| Background and AimThe initiation and progression of esophageal cancer is the result of multiple factors, multiple genes and multiple pathways coaction. Quercetin is one of most effective flavonoid polyphenols. It exhibits anti-allergic, anti-inflammatory and anti-diabetic effects, and has been proposed to be a potential anti-neoplastic agent. Sodium butyrate (SB) as the HDAC inhibitor is an example of the class of short-chain fatty acids and it is a basic human physiological deacetylase inhibitor. Many studies have confirmed that SB can inhibit a variety types of tumor cells proliferation, induce cells differentiation and promote cells apoptosis. The present study was designed to oberseved the effects of nanoliposomal quercetin(nLQ), SB and nLQ+SB on EC9706 cells proliferation and apoptosis, and was detected to regulate the expressions of p16, p21walf1, DNMT1, HDAC, NF-κB, Caspase 3, Cyclin D1 and E-cadherin. Furthermore, it was to explore the interaction mechanism between nLQ and SB. The human cytokine interferon-inducible protein 10 (IP-10) is a member of the-C-X-C- chemokine superfamily of proinflammatory cytokines whose secretion is induced by interferon-γ(IFN-γ). The reaserch was to detected the effects of nLQ, IP-10 and nLQ+IP-10 on EC9706 cell proliferation, and to explore the interaction mechanism between nLQ and IP-10.Methods1. Growth suppression rate detected by MTT assay(1) The cultured EC9706 cells were divided into four groups:①. nLQ group:20μmol/L,40μmol/L,60μmol/L.②. SB group:1mmol/L,2mmol/L,4mmol/L.③. Combined group:40μmol/L nLQ+2mmol/L SB.④. Control group:treated with no any drug for 48h.(2) The cultured EC9706 cells were divided into four groups①. nLQ group:20μmol/L,40μmol/L,60μmol/L.②. IP-10 group:10ng/ml,20ng/ml,40ng/ml.③. Combined group:40μmol/L nLQ+20ng/ml IP-10.④. Control group:treated with no any drug for 48h.2. Apoptotic rateThe cultured respective groups of EC9706 cells were examined with TUNEL assay in control group; nLQ group treated with 40μmol/L; the SB group treated with 2mM; the combined group,40μmol/L nLQ+2 mmol/L SB.3. Immunocytochemistry staining(1) Double immunocytochemical stainingThe monoclonal anti-p16 or anti- p21walf1 combined with polyclonal anti-DNA methyltransferase 1 (DNMT1) or anti-histone deacetylasel (HDAC1) were used as the primary antibodies. The HRP/AP (alkaline phosphatase)-conjugated monoclonal IgG/polyclonal IgG was used as the secondary antibody. Finally, the brown signal developed under the substrate of DAB of HRP; while the red signal, as the substrate of AEC of HRP; meanwhile, the dark blue signal developed under NBT-BCIP as the AP substrate.(2) Single immunocytochemical stainingThe expression level and location in each group of EC9706 cells were immunostained on the 4% paraformaldehyde fixed slides. Respective E-cadherin, Caspase3, NF-κB and Cyclin D1 primary antibodies, followed by the SP kits were incubated on the slides. At the same time the primary antibody substituted for PBS was set as the negative control.4. Western blottingThe total protein was extracted from each group of EC9706 cells and the SDS-PAGE was performed onto the nitrocellulose membrane (NCM). The expressions of DNMT1, HDAC1, NF-κB, Cyclin D1, p21walf1 and p16 were determined by Western blotting (β-actin as internal reference).5. Statistical analysisThe data obtained were statistically treated with SPSS 16 soft ware. The data of double immunocytochemical staining were analyzed with chi-square test, andα=0.0083 was considered as significance level.The data shown as (x+s) of immunocytochemical staining and immunoblotting were analyzed with chi-square test and ANOVA among groups, and a=0.05 was considered as significance level.Results1. MTT assay(1) The cell growth suppression by nLQ and SB respectively showed in dose-dependent manner (P<0.01). The nLQ+SB group was more significant than the single drug group(P<0.01).