Aberrant Epigenetic Alteration in Eca 9706 Cells Modulated by Nanoliposomal Quercetin Combined with Butyrate Mediated via Epigenetic-NF-κB Signaling

There is close relationship between neoplasia and inflammation. Many cancers arise from sites of chronic inflammation or irritation, and an oncogenic change always induces an inflammatory microenvironment to promote development of tumors. Inflammatory conditions in selected organs increase the risk of cancer, and in the tumor microenvironment smoldering inflammation contributes to proliferation and cancer progression. Natural bioactive compounds exhibit anti-inflammatory modulation (Pan et al., 2009; Gupta et al., 2011). The dietary flavonoid quercetin and resveratrol have nonsteroidal anti-inflammatory activity that may have applications for anti-inflammation treatment (Donnelly et al., 2004; Tuñón et al., 2009). Our other experiment indicates that the pro-inflammatory cytokine IP-10 in co-cultured human esophageal cancer 9706 cells can be inhibited by the polyphenol quercetin. Prevention of cancer has to be associated with prevention of inflammation, especially chronic inflammation. Flavonoid quercetin is one kind of yellowish powdered crystalline compound, (C15H10O7 in structure with five OH groups), widespread in vegetables, fruits and some


Introduction
There is close relationship between neoplasia and inflammation.Many cancers arise from sites of chronic inflammation or irritation, and an oncogenic change always induces an inflammatory microenvironment to promote development of tumors.Inflammatory conditions in selected organs increase the risk of cancer, and in the tumor microenvironment smoldering inflammation contributes to proliferation and cancer progression.Natural bioactive compounds exhibit anti-inflammatory modulation (Pan et al., 2009;Gupta et al., 2011).The dietary flavonoid quercetin and resveratrol have nonsteroidal anti-inflammatory activity that may have applications for anti-inflammation treatment (Donnelly et al., 2004;Tuñón et al., 2009).Our other experiment indicates that the pro-inflammatory cytokine IP-10 in co-cultured human esophageal cancer 9706 cells can be inhibited by the polyphenol quercetin.Prevention of cancer has to be associated with prevention of inflammation, especially chronic inflammation.
Flavonoid quercetin is one kind of yellowish powdered crystalline compound, (C15H10O7 in structure with five OH groups), widespread in vegetables, fruits and some

Aberrant Epigenetic Alteration in Eca9706 Cells Modulated by Nanoliposomal Quercetin Combined with Butyrate Mediated via Epigenetic-NF-κB Signaling
Nai-Gang Zheng 1,2 *, Jun-Ling Wang 1,2 , Sheng-Li Yang 1 , Jing-Lan Wu 2 beverages.It also is one kind of chemopreventive agent with both anti-neoplasm and anti-inflammation effects.Earlier report about the activity of protein kinase C and tyrosine kinase could be inhibited by quercetin in HL-60 cells (Kang et al., 1997).Our previous experiment showed that the natural polyphenol quercetin could arrest cancer cell cycle progression in G2-M phase and induce cells toward apoptosis in Eca109 cells (Zhang et al., 2007).Further research indicates that the tea polyphenols offer cancer prevention against lipid oxidation or adhesion molecule expression (Pandurangan et al., 2012;Zheng et al., 2012).The genetic mutations along with epigenetic alterations, mainly including DNA promoter hypermethylation of tumor suppressor genes, aberrant histone acetylation modification or micro RNAs, can be found in the cancer cells.The epigenetic alteration is reversible under certain condition induced by the natural polyphenols, which can be used for therapy as well as chemoprevention agents (Link et al., 2010).The quercetin can exert anti-tumor properties mediated via demethylation of p16INK4α gene promoter (Tan et al., 2009).When quercetin combined with trichostatin A (TSA, histone deacytylase (HDAC) inhibitor), they cooperatively kill human leukemia cells (Chen et al, 2005).
Butyrate is a short chain fatty acid (CH 3 CH 2 CH 2 COOH) generated from dietary fiber in body.There are lots of papers about the butyrate against various kinds of tumor cells, including cancer prevention as well as anti-cancer treatment, acting as HDAC inhibitor (HDI).The oral cancer cell proliferation can be inhibited by 2 mM -3 mM sodium butyrate (NaBu), while 1 mM NaBu displays no inhibitory effect (Miki et al., 2007).HDI butyrate can cause G2 cell-cycle arrest and up-regulation of p21/ waf1/cip1 protein, and also can activate the p53 protein via hyper-acetylation and nuclear re-localization in neuroblastoma cells (Condorelli et al., 2008).The butyrate can induce apoptosis through JNK MAPK activation in colon cancer RKO cells (Zhang et al., 2010).A butyrate derivative combined with doxorubincin may be beneficial in future therapy of HL-60 cells (Abizadeh et al., 2001).The plant phonolic compounds combined with sodium butyrate can effectively induce anti-tumour activity (Indap et al., 2003).Combined agents contribute to anti-tumor therapy and chemoprevention, especially combined with natural agent.However, So far, the information about the chemoprenventive effect induced by polyphenol quercetin combined with butyrate has been limited.We prepared nanoliposomal quercetin (nLQ/Q) to make it dissoluble into PBS, since the quercetin is hardly dissoluble into water.We designed to explore whether the reversibility of aberrant epigenetic modification induced by Q combined with B would be further promoted than Q or B alone in human esophageal cancer Eca9706 cells.

