调控食管鳞癌细胞CAR的表达对溶瘤腺病毒H101抗癌作用影响
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摘要
背景和目的
我国食管癌的发病率居全世界之首,由于食管癌本身具有进展快、侵袭性强等特点,大多数患者被发现时已处于中晚期,传统手术加化疗、放疗的治疗方案效果不佳,需要进一步寻找新的治疗方法。H101病毒是以人类C组5型腺病毒(adenovirus, Ad)为基础,利用基因重组技术得到的一种删除了E1B-55KD和E3区片段的溶瘤腺病毒,使病毒相对选择性的在肿瘤细胞中复制。在临床试验中联合化疗,H101已经取得了令人鼓舞的疗效。但是,临床试验表明作为单一制剂疗效不理想,并且在不同肿瘤中使用效果存在显著差异。
单独应用H101效果不佳和肿瘤细胞表达柯萨奇-腺病毒受体(coxsackievirus-. adenovirus receptor, CAR)水平密切相关。以5型Ad为基础构建的H101病毒有效感染肿瘤细胞,需要通过细胞表面CAR受体黏附和通过整合素家族的整合素αvβ3和αvβ5的内化。肿瘤细胞表面CAR作为腺病毒感染的第一受体完成病毒-细胞的识别,CAR表达水平决定了靶细胞对腺病毒(adenovirus, Ad)的感染效率。研究显示许多肿瘤细胞如卵巢癌、肺癌、乳腺癌及膀胱癌等CAR的表达降低甚至缺乏。
有资料表明CAR表达转录调控是通过乙酰化修饰重塑核小体调节进行的,组蛋白去乙酰化酶抑制剂(histone deacetylase inhibitor, HDACi)通过抑制组蛋白去乙酰化酶(histone deacetylase, HDACs)的活性来提高CAR基因的表达,从而使肿瘤细胞表面CAR受体表达增加,但是HDACi通过何种信号通路影响哪些分子从而改变细胞中CAR基因的转录、翻译,至今尚未见报道。曲古菌素A(trichostatin A, TSA)是一种强大的组蛋白去乙酰化酶抑制剂,研究发现TSA能使一些肿瘤细胞如乳腺癌、肺癌、卵巢癌中CAR表达上调表达,对食管鳞癌CAR的表达的影响未见报道。
目前关于食管鳞癌细胞CAR的表达情况及其与溶瘤腺病毒H101的抗癌效果的关系并不清楚,本研究首先以食管鳞癌(esophageal squamous cell carcinoma, ESCC)细胞系EC9706、EC1为研究对象,分析食管鳞癌细胞EC9706、EC1中CAR的表达情况以及H101病毒对二者的抗肿瘤效应,然后利用TSA来干扰肿瘤细胞CAR的表达,观察食管鳞癌细胞表面CAR表达变化后溶肿瘤腺病毒的抗肿瘤效果,并探索其通过影响MAPK/ERK1/2信号通路对CAR表达影响的机制,同时探讨TSA对溶瘤腺病毒H101抗食管鳞癌ECl细胞裸鼠移植瘤的作用影响,为进一步提高H101临床治疗食管癌效果奠定基础。
第一部分CAR在食管鳞癌细胞中的表达及对H101病毒的抗肿瘤效果的研究
方法
采用RT-PCR检测Hela、EC9706、EC1细胞系CAR mRNA水平；间接免疫荧光标记流式细胞术检测细胞表面CAR的表达阳性率；采用免疫细胞化学法检测、免疫荧光法和Western Blotting检测细胞CAR蛋白表达；以MOI=1 H101分别感染细胞株细胞后,以TCDI50的方法检测48小时测后病毒的增殖倍数,通过病毒增殖实验检测H101病毒在细胞的增殖倍数,采用MTS实验检测H101对肿瘤细胞增殖抑制作用。
结果1.食管鳞癌细胞EC9706、EC1细胞的CAR mRNA相对表达量分别为0.98±0.07、0.75±0.07,明显低于阳性对照细胞Hela细胞CAR mRNA相对表达量1.73±0.08(P<0.01)。
2.流式细胞术检测三株细胞CAR的表达,EC9706、EC1细胞表面CAR表达阳性率分别为21.00±2.00%和9.67±3.05%,明显低于Hela的CAR表达阳性率74.67±9.45%(P<0.01),并且EC1细胞表面CAR表达明显低于EC9706细胞CAR表达(P<0.05)。
3.免疫细胞化学结果显示,在三株细胞的细胞膜上均有棕黄色颗粒,和阳性对照Hela细胞相比,食管鳞癌EC9706、EC1细胞CAR的表达明显弱于Hela细胞。免疫荧光细胞化学检测结果EC9706、EC1细胞表面绿色荧光颗粒明显弱于Hela细胞。Western blotting结果显示,EC9706、EC1细胞CAR蛋白相对表达量为明显低于Hela细胞(P<0.01)。
