姜黄素对肺癌细胞增殖、凋亡、促血管生成和放射敏感性的作用及其机制的体外研究
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摘要
前言
肺癌是世界上发病率及死亡率较高的恶性肿瘤之一，手术是Ⅰ-Ⅱ期非小细胞肺癌的主要治疗手段，但至少有50％的术后患者会发生局部复发或远处转移，有70％的患者不适合再次手术治疗，放化疗及分子靶向治疗成为其主要的治疗手段，但长期使用毒性大费用高又影响其疗效。人们试图从中药有效成分中寻找一种高效、低毒，能抑制肿瘤细胞增殖诱导其凋亡且对放射具有增敏作用的药物，从而为肺癌的临床治疗提供一种新途径。
姜黄素(Curcumin，Cur)是从姜科姜黄属植物姜黄(curcumalonga)根茎中提取的一种酚类色素，大量研究表明，姜黄素具有抗炎、抗氧化、抗肿瘤等多种生物学功能，且价格低廉，毒性很低(小鼠LD_(50)为2g／Kg)，目前对其抗肿瘤作用的研究主要集中在肿瘤的化学预防方面。最近美国NCI已将其列为第三代防癌药进行临床研究。国内外对姜黄素抗促癌机制的研究主要集中在以下7个方面：①抗诱变及抗NO作用。②对表皮生长因子受体EGFR、PKC信号转导通路的影响。③下调核转录因子，抑制iNOS、COX-2活性。④调控癌基因和抑癌基因的表达。⑤诱导细胞周期阻滞。⑥抑制脲激酶(uPA)活性和MMP-9的分泌。⑦抑制肿瘤血管生成。
有关姜黄素对肺癌抗癌作用的研究国外仅限于通过调控肺癌细胞癌基因和抑癌基因表达促其凋亡及抑制血管生成因子表达来抑制肺癌血管生成等；而国内未见相关报道。本课题从诱导凋亡、抑制肿瘤血管生成、放射增敏三个方面观察姜黄素在体外对肺癌A2细胞的作用并通过检测Bcl-2、Bax、survivin、P53、Fas、VEGF、MMP-9、cyclinB_1、P34~(cdc2)、phos-P34~(cdc2)、P21及NF-kB蛋白表达活性改变及Ang-1、Ang-2和TSP的mRNA水平的变化从分子水平探讨其机制。
一、姜黄素抑制人肺癌细胞(A2)生长、诱导凋亡的作用及其机制
(一)实验方法
1、细胞培养
用含10％(vol／vol)56℃灭活胎牛血清的RPMI1640培养基，37℃饱和湿度、5％CO2的培养箱中培养，细胞呈单层贴壁生长，实验取对数生长期细胞。
2、MTT法药物敏感试验
消化细胞接种于96孔培养板，培养24h后，分别加入含不同浓度姜黄素的RPMI1640培养液100μl，使姜黄素的终浓度为5、10、20、40，60、80、100和160μmol／L，培养48h后，每孔加入5mg／mlMTT20μl，用酶标仪检测OD值，检测波长为490nm。采用SPSS统计分析软件，应用直线回归法计算IC_(50)。
3、Annexin-FITC标记法定量检测细胞凋亡
收集细胞，清洗，离心，加入结合buffer，重悬细胞。取195μl细胞重悬液，加5μl Annexin-FITC，室温孵育10分钟。清洗细胞，离心，弃上清，加入190μl结合buffer，重悬细胞。加入10μl 20μg／ml的Propidium Iodide。用FACScan检测细胞膜外表面磷脂酰丝氨酸结合蛋白的表达及死亡细胞DNA含量。
4、细胞凋亡的形态学检测
细胞经2.5％戊二醛、1％锇酸双固定，梯度乙醇和丙酮脱水，Epon812树脂包埋超薄切片机(AO型)切片，醋酸钠和柠檬酸铅双重染色，JEM-1200EX和H-600型透射电镜下观察并拍照。
5、细胞总DNA的提取及琼脂糖凝胶电泳
收集细胞，重悬，离心，去上清，重复一次；加入细胞裂解液500μl重悬细胞，50℃水浴3-5小时震荡，离心，吸取水相于另一离心管中，加入1／10体积3MNaCL和两倍体积乙醇混匀，沉淀DNA，去上清，70％乙醇漂洗1-2次，风扇吹干残存液体，加入50μl含100μg／mlRNAseA的TE缓冲液，65℃30 min；取8μl加上样缓冲液上样，1.8％琼脂糖凝胶电泳，采用1-5V／cm电压降，用凝胶成像系统观察和扫描成像。
6、Western blot检测A2细胞凋亡相关蛋白Bcl-2、Bax、P53、survivin、Fas
收集细胞，裂解，测蛋白浓度，定蛋白，10％SDS-PAGE电泳，转印，TBS封闭，转到Bcl-2、Bax、P53、Fas及survivin一抗4℃，过夜。二抗孵育。TTBS冲洗两次，每次15分钟，转到HRP-标记的羊抗鼠IgG(1：5000)、羊抗兔IgG(1：5000)、马抗羊IgG(1：5000)的二抗中，孵育2小时。用ECL化学发光检测，X-片感光。
(二)结果
1、MTT法药物敏感试验
姜黄素对A2细胞的抑制作用呈浓度依赖性，计算48小时的IC50值为40μmol／L。
2、细胞凋亡的形态学检测
透射电镜下观察，对照组A2细胞中细胞核及线粒体、内质网等结构清晰，核占细胞体积的大部分。姜黄素作用于A2细胞后可见细胞核固缩、碎裂、无核膜包被；核染色质边集于胞膜，核孔消失；正在形成的凋亡小体和已形成的凋亡小体等超微结构改变。
3、Annexin-FITC标记法定量检测细胞凋亡
分别收集姜黄素40μmol／L作用24小时、48小时、72小时及正常对照组的细胞进行Annexin V-FITC标记结合流式细胞仪检测细胞凋亡。发现姜黄素作用24小时、48小时，A2细胞凋亡比例较低，分别为9.45％、16.89％；72小时细胞凋亡比例较高，为21.05％。
4、DNA琼脂糖凝胶电泳
分别收集姜黄素40μmol／L作用24小时、48小时、72小时及正常对照组的细胞进行DNA琼脂糖电泳。48小时，凋亡细胞的DNA由于DNA降解成规则的大片段出现凋亡特有的“梯状”条带，72小时，细胞大多已经坏死，坏死细胞由于其DNA的不规则降解显现一条连续的膜状条带。而正常活细胞DNA基因组条带由于分子量大，迁移距离短，所以停留在加样孔附近
5、Western blotting
分别收集姜黄素40μmol／L作用6小时、12小时、24小时、48小时及正常对照组的细胞，提取总蛋白，检测凋亡相关蛋白P53、Bcl-2、Bax、Fas、survivin的表达，发现随时间的延长，Bax、P53、Fas蛋白表达水平逐渐升高。而Bcl-2、survivin蛋白表达水平逐渐降低。
总之：姜黄素诱导肺癌细胞株A2细胞凋亡是多靶点，多途径的，其与凋亡相关基因的改变、细胞因子的分泌等密切相关。
二、姜黄素对血管生成的作用及其机制的研究
(一)实验方法
1、细胞培养
用含10％(vol／vol)56℃灭活胎牛血清的RPMI1640培养基，37℃饱和湿度、5％CO2的培养箱中培养，HUVEC、A2细胞呈单层贴壁生长，实验取对数生长期细胞。
2、MTT法观察姜黄素对HUVECs增殖的影响
细胞接种于96孔培养板，培养24h后，分别加入含不同浓度姜黄素的RPMI1640培养液100μl，使姜黄素的终浓度为5、10、20、40，60、80、100和160μmol／L，每组设三个复孔，培养48h后，每孔加入5mg／ml MTT 20μl，继续培养4h后，吸去上清液，加入150μl DMSO液，震荡培养板10min，用酶标仪检测OD值，检测波长为490nm。细胞增殖抑制率(％)=[1-(实验组的OD值-空白组的OD值)／(阴性对照组的OD值-空白对照组的OD值)×100％
3、Annexin-FITC标记法定量检测细胞凋亡
消化收集细胞，加入结合buffer，重悬细胞，调整细胞数为5×10~5／ml。取195μl细胞重悬液，加5μl Annexin-FITC，室温孵育，清洗细胞，离心后弃上清，加入190μl结合buffer，重悬细胞。加入10μl 20μg／ml的Propidium Iodide。用FACScan检测细胞膜外表面磷脂酰丝氨酸结合蛋白的表达及死亡细胞DNA含量。
4、逆转录PCR检测血管生成素1和血管生成素2(Ang-1、Ang-2)和血小板反应素(TSP)的mRNA水平
细胞总RNA提取，采用紫外光谱扫描仪检测总RNA纯度；逆转录；PCR产物分析：取PCR产物在2％琼脂糖凝胶上电泳，电泳结果以Ang-1、Ang-2和TSP分别与β-actin条带的吸光度比值表示Ang-1、Ang-2和TSP的mRNA的相对表达强度。
5、Western blot检测VEGF、MMP-9蛋白的表达
收集细胞，裂解，测蛋白浓度，定蛋白，10％SDS-PAGE电泳，转印，TBS封闭，转到VEGF、MMP-9一抗4℃，过夜。二抗孵育。TTBS冲洗两次，每次15分钟，转到HRP-标记的羊抗鼠IgG(1：5000)、羊抗兔IgG(1：5000)、马抗羊IgG(1：5000)的二抗中，孵育2小时。用ECL化学发光检测，X-片感光。
(二)结果
1、姜黄素处理HUVEC细胞后MTT法检测结果
姜黄素对HUVEC细胞增殖抑制作用呈量效关系：不同浓度姜黄素对培养的人血管内皮细胞的增殖有显著抑制作用。随着姜黄素浓度的增加，作用时间的延长，其抑制作用增强。
2、姜黄素对HUVECs凋亡的影响
流式细胞术检测浓度为5，10，20，40，80，160μmol／L姜黄素对HUVECs凋亡的影响，结果显示，作用24h后HUVECs细胞凋亡率分别为4.12％，9.44％，15.91％，17.98％，21.05％，22.21％，明显高于对照组1.64％的凋亡率。
3、RT-PCR
分别收集姜黄素40μmol／L作用6小时、12小时、24小时、48小时及正常对照组的细胞，提取RNA，检测血管生成素-1(Ang-1)、血管生成素-2(Ang-2)及血小板反应素(TSP)的mRNA的表达，可见随姜黄素作用时间的延长Ang-1、Ang-2 mRNA的表达明显低于对照组；TSP的mRNA的表达高于对照组。
4、Western blotting
分别收集姜黄素40μmol／L作用6小时、12小时、24小时、48小时及正常对照组的细胞，提取总蛋白，检测VEGF及MMP9蛋白的表达显著低于对照组。
总之，姜黄素可能通过多种信号途径抑制血管生成：①降低肿瘤细胞血管生成因子(VEGF，Ang-1，Ang-2)和(或)提高抑制因子(TSP-1)的表达水平来调控血管生成因子的平衡，抑制血管生成的表型转换。②抑制血管内皮细胞的增殖，促进内皮细胞的凋亡。③减少基质金属蛋白酶(MMP-9)的表达从而抑制基底膜及细胞外基质的降解。
三、姜黄素对人肺癌(A2)细胞放疗增敏的体外实验研究
(一)实验方法
1、集落形成试验
接种细胞于培养皿中，培养24小时后更换含不同浓度的姜黄素(1、5、10μmol／L)培养液继续培养24小时和48小时后，在室温下用~(137)Cs放射源照射，照射剂量分别为0Gy、2Gy、4Gy、6Gy、8Gy、100y，照射后用药组更换为不含药物的培养液与单纯照射组一起继续培养9天。9天后细胞用95％乙醇固定，姬母萨染色，≥50个细胞记数为一个存活集落，对每个测量点实验均重复三次。集落实验被换算成存活分数，单纯加药组的存活分数为单纯加药组的集落形成率与空白对照组的集落形成率之比，以单纯加药组的存活分数(SF)为基准，对加药照射组的SF进行校正从而得到药物的放射增敏作用。以多靶单击数学模型拟合细胞存活曲线，计算出单纯照射组(对照组)和加药照射组(药物处理组)的Dq值。放射增敏效应以放射增敏比SER_(Dq)表示。
2、FACScan检测细胞周期
消化收集细胞，在PBS中清洗，离心，弃上清，加入70％(vol／vol)的冷乙醇，于-20℃放置，PBS洗两次，细胞中加入含RN aseA(0.1mg／ml)的PBS 1ml，置培养箱中，37℃、30分钟。然后细胞在含有50μg／ml propidium iodie的PBS 1ml中染色。用FACScan进行细胞DNA含量和细胞周期分析，所用软件为CELLQEST。
3、细胞凋亡检测
细胞接种于6孔培养板中，设空白对照(即单纯放射组)，实验组(即药物加放射组)浓度分别为5、10、15μmol／L的姜黄素，培养24h接受2、4、6、8Gy照射后，继续培养24h，取100μl细胞悬液，加入DNA-PREPTMLPR 200μl混匀，30s后加入DNA-PREP-TM染色剂(PI染色)2ml混匀。流式细胞仪检测细胞凋亡情况。利用Coulter公司的SystemⅡTM软件处理结果，每次实验均设阴性和阳性对照。
4、蛋白免疫印迹法(Western-blot)检测周期调控因子及NF-kB蛋白水平的表达
收集细胞，裂解，测蛋白浓度，定蛋白，样品加入样品缓冲液，10％SDS-PAGE电泳，转印，TBS封闭，封闭后TTBS洗2次，每次5分钟，转到cyclinB_1、P34~(cdc2)、phos-P34~(cdc2)、P21、survivin及NF-kB一抗4℃，过夜。二抗孵育。TTBS冲洗两次，每次15分钟，转到HRP-标记的羊抗鼠IgG(1：5000)、羊抗兔IgG(1：5000)、马抗羊IgG(1：5000)的二抗中，孵育2小时。用ECL化学发光检测，X-片感光。
(二)结果
1、细胞周期检测
血清饥饿法同步在G0期的细胞，弃无血清RPMI1640培养基，加入含10％胎牛血清RPMI1640培养基继续培养。每3小时作一次流式细胞仪检测。在经过2小时恢复期后，约在17小时到达G2期，并在22小时到达G2末期。整个细胞周期大约23个小时左右。
2、细胞周期阻滞
取血清饥饿法同步在G0期细胞，经2小时恢复期后，分别向实验组的3瓶细胞每瓶中加入姜黄素40μmol／L。再取恢复期后18小时的3瓶细胞，每瓶细胞中加入姜黄素，使终浓度为40μmol／L。每3小时收集一瓶细胞，进行流式细胞仪检测。加入姜黄素后，作用于G1期引起轻度G1期阻滞；作用于G2期，引起G2期阻滞，细胞停留在G2期，不发生G2／M期转换。各时间均可见凋亡标志的亚二倍体峰。
3、不同处理因素对A2细胞凋亡的影响
单纯放射组和药物加放射组分别处理A2细胞后，药物加放射组及单纯放射组均在G0+G1峰之前出现一个明显的亚二倍体峰(凋亡峰)，但以药物加放射组更为明显，各处理因素诱导A2细胞凋亡作用强度的顺序为：姜黄素+照射＞姜黄素或照射＞对照。
4、Western blotting
取同步化恢复期后18小时的细胞，加入姜黄素使终浓度为10μmol／L。作用3小时，再分别收集恢复期后18小时、21小时的细胞，检测cyclinB_1、P34~(cdc2)、phos-P34~(cdc2)、P21、survivin及NF-kB、蛋白印迹，在姜黄素处理3小时后，P34~(cdc2)、phos-P34~(cdc2)表达量没有改变，cyclinB_1、survivin及NF-kB蛋白表达量减少，P21蛋白表达量增加。
总之，姜黄素能增加肺癌细胞的放射敏感性，其机制可能为使A2细胞发生G2／M期阻滞，及抑制NF-kB蛋白的表达；细胞发生周期阻滞与cyclinB_1、survivin蛋白表达量减少，P21蛋白表达量增加有关。
1、姜黄素能诱导A2细胞凋亡，凋亡发生可能与c-myc、bcl-2、survivin蛋白表达下降；而bax、fas、p53、Caspase-3的蛋白表达升高相关。
2、姜黄素能抑制肿瘤血管生成其作用通过(1)降低肿瘤细胞血管生成因子(VEGF，Ang-1，Ang-2)和(或)提高抑制因子(TSP-1)的表达水平来调控血管生成因子的平衡，抑制血管生成的表型转换。(2)抑制血管内皮细胞的增殖，促进内皮细胞的凋亡。(3)减少基质金属蛋白酶(MMP-9)的表达从而抑制基底膜及细胞外基质的降解。
3、姜黄素能提高A2细胞的放射敏感性，可能通过相对特异地使A2细胞发生G2／M期阻滞及降低NF-kB蛋白表达来实现的。
Pulmonary carcinoma is one of the malignant tumors with high attack rate andmortality rate. For phaseⅠ-Ⅱof the non-small cell carcinoma, the main method fortreatment is operation. But the local recurrent or the distal metastasis may happen in atleast 50% of the patients after the operations, and 70% of these patients are not able totake the operation for the second time, then radiotherapy, the chemo, and the targetmolecule have to be the main treatment methods. But we know that when the patientstake these methods for a long time, there will be serious toxicity, large payment but badefferent. So now we are trying to get the drug with the better effect, lower toxicity,sensitization to the radiotherapy and can also inhibit the proliferation, indicate thenecrosis of the tumor cells from the traditional Chinese drugs, to find a new way for theclinical treatment of the pulmonary carcinoma.
