5-氨基乙酰丙酸—光动力疗法对人食管癌效应及其机制研究
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摘要
研究背景
食管癌是人类常见的消化道恶性肿瘤,我国是全世界该肿瘤发病率及死亡率的高发国家。其死亡率在我国恶性肿瘤中居第四位。其中约90%的食管癌病理类型为鳞癌。而在西方国家,该发病率亦日益增加,但病理类型主要为Barrett's食管发展而来的腺癌。由于大部分患者确诊时已是晚期,甚至存在远处转移或食管梗阻,使这些患者失去了手术的最佳时机,甚至不能耐受放化疗。因此,一种新的治疗手段—光动力疗法(PDT),成为这些患者的迫切需求。目前,光动力治疗已经被证实是食管癌的一种有前景的治疗模式。
光动力治疗是通过定位在肿瘤组织的光敏剂在特定波长的激光照射下产生单态氧及其他的活性氧成分,从而导致该部位的肿瘤细胞及组织的光损伤。与常规的手术、放化疗等疗法相比,光动力疗法具有创伤小,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,目前已开始应用于多种恶性肿瘤的治疗,并日渐成为肿瘤治疗的新手段。在食管癌的治疗上,光动力疗法最早是应用于食管癌的癌前病变如Barrett's食管及早期食管癌的治疗。现已开始应用于中晚期食管癌的减瘤治疗。通过选择最佳的光动力治疗参数,早期食管癌可达到根治而晚期食管癌亦可达到较好的治疗效果。但于临床治疗中,食管癌光动力治疗的最佳参数仍未明确,因此其疗效仍不稳定。
光动力疗法对肿瘤细胞的损伤主要通过凋亡和／坏死两种死亡方式,而哪种方式为主则主要取决于肿瘤细胞的类型,激光照射剂量,光敏剂的浓度及其亚细胞定位等。目前认为,定位于线粒体的光敏剂,如5-氨基乙酰丙酸(ALA),可导致细胞以凋亡为主的死亡方式,并导致其相关凋亡基因及蛋白的表达发生变化。其中,Bcl-2家族在肿瘤细胞凋亡过程中起着非常重要的作用。以往研究表明,通过上调或下调其表达可增强光动力效应。但其在ALA-PDT食管癌光动力治疗中的作用机制尚不明确。
5-氨基乙酰丙酸是一种二代光敏剂,已于2000年被美国FDA批准用于临床实验治疗。它是一种内源性光敏剂,是活细胞血红素的前体,可产生在光动力治疗中发挥光敏剂作用的血卟啉-Ⅸ。这种光敏剂相比Photofrin其光毒性持续时间较短(大约24小时),远远短于现在临床广泛应用的Photofrin。
因此,本课题选择二代光敏剂ALA,通过研究ALA-PDT在食管癌的体内外效应探讨其最佳作用参数,为临床光动力治疗最佳参数的选择提供一定依据。通过研究ALA-PDT对食管癌细胞凋亡、细胞周期及线粒体膜电位的影响、ALA的亚细胞定位以及一些相关基因及蛋白的变化以探讨其主要作用机制。
研究目的
1.通过研究ALA-PDT在食管癌的体内外效应探讨其最佳作用参数；
2.通过研究ALA的亚细胞定位、ALA-PDT对食管癌细胞的凋亡、细胞周期及线粒体膜电位的影响初步探讨ALA-PDT在食管癌的凋亡机制；
3.通过研究ALA-PDT对食管癌细胞凋亡相关基因Bcl-2、Bax、Bcl-xL, Bad、Bak-1、FADD、Bid表达的变化探讨其可能作用的主要凋亡通路；
4.通过研究Bcl-2、cytochrome c (cytc)的蛋白表达变化以探讨ALA-PDT凋亡通路的终末效应机制。
研究方法及内容
1.MTT法检测ALA-PDT对食管癌细胞Eca-109的光动力效应：实验分为对照组和光动力治疗组,光动力治疗组给予不同浓度的ALA (0.01mM,0.05mM, 0.1mM,0.25mM,0.5mM,1mM,2mM)和不同孵育时间(分别孵育4 h、6 h、12 h、24 h)后于饱和光剂量下((30J/cm2)进行光照,同时设立另一治疗组,给予不同浓度的ALA (0.01mM,0.05mM,0.1mM,0.25mM,0.5mM, 1mM,2mM)后于三种不同光剂量(10J/cm2,30J/cm2,50J/cm2)下进行光照,光源采用DIOMED 630PDT系统,光照后孵育24小时,通过MTT法检测细胞生存率。
2.ALA-PDT对人食管癌细胞Eca-109荷瘤裸鼠的增殖抑制：建立人食管鳞癌裸鼠种植瘤模型,每3天监测肿瘤瘤径,待肿瘤大小为150-350mm3时,开始给予行光动力治疗。实验分对照组、ALA-PDT组(给予光敏剂及光照),单纯光照组(只给予光照)、单纯光敏剂组(只给予光敏剂)(每组6只),ALA-PDT组及单纯光敏剂组按100 mg/Kg腹腔给药,给药4小时后,ALA-PDT组与单纯光照组开始进行光动力治疗。光源采用DIOMED 630PDT系统,按120J/cm2光剂量进行光照,光照后监测肿瘤体积及观察肿瘤表面皮肤变化,并于治疗后第21天处死裸鼠,取瘤组织进行H&E染色的检测。
3.荧光共聚焦显微镜检测光敏剂及线粒体探针在细胞内的定位：予细胞加入ALA(终浓度为2mM)孵育4小时后予线粒体绿色荧光探针(MTG)(终浓度为100nM)避光孵育30分钟后于荧光共聚焦显微镜FV1000检测其光敏剂及线粒体探针的亚细胞定位。
4.AnnexinV-FITC/PI双标检测ALA-PDT对Eca-109细胞凋亡的影响：实验分为对照组和光动力治疗组。光动力治疗组加入ALA并使其终浓度分别为0.05mM和0.5mM,避光培养6小时后于630nm Diomed光动力治疗系统下进行光照(功率200mW/cm2,照光150 s,光剂量30J/cm2)。光照24小时后收集细胞,加入AnnexinV-FITC (5ul)和PI(5ul)避光孵育10分钟后于流式细胞仪上检测其凋亡情况。
5.PI检测ALA-PDT对Eca-109细胞周期的影响：实验分为对照组和光动力治疗组。光动力治疗组加入ALA并使其终浓度分别为0.05mM、0.1mM和0.5mM,避光培养6小时后于630nm Diomed光动力治疗系统下进行光照(功率200mW/cm2,照光150 s,光剂量30J/cm2)。光照24小时后收集细胞,70%的酒精于4℃固定至少12小时后加入PI(终浓度为1mg/ml),避光孵育10分钟后于流式细胞仪上检测其细胞周期情况。
6. Hoechst 33342/PI检测ALA-PDT作用后Eca-109细胞凋亡情况：实验分为对照组和光动力治疗组。光动力治疗组加入ALA并使其终浓度分别为0.05mM、0.1mM和0.5mM,避光培养6小时后于630nm Diomed光动力治疗系统下进行光照(功率200mW/cm2,照光150 s,光剂量30J/cm2)。光照24小时后收集细胞,加入Hoechst 33342及PI,避光孵育15分钟后于荧光显微镜BX51观察其凋亡情况。
7、罗丹明123染色检测ALA-PDT对Eca-109细胞线粒体膜电位的影响：实验分为对照组和光动力治疗组。光动力治疗组加入ALA并使其终浓度分别为0.05mM、0.1mM和0.25mM,避光培养6小时后于630nm Diomed光动力治疗系统下进行光照(功率200 mW/cm2,照光150 s,光剂量30J/cm2)。光照1小时后收集细胞,加入罗丹明123染液,避光孵育30分钟后用培养基重悬细胞,继续孵育60min后于荧光显微镜BX51观察。
8.实时定量PCR法检测Bcl-2、Bax、Bad、Bcl-xL、Bak-1、FADD、Bid的基因表达变化：实验分为对照组和ALA-PDT组。治疗组细胞给予ALA(终浓度为0.25mM)孵育6小时后于630nm Diomed光动力治疗系统下进行光照(功率200mW/cm2,照光150 s,光剂量30J/cm2)。于光照后24 h收集细胞,并提取细胞总RNA,逆转录为cDNA后采用SYBR荧光定量试剂于ABI7500PCR仪上检测Bcl-2、Bax、Bad、Bcl-xL、Bak-1、FADD、Bid的基因表达变化情况。
9.Western Blotting法检测Bcl-2、cytc(胞浆)蛋白含量的变化：实验分为对照组和ALA-PDT组。治疗组细胞给予ALA(终浓度为0.25mM)孵育6小时后进行于630nm Diomed光动力治疗系统下进行光照(功率200mW/cm2,照光150 s,光剂量30J/cm2)。于光照后24 h收集细胞,并分别提取细胞总蛋白和胞浆蛋白,采用Western Blotting法检测Bcl-2、cytc蛋白含量的变化。
