Emodin reverses sorafenib resistance in hepatocellular carcinoma by inhibiting epithelial-mesenchymal transition via the Akt signaling pathway

Introduction

Globally, hepatocellular carcinoma (HCC) ranks as the sixth most prevalent malignant neoplasm and the third highest contributor to cancer-related mortality (1). A significant proportion of HCC cases are identified in advanced stages, limiting the possibility of surgical treatment. Chemotherapy is the primary treatment for advanced cancer. Sorafenib is the first Food and Drug Administration (FDA)-approved first-line molecular-targeted drug used for the treatment of advanced liver cancer. Notably, this drug inhibits tumor growth, angiogenesis, and metastasis (2). However, acquired resistance to sorafenib results in unsatisfactory clinical outcomes (3,4). Therefore, further exploration of adjuvant therapies to reverse sorafenib resistance is crucial.

The initiation of epithelial-mesenchymal transition (EMT) has been correlated with tumor metastasis and resistance to therapeutics in various diseases, including HCC. Abnormal activation of the PI3K/Akt pathway is also associated with cancer drug resistance. Zhuang et al. (5) found that BEX1 regulates AKT signaling pathway, thereby mediating sorafenib resistance in HCC. Similarly, Li et al. (6) reported that the circular RNA ITCH increases sorafenib sensitivity by sequestering miR-20b-5p and modifying the downstream PTEN-PI3K/Akt pathway in HCC. Overall, targeting the PI3K/Akt signaling pathway appears to be a promising therapeutic strategy to overcome sorafenib resistance in HCC.

The natural anthraquinone compound emodin (structural formula shown in Figure 1A) is derived from various Chinese herbs, including Rheum palmatum L. Notably, studies have shown that emodin has potent anticancer activity for lung cancer, endometrial cancer, myeloid leukemia, and HCC (7-10). Additionally, several studies have demonstrated the potential of emodin to alleviate resistance in colorectal and chronic myeloid leukemia cells (11,12), indicating its potential regulatory role in drug resistance. Although Kim et al. suggested that emodin enhances sorafenib’s anticancer impact on liver cancer by inhibiting cholesterol metabolism (13), its role in reversing drug resistance by EMT in resistant HCC cells is not entirely clear.

Figure 1 Emodin resensitizes Huh7SR cells to sorafenib. (A) Chemical structure of emodin. (B) CCK-8 assay for Huh7 and Huh7SR cells treated with varying sorafenib concentrations (0, 1, 2, 4, 8, 16, and 32 µM). (C) CCK-8 assay for Huh7 and Huh7SR cells treated with varying emodin concentrations (0, 10, 20, 40, and 80 µM). (D) Impact of treatment with sorafenib (0, 1, 2, 4, 8, 16 and 32 µM) and sorafenib combined with emodin (20 µM) on the proliferation of Huh7SR cells. (E) Clonogenic assay showing Huh7SR cell colonies stained with 0.01% crystal violet after different treatments (magnification, 10×). (F) Quantitative analysis of colony number in each treatment group. Each experiment was repeated at least three times. Results are presented as a mean ± SD (n=3). **, P<0.01, *, P<0.05 compared to the control group; ^##, P<0.01 compared to the sorafenib group. CCK-8, Cell Counting Kit-8; SD, standard deviation.

Therefore, this study aimed to determine whether emodin could reverse sorafenib resistance in HCC. Ultimately, these findings offer new insights into overcoming drug resistance in this cancer type. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-24-1260/rc).

