阿司匹林联合喜泊分-PDT体外对人食管癌Eca-109细胞的影响及凋亡机制的实验研究
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摘要
研究背景
光动力疗法(photodynamic therapy, PDT)是利用激光的光化学原理,通过特定波长的激光激活肿瘤组织内滞留的光敏剂,与肿瘤组织内的氧发生作用,产生一些活性氧分子(radical oxygen species, ROS),并通过氧化作用攻击细胞结构,造成细胞膜或蛋白质的氧化损伤,当氧化损伤的积累超过一定的阈值时,细胞便开始死亡。另外,还可造成肿瘤微血管急性损伤。光动力疗法的作用基础是光动力效应,这是一种有氧分子参与的伴随生物效应的光敏化反应。光敏剂、激光和氧分子构成光动力效应的三要素。目前临床上应用最广泛的光敏剂为血卟啉衍生物。因此本研究采用喜泊分,一种中国生产的血卟啉衍生物,已在临床上用于多种肿瘤的光动力治疗,如肝癌、食管癌、胃癌、膀肤癌、妇科肿瘤、皮肤癌、头颈部肿瘤等。由于630nm为喜泊分治疗的最适波长,因此我们选用波长为630nm的DIOMED630PDT系统。
在光动力剂量学研究中,不但要考虑光敏剂和照射光的质和量,还要考虑光敏剂的体内分布特性和受照射组织的光学特性,同时兼顾组织中血流变化对氧含量的影响。光动力剂量的简要表达式为：D=(1／α)Cφt。式中,D是光动力剂量；C是光敏剂浓度；中是光能流率；t是光照时间；α是组织对光敏毒性的耐受参数,它决定于组织的生物特性和组织内的氧浓度。然而,在临床应用中,光敏剂的剂量和光照剂量是光动力剂量学的两个最重要可控参量。本研究采用的光能流率(2000mw)是固定的,而光敏剂浓度和光照时间是可变的,从而探讨不同光动力剂量对细胞的体外效应。
PDT目前已开始应用于多种恶性肿瘤的治疗,日渐成为肿瘤治疗新手段。它与常规的手术、化疗等疗法相比,PDT具有创伤小,毒性小,选择性、适用性、重复性好,可姑息治疗,可消灭隐性病灶,可保护容貌及重要器官等优点。
食管癌是人类常见的消化道恶性肿瘤,而我国是高发国家。随着新型光敏剂的发展及半导体激光发生系统的开发,PDT在食管癌尤其是早期食管癌及癌前病变的治疗上已取得较大进展。但临床治疗中发现,肿瘤光动力治疗后近期疗效较好,但肿瘤复发率较高。PDT引起的直接肿瘤细胞杀伤、微血管损伤、缺氧、炎症与血管内皮生长因子(VEGF)、环氧化酶-2(COX-2)、金属蛋白酶(MMP)、细胞因子和前列腺素的表达增高有关。这些分子的增加减弱了PDT的效应。环氧化酶(COX)是前列腺素代谢中重要的酶,前列腺素是血管生成的重要调节剂。非甾体抗炎药物阿司匹林(aspiprin, ASA)通过阻断环氧合酶COX途径表现出抗血管生成功能。改变PDT介导的肿瘤微环境可调节PDT疗效。
研究目的
本实验以血卟啉衍生物喜泊分为光敏剂,波长630nm的DIOMED630PDT系统为激发光源,光敏剂浓度和光照剂量为两个可变因素,观察喜泊分-PDT、喜泊分-PDT联合ASA体外对人食管鳞癌细胞Eca-109的增殖和COX-2蛋白表达的影响。
研究方法、结果和结论
一、不同喜泊分浓度和光照剂量介导的PDT体外对Eca-109细胞增殖和COX-2蛋白表达的影响
(一)、方法
1、实验分为4组：单纯喜泊分组,单纯光照组,PDT组,对照组。
2、MTT法检测不同喜泊分浓度(0 ug/ml,2.5 ug/ml,5 ug/ml,10 ug/ml, 15 ug/ml,20 ug/ml)和不同光照时间(0s,30s,60s,90s)下的喜泊分-PDT对食管癌Eca-109细胞的增殖抑制作用。
3、In cell western blot技术检测不同喜泊分浓度(0 ug/ml,2.5 ug/ml,10 ug/ml,20 ug/ml)和不同光照时间(0s,30s,60s,90s)下的喜泊分-PDT作用于食管癌Eca-109细胞后的COX-2蛋白的表达情况。
4、统计学处理采用Spss13.0统计软件,实验结果以“均数±标准差”表示,统计方法采用析因设计的方差分析,多组间比较采用单向方差分析。限定p<0.05为差异有统计学意义。
(二)结果
1.不同浓度和不同光照时间下的喜泊分-PDT对Eca-109细胞存在显著的增殖抑制,并且随着喜泊分浓度和光照时间的增加,喜泊分-PDT对细胞增殖的抑制作用越强。
2.喜泊分-PDT作用后Eca-109细胞的COX-2蛋白相对表达量较对照组增高并且随着喜泊分浓度和光照时间的增加,Eca-109细胞的COX-2蛋白相对表达量呈增高的趋势。
(三)结论
1.本研究发现喜泊分-PDT可明显抑制人食管癌Eca-109细胞增殖并诱导其凋亡;
2.喜泊分-PDT可上调人食管癌Eca-109细胞COX-2蛋白表达。
二、不同浓度ASA对Eca-109细胞增殖和COX-2蛋白表达的影响
(一)、方法
1、实验分为2组：单用ASA组,对照组。
2、MTT法检测不同浓度ASA (0 mmol/L,2.5 mmol/L,5 mmol/L, 7.5mmol/L,10mmol/L,12.5 mmol/L)对食管癌Eca-109细胞的增殖影响。
3、In cell western blot技术检测不同浓度ASA(0 mmol/L,2.5 mmol/L, 5 mmol/L,12.5 mmol/L)对食管癌Eca-109细胞COX-2蛋白表达影响。