Preparation of nanoliposomal quercetin and sodium butyrate
The quercetin powder (Sigma US), cholesterol, lecithin, encephalin and polyglycol-4000 in ratio of 6:4:9:5:1 were dissolved in chloroform and methanol (3:1) which was processed by revolutionary-evaporator under vacuum for 4-8h, finally dissolved into PBS.The liposomal quercetin was cleaved by Sonics repeatedly until passing through a filter membrane with 80 nm pore size (Sigma, USA) to prepare the nanoliposomal quercetin (Q) in 4.8mg/ml in PBS as stocking solution.The water soluble powder of sodium butyrate (B) was purchased from Sigma USA and 0.227 mol/L butyrate dissolved in PBS as stocking solution.

MTT assay
After each group of Eca9706 cells, including Q group (20 µM, 40 µM 60 µM), B group (1 mM, 2 mM, 4 mM), Q+B group (40 µM Q+2 mM B) and no drug control group cultivated into repeated 5 wells of one 96-well plate (Corning, USA) for 48h, 5 mg/ml MTT (Amersco, USA) in 20 µl was added and re-incubated for 4h.The supernatant was removed, followed by recruitment of 150µl DMSO to dissolve the formazone product, and OD value in each well was determined by Label Sensors (Sunrise, China) under 492nm wave length.We collected each group of Eca9706 cells for MTT assay after 48h incubation, since floating dead cells appeared apparently after incubation for 72h and 48h incubation growth potential was superior over 24h observed under inverted microscope.

Grouping of cultured Eca9706 cells
The Eca9706 cancer cells were cultured with DMEM medium plus 10% FBS for 48h routinely.The cultured Eca9706 cells (1×10 7 )were divided into 4 groups based on the detected MTT assay: 1 Q group: with 40 µmol/L Q; 2 B group: cultivated with 2mmol/L sodium butyrate; 3Q+B group, cultivated with 40 µmol/L Q combined with 2mmol/L B (doses with 27%-28% growth suppressing rates) and 4 C group: cultured for 48h with neither Q nor B.

