Curcumin Derivative Epigenetically Reactivates Nrf2 Antioxidative

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Curcumin Derivative Epigenetically Reactivates Nrf2 Antioxidative Stress Signaling in Mouse Prostate Cancer TRAMP C1 Cells Wenji Li, Zheng-Yuan Su, Yue Guo, Chengyue Zhang, RenYi Wu, Linbo Gao, Xi Zheng, Zhi-Yun Du, Kun Zhang, and Ah-Ng Kong Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.7b00248 • Publication Date (Web): 11 Dec 2017 Downloaded from http://pubs.acs.org on December 13, 2017

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Chemical Research in Toxicology

Curcumin Derivative Epigenetically Reactivates Nrf2 Antioxidative Stress Signaling in Mouse Prostate Cancer TRAMP C1 Cells

Wenji Li §,¶, Zheng-Yuan Su§,¤, Yue Guo §,¶,œ, Chengyue Zhang ZhengƐ, Zhi-Yun Duƾ, Kun Zhangƺ, and Ah-Ng Kong §,¶,*

§,¶,œ

, Renyi Wu §,¶ , Linbo Gao §,¶, Xi

§

Center for Phytochemical Epigenome Study, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA, ¶ Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA, ¤ Department of Bioscience Technology, Chung Yuan Christian University, 200 Chung Pei Road, Chung Li District, Taoyuan City, Taiwan 32023 R.O.C. œ Graduate Program in Pharmaceutical Sciences, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA. Ɛ Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey,164 Frelinghuysen Road, Piscataway, NJ 08854, USA ƾ Allan H. Conney Laboratory for Anticancer Research, Guangdong University of Technology, Guangzhou, P.R. China ƺ Laboratory of Natural Medicinal Chemistry & Green Chemistry, Guangdong University of Technology, Guangzhou, P.R. China *Corresponding Author: Ah-Ng Tony Kong, Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, New Jersey 08854, Phone: 732-455-3831; Fax:732-455-3134; E-mail: [email protected]

Key Words Nrf2, Prostate cancer, curcumin, H3k27me3, DNA methylation, TRAMP

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Table of Contents Graphic

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Abstract The carcinogenesis of prostate cancer (PCa) in TRAMP model is highly correlated with hypermethylation in the promoter region of Nrf2 and the accompanying reduced transcription of Nrf2 and its regulated detoxifying genes. We aimed to investigate the effects of (3E,5E)-3,5-Bis(3,4,5-trimethoxybenzylidene) tetrahydrothiopyran-4-one (F10) and (3E,5E)-3,5-Bis(3,4,5-trimethoxybenzylidene) -tetrahydropyran-4one (E10), two synthetic curcumin derivatives, on restoring Nrf2 activity in TRAMP C1 cells. HepG2-C8 cells transfected with an antioxidant-response element (ARE)-luciferase vector were treated with F10, E10, curcumin and sulforaphane (SFN) to compare their effects on Nrf2-ARE pathways. We performed Real-time quantitative PCR and western blotting to investigate the effects of F10 and E10 on Nrf2, correlated phase II detoxification genes. We also measured expression and activity of DNMTand HDAC enzymes. Enrichment of H3K27me3 on the promoter region of Nrf2 was explored with a chromatin immunoprecipitation (ChIP) assay. Methylation of the CpG region in Nrf2 promoter were doubly examined by bisulfite genomic sequencing (BGS) and methylation DNA immunoprecipitation (MeDIP). Compared with curcumin and SFN, F10 are more potent in activating Nrf2-ARE pathways. Both F10 and E10 enhanced level of Nrf2 and the correlated phase II detoxifying genes. BGS and MeDIP assays indicated that F10 but not E10 hypomethylated the Nrf2 promoter. F10 also downregulated the protein level of DNMT1, DNMT3a, DNMT3b, HDAC1, HDAC4, and HDAC7 and the activity of DNMTs and HDACs. F10 but not E10 effectively reduced the accumulation of H3k27me3 on the promoter of Nrf2. F10 and E10 can activate the Nrf2-ARE pathway and increase the level of Nrf2 and correlated phase II detoxification genes. The reactivation effect on Nrf2 by F10 in TRAMP C1 may come from demethylation, decrease of HDACs and inhibition of H3k27me3 accumulation.