4、统计学处理采用Spss13.0统计软件,实验结果以“均数±标准差”表示,统计方法采用单向方差分析。限定p<0.05为差异有统计学意义。
(二)结果
1.阿司匹林对Eca-109细胞增殖的抑制作用：采用MTT法检测细胞增殖抑制率,描绘曲线图可知,经不同浓度阿司匹林处理后的Eca-109细胞生长缓慢,表现出明显的生长抑制,并呈作用浓度依赖性,而对照组细胞生长良好；
2.阿司匹林对Eca-109细胞COX-2表达的影响：阿司匹林作用于Eca-109细胞后,细胞中的COX-2蛋白相对表达量下调。
(三)结论：
1.本研究发现阿司匹林可抑制人食管癌Eca-109细胞增殖；
2.阿司匹林可下调Eca-109细胞的COX-2蛋白表达。
三、喜泊分-PDT联合ASA体外对Eca-109细胞增殖和COX-2表达的影响
(一)方法
1.实验分组分为4组：PDT组,PDT后ASA组,PDT前ASA组,对照组。根据ASA对Eca-109细胞作用的MTT实验结果,选择IC50的1个ASA浓度,即ASA浓度5 mmol/L,进行该项实验。
2.MTT法检测5种不同喜泊分浓度(2.5 ug/ml,5 ug/ml,10 ug/ml, 15 ug/ml,20 ug/ml)介导的PDT单独和联合ASA (PDT后ASA组,PDT前ASA组)对食管癌Eca-109细胞的增殖抑制作用。
3. In cell western blot技术检测3种不同喜泊分浓度(2.5 ug/ml,10 ug/ml,20 ug/ml)介导的PDT单独和联合ASA (PDT后ASA组,PDT前ASA组)下食管癌Eca-109细胞COX-2蛋白的表达情况。
4.统计学处理采用Spssl3.0统计软件,实验结果以“均数±标准差”表示,统计方法采用析因设计的方差分析,多组间比较采用单向方差分析。。p<0.05为差异有统计学意义。
(二)结果
1.喜泊分-PDT联合ASA均对Eca-109细胞存在明显的生长抑制。随着喜泊分浓度的升高,细胞的抑制率呈升高的趋势。PDT后ASA组(均数为0.79)的抑制率高于单用PDT组(均数为0.40)和PDT前ASA组(均数为0.41)。以细胞抑制率绘制细胞生存曲线,结果示,当喜泊分2.5ug/ml、作用方式为单用PDT时,细胞抑制率最低(均数=0.06),当喜泊分20ug/ml、作用方式为PDT后ASA时,细胞抑制率最高(均数=0.97)。
2.ASA联合喜泊分-PDT后降低细胞的COX-2蛋白相对表达量。PDT后ASA组的COX-2蛋白相对表达量最低(均数=0.06),单用PDT组的COX-2蛋白相对表达量最高(均数=0.11)。
(三)结论
1.本研究发现喜泊分-PDT联合ASA可增强喜泊分-PDT对Eca-109细胞增殖的抑制作用；
2.喜泊分-PDT联合ASA增强喜泊分-PDT对Eca-109细胞增殖的抑制作用的机制可能与下调COX-2表达有关；
3.ASA联合人食管癌喜泊分-PDT可能会减少PDT后的复发率。
Background:
Photodynamic Therapy (PDT) is a kind of photochemistry therapy. The PDT mechanism is that the resident photosensitizer in tumor tissue is activated by certain wavelength laser and takes action on oxygen in tumor tissue and produce some radical oxygen species (ROS), then attack the tumor cell through peroxidization. This may make the cellular membrane or oxidative protein damage. And the tumor cell will apoptosis after the oxidative damage get to the leak point. Also, it can damage to the tumor capillary. As contrast to conventional therapy such as operation and chemotherapy, PDT has the advantages as minimally invasive、little toxicity、high selectivity、wide applicability、well repeatability、be used as pamper treatment、maintain features and important organ function and can cure precancerous lesion. Nowadays, PDT has been used in curing some malignant tumor, and gradually to become a new method for tumor therapy.