4.H101在EC9706、EC1细胞中的增殖倍数分别为538.33±69.23和240.55±19.59,明显低于Hela细胞中的增殖倍数1825±256.72(P<0.05)
5.MTS实验检测H101对肿瘤细胞增殖抑制作用,H101对EC9706、EC1细胞增殖抑制作用明显弱于对Hela细胞抑制作用(P<0.05)。
第二部分TSA对食管鳞癌CAR表达的影响及其机制的研究一、TSA对食管鳞癌EC9706、EC1细胞后生物学特性的影响
方法
0.1,0.3,0.5,1.0,3.0μmol/L TSA作用EC9706、EC1细胞,用MTS细胞毒性实验检测TSA对细胞增殖抑制作用；0.3,0.5,1.0μmol/L TSA作用EC9706、EC1细胞,用流式细胞术检测对细胞周期、细胞凋亡的影响；同时用Westernblotting检测TSA处理EC9706、EC1细胞后p21WAF1/CIP1、Bax、Bcl-2表达的变化。
结果
1.细胞增殖实验显示,0.5μmol/l以上浓度TSA可引起EC1细胞增殖抑制而1.0μmol/L TSA以上才可抑制以EC9706细胞的增殖。
2. 0.3μmol/LTSA和0.5μmol/L TSA组EC9706细胞周期与对照组相比,无明显变化(P>0.05)。1.0μmol/L TSA组G0/G1期细胞百分比显著增加,同时S期细胞显著减少,与对照组相比有显著差异(P<0.05); 0.3μmol/L TSA处理EC1细胞后细胞周期与对照组相比,无明显变化(P>0.05)。随TSA浓度增加,G0/G1期EC1细胞百分比逐渐增大,而S期细胞百分比降低,且呈浓度依赖关系(P<0.05)。
3. 1.0μmol/L TSA作用于EC9706细胞后细胞凋亡率明显增加(P<0.05)；0.5μmol/L TSA以上随着TSA浓度升高,细胞凋亡率显著增加升高(P<0.05)。
4. 1.0μmol/L TSA作用EC9706细胞后P21 WAF1/CIP1蛋白、Bax蛋白表达明显增加,而Bcl-2蛋白表达明显减少(P<0.05)；EC1细胞0.5μmol/L TSA以上浓度作用后P21 WAF1/CIP1蛋白、Bax蛋白表达明显增加,而Bcl-2蛋白表达明显减少(P<0.05,图2.1.10)。
二、TSA上调食管鳞癌细胞CAR表达增强H101病毒抗肿瘤效应
方法
以0.3,0.5,1.0μmol/L TSA作用EC9706、EC1细胞48h,采用RT-PCR检测CAR mRNA的表达；间接免疫荧光标记流式细胞术检测细胞表面CAR的表达阳性率；采用免疫细胞化学法检测和免疫荧光法检、Western Blotting检测细胞CAR蛋白表达；以MOI=1 H101分别0.3μmol/L TSA处理的EC1细胞后,以TCDI50的方法检测48小时测后病毒的增殖倍数,采用MTS实验检测H1O1对0.3μmol/L TSA处理48h的EC1细胞后对细胞增殖抑制作用。
结果
1. 0.3,0.5,1.0μmol/L TSA作用EC9706细胞CAR mRNA相对表达量分别为0.71±0.09、0.91±0.03和1.13±0.05,较对照组(0.53±0.04)明显增加(P<0.05); 0.3,0.5,1.0μmol/L TSA作用EC1细胞RT-PCR检测CAR mRNA相对表达量分别为0.67±0.03、0.77±0.03和0.89±0.06,较对照组(0.47±0.03)明显增加(P<0.05)。
2. 0.3,0.5,1.0μmol/L TSA作用EC9706细胞,间接免疫荧光标记流式细胞术检测细胞表面CAR的表达阳性率分别为29.00±0.55%、32.36±1.02%和52.48±1.25%,较对照组(19.57±1.39%)明显增加(P<0.05)；0.3,0.5,1.0μmol/LTSA作用EC1细胞CAR的表达阳性率分别为19.02±0.35%、27.77±0.65%和40.63±1.06%,较对照组(9.58±0.89%)明显增加(P<0.05)。
3.免疫细胞化学法检测、免疫荧光法和Western Blotting检测EC9706和EC1细胞,CAR蛋白表达以剂量依赖方式增加(P<0.05)。