Curcumin (Cur) is a kind of phenols pigment which is extracted from thecurcumalonga of the curcuma category of the ginger family. Quite a lot of researches have indicated that the Cur has several biological functions such as anti-inflammation,antioxygen, anti-oncoma, and also, it is cheap-price, low-toxicity (LD_(50) of mouses is2g/Kg). The researches at present mostly focus on the chemical prevention. Recently,the NCI of America has already studied it in clinic as the third generation of theanti-oncoma medicines. The researches on the Cur all over the world are most about the7 aspects below: 1. Anti mutation and anti-NO 2. Effecting on the pathway of the signaltransduction of the epithelial grow factor receptors EGFR, PKC. 3. Down-graduatingthe nuclear factor, inhibiting the activity of iNOS, COX-2.4. Graduating the expressionof the oncogenes and anti-oncogenes. 5. Inducing the block of the cell cycle. 6.Inhibiting the activity of uPA and the secretion of MMP-9. 7. Inhibiting the formationof blood vessels.
The researches of the Cur on the anti-oncoma effection to the pulmonarycarcinoma have been limited within the regulation of the expression of the oncogeneand anti-oncogene to induce apoptosis, or inhibiting angiogenesis of pulmonarycarcinoma by reducing the expression of the angiogenesis factors. But nocorresponding reports in China. We observe the Cru of its functions to the A2pulmonary carcinoma cells in vitro in 3 aspects: reducing the apoptosis, inhibiting theangiogenesis of pulmonary carcinoma, and radiosensitization. Also, in this subject, wewill discus the mechanism on the molecular level by detecting the active changes of the protein expression of the Bcl-2, Bax, surviving, P53, C-myc, VEGF, MMP-9, cyclinB_1,P34~(cdc2), phos-P34~(cdc2), P21 and NF-kB, and the change of the Ang-1, Ang-2, and TSP.
Part One The function and mechanism of the Curinhibiting the growth and reducing the apoptosisof the human pulmonary carcinoma cells (A2 cells)
Method
1、Cell culture
The RPMI1640 medium with 10% (vol/vol), 56℃inactivated fetal bovine serum,have been cultured in the 37℃saturated humidity, 5%CO2 incubatons. The cells withmonolayer adherence grew. We took the logarithm of the cells in the growing period inthe experiment.
2、Drug sensitivity test in MTT
The cells in the growing period had been collected and digested, and regulated intothe 1*105/ml monolayer-cell suspension, inoculated into the cultivating plates with 96holes. After 24 hours, by adding the 100μl RPMI1640 culture fluid within different Crudensities, the terminal densities of the Cru were 5, 10, 20, 40, 60, 80,100 and 160μl, 3replies holes in each group. 48 hours later, 5mg/ml MTT20μl were added into the holes,then after 4 hours, blotting the clear superstratum lipid away, adding the DMSO fluid 150μl, shaking the plates for 10 min, then the OD value had been detected by enzymemark, getting the wave length 490nm. We had to repeat the experiments for 3 times.Analyzing the results by SPSS, and then calculated the IC_(50) by the method of linearregession.
3、etecting the cell apoptosis by Annexin-FITC marking method
The cells had been digested by the 0.25% trypsin, and collected after boasting, thenwashed in the PBS for 1 time, and 1000rpm, 10 min centrifugalizated. We threw thesuperstratum, added the combining buffer, resuspense the cells, and modulated the cellsinto 5*10~5/ml, then took 195μl resuspensory fluid, added Annexin-FITC 5μl, mixed inthe room temperature, incubated for 10 min. Next, we took the combining buffer 200μl,washed the cells, 1000rpm, 10 min centrifugalizated. Throwing the superstratum away,we added the combining buffer 190μl, resuspense the cells again, then added the20μg/ml Propidium Iodide 10μl. Then we detected the expression of thediacylglyceryl-phosphorylserine binding protein in the outer surface of the cellularmembrane and the content of the DNA in the death cells.
4、Morphology detection of the cell apoptosis
The cells had been fixed by the 2.5% glutaraldhyde, 1% osmium tetroxide,dehydrated by the gradient ethanol, and the acetone, cutted by the Epon812 resinembedding ultrathin section cutter (AO type), stained by natrium aceticum and Lead Citrate, then observed and photographed under the TME of JEM-1200EX and H-600.
5、Total DNA abstraction of cells and agarose gel electrophoresis
After collecting cells (5×10~6-1×10~8), we threw away the superstratum, used thecold PBS 1-10ml to resuspense the cells, 500-1000rpm, 5min, centrifugation, then gotrid of the superstratum, and repeated it again. The cells were resuspend with 500μlnucleus splitting fluid, 50℃aqueous bathed and shocked for 3-5 hours, then13000rpm/min centrifugated, extrated the aqueous phase into another centrifuge tube,added 1/10 volumed 3MNaCl and double volumed ethanol, reversed the mixture forseveral times. Centrifugated for 10min, 12000rpm/min, then deposit the DNA,abandoned the superstratum, and rinsed for 1-2 times by 70% etnanil, dried theremaining fluid. 50μl TE buffer with 100μg/ml RNAseA was added for 30min in 65℃.Get the fluid 8μl, added by the buffer sample, 1.8% agarose electrophoresis (voltage1-5V/cm), then we used automatic electrophoresis gel image analysis and tookphotographs.
6、Western blotting detected GAP-associated protein Bcl-2, Bax, P53,survivin of A2 cells
Treated cells were rinsed with PBS, centrifuged and added cracked buffer of 700μl.After 12000rpm, 4℃centrifuged for 1 hours, superstratum was collected and thedensity of the protein was detected. After adding the sample buffer into the sample, boiling for 5 rain in bulliens aqua. Used 10% SDS-PAGE to have an electrophoresiswith BIORAD electrophoresis board, the condition of double board is: 150V, 40mA.
Colloxylin equilibrated in the met-stamp liquid for 10min, then following the rulethat gel is in negative pole while membrane is in the other pole, after the 50V, 2 hoursmet-stamp, we rinsed it 5 min with TBS for 2 times. Blocked it with the TBS whichcontaining the 5% degreased milk powder, then rinsed with TTBS 2 times, each timefor 5min, then transferred into Bcl-2, Bax, P53, and survivin, first antibody -4℃overnight. Incubated with the secondary antibody, then washed for 2 times, each time15min, transferred into the secondary antibody of Sheep anti-Mouse IgG-HRP (1:5000), Sheep anti-Rabbit IgG (1:5000), Horse anti-Sheep IgG (2:5000) andincubated for 2 hours. Detection was carried out using ECL chemiluminescence system,X-ray sensitization. Mixed the A and B liquid according to the concentration of0.125ml/cm~2. Exposed the X-ray film in a dark room, the developed 5min and fixed for5min.
Result
1、MTT-medicine sensitivity test
The inhibition of the Cru to A2 cells was concentration dependent. The value of theIC50 for 48 hours was 40μmol/L.
2、Morphology of apoptosis cells
We observed the cells under the TEM, founded the structures of nucleus,chondrosome and the endoplasmic reticulum in the A2 cells of the control group clearly,and also we could see the nucleus have taken the most part of the cell. After adding theCru into the cells, the nucleus crimpled, cracked without karyon membrane embedded,chromosome gathered to cell membrane, and the changes of the ultramicrostructure ofthe cell transformed to apoptosis bodies which were forming or have already formed.