Esophageal carcinoma is a kind of common human alimentary canal tumor and it has high death rate in our country. As the development of the new type photosensitizers and semiconductor laser generator, PDT has made great progression in curing esophageal carcinoma especially in the earlier period and precancerous lesion. But in clinical curing, we find it is common that the photodynamic therapy effect is instability. Some human esophageal carcinoma patients can be clinical curative, while some is negative. Several laboratories have also shown that PDT can induce expression and/or activation of additional pro-angiogenic molecules including COX-2 and prostaglandins,TNF-a, matrix metalloproteinases (MMPs), integrins, IL-6,and IL-8 within the tumor microenvironment [11-18],which significantly decrease PDT. Nonsteroidal anti-inflammatory drugs (NSAIDs) exhibit antiangiogenic properties by blocking cyclooxygenase (COX) pathways that lead to the production of prostaglandins, which are powerful angiogenic mediators. Preclinical investigations indicate that combining PDT with targeted therapies directed at attenuating the pro-survival actions of the tumor microenvironment can enhance the therapeutic potential of PDT[11-15].
Objectives
The objective of this part of experiment is to investigate the effect of different combinded ways of aspirin and PDT on the human esophageal carcinoma cell line Eca-109 and its mechanism in vitro.
1、Study on the effect of different concentrations and light time of xibofen-PDT on proliferation and COX-2 expression of Eca-109 cell in vitro.
Methods:
1) There are four groups which contain only light, only Xibofen, PDT, and control respectively.
2) Inhibitory effect of Eca-109 cell Proliferation was detected at different concentrations (0ug/ml,2.5 ug/ml,5ug/ml, 10ug/ml,15ug/ml,20 ug/ml) and light time (0s,30s,60s,90s) of xibofen-PDT with MTT.
3) COX-2 andβ-actin expression of Eca-109 cell was detected at different concentrations (0 ug/ml,2.5 ug/ml,10 ug/ml,20 ug/ml) and light time (0s, 30s,60s,90s) of xibofen-PDT with In cell western blot.
4) The results of experiment express by the way of mean±SD. Survival rate of each group apply the way of General linear Models of Univariate and one-way ANOVA. We defined statistical significance as P<0.05.