4. 0.3μmol/L TSA作用EC1细胞后H101的增殖倍数为527.46±11.70,较对照组260.75±9.64明显增加(P<0.05)。
5.H101病毒的不同滴度对0.3μmol/L TSA组作用的EC1细胞增殖抑制作用明显高于对照组(P<0.05)。
三、TSA上调食管鳞癌细胞CAR表达机制的探讨
方法
1. Western blotting检测1.0μmol/L TSA作用1h、6h、12h、24h、48 h后EC1、EC9706 p-ERK和CAR的表达,分析p-ERK和CAR的相关性；
2.为了研究TSA可以通过MAPK/ERK1/2信号通路影响HDAC4的磷酸化水平,1.0μmol/L TSA分别作用EC1和EC9706细胞48h,用免疫共沉淀检测p-ERK1/2和HDAC4的相互作用,免疫共沉淀产物用Western blotting检测1.0μmol/L作用EC1和EC970后引起的和HDAC4作用ERK1/2的磷酸化水平的变化
3.为了研究TSA引起组蛋白H4乙酰化水平的改变对CAR基因启动子活性的影响,1.0μmol/L TSA分别作用ECl和EC9706细胞48h,应用乙酰化组蛋白H4抗体进行染色质免疫共沉淀,根据Gene bank中CAR启动子基因序列设计上、下游引物,通过PCR.扩增鉴定乙酰化组蛋白H4和CAR启动子之间的相互作用,Western blotting检测共沉淀产物中,和CAR启动子结合的组蛋白H4乙酰化水平的变化。
结果
1.TSA以时间依赖方式上调食管鳞癌细胞CAR的表达和抑制ERK1/2磷酸化,采用Pearson法双因素相关分析显示,1.0μmol/L TSA作用于食管鳞癌细胞引起CAR表达水平增加和p-ERK水平呈显著负相关(P<0.01)。
2.免疫共沉淀反应结果显示1.0μmol/L TSA处理组和对照组食管鳞癌细胞中p-ERK1/2均和HDAC4之间均存在相互作用。但是1.0μmol/L TSA处理细胞后和HDAC4作用的p-ERK1/2水平较对照组降低(P<0.05)。
3.以乙酰化的组蛋白H4进行CHIP反应,PCR扩增产物经1.5%琼脂糖凝胶电泳,显示在180bp左右有清晰的特异条带,与设计目的基因片段一致。说明乙酰化组蛋白H4和CAR基因启动子之间的相互作用,证明了乙酰化的组蛋白H4和CAR基因启动子之间存在相互作用；1.0μmol/L TSA作用于食管鳞癌细胞后,和CAR基因启动子的作用的乙酰化组蛋白H4水平较未处理组明显升高(P<0.05)。
第三部分TSA对溶瘤腺病毒H101抗食管鳞癌EC1细胞裸鼠移植瘤的作用影响
方法
每只裸鼠皮下注射6×106食管鳞癌细胞EC1,建立食管鳞癌EC1细胞皮下移植瘤模型,分为(1)PBS对照组,以PBS代替药物瘤内注射；(2)TSA组,0.3μmol/L的TSA 200μl瘤内注射,每3d一次；(3)H101组,在TSA组第二次注射后24h后,每只裸鼠瘤内注射1.0×109IU/100μl H101;(4) H101+TSA组,0.3μmol/L的TSA200μl瘤内注射,每3d一次,第二次注射TSA后24h后,每只裸鼠瘤内注射1.0×109IU/100μl H101,治疗3w后处死裸鼠,观察肿瘤体积的变化,测定各组裸鼠皮下移植瘤平均瘤重及抑瘤率；用免疫组化和Western blotting检测各组移植瘤组织CAR的变化。
结果
1. H101+TSA组和PBS对照组、TSA组、H101组相比裸鼠移植瘤体积、移植瘤重量均明显减少(P<0.05); H101+TSA组对肿瘤生长抑制率为80.32%,明显高于TSA组(2.18%)和H101组(28.42%)。
2.免疫组化和Western blotting检测EC1细胞裸鼠移植瘤组织CAR的表达,TSA组和TSA+H101组CAR蛋白的表达明显高于对照组和H101组(P<0.05),说明移植瘤瘤内注射TSA可提高移植瘤组织的CAR水平。
结论
1.食管鳞癌细胞系EC9706和ECl细胞CAR的表达水平明显低于宫颈癌Hela细胞表面CAR的表达；食管鳞癌细胞低表达CAR,影响H101病毒溶瘤效果。
2.TSA上调食管鳞癌细胞CAR的表达从而提高H101病毒抗肿瘤效应。
3.TSA通过抑制MAPK/ERK1/2信号通路,导致HDAC4的活性降低,使得组蛋白H4乙酰化水平提高,激活CAR基因启动子而增加CAR表达。