3、Annexin-FITC detected quantificationally apoptosis cells
We collected the cells treated with 40μmol/L Cru at 24h, 48h, 72h and normalcontrols, then marked Annexin-FITC and analyzed by flow cytometry to examine theapoptosis of the cells. We found that the proportion of A2 apoptosis cells were lowerafter Cru was added for 24h, 48h, (9.45%, 16.89%), while the proportion at 72h washigher (21.05%).
4、DNA agarose gel electrophoresis
We collected the cells treated with 40μmol/L Cru about 24h, 48h, 72h and normalcontrols, then DNA agarose gel electrophoresis was executed. There was a "ladderstrip" appearance. At 72h, most of cells were necrosis and DNA of these cellsdegraded unregularly, then a continuous "membrane strip" appeared. However,because the molecular of DNA of normal cells was large and moved short distance, so they remained near the adding holes.
5、Western blotting
The cells which were treated with 40μmol/L Cru at 24h, 48h, 72h and normalcontrols were collected, then exacted the total protein, detected the expression of theapoptosis associated protein P53, Bcl-2, Bax, survivin, and we found that Bax and P53were increased gradually with time extending, while Bcl-2, surviving were decreased.
Part Two Researches of the effection andmechanism of the Cru for the angiogenesis
Method
1、Cell culture
The RPMI1640 medium with 10% (vol/vol), 56℃inactivated fetal bovine serum,have been cultured in the 37℃saturated humidity, 5%CO2 incubatons. The HUVEC,A2cells with monolayer adherence grew. We took the logarithm of the cells in thegrowing period in the experiment.
2、Observing the effection of the Cru to the proliferation of HUVECsThe cells in the growing period had been collected and digested, and regulated into the1*105/ml monolayer-cell suspension, inoculated into the cultivating plates with 96holes. After 24 hours, by adding the 100μl RPMI1640 culture fluid within different Crudensities, the terminal densities of the Cru were 5, 10, 20, 40, 60, 80,100 and 160μl, 3 replies holes in each group. 48 hours later, 5mg/ml MTT20μl were added into the holes,then after 4 hours, blotting the clear superstratum lipid away, adding the DMSO fluid150μl, shaking the plates for 10min, then the OD value had been detected by enzymemark, getting the wave length 490nm. We had to repeat the experiments for 3 times.Cell proliferation inhibited rate (%)=[1-(OD value of the experimental group-ODvalue of the blank group)/(OD value of the negative control group-OD value blankgroup)]×100%
3、Annexin-FITC detected quantificationally apoptosis cells
The cells had been digested by the 0.25% trypsin, and collected after boasting, thenwashed in the PBS for 1 time, and 1000rpm, 10 min centrifugalizated. We threw thesuperstratum, added the combining buffer, resuspense the cells, and modulated the cellsinto 5×10~5/ml, then took 195μl resuspensory fluid, added Annexin-FITC 5μl, mixed inthe room temperature, incubated for 10min. Next, we took the combining buffer 200μl,washed the cells, 1000rpm, 10 min centrifugalizated. Throwing the superstratum away,we added the combining buffer 190μl, resuspense the cells again, then added the20μg/ml Propidium Iodide 10μl. Then we detected the expression of thediacylglyceryl-phosphorylserine binding protein in the outer surface of the cellularmembrane and the content of the DNA in the death cells.
Cell cycle synchronization
4、Detecting the mRNA level of the Ang-1, Ang-2 and the TSP byusing the reverse transcription PCR
After throwing the medium away, we added the Tripure in the bottles, and thenflew afloat the cells for several times, until the cells had dropped completely, thenmoved the fluid into the centrifuge tubes, placed for 5min. Added the chloroform as0.2ml per 1ml Tripure, shocked intensely for 15 seconds, then placed for 5min. Aftercentrifugating in 4℃, 12000rpm for 15rain, we took the upper colorless aqueous phase,added the -20℃dimethylcarbinol which had been colded as the ratio that 0.5ml perlml Tripure, placed upside down for several times, then placed in the environment of-20℃for 10min, centrifugated and threw the superstratum away. Then we added the-20℃, 75% ethanol which had been already colded as the ratio lml per 1ml Tripure,shocked turbulencely for 5min, then centriguagted for 5min. Waiting for the ethanolvolatilized spontaneously, we moved the total RNA into the 80ml DEPC, 55℃waterbath for 15min to help the dissolved to resuspense. Detected the purity of the total RNAusing the ultrabiolet apectra scanner. Reverse transcription: 2μl total RNA in each tubeto be taken as the template, with the reaction volume was 25μl. Added the up anddown-stream primers of Ang-1, Ang-2 and TSP separately, and the up anddown-stream primers ofβ-actin protein 1μl, 10 times density of the PCR buffer 25μl,TaqDNA polymerase 0.5μl in each tube, then added the water till 25μl. The conditions of amplification of the TSP were degeneration in 94℃for 30 seconds, regnaturation in60℃for 40 seconds, extension in 72℃for 30 seconds, took the process for 30 times.The conditions of amplification of the Ang-1, Ang-2 were degeneration in 94℃for 30seconds, regnaturation in 60℃for 30 seconds, extension in 72℃for lmin, also 30times. Analyzed the production of the PCR: taking the PCR production electrophoresisin the 2% agarose gel, then ratio of the result of the electrophoresis of the Ang-1,Ang-2 and the TSP to the absorbance of theβ-actin strap were used to indicate therelative expression intensities of the mRNA of the Aug-1, Ang-2 and TSP.
5、Detecting the expression of the protein VEGF, MM-9 by usingWestern blot
Treated cells were rinsed with PBS, centrifuged and added cracked buffer of 700μl.After 12000rpm, 4℃centrifuged for 1 hours, superstratum was collected and thedensity of the protein was detected. After adding the sample buffer into the sample,boiling for 5 min in bulliens aqua. Used 10% SDS-PAGE to have an electrophoresiswith BIORAD electrophoresis board, the condition of double board is: 150V, 40mA.
Colloxylin equilibrated in the met-stamp liquid for 10min, then following the rulethat gel is in negative pole while membrane is in the other pole, after the 50V, 2 hoursmet-stamp, we rinsed it 5 min with TBS for 2 times. Blocked it with the TBS whichcontaining the 5% degreased milk powder, then rinsed with TTBS 2 times, each time for 5min, then transferred into VEGF, MMP-9, first antibody -4℃ovemight. Incubatedwith the secondary antibody, then washed for 2 times, each time 15min, transferred intothe secondary antibody of Sheep anti-Mouse IgG-HRP (1:5000), Sheep anti-RabbitIgG (1:5000), Horse anti-Sheep IgG (1:5000) and incubated for 2 hours. Detectionwas carried out using ECL chemiluminescence system, X-ray sensitization. Mixed theA and B liquid according to the concentration of 0.125ml/cm~2. Exposed the X-ray filmin a dark room, the developed 5min and fixed for 5min.
Result
1、Detect the result of HUVEC cells which had been treated by theCru by using MTT
It gave us a dose-effect relationship of the proliferation-inhibiting effection of the Cruto the HUVEC: there was significant inhibitory action of the Cru in different densitiesto the cultured-human vascular endothelial cells. With the increasing of the density andthe prolonging of the time, the inhibitory action was more obviously.
2、The influence of the Cru to the apoptosis of HUVECs
We detected the effections of the Cru to the apoptosis of the HUVECs in 5, 10, 20, 40,80, 160μmol/L by flowing cytometry. It was shown that the apoptosis rate were 4.12%,9.44%, 15.91%, 17.98%, 21.05%, 22.21% of the HUVECs 24 hours later, much morehigher than the 1.64% apoptosis rate in the control group.
3、RT-PCR
We collected the cells 6h, 12h, 24h, 48h after adding the 40μmol/L Cru and thecells in the control group, and excreted the RNA, then detected the expression of themRNA of the Ang-1, Ang-2 and TSP. We could see that the longer the action time ofthe Cru, the lower expression the mRNA of the Ang-1, Ang-2 than the control group,while the higher expression of the mRNA of TSP.
4、Western blotting
We collected the cells 6h, 12h, 24h, 48h after adding the 40μmol/L Cru and thecells in the control group, extracted the total protein, then detected, and we could seethat the expressions of VEGF and MMP-9 were observely lower than that of the controlgroup.
In short, the Cru could inhibit the angiogenesis by many signal transductions: (1)decreasing the tumor cell angiogenesis factors (VEGF, Ang-1, Ang-2) and/orincreasing the expression of the inhibitory factors (TSP-1) to regulate the balance ofthe angiogenesis factors, and inhibit the phenotype transformation of angiogenesis. (2)inhibiting the proliferation of the vascular endothelium, and promoting the apoptosis ofthem. (3) reducing the expression of the matrix metalloproteinase (MMP-9) to inhibitthe degradation of the basal membrane and the extracellular matrix.
Part Three The in vitro experimental researchof the radiotherapy sensitization of the humanpulmonary carcinoma cells (A2) effected by theCru
Method
1、Colony forming test
We got the cells which grew in the log phase, made them into the suspension bythe digestion of 0.25%trypase and 0.02%EDTA, then according to the different exposedoses, we inoculated different number of cells (cell densities) with fresh culture fluidinto the culture capsules. 24 hours later, we changed the culture fluid into the Cru with-different densities (1, 5, 10μmol/L ) cultured for another 24 and 48 hours, then in theroom temperature, we used the ~(137)Cs radioactive source with the doses were 0Gy, 2Gy,4Gy, 6Gy, 8Gy, 10Gy to irradiate, then changed the culture fluid of the medicine-usedgroup into the fluid that without the medicine and cultured for another 9 hours togetherwith the irradiate-only group. After 9 days, the cells were fixed with the 95% ethanoland stained by the Giemsa, then≥50 cells were marked as a survival colony, for eachmeasuring point, we took 3 times experiments. The colony experiments were convertedinto the survival fraction, the survival fraction of the simple medicine-used groupmeant the rate of the colony-formation-rate of the group comparing with colony-formation-rate of the blank group, then we took the survival fraction (SF) asthe standard, and rectified the SF of the medicin-irradiation group to get theradiosensitization effection of the medicines, then we calculated the Dq values of theirradiation-only (control) group and the medicine-used group by the multi-targetssingle knocked mathematical model fitting cell survival curve. The effection ofradiosensitization was indicated by the SER_(Dq).
2、Detecting the cell cycle by FACScan
The cells had been digested by the 0.25% trypsin, and collected after boasting, thenwashed in the PBS for 2 times, and 1000rpm, 10 min centrifugalizated. We threw thesuperstratum away, added the 70% (vol/vol) cold-ethanol, leaving them in -20℃, thenremoved the ethanol, washed for 2 times in PBS. Then, we added the 1 ml PBS withRNaseA (0.1mg/ml) in the cells, putting them into the 37℃incubatons for 30 min, andstained for 30 min in 1 ml PBS with 50μg/ml propidium iodie, 4℃, away from light. Atlast we analyzed the content of the DNA in the cells and the cell cycle by the method ofFACScan using the CELLQEST.
3、Detecting the cell apoptosis
When the cells were almost in the log phase, we digested the cells as a rule to makethem into the 0.5~*10~6 cell suspensions and then inoculated into the 6 holes inoculatingplates. Then we took one hole in a plate to add the DNSO culture fluid as the blank (the simple-irradiation group), while 5, 10, 15μmol/L Cru as the experimental groups(medicine and irradiation groups). 24 hours later, irradiated by dose sources of 2, 4, 6,8Gy, the cells were cultured for another 24 hours, then washed by the PBS for 2 times,digested by the trypsin completely, 1000r/min centrifuged for 5min and then we got thedensity of the cells into 10~6/ml by adding the PBS. Joined 200μl DNA-PREPTMLPRinto 100μl cell suspension, 30s later, added 2ml DNA-PREP-TM Stain and mixed.Avoided the light for 30min in room temperature, we detected the apoptosis of the cellsby the flowing cytometry, and deal with the results by the software of SystemⅡTMinvented by the Coulter Company. In each experiments, we took the negative andpositive controls.
4、DNA segments analysis
Took the method as we talked above, we culture the cells for another 24 hoursafter the A2 cells had been in the 10μmol/L Cru (medicine-irradiation group)or DMSO(simple irradiation group) for 24 hours and had been irradiated by the 4Gy. Then wedigested the cells by the trypsin and collected them, washed in PBS for 3 times,collected them after the centrifugalization, then extracted the genome DNA in thenormal way. Took 2μg sample DNA, then took the electrophoresis in the agarose gel(promeaga, USA) which containing 1.5% ethidium bromide, then took the photos andanalysed the DNA segments under the violet-perspection.
