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. 2018 Apr;57(4):512-521.
doi: 10.1002/mc.22776. Epub 2018 Jan 11.

The triterpenoid corosolic acid blocks transformation and epigenetically reactivates Nrf2 in TRAMP-C1 prostate cells

Jie Yang  1   2 , Renyi Wu  2 , Wenji Li  2 , Linbo Gao  2 , Yuqing Yang  2   3 , Ping Li  1 , Ah-Ng Kong  2
Affiliations

The triterpenoid corosolic acid blocks transformation and epigenetically reactivates Nrf2 in TRAMP-C1 prostate cells

Jie Yang et al. Mol Carcinog. 2018 Apr.

Abstract

Corosolic acid (CRA) is found in various plants and has been used as a health food supplement worldwide. Although it has been reported that CRA exhibits significant anticancer activity, the effect of this compound on prostate cancer remains unknown. In this study, we investigated the effect of CRA on cellular transformation and the reactivation of nuclear factor erythroid 2-related factor 2 (Nrf2) through epigenetic regulation in TRAMP-C1 prostate cells. Specifically, we found that CRA inhibited anchorage-independent growth of prostate cancer TRAMP-C1 cells but not Nrf2 knockout prostate cancer TRAMP-C1 cells. Moreover, CRA induced mRNA and protein expression of Nrf2, heme oxygenase-1 (HO-1) and NAD(P)H Quinone Oxidoreductase 1 (NQO1). Bisulfite genomic sequencing and methylated DNA immunoprecipitation results revealed that CRA treatment decreased the level of methylation of the first five CpG sites of the Nrf2 promoter. Histone modification was analyzed using a chromatin immunoprecipitation (ChIP) assay, which revealed that CRA treatment increased the acetylation of histone H3 lysine 27 (H3K27ac) while decreasing the trimethylation of histone H3 lysine 27 (H3K27me3) in the promoter region of Nrf2. Furthermore, CRA treatment attenuated the protein expression of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs). These findings indicate that CRA has a significant anticancer effect in TRAMP-C1 cells, which could be partly attributed to epigenetics including its ability to epigenetically restore the expression of Nrf2.

Keywords: DNMT; HDAC; Nrf2; corosolic acid; methylation.

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Conflict of interest statement

Conflict of Interest

The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1. Chemical structure of corosolic acid (CRA, A) and the cytotoxicity of CRA to TRAMP-C1 cells (B)
Cells were treated with various concentrations of CRA for 3 or 5 days. The MTS assay was performed to assess cell viability. The data are presented as the mean ± SEM.
Figure 2
Figure 2. Nrf2 knockdown attenuated the inhibitory effects of CRA on the anchorage-independent growth of TRAMP-C1 cells
Stable mock (scramble-sequence control, sh-Mock) and Nrf2 knockdown (sh-Nrf2) TRAMP-C1 cells were established using lentivirus mediated short hairpin RNAs and were selected with puromycin for 3 weeks. (A) Reduced mRNA expression of Nrf2 in knockdown cells was confirmed by qPCR. (B) Anchorage-independent growth of sh-Mock and sh-Nrf2 with or without the presence of CRA for 14 days. (C) Representative images of each group under a microscope. All the data are presented as the mean ± SEM. *P < 0.05 compared to the control.
Figure 3
Figure 3. CRA increased the mRNA expression levels of Nrf2 and Nrf2 target genes in TRAMP-C1 cells
TRAMP-C1 cells were treated with CRA (2, 4 and 8 µM) for 5 days. After treatment, mRNA was isolated from the TRAMP-C1 cells, and the expression levels of target genes were determined using real-time qPCR assays with β-actin as an internal control. The graphical data are expressed as the mean ± SEM from three independent experiments. *P < 0.05 compared to the control.
Figure 4
Figure 4. CRA increased the expression levels of Nrf2, HO-1, and NQO1 in TRAMP-C1 cells
TRAMP-C1 cells were treated with CRA (2, 4 and 8 µM) for 5 days. After treatment, the cells were lysed, and the expression levels of proteins were determined by western blot analysis. The relative protein levels in the treatment groups were quantified and compared to that of the control group. β-actin was used as an internal control to normalize loading. The data are presented as the mean ± SEM from 3 independent experiments. *P < 0.05 compared to the control.
Figure 5
Figure 5. CRA altered the DNA methylation of the Nrf2 promoter and regulated its histone modification in TRAMP-C1 cells
TRAMP-C1 cells were treated with various concentrations of CRA or 5-AZA/TSA for 5 days. The analyzed CpG sites were located at positions −1085 bp to −1226 bp in the promoter region of Nrf2. (A and B) BGS was used to analyze DNA methylation. Black spots denote methylated CpGs, and hollow dots denote unmethylated CpGs. (C) An MeDIP assay was used to estimate the enrichment of methylated DNA fragments captured by the anti-5-methylcytosine antibody. A ChIP assay was used to assess the enrichment of H3K27AC (D) and H3K27Me3 (E) at the promoter of Nrf2. The graphical data are expressed as the mean ± SEM. *P < 0.05 compared to the control.
Figure 6
Figure 6. CRA decreased the protein levels and activities of DNMTs (A and C) and HDACs (B and D)
DNMT and HDAC protein levels were determined by a western blot analysis. The relative expression was calculated according to the intensity of each band normalized to β-actin. *P < 0.05 compared to the control.
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