Total ginsenosides induce autophagic cell death in cervical cancer cells accompanied by downregulation of bone marrow stromal antigen-2

Abstract

Ginsenosides are important active components in Panax ginseng. In the present study, total ginsenosides (TGNs) were demonstrated to enhance autophagy by promoting acidic vacuole organelle formation, recruitment of enhanced green fluorescent protein-microtubule-associated protein light chain 3 and expression of autophagy-related factors in cervical cancer cell lines. TGN markedly increased the expression of p62 at the transcriptional level, but decreased p62 protein expression in the presence of actinomycin D. The autophagic regulatory effect was reversible. TGN (≤120 µg/ml) did not affect the proliferation of cervical cancer cells under normal culture conditions, but markedly inhibited the growth of serum-deprived cells. Treatment with an inhibitor of autophagy (3-methyladenine) impaired TGN-induced cell death. This suggested that TGN caused autophagic cell death. In addition, western blot analysis demonstrated that the protein level of bone marrow stromal antigen-2 (BST-2) was downregulated by TGN. Upregulation of BST-2 reduced cell death. The results of the combined actions of various monomeric ginsenosides in TGN provide the molecular basis to develop TGN as a promising candidate for cancer therapy.

Keywords: autophagic cell death; autophagy; bone marrow stromal antigen-2; cervical cancer; total ginsenosides.

Figure 1

TGN induces cell autophagy in cervical cancer cell lines, HeLa, MS751 and C-33A in a (A) dose- and (B) time-dependent manner. Western blot analysis was performed with antibodies specific for Beclin-1, LC3 I/II and control protein, tubulin, followed by statistical analysis of the results; the values are presented as means ± SD. ^*P<0.05, ^**P<0.01, ^***P<0.001 and ^****P<0.0001 vs. DMSO. DMSO, dimethylsulfoxide; TGN, total ginsenoside; LC3, microtubule-associated protein light chain 3.

Figure 2

TGN increased EGFP-LC3 puncta and the quantity of AVOs. (A) TGN treatment markedly increased EGFP-LC3 puncta as observed by fluorescence microscopy. (B) AVOs were examined by incubating HeLa, MS751 and C-33A cells with acridine orange and observed by fluorescence microscopy. (C) Quantification of EGFP-LC3 puncta observed by fluorescence microscopy. (D) Quantification of AVOs stained with acridine orange and observed by fluorescence microscopy. Values are presented as means ± SD. ^*P<0.05, ^**P<0.01 and ^****P<0.0001 vs. DMSO. TGN, total ginsenoside; EGFP-LC3, enhanced green fluorescent protein microtubule-associated protein light chain 3; AVO, acidic vesicular organelle; DMSO, dimethylsulfoxide.

Figure 3

Autophagy is maintained for a long duration even after removing TGN. Western blots of (A) HeLa, (B) MS751 and (C) C-33A cells following treatment with 80 µg/ml TGN for 16 h or EBSS for 3 h. The compounds were washed out and proteins were extracted at the indicated time points. Statistical analysis of the western blots is also presented. (D) HeLa cells were treated with different ginsenosides for 16 h (60 µg/ml TGN; 70 µM ginsenosides monomer), cell lysates were detected with an LC3 antibody and tubulin served as the loading control. Values are presented as means ± SD. ^*P<0.05, ^**P<0.01, ^***P<0.001 and ^****P<0.0001 vs. DMSO or Mock; ns, not significant. TGN, total ginsenoside; LC3, microtubule-associated protein light chain 3; DMSO, dimethylsulfoxide; EBSS, Earle's Balanced Salt Solution.

Figure 4

TGN induces autophagic cell death of cervical cancer cells under serum deprivation. (A) Effects of treatment with TGN at various concentrations on HeLa, MS751 and C-33A cells under normal conditions. (B) Effects of treatment with TGN at various concentrations on HeLa, MS751 and C-33A cells under serum-deprived conditions. (C) HeLa, MS751 and C-33A cells were treated with 80 µg/ml TGN with 3-MA for 24 h and then cell viability was detected via Cell Counting Kit-8 assays. ^*P<0.05, ^**P<0.01, ^***P<0.001 and ^****P<0.0001 vs. DMSO. TGN, total ginsenoside; 3-MA, 3-methyladenine; DMSO, dimethylsulfoxide.

Figure 5

TGN induced downregulation of BST-2 and promoted cervical cancer cell death in serum-deprived cultures. (A) After cells were treated with 80 µg/ml TGN for 16 h, the protein expression levels of BST-2 was determined by western blotting. (B) Cell viability in the serum-deprived culture was detected via Cell Counting Kit-8 assays following BST-2 overexpression. ^*P<0.05, ^**P<0.01, ^***P<0.001 vs. DMSO. TGN, total ginsenoside; BST-2, bone marrow stromal antigen-2; DMSO, dimethylsulfoxide.