5、Detecting the cycle regulating factors and the expression of theNF-kB by the method of Western-blot
Treated cells were rinsed with PBS, centrifuged and added cracked buffer of 700μl.After 12000rpm, 4℃centrifuged for 1 hours, superstratum was collected and thedensity of the protein was detected. After adding the sample buffer into the sample,boiling for 5 min in bulliens aqua. Used 10% SDS-PAGE to have an electrophoresiswith BIORAD electrophoresis board, the condition of double board is: 150V, 40mA.Colloxylin equilibrated in the met-stamp liquid for 10min, then following the rule thatgel is in negative pole while membrane is in the other pole, after the 50V, 2 hoursmet-stamp, we rinsed it 5 min with TBS for 2 times. Blocked it with the TBS whichcontaining the 5% degreased milk powder, then rinsed with TTBS 2 times, each timefor 5min, then transferred into cyclinB1、P34cdc2、phos-P34cdc2、P21、survivin andNF-kB, first antibody -4℃overnight. Incubated with the secondary antibody, thenwashed for 2 times, each time 15min, transferred into the secondary antibody of Sheepanti-Mouse IgG-HRP (1:5000), Sheep anti-Rabbit IgG (1:5000), Horse anti-SheepIgG (1:5000) and incubated for 2 hours. Detection was carried out using ECLchemiluminescence system, X-ray sensitization. Mixed the A and B liquid according tothe concentration of 0.125ml/cm~2. Exposed the X-ray film in a dark room, thedeveloped 5min and fixed for 5min.
We maked the A2 cells synchronizated in the G_0 phase by the serum starvationmethod. Then we diluted the log phase growth cells in the culture flask into 1~*10~6/ml,adding the RPMI1640 medium without the serum, after 48 hours cultured in the 37℃,CO2 incubatons, the cells stayed in the G_0 phase. Then we threw the RPMI1640medium without the serum away, and got the medium with 10% fetal bovine serum in,then did the relative experiments after recovery phase.
Result
1、Cell cycle synchronization
Abandoned RPMI 1640 without serum, we obtained the cells synchronized at G_0 phrasethrough serum hungry, and cultured the cells with RPMI1640 containing 10% FBS. Weexamined the cells by flow cytometry every 3 hours. After 2 hours' convalescence, thecells reached the G_2 phase after 17 hours, and arrived at the end of the G_2 phase after 22hours. The whole cell cycle was about 23 hours.
2、Cell cycle block
40μmol/L Cru were added into each of 3 bottles of experimental groups of cellssynchronized at G_0 phase by serum hungry and convalesced for 2 hours. Then took 3bottles of cells 18 hours after the convalesced, added the Cru in all of them, getting thefinal concentration was 40μmol/L. A bottle of cells was collected every 3 hours,measured by flow cytometry. Cru acted on G1 phase inducing Glblocked slightly, acted on G2 phase causing G2 block, the cells staying at G2 no longer transiting G2/M.We could see the subdiploid peak of apoptosis at any period.
3、The influences to the A2 cells from the different factors
There were obviously subdiploid peaks of apoptosis before the G_0+G_1 peak in both themedicine-irradiation group and the simple irradiation group after the A2 cells had beentreated in these 2 groups, but in the medicine-irradiation group, the peak was moreobviously. So the intensities of the effection dealing with the apoptosis of A2 cells ofthese factors were: Cru+irradiation＞Cru or irradiation＞the contrast.
4、DNA agarose gel electrophoresis
We collected the cells treated with 40μmol/L Cru about 24h, 48h, 72h and normalcontrols, then DNA agarose gel electrophoresis was executed. There was a "ladderstrip" appearance. At 72h, most of cells were necrosis and DNA of these cellsdegraded unregularly, then a continuous " membrane strip " appeared. However,because the molecular of DNA of normal cells was large and moved short distance, sothey remained near the adding holes.
5、Western blotting
Took the cells 18 hours after the synchronization recovery phase, added the Cru to getthe final concentration 10μmol/L, 3 hours later, we collected the cells 18 and 21 hoursafter the recovery phase, then detected the cyclinB_1,P34~(cdc2), phos-P34~(cdc2), P21, survivin, NF-kB, and Western blotting. We found that the there were no change of theexpressions of the P34~(cdc2)、phos-P34~(cdc2), while the expressions of the cyclinB_1,survivin and NF-kB decreased and the P21 increased.
In a word, the Cru could increase the radiosensitiveness of the pulmonary carcinomacells, with the mechanism was that blocking the G_2/M of the A2 cells, inhibiting theexpression of NF-kB. The block of the cell cycle had relationship with the decreasedexpressions of cyclinB_1, survivin and the increased expression of P21.
Conclusion
1、Cru could induce the apoptosis of A2 cells. The apoptosis had the relationshipwith the decreased expressions of c-myc、bcl-2、survivin, and the increased expressionsof bax、fas、p53、Caspase-3.
2、Cru could inhibit the tumor angiogenesis by (1) decreasing the tumor cellangiogenesis factors (VEGF, Ang-1, Ang-2) and/or increasing the expression of theinhibitory factors (TSP-1) to regulate the balance of the angiogenesis factors, andinhibit the phenotype transformation of angiogenesis. (2) inhibiting the proliferation ofthe vascular endothelium, and promoting the apoptosis of them. (3) reducing theexpression of the matrix metalloproteinase (MMP-9) to inhibit the degradation of thebasal membrane and the extracellular matrix.
3、Cru could block the G_2/M of A2 cells specifically and decrease the expressionof NF-kB to increase the radiosensitiveness of the A2 cells.
肺癌是世界上发病率及死亡率较高的恶性肿瘤之一，手术是Ⅰ-Ⅱ期非小细胞肺癌的主要治疗手段，但至少有50％的术后患者会发生局部复发或远处转移，有70％的患者不适合再次手术治疗，放化疗及分子靶向治疗成为其主要的治疗手段，但长期使用毒性大费用高又影响其疗效。人们试图从中药有效成分中寻找一种高效、低毒，能抑制肿瘤细胞增殖诱导其凋亡且对放射具有增敏作用的药物，从而为肺癌的临床治疗提供一种新途径。
姜黄素(Curcumin，Cur)是从姜科姜黄属植物姜黄(curcumalonga)根茎中提取的一种酚类色素，大量研究表明，姜黄素具有抗炎、抗氧化、抗肿瘤等多种生物学功能，且价格低廉，毒性很低(小鼠LD_(50)为2g／Kg)，目前对其抗肿瘤作用的研究主要集中在肿瘤的化学预防方面。最近美国NCI已将其列为第三代防癌药进行临床研究。国内外对姜黄素抗促癌机制的研究主要集中在以下7个方面：①抗诱变及抗NO作用。②对表皮生长因子受体EGFR、PKC信号转导通路的影响。③下调核转录因子，抑制iNOS、COX-2活性。④调控癌基因和抑癌基因的表达。⑤诱导细胞周期阻滞。⑥抑制脲激酶(uPA)活性和MMP-9的分泌。⑦抑制肿瘤血管生成。
有关姜黄素对肺癌抗癌作用的研究国外仅限于通过调控肺癌细胞癌基因和抑癌基因表达促其凋亡及抑制血管生成因子表达来抑制肺癌血管生成等；而国内未见相关报道。本课题从诱导凋亡、抑制肿瘤血管生成、放射增敏三个方面观察姜黄素在体外对肺癌A2细胞的作用并通过检测Bcl-2、Bax、survivin、P53、Fas、VEGF、MMP-9、cyclinB_1、P34~(cdc2)、phos-P34~(cdc2)、P21及NF-kB蛋白表达活性改变及Ang-1、Ang-2和TSP的mRNA水平的变化从分子水平探讨其机制。
一、姜黄素抑制人肺癌细胞(A2)生长、诱导凋亡的作用及其机制
(一)实验方法
1、细胞培养
用含10％(vol／vol)56℃灭活胎牛血清的RPMI1640培养基，37℃饱和湿度、5％CO2的培养箱中培养，细胞呈单层贴壁生长，实验取对数生长期细胞。
2、MTT法药物敏感试验
消化细胞接种于96孔培养板，培养24h后，分别加入含不同浓度姜黄素的RPMI1640培养液100μl，使姜黄素的终浓度为5、10、20、40，60、80、100和160μmol／L，培养48h后，每孔加入5mg／mlMTT20μl，用酶标仪检测OD值，检测波长为490nm。采用SPSS统计分析软件，应用直线回归法计算IC_(50)。
3、Annexin-FITC标记法定量检测细胞凋亡
收集细胞，清洗，离心，加入结合buffer，重悬细胞。取195μl细胞重悬液，加5μl Annexin-FITC，室温孵育10分钟。清洗细胞，离心，弃上清，加入190μl结合buffer，重悬细胞。加入10μl 20μg／ml的Propidium Iodide。用FACScan检测细胞膜外表面磷脂酰丝氨酸结合蛋白的表达及死亡细胞DNA含量。
4、细胞凋亡的形态学检测
细胞经2.5％戊二醛、1％锇酸双固定，梯度乙醇和丙酮脱水，Epon812树脂包埋超薄切片机(AO型)切片，醋酸钠和柠檬酸铅双重染色，JEM-1200EX和H-600型透射电镜下观察并拍照。
5、细胞总DNA的提取及琼脂糖凝胶电泳
收集细胞，重悬，离心，去上清，重复一次；加入细胞裂解液500μl重悬细胞，50℃水浴3-5小时震荡，离心，吸取水相于另一离心管中，加入1／10体积3MNaCL和两倍体积乙醇混匀，沉淀DNA，去上清，70％乙醇漂洗1-2次，风扇吹干残存液体，加入50μl含100μg／mlRNAseA的TE缓冲液，65℃30 min；取8μl加上样缓冲液上样，1.8％琼脂糖凝胶电泳，采用1-5V／cm电压降，用凝胶成像系统观察和扫描成像。
6、Western blot检测A2细胞凋亡相关蛋白Bcl-2、Bax、P53、survivin、Fas
收集细胞，裂解，测蛋白浓度，定蛋白，10％SDS-PAGE电泳，转印，TBS封闭，转到Bcl-2、Bax、P53、Fas及survivin一抗4℃，过夜。二抗孵育。TTBS冲洗两次，每次15分钟，转到HRP-标记的羊抗鼠IgG(1：5000)、羊抗兔IgG(1：5000)、马抗羊IgG(1：5000)的二抗中，孵育2小时。用ECL化学发光检测，X-片感光。
(二)结果