10.统计分析：所有数据采用spss 13.0统计软件进行统计处理。实验结果以“均数±标准差”表示。细胞生存率的分析采用析因设计的方差分析。各组体积的比较采用重复测量数据的方差分析。细胞凋亡、细胞周期以及细胞生存率、肿瘤体积的多组间比较均采用单向方差分析,多重比较在方差齐性时采用LSD检验,方差不齐时采用近似F检验(Welch方法)及多重比较的Dunnett'sT3方法。对照组与治疗组之间基因及蛋白表达相对量的差异分析均采用独立样本t检验。P值<0.05时其差异有统计学意义。
结果
1.ALA-PDT对Eca-109细胞有显著的增殖抑制。不同光剂量及不同浓度其生存率间均有显著差异(P均<0.01)。同一光剂量下不同浓度其生存率间均有显著差异(P均<0.01),而同一浓度下不同光剂量的生存率间,则除了浓度为0.01mM及0.05mM间无明显差异外,其余均有显著差异(P均<0.05)。当孵育浓度由0.5mM增至2.0mM,光剂量由30J/cm2增加至50J/cm2时不能明显提高杀伤效应。当孵育浓度为0.5mM,光照剂量为50J/cm2(孵育时间为24 h)时其杀伤效应最强。不同孵育时间及不同浓度的生存率间均有显著差异(P均<0.01)。同一孵育时间不同浓度其生存率间均有显著差异(P均<0.01),而同一浓度下不同孵育时间的生存率,则除了浓度为0.01mM、0.05mM、0.1mM及1.0mM间无显著差异外,其余均有显著差异(P均<0.05)。当孵育浓度为2.0mM,孵育时间为24小时(光剂量为30J/cm2)其杀伤效应最强。当孵育浓度由0.5mM增至2.0mM,孵育时间由6小时增至24小时不能明显提高杀伤效应。
2.四个组治疗前后的体积间有显著差异(P<0.01)。各个组治疗前后不同时间点体积间有显著差异(P<0.01))。四个组治疗前的体积之间无显著差异(P>0.05),治疗后四个时间点体积之间均有显著差异(P均<0.05)。ALA-PDT组治疗后2周体积增长较缓慢,治疗后第7天体积抑制最明显,第14天增长速度开始增快,但治疗后四个时间点体积仍明显小于其余三组。
3.H&E染色于光镜下对照组、单纯光照组及单纯光敏剂组均可见大量异型瘤细胞,可见癌巢,血管内皮细胞完整。光动力治疗组则可见均质红染坏死物质,并有淋巴细胞侵润。
4.荧光共聚焦显微镜下可见,线粒体探针与ALA-PpⅨ存在共定位,即光敏剂ALA-PpⅨ部分定位于细胞内线粒体上。
5.FCM结果示：对照组和浓度为0.05mM和0.5mM ALA-PDT组间的凋亡率有显著差异(P<0.05)。当ALA为极低浓度(0.05mM)时,其凋亡率与对照组间无显著差异(P>0.05)。当其浓度提高为0.5mM时,其凋亡率间有显著差异(P<0.01)。
6.细胞周期分析结果可见,对照组和0.05mM、0.1mM和0.5mM ALA-PDT组G0/G1期,S期,G2/M期的比率间有显著差异(P均<0.05)。G0/G1期随着浓度增加比率呈上升趋势,而G2/M期,S期则相反,呈下降趋势。
7.荧光显微镜下可见,Hoechst 33342/PI双染的ALA-PDT组细胞出现明显的变化(染色质凝聚,核皱缩,高蓝荧光的凋亡细胞)及细胞坏死(高红荧光)。而对照组未见明显变化。
8、荧光显微镜下可见,罗丹明123染色的ALA-PDT组均可见强黄绿色荧光,且荧光强度随着ALA浓度增加而增强,而对照组细胞荧光非常弱甚至没有荧光。
9.Bcl-2、Bcl-xL、FADD在ALA-PDT作用于Eca-109细胞24 h后其表达均均显著下调,而Bax基因的表达则显著上调,与对照组相比,其差异均有统计学意义(P均<0.05)。Bad、Bid、Bak-1表达均轻微上调,但与对照组相比其差异无统计学意义(P均>0.05)。
10.Bcl-2蛋白在ALA-PDT作用于Eca-109细胞24 h后其表达显著下调,而胞浆中cytc蛋白的表达则显著上调,且与对照组相比,其差异均有统计学差异(P均<0.05)
结论
1.ALA-PDT对人食管癌细胞Eca-109有显著的增殖抑制作用。在特定光源状态下,光照剂量、光敏剂孵育浓度及孵育时间是Eca-109细胞体外光动力效应的主要影响因素。在特定孵育时间下(孵育时间为24 h),当孵育浓度为0.5mM,光照剂量为50J/cm2时其杀伤效应最强。而当孵育浓度由0.5mM增至2.0mM时,光剂量由30J/cm2增加至50J/cm2时杀伤效应达到平台期。特定光剂量下(光剂量为30J/cm2),当孵育浓度为2.0mM,孵育时间为24小时其杀伤效应最强。而当孵育浓度由0.5mM增至2.0mM时,孵育时间由6小时增至24小时其杀伤效应亦达到平台期。以上结果表明,要达到ALA-PDT对人食管癌细胞Eca-109细胞的最佳杀伤效应,需选择最佳作用参数。
2.ALA-PDT对Eca-109荷瘤裸鼠有显著增殖抑制作用。其效应在治疗后第7天最明显,第14天开始增长加快,因此治疗后第14天可作为复照的参考时间。
3.H&E染色、Hoechst 33342/PI染色从组织学的形态上进一步证明了ALA-PDT对人食管癌细胞的杀伤作用。
4.ALA-PpIX部分定位于线粒体上,ALA-PDT使Eca-109细胞线粒体膜电位发生改变可能为其使细胞死亡的机制之一。
5.ALA-PDT可使Eca-109细胞发生凋亡；随着ALA浓度增加,凋亡率呈上升趋势。
6.ALA-PDT可使Eca-109细胞明显阻滞于G0/G1期,且随着ALA浓度增加呈上升趋势。ALA-PDT对细胞周期的阻滞可能为其凋亡作用机制之一
7.外源性通路不是ALA-PDT诱导Eca-109细胞凋亡的主要通路,其主要凋亡通路为线粒体诱导的凋亡通路。Bcl-2基因及蛋白的表达在PDT后24 h均显著下调,表明Bcl-2可能为该凋亡通路的靶基因之一。
8.释放cytc进而诱导细胞凋亡可能为ALA-PDT诱导Eca-109细胞凋亡的线粒体通路终末效应之一。
Background:
Esophageal cancer is one of the most common cancers. The morbidity and mortality of esophageal cancer in China is very high, and it ranks the fourth killer among all cancer death in China. More than 90% of esophageal cancer in China is squamous cell carcinoma. The incidence of this cancer is increasing rapidly in the Western countries, however, there are considerably many adenocarcinoma of esophagus, most of which are developed from Barrett's esophagus. A great many of patients with this cancer are diagnosed in an advanced stage, with distant metastasis and esophageal obstruction. Many patients often lost the optimal chance for surgery, or even could not undergo chemotherapy or radiotherapy. It is highly demanded for an effective medical treatment such as photodynamic therapy (PDT). PDT has been found to be a promising treatment modality for esophageal cancer.
