The Yunnan national medicine Maytenus compound inhibits the proliferation of hepatocellular carcinoma (HCC) by suppressing the activation of the EGFR-PI3K-AKT signaling pathway

Abstract

Objective: To investigate the effects of Maytenus compound on the proliferation of hepatocellular carcinoma (HCC) cells in vitro and in vivo and to explore the underlying mechanism. Methods: The half maximal inhibitory concentration (IC50) values of Maytenus compound in HepG2 and BEL-7402 cells were determined by the MTS assay. HepG2 and BEL-7402 cells were treated with different concentrations of Maytenus compound. MTS assays, colony formation assays and cell cycle analyses were performed to clarify the inhibitory effect of Maytenus compound on the proliferation of HepG2 and BEL-7402 cells in vitro. After subcutaneous injection of HepG2 cells, nude mice were randomly divided into a vehicle control group and a drug intervention group, which were intragastrically administered ddH₂O or Maytenus compound, respectively. The inhibitory effect of Maytenus compound on the proliferation of HepG2 cells in vivo was analyzed using subcutaneous tumor growth curves, tumor weight, the tumor growth inhibition rate and the immunohistochemical detection of BrdU-labeled cells in S phase. The organ toxicity of Maytenus compound was initially evaluated by comparing the weight difference and organ index of the two groups of nude mice. The main proteins in the EGFR-PI3K-AKT signaling pathway were detected by Western blot after Maytenus compound intervention in vivo and in vitro. Results: Maytenus compound showed favorable antiproliferation activity against HepG2 and BEL-7402 cells with IC50 values of 79.42±11.71 µg/mL and 78.48±8.87 µg/mL, respectively. MTS assays, colony formation assays and cell cycle analyses showed that Maytenus compound at different concentration gradients within the IC50 concentration range significantly suppressed the proliferation of HepG2 and BEL-7402 cells in vitro and inhibited cell cycle progression from G1 to S phase. Additionally, Maytenus compound, at an oral dose of 2.45 g/kg, dramatically inhibited, without obvious organ toxicity, the proliferation of subcutaneous tumors formed by HepG2 cells in nude mice. In addition, the tumor growth inhibition rate for Maytenus compound was 66.94%. Furthermore, Maytenus compound inhibited the proliferation of liver orthotopic transplantation tumors in nude mice. Western blot analysis showed that Maytenus compound significantly downregulated the expression of p-EGFR, p-PI3K, and p-AKT and upregulated the expression of p-FOXO3a, p27, and p21 in vivo and in vitro. Conclusion: Maytenus compound significantly inhibited the proliferation of HCC cells in vitro and in vivo. The downregulation of the EGFR-PI3K-AKT signaling pathway and subsequent inhibition of cell cycle progression from G1 to S phase is one of the possible mechanisms. Maytenus compound has a high tumor growth inhibition rate and has no obvious organ toxicity, which may make it a potential anti-HCC drug, but the results from this study need to be confirmed by further clinical trials in HCC patients.

Keywords: EGFR-PI3K-AKT signaling pathway; Hepatocellular Carcinoma (HCC); Maytenus compound; Proliferation.

Figure 1

Effect of Maytenus compound on HepG2 and BEL-7402 cells in vitro. (A-B) IC50 of the Maytenus compound against HepG2 and BEL-7402 cells. (C-D) Cell proliferation of HepG2 and BEL-7402 cells treated with different concentrations of Maytenus compound was detected by MTS assay (**P < 0.01, ***P < 0.001).

Figure 2

Maytenus compound inhibits the proliferation of HCC cells in vitro. (A-C) Maytenus compound inhibited clone formation in HepG2 and BEL-7402 cells with a dose-dependent mode. (D-E) Maytenus compound inhibited cell cycle progression from G1 phase to S phase in HepG2 cells with a dose-dependent mode (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 3

Maytenus compound inhibits HCC cell xenograft growth in nude mice. (A) Representative pictures of xenograft tumors treated with Maytenus compound and ddH2O, respectively (n=6/group). (B) Tumor growth curves of control group and Maytenus compound treatment group. (C) Tumor weight of control group and Maytenus compound treatment group. (D) Representative images of HE and BrdU staining of xenograft tumors of control group and Maytenus compound treatment group. The red arrow indicates cell necrosis. (E) The body weight of the nude mice in control group and Maytenus compound treatment group. (F) The viscera index of control group and Maytenus compound treatment group. (G) Representative pictures of liver orthotopic transplantation tumor treated with Maytenus compound and ddH₂O, respectively. (H) Representative images of HE staining of liver orthotopic transplantation tumor of control group and Maytenus compound treatment group. The red arrow indicates the tumor formed in liver. (I) Liver tumor formation of the liver orthotopic transplantation tumor model after Maytenus compound treatment (**P < 0.01, ***P < 0.001).

Figure 4

Maytenus compound promotes apoptosis of HCC cells in vitro. (A) Apoptotic cell ratio was detected by flow cytometry. (B) Maytenus compound increased the early and late apoptotsis rate of HepG2 cells with a dose-dependent mode (**P < 0.01, ***P < 0.001).

Figure 5

The EGFR-PI3K-AKT signaling pathway is down-regulated by Maytenus compound at the cell level in vitro and the animal level in vivo. (A) Maytenus compound suppresses EGFR-PI3K-AKT signaling pathway in subcutaneous tumor in nude mice. (B) Maytenus compound inhibits EGFR-PI3K-AKT signaling pathway in a dose-dependent manner in HepG2 cells. (C) A schematic diagram of the signal pathway that Maytenus compound inhibits HCC cell proliferation and induces cell cycle arrest via suppression of EGFR-PI3K-AKT pathway.