4.TSA通过提高CAR的表达增强H101病毒对食管鳞癌EC1细胞裸鼠移植瘤的抗肿瘤效果。
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.
Methods
1. 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).
Results
1. 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 TSA
ChapterⅠBiological characteristic affects on ESCC cells induced with TSA
Methods
1. 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.
Results
1. 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.
Methods
1. 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).
Results
1. 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 TSA
Methods
1. 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.
Results
1. 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 mice
Methods
1. 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.
Results
1. 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).
Conclusions
1. 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.
我国食管癌的发病率居全世界之首,由于食管癌本身具有进展快、侵袭性强等特点,大多数患者被发现时已处于中晚期,传统手术加化疗、放疗的治疗方案效果不佳,需要进一步寻找新的治疗方法。H101病毒是以人类C组5型腺病毒(adenovirus, Ad)为基础,利用基因重组技术得到的一种删除了E1B-55KD和E3区片段的溶瘤腺病毒,使病毒相对选择性的在肿瘤细胞中复制。在临床试验中联合化疗,H101已经取得了令人鼓舞的疗效。但是,临床试验表明作为单一制剂疗效不理想,并且在不同肿瘤中使用效果存在显著差异。
单独应用H101效果不佳和肿瘤细胞表达柯萨奇-腺病毒受体(coxsackievirus-. adenovirus receptor, CAR)水平密切相关。以5型Ad为基础构建的H101病毒有效感染肿瘤细胞,需要通过细胞表面CAR受体黏附和通过整合素家族的整合素αvβ3和αvβ5的内化。肿瘤细胞表面CAR作为腺病毒感染的第一受体完成病毒-细胞的识别,CAR表达水平决定了靶细胞对腺病毒(adenovirus, Ad)的感染效率。研究显示许多肿瘤细胞如卵巢癌、肺癌、乳腺癌及膀胱癌等CAR的表达降低甚至缺乏。
有资料表明CAR表达转录调控是通过乙酰化修饰重塑核小体调节进行的,组蛋白去乙酰化酶抑制剂(histone deacetylase inhibitor, HDACi)通过抑制组蛋白去乙酰化酶(histone deacetylase, HDACs)的活性来提高CAR基因的表达,从而使肿瘤细胞表面CAR受体表达增加,但是HDACi通过何种信号通路影响哪些分子从而改变细胞中CAR基因的转录、翻译,至今尚未见报道。曲古菌素A(trichostatin A, TSA)是一种强大的组蛋白去乙酰化酶抑制剂,研究发现TSA能使一些肿瘤细胞如乳腺癌、肺癌、卵巢癌中CAR表达上调表达,对食管鳞癌CAR的表达的影响未见报道。
