Ginsenoside PPD's Antitumor Effect via Down-Regulation of mTOR Revealed by Super-Resolution Imaging

Abstract

Derived from Panax ginseng, the natural product 20(S)-Protopanaxadiol (PPD) has been reported for its cytotoxicity against several cancer cell lines. The molecular mechanism is, however, not well understood. Here we show that PPD significantly inhibits proliferation, induces apoptosis and causes G2/M cell cycle arrest in human laryngeal carcinoma cells (Hep-2 cells). PPD also decreases the levels of proteins related to cell proliferation. Moreover, PPD-induced apoptosis is characterized by a dose-dependent down-regulation of Bcl-2 expression and up-regulation of Bax, and is accompanied by the activation of Caspase-3 as well. Further molecular mechanism is revealed by direct stochastic optical reconstruction microscopy (dSTORM)-a novel high-precision localization microscopy which enables effective resolution down to the order of 10 nm. It shows the expression and spatial arrangement of mTOR and its downstream effectors, demonstrating that this ginsenoside exerts its excellent anticancer effects via down-regulation of mTOR signaling pathway in Hep-2 cells. Taken together, our findings elucidate that the antitumor effect of PPD is associated with its regulation of mTOR expression and distribution, which encourages further studies of PPD as a promising therapeutic agent against laryngeal carcinoma.

Keywords: PPD; antitumor; dSTORM; laryngeal cancer; mTOR.

Figure 1

Inhibitory effect of 20(S)-Protopanaxadiol (PPD) on the proliferation of Hep-2 cells. (A) The chemical structure of PPD; (B) MTT assay measured the viability of Hep-2 cells treated with different concentrations of PPD for 24 h. Data are expressed as mean ± S.D., * p < 0.05, ** p < 0.01, for PPD-treated cells vs. DMSO (Dimethyl Sulphoxide)-treated cells, n = 6; (C) Morphological images of Hep-2 cells in the presence of treatment as illustrated; (D) Confocal images of Ki67 and nucleus in Hep-2 cells with or without PPD treatment to detect cell proliferation. Ki67 was labeled with primary antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG. Nuclei were stained with Hoechst; (E) The relative optical density of Ki67, which presents the ability of cell proliferation. Data are expressed as mean ± S.D., ** p < 0.01, compared to control, n = 3.

Figure 2

G2/M-phase arrest induced by PPD in Hep-2 cells. (A) FACS analysis of PPD inhibiting cell cycle in G2/M phase in Hep-2 cells. Cells were treated with 0, 40, 80 and 160 µM PPD for 24 h, respectively, and then fixed, labeled with PI (Propidium Iodide), and subjected to flow cytometry; (B) The percentage of cells in different phase as shown in (A). Data are expressed as mean ± S.D., * p < 0.05, ** p < 0.01, compared to control, n = 3; (C) Western blot analyses of the expression of cell cycle related proteins in Hep-2 cells treated with different concentration of PPD; (D) The quantization of the Western blot data after correction for the β-actin loading control. Data are expressed as mean ± S.D., * p < 0.05, ** p < 0.01, compared to control, n = 3.

Figure 3

PPD induces the apoptosis of Hep-2 cells and affects the expression of apoptosis-related proteins. (A) Annexin V/PI analysis of the apoptosis of Hep-2 cells treated with different concentrations of PPD (0, 40, 80 and 160 μM). Viable cells with both negative Annexin V and PI are in Q4. Apoptotic cells with positive Annexin V and negative PI are in Q3. Secondary necrotic cells with both positive Annexin V and PI are in Q2. Mechanically injured cells with negative Annexin V and positive PI are in Q1; (B) Apoptosis rate of Hep-2 cells statistics from Annexin V/PI analysis. Data are expressed as mean ± S.D., ** p < 0.01, compared to control, n = 3; (C) TUNEL analysis of the apoptosis of Hep-2 cells treated with or without PPD; (D) Confocal images of nuclei stained with Hoechst 33342 in control and PPD treated Hep-2 cells; (E) Western blot analyses of the expression of various apoptosis-related proteins in Hep-2 cells with or without PPD treatment; (F) The quantization of the Western blot data after correction for the β-actin loading control. Data are expressed as mean ± S.D., ** p < 0.01, compared to control, n = 3.

Figure 4

PPD affects the transcription and expression of mTOR and its downstream effectors. (A) Quantitative RT-PCR analyses of the effects of PPD on mTOR, 4EBP1 and eIF4E transcription. The relative levels of their mRNA are displayed in a histogram; (B) Western blot analyses of expression levels of mTOR, 4EBP1 and eIF4E in Hep-2 cells with different doses of PPD treatment; (C) The quantization of the Western blot data after correction for the β-actin loading control. Data in (A,C) are expressed as mean ± S.D., * p < 0.05, ** p < 0.01, compared to control, n = 3.

Figure 5

PPD regulates the distribution pattern of mTOR and its downstream effectors. (A) direct Stochastic Optical Reconstruction Microscopy (dSTORM) imaging of mTOR, 4EBP1 and eIF4E in Hep-2 cells treated with different doses of PPD. mTOR, 4EBP1 and eIF4E were labeled with individual primary antibodies and Alexa Fluor 647 secondary antibodies, nuclei were labeled with Hoechst 33342. Scale bars are 10 μm; (B,E,H) Normalized total localizations of reconstructed dSTORM images of mTOR (B), 4EBP1 (E) and eIF4E (H) in Hep-2 cells with different concentrations of PPD; (C,F,I) The cluster number of mTOR (C), 4EBP1 (F) and eIF4E (I) per μm² in Hep-2 cells with or without PPD treatment; (D,G,J) The average cluster area of mTOR (D), 4EBP1 (G) and eIF4E (J) in Hep-2 cells with or without PPD treatment. Data in (B–J) are obtained from 20 cells in four independent experiments. Data are expressed as mean ± S.D., * p < 0.05, *** p < 0.001, compared to control, n = 10.