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Introduction

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According to epidemiology studies, the frequency of diagnosis of prostate cancer (PCa) ranks

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second among all cancers in the USA.1 Like many other cancer types, it progresses from the benign to

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malignant stage due to genetic and epigenetic alterations.2 Compared to genetic factors, epigenetic

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changes are relatively reversible. In addition, epigenetic alterations are accepted as a major predictor

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of PCa significance by an increasing number of scientists.3 Aberrant methylation of many genes, such

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as GSTP1, RASSF1A, RARβ2 and galectin-3, is highly involved in PCa progression and can be

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found by early detection in tissue biopsies, serum and urine, and thus can be promising markers for

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PCa diagnosis and targeted therapies.4, 5

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The antioxidant defense system normally exerts a positive cancer prevention effect; however,

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when this system is deregulated, it is one of the major promoting factors for toxicity and neoplastic

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progression of PCa.6, 7 Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a basic-region leucine

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zipper (bZIP) transcription factor, which has been found to be a vital mediator in upregulating

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antioxidant-response element (ARE)-related phase II detoxifying and antioxidation gene

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transcription.8 These genes are closely regulated by Nrf2 and contribute to preservation from cellular

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invasion of ROS/RNS and active metabolites of carcinogens.

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In a TRAMP model, we found that PCa carcinogenesis is highly associated with

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hypermethylation of the promoter region of Nrf2 and the consequent silencing of Nrf2 and the

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correlated phase II detoxification genes, comprising of NAD(P)H quinone dehydrogenase 1 (NQO1)

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and heme oxygenase-1 (HO-1) .9, 10 Many phytochemicals, including 3,3'-diindolylmethane (DIM) ,11

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sulforaphane (SFN)12, and tocopherols 13 have been demonstrated to restore Nrf2 expression by

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demethylating the CpG regions on the Nrf2 promoter and thus increasing its downstream phase II

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detoxifying and antioxidation genes and preventing PCa in the TRAMP model.

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F10 ((3E,5E)-3,5-bis(3,4,5-trimethoxybenzylidene)-tetrahydrothiopyran-4-one) and E10 ((3E,5E)-3,5-bis(3,4,5-trimethoxybenzylidene) tetrahydropyran-4-one) are synthetic curcumin


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derivatives (Figure 1). They exhibited higher growth inhibiting efficacy against human prostate

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cancer cells than curcumin through inhibition of androgen receptor activity.14 Structure activity

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relationship (SAR) analysis indicated the more potent anti-PCa cell effects of E10 and F10 may be

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related to the heteroatom structure.14 In addition, some additional groups on the aromatic rings such

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as a sulfur or oxygen heterocyclic ketone group, distal benzene rings and methoxy groups could

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enhance the effects further.15 However, the mechanism of E10 and F10 in inhibition of PCa

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progression is not clear. In our previous study, curcumin exerted its PCa preventive effect by

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epigenetically reactivating Nrf2 transcription and activating its downstream antioxidative pathway.9

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In this project, we aimed to explore the potential mechanism of F10 and E10 in restoring Nrf2

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expression via epigenetic regulation in TRAMP C1.

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Material & Methodology

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Material

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Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-

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streptomycin (10,000 U/ml), versene and trypsin-EDTA were purchased from Gibco (Grand Island, NY,

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USA). Antibodies against Nrf2, HO-1, UGT1A1, and beta-actin (I-19) were obtained from Santa Cruz

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Biotechnology (Santa Cruz, CA, USA). Anti-HDAC1 and anti-HDAC4 antibody were supplied by Cell

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Signaling Technology (Beverly, MA). The anti-NQO1, anti-HDAC7, anti-H3, anti-DNMT3a and anti-

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DNMT3b antibodies were from Abcam (Cambridge, MA,USA). Anti-DNMT1 was supplied by Novus

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Biologicals (Littleton, CO, USA). E10 and F10 (purity >95%) were obtained from Dr. Kun Zhang’s

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laboratory, which was synthesized by aldehyde and ketone in glacial acetic acid following published

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method.15 Dimethyl sulfoxide (DMSO), 5-aza deoxycytidine (5-aza), and trichostatin A (TSA) and all

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others were ordered from Sigma (St. Louis, MO, USA).

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Cell Culture 5 ACS Paragon Plus Environment


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Mouse prostate cancer TRAMP-C1 cells (ATCC- CRL-2730) were purchased from ATCC

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(Rockville, Maryland, USA) with certificate of analysis. The cells were grown in DMEM medium

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containing 10% FBS. HepG2-C8 cells stably transfected with an ARE-luciferase construct16 were cultured

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in DMEM medium containing 10% FBS and 1% penicillin-streptomycin. For all the cell experiments, the

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treatment medium including 0.1% DMSO and drugs were changed every 2 days.

