by IHC. As shown in Figure 2B, there was no significant difference in Ki-67 and PCNA positive cells in the Sch. B-L group (P>0.05). Interestingly, the number of Ki-67 and PCNA positive cells in tumor tissue of the Sch. B-M and Sch. B-L groups was less than that in Con group.

Sch. B blocks the RhoA/ROCK1 pathway in tumor tissues

To further uncover the regulatory mechanism of Sch. B on the RhoA/ROCK1 pathway, RhoA and ROCK1 expression levels were determined by western blot. As shown in Figure 3, the expression of RhoA and ROCK1 in the tumor tissues of the Sch. B-L group was not significant (P>0.05). However, RhoA and ROCK1 expression in the Sch. B-M and Sch. B-H groups was significantly reduced (P<0.05). The above results indicated Sch. B promoted cell apoptosis and inhibited proliferation of tumor cells in tumor-bearing mice, which may have been related to inhibition of the RhoA/ROCK1 pathway.

Figure 3 Levels of RhoA and ROCK1 expression in tumor tissues under Sch. B treatment (n=3). *P<0.05 vs. Con group; ^#P<0.05 vs. Sch. B-L group; ns, P>0.05 vs. Con group.

The inhibitory effect of different Sch. B concentrations on HUH-7 cells

After HUH-7 cells were incubated in different concentrations of Sch. B for 24 hours, the results of the CCK-8 assay showed that cell survival rate was decreased significantly (Figure 4). Cell viability under 1 and 5 µM Sch. B treatment was not significantly different compared with 0 µM Sch. B treatment (P>0.05). However, cell viability in the 10 µM, 20 µM, 30 µM, and 40 µM Sch. B groups was significantly lower than in the non-Sch. B treatment group (P<0.05). Therefore, 10 µM was chosen as the experimental concentration for subsequent experiments.

Figure 4 CCK-8 results of Huh-7 cells with different amounts of Sch. B treatment (n=3). *P<0.05 vs. 0 µM group; ns, no significant. CCK-8, Cell Counting Kit-8.

Sch. B regulates the RhoA/ROCK1 signaling pathway in Huh-7

To further uncover the detailed mechanism of Sch. B on Huh-7 cells, RhoA overexpression cells were constructed to upregulate intracellular RhoA and ROCK1 levels. As shown in Figure 5, Sch. B markedly reduced RhoA and ROCK1 levels in vitro (P<0.05). Moreover, overexpression of RhoA could induce the expression of ROCK1 in Huh-7 (P<0.05).

Figure 5 The levels of RhoA and ROCK1 in Huh-7 cells (n=3). The final concentration of Sch. B is 10 µM. *, P<0.05 vs. Con group; ^#, P<0.05 vs. Sch. B group; ^, P<0.05 vs. RhoA group.

Sch. B affects the proliferative capacity of Huh-7 cells through the RhoA/ROCK1 pathway

Figure 6 shows the number of cell colonies in the Sch. B group was significantly reduced (P<0.05). However, colony formation results also demonstrated that cell colonies in the Sch. B + RhoA group were remarkably increased compared with Sch. B treatment (P<0.05).

Figure 6 Colony formation results of Huh-7 cells in each group stained by 0.1% crystalline violet staining (n=3). *P<0.05. The final concentration of Sch. B is 10 µM.

Sch. B promotes Huh-7 apoptosis via the RhoA/ROCK1 pathway

The flow cytometry data are shown in Figure 7. These data demonstrated Sch. B treatment markedly promoted the apoptosis of Huh-7 cells (P<0.05). Interestingly, RhoA overexpression could partially block apoptosis induction under Sch. B treatment (P<0.05). These data also indicated that Sch. B increased HUH-7 cell apoptosis dependance on the inhibition of RhoA.

Figure 7 Flow cytometry results of Huh-7 (n=3). *P<0.05. The final concentration of Sch. B is 10 µM. PI, propidium iodide staining; FITC, annexin V-FITC staining.

Sch. B inhibits the metastasis of Huh-7 cells via the RhoA/ROCK1 pathway

We also evaluated the impact of Sch. B on migration and invasion capability of HCC cells. Figure 8A shows Sch. B significantly reduced Huh-7 cell migration capability (P<0.05). Interestingly, the cell migration rate of the Sch. B + RhoA overexpression group was reversed than that the inhibitory effect in Sch. B treatment (P<0.05). In addition, the Transwell assay results (Figure 8B) show Huh-7 cell invasion under Sch. B treatment was significantly reduced (P<0.05). The invasion capability in the Sch. B + RhoA group was significantly higher than that in Sch. B treatment group (P<0.05).

Figure 8 The metastasis capability of Huh-7 under Sch. B treatment. (A) The results of scratch wound healing rate assay experiments (n=3). Cells were photographed by a light microscope (Olympus) at 0 hours and 24 hours after drug administration. Magnification, ×400. (B) The results of Transwell assay experiments stained by crystal violet solution (n=3). Cells were imaged by a light microscope (Olympus). Magnification, ×400; *P<0.05.