1、MTT法药物敏感试验
姜黄素对A2细胞的抑制作用呈浓度依赖性，计算48小时的IC50值为40μmol／L。
2、细胞凋亡的形态学检测
透射电镜下观察，对照组A2细胞中细胞核及线粒体、内质网等结构清晰，核占细胞体积的大部分。姜黄素作用于A2细胞后可见细胞核固缩、碎裂、无核膜包被；核染色质边集于胞膜，核孔消失；正在形成的凋亡小体和已形成的凋亡小体等超微结构改变。
3、Annexin-FITC标记法定量检测细胞凋亡
分别收集姜黄素40μmol／L作用24小时、48小时、72小时及正常对照组的细胞进行Annexin V-FITC标记结合流式细胞仪检测细胞凋亡。发现姜黄素作用24小时、48小时，A2细胞凋亡比例较低，分别为9.45％、16.89％；72小时细胞凋亡比例较高，为21.05％。
4、DNA琼脂糖凝胶电泳
分别收集姜黄素40μmol／L作用24小时、48小时、72小时及正常对照组的细胞进行DNA琼脂糖电泳。48小时，凋亡细胞的DNA由于DNA降解成规则的大片段出现凋亡特有的“梯状”条带，72小时，细胞大多已经坏死，坏死细胞由于其DNA的不规则降解显现一条连续的膜状条带。而正常活细胞DNA基因组条带由于分子量大，迁移距离短，所以停留在加样孔附近
5、Western blotting
分别收集姜黄素40μmol／L作用6小时、12小时、24小时、48小时及正常对照组的细胞，提取总蛋白，检测凋亡相关蛋白P53、Bcl-2、Bax、Fas、survivin的表达，发现随时间的延长，Bax、P53、Fas蛋白表达水平逐渐升高。而Bcl-2、survivin蛋白表达水平逐渐降低。
总之：姜黄素诱导肺癌细胞株A2细胞凋亡是多靶点，多途径的，其与凋亡相关基因的改变、细胞因子的分泌等密切相关。
二、姜黄素对血管生成的作用及其机制的研究
(一)实验方法
1、细胞培养
用含10％(vol／vol)56℃灭活胎牛血清的RPMI1640培养基，37℃饱和湿度、5％CO2的培养箱中培养，HUVEC、A2细胞呈单层贴壁生长，实验取对数生长期细胞。
2、MTT法观察姜黄素对HUVECs增殖的影响
细胞接种于96孔培养板，培养24h后，分别加入含不同浓度姜黄素的RPMI1640培养液100μl，使姜黄素的终浓度为5、10、20、40，60、80、100和160μmol／L，每组设三个复孔，培养48h后，每孔加入5mg／ml MTT 20μl，继续培养4h后，吸去上清液，加入150μl DMSO液，震荡培养板10min，用酶标仪检测OD值，检测波长为490nm。细胞增殖抑制率(％)=[1-(实验组的OD值-空白组的OD值)／(阴性对照组的OD值-空白对照组的OD值)×100％
3、Annexin-FITC标记法定量检测细胞凋亡
消化收集细胞，加入结合buffer，重悬细胞，调整细胞数为5×10~5／ml。取195μl细胞重悬液，加5μl Annexin-FITC，室温孵育，清洗细胞，离心后弃上清，加入190μl结合buffer，重悬细胞。加入10μl 20μg／ml的Propidium Iodide。用FACScan检测细胞膜外表面磷脂酰丝氨酸结合蛋白的表达及死亡细胞DNA含量。
4、逆转录PCR检测血管生成素1和血管生成素2(Ang-1、Ang-2)和血小板反应素(TSP)的mRNA水平
细胞总RNA提取，采用紫外光谱扫描仪检测总RNA纯度；逆转录；PCR产物分析：取PCR产物在2％琼脂糖凝胶上电泳，电泳结果以Ang-1、Ang-2和TSP分别与β-actin条带的吸光度比值表示Ang-1、Ang-2和TSP的mRNA的相对表达强度。
5、Western blot检测VEGF、MMP-9蛋白的表达
收集细胞，裂解，测蛋白浓度，定蛋白，10％SDS-PAGE电泳，转印，TBS封闭，转到VEGF、MMP-9一抗4℃，过夜。二抗孵育。TTBS冲洗两次，每次15分钟，转到HRP-标记的羊抗鼠IgG(1：5000)、羊抗兔IgG(1：5000)、马抗羊IgG(1：5000)的二抗中，孵育2小时。用ECL化学发光检测，X-片感光。
(二)结果
1、姜黄素处理HUVEC细胞后MTT法检测结果
姜黄素对HUVEC细胞增殖抑制作用呈量效关系：不同浓度姜黄素对培养的人血管内皮细胞的增殖有显著抑制作用。随着姜黄素浓度的增加，作用时间的延长，其抑制作用增强。
2、姜黄素对HUVECs凋亡的影响
流式细胞术检测浓度为5，10，20，40，80，160μmol／L姜黄素对HUVECs凋亡的影响，结果显示，作用24h后HUVECs细胞凋亡率分别为4.12％，9.44％，15.91％，17.98％，21.05％，22.21％，明显高于对照组1.64％的凋亡率。
3、RT-PCR
分别收集姜黄素40μmol／L作用6小时、12小时、24小时、48小时及正常对照组的细胞，提取RNA，检测血管生成素-1(Ang-1)、血管生成素-2(Ang-2)及血小板反应素(TSP)的mRNA的表达，可见随姜黄素作用时间的延长Ang-1、Ang-2 mRNA的表达明显低于对照组；TSP的mRNA的表达高于对照组。
4、Western blotting
分别收集姜黄素40μmol／L作用6小时、12小时、24小时、48小时及正常对照组的细胞，提取总蛋白，检测VEGF及MMP9蛋白的表达显著低于对照组。
总之，姜黄素可能通过多种信号途径抑制血管生成：①降低肿瘤细胞血管生成因子(VEGF，Ang-1，Ang-2)和(或)提高抑制因子(TSP-1)的表达水平来调控血管生成因子的平衡，抑制血管生成的表型转换。②抑制血管内皮细胞的增殖，促进内皮细胞的凋亡。③减少基质金属蛋白酶(MMP-9)的表达从而抑制基底膜及细胞外基质的降解。
三、姜黄素对人肺癌(A2)细胞放疗增敏的体外实验研究
(一)实验方法
1、集落形成试验
接种细胞于培养皿中，培养24小时后更换含不同浓度的姜黄素(1、5、10μmol／L)培养液继续培养24小时和48小时后，在室温下用~(137)Cs放射源照射，照射剂量分别为0Gy、2Gy、4Gy、6Gy、8Gy、100y，照射后用药组更换为不含药物的培养液与单纯照射组一起继续培养9天。9天后细胞用95％乙醇固定，姬母萨染色，≥50个细胞记数为一个存活集落，对每个测量点实验均重复三次。集落实验被换算成存活分数，单纯加药组的存活分数为单纯加药组的集落形成率与空白对照组的集落形成率之比，以单纯加药组的存活分数(SF)为基准，对加药照射组的SF进行校正从而得到药物的放射增敏作用。以多靶单击数学模型拟合细胞存活曲线，计算出单纯照射组(对照组)和加药照射组(药物处理组)的Dq值。放射增敏效应以放射增敏比SER_(Dq)表示。
2、FACScan检测细胞周期
消化收集细胞，在PBS中清洗，离心，弃上清，加入70％(vol／vol)的冷乙醇，于-20℃放置，PBS洗两次，细胞中加入含RN aseA(0.1mg／ml)的PBS 1ml，置培养箱中，37℃、30分钟。然后细胞在含有50μg／ml propidium iodie的PBS 1ml中染色。用FACScan进行细胞DNA含量和细胞周期分析，所用软件为CELLQEST。
3、细胞凋亡检测
细胞接种于6孔培养板中，设空白对照(即单纯放射组)，实验组(即药物加放射组)浓度分别为5、10、15μmol／L的姜黄素，培养24h接受2、4、6、8Gy照射后，继续培养24h，取100μl细胞悬液，加入DNA-PREPTMLPR 200μl混匀，30s后加入DNA-PREP-TM染色剂(PI染色)2ml混匀。流式细胞仪检测细胞凋亡情况。利用Coulter公司的SystemⅡTM软件处理结果，每次实验均设阴性和阳性对照。
4、蛋白免疫印迹法(Western-blot)检测周期调控因子及NF-kB蛋白水平的表达
收集细胞，裂解，测蛋白浓度，定蛋白，样品加入样品缓冲液，10％SDS-PAGE电泳，转印，TBS封闭，封闭后TTBS洗2次，每次5分钟，转到cyclinB_1、P34~(cdc2)、phos-P34~(cdc2)、P21、survivin及NF-kB一抗4℃，过夜。二抗孵育。TTBS冲洗两次，每次15分钟，转到HRP-标记的羊抗鼠IgG(1：5000)、羊抗兔IgG(1：5000)、马抗羊IgG(1：5000)的二抗中，孵育2小时。用ECL化学发光检测，X-片感光。
(二)结果
1、细胞周期检测
血清饥饿法同步在G0期的细胞，弃无血清RPMI1640培养基，加入含10％胎牛血清RPMI1640培养基继续培养。每3小时作一次流式细胞仪检测。在经过2小时恢复期后，约在17小时到达G2期，并在22小时到达G2末期。整个细胞周期大约23个小时左右。
2、细胞周期阻滞
取血清饥饿法同步在G0期细胞，经2小时恢复期后，分别向实验组的3瓶细胞每瓶中加入姜黄素40μmol／L。再取恢复期后18小时的3瓶细胞，每瓶细胞中加入姜黄素，使终浓度为40μmol／L。每3小时收集一瓶细胞，进行流式细胞仪检测。加入姜黄素后，作用于G1期引起轻度G1期阻滞；作用于G2期，引起G2期阻滞，细胞停留在G2期，不发生G2／M期转换。各时间均可见凋亡标志的亚二倍体峰。
3、不同处理因素对A2细胞凋亡的影响
单纯放射组和药物加放射组分别处理A2细胞后，药物加放射组及单纯放射组均在G0+G1峰之前出现一个明显的亚二倍体峰(凋亡峰)，但以药物加放射组更为明显，各处理因素诱导A2细胞凋亡作用强度的顺序为：姜黄素+照射＞姜黄素或照射＞对照。
4、Western blotting
取同步化恢复期后18小时的细胞，加入姜黄素使终浓度为10μmol／L。作用3小时，再分别收集恢复期后18小时、21小时的细胞，检测cyclinB_1、P34~(cdc2)、phos-P34~(cdc2)、P21、survivin及NF-kB、蛋白印迹，在姜黄素处理3小时后，P34~(cdc2)、phos-P34~(cdc2)表达量没有改变，cyclinB_1、survivin及NF-kB蛋白表达量减少，P21蛋白表达量增加。
总之，姜黄素能增加肺癌细胞的放射敏感性，其机制可能为使A2细胞发生G2／M期阻滞，及抑制NF-kB蛋白的表达；细胞发生周期阻滞与cyclinB_1、survivin蛋白表达量减少，P21蛋白表达量增加有关。
1、姜黄素能诱导A2细胞凋亡，凋亡发生可能与c-myc、bcl-2、survivin蛋白表达下降；而bax、fas、p53、Caspase-3的蛋白表达升高相关。
2、姜黄素能抑制肿瘤血管生成其作用通过(1)降低肿瘤细胞血管生成因子(VEGF，Ang-1，Ang-2)和(或)提高抑制因子(TSP-1)的表达水平来调控血管生成因子的平衡，抑制血管生成的表型转换。(2)抑制血管内皮细胞的增殖，促进内皮细胞的凋亡。(3)减少基质金属蛋白酶(MMP-9)的表达从而抑制基底膜及细胞外基质的降解。
3、姜黄素能提高A2细胞的放射敏感性，可能通过相对特异地使A2细胞发生G2／M期阻滞及降低NF-kB蛋白表达来实现的。
Pulmonary carcinoma is one of the malignant tumors with high attack rate andmortality rate. For phaseⅠ-Ⅱof the non-small cell carcinoma, the main method fortreatment is operation. But the local recurrent or the distal metastasis may happen in atleast 50% of the patients after the operations, and 70% of these patients are not able totake the operation for the second time, then radiotherapy, the chemo, and the targetmolecule have to be the main treatment methods. But we know that when the patientstake these methods for a long time, there will be serious toxicity, large payment but badefferent. So now we are trying to get the drug with the better effect, lower toxicity,sensitization to the radiotherapy and can also inhibit the proliferation, indicate thenecrosis of the tumor cells from the traditional Chinese drugs, to find a new way for theclinical treatment of the pulmonary carcinoma.
Curcumin (Cur) is a kind of phenols pigment which is extracted from thecurcumalonga of the curcuma category of the ginger family. Quite a lot of researches have indicated that the Cur has several biological functions such as anti-inflammation,antioxygen, anti-oncoma, and also, it is cheap-price, low-toxicity (LD_(50) of mouses is2g/Kg). The researches at present mostly focus on the chemical prevention. Recently,the NCI of America has already studied it in clinic as the third generation of theanti-oncoma medicines. The researches on the Cur all over the world are most about the7 aspects below: 1. Anti mutation and anti-NO 2. Effecting on the pathway of the signaltransduction of the epithelial grow factor receptors EGFR, PKC. 3. Down-graduatingthe nuclear factor, inhibiting the activity of iNOS, COX-2.4. Graduating the expressionof the oncogenes and anti-oncogenes. 5. Inducing the block of the cell cycle. 6.Inhibiting the activity of uPA and the secretion of MMP-9. 7. Inhibiting the formationof blood vessels.
The researches of the Cur on the anti-oncoma effection to the pulmonarycarcinoma have been limited within the regulation of the expression of the oncogeneand anti-oncogene to induce apoptosis, or inhibiting angiogenesis of pulmonarycarcinoma by reducing the expression of the angiogenesis factors. But nocorresponding reports in China. We observe the Cru of its functions to the A2pulmonary carcinoma cells in vitro in 3 aspects: reducing the apoptosis, inhibiting theangiogenesis of pulmonary carcinoma, and radiosensitization. Also, in this subject, wewill discus the mechanism on the molecular level by detecting the active changes of the protein expression of the Bcl-2, Bax, surviving, P53, C-myc, VEGF, MMP-9, cyclinB_1,P34~(cdc2), phos-P34~(cdc2), P21 and NF-kB, and the change of the Ang-1, Ang-2, and TSP.
