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. 2019 May 15;14(5):e0216759.
doi: 10.1371/journal.pone.0216759. eCollection 2019.

The ginsenoside Rk3 exerts anti-esophageal cancer activity in vitro and in vivo by mediating apoptosis and autophagy through regulation of the PI3K/Akt/mTOR pathway

Huanhuan Liu  1   2   3 , Jiaqi Zhao  4 , Rongzhan Fu  1   2   3 , Chenhui Zhu  1   2   3 , Daidi Fan  1   2   3
Affiliations

The ginsenoside Rk3 exerts anti-esophageal cancer activity in vitro and in vivo by mediating apoptosis and autophagy through regulation of the PI3K/Akt/mTOR pathway

Huanhuan Liu et al. PLoS One. .

Abstract

The rare ginsenoside Rk3 is a bioactive component derived from ginseng and Panax notoginseng that has been proven to possess anti-lung cancer activity. However, the effect of Rk3 on human esophageal cancer has not yet been reported. In this study, we aimed to explore its anticancer curative effect and potential molecular mechanisms in the Eca109 and KYSE150 cell lines. We found that Rk3 was able to significantly repress cell proliferation and colony formation in both Eca109 and KYSE150 cells in vitro. In the KYSE150 xenograft model, Rk3 obviously inhibited tumor growth and exhibited little toxicity in organs. Moreover, Rk3 could trigger G1 phase arrest and induce apoptosis and autophagy. Interestingly, apoptosis induced by Rk3 could be partly abrogated by 3-MA (an autophagy inhibitor), implying that autophagy could enhance apoptosis. Further studies indicated that pretreatment with the Akt inhibitor GSK690693 or the mTOR inhibitor rapamycin promoted Rk3-induced apoptosis and autophagy, demonstrating that the PI3K/Akt/mTOR pathway is related to Rk3-induced apoptosis and autophagy. In conclusion, the present study is the first to clarify that Rk3 can inhibit Eca109 and KYSE150 cell proliferation through activating apoptosis and autophagy by blocking the PI3K/Akt/mTOR pathway, suggesting that Rk3 may be a promising antitumor agent for esophageal cancer. In addition, this study provides ideas and an experimental basis for further research on the anti-esophageal cancer effects of the ginsenoside Rk3 and its mechanism.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Anti-esophageal cancer effects of the ginsenoside Rk3 in vitro and in vivo.
(A) The chemical structure of the ginsenoside Rk3. (B) Cell viability was measured by the MTT assay. (C) Colony formation. (D) Representative image of the tumor. (E) Tumor volume. (F) Body weight of mice. (G) H&E staining of major organs and tumors. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with control.
Fig 2
Fig 2. Rk3 triggers G1 arrest in Eca109 and KYSE150 cells.
(A) Cell cycle distribution analyzed by flow cytometry. (B) The expression level of G1 transition-related proteins was evaluated by western blotting. *p < 0.05, **p < 0.01 compared with control.
Fig 3
Fig 3. Rk3 induces apoptosis in Eca109 and KYSE150 cells.
(A) Hoechst 33342 staining. Scale bar: 100 μm. (B) Ultrastructure of apoptotic cells was observed by TEM. Scale bar: 1 μm. (C) Apoptosis was quantified by flow cytometry. (D) Effect of different doses of Rk3 on the expression of apoptosis-related proteins after treatment for 24 h. (E) Immunohistochemistry staining of tumor tissue. *p < 0.05, **p < 0.01 compared with control.
Fig 4
Fig 4. Rk3 induces autophagy in esophageal cancer cells.
(A) Autophagosomes were observed under TEM. Red arrows represent autophagosomes (scale bar = 1 μm). (B) Effect of different doses of Rk3 on the expression of autophagy-related proteins after treatment for 24 h. (C) Immunohistochemical staining of tumor tissue. (D) Cells were pretreated with 3-MA (5 mM) for 2 h and then incubated with the indicated dose of Rk3 for 24 h. The inhibition of cell proliferation was measured by MTT assays. (E) Cells were treated as state above, and Atg5, p62, LC3, Bax, c-Casp 3 and c-Casp 9 were analyzed by western blotting. *p < 0.05, **p < 0.01 compared with the control; #p < 0.05, ##p < 0.01 compared with the Rk3-treated group.
Fig 5
Fig 5. Rk3 suppresses the PI3K/Akt/mTOR pathway in Eca109 and KYSE150 cells.
(A) Cells were treated with differernt concentrations of Rk3 for 24 h, and the expression of PI3K, p-PI3K, Akt, p-Akt, mTOR and p-mTOR was determined by western blotting. (B) Immunohistochemical staining of tumor tissue. *p < 0.05, **p < 0.01 compared with the control.
Fig 6
Fig 6. Rk3 induces apoptosis and autophagy by blocking the PI3K/Akt/mTOR pathway in esophageal cancer cells.
(A) Cells were pretreated with GSK690693 (25 nM) or rapamycin (50 nM) for 2 h and then incubated with 150 μM Rk3 for 24 h. The inhibition of cell proliferation was detected using MTT assays. (B) and (C) The expression of apoptosis- and autophagy-related proteins was analyzed by western blotting after pretreatment with inhibitors. Cells were treated as stated above. *p < 0.05 compared with the control; #p < 0.05, ##p < 0.01 compared with the Rk3-treated group.
Fig 7
Fig 7. Proposed molecular mechanism of the anti-esophageal cancer activity of Rk3.
The PI3K/Akt/mTOR signaling pathway is involved in apoptosis and autophagy induced by Rk3 in esophageal cancer.
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Grants and funding

This study was financially supported by the National Natural Science Foundation of China (21576222 to CZ, 21576223, 21576160, 21878247 to CZ, 21838009 to DF, 21808184 to RF) and Shaanxi Key Laboratory of Degradable Biomedical Materials Program (16JS106, 2016SZSj-35 to DF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.