Discussion

Used for thousands of years to treat pandemics and endemic diseases, traditional Chinese medicine (TCM) is widely considered as the jewel of the nation (12). Sch. B is an active monomeric component of TCM that demonstrates antitumor activity (13). Liu et al. found Sch. B performed an inhibitory role in gastric cancer (14). He et al. reported that Sch. B increased the efficacy of chemotherapy drug 5-FU in gastric cancer cell (7). In addition, Sch. B has been shown to inhibit epithelial-mesenchymal transition and stemness in lung cancer by regulating the NF-κB and p38 MAPK pathways, thereby exerting an inhibitory effect (15). Sch. B has also been reported to block the Wnt-β-catenin and PI3K-Akt pathways to inhibit progression in osteosarcoma cells together with inducing apoptosis (8). Furthermore, in prostate cancer cells, Sch. B promotes cell apoptosis and oxidative stress as an effective agent for cancer therapy (16). Dai et al. found that Sch. B was effective for inhibiting triple negative breast cancer through regulation of STAT3 (6). Jiang et al. demonstrated that Sch. B could block the progression of glioma cells by regulating the HOTAIR–micoRNA-125a–mTOR axis (17). In our research, the therapeutic role of Sch. B was demonstrated by constructing mice xenograft models with Huh-7 cells. Our data demonstrated that tumor weight and volume in tumor-bearing mice were markedly reduced, cell numbers were markedly reduced, and the apoptotic index was also promoted under Sch. B treatment, indicating that Sch. B performed a promising anticancer role.

The RhoA/ROCK1 pathway is one of most important pathways in tumor cell motility and morphogenesis, which is associated with cell metastasis (18,19). RhoA belongs to the Rho family of GTPases, which contributes to F-actin polymerization forming stress fibers to regulate actin movement and cellular contractility. In addition, the activation of ROCK1 by RhoA has been shown to promote the formation of local adhesion of tumor cells, thus allowing their movement, with the result that the RhoA/ROCK1 signaling pathway affects tumor cell migration and invasion (10,19). During tumor cell migration and invasion, cell adhesion capacity, cytoskeletal alterations, and the polarization of cells are associated with a stimulated RhoA/ROCK1 pathway. Many studies have shown that over activating the RhoA/ROCK1 pathway in HCC could induce migration and invasion ability (10,20). Similar results were reported in breast and gastric cancer cells, where the stimulation of the RhoA/ROCK1 signaling pathway was found to promote cell migration (21,22). Therefore, the inhibition of RhoA/ROCK1 signaling pathway activity is considered an effective way to inhibit HCC progression.

Our data demonstrated RhoA and ROCK1 expression levels were promoted in tumor tissues of mice, indicating the RhoA/ROCK1 pathway was markedly activated, which is consistent with the results of a previous study (9). After Sch. B treatment, RhoA and ROCK1 levels in tumor tissues were significantly reduced, suggesting Sch. B treatment in liver cancer could be related to inactivation of the RhoA/ROCK1 pathway. You et al. investigated that Schisandrin A inhibit the RhoA/ROCK1 pathway to promote pulmonary capillary endothelial (23). Other study found that Schisandrin A recovered erectile dysfunction through regulating RhoA/ROCK1 pathway in type 1 diabetes mice (24). However, the regulatory role of Sch. B on RhoA/ROCK1 pathway was not reported. To further verify the detailed molecular mechanism of Sch. B treatment in HCC, we constructed Huh-7 cells overexpressing RhoA in vitro. Our data demonstrated that RhoA and ROCK1 levels were markedly increased in Huh-7 cells after transfection with RhoA overexpression viral-based vector, indicating that the RhoA/ROCK1 signaling pathway was markedly activated. After Sch. B treatment, RhoA and ROCK1 levels were significantly reduced, which was consistent with the results in vivo. Interestingly, Sch. B could partially reverse the activation of RhoA/ROCK1 signaling pathway by overexpression of RhoA. In addition, the proliferation, migration, and invasion abilities under Sch. B treatment were significantly reduced, and apoptotic cells were also increased. These results suggested that Sch. B could partially reverse the impact of RhoA overexpression in HCC cell proliferation, apoptosis, migration, and invasion, indicating that Sch. B suppressed the proliferation and metastasis of HCC by downregulating the RhoA/ROCK1 pathway. There is a limitation that we did not examine other mechanisms in Sch. B regulating on HCC cell. And we will explore this in a follow-up study.

Conclusions

Sch. B regulates the migratory, invasion, and proliferation processes of HCC by inhibiting the RhoA/ROCK1 signaling pathway. Our results have provided a new therapeutic approach for effectively slowing down the progression of HCC. However, the specific molecular mechanism needs to be further investigated and confirmed.

Acknowledgments

Funding: None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-23-87/rc

Data Sharing Statement: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-23-87/dss

Peer Review File: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-23-87/prf

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-23-87/coif). HY is from OBiO Technology (Shanghai) Corp., Ltd. The other author has no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The entire animal protocol was approved by the Institutional Animal Care and Use Committee of China Medical University (No. CMU2021633), in compliance with institutional guideline for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: A. Muijlwijk)