Immunoblotting of Eca9706 cells
The total protein was extracted from each group of 9706 cells by using CelLyticTM Cell Lysis Reagent (Sigma, USA), and the protein concentration was detected with Bradford method.The performed SDS-PAGE gel was electro-transferred onto the pre-treated nitrocellulose membrane (NCM, Protran, USA).The primary antibodies, including anti-DNMT1 (polyclonal, Boisynthesis, China), anti-HDAC1 (polyclonal, Santa Cruz, USA), anti-NF-κBp65 (monoclonal, Santa Cruz, USA), anti-cyclin D1 (monoclonal, Zymed, USA), anti-caspase-3 (polyclonal, Zymed, USA) and anti-β-actin (Santa Cruz, USA) was added into each lane as loading control.The primary anti-sera were incubated with corresponding NCM on a vibrator under room temperature for 2.5 h.The TTBS buffer (0.15 mol/L NaCl and 0.05% Tween 20 added into 0.1 mol/L Tris-Cl buffer, pH 7.5) was used as blocking or washing solution.The goat or mouse IgG-HRP (monoclonal ZSGB, China) was used as the secondary antibody to incubate the NCM on a vibrator under room temperature for 1.5h, finally the brownish color developed on the NCM under DAB substrate for 20min.The grey scaled means (GSM) of target bands were determined by an Image-Scanner (ShanFu, China).

Methylation specific PCR (MSP) of p16INK4a gene promoter
The genome DNA was extracted from each group of Eca9706 cells by using a kit (Tiangen, China).The procedures from cell lysis to DNA eluted from column filter were performed according to the instruction.After the DNA purity and concentration was determined, the MSP was performed by using Methylamp™ DNA Modification Kit (Epigentek, USA) strictly according to user guide.Firstly the DNA is chemically denatured to allow bisulfite reagent to react specifically with single-stranded DNA to convert C into U.The modified DNA captured onto a column filter was collected through cleaning and elution.The methylation-specific primers for p16INK4α promoter included forward primer, 5' -ttattagagggtggggcggatcgc-3' and reverse primer, 5' -gaccccgaaccgcgaccgtaa-3'. The real time MS-PCR procedures for p16INK4α were performed as follows: 95℃ 5min, 95℃ 50s 35cycles, 60 ℃ 50s 35cycles, 72℃ 50s 35cycles, 72℃ 5min.The DNA 100 ladder marker (Solarbio Biotech, China) was used in 2% agarose gel electrophoresis and the MSP results were analyzed by Image-Scanner (ShanFu, China).

Immunocytochemical reactivity of E-cadherin and HDAC1
The E-cadherin antiserum (monoclonal, Zemed, USA) or HDAC1 antiserum was used as primary antibody, the SP kit from ZhongShan (China) was used as secondary and tertiary anti-sera.The primary antibody replaced by PBS was performed as the negative control.The grey scale means (GSM) for target bands were determined by an Image-Scanner (ShanFu, China).

TUNEL assay
The assay was performed according to the user guide from Promega USA.The TdT buffer substituted for TdT enzyme was performed as the negative control.The apoptotic cells with bluish-violet apoptotic signals were enumerated per two hundred Eca9706 cells in each group as the apoptotic rate.

Statistical analysis
Statistical analyses were performed by use of SPSS 12.0  .05 as significant level; while the difference in complex parameters was analyzed with LSD-t test, P<0.008 was considered as significant level.

MTT assay
The MTT assay showed that Q or B suppressed cell growth in dose dependent manner, Q+B further inhibited the cell growth compared with Q/B or C group, P<0.008.However, there was no significant difference in suppressing cell growth between Q and B, P>0.05 (Table 1).The growth suppressing rate (GSR) = 1-experimental OD value/ control OD value×100

Immunoblotting
The immunobkotting showed that the expressions of global DNMT1, NF-κBp65, HDAC1, and cyclin D1 were down-regulated by Q or B, and further by Q+B, compared with C, Q or B, P<0.008.However, the expressions of caspase-3 and p16INK4α were up-regulated, and further by Q+B compared with C, Q or B, P<0.008 (Table 2, Figure 1).

