Regulating The Coxsackie And Adenovirus Receptor In ESCC To Anti-tumor Activity Of Oncolytic Adenovirus H101

The morbidity of the esophageal carcinoma (EC) of our country occupies the first place in whole world. EC is an intractable disease and curative surgical operation is often difficult for aged and extended cases. Another characteristic of EC is its high mortality, although the patients can get radical excision by surgical intervention operation and comprehensive treatments, most of the patients still die of metastatic dieases. New approaches are urgently needed for better EC. H101 is based on human species C adenovirus serotype 5 (Ad5), utilize genetic recombinant technique to delete the E1B-55KD and E3 fragment which can make the virus replicate relative selectivity in the tumor cells only. H101 have been clinically examined for feasibility. Although there are some promising early clinical results, single-agent efficacy has been mostly unimpressive.Concurrently, it has become apparent that a major determinant of gene transfer efficacy with adenovirus (Ad) is expression of its primary receptor, Coxsackievirus and adenovirus receptor (CAR), on target cells. To infect tumor cells efficiently, H101 requires CAR for attachment and av integrin for internalization. However, it has become evident that CAR expression is often low on various types of advanced clinical tumors including ovarian, lung, breast, bladder, and others. It was reported that histone deacetylase inhibitor (HDACi) induced enhancing the levels of CAR on some tumor cell surface via accumulation of hyperacetylated nucleosome core histonesand results in transcriptional activation of genes. However, it is not clear about signaling pathway that HDACi induces the expression of CAR on tuomr cells. Trichostatin A (TSA) is one of the most promising HDACi that can increase the levels of CAR on some tumor cells such as breast, lung, ovarian and others. But it is not reported that TSA influences on the expression of CAR on EC cells.In this study, firstly, to detect the expression of CAR on esophageal squamous cell carcinoma (ESCC) cell lines, EC9706 and EC1, and analyze the relationship between CAR and the anti-tumor activity of oncolytic adenovirus H101. Secondly, to investigate TSA to increase the expression of CAR and the influence on the anti-tumor activity of H101, to study the mechanism of increased CAR gene expression through the MAPK/ERK1/2 signaling pathway induced with TSA. Thirdly, to detect the role of the expression of CAR induced with TSA to the anti-EC1 activity of oncolytic adenovirus H101 in transplanted tumors of nude mice.PartⅠ:Detecting the expression of CAR on ESCC cell lines and analyzing the relationship between CAR and the anti-tumor activity of oncolytic adenovirus H101.Methods1. The expression of CAR mRNA in two ESCC cells lines (EC9706 and EC1) and the control positive cells, Hela cells were investigated with reverse transcription-polymerase chain reaction (RT-PCR).2. The positive rate of CAR expression in two ESCC cells lines (EC1, EC9706) and the control positive cells were analyzed by flow-cytometry. 3. The expression of CAR protein in ESCC cells lines and Hela cells were detected with immunocytochemistry, immunofluorescence and western blotting.4. The reproductive activity of H101 in ESCC cells lines and Hela cells were detected with multiplication experiment and finally investigated the killing effect of H101 via cytotoxic experiment (MTS assay).Results1. The CAR mRNA expression level of EC9706 cells, EC1 cells and Hela cells were 0.98±0.07 and 0.75±0.07,1.73±0.08. The CAR mRNA expression of ESCC cells were obviously low compared with control cells'(P<0.01).2. The positive rate of CAR expression of EC9706 cells, EC1 cells and Hela cells were 21.00±2.00%,67±3.05% and 74.67±9.45%. The positive rate of CAR expression in two ESCC cells line was significantly low than the control cells detected with FCM (P<0.01).3. The result of immunocytochemistry, Immunofluorescence and Western blotting indicated that the expression of CAR protein in ESCC cells lines decreased, especially in EC1 cells (P<0.05).4. The reproductive multiple of H101 EC9706 cells, EC1 cells and Hela cells were 538.33±69.23,240.55±19.59 and 1825±256.72. The reproductive multiple of H101 was obviously low in two ESCC cells compared with Hela