Antrodia camphorata extract (ACE)-induced apoptosis is associated with BMP4 expression and p53-dependent ROS generation in human colon cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE

Antrodia camphorata (AC) is a rare functional fungus in Taiwan and is known as traditional Chinese medicine. It has been reported to inhibit proliferation and promote apoptosis in human cancer cells.

AIM OF THE STUDY

To investigate the potential mechanism of apoptosis induced in colon cancer cells by Antrodia camphorata extract (ACE).

MATERIALS AND METHODS

The MTT assay and crystal violet staining were used to determine relative cell viability in vitro at 24 and 48 h. The effects of ACE on apoptosis were determined by Hoechst 33342 staining and flow cytometric analysis following Annexin V-FITC/PI staining. The gene expression profile of HCT116 cells was assessed by the RNA sequencing system. In combination with RNA-seq data and qRT-PCR, Western blot analysis was used to evaluate expression of proteins. The intracellular ROS of HCT116 cells were determined using a DCFH-DA fluorescence probe.

RESULTS

ACE significantly reduces cell viability in a dose-dependent manner and triggers apoptosis. To explore the underlying mechanism, we performed transcriptome analysis of ACE-treated colon cancer HCT116 cells. Bioinformatics analyses showed that ACE treatment is associated with pathways in cancer. We further used Cytoscape to analyze hub genes in this network. Among them, BMP4, which is associated with cancer cell death through regulation of the tumor suppressor p53, was significantly decreased at both mRNA and protein levels in ACE treatment groups. We found that cell death is reversible via inactivation or knockdown of p53 gene and reduction of reactive oxygen species (ROS) generation in response to ACE exposure, indicating that p53 plays an important role in ROS generation induced by ACE. Meanwhile, ROS scavenger NAC was used to verify that cell death is reversible via reduction of ROS.

CONCLUSION

Our findings demonstrate that ACE has potential as an anticancer agent that induces apoptosis through BMP4 and p53-dependent response to ROS in human colon cancer.