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. 2017 Sep;14(3):2469-2476.
doi: 10.3892/etm.2017.4840. Epub 2017 Jul 25.

Gypenosides Induce Cell Death and Alter Gene Expression in Human Oral Cancer HSC-3 Cells

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Free PMC article

Gypenosides Induce Cell Death and Alter Gene Expression in Human Oral Cancer HSC-3 Cells

Kung-Wen Lu et al. Exp Ther Med. .
Free PMC article

Abstract

Gypenosides (Gyp), the primary components of Gynostemma pentaphyllum Makino, have long been used as a Chinese herbal medicine. In the present study, the effects of Gyp on cell viability, the cell cycle, cell apoptosis, DNA damage and chromatin condensation were investigated in vitro using human oral cancer HSC-3 cells. The results of the present study indicated that Gyp induces cell death, G2/M phase arrest and apoptosis in HSC-3 cells in a dose-dependent manner. It was also demonstrated that Gyp decreased the depolarization of mitochondrial membrane potential in a time-dependent manner. A cDNA microarray assay was performed and the results indicated that a number of genes were upregulated following Gyp treatment. The greatest increase was a 75.42-fold increase in the expression of GTP binding protein in skeletal muscle. Levels of the following proteins were also increased by Gyp: Serpine peptidase inhibitor, clade E, member 1 by 20.25-fold; ras homolog family member B by 18.04-fold, kelch repeat and BTB domain containing 8 by 15.22-fold; interleukin 11 by 14.96-fold; activating transcription factor 3 by 14.49-fold; cytochrome P450, family 1 by 14.44-fold; ADP-ribosylation factor-like 14 by 13.88-fold; transfer RNA selenocysteine 2 by 13.23-fold; and syntaxin 11 by 13.08-fold. However, the following genes were downregulated by GYP: Six-transmembrane epithelial antigen of prostate family member 4, 14.19-fold; γ-aminobutyric acid A receptor by 14.58-fold; transcriptional-regulating factor 1 by 14.69-fold; serpin peptidase inhibitor, clade B, member 13 by 14.71-fold; apolipoprotein L 1 by 14.85-fold; follistatin by 15.22-fold; uncharacterized LOC100506718; fibronectin leucine rich transmembrane protein 2 by 15.61-fold; microRNA 205 by 16.38-fold; neuregulin 1 by 19.69-fold; and G protein-coupled receptor 110 by 22.05-fold. These changes in gene expression illustrate the effects of Gyp at the genetic level and identify potential targets for oral cancer therapy.

Keywords: cDNA microarray; gene expression; gypenosides; human oral cancer.

Figures

Figure 1.
Figure 1.
Gyp decreased the percentage of viable cells in human oral cancer HSC-3 cells. Cells were treated with 0, 60, 90, 120, 150 and 180 µg/ml Gyp for 12, 24, 48 and 72 h and cell viability was assessed using flow cytometry. Data are presented as the mean ± standard deviation. Gyp, Gypenosides.
Figure 2.
Figure 2.
Gyp affects the cell cycle in human oral cancer HSC-3 cells. Cells were exposed to 120 µg/ml Gyp for 0, 6, 12, 24, 48 and 72 h, and subsequently underwent analysis of (A) cell cycle distribution and (B) the proportion of cells in the sub-G1 phase (apoptotic cells) via flow cytometry. Data are presented as the mean ± standard deviation. **P<0.01 vs. C. Gyp, Gypenosides; C, control cells.
Figure 3.
Figure 3.
Gyp induces chromatin condensation in human oral cancer HSC-3 cells. Cells were exposed to 0, 60, 90, 120, 150 and 180 µg/ml Gyp for 24 h, harvested and stained with 6-diamidino-2-phenylindole. Cells were examined and photographed using a fluorescence microscope (magnification, ×200). Gyp, Gypenosides; C, control cells.
Figure 4.
Figure 4.
Gyp induces DNA damage in human oral cancer HSC-3 cells. Cells were exposed to 0, 60, 90, 120, 150 and 180 µg/ml Gyp for 24 h and harvested. DNA damage was examined by Comet assay. Cells were examined and photographed using a fluorescence microscope (magnification, ×200). Gyp, Gypenosides; C, control cells.
Figure 5.
Figure 5.
Gyp induced changes in the mitochondrial membrane potential in human oral cancer HSC-3 cells. Cells were treated with 120 µg/ml Gyp for 0.25, 0.5, 1, 3, 6, 12, 24 and 48 h, collected and stained with DiOC6. Data are presented as the mean ± standard deviation. *P<0.05, **P<0.01 vs. C. Gyp, Gypenosides; C, control cells.
Figure 6.
Figure 6.
The top scored (by the number of pathways) network from Gyp (120 µg/ml) vs. control using the Analyze Networks algorithm on GeneGo software. Thick cyan lines indicate the fragments of canonical pathways. Red circles indicate upregulated gene expression. Blue circles indicate downregulated gene expression.
Figure 7.
Figure 7.
The second scored (by the number of pathways) network from Gyp (120 µg/ml) vs. control using the Analyze Networks algorithm on GeneGo software. Thick cyan lines indicate the fragments of canonical pathways. Red circles indicate upregulated gene expression. Blue circles indicate downregulated gene expression.
Figure 8.
Figure 8.
The third scored (by the number of pathways) network from Gyp (120 µg/ml) vs. control using the Analyze Networks algorithm on GeneGo software. Thick cyan lines indicate the fragments of canonical pathways. Red circles indicate upregulated gene expression. Blue circles indicate downregulated gene expression.

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