cells (P<0.05); and the cytotoxic activity of H101 in two ESCC cells was more weak than in controls'.PartⅡ:Influence on the CAR expression on ESCC cells and mechanism of increased CAR induced by TSAChapterⅠBiological characteristic affects on ESCC cells induced with TSA Methods1. ESCC cells in logarithmic growth phase were treated at the dosage of 0.1,0.3, 0.5,1.0,3.0μmol/L TSA and treated with DMSO (0.25%) for 24 h, then the effects of TSA on cell proliferations were assessed by MTS.2. After treatment with TSA at the dosage of 0.3,0.5,1.0μmol/L for 24 h, cell cycle distributions and apoptosis were detected by flow cytometric assay(FCM), and the expression of p21 WAF1/CIP1, Bax and Bcl-2 protein in ESCC cells lines were detected with Western blotting.Results1. TSA inhibited the growth of human EC9706 above the dosage of 1.0μmol/L, while TSA inhibited the growth of EC1 above the dosage of 0.5μmol/L TSA. In comparison with the control group, the proportion of EC9706 cells in G1/G0 phase was increased (P<0.05), while the proportion of cells in S phase was decreased (P<0.05) induced by 1.0μmol/L TSA; The proportion of EC1 cells in G1/G0 phase was increased (P<0.05), while the proportion of cells in S phase was decreased (P<0.05) induced by 0.5 and 1.0μmol/L TSA.2. The apoptosis rate of EC9706 cells induced with 1.0μmol/L TSA was significant increase compared with the control group's (P<0.05); The apoptosis rate of EC1 cells induced with 0.5μmol/L TSA was significant increase compared with the control group's (P<0.05).3. The P21 WAF1/CIP1 and Bax protein expression of EC9706 cells was significantly increased, while Bcl-2 expression significantly decreased compared with the control group after treatment with 1.0μmol/L TSA; The P21 WAF1/CIP1 and Bax protein expression of EC1 cells in trial groups (0.5μmol/L, 1.0μmol/L) was significantly increased, while Bcl-2 expression significantly decreased compared with the control group. Chapter 2 Modulation of coxsackie-adenovirus receptor expression for increased the anti-tumor activity of oncolytic adenovirus H101.Methods1. ESCC cells were treated 0.3,0.5,1.0μmol/L TSA for 48h, the expression of CAR mRNA on EC9706. cells and EC 1 cells were investigated with RT-PCR.2. The positive rate of CAR expression evoked by different concentrations of TSA on EC9706 cells and EC1 cells were analyzed by flow-cytometry.3. After treated with different concentrations of TSA, the expression of CAR protein on EC9706 cells and EC1 cells were detected with immunocytochemistry, immuno-fluorescence and Western blotting.4. The reproductive activity of H101 in EC1 cells induced by 0.3μmol/L TSA was detected with multiplication experiment and finally investigated the killing effect of H101 via cytotoxic experiment (MTS assay).Results1. The CAR mRNA expression level of EC9706 cell trial groups (0.3μmol/L, 0.5μmol/L, 1.0μmol/L) were 0.71±0.09,0.91±0.03 and 1.13±0.05, and control group was 0.53±0.04. There was significant increase between trial groups and control group (P<0.05); The CAR mRNA expression level of EC1 cell trial groups (0.3μmol/L,0.5μmol/L, 1.0μmol/L) were 0.67±0.03,0.77±0.0 and 0.89±0.06, and control group was 0.47±0.03. There was significant increase between trial groups and control group (P<0.05).2. The positive rate of CAR expression of EC9706 cell trial groups (0.3μmol/L, 0.5μmol/L,1.0μmol/L) were 29.00±0.55%,32.36±1.02% and 52.48±1.25%, and control group was 19.57±1.39%. There was significant increase between trial groups and control group (P<0.05); The positive rate of CAR expression of EC1 cell trial groups (0.3μmol/L,0.5μmol/L, 1.0μmol/L) were 19.02±0.35%, 27.77±0.65% and 40.63±1.06%, and control group was 9.58±0.89%. There was significant increase between trial groups and control group (P<0.05).3. The expression of CAR protein in ESCC cells induced by different concentrations of TSA was increased with treatment of 0.3,0.5,1.0μmol/LTSA for 48h, which was a concentration-dependent manner (P<0.05).4. The reproductive multiple of EC1 cells induced with 0.3μmol/LTSA and control group cells were 527.46±11.70 and 260.75±9.64. The reproductive multiple of H101 was obviously higher in 0.3μmol/L TSA group compared with control group cells'(P<0.05).5. The cytotoxic activity of H101 was more powerful in EC1 cells induced by 0.3μmol/L TSA compared with control group