Methods

Reagents and chemicals

Emodin (Cat No. BP0532) was purchased from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China). Sorafenib (Cat No. HY-10201) was procured from MedChemExpress (Shanghai, China). Both drugs were dissolved in dimethyl sulfoxide, which did not exceed 0.1% (v/v) during use. The Cell Counting Kit-8 (CCK-8; Cat No. CK04) was procured from Dojindo Molecular Technologies, Inc. (Tokyo, Japan). Primary antibodies acquired from Cell Signaling Technology (Shanghai, China) included: N-cadherin rabbit monoclonal antibody (mAb) (D4R1H; Cat No. 13116), vimentin rabbit mAb (D21H3; Cat No. 5741), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rabbit mAb (D16H11; Cat No. 5174), phosphor-Akt rabbit mAb (Ser473; D9E; Cat No. 4060), Akt rabbit mAb (pan; C67E7), Bax rabbit mAb (D2E11; Cat No. 5023T), cleaved-caspase-3 rabbit mAb (Asp175; Cat No. 9661T), and cleaved poly(ADP-ribose) polymerase (cleaved-PARP) rabbit mAb (Asp214; D64E10; Cat No. 5625T). E-cadherin rabbit mAb (Cat No. 20874-1-AP) was purchased from Proteintech Group, Inc. (Wuhan, China) and anti-rabbit IgG (H+L) horseradish peroxidase-linked antibody (Cat No. 70-GAR0072) was procured from Multi Sciences (Hangzhou, China).

Cell culture

The Huh7 cell line (Cat No. TCHu182) was purchased from the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) in a 37 ℃ incubator with 5% CO₂. A sorafenib-resistant cell line [sorafenib-resistant Huh7 (Huh7SR)] was obtained by exposing Huh7 cells to increasing doses of sorafenib for nearly 6 months.

Cell viability assay

Huh7SR cells (5×10⁴ cells/mL) were plated in 96-well plates and confluence reached within 24 h. Subsequently, each well was exposed to 10 µL CCK-8 test solution at 37 ℃. After 1 h, the optical density (OD) was assessed at 450 nm with a microplate reader. Each experimental group consisted of five replicates. A cell viability curve was then constructed using GraphPad Prism 5.0 software.

Clonogenic colony formation assay

Huh7SR cells (150 cells/well) were distributed into 6-well plates. Cells were subjected to various treatments, including exposure to emodin (20 µM), sorafenib (2 µM), or a combination of both, for 12 days to facilitate colony formation. Following treatment, the cells were fixed with methanol for 20 min and stained with 0.01% crystal violet for 20 min at room temperature. Colonies containing >50 cells were quantified using an inverted microscope.

Transwell assay

Cell invasion and migration were measured using the transwell assay. For the invasion assay, Matrigel (BD Sciences, San Jose, CA, USA) was combined with DMEM at a ratio of 1:5 to produce a solution that covered the upper chambers of the transwell (pore size 8 µm; Corning, NY, USA). Cells (5×10⁴ cells/well) were suspended in serum-free media and added to the upper chambers of the transwell inserts. The cells were then treated with emodin, sorafenib, or a combination of both for 48 h. Additionally, 500 µL DMEM supplemented with 15% FBS was placed in the lower chambers of the transwell to serve as a chemoattractant. After 48 h, uninvaded cells were removed, and the remaining cells that infiltrated the basal region of the membrane in the lower chamber compartment were fixed using methanol and stained with crystal violet. Subsequently, the invaded cells were imaged using a microscope at 100× magnification and five randomly chosen fields were quantified. For the migration assay, the upper chambers of the transwell inserts were devoid of any matrix, and a total of 2×10⁴ cells were introduced per well. All other procedures remained the same as the invasion assay.

Apoptosis analysis using flow cytometry

Cellular apoptosis was assessed using the Annexin V-FITC cell apoptosis assay kit (Multi Sciences, Hangzhou, China) according to the manufacturer’s guidelines. Briefly, Huh7SR cells were subjected to treatment with emodin, sorafenib, or a combination of both for a duration of 48 h, followed by harvesting and washing with cold phosphate-buffered saline (PBS) thrice. Cells were then resuspended in 500 µL binding buffer, 5 µL V-FITC, and 5 µL propidium iodide (PI) and incubated for 30 min. BD Fortessa flow cytometer was then used to detect apoptotic cells.

Hoechst staining

To analyze apoptosis and morphological changes in Huh7SR cells, the Hoechst staining kit (Beyotime, Shanghai, China) was used, according to the manufacturer’s instruction. Briefly, cells were fixed for 10 min at room temperature, stained with Hoechst 33258 for 5 min in the dark, washed with PBS thrice, and sealed with anti-fluorescence quenching solution. Apoptotic cells were identified by nuclear staining using a fluorescence microscope.

