Eupalinolide A induces autophagy via the ROS/ERK signaling pathway in hepatocellular carcinoma cells in vitro and in vivo

Abstract

Hepatocellular carcinoma is the most common primary malignancy of the liver. The current systemic drugs used to treat hepatocellular carcinoma result in low overall survival time. It has therefore been suggested that new small‑molecule drugs should be developed for treating hepatocellular carcinoma. Eupatorium lindleyanum DC. (EL) has been used to treat numerous diseases, particularly respiratory diseases; however, to the best of our knowledge, studies have not yet fully elucidated the effect of EL on hepatocellular carcinoma. In the present study, the effect of eupalinolide A (EA), one of the extracts of EL, was evaluated on tumor growth in a xenograft model of human hepatocellular carcinoma cells, and on the proliferation and migration of hepatocellular carcinoma cell lines. Cell cycle progression and the type of cell death were then evaluated using the Cell Counting Kit 8 assay, flow cytometry, electron microscopy and western blotting. EA significantly inhibited cell proliferation and migration by arresting the cell cycle at the G₁ phase and inducing autophagy in hepatocellular carcinoma cells. EA‑induced autophagy was mediated by reactive oxygen species (ROS) and ERK signaling activation. Specific inhibitors of ROS, autophagy and ERK inhibited EA‑induced cell death and migration. In conclusion, the present study revealed that EA may inhibit the proliferation and migration of hepatocellular carcinoma cells, highlighting its potential as a promising antitumor compound for treating hepatocellular carcinoma.

Keywords: ERK; autophagy; eupalinolide A; hepatocellular carcinoma; reactive oxygen species.

Figure 1

Effect of EA on the proliferation of human hepatocellular carcinoma cells in vitro. (A) Morphology of MHCC97-L and HCCLM3 cells after EA treatment for 48 h. Arrows indicate morphologically altered cells. Scale bar, 100 µm. (B) Proliferation rate of MHCC97-L and HCCLM3 cells after EA treatment for 48 h. Scale bar, 100 mm. Representative of biologically independent samples, n=5. (C) Viability of MHCC97-L and HCCLM3 cells after EA treatment for 1, 2 or 3 days. At each time point, DMSO was regarded as the control group. n=6. (D) Image of MHCC97-L and HCCLM3 cells positive for BrdU staining after EA treatment for 48 h. Scale bar, 100 µm. Representative of biologically independent samples, n=3. (E) Quantification of BrdU-positive MHCC97-L and HCCLM3 cells in (D). (F) Effects of EA on colony formation in MHCC97-L and HCCLM3 cells. (G) Colony numbers in (F) were quantified. Scale bar, 100 µm. n=3. All data are shown as the mean ± SD. ^*P<0.05, ^**P<0.01, ^***P<0.001. EA, eupalinolide A; BrdU, 5-bromo-2-deoxyuridine.

Figure 2

Effect of EA on the migration of human hepatocellular carcinoma cells. (A and B) Migration of MHCC97-L and HCCLM3 cells following treatment with EA for the indicated durations, as determined by wound-healing assay. Scale bar, 250 µm. Representative of biologically independent samples, n=3. (C and D) Migration of MHCC97-L and HCCLM3 cells following treatment with EA for 24 h, as determined by Transwell migration assay. Scale bar, 100 µm. Representative of biologically independent samples, n=5. (E and F) Western blot analysis of the expression levels of EMT-related proteins at 48 h in MHCC97-L and HCCLM3 cells. β-actin was used as a control. All data are shown as the mean ± SD. ^*P<0.05, ^**P<0.01, ^***P<0.001. EA, eupalinolide A.

Figure 3

Effect of EA on the cell cycle progression of human hepatocellular carcinoma cells. (A) Cell cycle progression of MHCC97-L and HCCLM3 cells was analyzed by flow cytometry following EA treatment. (B) Percentage of MHCC97-L and HCCLM3 cells in each phase of the cell cycle. n=5. (C and D) Western blot analysis of the expression levels of cell cycle-related proteins at 48 h in MHCC97-L and HCCLM3 cells. β-actin was used as a control. All data are shown as the mean ± SD. ^*P<0.05, ^**P<0.01, ^***P<0.001. EA, eupalinolide A.