Results:
1) The effects of xibofen-PDT on proliferation:MTT assay showed that Eca-109 cell proliferation was significant inhibited by xibofen-PDT in time and dose dependent manner.
2) The cell survival rate and the expression rate of COX-2/β-actin between different Xibofen incubating concentration and three different light time and under the same light time with different incubating concentrations are significant different and also significant interaction, all P<0.01.The cell survival rate is the lowest as xibofen concentration is 20.0ug/ml and the light time was 90s. The expression rate of COX-2/p-actin is the highest as xibofen concentration is 10.0ug/ml and the light time was 90s.
Conclusions:
1) The study showed xibofen-PDT can inhibited the proliferation of Eca-109 cells and induct apoptosis of them.
2) The study also showed that related molecular mechanism of treating Eca-109 cells using xibofen-PDT were up-regulation of the expression of COX-2.
2、Study on the effect of different concentrations of ASA on proliferation and COX-2 expression of Eca-109 cell in vitro
Methods:
1) There are two groups which contain ASA and control respectively.
2) Inhibitory effect of Eca-109 cell Proliferation were detected at different concentrations(0 mmol/L,2.5 mmol/L,5 mmol/L,7.5mmol/L, 10mmol/L, 12.5mmol/L) of ASA with MTT.
3) COX-2 andβ-actin expression of Eca-109 cell was detected at different concentrations (0 mmol/L,2.5 mmol/L,5 mmol/L,12.5 mmol/L) of ASA with In cell western blot.
4) The results of experiment express by the way of mean±SD. Survival rate of each group apply the way of one-way ANOVA. We defined statistical significance as P<0.05.
Results:
1) The effects of ASA on proliferation:MTT assay showed that Eca-109 cell proliferation was significant inhibited by ASA in time dependent manner.
2) The expression rate of COX-2/β-actin descent as the ASA incubating concentration increase, this means that as the incubating concentration increase,the expression rate of COX-2/β-actin of Eca-109 cells decrease.
Conclusions:
1) The study showed aspirin can inhibited the proliferation of Eca-109 cells,and induct apoptosis of them.
2) The study also showed that related molecular mechanism of treating Eca-109 cells using aspirin were down-regulation of the expression of COX-2.
3、Study on the effect of different concentrations and joint modals of xibofen-PDT with ASA on proliferation and COX-2 expression of Eca-109 cell in vitro
Methods:
1) There are four groups which contain PDT,PDT beofre ASA, ASA beofre PDT,and control respectively.This part is on the role of the ASA concentration(5 mmol/L) IC50 effect on Proliferation of Eca-109 cells.
2) Inhibitory effect of Eca-109 cell Proliferation was detected at different concentrations (2.5ug/ml,5ug/ml, 10ug/ml,15ug/ml, 20ug/ml) and different joint modals (PDT,PDT beofre ASA,ASA beofre PDT) of xibofen-PDT and ASA with MTT.
3) COX-2 andβ-actin expression of Eca-109 cell was detected at different concentrations (2.5 ug/ml,10 ug/ml,20 ug/ml) and different joint modals (PDT,PDT beofre AS A, AS A beofre PDT) of xibofen-PDT and ASA with In cell western blot.
4) The results of experiment express by the way of mean±SD. Survival rate of each group apply the way of General linear Models of Univariate and one-way ANOVA. We defined statistical significance as P<0.05.
Results:
The cell survival rate and the expression rate of COX-2/β-actin between different Xibofen incubating concentrations and different joint modals、under the same joint modals with different incubating concentrations and under the same incubating concentration with different joint modals are significant different and also significant interaction, all P<0.01.The cell survival rate is the lowest as xibofen concentration is 20.0ug/ml and the PDT before ASA. The expression rate of COX-2/β-actin is the lowest as PDT before ASA.
Conclusions:
1) The study showed ASA can Enhance the killing effect of Xibofen-PDT on the Eca-109 cells.
2) The study also showed that related molecular mechanism of ASA enhancing the killing effect of Xibofen-PDT on the Eca-109 cells were down-regulation of the expression of COX-2.
