Ellagic acid inhibits tumor growth and potentiates the therapeutic efficacy of sorafenib in hepatocellular carcinoma

Abstract

Background: Sorafenib is a classic molecular targeted drug approved for hepatocellular carcinoma (HCC) therapy. However, a poor response rate and increasing resistance to sorafenib make its therapeutic efficacy suboptimal. Combination treatment with an agent capable of potentiating sorafenib sensitivity may be a promising solution.

Aim: The aim of this study was to determine the synergistic effect of ellagic acid (EA), a natural polyphenol, and sorafenib on HCC.

Methods: CCK-8, EdU incorporation and colony formation assays were used to study the effect of EA on HCC cell proliferation. Apoptosis was detected by flow cytometry in HCC cells and TUNEL assay in xenograft tumors. Transcriptome analysis was utilized to investigate alterations in signaling pathways with EA treatment. A xenograft mouse model was used to confirm the synergistic effect of sorafenib and EA on HCC tumors in vivo.

Results: We found that EA inhibited growth and induced apoptosis in both HCC cells and xenograft tumors. Mechanistically, EA treatment reduced the activation of the MAPK and Akt/mTOR signaling pathways in HCC cells. Furthermore, combined EA and sorafenib treatment further inhibited the MAPK and Akt/mTOR signaling pathways compared to EA or sorafenib alone. EA synergistically potentiated the anticancer activity of sorafenib against HCC both in vitro and in vivo.

Conclusion: EA inhibits HCC growth by inducing apoptosis through attenuation of the MAPK and Akt/mTOR signaling pathways. EA potentiates the response of HCC tumors to sorafenib both in vitro and in vivo, an effect that may be attributed to further inhibition of the MAPK and Akt/mTOR signaling pathways. These results suggest that EA is an effective adjuvant option for sorafenib therapy.

Keywords: Akt/mTOR; Ellagic acid; Hepatocellular carcinoma; MAPK; Sorafenib.

Fig. 1

EA reduces HCC growth both in vitro and in vivo. (A) Representative drug response curves of Huh7 and Hep3B cells to EA treatment. (B) EA suppresses colony formation by Huh7 and Hep3B cells. (C) Quantification of the colony formation efficiency with EA treatment. (D) EdU incorporation assay shows reduced DNA replication with EA treatment. (E) Quantification of the EdU incorporation rate in (D). (F) Growth curves of subcutaneous tumors in nude mice administered EA or vehicle. (G) Graph of tumors 9 d after EA or vehicle administration. (H) Quantification of tumor weight in (G). Results are expressed as the means ± SDs. P values were determined by one-way ANOVA with multiple comparison test in (C, E); two-way ANOVA with multiple comparison test in (F); unpaired two-tailed t-test in (H). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2

EA induces apoptosis in HCC cells both in vitro and in vivo. (A) Representative images showing increased apoptosis rates in Huh7 and Hep3B cells after 4 days of EA treatment. (B) Quantification of the apoptosis rates in (A). (C) Double staining of TUNEL and AFP showing an increased apoptosis rate in HCC cells in the EA-treated xenograft mouse model. (D) Quantification of TUNEL positivity in (C). (E) Western blot analysis showing increased levels of the apoptotic proteins C-PARP, C-Caspase3 and Bax in both EA-treated cells and the xenograft mouse model. Results are expressed as the means ± SDs. (F) Quantification of protein expression level in (E). P values were determined by one-way ANOVA with multiple comparison test in (B, F); unpaired two-tailed t-test in (D). *P < 0.05, **P < 0.01, ****P < 0.0001.

Fig. 3

EA inhibits the MAPK and Akt/mTOR signaling pathways in HCC cells. (A) Heatmap showing the differentially expressed genes in Huh7 cells treated with EA for 4 days. (B) Volcano plot showing upregulated and downregulated genes. (C) KEGG analysis results showing the enriched signaling pathways. (D) Western blot analysis showing inhibition of the MAPK and Akt/mTOR signaling pathways. (E) Quantification of protein expression level in (D). P values were determined by one-way ANOVA with multiple comparison test in (E). Results are expressed as the means ± SDs.

Fig. 4

EA potentiates the response of HCC cells to sorafenib. (A) Representative drug response curves of Huh7 and Hep3B cells to sorafenib. (B) EA and sorafenib show synergistic effects on inhibiting HCC cell growth at 4 days. IC₇₅ and IC₅₀ of EA are 2 and 5 μM in both cell lines; IC₇₅ and IC₅₀ of sorafenib is 10 and 40 μM in both cell lines. (C) Western blot analysis showing synergistic inhibition of the MAPK and Akt/mTOR signaling pathways. Cells were incubated with sorafenib (5 μM), EA (20 μM) or both for 48 h. (E) Quantification of protein expression level in (D). Results are expressed as the means ± SDs. P values were determined by one-way ANOVA with multiple comparison test in (B, E). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5

EA potentiates the response of HCC to sorafenib in a xenograft mouse model. (A) Growth curves of subcutaneous tumors in nude mice administered EA, sorafenib, EA combined with sorafenib, or vehicle. (B) Graph of tumor weights 9 days after administration of EA, sorafenib, EA combined with sorafenib or vehicle. (C) Double staining of TUNEL and AFP showing an increased apoptosis rate in HCC cells in xenograft tumors from mice administered the combination of EA and sorafenib, which is quantified in (D). (E) Western blot analysis showing synergistic inhibition of the MAPK and Akt/mTOR signaling pathways in xenograft tumors. (F) Quantification of protein expression level in (E). Results are expressed as the means ± SDs. P values were determined by two-way ANOVA with multiple comparison test in (A); one-way ANOVA with multiple comparison test in (C, E, F). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.