Part One The function and mechanism of the Curinhibiting the growth and reducing the apoptosisof the human pulmonary carcinoma cells (A2 cells)
Method
1、Cell culture
The RPMI1640 medium with 10% (vol/vol), 56℃inactivated fetal bovine serum,have been cultured in the 37℃saturated humidity, 5%CO2 incubatons. The cells withmonolayer adherence grew. We took the logarithm of the cells in the growing period inthe experiment.
2、Drug sensitivity test in MTT
The cells in the growing period had been collected and digested, and regulated intothe 1*105/ml monolayer-cell suspension, inoculated into the cultivating plates with 96holes. After 24 hours, by adding the 100μl RPMI1640 culture fluid within different Crudensities, the terminal densities of the Cru were 5, 10, 20, 40, 60, 80,100 and 160μl, 3replies holes in each group. 48 hours later, 5mg/ml MTT20μl were added into the holes,then after 4 hours, blotting the clear superstratum lipid away, adding the DMSO fluid 150μl, shaking the plates for 10 min, then the OD value had been detected by enzymemark, getting the wave length 490nm. We had to repeat the experiments for 3 times.Analyzing the results by SPSS, and then calculated the IC_(50) by the method of linearregession.
3、etecting the cell apoptosis by Annexin-FITC marking method
The cells had been digested by the 0.25% trypsin, and collected after boasting, thenwashed in the PBS for 1 time, and 1000rpm, 10 min centrifugalizated. We threw thesuperstratum, added the combining buffer, resuspense the cells, and modulated the cellsinto 5*10~5/ml, then took 195μl resuspensory fluid, added Annexin-FITC 5μl, mixed inthe room temperature, incubated for 10 min. Next, we took the combining buffer 200μl,washed the cells, 1000rpm, 10 min centrifugalizated. Throwing the superstratum away,we added the combining buffer 190μl, resuspense the cells again, then added the20μg/ml Propidium Iodide 10μl. Then we detected the expression of thediacylglyceryl-phosphorylserine binding protein in the outer surface of the cellularmembrane and the content of the DNA in the death cells.
4、Morphology detection of the cell apoptosis
The cells had been fixed by the 2.5% glutaraldhyde, 1% osmium tetroxide,dehydrated by the gradient ethanol, and the acetone, cutted by the Epon812 resinembedding ultrathin section cutter (AO type), stained by natrium aceticum and Lead Citrate, then observed and photographed under the TME of JEM-1200EX and H-600.
5、Total DNA abstraction of cells and agarose gel electrophoresis
After collecting cells (5×10~6-1×10~8), we threw away the superstratum, used thecold PBS 1-10ml to resuspense the cells, 500-1000rpm, 5min, centrifugation, then gotrid of the superstratum, and repeated it again. The cells were resuspend with 500μlnucleus splitting fluid, 50℃aqueous bathed and shocked for 3-5 hours, then13000rpm/min centrifugated, extrated the aqueous phase into another centrifuge tube,added 1/10 volumed 3MNaCl and double volumed ethanol, reversed the mixture forseveral times. Centrifugated for 10min, 12000rpm/min, then deposit the DNA,abandoned the superstratum, and rinsed for 1-2 times by 70% etnanil, dried theremaining fluid. 50μl TE buffer with 100μg/ml RNAseA was added for 30min in 65℃.Get the fluid 8μl, added by the buffer sample, 1.8% agarose electrophoresis (voltage1-5V/cm), then we used automatic electrophoresis gel image analysis and tookphotographs.
6、Western blotting detected GAP-associated protein Bcl-2, Bax, P53,survivin of A2 cells
Treated cells were rinsed with PBS, centrifuged and added cracked buffer of 700μl.After 12000rpm, 4℃centrifuged for 1 hours, superstratum was collected and thedensity of the protein was detected. After adding the sample buffer into the sample, boiling for 5 rain in bulliens aqua. Used 10% SDS-PAGE to have an electrophoresiswith BIORAD electrophoresis board, the condition of double board is: 150V, 40mA.
Colloxylin equilibrated in the met-stamp liquid for 10min, then following the rulethat gel is in negative pole while membrane is in the other pole, after the 50V, 2 hoursmet-stamp, we rinsed it 5 min with TBS for 2 times. Blocked it with the TBS whichcontaining the 5% degreased milk powder, then rinsed with TTBS 2 times, each timefor 5min, then transferred into Bcl-2, Bax, P53, and survivin, first antibody -4℃overnight. Incubated with the secondary antibody, then washed for 2 times, each time15min, transferred into the secondary antibody of Sheep anti-Mouse IgG-HRP (1:5000), Sheep anti-Rabbit IgG (1:5000), Horse anti-Sheep IgG (2:5000) andincubated for 2 hours. Detection was carried out using ECL chemiluminescence system,X-ray sensitization. Mixed the A and B liquid according to the concentration of0.125ml/cm~2. Exposed the X-ray film in a dark room, the developed 5min and fixed for5min.
Result
1、MTT-medicine sensitivity test
The inhibition of the Cru to A2 cells was concentration dependent. The value of theIC50 for 48 hours was 40μmol/L.
2、Morphology of apoptosis cells
We observed the cells under the TEM, founded the structures of nucleus,chondrosome and the endoplasmic reticulum in the A2 cells of the control group clearly,and also we could see the nucleus have taken the most part of the cell. After adding theCru into the cells, the nucleus crimpled, cracked without karyon membrane embedded,chromosome gathered to cell membrane, and the changes of the ultramicrostructure ofthe cell transformed to apoptosis bodies which were forming or have already formed.
3、Annexin-FITC detected quantificationally apoptosis cells
We collected the cells treated with 40μmol/L Cru at 24h, 48h, 72h and normalcontrols, then marked Annexin-FITC and analyzed by flow cytometry to examine theapoptosis of the cells. We found that the proportion of A2 apoptosis cells were lowerafter Cru was added for 24h, 48h, (9.45%, 16.89%), while the proportion at 72h washigher (21.05%).
4、DNA agarose gel electrophoresis
We collected the cells treated with 40μmol/L Cru about 24h, 48h, 72h and normalcontrols, then DNA agarose gel electrophoresis was executed. There was a "ladderstrip" appearance. At 72h, most of cells were necrosis and DNA of these cellsdegraded unregularly, then a continuous "membrane strip" appeared. However,because the molecular of DNA of normal cells was large and moved short distance, so they remained near the adding holes.
5、Western blotting
The cells which were treated with 40μmol/L Cru at 24h, 48h, 72h and normalcontrols were collected, then exacted the total protein, detected the expression of theapoptosis associated protein P53, Bcl-2, Bax, survivin, and we found that Bax and P53were increased gradually with time extending, while Bcl-2, surviving were decreased.
Part Two Researches of the effection andmechanism of the Cru for the angiogenesis
Method
1、Cell culture
The RPMI1640 medium with 10% (vol/vol), 56℃inactivated fetal bovine serum,have been cultured in the 37℃saturated humidity, 5%CO2 incubatons. The HUVEC,A2cells with monolayer adherence grew. We took the logarithm of the cells in thegrowing period in the experiment.
2、Observing the effection of the Cru to the proliferation of HUVECsThe cells in the growing period had been collected and digested, and regulated into the1*105/ml monolayer-cell suspension, inoculated into the cultivating plates with 96holes. After 24 hours, by adding the 100μl RPMI1640 culture fluid within different Crudensities, the terminal densities of the Cru were 5, 10, 20, 40, 60, 80,100 and 160μl, 3 replies holes in each group. 48 hours later, 5mg/ml MTT20μl were added into the holes,then after 4 hours, blotting the clear superstratum lipid away, adding the DMSO fluid150μl, shaking the plates for 10min, then the OD value had been detected by enzymemark, getting the wave length 490nm. We had to repeat the experiments for 3 times.Cell proliferation inhibited rate (%)=[1-(OD value of the experimental group-ODvalue of the blank group)/(OD value of the negative control group-OD value blankgroup)]×100%
3、Annexin-FITC detected quantificationally apoptosis cells
The cells had been digested by the 0.25% trypsin, and collected after boasting, thenwashed in the PBS for 1 time, and 1000rpm, 10 min centrifugalizated. We threw thesuperstratum, added the combining buffer, resuspense the cells, and modulated the cellsinto 5×10~5/ml, then took 195μl resuspensory fluid, added Annexin-FITC 5μl, mixed inthe room temperature, incubated for 10min. Next, we took the combining buffer 200μl,washed the cells, 1000rpm, 10 min centrifugalizated. Throwing the superstratum away,we added the combining buffer 190μl, resuspense the cells again, then added the20μg/ml Propidium Iodide 10μl. Then we detected the expression of thediacylglyceryl-phosphorylserine binding protein in the outer surface of the cellularmembrane and the content of the DNA in the death cells.
Cell cycle synchronization
4、Detecting the mRNA level of the Ang-1, Ang-2 and the TSP byusing the reverse transcription PCR
After throwing the medium away, we added the Tripure in the bottles, and thenflew afloat the cells for several times, until the cells had dropped completely, thenmoved the fluid into the centrifuge tubes, placed for 5min. Added the chloroform as0.2ml per 1ml Tripure, shocked intensely for 15 seconds, then placed for 5min. Aftercentrifugating in 4℃, 12000rpm for 15rain, we took the upper colorless aqueous phase,added the -20℃dimethylcarbinol which had been colded as the ratio that 0.5ml perlml Tripure, placed upside down for several times, then placed in the environment of-20℃for 10min, centrifugated and threw the superstratum away. Then we added the-20℃, 75% ethanol which had been already colded as the ratio lml per 1ml Tripure,shocked turbulencely for 5min, then centriguagted for 5min. Waiting for the ethanolvolatilized spontaneously, we moved the total RNA into the 80ml DEPC, 55℃waterbath for 15min to help the dissolved to resuspense. Detected the purity of the total RNAusing the ultrabiolet apectra scanner. Reverse transcription: 2μl total RNA in each tubeto be taken as the template, with the reaction volume was 25μl. Added the up anddown-stream primers of Ang-1, Ang-2 and TSP separately, and the up anddown-stream primers ofβ-actin protein 1μl, 10 times density of the PCR buffer 25μl,TaqDNA polymerase 0.5μl in each tube, then added the water till 25μl. The conditions of amplification of the TSP were degeneration in 94℃for 30 seconds, regnaturation in60℃for 40 seconds, extension in 72℃for 30 seconds, took the process for 30 times.The conditions of amplification of the Ang-1, Ang-2 were degeneration in 94℃for 30seconds, regnaturation in 60℃for 30 seconds, extension in 72℃for lmin, also 30times. Analyzed the production of the PCR: taking the PCR production electrophoresisin the 2% agarose gel, then ratio of the result of the electrophoresis of the Ang-1,Ang-2 and the TSP to the absorbance of theβ-actin strap were used to indicate therelative expression intensities of the mRNA of the Aug-1, Ang-2 and TSP.
5、Detecting the expression of the protein VEGF, MM-9 by usingWestern blot
Treated cells were rinsed with PBS, centrifuged and added cracked buffer of 700μl.After 12000rpm, 4℃centrifuged for 1 hours, superstratum was collected and thedensity of the protein was detected. After adding the sample buffer into the sample,boiling for 5 min in bulliens aqua. Used 10% SDS-PAGE to have an electrophoresiswith BIORAD electrophoresis board, the condition of double board is: 150V, 40mA.
Colloxylin equilibrated in the met-stamp liquid for 10min, then following the rulethat gel is in negative pole while membrane is in the other pole, after the 50V, 2 hoursmet-stamp, we rinsed it 5 min with TBS for 2 times. Blocked it with the TBS whichcontaining the 5% degreased milk powder, then rinsed with TTBS 2 times, each time for 5min, then transferred into VEGF, MMP-9, first antibody -4℃ovemight. Incubatedwith the secondary antibody, then washed for 2 times, each time 15min, transferred intothe secondary antibody of Sheep anti-Mouse IgG-HRP (1:5000), Sheep anti-RabbitIgG (1:5000), Horse anti-Sheep IgG (1:5000) and incubated for 2 hours. Detectionwas carried out using ECL chemiluminescence system, X-ray sensitization. Mixed theA and B liquid according to the concentration of 0.125ml/cm~2. Exposed the X-ray filmin a dark room, the developed 5min and fixed for 5min.
Result
1、Detect the result of HUVEC cells which had been treated by theCru by using MTT
It gave us a dose-effect relationship of the proliferation-inhibiting effection of the Cruto the HUVEC: there was significant inhibitory action of the Cru in different densitiesto the cultured-human vascular endothelial cells. With the increasing of the density andthe prolonging of the time, the inhibitory action was more obviously.