Figure 4

Effect of EA on apoptosis and necroptosis of human hepatocellular carcinoma cells. (A and B) Western blot analysis of the expression levels of apoptosis and necroptosis-related proteins at 48 h in MHCC97-L and HCCLM3 cells. β-actin was used as a control. (C and D) Effect of EA on apoptosis in MHCC97-L and HCCLM3 cells, as determined by flow cytometry. n=5. (E) Viability of MHCC97-L and HCCLM3 cells treated with or without apoptosis or necroptosis inhibitors was measured by Cell Counting Kit 8 assays. n=6. All data are shown as the mean ± SD. ^***P<0.001. EA, eupalinolide A; C-, cleaved; p-, phosphorylated.

Figure 5

Effect of EA on the autophagy of human hepatocellular carcinoma cells. (A-F) Western blot analysis of the expression levels of autophagy-related proteins at 48 h in MHCC97-L and HCCLM3 cells. β-actin was used as a control. (G) Transmission electron microscopy of MHCC97-L and HCCLM3 cells following EA treatment for 48 h. Arrow indicates autophagosomes. Scale bar, 500 nm. (H and I) ROS production of MHCC97-L and HCCLM3 cells was measured by DFC fluorescence following EA treatment. Representative of biologically independent samples, n=6. (J) Viability of MHCC97-L and HCCLM3 cells treated with or without ROS or autophagy inhibitors, as measured by Cell Counting Kit 8 assays. Representative of biologically independent samples, n=6. (K) Migratory ability of MHCC97-L and HCCLM3 cells treated with or without ROS or autophagy inhibitors, as measured by Transwell migration assays after EA treatment. All data are shown as the mean ± SD. ^*P<0.05, ^**P<0.01, ^***P<0.001. EA, eupalinolide A; Atg5, autophagy-related 5; 3-MA, 3-methyladenine; NAC, N-acetylcysteine; ROS, reactive oxygen species.

Figure 6

Effect of EA on MAPK signaling in human hepatocellular carcinoma cells. (A) Heatmap showing differential gene expression in HCCLM3 cells with or without 28 µM EA treatment for 48 h. (B) KEGG analysis of genes in HCCLM3 cells. The top 10 KEGG pathways based on fold enrichment are shown. The arrow indicates the 'MAPK signaling pathway'. (C) Western blot analysis of the expression levels of MAPK-related protein at 48 h in MHCC97-L and HCCLM3 cells. β-actin was used as a control. (D) Viability of MHCC97-L and HCCLM3 cells treated with or without an ERK inhibitor was measured by Cell Counting Kit 8 assay. n=6. (E and F) Migratory ability of MHCC97-L and HCCLM3 cells treated with or without an ERK inhibitor, as measured by Transwell migration assays after EA treatment. All data are shown as the mean ± SD. ^***P<0.001. EA, eupalinolide A; p-, phosphorylated; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 7

Effect of EA on the ROS/ERK signaling pathway in human hepatocellular carcinoma cells. (A-D) Western blot analysis of the protein expression levels of p-ERK and LC3 II/I at 48 h in MHCC97-L and HCCLM3 cells treated with or without a ROS inhibitor. β-actin was used as a control. (E-H) Western blot analysis of the protein expression levels of p-ERK and LC3 II/I at 48 h in MHCC97-L and HCCLM3 cells treated with or without the ERK inhibitor PD98059. β-actin was used as a control. All data are shown as the mean ± SD. ^*P<0.05. EA, eupalinolide A; NAC, N-acetylcysteine; p-, phosphorylated.

Figure 8

Effect of EA on tumor growth in a xenograft model of human hepatocellular carcinoma cells in vivo. (A) MHCC97-L and HCCLM3 xenograft tumor volume. n=6. (B) Weight of xenograft tumors formed by MHCC97-L and HCCLM3 cells. n=6. (C) Images of MHCC97-L and HCCLM3 cell xenograft tumors treated with or without EA treatment. All data are shown as the mean ± SD. ^**P<0.01, ^***P<0.001. EA, eupalinolide A.

Figure 9

Diagram showing the mechanism in which EA inhibits hepatocellular carcinoma by inducing autophagy and blocking the cell cycle. NAC, N-acetylcysteine; 3-MA, 3-methyladenine; ROS, reactive oxygen species.