Discussion
NF-κB is activated by response to various stimuli, such as viral or bacterial infection, oxidant stress, immune response and carcinogenesis.Transcription factor NF-κB is a common final target for many cell proliferation and inflammatory pathways (Wilczynsk et al., 2011).The activated NF-κBp65 (RelA) and the aberrant HDAC activity play the pivotal role of tumorigenesis.There is link between RelA/p65 and class I HDACs in nuclear translocation as well as RelA/p65 DNA binding activity which can be markedly diminished by HDAC inhibitor (Lehmann et al., 2009).In prostate cancer cells the apoptosis is induced through inactivation of nuclear factor-kappaB and activation of caspase pathway after anti-cancer treatment (Park et al., 2011).Butyrate induces human colon cancer RKO cell apoptosis through activation of caspase pathway (Zhang et al., 2010).In present experiment the down-regulated HDAC1 with altered nuclear translocation in treated groups to inhibit Rel A/p65 activity, and down-regulated HDAC1, Rel A and cyclin D1, and up-regulated caspase-3 and p16INK4α, the growth suppressing rates (GSR) were induced by Q/B and further by Q+B, acting as HDAC inhibitor via HDAC-NF-κB cascade signaling.Besides, the up-regulated expression of p16INK4α by Q/B and further by Q+B in immunoblotting was associated with the local hypermethylation of p16INK4α gene promoter attenuated by Q/B and further by Q+B in MS-PCR.The NF-κBp65 activity and HDAC are important inducers of epithelial-mesenchymal transition (EMT), cell metastasis and invasiveness (Strippoli et al., 2010;Lei et al., 2010).In this experiment the E-cadherin expression was increased, suggesting that the reversal EMT may be contributed to inhibiting migration/invasive potency of Eca9706 cells mediated via HD1 or inactivation of NF-κBp65 induced by Q/B, especially by Q+B.Thus, the promoted anti-cancer effect by Q/B and further by Q+B may be mediated through targeting both altered DNA methylation and histone acetylation, mediated via epigenetic-NF-κBsignaling. Flavonoids derived from fruits, vegetables, etc, can suppress the proinflammatory cell signaling (such as activated NF-κB) pathways and thus can prevent and even treat the cancer (Prasad et al., 2010).
Screening of potential cancer chemopreventive agents mainly based on anti-oxidant/anti-inflammatory mechanism in murine hepatoma cell cultures was induced by sodium butyrate or polyphenols, including quercetin, EGCG, resveratrol, curcumin, etc. (Gerhauser et al., 2003).The polyphenol quercetin may prevent cardiovascular disease due to its anti-oxidative stress; the healthy male subjects consumed 150 mg/d quercetin for 8 wks intermitted by a three-week washout phase (Pfeuffer, 2013).The cancer chemopreventive experiment on the Wistar rat hepatocarcinogenesis model treated with histone deacetylase inhibitor, tributyrin (butyric prodrug, 2g/kg body wt) daily for 8 wks resulted in lower HDAC activity and Hdac3 and Hdac4 gene expression, and etc (de Corti, 2013).Moreover, Steliou k et al indicate that the histone deacetylase inhibitor butyrate is not only capable to suppress cancer growth, but also to regulate other medical disorders, such as neurological,

Figure 1 .
Figure 1. A. Immunoblotting Showed Down-regulated Expressions of DNMT1, NF-κBp65, HDAC1, and Cyclin D1 and Up-regulated Expressions of Caspase-3 and p16INK4α in Eca9706 Cells, Q+B> Q/B, Compared with C Group, P<0.008; B. Diagram Showed Decreased Global DNMT1, NF-κBp65, HDAC1, Cyclin D1 and Increased Caspase 3, p16INK4α Levels in Eca9706 Cells Treated by Q/B or Q+B Compared with C. The Increased/decreased Sequence was Q+B>Q/B>C, P<0.05

Figure 2 .
Figure 2. A. p16INK4α MS-PCR in Each Group of Eca9706 Cells; Lane 1: Q Group; Lane 2: C Group; Lane 3: Q+B Group; Lane 4: B Group; M Lane: DNA Marker.The Methylated p16 INK4α Located at 150bp.B. Diagram of Methylated p16INK4α in Each Group of Eca9706 Cells.Diagram of p16INK4α MS-PCR Showed That the MSP Order was C>B/Q>Q+B, P<0.05