目前关于食管鳞癌细胞CAR的表达情况及其与溶瘤腺病毒H101的抗癌效果的关系并不清楚,本研究首先以食管鳞癌(esophageal squamous cell carcinoma, ESCC)细胞系EC9706、EC1为研究对象,分析食管鳞癌细胞EC9706、EC1中CAR的表达情况以及H101病毒对二者的抗肿瘤效应,然后利用TSA来干扰肿瘤细胞CAR的表达,观察食管鳞癌细胞表面CAR表达变化后溶肿瘤腺病毒的抗肿瘤效果,并探索其通过影响MAPK/ERK1/2信号通路对CAR表达影响的机制,同时探讨TSA对溶瘤腺病毒H101抗食管鳞癌ECl细胞裸鼠移植瘤的作用影响,为进一步提高H101临床治疗食管癌效果奠定基础。
第一部分CAR在食管鳞癌细胞中的表达及对H101病毒的抗肿瘤效果的研究
方法
采用RT-PCR检测Hela、EC9706、EC1细胞系CAR mRNA水平；间接免疫荧光标记流式细胞术检测细胞表面CAR的表达阳性率；采用免疫细胞化学法检测、免疫荧光法和Western Blotting检测细胞CAR蛋白表达；以MOI=1 H101分别感染细胞株细胞后,以TCDI50的方法检测48小时测后病毒的增殖倍数,通过病毒增殖实验检测H101病毒在细胞的增殖倍数,采用MTS实验检测H101对肿瘤细胞增殖抑制作用。
结果1.食管鳞癌细胞EC9706、EC1细胞的CAR mRNA相对表达量分别为0.98±0.07、0.75±0.07,明显低于阳性对照细胞Hela细胞CAR mRNA相对表达量1.73±0.08(P<0.01)。
2.流式细胞术检测三株细胞CAR的表达,EC9706、EC1细胞表面CAR表达阳性率分别为21.00±2.00%和9.67±3.05%,明显低于Hela的CAR表达阳性率74.67±9.45%(P<0.01),并且EC1细胞表面CAR表达明显低于EC9706细胞CAR表达(P<0.05)。
3.免疫细胞化学结果显示,在三株细胞的细胞膜上均有棕黄色颗粒,和阳性对照Hela细胞相比,食管鳞癌EC9706、EC1细胞CAR的表达明显弱于Hela细胞。免疫荧光细胞化学检测结果EC9706、EC1细胞表面绿色荧光颗粒明显弱于Hela细胞。Western blotting结果显示,EC9706、EC1细胞CAR蛋白相对表达量为明显低于Hela细胞(P<0.01)。
4.H101在EC9706、EC1细胞中的增殖倍数分别为538.33±69.23和240.55±19.59,明显低于Hela细胞中的增殖倍数1825±256.72(P<0.05)
5.MTS实验检测H101对肿瘤细胞增殖抑制作用,H101对EC9706、EC1细胞增殖抑制作用明显弱于对Hela细胞抑制作用(P<0.05)。
第二部分TSA对食管鳞癌CAR表达的影响及其机制的研究一、TSA对食管鳞癌EC9706、EC1细胞后生物学特性的影响
方法
0.1,0.3,0.5,1.0,3.0μmol/L TSA作用EC9706、EC1细胞,用MTS细胞毒性实验检测TSA对细胞增殖抑制作用；0.3,0.5,1.0μmol/L TSA作用EC9706、EC1细胞,用流式细胞术检测对细胞周期、细胞凋亡的影响；同时用Westernblotting检测TSA处理EC9706、EC1细胞后p21WAF1/CIP1、Bax、Bcl-2表达的变化。
结果
1.细胞增殖实验显示,0.5μmol/l以上浓度TSA可引起EC1细胞增殖抑制而1.0μmol/L TSA以上才可抑制以EC9706细胞的增殖。
2. 0.3μmol/LTSA和0.5μmol/L TSA组EC9706细胞周期与对照组相比,无明显变化(P>0.05)。1.0μmol/L TSA组G0/G1期细胞百分比显著增加,同时S期细胞显著减少,与对照组相比有显著差异(P<0.05); 0.3μmol/L TSA处理EC1细胞后细胞周期与对照组相比,无明显变化(P>0.05)。随TSA浓度增加,G0/G1期EC1细胞百分比逐渐增大,而S期细胞百分比降低,且呈浓度依赖关系(P<0.05)。
3. 1.0μmol/L TSA作用于EC9706细胞后细胞凋亡率明显增加(P<0.05)；0.5μmol/L TSA以上随着TSA浓度升高,细胞凋亡率显著增加升高(P<0.05)。
4. 1.0μmol/L TSA作用EC9706细胞后P21 WAF1/CIP1蛋白、Bax蛋白表达明显增加,而Bcl-2蛋白表达明显减少(P<0.05)；EC1细胞0.5μmol/L TSA以上浓度作用后P21 WAF1/CIP1蛋白、Bax蛋白表达明显增加,而Bcl-2蛋白表达明显减少(P<0.05,图2.1.10)。