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MTS Assay

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One thousand TRAMP-C1 cells were plated in each well of ninety-six-well plates overnight. The

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culture medium was changed to E10 and F10 at different concentrations (from 0 to 800nM) and the cells

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were incubated for 1, 3, or 5 days. The treatment medium was changed every other day. MTS assay was

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done to compare the cell viability according to the kit’s protocol (Promega, Madison, WI, USA).

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ARE-Luciferase Assay

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ARE-Luciferase activity assay in evaluating the potency of E10, F10, curcumin, and SFN in

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activating Nrf2 were evaluated in HepG2-C8 cells which stably expressed ARE-luciferase. One million

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HepG2-ARE-C8 cells were grown in each well in 12-well plates overnight, and then incubated with E10,

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F10, curcumin, and SFN for 1 day. Ten microliter cell lysate supernatant was used for ARE-Luciferase

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Assay following the kit’s protocol (Promega, Madison, WI, USA) by using a Sirius luminometer

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(Berthold Detection System GmbH, Pforzheim, Germany). The luciferase activity was normalized by cell

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lysate protein concentration calculated by using a BCA kit (Pierce Biotech, Rockford, IL, USA).

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RNA extraction and quantitative real-time PCR (qPCR)

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TRAMP-C1 cells at density of one million / culture plate (10-cm diameter) were treated with E10

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(50 nM and 100 nM), F10 (50 nM and 100 nM) and 0.1% DMSO (control) for 3 days. Total RNA was

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extracted from the cells using an RNeasy Mini Kit (QIAGEN, Valencia, CA). cDNA was synthesized by

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a SuperScript III First-Strand Synthesis System (Invitrogen, Grand Island, NY, USA) according to the


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manufacturer’s protocol. mRNA expression levels were determined with qPCR using Power SYBR Green

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PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA). The primer sequences for Nrf2, HO-1,

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NQO1, and UGT1A1 were same with our previous reports. 17

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Western Blotting

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After three days treatment of E10 or F10 at the same concentration, TRAMP-C1 cells were

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washed with phosphate-buffered saline (PBS) and collected in radioimmunoprecipitation assay (RIPA)

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buffer (Cell Signaling Technology, Danvers, MA, USA) containing a protease inhibitor cocktail (Sigma).

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Protein concentrations were measured using the bicinchoninic acid (BCA) method (Pierce, Rockford, IL,

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USA). Twenty microgram protein was loaded and separated by a 4 to 15% SDS–polyacrylamide gel (Bio-

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Rad, Hercules, CA, USA) electrophoresis (SDS–PAGE) and then transferred to PVDF membranes

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(Millipore, Billerica, MA, USA). The membranes were blocked with 5% BSA and incubated with

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corresponding primary antibodies and HRP-conjugated secondary antibodies in sequence. The bands were

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visualized using the SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific, Rockford,

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IL, USA) and analyzed with a Gel Documentation 2000 system (Bio-Rad).

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Bisulfite Genomic Sequencing (BGS)

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BGS method is elaborated in detail in our previous report. 17 Briefly, after three days treatment of

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E10 or F10 at the same concentration and combination of 5-aza (500 nM) and TSA (100 nM) for 3 days,

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genomic DNA was extracted from TRAMP-C1 cells with a QIAamp DNA Mini kit (Qiagen, Valencia,

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CA, USA), subjected to bisulfite conversion with an EZ DNA Methylation-Gold Kit (Zymo Research

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Corp., Orange, CA, USA) and amplified with Platinum Taq DNA polymerase (Invitrogen, Grand Island,

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NY, USA) using the same primers targeted the mouse Nrf2 promoter. 17 The PCR products were purified

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and cloned into a pCR4 TOPO vector using the TOPO TA Cloning Kit (Thermo Fisher

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Scientific,Rockford, IL, USA). Plasmid DNA from randomly selected clones was isolated and sequenced

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(Genewiz, Piscataway, NJ, USA). 7 ACS Paragon Plus Environment


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Nuclear Extraction, DNMTs and HDACs activity Assay

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After three days treatment of E10 or F10 at the same concentration, nuclear extracts were

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isolated from control and treated cells by the EpiQuik Nuclear Extraction Kit (Epigentek, Brooklyn, NY)

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in line with the manufacturer's protocol. The protein concentration of nuclear extracts was measured by

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BCA Protein Assay Reagent (Pierce, Rockford, IL).