3) Xibofen-PDT with ASA can reduce the recurrence rate of PDT.
光动力疗法(photodynamic therapy, PDT)是利用激光的光化学原理,通过特定波长的激光激活肿瘤组织内滞留的光敏剂,与肿瘤组织内的氧发生作用,产生一些活性氧分子(radical oxygen species, ROS),并通过氧化作用攻击细胞结构,造成细胞膜或蛋白质的氧化损伤,当氧化损伤的积累超过一定的阈值时,细胞便开始死亡。另外,还可造成肿瘤微血管急性损伤。光动力疗法的作用基础是光动力效应,这是一种有氧分子参与的伴随生物效应的光敏化反应。光敏剂、激光和氧分子构成光动力效应的三要素。目前临床上应用最广泛的光敏剂为血卟啉衍生物。因此本研究采用喜泊分,一种中国生产的血卟啉衍生物,已在临床上用于多种肿瘤的光动力治疗,如肝癌、食管癌、胃癌、膀肤癌、妇科肿瘤、皮肤癌、头颈部肿瘤等。由于630nm为喜泊分治疗的最适波长,因此我们选用波长为630nm的DIOMED630PDT系统。
在光动力剂量学研究中,不但要考虑光敏剂和照射光的质和量,还要考虑光敏剂的体内分布特性和受照射组织的光学特性,同时兼顾组织中血流变化对氧含量的影响。光动力剂量的简要表达式为：D=(1／α)Cφt。式中,D是光动力剂量；C是光敏剂浓度；中是光能流率；t是光照时间；α是组织对光敏毒性的耐受参数,它决定于组织的生物特性和组织内的氧浓度。然而,在临床应用中,光敏剂的剂量和光照剂量是光动力剂量学的两个最重要可控参量。本研究采用的光能流率(2000mw)是固定的,而光敏剂浓度和光照时间是可变的,从而探讨不同光动力剂量对细胞的体外效应。
PDT目前已开始应用于多种恶性肿瘤的治疗,日渐成为肿瘤治疗新手段。它与常规的手术、化疗等疗法相比,PDT具有创伤小,毒性小,选择性、适用性、重复性好,可姑息治疗,可消灭隐性病灶,可保护容貌及重要器官等优点。
食管癌是人类常见的消化道恶性肿瘤,而我国是高发国家。随着新型光敏剂的发展及半导体激光发生系统的开发,PDT在食管癌尤其是早期食管癌及癌前病变的治疗上已取得较大进展。但临床治疗中发现,肿瘤光动力治疗后近期疗效较好,但肿瘤复发率较高。PDT引起的直接肿瘤细胞杀伤、微血管损伤、缺氧、炎症与血管内皮生长因子(VEGF)、环氧化酶-2(COX-2)、金属蛋白酶(MMP)、细胞因子和前列腺素的表达增高有关。这些分子的增加减弱了PDT的效应。环氧化酶(COX)是前列腺素代谢中重要的酶,前列腺素是血管生成的重要调节剂。非甾体抗炎药物阿司匹林(aspiprin, ASA)通过阻断环氧合酶COX途径表现出抗血管生成功能。改变PDT介导的肿瘤微环境可调节PDT疗效。
研究目的
本实验以血卟啉衍生物喜泊分为光敏剂,波长630nm的DIOMED630PDT系统为激发光源,光敏剂浓度和光照剂量为两个可变因素,观察喜泊分-PDT、喜泊分-PDT联合ASA体外对人食管鳞癌细胞Eca-109的增殖和COX-2蛋白表达的影响。
研究方法、结果和结论
一、不同喜泊分浓度和光照剂量介导的PDT体外对Eca-109细胞增殖和COX-2蛋白表达的影响
(一)、方法
1、实验分为4组：单纯喜泊分组,单纯光照组,PDT组,对照组。
2、MTT法检测不同喜泊分浓度(0 ug/ml,2.5 ug/ml,5 ug/ml,10 ug/ml, 15 ug/ml,20 ug/ml)和不同光照时间(0s,30s,60s,90s)下的喜泊分-PDT对食管癌Eca-109细胞的增殖抑制作用。
3、In cell western blot技术检测不同喜泊分浓度(0 ug/ml,2.5 ug/ml,10 ug/ml,20 ug/ml)和不同光照时间(0s,30s,60s,90s)下的喜泊分-PDT作用于食管癌Eca-109细胞后的COX-2蛋白的表达情况。
4、统计学处理采用Spss13.0统计软件,实验结果以“均数±标准差”表示,统计方法采用析因设计的方差分析,多组间比较采用单向方差分析。限定p<0.05为差异有统计学意义。
(二)结果
1.不同浓度和不同光照时间下的喜泊分-PDT对Eca-109细胞存在显著的增殖抑制,并且随着喜泊分浓度和光照时间的增加,喜泊分-PDT对细胞增殖的抑制作用越强。
2.喜泊分-PDT作用后Eca-109细胞的COX-2蛋白相对表达量较对照组增高并且随着喜泊分浓度和光照时间的增加,Eca-109细胞的COX-2蛋白相对表达量呈增高的趋势。
(三)结论
1.本研究发现喜泊分-PDT可明显抑制人食管癌Eca-109细胞增殖并诱导其凋亡;
2.喜泊分-PDT可上调人食管癌Eca-109细胞COX-2蛋白表达。
二、不同浓度ASA对Eca-109细胞增殖和COX-2蛋白表达的影响
(一)、方法
1、实验分为2组：单用ASA组,对照组。
2、MTT法检测不同浓度ASA (0 mmol/L,2.5 mmol/L,5 mmol/L, 7.5mmol/L,10mmol/L,12.5 mmol/L)对食管癌Eca-109细胞的增殖影响。
3、In cell western blot技术检测不同浓度ASA(0 mmol/L,2.5 mmol/L, 5 mmol/L,12.5 mmol/L)对食管癌Eca-109细胞COX-2蛋白表达影响。