二、TSA上调食管鳞癌细胞CAR表达增强H101病毒抗肿瘤效应
方法
以0.3,0.5,1.0μmol/L TSA作用EC9706、EC1细胞48h,采用RT-PCR检测CAR mRNA的表达；间接免疫荧光标记流式细胞术检测细胞表面CAR的表达阳性率；采用免疫细胞化学法检测和免疫荧光法检、Western Blotting检测细胞CAR蛋白表达；以MOI=1 H101分别0.3μmol/L TSA处理的EC1细胞后,以TCDI50的方法检测48小时测后病毒的增殖倍数,采用MTS实验检测H1O1对0.3μmol/L TSA处理48h的EC1细胞后对细胞增殖抑制作用。
结果
1. 0.3,0.5,1.0μmol/L TSA作用EC9706细胞CAR mRNA相对表达量分别为0.71±0.09、0.91±0.03和1.13±0.05,较对照组(0.53±0.04)明显增加(P<0.05); 0.3,0.5,1.0μmol/L TSA作用EC1细胞RT-PCR检测CAR mRNA相对表达量分别为0.67±0.03、0.77±0.03和0.89±0.06,较对照组(0.47±0.03)明显增加(P<0.05)。
2. 0.3,0.5,1.0μmol/L TSA作用EC9706细胞,间接免疫荧光标记流式细胞术检测细胞表面CAR的表达阳性率分别为29.00±0.55%、32.36±1.02%和52.48±1.25%,较对照组(19.57±1.39%)明显增加(P<0.05)；0.3,0.5,1.0μmol/LTSA作用EC1细胞CAR的表达阳性率分别为19.02±0.35%、27.77±0.65%和40.63±1.06%,较对照组(9.58±0.89%)明显增加(P<0.05)。
3.免疫细胞化学法检测、免疫荧光法和Western Blotting检测EC9706和EC1细胞,CAR蛋白表达以剂量依赖方式增加(P<0.05)。
4. 0.3μmol/L TSA作用EC1细胞后H101的增殖倍数为527.46±11.70,较对照组260.75±9.64明显增加(P<0.05)。
5.H101病毒的不同滴度对0.3μmol/L TSA组作用的EC1细胞增殖抑制作用明显高于对照组(P<0.05)。
三、TSA上调食管鳞癌细胞CAR表达机制的探讨
方法
1. Western blotting检测1.0μmol/L TSA作用1h、6h、12h、24h、48 h后EC1、EC9706 p-ERK和CAR的表达,分析p-ERK和CAR的相关性；
2.为了研究TSA可以通过MAPK/ERK1/2信号通路影响HDAC4的磷酸化水平,1.0μmol/L TSA分别作用EC1和EC9706细胞48h,用免疫共沉淀检测p-ERK1/2和HDAC4的相互作用,免疫共沉淀产物用Western blotting检测1.0μmol/L作用EC1和EC970后引起的和HDAC4作用ERK1/2的磷酸化水平的变化
3.为了研究TSA引起组蛋白H4乙酰化水平的改变对CAR基因启动子活性的影响,1.0μmol/L TSA分别作用ECl和EC9706细胞48h,应用乙酰化组蛋白H4抗体进行染色质免疫共沉淀,根据Gene bank中CAR启动子基因序列设计上、下游引物,通过PCR.扩增鉴定乙酰化组蛋白H4和CAR启动子之间的相互作用,Western blotting检测共沉淀产物中,和CAR启动子结合的组蛋白H4乙酰化水平的变化。
结果
1.TSA以时间依赖方式上调食管鳞癌细胞CAR的表达和抑制ERK1/2磷酸化,采用Pearson法双因素相关分析显示,1.0μmol/L TSA作用于食管鳞癌细胞引起CAR表达水平增加和p-ERK水平呈显著负相关(P<0.01)。
2.免疫共沉淀反应结果显示1.0μmol/L TSA处理组和对照组食管鳞癌细胞中p-ERK1/2均和HDAC4之间均存在相互作用。但是1.0μmol/L TSA处理细胞后和HDAC4作用的p-ERK1/2水平较对照组降低(P<0.05)。
3.以乙酰化的组蛋白H4进行CHIP反应,PCR扩增产物经1.5%琼脂糖凝胶电泳,显示在180bp左右有清晰的特异条带,与设计目的基因片段一致。说明乙酰化组蛋白H4和CAR基因启动子之间的相互作用,证明了乙酰化的组蛋白H4和CAR基因启动子之间存在相互作用；1.0μmol/L TSA作用于食管鳞癌细胞后,和CAR基因启动子的作用的乙酰化组蛋白H4水平较未处理组明显升高(P<0.05)。