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Total DNMTs activities of isolated nuclear proteins were quantified using a fluorimetric

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EpiQuickTM DNMT Activity/Inhibition Assay Ultra kit (Epigentek, Brooklyn, NY) . One microliter (5–10

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µg) of nuclear extracts and 49 microliter assay reagent were applied to each well to test the activity of

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HDACs according to manufacturer instructions using a Tecan microplate reader plate reader (Infinite

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m200 pro; Männedorf Switzerland) with an excitation wavelength of 530 nm and an emission wavelength

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of 590 nm. DNMTs activities were normalized by protein amount of each sample.

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Total HDACs activities of isolated nuclear proteins were measured using a fluorometric

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EpigenaseTM HDAC Activity/Inhibition Direct Assay Kit (Epigentek, Brooklyn, NY) . One microliter (5–

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10 µg) of nuclear extracts and 49 microliter assay reagent were applied to each well to test the activity of

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HDACs according to manufacturer instructions using a Tecan microplate reader plate reader (Infinite

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m200 pro; Männedorf Switzerland) with an excitation wavelength of 530 nm and an emission wavelength

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of 590 nm. HDAC activities were normalized by protein amount of each sample.

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Methylation DNA Immunoprecipitation (MeDIP)

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To confirm the BGS results, MeDIP assay was implemented by a Methylamp Methylated DNA

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Capture Kit (EpiGentek, Farmingdale, NY, USA) as described in our previous work.12, 17 In brief, the

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genomic DNA that was isolated from the treated cells was subjected to sonication with a Bioruptor

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sonicator (Diagenode Inc., Sparta, NJ, USA) to generate 200- to 1000-bp long DNA fragments. The DNA

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fragments were incubated at 95 °C for two minutes, and immunoprecipitated with anti-5-methylcytosine 8 ACS Paragon Plus Environment

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at room temperature for two hours. After purification, the methylation ratio of DNA was calculated by

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qPCR.

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Chromatin Immunoprecipitation (ChIP) Assay

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The ChIP assay was performed using a MAGnify TM Chromatin Immunoprecipitation System

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(ThermoFisher Scientific, Waltham, MA) according to the product protocol. In brief, after three days

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treatment of E10 or F10 at the same concentration above, TRAMP-C1 cells were washed with PBS and

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trypsinized. After a PBS wash, the chromatin in these cells (one hundred thousand cells total were used

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per IP) were then cross-linked with 1% formaldehyde for ten minutes at room temperature, sheared to an

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average length of 200–500 bp via sonication at 4 °C in lysis buffer. The diluted chromatin solution was

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immunoprecipitated with 2 µg of anti-trimethyl-histone H3-Lys27 (H3K27me3) antibody (Abcam) or

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mouse immunoglobulin G. After washing, cross-link reversal, DNA elution and DNA purification, the

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relative amount of immunoprecipitated DNA was quantified via qPCR using primer 1 with 5’-

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GTATCACTTCATCCACCCAGAG-3’(forward) and 5’-GTACGTGTAAAGGAACCCTGAG-

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3’(reverse) and primer 2, 5’-GGGTTCCTTTACACGTACTTACTC-3’(forward) and 5’-

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GGTCACCACAACACGAACTAT-3’(reverse), which cover the promoter regions of Nrf2. The

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enrichment of the precipitated DNA was calibrated using the standard curve from the serial dilution of the

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inputs, and the data are presented as the fold changes in the signal-to-input ratio normalized to the control.

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Statistical Analysis

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All statistical analyses were carried out using SPSS software, version 22.0, (IBM, Armonk, NY).

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Data are presented as the mean ± standard deviation (SD). The statistical analyses were carried out using

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one-way analysis of variance (ANOVA) or Student's t-test. P values less than 0.05 were considered as

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statistically significant.

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Results 9 ACS Paragon Plus Environment


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E10 and F10 Enhanced Nrf2 Expression by ARE-Luciferase Reporter Assay

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The relative luciferase activity was analyzed in ARE-luciferase reporter vector transfected

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HepG2-C8 cells (Figure 2) by luciferase fluorescence signal normalized by protein expression. E10 and

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F10 both enhanced greater luciferase activity than the negative control (0.1% DMSO in medium) in direct

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proportion to concentration ranging from 50 to 1000 nM, which suggests that these two compounds can

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activate Nrf2 expression and hence increase the expression of the antioxidant /detoxification genes with

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an ARE sequence on their promoter regions. When comparing the activation efficacy in terms of

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luciferase signal normalized by protein concentration, F10 produced a much more powerful effect at 1000

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nM than E10, curcumin and SFN.