PDT process involves the photosensitiser localized in tumor tissue and irradiation of the tumor site by visible light of specific wavelength, to produce singlet oxygen and other reactive oxygen species, leading to photodamage in cancer cells and cancer destruction. 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. The first PDT used in esophageal carcinoma treatment was to cure esophageal precancerous lesion such as Barrett's of esophagus and early esophageal cancer, and now began to be used in diminish treatment of advanced stage. By properly chosen optimal parameters of PDT, complete cure of early esophageal cancer and a good therapeutic result in advanced stage of the disease can be frequently achieved. However, in clinical application, the optimal parameters have not been established that the effects for esophageal patients were instability.
The tumor cell death by PDT is induced via apoptosis and/or necrosis, depending on various conditions, such as tumor cell type, laser irradiation dose, photosensitizer concentration and subcellular localization, et.al. It has been reported that PDT with mitochondria-localizing photosensitizers, such as 5-aminolevulinic acid (ALA), can induce rapid cell death via apoptosis and change the expression of apoptotic related gene and protein. Expression of some genes related with apoptosis pathway, such as Bcl-2 family, is involved and plays an important role in this photodynamic process. It has been reported that up-regulation or down-regulation some genes of Bcl-2 family can enhance the effect of PDT. However, their roles in ALA-PDT for esophageal cancer have still not been fully investigated.
ALA is one of the second-generation photosensitizers, and has been approved to be used in clinical trial by the USA FDA since 2000. ALA is an endogenous material and a precursor of heme in living cells, thus the induced protoporphyrinⅨ(PpⅨ) can be employed as a photosensitizer in PDT. The ALA-produced PpⅨ(ALA-PpⅨ) induces a short-lasting phototoxicity about 24 hours, much shorter compared with that induced by photofrin which was used generally in clinical treatment。
Thus, we choosed the second-generation photosensitizers ALA, and investigated the effects of ALA-PDT on esophageal cancer cells in vitro and vivo, to explore optimal parameters in PDT for esophageal cancer and provided some evidence for PDT optinal clinical premeters established. Also, we investigated the role of cell apoptosis, cell cycle and mitochondria membrane potential changes, subcellular localization of ALA-PpⅨ, and some molecular changes to understand the mechanism of ALA-PDT for esophageal cancer.
Objectives:
1. Investigate ALA-PDT effect in the human esophageal carcinoma both in intro and vivo to explore optimal parameters in PDT for esophageal cancer;
2. Investigate the subcellular localization of ALA, the role of cell apoptosis, cell cycle and mitochondria membrane potential changes by ALA-PDT on Eca-109 cells to understand apoptotic mechanism for esophageal carcinoma;
3. Investigate the expression changes in the apoptoptic related genes of Bcl-2, Bax、Bcl-xL, Bad, Bak-1, FADD and Bid to explore the main apoptosis pathway in Eca-109 cells after ALA-PDT;
4. Investigate the expression changes in the proteins of Bcl-2 and cytochrome c (cytc) to explore the final effect pathway in Eca-109 cells after ALA-PDT.
Methods:
1. Use the MTT assay to detect ALA-PDT effect on the human esophageal carcinoma cell line Eca-109:The experiment was divided into two groups:the control group and PDT group. Cells of PDT group were incubated in vitro with ALA incubating concentration (0.01 mM,0.05 mM,0.1 mM,0.25 mM,0.5 mM,1 mM,2 mM) and different incubating time (for 4,6,12 and 24 h, respectively), and then irradiated in DIOMED 630PDT systems under 30 J/m2 saturated laser dose, meanwhile make other tests that cells were incubated in vitro with different ALA incubating concentration(0.01mM,0.05 mM,0.1 mM,0.25 mM,0.5 mM,1 mM and 2 mM) and then irradiated in DIOMED 630PDT systems under three different laser dose (10 J/cm2,30 J/cm2 and 50 J/cm2, respectively), and survival rate of each concentration was measured by MTT assay after 24 hours'incubation.
2, The inhibition of tumor growth in the human esophageal carcinoma Eca-109 cells bearing mice:Raise the Eca-109 cells bearing mice model, and monitor the tumor volumes every three days. PDT therapy to the model was initiated.when the tumor volume reached 150-350 mm3. The mice were randomly assigned into four groups: control group, PDT group (given ALA and light irradiation), lighted group (given light irradiation alone) and ALA group (given ALA alone) (6 nude mice in each group). The PDT and ALA group were injected i.p. with ALA in a dose of 100 mg/kg. The mice of PDT and lighted groups were irradiated with 120 J/cm2 630 nm-light from the Diomed 630-PDT system 4 h after the photosensitizer injected. Then continue to monitor the tumor volume and observe the change of the tumor skin. Kill mice of each group 21 days after PDT. Then dislodged the tumor tissue and detected the H&E staining in the tumor tissue.
3. Subcellular localization of ALA and Mito-Tracker Green by confocal laser scanning microscope:After the Eca-109 cells were incubated with ALA (to the final concentration of 2 mM) for 4 h, followed by stained with 100 nM of MitoTracker Green for 30 min, the cells were examined by confocal laser scanning microscope FV1000 to detect the subcellular localization of ALA and Mito-Tracker Green.
4. Apoptosis determined by AnnexinV-FITC/PI binding assay:The experiment was divided into two groups:the control group and PDT group. After culture for 24 h, the cells of PDT group were incubated with ALA to the final concentration of 0.05 and 0.5 mM for 6 h in dark, and irradiated with a light dose of 30 J/cm2 (200 mW/cm2,150 s) under the 630 nm laser system. After the treatment, cells were further cultured for 24 h. Then cells were harvested and stained with 5μl of Annexin V-fluorescein isothiocyanate (AnnexinV-FITC) and 5μl of propidium iodide (PI) for 10 min in dark. The cell apoptosis were analyzed by fluorescence activated cell sorting (FACS) with flow cytometry(FCM)
5. Cell cycle analysis by PI:The experiment was divided into two groups:the control group and PDT group. After culture for 24 h, the cells of PDT group were incubated with ALA to the final concentration of 0.05,0.1 and 0.5 mM for 6 h in dark, and irradiated with a light dose of 30 J/cm2 (200 mW/cm2,150 s) under the 630 nm laser system. Cells were harvested when were further cultured for 24 h after treatment and fixed with 70% ethanol at least 12 h at 4℃and then stained with propidium iodide (PI) (to final concentration of 1 mg/ml) for 10 min in dark. The cell clycle were analyzed by fluorescence activated cell sorting (FACS) in a
FACS-SCAN system.
6. Determine of morphologic changes by Hoechst 33342/PI staining:The experiment was divided into two groups:the control group and PDT group. After culture for 24 h, the cells of PDT group were incubated with ALA to the final concentration of 0.05 mM、0.1 mM and 0.5 mM for 6 h in dark, and irradiated with a light dose of 30 J/cm2 (200 mW/cm2,150 s) under the 630 nm laser system. After the PDT treatment, the cells were further cultured for 24 h. Then the cells were harvested and stained with Hoechst 33342 and propidium iodide (PI) for 15 min in dark. The cells were observed under the fluorescence microscope BX51 to detect apoptosis in the Eca-109 cells.
7. Determine of mitochondria membrane potential by rhodamine123 staining:The experiment was divided into two groups:the control group and PDT group. After culture for 24 h, the cells of PDT group were incubated with ALA to the final concentration of 0.05 mM、0.1 mM and 0.25 mM for 6 h in dark, and irradiated with a light dose of 30 J/cm2 (200 mW/cm2,150 s) under the 630 nm laser system. After the treatment, the cells were further cultured for 1 h. Then the cells were harvested and stained with rhodaminel23 for 30 min in dark. The cells were resuspended in medium and after the cells were incubated for 60 min, observed under the fluorescence microscope BX51.