(2) The cell growth suppression by nLQ or the cell growth facilitation by IP-10 showed in dose-dependent manner(P<0.01). However, there was no significant difference between the control group and nLQ+IP-10 group (P>0.05).2. ApoptosisThe TUNEL apoptotic signal in bluish-violet color showed in the cells. Apoptotic rates of control group, nLQ group, SB group and nLQ+SB group were 4%, 23.6%,22.3%,43.8% respectively.Compard with the control group, the apoptotic rate of each drug group was significant (P<0.01).And there was significant different between the nLQ+SB group and singular drug group(P<0.01).3. Immunohistochemical staining(1) The cells treated with nLQ+SB①. Double immunocytochemical staining:When compared with the control group up-regulation of pl6/p21walfl gene expression in brown signals and down-regulation of DNMT1/HDAC1 in blue/red signals shown in the nLQ group, SB group and nLQ+SB group were significant (P<0.0083). There was significant difference between the nLQ+SB group and singular drug group (P<0.0083).②. Single immunocytochemical staining:Immunohistochemical reactivity (IR) appeared as brownish granules. When compared with the control group, the enhanced E-cadherin IR signal and attenuated Cyclin D1 and NF-κB IR signals in the drug group were significant (P<0.01). The nLQ+SB group exhibited more significant than the singular drug group (P<0.01).(2) Cells treated with nLQ and IP-10①. Double immunocytochemical staining:The down-regulated DNA methylation and up-regulated histone acetylation of p16/p21walf1 displayed in the 40μM nLQ group in comparison with the C group (P<0.0083). The epigenetic modification effect induced by IP-10 could be counteracted by that of nLQ+IP-10 group (P<0.0083).②. Single immunocytochemical staining:The immunohistochemical reactivity (IR) appeared as brownish granules. Compared with the control group, the Cyclin D1 and NF-κB IR signals were attenuated in the nLQ group (P<0.05), while those IR signals in the IP-10 group were enhanced (P<0.05). There was no significance between the control group and nLQ+IP-10 group (P>0.05), but There was significance between the IP-10 group and nLQ+IP-10 group4. Immunoblotting(1) The cells treated with nLQ and SB Compared with the control group, the immunoblotting signals of p21walf1 and p16 were enhanced(P<0.01), and those of HDAC1, NF-κB, Cyclin D1 were decreased in each group (P<0.01). There was significant difference between the drugs nLQ+SB group and the single drug group (P<0.01). However, the immunoblotting signals of DNMTlwas not significant in the SB group(P>0.05).(2) The cells treated with nLQ and IP-10 The immunoblotting showed that the expressions of HDAC1, NF-κB and Cyclin D1 were down-regulated by nLQ (P<0.05); while there was contrary effect induced by IP-10. The immunoblotting signals of DNMT1 was not significant in the IP-10 group(P>0.05). There was significant difference between the nLQ+IP-10 group and IP-10 group (P<0.01).Conclusion1. The nLQ or SB can inhibit proliferation of EC9706 cells, and the inhibition effect can be enhanced by nLQ combined with SB. Both of them can up-regulate the expression of p16/p21walf1 via down-regulating the activity of DNMT1 and HDAC1, and down-regulate expression of NF-κB or Cyclin D1. It may suggest that the nanaoliposomal quercetin combined with sodium butyrate may inhibit the cancer cell proliferation and reverse its malignancy.2. IP-10 can promote EC9706 cell proliferation. The mechanism may be that IP-10 can down-regulate the expression of p16/p21walf1 via up-regulating the activity of DNMT1 and HDAC1, and can up-regulate the expression of NF-κB, Cyclin D1. However, The nLQ can counteract the effect of IP-10 in certain extent. |
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