cells'(P<0.05).Chapter 3 Mechanism of up-regulating the expression of CAR on ESCC cells induced by TSAMethods1. The ESCC cells were respectively exposed by 1.0μmol/L TSA for 0,1,6,12,24, 48h, the expression of CAR and phosphorylation of MAPK/ERK1/2 were investigated with Western blotting. The correlation between the activity of phosphorylation of ERK and the expression was analyzed.2. In order to identify the effect on association with HDAC4 and ERK1/2 phosphorylating, HDAC4 antibody was used to CO-immunoprecipition to analyze the activation between HDAC4 and ERK1/2 phosphorylation induced by 1.0μmol/LTSA in ESCC cells.3. Based on the CAR promoter gene sequence reported by GenBank, use DNASIS,OLIGO software to design a pair of primer for the CAR promoter full length genome. Chromatin immunoprecipitation (CHIP) assay was done to detect the effect of TSA on the level of acetylated histone H4 at the CAR promoter region. Results1. Western blotting results showed that the expression of CAR protein increased and the level of phosphorylation of MAPK/ERK1/2 decreased in a time dependent manner. There were significant negative correlations between the activities of p-ERK and the expression of CAR (P<0.01).2. The CO-immunoprecipition results showed that phosphorylation of ERK1/2 are associated with HDAC4 both in ESCC cells induced by TSA and the controls, but the level of phosphorylation of ERK1/2 in ESCC cells treated by TSA significantly decreased.3. Chromatin immunoprecipitation results showed that acetylated histone H4 were associated with CAR gene promoter and an increased histone H4 acetylation in the CAR promoter gene induced with TSA was detected, which are due to the suppression of histone deacetylase activity and promote the CAR gene expression.PartⅢ:The effect of oncolytic adenovirus H101 under the condition of CAR gene up-regulation on ESCC transplanted subcutaneously in nude miceMethods1. EC1 cells were injected into the left flank area of BALB/c nude mice aged 5 weeks old. Once tumors reached 100-200 mm3, mice were treated in the following, TSA only, H101 only and H101 combination with TSA. TSA was intratumorally injected every three day for 3 week. For determining the effect of H101, a single dose of 1.0×109 IU/100μl H101 was given intratumorally 24 hours after the second injection of TSA. The respective control groups received corresponding PBS injection. 2. The mice weight and tumor volume were detected following the treatment. The nude mice were sacrificed until the 5th weekend and removed the tumor masses. Immunohistochemistry and Western blotting were adopted to analyze the expression of CAR.Results1. Animal experiment suggested that the average tumor volume and weight of H101 combination with TSA group were smaller than the TSA group and H101 group, transplanted growth tumor inhibition of transplanted tumor of H101 combination with TSA group was much better than the other three groups (P<0.05).2. Immunocytochemistry and Western blotting results indicated that the expression of CAR on transplanted tumor of TSA group and H101 combination with TSA group significantly increased compared with the other groups(P<0.05), and had no obviously difference between the TSA group and H101 combination with TSA group(P>0.05).Conclusions1. The expression of CAR protein in ESCC cells lines is down-regulatedand compared with Hela cells. The anti-tumor activity of oncolytic adenovirus H101 is related to the expression of CAR on tumor cells. There were significanttly positive correlations between the CAR expression on tumor cells and the anti-tumor activity of oncolytic adenovirus H101.2. TSA can increase the expression of CAR on human ESCC cells and can increase the anti-tumor activity of oncolytic adenovirus H101 to ESCC cells through enhancing the expression of CAR.3. The mechanisms maybe involve the interruption of ERK/MAPK pathway. Protein kinase activity ERK1/2 is present in a protein complex with HDAC4, TSA can inhibits the level of phosphorylation of ERK1/2 and decrease the activity of HDAC4 to up-regulate the acetylation level of histone H4 associated with CAR gene promoter.4. Up-regulation the expression of CAR on ESCC tissues of nude mice induced by TSA can enhance the anti-tumor activity of oncolytic adenovirus H101, which may be become a novel therapeutic strategies of esophageal carcinoma.