8. The gene expression changes of Bcl-2, Bax, Bcl-xL, Bad, Bak-1, FADD and Bid by quantitative real-time polymerase chain reaction ((QRT-PCR):The experiment was divided into two groups:the control group and PDT group. The cells of PDT group werer incubated with ALA to the final concentration of 0.25 mM for 6 h in dark, and irradiated with a light dose of 30 J/cm2 (200 mW/cm2,150 s) under the 630 nm laser system. RNA was extracted from Eca-109 cells at 24 h post-PDT.and synthesized cDNA.Then the cDNA was amplified and analyzed for Bcl-2, Bax、Bcl-xL, Bad, Bak-1, FADD, Bid andβ-actin on ABI 7500 PCR detector using QuantiTect SYBR Green kits.
9. The protein expression changes of Bcl-2 and cytc (cytoplasm):The experiment was divided into two groups:the control group and PDT group. The cells of PDT group were incubated with ALA to the final concentration of 0.25 mM for 6 h in dark, and irradiated with a light dose of 30 J/cm2 (200 mW/cm2,150 s) under the 630 nm laser system. Total protein and cytoplasm protein were extracted from Eca-109 cells at 24 h after ALA-PDT. Then Western Blotting assay was used to detect the protein expression changes of Bcl-2 and cytc.
10. Statistical analysis:All datas were analysised by SPSS 13.0 statistical softwire. The results of experiment were expressed by the way of mean±SD. Survival rate of each group was analyzed apply by the way of General linear Models of Univariate. The tumor tissue volume analysis used the way of General Linear Models of Repeated Measures. Use one-way ANOVA followed by LSD multiple comparison test when the data was equal variance to analysis cell apoptosis rate, cell cycle and the difference between the tumor volume of different groups. Multiple comparison test used F test (Welch) and Dunnett's T3 test if the data was not equal variance. The relative gene and protein expression difference between the control group and the PDT group were analysis using independent t test. P-Values were considered to be significant at<0.05.
Results:
1. ALA-PDT significantly inhibited the growth of Eca-109 cells The cell survival rate between different ALA concentration and three different laser dose and under the same laser dose with different incubating concentration are all significant different (all P<0.01). While under the same concentration, the cell survival rate is also significant different with different laser dose except that the concentration is 0.01 mM and 0.05 mM. The cell survival rate is the lowest as ALA concentration is 0.5 mM and the laser dose is 50 J/m2. While ALA concentration increases from 0.5 mM to 2.0 mM, the cell survival rate is not decrease as the laser dose increase from 30 J/m2 to 50 J/m2. The killing effect is the best as ALA concentration is 0.5 mM and the laser dose was 50 J/m2. The cell survival rate between different ALA concentration and four different incubating time and under the same incubating time with different ALA concentrations are all significant different (P<0.01). While under the same concentration, the cell survival rate is also significant different with different incubating time except that the concentration is 0.01 mM, 0.05 mM,0.1 mM and 1.0 mM. The cell survival rate is the lowest as ALA concentration is 2.0 mM and the incubating time is 24 hours. While ALA concentration increases from 0.5 mM to 2.0 mM, the killing effect is not enhanced as the incubating time increase from 6 hours to 24 hours.
2. The tumor volume before and after PDT between the four groups were significant different (P<0.01) There were significant different between volumes of the four groups of the different period before and after therapy (P<0.01). The volumes before PDT in the four groups were not significant different (P>0.05).There are significant different between the four different time point after therapy (all P< 0.05), The tumor growth in the ALA-PDT group was more slowly in 2 weeks after PDT, and the best inhibited effect was at the seventh day after PDT. The tumor in ALA-PDT group began to increase faster at the 14 th day after PDT., while the tumor volumes were smaller than the other groups at the four time points after PDT.
3. Under microscope from H&E staining, we can see allotype tumor cells and caner nest, the blood vessel endothelium is complete in the control group, ALA group and lighted group. While the PDT group, we can observed some homogen eryth-dyeing necrosis, and with some lymphocyte infiltrated.
4. Under confocal laser scanning microscope, there was co-localization of MitoTracker Green and ALA-PpⅨ, that meaned ALA-PpⅨwas partially localized in mitochondria.
5. The date of FCM analysis indicated that there was significant different apoptosis rate between the control group and the ALA-PDT (with ALA concentration of 0.05 mM and 0.5 mM) (P<0.05). When the concentration was 0.05 mM, there was not significant different between the PDT group and the control group (P>0.05), while when the concentration was up to 0.5 mM, the different between the control group and PDT group was significant (P<0.01).
6. The result of cell cycle analysis indicated that there were significant different in the cell rate of G0/G1, S and G2/M phase between the control group and the PDT group (all P<0.05). The cell rate of G0/G1 phase was increase as ALA concentration increased, while the proportion of cells in S and G2/M phase was decreased.
7. Fluorescence microscopy with Hoechst 33342/PI staining detected typical apoptotic changes (condensed chromatin, shrunken nuclei and high blue fluorescence by Hoechst 33342 staining) and necrotic cells (high red fluorescence) in the PDT-treated cells. In contrast, few apoptotic and necrotic cells were observed in the cells of control group.
8. Fluorescence microscopy with rhodamine123 staining detected mitochondria membrane potential changes, with high flavovirens fluorescenc in all the ALA-PDT groups, and the fluorescenc enhanced as ALA concentration increased, while in the control group, the fluorescenc is thinness or even with no fluorescenc.
9. The expression of Bcl-2, Bcl-xL and FADD genes were all significant down regulation at 24 h after ALA-PDT on the Eca-109 cell, while Bax was significant up-regulation (all P<0.05). Bad, Bid and Bak-1 were all slightly up-regulation, but the difference contrast to the control group were all not significant (all P> 0.05).
10. The expression of Bcl-2 protein was significant down regulation at 24 h psot-PDT in the Eca-109 cell, while the expression of cytc (cytoplasm) was significant up-regulation (all P<0.05).
Conclusion:
1. ALA-PDT significantly inhibited the growth of Eca-109 cells. Under the special light source, the laser dose, the kinds of photosensitizers, the photosensitizers incubating concentration and incubating time, were the main effect to PDT. Under the specific incubating time (24 h), the cell survival rate is the lowest as ALA concentration is 0.5 mM and the laser dose was 50 J/m2. While ALA concentration increases from 0.5 mM to 2.0 mM, the cell survival rate reached a plateau as the laser dose increase from 30 J/m2 to 50 J/m2. Under specific laser dose (30 J/m2.), the cell survival rate is the lowest as ALA concentration is 2.0 mM and the incubating time is 24 hours. While ALA concentration increases from 0.5 mM to 2.0 mM, the killing effect also reached a plateau as the incubating time increase from 6 hours to 24 hours. These results indicated that it is demanded to choose the optimal premeters to achieve the best effect in ALA-PDT of esophageal carcinoma.
2. ALA-PDT inhibited the tumor growth significantly. The best inhibited effect was at the seventh day after PDT. The tumor in ALA-PDT group began to grow faster at the 14 th day post-PDT., thus 14 th day after PDT can be choosen as the date to take the second treatment.
3. The results of H&E and Hoechst 33342/PI staining indicated the morphologic changes in the human esophageal carcinoma after ALA-PDT.
4. ALA-PpIX was partially localized in mitochondria. ALA-PDT changed the mitochondria membrane potential of Eca-109 cells and it maybe one of the mechanisms for ALA-PDT to induce cell death.
5. ALA-PDT induced the Eca-109 cells to apoptosis, and the apoptosis rate was increase as ALA concentration increased.
6. ALA-PDT arrested the Eca-109 cells at G0/G1 phase, and the cell rate of G0/G1 phase was increased as ALA concentration increased. The arrest of cell cycle maybe one of the mechanism that to induce apoptosis.
7. The extrinsic apoptosis pathway was not the main apoptosis way in ALA-PDT, The main way was mitochondria-dependent apoptosis pathway. The protein and gene expression of Bcl-2 were all significant down regulation in Eca-109 cells at 24 h after ALA-PDT indicated that Bcl-2 maybe one of the target of photo-damage during the PDT process
8. Release cytc from mitochondria to induce apoptosis maybe one of the final reaction in the mitochondria-dependent apoptosis pathway.