Western blotting

Huh7SR cells were subjected to various treatments and harvested after 48 h. Cells were washed with cold PBS thrice, lysed with Radio Immunoprecipitation Assay (RIPA) Lysis buffer for 30 min on ice, and centrifuged at 12,000 rpm for 15 min at 4 ℃. The supernatant was collected and the protein concentration measured using a Bicinchoninic Acid Assay (BCA) assay kit. Proteins (30 µg per sample) were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis for 2 h at 100 V, and transferred to a nitrocellulose membrane for 90 min at 300 mA. Membranes were blocked with skim milk for 1 h before incubating with primary antibody overnight at 4 ℃. After incubation, membranes were washed with PBS with Tween thrice and incubated with secondary antibody for 1 h at room temperature. Immunoreactive bands were visualized utilizing a ChemiDoc imaging system (Bio-Rad, USA), and subsequent grayscale analysis of the protein blots was performed using ImageJ software (https://imagej.net/ij/).

Statistical analyses

Data were expressed as the mean ± standard deviation (SD). Statistical analyses were performed using Student’s t-test and one-way analysis of variance (ANOVA) in SPSS software (version 17.0, SPSS Inc., Chicago, IL, USA). A P value <0.05 was considered statistically significant.

Results

Emodin resensitizes Huh7SR cells to sorafenib

We initially constructed a Huh7SR cell line according to previously established methods. Notably, CCK-8 assay results demonstrated that these Huh7SR cells exhibited significant chemoresistance to sorafenib compared to the parental Huh7 cells (Figure 1B). Emodin treatment showed a dose-dependent reduction in cell proliferation, with Huh7SR cells maintaining higher viability than Huh7 cells at the same concentrations (Figure 1C). This dose-dependent relationship was observed at a concentration of 20 µM, in which the inhibition ratio remained <10%, indicating non-toxicity; therefore, this concentration was selected as the in vitro concentration for further study. Emodin significantly resensitized Huh7SR cells to sorafenib at 1, 2, 4, and 8 µM, ultimately enhancing the inhibitory effects of sorafenib on proliferation (Figure 1D). Sorafenib and emodin suppressed colony numbers, with the combination treatment exhibiting an enhanced inhibitory effect (Figure 1E,1F).

Emodin combined with sorafenib significantly suppresses cell migration and invasion

A key characteristic of cellular resistance is the increased capacity for invasion. Therefore, we investigated the migration and invasion properties of Huh7SR cells after treatment with either emodin, sorafenib, or a combination of both. Following sorafenib or emodin treatment, Huh7SR cells exhibited significantly reduced migration and invasion abilities (Figure 2). The combination treatment demonstrated a more pronounced effect, greatly reducing migration and invasion capabilities compared to either drug alone. Therefore, the combined use of emodin and sorafenib significantly attenuates the invasion and migration of HCC Huh7SR cells.

Figure 2 Emodin combined with sorafenib treatment significantly suppresses cell migration and invasion. Transwell assay of Huh7SR cells treated with sorafenib (2 µM), emodin (20 µM), or a combination of both. (A) Migration and (B) invasion of Huh7SR cells was stained with crystal violet and then observed at ×100 magnification. Quantitative analysis of migrated cells number (C) and invaded cells number (D) in each treatment group. Each experiment was repeated at least three times. Results are presented as a mean ± SD (n=3). **, P<0.01, *, P<0.05 compared to the control group; ^##, P<0.01, ^#, P<0.05 compared to the sorafenib group. SD, standard deviation.

Emodin combined with sorafenib significantly facilitates apoptosis

To assess the efficacy of emodin and sorafenib combination treatment in inducing apoptosis in Huh7SR cells, we performed Annexin V-FITC/PI apoptosis and Hoechst staining assays. After treatment for 48 h, apoptosis rates in the sorafenib and emodin groups, encompassing both early- and late-stage apoptotic cells, were 10.8% and 12.6%, respectively; meanwhile, the apoptosis rate of the combination treatment reached 21.1% (Figure 3A,3B). Furthermore, Hoechst staining revealed the combination treatment group had more pronounced changes in chromatin condensation and brightness compared with the individual treatment groups (Figure 3C). Overall, these findings indicate that the synergistic use of emodin and sorafenib significantly elevates apoptosis.

