Amarogentin Induces Apoptosis of Liver Cancer Cells via Upregulation of p53 and Downregulation of Human Telomerase Reverse Transcriptase in Mice

Abstract

Background and objective: Amarogentin has been reported to have a preventive effect on liver cancer via inducing cancer cell apoptosis. We attempted to elucidate the roles of p53-associated apoptosis pathways in the chemopreventive mechanism of amarogentin. The findings of this study will facilitate the development of a novel supplementary strategy for the treatment of liver cancer.

Materials and methods: The purity of amarogentin was assessed by high-performance liquid chromatography. The inhibitory ratios of the liver cell lines were determined using a Cell Counting Kit-8 following treatment with a gradient concentration of amarogentin. Cell apoptosis was detected by flow cytometry using annexin V-fluorescein isothiocyanate/propidium iodide kits. The gene and protein expression of p53-associated molecules, such as Akt, human telomerase reverse transcriptase, RelA, and p38, was detected by real-time quantitative polymerase chain reaction, Western blotting, and immunohistochemical staining in liver cancer cells and mouse tumor tissues after treatment with amarogentin.

Results: The inhibitory effect of amarogentin on cell proliferation was more obvious in liver cancer cells, and amarogentin was more likely to induce the apoptosis of liver cancer cells than that of normal liver cells. The gene and protein expression levels of Akt, RelA, and human telomerase reverse transcriptase were markedly higher in the control group than in the preventive group and treatment groups. Only the expression of human telomerase reverse transcriptase was downregulated, accompanied by the upregulation of p53.

Conclusion: The results of our study suggest that amarogentin promotes apoptosis of liver cancer cells by the upregulation of p53 and downregulation of human telomerase reverse transcriptase and prevents the malignant transformation of these cells.

Keywords: amarogentin; hepatocellular carcinoma; human telomerase reverse transcriptase; p53.

Figure 1.

The purity of amarogentin was detected by high-performance liquid chromatography (HPLC). The peak of amarogentin emerged at 13 to 15 minutes. A, The proportion of amarogentin in an extraction prepared from Swertia davidi Franch, as determined by HPLC. B, The purity of amarogentin after purification, as determined by HPLC.

Figure 2.

Effects of amarogentin on liver cell proliferation. The inhibitory effect of amarogentin was more obvious in cancer cells than in normal cells. A, The linear relationships of the LO2 cell concentration with the survival rate at 24, 48, and 72 hours were y_24h = 0.987 − 0.0028x (95% confidence interval [CI]: 0.969-1.006), y_48h = 0.923 − 0.003x (95% CI: 0.903-0.943), and y_72h = 0.916 − 0.0036x (95% CI: 0.871-0.962). The half-maximal inhibitory concentration (IC₅₀) values of amarogentin for the LO2 cells at 24, 48, and 72 hours were 173.93, 141, and 115.5 µg/mL, respectively. B, The linear relationships of the HepG2 cell concentration with the survival rate at 24, 48, and 72 hours were y_24h = 0.912 − 0.0031x (95% CI: 0.894-0.931), y_48h = 0.864 − 0.0033x (95% CI: 0.836-0.893), and y_72h = 0.783 − 0.0036x (95% CI: 0.739-0.827). The IC₅₀ values for the HepG2 cells at 24, 48, and 72 hours were 132.90, 110.30, and 78.61 µg/mL, respectively. C, The linear relationships of the SMMC-7721 cell concentration with the survival rate at 24, 48, and 72 hours were y_24h = 0.971 − 0.0026x (95% CI: 0.946-0.995), y_48h = 0.958 − 0.0037x (95% CI: 0.934-0.982), and y_72h = 0.883 − 0.0039x (95% CI: 0.780-0.896). The IC₅₀ values for the SMMC-7721 cells at 24, 48, and 72 hours were 181.15, 123.78, and 86.67 µg/mL, respectively.

Figure 3.

Effects of amarogentin on liver cell apoptosis. Amarogentin was likely more effective at “killing” cancer cells than normal liver cells. A, The liver cell apoptosis rates were detected by flow cytometry. B, The apoptosis rates for the LO2 cells at 24, 48, and 72 hours were 19.32% ± 1.76%, 22.89% ± 2.37%, and 29.10% ± 1.54%, respectively; those for the HepG2 cells at 24, 48, and 72 hours were 35.00% ± 1.56%, 39.43% ± 1.35%, and 48.00% ± 1.73%, respectively; those for the SMMC-7721 cells at 24, 48, and 72 hours were 32.25% ± 1.17%, 36.01% ± 0.17%, and 44.21% ± 2.00%, respectively. *The apoptosis rate for LO2 cells at 24, 48, and 72 hours were significantly lower than those for the HepG2 and SMMC-7721 cells (P < .05).

Figure 4.

Effects of amarogentin on liver cancer cell migration. A, Liver cancer cell migration following treatment with amarogentin compared with that of control cells. B, The migrated distances of the treatment group were significantly shorter than those of the control groups. *P < .05 (magnification 10 × 10).

Figure 5.

Effects of amarogentin on liver cancer cell invasion. A, The invasion of liver cancer cells following treatment with amarogentin compared with that of control cells. B, The numbers of invading cells in the treatment group were significantly less than those of in the control group. *P < .05. (Magnification 10 × 20).

Figure 6.

Effects of amarogentin on p53-associated apoptosis signaling pathways in liver cancer cells. A, The p53 messenger RNA (mRNA) levels were significantly increased following treatment with amarogentin in both the HepG2 and SMMC-7721 cell lines, whereas Akt, RelA, and hTERT expression was significantly inhibited by amarogentin treatment in both of these cell lines (P < .05). However, the p38 levels were not influenced by amarogentin treatment in either the HepG2 or SMMC-7721 cells (P > .05). B, The Akt, RelA, and hTERT mRNA and protein levels showed similar changes; however, trend was not observed for p38 expression. C indicates control group; T, treatment group.

Figure 7.

Effects of amarogentin on p53-associated apoptosis signaling pathways in mouse models. A, The tumor weights of the preventive group were significantly lower than those of the control group but were only slightly lower than those of the treatment group. *P < .05, PG versus CG and PG versus TG. B, The p53 messenger RNA (mRNA) levels were significantly increased in both the preventive and treatment groups, whereas the Akt, RelA, and hTERT expression levels were significantly decreased in both the preventive and treatment groups (P < .05). However, the p38 levels were not influenced by amarogentin treatment in either the preventive or the treatment groups (P > .05). Additionally, only the hTERT expression was downregulated in accordance with the upregulation of p53. *, P < 0.05, PG versus TG. C, The p53, Akt, RelA, and hTERT protein levels changed in accordance with the changes in their mRNA levels, the same trend was not observed for p38 expression. Only hTERT expression was downregulated in accordance with the upregulation of p53. D, The results of hematoxylin-eosin (HE) staining (10×10 and 10×40) and immunohistochemistry (IHC) staining (10×10 and 10×40) were in agreement with the changes in mRNA and protein expression. Only the expression of human telomerase reverse transcriptase (hTERT) was downregulated in accordance with the upregulation of p53. CG indicates control group; PG, preventive group; TG, treatment group.

Figure 8.

Histological examination of amarogentin-treated mice. No observed adverse effects of amarogentin were observed in the mice in the preventive or treatment groups compared with those in the control group. A, Liver tissue. B, Cardiac muscle tissue. C, Renal tissue. D, Pulmonary tissue. CG indicates control group; PG, preventive group; TG, treatment group (magnification 10 × 20).