食管癌是人类常见的消化道恶性肿瘤,我国是全世界该肿瘤发病率及死亡率的高发国家。其死亡率在我国恶性肿瘤中居第四位。其中约90%的食管癌病理类型为鳞癌。而在西方国家,该发病率亦日益增加,但病理类型主要为Barrett's食管发展而来的腺癌。由于大部分患者确诊时已是晚期,甚至存在远处转移或食管梗阻,使这些患者失去了手术的最佳时机,甚至不能耐受放化疗。因此,一种新的治疗手段—光动力疗法(PDT),成为这些患者的迫切需求。目前,光动力治疗已经被证实是食管癌的一种有前景的治疗模式。
光动力治疗是通过定位在肿瘤组织的光敏剂在特定波长的激光照射下产生单态氧及其他的活性氧成分,从而导致该部位的肿瘤细胞及组织的光损伤。与常规的手术、放化疗等疗法相比,光动力疗法具有创伤小,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,目前已开始应用于多种恶性肿瘤的治疗,并日渐成为肿瘤治疗的新手段。在食管癌的治疗上,光动力疗法最早是应用于食管癌的癌前病变如Barrett's食管及早期食管癌的治疗。现已开始应用于中晚期食管癌的减瘤治疗。通过选择最佳的光动力治疗参数,早期食管癌可达到根治而晚期食管癌亦可达到较好的治疗效果。但于临床治疗中,食管癌光动力治疗的最佳参数仍未明确,因此其疗效仍不稳定。
光动力疗法对肿瘤细胞的损伤主要通过凋亡和／坏死两种死亡方式,而哪种方式为主则主要取决于肿瘤细胞的类型,激光照射剂量,光敏剂的浓度及其亚细胞定位等。目前认为,定位于线粒体的光敏剂,如5-氨基乙酰丙酸(ALA),可导致细胞以凋亡为主的死亡方式,并导致其相关凋亡基因及蛋白的表达发生变化。其中,Bcl-2家族在肿瘤细胞凋亡过程中起着非常重要的作用。以往研究表明,通过上调或下调其表达可增强光动力效应。但其在ALA-PDT食管癌光动力治疗中的作用机制尚不明确。
5-氨基乙酰丙酸是一种二代光敏剂,已于2000年被美国FDA批准用于临床实验治疗。它是一种内源性光敏剂,是活细胞血红素的前体,可产生在光动力治疗中发挥光敏剂作用的血卟啉-Ⅸ。这种光敏剂相比Photofrin其光毒性持续时间较短(大约24小时),远远短于现在临床广泛应用的Photofrin。
因此,本课题选择二代光敏剂ALA,通过研究ALA-PDT在食管癌的体内外效应探讨其最佳作用参数,为临床光动力治疗最佳参数的选择提供一定依据。通过研究ALA-PDT对食管癌细胞凋亡、细胞周期及线粒体膜电位的影响、ALA的亚细胞定位以及一些相关基因及蛋白的变化以探讨其主要作用机制。
研究目的
1.通过研究ALA-PDT在食管癌的体内外效应探讨其最佳作用参数；
2.通过研究ALA的亚细胞定位、ALA-PDT对食管癌细胞的凋亡、细胞周期及线粒体膜电位的影响初步探讨ALA-PDT在食管癌的凋亡机制；
3.通过研究ALA-PDT对食管癌细胞凋亡相关基因Bcl-2、Bax、Bcl-xL, Bad、Bak-1、FADD、Bid表达的变化探讨其可能作用的主要凋亡通路；
4.通过研究Bcl-2、cytochrome c (cytc)的蛋白表达变化以探讨ALA-PDT凋亡通路的终末效应机制。
研究方法及内容
1.MTT法检测ALA-PDT对食管癌细胞Eca-109的光动力效应：实验分为对照组和光动力治疗组,光动力治疗组给予不同浓度的ALA (0.01mM,0.05mM, 0.1mM,0.25mM,0.5mM,1mM,2mM)和不同孵育时间(分别孵育4 h、6 h、12 h、24 h)后于饱和光剂量下((30J/cm2)进行光照,同时设立另一治疗组,给予不同浓度的ALA (0.01mM,0.05mM,0.1mM,0.25mM,0.5mM, 1mM,2mM)后于三种不同光剂量(10J/cm2,30J/cm2,50J/cm2)下进行光照,光源采用DIOMED 630PDT系统,光照后孵育24小时,通过MTT法检测细胞生存率。
2.ALA-PDT对人食管癌细胞Eca-109荷瘤裸鼠的增殖抑制：建立人食管鳞癌裸鼠种植瘤模型,每3天监测肿瘤瘤径,待肿瘤大小为150-350mm3时,开始给予行光动力治疗。实验分对照组、ALA-PDT组(给予光敏剂及光照),单纯光照组(只给予光照)、单纯光敏剂组(只给予光敏剂)(每组6只),ALA-PDT组及单纯光敏剂组按100 mg/Kg腹腔给药,给药4小时后,ALA-PDT组与单纯光照组开始进行光动力治疗。光源采用DIOMED 630PDT系统,按120J/cm2光剂量进行光照,光照后监测肿瘤体积及观察肿瘤表面皮肤变化,并于治疗后第21天处死裸鼠,取瘤组织进行H&E染色的检测。
3.荧光共聚焦显微镜检测光敏剂及线粒体探针在细胞内的定位：予细胞加入ALA(终浓度为2mM)孵育4小时后予线粒体绿色荧光探针(MTG)(终浓度为100nM)避光孵育30分钟后于荧光共聚焦显微镜FV1000检测其光敏剂及线粒体探针的亚细胞定位。
4.AnnexinV-FITC/PI双标检测ALA-PDT对Eca-109细胞凋亡的影响：实验分为对照组和光动力治疗组。光动力治疗组加入ALA并使其终浓度分别为0.05mM和0.5mM,避光培养6小时后于630nm Diomed光动力治疗系统下进行光照(功率200mW/cm2,照光150 s,光剂量30J/cm2)。光照24小时后收集细胞,加入AnnexinV-FITC (5ul)和PI(5ul)避光孵育10分钟后于流式细胞仪上检测其凋亡情况。
5.PI检测ALA-PDT对Eca-109细胞周期的影响：实验分为对照组和光动力治疗组。光动力治疗组加入ALA并使其终浓度分别为0.05mM、0.1mM和0.5mM,避光培养6小时后于630nm Diomed光动力治疗系统下进行光照(功率200mW/cm2,照光150 s,光剂量30J/cm2)。光照24小时后收集细胞,70%的酒精于4℃固定至少12小时后加入PI(终浓度为1mg/ml),避光孵育10分钟后于流式细胞仪上检测其细胞周期情况。
6. Hoechst 33342/PI检测ALA-PDT作用后Eca-109细胞凋亡情况：实验分为对照组和光动力治疗组。光动力治疗组加入ALA并使其终浓度分别为0.05mM、0.1mM和0.5mM,避光培养6小时后于630nm Diomed光动力治疗系统下进行光照(功率200mW/cm2,照光150 s,光剂量30J/cm2)。光照24小时后收集细胞,加入Hoechst 33342及PI,避光孵育15分钟后于荧光显微镜BX51观察其凋亡情况。
7、罗丹明123染色检测ALA-PDT对Eca-109细胞线粒体膜电位的影响：实验分为对照组和光动力治疗组。光动力治疗组加入ALA并使其终浓度分别为0.05mM、0.1mM和0.25mM,避光培养6小时后于630nm Diomed光动力治疗系统下进行光照(功率200 mW/cm2,照光150 s,光剂量30J/cm2)。光照1小时后收集细胞,加入罗丹明123染液,避光孵育30分钟后用培养基重悬细胞,继续孵育60min后于荧光显微镜BX51观察。
8.实时定量PCR法检测Bcl-2、Bax、Bad、Bcl-xL、Bak-1、FADD、Bid的基因表达变化：实验分为对照组和ALA-PDT组。治疗组细胞给予ALA(终浓度为0.25mM)孵育6小时后于630nm Diomed光动力治疗系统下进行光照(功率200mW/cm2,照光150 s,光剂量30J/cm2)。于光照后24 h收集细胞,并提取细胞总RNA,逆转录为cDNA后采用SYBR荧光定量试剂于ABI7500PCR仪上检测Bcl-2、Bax、Bad、Bcl-xL、Bak-1、FADD、Bid的基因表达变化情况。
9.Western Blotting法检测Bcl-2、cytc(胞浆)蛋白含量的变化：实验分为对照组和ALA-PDT组。治疗组细胞给予ALA(终浓度为0.25mM)孵育6小时后进行于630nm Diomed光动力治疗系统下进行光照(功率200mW/cm2,照光150 s,光剂量30J/cm2)。于光照后24 h收集细胞,并分别提取细胞总蛋白和胞浆蛋白,采用Western Blotting法检测Bcl-2、cytc蛋白含量的变化。