Figure 3 Emodin and sorafenib treatment significantly facilitates apoptosis. (A) Flow cytometry results for Annexin V/PI stained Huh7SR cells treated with sorafenib (2 µM), emodin (20 µM), or a combination of both. (B) Histogram of the apoptosis percentages in each treatment group. (C) Hoechst staining showing the morphological changes in Huh7SR cells after different treatments (magnification, 100×). White arrows indicated condensed nuclei. Each experiment was repeated at least three times. Results are presented as a mean ± SD (n=3). **, P<0.01, *, P<0.05 compared to the control group; ^##, P<0.01 compared to the sorafenib group. SD, standard deviation; Annexin V/PI, Annexin V/propidium iodide.

Emodin combined with sorafenib significantly elevates apoptosis-related protein expression and reverses EMT through the AKT signaling pathway

To investigate the apoptotic mechanism in Huh7SR cells treated with a combination of emodin and sorafenib, western blotting was employed to assess the expression of apoptosis-associated proteins. Cleaved-caspase-3, cleaved-PARP and Bax expression were significantly elevated in the combination therapy group compared to the monotherapy groups (Figure 4A). While the expression of cleaved-caspase-3 and Bax were elevated in Huh7SR cells treated with sorafenib alone, this effect was further enhanced in the presence of emodin. Emodin alone also upregulated Bax and cleaved-caspase-3 expression, indicating a synergistic effect on the apoptotic signaling pathway.

Figure 4 Effects of emodin and sorafenib treatment, alone or combined, on expression of apoptotic, EMT marker-related, and AKT signaling pathway proteins. (A) Western blot results of Bax, cleaved-PARP, and cleaved-caspase-3 expression in Huh7SR cells following different treatments. (B) Western blot results of vimentin, N-cadherin, and E-cadherin expression in Huh7 and Huh7SR cells. (C) Western blot results of vimentin, N-cadherin, and E-cadherin expression in Huh7SR cells following different treatments. (D) Western blot results of p-AKT and AKT expression in Huh7 and Huh7SR cells. (E) Western blot results of p-AKT and AKT expression in Huh7SR cells after different treatments. Relative protein expression levels were normalized to GAPDH. Each experiment was repeated at least three times. Results are presented as a mean ± SD (n=3). **, P<0.01, *, P<0.05 compared to the control group; ^##, P<0.01, ^#, P<0.05 compared to the sorafenib group. EMT, epithelial-mesenchymal transition; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; cleaved-PARP, cleaved poly(ADP-ribose) polymerase.

Previous studies have established a correlation between drug resistance and EMT, a biological process known to induce migration and invasion during tumor development. As expected, the Huh7SR cells established in this study exhibited EMT. According to western blot analysis, Huh7 cells demonstrated markedly elevated E-cadherin levels and significantly diminished vimentin and N-cadherin levels compared to those observed in Huh7SR cells (Figure 4B). Treatment with either sorafenib or emodin individually increased the expression of E-cadherin and decreased the expression of N-cadherin and vimentin (Figure 4C). Notably, the administration of combination therapy resulted in a more pronounced downregulation of N-cadherin and vimentin, while concurrently inducing an upregulation of E-cadherin expression. Ultimately, the results indicate that the co-administration of emodin and sorafenib successfully induces the reversal of EMT in Huh7SR cells.

Our findings also indicated a significantly higher expression of p-AKT in Huh7SR cells than in Huh7 cells (Figure 4D). Notably, treatment with either sorafenib and emodin individually downregulated p-AKT protein levels (Figure 4E), while the combination treatment group exhibited a greater decrease in p-AKT expression levels than the monotreatment groups. However, no statistically significant variances in total levels of AKT expression were observed among the groups. These findings suggest that emodin effectively inhibits Akt signaling pathway, restoring Huh7SR cell sensitivity to sorafenib.