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E10 and F10 Induced TRAMP-C1 Cytotoxicity

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Based on MTS assays, E10 and F10 were found to diminish the viability of TRAMP-C1 cells in

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direct proportion to time and drug concentration after 24h, 72h, and 120h of incubation (Figure 3). Since

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the survival ratio of TRAMP-C1 cells incubated with E10 and F10 below 100 nM was above 80%, both

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50 and 100 nM E10 and F10 were selected for studying the epigenetic mechanism of Nrf2 restoration.

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E10 and F10 Enhanced Nrf2 expression and the correlated Antioxidant and Detoxification genes Nrf2 is a vital transcription factor for activating type II antioxidant and detoxification enzymes.7

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In TRAMP PCa model, previous reports demonstrated that Nrf2 expression is decreased due to its highly

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methylated promoter region.10 To evaluate the influence of E10 and F10 on Nrf2 and the correlated type

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II antioxidant and detoxification genes, we did qPCR to compare the change in Nrf2, HO-1, NQO1, and

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UGT1A1 mRNA levels in TRAMP-C1 cells upon a 3-day treatment with E10 and F10 (Figures 4A-D).

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E10 and F10 at 100 nM both significantly increased Nrf2 mRNA expression (Figure 4A); E10 (50, 100

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nM) and F10 (100 nM) induced a significant upregulation in HO-1 (Figure 4B); E10 (50, 100 nM) and

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F10 (100 nM) significantly upgraded NQO-1 (Figure 4C); however, neither E10 nor F10 induced any 10 ACS Paragon Plus Environment

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significant increase in UGT1A1 mRNA expression (Figure 4D). The protein expression of the above

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genes upon the same treatment was analyzed by western blotting. After a 3-day treatment, E10

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significantly enhanced the protein expression of Nrf2 and NQO1 at 50 and 100 nM and HO-1 and

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UGT1A1 at 100 nM (Figure 4E). Likewise, higher concentrations of F10 also significantly increased the

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protein level of Nrf2 (100 nM), HO-1 (50, 100 nM), NQO1 (50, 100 nM), and UGT1A1 (100 nM)

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(Figure 4F). Hence, both E10 and F10 are able to raise the level of Nrf2 and the correlated antioxidant

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and detoxification genes transcriptionally and post-transcriptionally in a dose-dependent manner.

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F10 but not E10 Reduced the Methylation Rate of the CpG Regions in the Nrf2 Promoter

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High methylation rate at positions relative (-1226 to -1086) to the transcription start site (TSS) of

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Nrf2 has indicated a close correlation with a decrease in the expression of Nrf2 in the TRAMP model.10

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Thus, we employed BGS to examine whether E10 or F10 can reverse the aberrant methylation status in

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TRAMP-C1 cells. The results showed a high methylation ratio (88.13%) of the CpGs region in the Nrf2

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gene promoter in the untreated cells (Figure 5A). In the positive control group, 3-day treatment of the 5-

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aza-TSA (combination of 5-aza (500 nM) and TSA (100 nM)), the methylation rate was reduced to 63.89%

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(Figure 5A). In E10 (100 nM for 3 days) and F10 (100 nM for 3 days) group, the methylation ratio was

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changed to 85.64%, and 76.80%, respectively (Figure 5A). Treatment with 5-aza and TSA or with F10 for

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3 days significantly induced demethylation in the Nrf2 promoter (p < 0.05). To further confirm the

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findings, we performed a MeDIP-qPCR test. After sonication and pull-down by anti-5-methylcytosine

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antibody, the first 5 CpG regions located at the positions relative (-1226 to -1086) to the TSS of the Nrf2

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promoter was expanded and analyzed by qPCR. The results showed that the 3-day treatment 5-aza -TSA

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group or F10 group (3-day treatment with 100 nM F-10) significantly decreased the methylation rate in

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the Nrf2 promoter region (p < 0.01, Figure 5B), while there does not exist much difference between E10

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and control (Figure 5B), which resonates with the BGS data. To sum up, F10 but not E10 can diminish

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the methylation rate in the Nrf2 promoter, which may contribute to restoration of Nrf2 expression.

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Regulation of Epigenetic Modification Enzymes by F10 and E10

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To analyze the influence of E10 or F10 on vital epigenetic regulation enzymes, the protein

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expression of DNMTs and HDACs with E10 or F10 treatment were examined. DNMTs (DNMT1,

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DNMT3a, and DNMT3b) were all significantly decreased only upon treatment with F10 (p < 0.05, Figure

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6B). In DNMT activity assay, F10 at 50nM and 100nM can both inhibit the total activity of DNMTs (p

Curcumin Derivative Epigenetically Reactivates Nrf2 Antioxidative

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