10.统计分析：所有数据采用spss 13.0统计软件进行统计处理。实验结果以“均数±标准差”表示。细胞生存率的分析采用析因设计的方差分析。各组体积的比较采用重复测量数据的方差分析。细胞凋亡、细胞周期以及细胞生存率、肿瘤体积的多组间比较均采用单向方差分析,多重比较在方差齐性时采用LSD检验,方差不齐时采用近似F检验(Welch方法)及多重比较的Dunnett'sT3方法。对照组与治疗组之间基因及蛋白表达相对量的差异分析均采用独立样本t检验。P值<0.05时其差异有统计学意义。
结果
1.ALA-PDT对Eca-109细胞有显著的增殖抑制。不同光剂量及不同浓度其生存率间均有显著差异(P均<0.01)。同一光剂量下不同浓度其生存率间均有显著差异(P均<0.01),而同一浓度下不同光剂量的生存率间,则除了浓度为0.01mM及0.05mM间无明显差异外,其余均有显著差异(P均<0.05)。当孵育浓度由0.5mM增至2.0mM,光剂量由30J/cm2增加至50J/cm2时不能明显提高杀伤效应。当孵育浓度为0.5mM,光照剂量为50J/cm2(孵育时间为24 h)时其杀伤效应最强。不同孵育时间及不同浓度的生存率间均有显著差异(P均<0.01)。同一孵育时间不同浓度其生存率间均有显著差异(P均<0.01),而同一浓度下不同孵育时间的生存率,则除了浓度为0.01mM、0.05mM、0.1mM及1.0mM间无显著差异外,其余均有显著差异(P均<0.05)。当孵育浓度为2.0mM,孵育时间为24小时(光剂量为30J/cm2)其杀伤效应最强。当孵育浓度由0.5mM增至2.0mM,孵育时间由6小时增至24小时不能明显提高杀伤效应。
2.四个组治疗前后的体积间有显著差异(P<0.01)。各个组治疗前后不同时间点体积间有显著差异(P<0.01))。四个组治疗前的体积之间无显著差异(P>0.05),治疗后四个时间点体积之间均有显著差异(P均<0.05)。ALA-PDT组治疗后2周体积增长较缓慢,治疗后第7天体积抑制最明显,第14天增长速度开始增快,但治疗后四个时间点体积仍明显小于其余三组。
3.H&E染色于光镜下对照组、单纯光照组及单纯光敏剂组均可见大量异型瘤细胞,可见癌巢,血管内皮细胞完整。光动力治疗组则可见均质红染坏死物质,并有淋巴细胞侵润。
4.荧光共聚焦显微镜下可见,线粒体探针与ALA-PpⅨ存在共定位,即光敏剂ALA-PpⅨ部分定位于细胞内线粒体上。
5.FCM结果示：对照组和浓度为0.05mM和0.5mM ALA-PDT组间的凋亡率有显著差异(P<0.05)。当ALA为极低浓度(0.05mM)时,其凋亡率与对照组间无显著差异(P>0.05)。当其浓度提高为0.5mM时,其凋亡率间有显著差异(P<0.01)。
6.细胞周期分析结果可见,对照组和0.05mM、0.1mM和0.5mM ALA-PDT组G0/G1期,S期,G2/M期的比率间有显著差异(P均<0.05)。G0/G1期随着浓度增加比率呈上升趋势,而G2/M期,S期则相反,呈下降趋势。
7.荧光显微镜下可见,Hoechst 33342/PI双染的ALA-PDT组细胞出现明显的变化(染色质凝聚,核皱缩,高蓝荧光的凋亡细胞)及细胞坏死(高红荧光)。而对照组未见明显变化。
8、荧光显微镜下可见,罗丹明123染色的ALA-PDT组均可见强黄绿色荧光,且荧光强度随着ALA浓度增加而增强,而对照组细胞荧光非常弱甚至没有荧光。
9.Bcl-2、Bcl-xL、FADD在ALA-PDT作用于Eca-109细胞24 h后其表达均均显著下调,而Bax基因的表达则显著上调,与对照组相比,其差异均有统计学意义(P均<0.05)。Bad、Bid、Bak-1表达均轻微上调,但与对照组相比其差异无统计学意义(P均>0.05)。
10.Bcl-2蛋白在ALA-PDT作用于Eca-109细胞24 h后其表达显著下调,而胞浆中cytc蛋白的表达则显著上调,且与对照组相比,其差异均有统计学差异(P均<0.05)
结论
1.ALA-PDT对人食管癌细胞Eca-109有显著的增殖抑制作用。在特定光源状态下,光照剂量、光敏剂孵育浓度及孵育时间是Eca-109细胞体外光动力效应的主要影响因素。在特定孵育时间下(孵育时间为24 h),当孵育浓度为0.5mM,光照剂量为50J/cm2时其杀伤效应最强。而当孵育浓度由0.5mM增至2.0mM时,光剂量由30J/cm2增加至50J/cm2时杀伤效应达到平台期。特定光剂量下(光剂量为30J/cm2),当孵育浓度为2.0mM,孵育时间为24小时其杀伤效应最强。而当孵育浓度由0.5mM增至2.0mM时,孵育时间由6小时增至24小时其杀伤效应亦达到平台期。以上结果表明,要达到ALA-PDT对人食管癌细胞Eca-109细胞的最佳杀伤效应,需选择最佳作用参数。
2.ALA-PDT对Eca-109荷瘤裸鼠有显著增殖抑制作用。其效应在治疗后第7天最明显,第14天开始增长加快,因此治疗后第14天可作为复照的参考时间。
3.H&E染色、Hoechst 33342/PI染色从组织学的形态上进一步证明了ALA-PDT对人食管癌细胞的杀伤作用。
4.ALA-PpIX部分定位于线粒体上,ALA-PDT使Eca-109细胞线粒体膜电位发生改变可能为其使细胞死亡的机制之一。
5.ALA-PDT可使Eca-109细胞发生凋亡；随着ALA浓度增加,凋亡率呈上升趋势。
6.ALA-PDT可使Eca-109细胞明显阻滞于G0/G1期,且随着ALA浓度增加呈上升趋势。ALA-PDT对细胞周期的阻滞可能为其凋亡作用机制之一
7.外源性通路不是ALA-PDT诱导Eca-109细胞凋亡的主要通路,其主要凋亡通路为线粒体诱导的凋亡通路。Bcl-2基因及蛋白的表达在PDT后24 h均显著下调,表明Bcl-2可能为该凋亡通路的靶基因之一。
8.释放cytc进而诱导细胞凋亡可能为ALA-PDT诱导Eca-109细胞凋亡的线粒体通路终末效应之一。
Background:
Esophageal cancer is one of the most common cancers. The morbidity and mortality of esophageal cancer in China is very high, and it ranks the fourth killer among all cancer death in China. More than 90% of esophageal cancer in China is squamous cell carcinoma. The incidence of this cancer is increasing rapidly in the Western countries, however, there are considerably many adenocarcinoma of esophagus, most of which are developed from Barrett's esophagus. A great many of patients with this cancer are diagnosed in an advanced stage, with distant metastasis and esophageal obstruction. Many patients often lost the optimal chance for surgery, or even could not undergo chemotherapy or radiotherapy. It is highly demanded for an effective medical treatment such as photodynamic therapy (PDT). PDT has been found to be a promising treatment modality for esophageal cancer.
