Epigenetic reactivation of RASSF1A by phenethyl isothiocyanate (PEITC) and promotion of apoptosis in LNCaP cells

Abstract

Epigenetic silencing of tumor suppressor genes is a phenomenon frequently observed in multiple cancers. Ras-association domain family 1 isoform A (RASSF1A) is a well-characterized tumor suppressor that belongs to the Ras-association domain family. Several studies have demonstrated that hypermethylation of the RASSF1A promoter is frequently observed in lung, prostate, and breast cancers. Phenethyl isothiocyanate (PEITC), a phytochemical abundant in cruciferous vegetables, possesses chemopreventive activities; however, its potential involvement in epigenetic mechanisms remains elusive. The present study aimed to examine the role of PEITC in the epigenetic reactivation of RASSF1A and the induction of apoptosis in LNCaP cells. LNCaP cells were treated for 5 days with 0.01% DMSO, 2.5 or 5 μM PETIC or 2.5 μM azadeoxycytidine (5-Aza) with 0.5 μM trichostatin A (TSA). We evaluated the effects of these treatments on CpG demethylation using methylation-specific polymerase chain reaction (MSP) and bisulfite genomic sequencing (BGS). CpG demethylation was significantly enhanced in cells treated with 5 μM PEITC and 5-Aza+TSA; therefore, the latter treatment was used as a positive control in subsequent experiments. The decrease in RASSF1A promoter methylation correlated with an increase in expression of the RASSF1A gene in a dose-dependent manner. To confirm that promoter demethylation was mediated by DNA methyltransferases (DNMTs), we analyzed the expression levels of DNMTs and histone deacetylases (HDACs) at the gene and protein levels. PEITC reduced DNMT1, 3A and 3B protein levels in a dose-dependent manner, and 5 μM PEITC significantly reduced DNMT3A and 3B protein levels. HDAC1, 2, 4 and 6 protein expression was also inhibited by 5 μM PEITC. The combination of 5-Aza and TSA, a DNMT inhibitor and a HDAC inhibitor, respectively, was used as a positive control as this treatment significantly inhibited both HDACs and DNMTs. The function of RASSF1A reactivation in promoting apoptosis and inducing G2/M cell cycle arrest was analyzed using flow-cytometry analysis with Annexin V and propidium iodide (PI). Growth inhibition effect on LNCaP cells were investigated by colony formation assay. In addition, we analyzed p21, caspase-3 and 7, Bax, and Cyclin B1 protein levels. Flow-cytometry analysis of cells stained with PI alone demonstrated that 5 μM PEITC promotes early apoptosis and G2/M cell cycle arrest. Flow cytometry analysis of cells stained with Annexin V and PI also demonstrated an increased proportion of cells in early apoptosis in cells treated with 5 μM PEITC or 5-Aza with TSA. PEITC and efficiently inhibit colony numbers and total area. In addition, 5 μM PEITC significantly enhanced p21, caspase-3, 7 and Bax levels and reduced Cyclin B1 expression compared with the control group. Collectively, the results of our study suggest that PEITC induces apoptosis in LNCaP cells potentially by reactivating RASSF1A via epigenetic mechanisms.

Phenethyl Isothiocyanate, a Dual Activator of Transcription Factors NRF2 and HSF1

“…Interestingly, PEITC treatment in LNCaP cells causes the epigenetic reactivation of Ras‐association domain family 1 isoform A ( RASSF1A ) gene by enhancing CpG demethylation at its promoter. In this study, PEITC also reduced the expression of the DNMTs 1, 3A, and 4 as well as HDACs 1, 2, 4, and 6 66. Additionally, in LPS‐stimulated human colon epithelial cells SW480, PEITC treatment decreased the expression of several genes (e.g., interleukin 8 [IL‐8], MMP7, signal transducer and activator of transcription 1 [STAT1], and nuclear factor of kappa light chain gene enhancer in B cells 1, p105 [NF‐κB1]) by causing the trimethylation of hH3 at lysine 27 (H3K27me3) 67.…”

PTEN deletion drives aberrations of DNA methylome and transcriptome in different stages of prostate cancer

“…9,10 DNA methylation is the most well-studied epigenetic mechanism associated with aberrant gene expression in cancer and consists of the addition of a methyl group by DNA methyltransferases to the 5-cytosine residue within the CpG dinucleotide. [22][23][24] However, it is not clear how silencing of tumor suppressor gene such as PTEN deletion would impact the CpG methylome, and transcriptome during PCa development. 12 Specifically, DNA hypermethylation in promoter regions plays a critical role in suppressing tumor suppressor gene expression partly by blocking the binding of transcription factors.…”

“…[16][17][18][19][20][21] Evidence suggests that in PCa, tumor suppressor genes silenced by promoter hypermethylation are related to many important cellular events, including cell proliferation, apoptosis, DNA repair, hormonal responses, and cancer initiation and progression. [22][23][24] However, it is not clear how silencing of tumor suppressor gene such as PTEN deletion would impact the CpG methylome, and transcriptome during PCa development.…”

Phytochemicals based chemopreventive and chemotherapeutic strategies and modern technologies to overcome limitations for better clinical applications

“…Phytochemicals, both dietary and nondietary, have been extensively evaluated for their chemopreventive or therapeutic efficacy both in vitro and in vivo (Pande et al, 2017;Panjamurthy, Manoharan, Nirmal, & Vellaichamy, 2009;Weng & Yen, 2012). Phytochemicals drive their chemopreventive effect via multiple mechanisms such as acting as antioxidants, antiinflammatory, antimutagenic, and epigenetic modulators (Boyanapalli et al, 2016;Link, Balaguer, & Goel, 2010). Phytochemicals can efficiently kill rapidly proliferating cells by modulating growth signaling, suppression of angiogenesis, and activation of apoptosis.…”