2、The influence of the Cru to the apoptosis of HUVECs
We detected the effections of the Cru to the apoptosis of the HUVECs in 5, 10, 20, 40,80, 160μmol/L by flowing cytometry. It was shown that the apoptosis rate were 4.12%,9.44%, 15.91%, 17.98%, 21.05%, 22.21% of the HUVECs 24 hours later, much morehigher than the 1.64% apoptosis rate in the control group.
3、RT-PCR
We collected the cells 6h, 12h, 24h, 48h after adding the 40μmol/L Cru and thecells in the control group, and excreted the RNA, then detected the expression of themRNA of the Ang-1, Ang-2 and TSP. We could see that the longer the action time ofthe Cru, the lower expression the mRNA of the Ang-1, Ang-2 than the control group,while the higher expression of the mRNA of TSP.
4、Western blotting
We collected the cells 6h, 12h, 24h, 48h after adding the 40μmol/L Cru and thecells in the control group, extracted the total protein, then detected, and we could seethat the expressions of VEGF and MMP-9 were observely lower than that of the controlgroup.
In short, the Cru could inhibit the angiogenesis by many signal transductions: (1)decreasing the tumor cell angiogenesis factors (VEGF, Ang-1, Ang-2) and/orincreasing the expression of the inhibitory factors (TSP-1) to regulate the balance ofthe angiogenesis factors, and inhibit the phenotype transformation of angiogenesis. (2)inhibiting the proliferation of the vascular endothelium, and promoting the apoptosis ofthem. (3) reducing the expression of the matrix metalloproteinase (MMP-9) to inhibitthe degradation of the basal membrane and the extracellular matrix.
Part Three The in vitro experimental researchof the radiotherapy sensitization of the humanpulmonary carcinoma cells (A2) effected by theCru
Method
1、Colony forming test
We got the cells which grew in the log phase, made them into the suspension bythe digestion of 0.25%trypase and 0.02%EDTA, then according to the different exposedoses, we inoculated different number of cells (cell densities) with fresh culture fluidinto the culture capsules. 24 hours later, we changed the culture fluid into the Cru with-different densities (1, 5, 10μmol/L ) cultured for another 24 and 48 hours, then in theroom temperature, we used the ~(137)Cs radioactive source with the doses were 0Gy, 2Gy,4Gy, 6Gy, 8Gy, 10Gy to irradiate, then changed the culture fluid of the medicine-usedgroup into the fluid that without the medicine and cultured for another 9 hours togetherwith the irradiate-only group. After 9 days, the cells were fixed with the 95% ethanoland stained by the Giemsa, then≥50 cells were marked as a survival colony, for eachmeasuring point, we took 3 times experiments. The colony experiments were convertedinto the survival fraction, the survival fraction of the simple medicine-used groupmeant the rate of the colony-formation-rate of the group comparing with colony-formation-rate of the blank group, then we took the survival fraction (SF) asthe standard, and rectified the SF of the medicin-irradiation group to get theradiosensitization effection of the medicines, then we calculated the Dq values of theirradiation-only (control) group and the medicine-used group by the multi-targetssingle knocked mathematical model fitting cell survival curve. The effection ofradiosensitization was indicated by the SER_(Dq).
2、Detecting the cell cycle by FACScan
The cells had been digested by the 0.25% trypsin, and collected after boasting, thenwashed in the PBS for 2 times, and 1000rpm, 10 min centrifugalizated. We threw thesuperstratum away, added the 70% (vol/vol) cold-ethanol, leaving them in -20℃, thenremoved the ethanol, washed for 2 times in PBS. Then, we added the 1 ml PBS withRNaseA (0.1mg/ml) in the cells, putting them into the 37℃incubatons for 30 min, andstained for 30 min in 1 ml PBS with 50μg/ml propidium iodie, 4℃, away from light. Atlast we analyzed the content of the DNA in the cells and the cell cycle by the method ofFACScan using the CELLQEST.
3、Detecting the cell apoptosis
When the cells were almost in the log phase, we digested the cells as a rule to makethem into the 0.5~*10~6 cell suspensions and then inoculated into the 6 holes inoculatingplates. Then we took one hole in a plate to add the DNSO culture fluid as the blank (the simple-irradiation group), while 5, 10, 15μmol/L Cru as the experimental groups(medicine and irradiation groups). 24 hours later, irradiated by dose sources of 2, 4, 6,8Gy, the cells were cultured for another 24 hours, then washed by the PBS for 2 times,digested by the trypsin completely, 1000r/min centrifuged for 5min and then we got thedensity of the cells into 10~6/ml by adding the PBS. Joined 200μl DNA-PREPTMLPRinto 100μl cell suspension, 30s later, added 2ml DNA-PREP-TM Stain and mixed.Avoided the light for 30min in room temperature, we detected the apoptosis of the cellsby the flowing cytometry, and deal with the results by the software of SystemⅡTMinvented by the Coulter Company. In each experiments, we took the negative andpositive controls.
4、DNA segments analysis
Took the method as we talked above, we culture the cells for another 24 hoursafter the A2 cells had been in the 10μmol/L Cru (medicine-irradiation group)or DMSO(simple irradiation group) for 24 hours and had been irradiated by the 4Gy. Then wedigested the cells by the trypsin and collected them, washed in PBS for 3 times,collected them after the centrifugalization, then extracted the genome DNA in thenormal way. Took 2μg sample DNA, then took the electrophoresis in the agarose gel(promeaga, USA) which containing 1.5% ethidium bromide, then took the photos andanalysed the DNA segments under the violet-perspection.
5、Detecting the cycle regulating factors and the expression of theNF-kB by the method of Western-blot
Treated cells were rinsed with PBS, centrifuged and added cracked buffer of 700μl.After 12000rpm, 4℃centrifuged for 1 hours, superstratum was collected and thedensity of the protein was detected. After adding the sample buffer into the sample,boiling for 5 min in bulliens aqua. Used 10% SDS-PAGE to have an electrophoresiswith BIORAD electrophoresis board, the condition of double board is: 150V, 40mA.Colloxylin equilibrated in the met-stamp liquid for 10min, then following the rule thatgel is in negative pole while membrane is in the other pole, after the 50V, 2 hoursmet-stamp, we rinsed it 5 min with TBS for 2 times. Blocked it with the TBS whichcontaining the 5% degreased milk powder, then rinsed with TTBS 2 times, each timefor 5min, then transferred into cyclinB1、P34cdc2、phos-P34cdc2、P21、survivin andNF-kB, first antibody -4℃overnight. Incubated with the secondary antibody, thenwashed for 2 times, each time 15min, transferred into the secondary antibody of Sheepanti-Mouse IgG-HRP (1:5000), Sheep anti-Rabbit IgG (1:5000), Horse anti-SheepIgG (1:5000) and incubated for 2 hours. Detection was carried out using ECLchemiluminescence system, X-ray sensitization. Mixed the A and B liquid according tothe concentration of 0.125ml/cm~2. Exposed the X-ray film in a dark room, thedeveloped 5min and fixed for 5min.
We maked the A2 cells synchronizated in the G_0 phase by the serum starvationmethod. Then we diluted the log phase growth cells in the culture flask into 1~*10~6/ml,adding the RPMI1640 medium without the serum, after 48 hours cultured in the 37℃,CO2 incubatons, the cells stayed in the G_0 phase. Then we threw the RPMI1640medium without the serum away, and got the medium with 10% fetal bovine serum in,then did the relative experiments after recovery phase.
Result
1、Cell cycle synchronization
Abandoned RPMI 1640 without serum, we obtained the cells synchronized at G_0 phrasethrough serum hungry, and cultured the cells with RPMI1640 containing 10% FBS. Weexamined the cells by flow cytometry every 3 hours. After 2 hours' convalescence, thecells reached the G_2 phase after 17 hours, and arrived at the end of the G_2 phase after 22hours. The whole cell cycle was about 23 hours.
2、Cell cycle block
40μmol/L Cru were added into each of 3 bottles of experimental groups of cellssynchronized at G_0 phase by serum hungry and convalesced for 2 hours. Then took 3bottles of cells 18 hours after the convalesced, added the Cru in all of them, getting thefinal concentration was 40μmol/L. A bottle of cells was collected every 3 hours,measured by flow cytometry. Cru acted on G1 phase inducing Glblocked slightly, acted on G2 phase causing G2 block, the cells staying at G2 no longer transiting G2/M.We could see the subdiploid peak of apoptosis at any period.
3、The influences to the A2 cells from the different factors
There were obviously subdiploid peaks of apoptosis before the G_0+G_1 peak in both themedicine-irradiation group and the simple irradiation group after the A2 cells had beentreated in these 2 groups, but in the medicine-irradiation group, the peak was moreobviously. So the intensities of the effection dealing with the apoptosis of A2 cells ofthese factors were: Cru+irradiation＞Cru or irradiation＞the contrast.
4、DNA agarose gel electrophoresis
We collected the cells treated with 40μmol/L Cru about 24h, 48h, 72h and normalcontrols, then DNA agarose gel electrophoresis was executed. There was a "ladderstrip" appearance. At 72h, most of cells were necrosis and DNA of these cellsdegraded unregularly, then a continuous " membrane strip " appeared. However,because the molecular of DNA of normal cells was large and moved short distance, sothey remained near the adding holes.
5、Western blotting
Took the cells 18 hours after the synchronization recovery phase, added the Cru to getthe final concentration 10μmol/L, 3 hours later, we collected the cells 18 and 21 hoursafter the recovery phase, then detected the cyclinB_1,P34~(cdc2), phos-P34~(cdc2), P21, survivin, NF-kB, and Western blotting. We found that the there were no change of theexpressions of the P34~(cdc2)、phos-P34~(cdc2), while the expressions of the cyclinB_1,survivin and NF-kB decreased and the P21 increased.
In a word, the Cru could increase the radiosensitiveness of the pulmonary carcinomacells, with the mechanism was that blocking the G_2/M of the A2 cells, inhibiting theexpression of NF-kB. The block of the cell cycle had relationship with the decreasedexpressions of cyclinB_1, survivin and the increased expression of P21.
Conclusion
1、Cru could induce the apoptosis of A2 cells. The apoptosis had the relationshipwith the decreased expressions of c-myc、bcl-2、survivin, and the increased expressionsof bax、fas、p53、Caspase-3.
2、Cru could inhibit the tumor angiogenesis by (1) decreasing the tumor cellangiogenesis factors (VEGF, Ang-1, Ang-2) and/or increasing the expression of theinhibitory factors (TSP-1) to regulate the balance of the angiogenesis factors, andinhibit the phenotype transformation of angiogenesis. (2) inhibiting the proliferation ofthe vascular endothelium, and promoting the apoptosis of them. (3) reducing theexpression of the matrix metalloproteinase (MMP-9) to inhibit the degradation of thebasal membrane and the extracellular matrix.
3、Cru could block the G_2/M of A2 cells specifically and decrease the expressionof NF-kB to increase the radiosensitiveness of the A2 cells.
引文
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54 Pal S, Choudhuri T, Chattopadhyay S, Bhattacharya A, Datta GK, Das T, Sa G. Mechanisms of curcumin-induced apoptosis of Ehrlich's ascites carcinoma cells. Biochem Biophys Res Commun.2001; 288(3): 658-665.
55 Gururaj AE, Belakavadi M, Venkatesh DA, Marme D, Salimath BP. Molccular mechanisms of anti-angiogenic effect of curcumin. Biochem Biophys Res Commun. 2002; 297(4): 934-942.
56 Goel A, Bolang CR, Chauhan DP, et al. Specific inhibition of cyclooxygenase-2(COX-2) expression by dietary curcumin in HT29 human colon cancer cells. Cancer Lett, 2001, 172(2): 111-118.
57 Sokoloski JA, Narayanan R, Sartoreli AC, et al. Enhancement by antisense oligomucleotides to NF-kappa B of the differentiation of HL-60 promyelocyt icleukemia cells induced by vitam in D3. Cancer Lett, 1998; 125: 157-164.
58 Biade Stobbe CC, Chaman JD, The instrinsic radiosensitivity of some tumor cells through their cell cycles.Radiation Res.1997; 147: 416
59 Owen DG, McNamee JP, Raaphorst GP, et al. Potentiation of cell killing by low-dose-rate radiation by camptothecin is related to an increase in the level of DNA double-strand breaks. Radiat Res. 2002; 157(2): 149-157.
60 Pocard M, Cherillard S, Villandy J .et al. Different P53 mutation produce distinct effects on the ability of colon carcinoma cells to become bloked at gils boundary after irradiation Oncogen. 1996; 12: 875
61 Meyn RE, Stephens LC, Ang KK, et al. Heterogeneity in the development of apoptosis in irradiated murine tumors of different histologies. Int J Radiat Biol Phys. 1993, 64: 583.