PDT process involves the photosensitiser localized in tumor tissue and irradiation of the tumor site by visible light of specific wavelength, to produce singlet oxygen and other reactive oxygen species, leading to photodamage in cancer cells and cancer destruction. 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. The first PDT used in esophageal carcinoma treatment was to cure esophageal precancerous lesion such as Barrett's of esophagus and early esophageal cancer, and now began to be used in diminish treatment of advanced stage. By properly chosen optimal parameters of PDT, complete cure of early esophageal cancer and a good therapeutic result in advanced stage of the disease can be frequently achieved. However, in clinical application, the optimal parameters have not been established that the effects for esophageal patients were instability.
The tumor cell death by PDT is induced via apoptosis and/or necrosis, depending on various conditions, such as tumor cell type, laser irradiation dose, photosensitizer concentration and subcellular localization, et.al. It has been reported that PDT with mitochondria-localizing photosensitizers, such as 5-aminolevulinic acid (ALA), can induce rapid cell death via apoptosis and change the expression of apoptotic related gene and protein. Expression of some genes related with apoptosis pathway, such as Bcl-2 family, is involved and plays an important role in this photodynamic process. It has been reported that up-regulation or down-regulation some genes of Bcl-2 family can enhance the effect of PDT. However, their roles in ALA-PDT for esophageal cancer have still not been fully investigated.
ALA is one of the second-generation photosensitizers, and has been approved to be used in clinical trial by the USA FDA since 2000. ALA is an endogenous material and a precursor of heme in living cells, thus the induced protoporphyrinⅨ(PpⅨ) can be employed as a photosensitizer in PDT. The ALA-produced PpⅨ(ALA-PpⅨ) induces a short-lasting phototoxicity about 24 hours, much shorter compared with that induced by photofrin which was used generally in clinical treatment。
Thus, we choosed the second-generation photosensitizers ALA, and investigated the effects of ALA-PDT on esophageal cancer cells in vitro and vivo, to explore optimal parameters in PDT for esophageal cancer and provided some evidence for PDT optinal clinical premeters established. Also, we investigated the role of cell apoptosis, cell cycle and mitochondria membrane potential changes, subcellular localization of ALA-PpⅨ, and some molecular changes to understand the mechanism of ALA-PDT for esophageal cancer.
Objectives:
1. Investigate ALA-PDT effect in the human esophageal carcinoma both in intro and vivo to explore optimal parameters in PDT for esophageal cancer;
2. Investigate the subcellular localization of ALA, the role of cell apoptosis, cell cycle and mitochondria membrane potential changes by ALA-PDT on Eca-109 cells to understand apoptotic mechanism for esophageal carcinoma;
3. Investigate the expression changes in the apoptoptic related genes of Bcl-2, Bax、Bcl-xL, Bad, Bak-1, FADD and Bid to explore the main apoptosis pathway in Eca-109 cells after ALA-PDT;
4. Investigate the expression changes in the proteins of Bcl-2 and cytochrome c (cytc) to explore the final effect pathway in Eca-109 cells after ALA-PDT.
Methods:
1. Use the MTT assay to detect ALA-PDT effect on the human esophageal carcinoma cell line Eca-109:The experiment was divided into two groups:the control group and PDT group. Cells of PDT group were incubated in vitro with ALA incubating concentration (0.01 mM,0.05 mM,0.1 mM,0.25 mM,0.5 mM,1 mM,2 mM) and different incubating time (for 4,6,12 and 24 h, respectively), and then irradiated in DIOMED 630PDT systems under 30 J/m2 saturated laser dose, meanwhile make other tests that cells were incubated in vitro with different ALA incubating concentration(0.01mM,0.05 mM,0.1 mM,0.25 mM,0.5 mM,1 mM and 2 mM) and then irradiated in DIOMED 630PDT systems under three different laser dose (10 J/cm2,30 J/cm2 and 50 J/cm2, respectively), and survival rate of each concentration was measured by MTT assay after 24 hours'incubation.
2, The inhibition of tumor growth in the human esophageal carcinoma Eca-109 cells bearing mice:Raise the Eca-109 cells bearing mice model, and monitor the tumor volumes every three days. PDT therapy to the model was initiated.when the tumor volume reached 150-350 mm3. The mice were randomly assigned into four groups: control group, PDT group (given ALA and light irradiation), lighted group (given light irradiation alone) and ALA group (given ALA alone) (6 nude mice in each group). The PDT and ALA group were injected i.p. with ALA in a dose of 100 mg/kg. The mice of PDT and lighted groups were irradiated with 120 J/cm2 630 nm-light from the Diomed 630-PDT system 4 h after the photosensitizer injected. Then continue to monitor the tumor volume and observe the change of the tumor skin. Kill mice of each group 21 days after PDT. Then dislodged the tumor tissue and detected the H&E staining in the tumor tissue.
3. Subcellular localization of ALA and Mito-Tracker Green by confocal laser scanning microscope:After the Eca-109 cells were incubated with ALA (to the final concentration of 2 mM) for 4 h, followed by stained with 100 nM of MitoTracker Green for 30 min, the cells were examined by confocal laser scanning microscope FV1000 to detect the subcellular localization of ALA and Mito-Tracker Green.
4. Apoptosis determined by AnnexinV-FITC/PI binding assay:The experiment was divided into two groups:the control group and PDT group. After culture for 24 h, the cells of PDT group were incubated with ALA to the final concentration of 0.05 and 0.5 mM for 6 h in dark, and irradiated with a light dose of 30 J/cm2 (200 mW/cm2,150 s) under the 630 nm laser system. After the treatment, cells were further cultured for 24 h. Then cells were harvested and stained with 5μl of Annexin V-fluorescein isothiocyanate (AnnexinV-FITC) and 5μl of propidium iodide (PI) for 10 min in dark. The cell apoptosis were analyzed by fluorescence activated cell sorting (FACS) with flow cytometry(FCM)
5. Cell cycle analysis by PI:The experiment was divided into two groups:the control group and PDT group. After culture for 24 h, the cells of PDT group were incubated with ALA to the final concentration of 0.05,0.1 and 0.5 mM for 6 h in dark, and irradiated with a light dose of 30 J/cm2 (200 mW/cm2,150 s) under the 630 nm laser system. Cells were harvested when were further cultured for 24 h after treatment and fixed with 70% ethanol at least 12 h at 4℃and then stained with propidium iodide (PI) (to final concentration of 1 mg/ml) for 10 min in dark. The cell clycle were analyzed by fluorescence activated cell sorting (FACS) in a
FACS-SCAN system.
6. Determine of morphologic changes by Hoechst 33342/PI staining:The experiment was divided into two groups:the control group and PDT group. After culture for 24 h, the cells of PDT group were incubated with ALA to the final concentration of 0.05 mM、0.1 mM and 0.5 mM for 6 h in dark, and irradiated with a light dose of 30 J/cm2 (200 mW/cm2,150 s) under the 630 nm laser system. After the PDT treatment, the cells were further cultured for 24 h. Then the cells were harvested and stained with Hoechst 33342 and propidium iodide (PI) for 15 min in dark. The cells were observed under the fluorescence microscope BX51 to detect apoptosis in the Eca-109 cells.
7. Determine of mitochondria membrane potential by rhodamine123 staining:The experiment was divided into two groups:the control group and PDT group. After culture for 24 h, the cells of PDT group were incubated with ALA to the final concentration of 0.05 mM、0.1 mM and 0.25 mM for 6 h in dark, and irradiated with a light dose of 30 J/cm2 (200 mW/cm2,150 s) under the 630 nm laser system. After the treatment, the cells were further cultured for 1 h. Then the cells were harvested and stained with rhodaminel23 for 30 min in dark. The cells were resuspended in medium and after the cells were incubated for 60 min, observed under the fluorescence microscope BX51.