第三部分TSA对溶瘤腺病毒H101抗食管鳞癌EC1细胞裸鼠移植瘤的作用影响
方法
每只裸鼠皮下注射6×106食管鳞癌细胞EC1,建立食管鳞癌EC1细胞皮下移植瘤模型,分为(1)PBS对照组,以PBS代替药物瘤内注射；(2)TSA组,0.3μmol/L的TSA 200μl瘤内注射,每3d一次；(3)H101组,在TSA组第二次注射后24h后,每只裸鼠瘤内注射1.0×109IU/100μl H101;(4) H101+TSA组,0.3μmol/L的TSA200μl瘤内注射,每3d一次,第二次注射TSA后24h后,每只裸鼠瘤内注射1.0×109IU/100μl H101,治疗3w后处死裸鼠,观察肿瘤体积的变化,测定各组裸鼠皮下移植瘤平均瘤重及抑瘤率；用免疫组化和Western blotting检测各组移植瘤组织CAR的变化。
结果
1. H101+TSA组和PBS对照组、TSA组、H101组相比裸鼠移植瘤体积、移植瘤重量均明显减少(P<0.05); H101+TSA组对肿瘤生长抑制率为80.32%,明显高于TSA组(2.18%)和H101组(28.42%)。
2.免疫组化和Western blotting检测EC1细胞裸鼠移植瘤组织CAR的表达,TSA组和TSA+H101组CAR蛋白的表达明显高于对照组和H101组(P<0.05),说明移植瘤瘤内注射TSA可提高移植瘤组织的CAR水平。
结论
1.食管鳞癌细胞系EC9706和ECl细胞CAR的表达水平明显低于宫颈癌Hela细胞表面CAR的表达；食管鳞癌细胞低表达CAR,影响H101病毒溶瘤效果。
2.TSA上调食管鳞癌细胞CAR的表达从而提高H101病毒抗肿瘤效应。
3.TSA通过抑制MAPK/ERK1/2信号通路,导致HDAC4的活性降低,使得组蛋白H4乙酰化水平提高,激活CAR基因启动子而增加CAR表达。
4.TSA通过提高CAR的表达增强H101病毒对食管鳞癌EC1细胞裸鼠移植瘤的抗肿瘤效果。
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.
Methods
1. 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).
Results
1. 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 TSA
ChapterⅠBiological characteristic affects on ESCC cells induced with TSA
Methods
1. 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.
Results
1. 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.
Methods
1. 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).
Results
1. 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 TSA
Methods
1. 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.
Results
1. 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 mice
Methods
1. 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.
Results
1. 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).
Conclusions
1. 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.
引文
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