62 Clark AJ, Chan DC, Chen MY, Fillmore H, Dos Santos WG, Van Meter TE, Graf MR, Broaddus WC.Down-regulation of Wilms' tumor 1 expression in glioblastoma cells increases radiosensitivity independently of p53. J Neuro oncol. 2007 Jan 6; [Epub ahead of print]
63 Lang FF, Yung WK, Raju U, Libunao F, Terry NH, Tofilon PJ. Enhancement of radiosensitivity of wild-type p53 human glioma cells by adenovirus-mediated delivery of the p53 gene.J Neurosurg. 1998; 89(1):125-132.
64 FW Wang, Bx Wen, WB Yin,et al.Relation between apoptosis and expression of two genes in nasopharyngeal carcinoma. J Euro Radiat Oncol, 1997, Sup2: 59.
65 Ozturk B, Egehan I, Atavci S, et al. Pentoxifylline in prevention of radiation-induced lung toxicity in patients with breast and lung cancer; a double-blind randomized trial. Int J Radiat Oncol Biol Phys. 2004,58(1):213-9.
66 杨光伟，毛志达．放射增敏剂的临床研究．癌症，1997，16：74-75．
67 Kuttan R. Potential anticancer activity of turmeric(Curcuma longa). Cancer Lett, 1985; 29(1):197.
68 Xu JH, Chen YZ, Huang XW et al.Curcumin inducing apoptosis and inhibiting expression of P210 ber/abl protein in human chronic myelogenous leukemia K562 cells.FAS EBJ, 2000, 14(8):1516
69 Moragoda L, Jaszewski R, Maju mdar AR Curcumin induced modulation of cell cycle and apoptosis in gastric and colon cancer cells.Anticancer Res, 2001, 21(2A): 873
70 Radhakrishna Pillai G, Srivastava AS, Hassanein TI, Chauhan DP, Carrier E. Induction of apoptosis in human lung cancer cells by curcumin.Cancer Lett. 2004 May 28; 208(2): 163-170.
71 陈文鹃、陈燕、吴裕丹等．姜黄素调节HL-60细胞周期蛋白的表达的研究[J]．临床血液学杂志．2001，14(5)：215-217．
72 Doree M. Control of M-phase by maturation-promoting factor. Curr Opin Cell Biol, 1990, 2(2):269-273.
73 Gautier J, Solomon MJ, Booher RN, et al.CDC25 is a specific tyrosine phosphatase that directly activates P34CDC2. Cell. 1991, 67(1): 197-211.
74 刘莹，于秉治，宗志红等．PKC对小鼠受精卵发育的调控作用。中国生物化学与分子生物学学报．2000，16(3)：368-371．
75 Kao GD, McKenna WG, Maity A, et al.cyclinB1 availability is a rate-limiting component of the radiation-induced G2 delay in HeLa cells. Cancer Res. 1997, 57(4): 753-8.
76 Harper JW, Adami GR, Wei N, et al.The P21 Cdk-interacting protein Cipl is a potent inhibitor of G1 cyclin-dependent kinases.Cell. 1993, 75(4): 805-816.
77 Barboule N, Chadebech P, Baldin V, et al.Involvement of P21 in mitotic exit after paclitaxel treatment in MCF-7 breast adenocarcinoma cell line. Oncogene, 1997, 15(23): 2867-75.
78 Li F, Ambrosini G, Chu EY, et al. Control of apoptosis and mitotic spindle checkpoint by surviving. Nature. 1998, 396(6711): 580-4.
79 Ambrosini G, Adida C, Sirugo G, et al. Induction of apoptosis and inhibition of cell proliferation by survivin gene targeting. J Biol Chem, 1998, 273(18), 11177-11182.
80 Reed JC. The Survivin saga gose in vivo.J Clin Invest, 2001,108: 965-969.
81 Sahidak UL, Lrich RK, Mikkelsen RB,et al.Radioation-induced proliferation of human A 431 squamous carcinoma cells is dependent on EGFR tyrosine phosphorylation [J]. Oncogene, 1997, 15(10): 1191-1197.
82 Ridder MD, Den Berge DV, Verovski VN, et al, NF-k b inhibition impairs the radioresponse of hypoxic EMT-6tumor cells through downregulation of inducible nitric oxide synthase. Br J Cancer, 2003, 88(1):120-124.
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2 Aggarwal BB, Kumar A, Bharti AC. Anticancer potential of curcumin: preclinical and clinical studies. Anticancer Res, 2003, 23(1A):363
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54 Pal S, Choudhuri T, Chattopadhyay S, Bhattacharya A, Datta GK, Das T, Sa G. Mechanisms of curcumin-induced apoptosis of Ehrlich's ascites carcinoma cells. Biochem Biophys Res Commun.2001; 288(3): 658-665.
55 Gururaj AE, Belakavadi M, Venkatesh DA, Marme D, Salimath BP. Molccular mechanisms of anti-angiogenic effect of curcumin. Biochem Biophys Res Commun. 2002; 297(4): 934-942.
56 Goel A, Bolang CR, Chauhan DP, et al. Specific inhibition of cyclooxygenase-2(COX-2) expression by dietary curcumin in HT29 human colon cancer cells. Cancer Lett, 2001, 172(2): 111-118.
57 Sokoloski JA, Narayanan R, Sartoreli AC, et al. Enhancement by antisense oligomucleotides to NF-kappa B of the differentiation of HL-60 promyelocyt icleukemia cells induced by vitam in D3. Cancer Lett, 1998; 125: 157-164.
58 Biade Stobbe CC, Chaman JD, The instrinsic radiosensitivity of some tumor cells through their cell cycles.Radiation Res.1997; 147: 416
59 Owen DG, McNamee JP, Raaphorst GP, et al. Potentiation of cell killing by low-dose-rate radiation by camptothecin is related to an increase in the level of DNA double-strand breaks. Radiat Res. 2002; 157(2): 149-157.
60 Pocard M, Cherillard S, Villandy J .et al. Different P53 mutation produce distinct effects on the ability of colon carcinoma cells to become bloked at gils boundary after irradiation Oncogen. 1996; 12: 875
61 Meyn RE, Stephens LC, Ang KK, et al. Heterogeneity in the development of apoptosis in irradiated murine tumors of different histologies. Int J Radiat Biol Phys. 1993, 64: 583.
62 Clark AJ, Chan DC, Chen MY, Fillmore H, Dos Santos WG, Van Meter TE, Graf MR, Broaddus WC.Down-regulation of Wilms' tumor 1 expression in glioblastoma cells increases radiosensitivity independently of p53. J Neuro oncol. 2007 Jan 6; [Epub ahead of print]
63 Lang FF, Yung WK, Raju U, Libunao F, Terry NH, Tofilon PJ. Enhancement of radiosensitivity of wild-type p53 human glioma cells by adenovirus-mediated delivery of the p53 gene.J Neurosurg. 1998; 89(1):125-132.
64 FW Wang, Bx Wen, WB Yin,et al.Relation between apoptosis and expression of two genes in nasopharyngeal carcinoma. J Euro Radiat Oncol, 1997, Sup2: 59.
65 Ozturk B, Egehan I, Atavci S, et al. Pentoxifylline in prevention of radiation-induced lung toxicity in patients with breast and lung cancer; a double-blind randomized trial. Int J Radiat Oncol Biol Phys. 2004,58(1):213-9.
66 杨光伟，毛志达．放射增敏剂的临床研究．癌症，1997，16：74-75．
67 Kuttan R. Potential anticancer activity of turmeric(Curcuma longa). Cancer Lett, 1985; 29(1):197.
68 Xu JH, Chen YZ, Huang XW et al.Curcumin inducing apoptosis and inhibiting expression of P210 ber/abl protein in human chronic myelogenous leukemia K562 cells.FAS EBJ, 2000, 14(8):1516
69 Moragoda L, Jaszewski R, Maju mdar AR Curcumin induced modulation of cell cycle and apoptosis in gastric and colon cancer cells.Anticancer Res, 2001, 21(2A): 873
70 Radhakrishna Pillai G, Srivastava AS, Hassanein TI, Chauhan DP, Carrier E. Induction of apoptosis in human lung cancer cells by curcumin.Cancer Lett. 2004 May 28; 208(2): 163-170.
71 陈文鹃、陈燕、吴裕丹等．姜黄素调节HL-60细胞周期蛋白的表达的研究[J]．临床血液学杂志．2001，14(5)：215-217．
72 Doree M. Control of M-phase by maturation-promoting factor. Curr Opin Cell Biol, 1990, 2(2):269-273.
73 Gautier J, Solomon MJ, Booher RN, et al.CDC25 is a specific tyrosine phosphatase that directly activates P34CDC2. Cell. 1991, 67(1): 197-211.
74 刘莹，于秉治，宗志红等．PKC对小鼠受精卵发育的调控作用。中国生物化学与分子生物学学报．2000，16(3)：368-371．
75 Kao GD, McKenna WG, Maity A, et al.cyclinB1 availability is a rate-limiting component of the radiation-induced G2 delay in HeLa cells. Cancer Res. 1997, 57(4): 753-8.
76 Harper JW, Adami GR, Wei N, et al.The P21 Cdk-interacting protein Cipl is a potent inhibitor of G1 cyclin-dependent kinases.Cell. 1993, 75(4): 805-816.
77 Barboule N, Chadebech P, Baldin V, et al.Involvement of P21 in mitotic exit after paclitaxel treatment in MCF-7 breast adenocarcinoma cell line. Oncogene, 1997, 15(23): 2867-75.
78 Li F, Ambrosini G, Chu EY, et al. Control of apoptosis and mitotic spindle checkpoint by surviving. Nature. 1998, 396(6711): 580-4.
79 Ambrosini G, Adida C, Sirugo G, et al. Induction of apoptosis and inhibition of cell proliferation by survivin gene targeting. J Biol Chem, 1998, 273(18), 11177-11182.
80 Reed JC. The Survivin saga gose in vivo.J Clin Invest, 2001,108: 965-969.
81 Sahidak UL, Lrich RK, Mikkelsen RB,et al.Radioation-induced proliferation of human A 431 squamous carcinoma cells is dependent on EGFR tyrosine phosphorylation [J]. Oncogene, 1997, 15(10): 1191-1197.
82 Ridder MD, Den Berge DV, Verovski VN, et al, NF-k b inhibition impairs the radioresponse of hypoxic EMT-6tumor cells through downregulation of inducible nitric oxide synthase. Br J Cancer, 2003, 88(1):120-124.
1 Surh YJ. Anti-tumor promoting potential of selected spice ingredients with antioxidative and anti-inflammatory activities: a short review. Food Chem Toxicol, 2002, 4 (8):1091
2 Aggarwal BB, Kumar A, Bharti AC. Anticancer potential of curcumin: preclinical and clinical studies. Anticancer Res, 2003, 23(1A):363
3 Ireson C, Orr S, Jones DJ, et al. Characterization of metabolites of the chemopreventive agent curcumin in human and rat hepatocytes and in the rat in vivo, and evaluation of their ability to inhibit phorbol ester-induced prosta glandin E2 production[J]. Cancer Res,2001,61(3): 1058
4 Chauhan DP. Chemotherapeutic potential of curcumin for colorectal cancer. Curr Pharm Des, 2002, 8(19):1695
5 Chen YS, Ho CC, Cheny CC, et al. Curcumin inhibited the arylamines N-acetyltransferase activity, gene expression and DNA adduct formation in human lung cancer cells (A549). Toxicol In Vitro, 2003,17(3):323
6 Lin CC, Lu YP, Lou YR, et al. Inhibition by dietary dibenzoylmethane of mammary gland proliferation, for mation of DMBA-DNA adducts in mammary glands, and mammary tumorigenesis in Sencar mice. Cancer Lett, 2001, 168(2):125
7 Pan MH, Chang WL, Lin Shiau SY, et al. Induction of apoptosis by garcinol and curcumin through cytochrome c release and activation of caspases in human leukemia HL 60 cells. J Agric Food Chem, 2001, 49(3):1464
8 Bush JA, Cheung KJ, Li G. Curcumin induces apoptosis in human melanoma cells through a Fas receptor/caspase-8 pathway independent of p53.Exp Cell Res, 2001, 271(2):305
9 Anto RJ, Mukhopadhyay A, Denning K, et al. Curcumin (diferuloylmethane) induces apoptosis through activation of caspase-8, BID cleavage and cytochrome c release: its suppression by ectopic expression of bcl-2 and bcl-xl. Carcinogenesis, 2002, 23(1):143
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