8. The gene expression changes of Bcl-2, Bax, Bcl-xL, Bad, Bak-1, FADD and Bid by quantitative real-time polymerase chain reaction ((QRT-PCR):The experiment was divided into two groups:the control group and PDT group. The cells of PDT group werer incubated with ALA to the final concentration of 0.25 mM for 6 h in dark, and irradiated with a light dose of 30 J/cm2 (200 mW/cm2,150 s) under the 630 nm laser system. RNA was extracted from Eca-109 cells at 24 h post-PDT.and synthesized cDNA.Then the cDNA was amplified and analyzed for Bcl-2, Bax、Bcl-xL, Bad, Bak-1, FADD, Bid andβ-actin on ABI 7500 PCR detector using QuantiTect SYBR Green kits.
9. The protein expression changes of Bcl-2 and cytc (cytoplasm):The experiment was divided into two groups:the control group and PDT group. The cells of PDT group were incubated with ALA to the final concentration of 0.25 mM for 6 h in dark, and irradiated with a light dose of 30 J/cm2 (200 mW/cm2,150 s) under the 630 nm laser system. Total protein and cytoplasm protein were extracted from Eca-109 cells at 24 h after ALA-PDT. Then Western Blotting assay was used to detect the protein expression changes of Bcl-2 and cytc.
10. Statistical analysis:All datas were analysised by SPSS 13.0 statistical softwire. The results of experiment were expressed by the way of mean±SD. Survival rate of each group was analyzed apply by the way of General linear Models of Univariate. The tumor tissue volume analysis used the way of General Linear Models of Repeated Measures. Use one-way ANOVA followed by LSD multiple comparison test when the data was equal variance to analysis cell apoptosis rate, cell cycle and the difference between the tumor volume of different groups. Multiple comparison test used F test (Welch) and Dunnett's T3 test if the data was not equal variance. The relative gene and protein expression difference between the control group and the PDT group were analysis using independent t test. P-Values were considered to be significant at<0.05.
Results:
1. ALA-PDT significantly inhibited the growth of Eca-109 cells The cell survival rate between different ALA concentration and three different laser dose and under the same laser dose with different incubating concentration are all significant different (all P<0.01). While under the same concentration, the cell survival rate is also significant different with different laser dose except that the concentration is 0.01 mM and 0.05 mM. The cell survival rate is the lowest as ALA concentration is 0.5 mM and the laser dose is 50 J/m2. While ALA concentration increases from 0.5 mM to 2.0 mM, the cell survival rate is not decrease as the laser dose increase from 30 J/m2 to 50 J/m2. The killing effect is the best as ALA concentration is 0.5 mM and the laser dose was 50 J/m2. The cell survival rate between different ALA concentration and four different incubating time and under the same incubating time with different ALA concentrations are all significant different (P<0.01). While under the same concentration, the cell survival rate is also significant different with different incubating time except that the concentration is 0.01 mM, 0.05 mM,0.1 mM and 1.0 mM. The cell survival rate is the lowest as ALA concentration is 2.0 mM and the incubating time is 24 hours. While ALA concentration increases from 0.5 mM to 2.0 mM, the killing effect is not enhanced as the incubating time increase from 6 hours to 24 hours.
2. The tumor volume before and after PDT between the four groups were significant different (P<0.01) There were significant different between volumes of the four groups of the different period before and after therapy (P<0.01). The volumes before PDT in the four groups were not significant different (P>0.05).There are significant different between the four different time point after therapy (all P< 0.05), The tumor growth in the ALA-PDT group was more slowly in 2 weeks after PDT, and the best inhibited effect was at the seventh day after PDT. The tumor in ALA-PDT group began to increase faster at the 14 th day after PDT., while the tumor volumes were smaller than the other groups at the four time points after PDT.
3. Under microscope from H&E staining, we can see allotype tumor cells and caner nest, the blood vessel endothelium is complete in the control group, ALA group and lighted group. While the PDT group, we can observed some homogen eryth-dyeing necrosis, and with some lymphocyte infiltrated.
4. Under confocal laser scanning microscope, there was co-localization of MitoTracker Green and ALA-PpⅨ, that meaned ALA-PpⅨwas partially localized in mitochondria.
5. The date of FCM analysis indicated that there was significant different apoptosis rate between the control group and the ALA-PDT (with ALA concentration of 0.05 mM and 0.5 mM) (P<0.05). When the concentration was 0.05 mM, there was not significant different between the PDT group and the control group (P>0.05), while when the concentration was up to 0.5 mM, the different between the control group and PDT group was significant (P<0.01).
6. The result of cell cycle analysis indicated that there were significant different in the cell rate of G0/G1, S and G2/M phase between the control group and the PDT group (all P<0.05). The cell rate of G0/G1 phase was increase as ALA concentration increased, while the proportion of cells in S and G2/M phase was decreased.
7. Fluorescence microscopy with Hoechst 33342/PI staining detected typical apoptotic changes (condensed chromatin, shrunken nuclei and high blue fluorescence by Hoechst 33342 staining) and necrotic cells (high red fluorescence) in the PDT-treated cells. In contrast, few apoptotic and necrotic cells were observed in the cells of control group.
8. Fluorescence microscopy with rhodamine123 staining detected mitochondria membrane potential changes, with high flavovirens fluorescenc in all the ALA-PDT groups, and the fluorescenc enhanced as ALA concentration increased, while in the control group, the fluorescenc is thinness or even with no fluorescenc.
9. The expression of Bcl-2, Bcl-xL and FADD genes were all significant down regulation at 24 h after ALA-PDT on the Eca-109 cell, while Bax was significant up-regulation (all P<0.05). Bad, Bid and Bak-1 were all slightly up-regulation, but the difference contrast to the control group were all not significant (all P> 0.05).
10. The expression of Bcl-2 protein was significant down regulation at 24 h psot-PDT in the Eca-109 cell, while the expression of cytc (cytoplasm) was significant up-regulation (all P<0.05).
Conclusion:
1. ALA-PDT significantly inhibited the growth of Eca-109 cells. Under the special light source, the laser dose, the kinds of photosensitizers, the photosensitizers incubating concentration and incubating time, were the main effect to PDT. Under the specific incubating time (24 h), the cell survival rate is the lowest as ALA concentration is 0.5 mM and the laser dose was 50 J/m2. While ALA concentration increases from 0.5 mM to 2.0 mM, the cell survival rate reached a plateau as the laser dose increase from 30 J/m2 to 50 J/m2. Under specific laser dose (30 J/m2.), the cell survival rate is the lowest as ALA concentration is 2.0 mM and the incubating time is 24 hours. While ALA concentration increases from 0.5 mM to 2.0 mM, the killing effect also reached a plateau as the incubating time increase from 6 hours to 24 hours. These results indicated that it is demanded to choose the optimal premeters to achieve the best effect in ALA-PDT of esophageal carcinoma.
2. ALA-PDT inhibited the tumor growth significantly. The best inhibited effect was at the seventh day after PDT. The tumor in ALA-PDT group began to grow faster at the 14 th day post-PDT., thus 14 th day after PDT can be choosen as the date to take the second treatment.
3. The results of H&E and Hoechst 33342/PI staining indicated the morphologic changes in the human esophageal carcinoma after ALA-PDT.
4. ALA-PpIX was partially localized in mitochondria. ALA-PDT changed the mitochondria membrane potential of Eca-109 cells and it maybe one of the mechanisms for ALA-PDT to induce cell death.
5. ALA-PDT induced the Eca-109 cells to apoptosis, and the apoptosis rate was increase as ALA concentration increased.
6. ALA-PDT arrested the Eca-109 cells at G0/G1 phase, and the cell rate of G0/G1 phase was increased as ALA concentration increased. The arrest of cell cycle maybe one of the mechanism that to induce apoptosis.
7. The extrinsic apoptosis pathway was not the main apoptosis way in ALA-PDT, The main way was mitochondria-dependent apoptosis pathway. The protein and gene expression of Bcl-2 were all significant down regulation in Eca-109 cells at 24 h after ALA-PDT indicated that Bcl-2 maybe one of the target of photo-damage during the PDT process
8. Release cytc from mitochondria to induce apoptosis maybe one of the final reaction in the mitochondria-dependent apoptosis pathway.
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