4、统计学处理采用Spss13.0统计软件,实验结果以“均数±标准差”表示,统计方法采用单向方差分析。限定p<0.05为差异有统计学意义。
(二)结果
1.阿司匹林对Eca-109细胞增殖的抑制作用：采用MTT法检测细胞增殖抑制率,描绘曲线图可知,经不同浓度阿司匹林处理后的Eca-109细胞生长缓慢,表现出明显的生长抑制,并呈作用浓度依赖性,而对照组细胞生长良好；
2.阿司匹林对Eca-109细胞COX-2表达的影响：阿司匹林作用于Eca-109细胞后,细胞中的COX-2蛋白相对表达量下调。
(三)结论：
1.本研究发现阿司匹林可抑制人食管癌Eca-109细胞增殖；
2.阿司匹林可下调Eca-109细胞的COX-2蛋白表达。
三、喜泊分-PDT联合ASA体外对Eca-109细胞增殖和COX-2表达的影响
(一)方法
1.实验分组分为4组：PDT组,PDT后ASA组,PDT前ASA组,对照组。根据ASA对Eca-109细胞作用的MTT实验结果,选择IC50的1个ASA浓度,即ASA浓度5 mmol/L,进行该项实验。
2.MTT法检测5种不同喜泊分浓度(2.5 ug/ml,5 ug/ml,10 ug/ml, 15 ug/ml,20 ug/ml)介导的PDT单独和联合ASA (PDT后ASA组,PDT前ASA组)对食管癌Eca-109细胞的增殖抑制作用。
3. In cell western blot技术检测3种不同喜泊分浓度(2.5 ug/ml,10 ug/ml,20 ug/ml)介导的PDT单独和联合ASA (PDT后ASA组,PDT前ASA组)下食管癌Eca-109细胞COX-2蛋白的表达情况。
4.统计学处理采用Spssl3.0统计软件,实验结果以“均数±标准差”表示,统计方法采用析因设计的方差分析,多组间比较采用单向方差分析。。p<0.05为差异有统计学意义。
(二)结果
1.喜泊分-PDT联合ASA均对Eca-109细胞存在明显的生长抑制。随着喜泊分浓度的升高,细胞的抑制率呈升高的趋势。PDT后ASA组(均数为0.79)的抑制率高于单用PDT组(均数为0.40)和PDT前ASA组(均数为0.41)。以细胞抑制率绘制细胞生存曲线,结果示,当喜泊分2.5ug/ml、作用方式为单用PDT时,细胞抑制率最低(均数=0.06),当喜泊分20ug/ml、作用方式为PDT后ASA时,细胞抑制率最高(均数=0.97)。
2.ASA联合喜泊分-PDT后降低细胞的COX-2蛋白相对表达量。PDT后ASA组的COX-2蛋白相对表达量最低(均数=0.06),单用PDT组的COX-2蛋白相对表达量最高(均数=0.11)。
(三)结论
1.本研究发现喜泊分-PDT联合ASA可增强喜泊分-PDT对Eca-109细胞增殖的抑制作用；
2.喜泊分-PDT联合ASA增强喜泊分-PDT对Eca-109细胞增殖的抑制作用的机制可能与下调COX-2表达有关；
3.ASA联合人食管癌喜泊分-PDT可能会减少PDT后的复发率。
Background:
Photodynamic Therapy (PDT) is a kind of photochemistry therapy. The PDT mechanism is that the resident photosensitizer in tumor tissue is activated by certain wavelength laser and takes action on oxygen in tumor tissue and produce some radical oxygen species (ROS), then attack the tumor cell through peroxidization. This may make the cellular membrane or oxidative protein damage. And the tumor cell will apoptosis after the oxidative damage get to the leak point. Also, it can damage to the tumor capillary. As contrast to conventional therapy such as operation and chemotherapy, PDT has the advantages as minimally invasive、little toxicity、high selectivity、wide applicability、well repeatability、be used as pamper treatment、maintain features and important organ function and can cure precancerous lesion. Nowadays, PDT has been used in curing some malignant tumor, and gradually to become a new method for tumor therapy.