Discussion

In recent years, there have been numerous advancements in therapeutic approaches for tumors; however, the emergence of drug resistance limits clinical efficacy. Targeted therapy is a key therapeutic strategy for HCC, owing to its high effectiveness, minimal side effects, and broad applicability. Notably, sorafenib is considered a primary targeted therapy for the management of advanced HCC. However, acquired sorafenib resistance diminishes its clinical efficacy, leading to a poor prognosis. Consequently, further investigations into adjunctive therapies to enhance the efficacy of chemotherapy are warranted. Previous studies have shown that emodin exerts strong anticancer effects and enhances the effectiveness of chemotherapy (13,14). Nevertheless, there remain limited data regarding the impact of emodin combined with sorafenib on proliferation, migration, invasion, and apoptosis activation in sorafenib-resistant HCC cells. Thus, the primary objective of the present study was to illustrate the potential of emodin in augmenting the efficacy of sorafenib in tumor cells resistant to chemotherapy.

The role of EMT in drug resistance has been established in various cancers, including HCC (15). Notably, the current investigation demonstrated a notable decrease in E-cadherin expression in Huh7SR cells relative to Huh7 cells, concomitant with an increase in vimentin and N-cadherin expression; ultimately indicating the occurrence of EMT in the drug-resistant cells. Additionally, we established that the emodin and sorafenib combination treatment effectively reversed EMT in vitro. In a prior study, Xu et al. (16) discovered that resveratrol can inhibit EMT in gastric cancer by regulating the PTEN/Akt pathway. Similarly, Wu et al. (17) demonstrated that the c-Src/PI3K/Akt pathway regulates EMT-mediated tamoxifen resistance in breast cancer. Therefore, we hypothesized that emodin and sorafenib combination treatment may reverse EMT and chemotherapy resistance by inhibiting Akt signaling pathway.

Previous studies have indicated that emodin triggers apoptosis (7,18). Using flow cytometry and Hoechst staining, our study revealed that combining sorafenib and emodin increased apoptosis in Huh7SR cells more than the control or single treatment groups. Moreover, western blot analysis revealed that emodin and sorafenib combination treatment resulted in a significant upregulation of apoptotic proteins, such as Bax, cleaved-PARP, and cleaved-caspase-3. Therefore, the results indicate that the co-administration of emodin and sorafenib can potentially enhance cellular apoptosis in vitro. Li et al. (19) revealed that S63845-induced apoptosis is triggered by AKT. Similarly, Yang et al. (20) found that 7-epitaxol triggers cell apoptosis in patients with cisplatin-resistant head and neck squamous cell carcinoma by inhibiting AKT and MAPK signaling. Therefore, we hypothesized that the emodin and sorafenib combination treatment may induce apoptosis and reverse chemoresistance through inhibition of the Akt signaling pathway.

The Akt signaling pathway is essential in regulating a variety of cellular processes such as apoptosis, proliferation, chemoresistance, and EMT (21,22). Our findings demonstrated a marked increase in p-AKT levels in Huh7SR cells compared to Huh7 cells; meanwhile, no significant differences in total AKT levels were observed. These findings indicated the AKT signaling pathway is activated in sorafenib-resistant cells. Similarly, Li et al. (11) discovered that emodin can overcome 5-Fu resistance in colorectal cancer by inhibiting the PI3K/Akt pathway. Our findings indicate that the concurrent administration of sorafenib and emodin effectively suppressed cell proliferation and decreased the expression of p-AKT in comparison to both control and individual treatment groups. Therefore, combined emodin and sorafenib treatment was found to reverse chemoresistance by inhibiting Akt signaling pathway.

Conclusions

Emodin increases Huh7SR cell sensitivity by promoting cell apoptosis and reducing cell growth, migration, and invasion. Furthermore, emodin can reverse EMT and promote apoptosis by inhibiting the Akt signaling pathway. Overall, a limitation of this study is its reliance on a single cell line, yet it also offers significant insights into the potential use of emodin as an adjunctive therapy to enhance the efficacy of chemotherapy.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-24-1260/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-24-1260/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-24-1260/prf

Funding: This work was supported by Medical Science and Technology Project of Zhejiang Province (No. 2021KY1101).

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-24-1260/coif). Both authors report that this work was supported by Medical Science and Technology Project of Zhejiang Province (No. 2021KY1101). The authors have no other 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.

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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