Amino Acid Derivatives of Ginsenoside AD-2 Induce HepG2 Cell Apoptosis by Affecting the Cytoskeleton

Abstract

AD-2 (20(R)-dammarane-3β, 12β, 20, 25-tetrol, 25-OH-PPD) was structurally modified to introduce additional amino groups, which can better exert its anti-tumor effects in MCF-7, A549, LoVo, HCT-116, HT -29, and U-87 cell lines. We investigated the cellular activity of 15 different AD-2 amino acid derivatives on HepG2 cells and the possible mechanism of action of the superior derivative 6b. An MTT assay was used to detect the cytotoxicity of the derivatives. Western blotting was used to study the signaling pathways. Flow cytometry was used to detect cell apoptosis and ghost pen peptide staining was used to identify the changes in the cytoskeleton. The AD-2 amino acid derivatives have a better cytotoxic effect on the HepG2 cells than AD-2, which may be achieved by promoting the apoptosis of HepG2 cells and influencing the cytoskeleton. The derivative 6b shows obvious anti-HepG2 cells activity through affecting the expression of apoptotic proteins such as MDM2, P-p53, Bcl-2, Bax, Caspase 3, Cleaved Caspase 3, Caspase 8, and NSD2. According to the above findings, the amino acid derivatives of AD-2 may be developed as HepG2 cytotoxic therapeutic drugs.

Keywords: AD-2; amino acid derivatives; apoptosis; cytotoxic; dammarane triterpene.

Figure 1

Chemical structure of AD-2 and amino acid modification site.

Figure 2

Toxicity determination of AD-2 amino acid derivative 6b (IC₅₀ ± SD values, μM, 24 h) on cells of HepG2, HSC-T6, BEAS-2B, and L929. Data are expressed as means ± SDs of triplicate experiments performed independently (*** p < 0.001).

Figure 3

AD-2 amino acid derivative 6b inhibited the colony formation of HepG2 cells. HepG2 cells were treated with 6b (0, 5, 10, 20 μM) for 14 days and stained with 0.1% crystal violet.

Figure 4

The expression levels of NSD2, MDM2, p53, P-p53, Bcl-2, Bax, Caspase 3, Cleaved Caspase 3, and Caspase 8 proteins were detected by Western blotting. (a) Protein band diagram; (b–h) protein quantification map. The data are expressed as the average standard deviation of three independent experiments. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 5

Detection of apoptosis of HepG2 cells induced by 6b administration by flow cytometry. The cell apoptosis ratio is calculated based on the sum of the number of Q2 and Q3 phase cells in the figure. (** p < 0.01, *** p < 0.001).

Figure 6

Mechanisms of derivative 6b inducing the HepG2 cytoskeleton. (a–f) 3a, 1c, 6b administration promotes the ablation of β-actin in HepG2 cells by Western blotting assay. Data are expressed as means ± SDs of triplicate experiments performed independently (** p < 0.01, *** p < 0.001).

Figure 7

Phalloidin staining to detect derivative 6b administration-induced ablation of HepG2 cytoskeleton. HepG2 cells were treated with 6b (0, 5, 10, 20 μM) for 24 h. Actin was observed by phalloidin, and the nucleus was stained by DAPI. These images were captured with a fluorescence microscope at a scale of 100 μm, and the combined images of two kinds of staining were displayed. The arrow in the figure represents the place where morphological changes occur after administration.

Figure 8

Proposed mechanism of compound 6b for promoting apoptosis of HepG2 cells. To enhance the anti-hepatoma activity of AD-2, the amino acid derivatives of AD-2 were introduced in the previous work. The amino acid derivative 6b with high efficacy and low toxicity was screened by MTT assay. It has an excellent ability to resist the proliferation of hepatoma cell HepG2. The mechanism study found that 6b could destroy the cytoskeleton, regulate the MDM2/p53 signaling pathway, increase the expression level of Bax/Bcl-2, and activate Caspase3/8 system, thus causing apoptosis, which was also verified by flow cytometry.