Esophageal carcinoma is a kind of common human alimentary canal tumor and it has high death rate in our country. As the development of the new type photosensitizers and semiconductor laser generator, PDT has made great progression in curing esophageal carcinoma especially in the earlier period and precancerous lesion. But in clinical curing, we find it is common that the photodynamic therapy effect is instability. Some human esophageal carcinoma patients can be clinical curative, while some is negative. Several laboratories have also shown that PDT can induce expression and/or activation of additional pro-angiogenic molecules including COX-2 and prostaglandins,TNF-a, matrix metalloproteinases (MMPs), integrins, IL-6,and IL-8 within the tumor microenvironment [11-18],which significantly decrease PDT. Nonsteroidal anti-inflammatory drugs (NSAIDs) exhibit antiangiogenic properties by blocking cyclooxygenase (COX) pathways that lead to the production of prostaglandins, which are powerful angiogenic mediators. Preclinical investigations indicate that combining PDT with targeted therapies directed at attenuating the pro-survival actions of the tumor microenvironment can enhance the therapeutic potential of PDT[11-15].
Objectives
The objective of this part of experiment is to investigate the effect of different combinded ways of aspirin and PDT on the human esophageal carcinoma cell line Eca-109 and its mechanism in vitro.
1、Study on the effect of different concentrations and light time of xibofen-PDT on proliferation and COX-2 expression of Eca-109 cell in vitro.
Methods:
1) There are four groups which contain only light, only Xibofen, PDT, and control respectively.
2) Inhibitory effect of Eca-109 cell Proliferation was detected at different concentrations (0ug/ml,2.5 ug/ml,5ug/ml, 10ug/ml,15ug/ml,20 ug/ml) and light time (0s,30s,60s,90s) of xibofen-PDT with MTT.
3) COX-2 andβ-actin expression of Eca-109 cell was detected at different concentrations (0 ug/ml,2.5 ug/ml,10 ug/ml,20 ug/ml) and light time (0s, 30s,60s,90s) of xibofen-PDT with In cell western blot.
4) The results of experiment express by the way of mean±SD. Survival rate of each group apply the way of General linear Models of Univariate and one-way ANOVA. We defined statistical significance as P<0.05.
Results:
1) The effects of xibofen-PDT on proliferation:MTT assay showed that Eca-109 cell proliferation was significant inhibited by xibofen-PDT in time and dose dependent manner.
2) The cell survival rate and the expression rate of COX-2/β-actin between different Xibofen incubating concentration and three different light time and under the same light time with different incubating concentrations are significant different and also significant interaction, all P<0.01.The cell survival rate is the lowest as xibofen concentration is 20.0ug/ml and the light time was 90s. The expression rate of COX-2/p-actin is the highest as xibofen concentration is 10.0ug/ml and the light time was 90s.
Conclusions:
1) The study showed xibofen-PDT can inhibited the proliferation of Eca-109 cells and induct apoptosis of them.
2) The study also showed that related molecular mechanism of treating Eca-109 cells using xibofen-PDT were up-regulation of the expression of COX-2.
2、Study on the effect of different concentrations of ASA on proliferation and COX-2 expression of Eca-109 cell in vitro
Methods:
1) There are two groups which contain ASA and control respectively.
2) Inhibitory effect of Eca-109 cell Proliferation were detected at different concentrations(0 mmol/L,2.5 mmol/L,5 mmol/L,7.5mmol/L, 10mmol/L, 12.5mmol/L) of ASA with MTT.
3) COX-2 andβ-actin expression of Eca-109 cell was detected at different concentrations (0 mmol/L,2.5 mmol/L,5 mmol/L,12.5 mmol/L) of ASA with In cell western blot.
4) The results of experiment express by the way of mean±SD. Survival rate of each group apply the way of one-way ANOVA. We defined statistical significance as P<0.05.
Results:
1) The effects of ASA on proliferation:MTT assay showed that Eca-109 cell proliferation was significant inhibited by ASA in time dependent manner.
2) The expression rate of COX-2/β-actin descent as the ASA incubating concentration increase, this means that as the incubating concentration increase,the expression rate of COX-2/β-actin of Eca-109 cells decrease.
Conclusions:
1) The study showed aspirin can inhibited the proliferation of Eca-109 cells,and induct apoptosis of them.
2) The study also showed that related molecular mechanism of treating Eca-109 cells using aspirin were down-regulation of the expression of COX-2.
3、Study on the effect of different concentrations and joint modals of xibofen-PDT with ASA on proliferation and COX-2 expression of Eca-109 cell in vitro
Methods:
1) There are four groups which contain PDT,PDT beofre ASA, ASA beofre PDT,and control respectively.This part is on the role of the ASA concentration(5 mmol/L) IC50 effect on Proliferation of Eca-109 cells.
2) Inhibitory effect of Eca-109 cell Proliferation was detected at different concentrations (2.5ug/ml,5ug/ml, 10ug/ml,15ug/ml, 20ug/ml) and different joint modals (PDT,PDT beofre ASA,ASA beofre PDT) of xibofen-PDT and ASA with MTT.
3) COX-2 andβ-actin expression of Eca-109 cell was detected at different concentrations (2.5 ug/ml,10 ug/ml,20 ug/ml) and different joint modals (PDT,PDT beofre AS A, AS A beofre PDT) of xibofen-PDT and ASA with In cell western blot.
4) The results of experiment express by the way of mean±SD. Survival rate of each group apply the way of General linear Models of Univariate and one-way ANOVA. We defined statistical significance as P<0.05.
Results:
The cell survival rate and the expression rate of COX-2/β-actin between different Xibofen incubating concentrations and different joint modals、under the same joint modals with different incubating concentrations and under the same incubating concentration with different joint modals are significant different and also significant interaction, all P<0.01.The cell survival rate is the lowest as xibofen concentration is 20.0ug/ml and the PDT before ASA. The expression rate of COX-2/β-actin is the lowest as PDT before ASA.
Conclusions:
1) The study showed ASA can Enhance the killing effect of Xibofen-PDT on the Eca-109 cells.
2) The study also showed that related molecular mechanism of ASA enhancing the killing effect of Xibofen-PDT on the Eca-109 cells were down-regulation of the expression of COX-2.
3) Xibofen-PDT with ASA can reduce the recurrence rate of PDT.
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