卵巢癌表观遗传学调控及其应用相关研究
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摘要
卵巢癌是导致美国妇女死亡第五位的妇科恶性肿瘤。卵巢癌致死性高的原因主要在于首次诊断即为晚期,病灶扩散至卵巢外,尽管手术后化疗可以取得较好的效果,但是只有30%患者在首次诊断后存活5年。目前卵巢癌面临的主要问题是缺乏早期诊断手段以及晚期远处转移并伴有化疗耐药。所以快速准确的早期诊断手段以及晚期有效安全的治疗策略是卵巢癌治疗的关键。
肿瘤的发生及发展由遗传学和表观遗传学共同决定。与遗传学不同的是,表观遗传学具有可逆性并能使细胞转换到正常状态,表观遗传调控网络的破坏可能是导致疾病的重要机制,包括染色体不稳定性引起的相关综合征以及精神发育迟缓等。通过对表观遗传学相关酶类位点药物研究发现,在表观遗传调控中,将表观遗传修饰改变作为治疗方法将和化疗一样成为肿瘤防治的重要手段。目前新的检测方法的发展对解释表观遗传改变引起的疾病提供了重要依据。随着表观遗传药物在治疗疾病过程中所发挥的作用,原来越多的表观修饰抑制剂的作用机理得到深入研究,特别是DNA甲基化转移酶抑制剂和组蛋白去乙酰化酶以及组蛋白去甲基化酶抑制剂。也有研究发现表观遗传药物对恶性肿瘤具有较好的抗肿瘤作用,
我们推测传统化疗药物联合表观遗传药物可以改善卵巢癌细胞的生物学行为,我们希望通过对卵巢癌细胞表观遗传修饰表达方式的研究,探讨联合用药和序贯用药的方法是否对卵巢癌分子生物学、细胞生物学、形态学及致瘤能力产生影响,以期为寻求新的卵巢癌治疗策略提供依据,从而达到有效抑制肿瘤生长的目的。
第一章表观遗传药物联合化疗药物对卵巢癌细胞株影响
目的：
通过表观遗传修饰药物及传统化疗药物对卵巢癌转移灶及腹水肿瘤细胞株生物学功能、表观遗传修饰、Spheroid形成的分析,试图寻找卵巢癌转移部位肿瘤对表观遗传修饰药物的反应,通过对表观遗传修饰规律的发现,为进一步改善传统化疗药物治疗效果,尤其是为有效腹水肿瘤及转移部位肿瘤的治疗提供依据。
方法：
1细胞准备及蛋白提取
1.1药物处理的细胞蛋白酶消化后,细胞转移到15ml离心管,将细胞用PBS冲洗一遍在1000转/分钟条件下离心5分钟,待提蛋白。
1.2总蛋白提取方案：准备1x提取液并将1ml提取液加入到4ml蒸馏水中,加入DTT溶液,并使DTT与提取液的比例为1：1000,将106粘附细胞重悬浮的细胞放入到100ul的冰冷1x提取液中,将含有细胞的溶液转移到1ml离心管中,冰上放置15分钟,并且每5分钟摇晃5秒,4-C条件下14000转/分钟离心细胞溶液10分钟,并将上清液转移到新的1ml离心管中,细胞蛋白提取液定量,将提取的细胞裂解液放置到-80℃冰箱备用。
1.3组蛋白提取方案：将细胞收取并在1000转/分钟4℃条件下离心5分钟,用蒸馏水将10x预裂解液稀释成1x,将106细胞重新悬浮在裂解液中放置在冰上10分钟,该过程中轻轻摇晃,然后在3000转4℃条件下离心5分钟,如果细胞裂解液的体积为1.5-2ml则需要在10,000转/分钟4℃条件下离心1分钟并丢弃上清液。将200ul的裂解液加入到含有离心沉淀的离心管中并在冰上放置30分钟,将离心管在12,000转/分钟4℃条件下放置5分钟,并将上清液转移到新的离心管中,准备DTT平衡溶液,将DTT溶液以1：500的比例配制(0.3mlDTT平衡液加入到1ml上清液中),用OD值进行蛋白定量,BSA作为标准对照,将获取的组蛋白提取液放置到-20℃中保存数天或者在-80℃中长期保存,避免反复冻融和冷冻。
1.4核蛋白提取方案：细胞消化待提取,按照1：20(例如3mlPBS加入到100mm培养板中)的比例将新鲜PBS加入到培养板中,将细胞刮掉并放置到15ml离心管中,在1000转/分钟条件下离心5分钟并丢弃上清液,将NE1溶液用水按照1：10比例稀释,将DTT和PIC以1：1000的比例加入到冰冷稀释1XNE1液中,将细胞按照106每100ul稀释NE1液的比例混合并转移到新的1ml离心管中,将离心管在冰上放置10分钟,每2分钟震荡10秒,随后在12,000转/分钟下离心1分钟,将细胞浆小心与细胞核沉淀分离,将DTT和PIC以1：1000的比例与NE2溶液混合,将2体积的含有DTT和PIC的NE2溶液加入到沉淀物中,将提取物在冰上放置15分钟并每3分钟震荡5秒,细胞提取物随后超声3x10秒为了增加核蛋白的提取效率,将提取液在14,000转4℃条件下离心10分钟,并将上清液转移到新的离心管中,对核蛋白进行定量,将提取液迅速的冷冻到-80℃冰箱中,避免反复冻融。
2 Western Blotting
2.1配胶：将灌胶架放置到试验台中,保持水平,将两块玻璃板放置到灌胶架中,保持短板面朝外,保证两块玻璃板在同一水平位置,将两块玻璃压紧,以保持密闭性,调整灌胶架以及玻璃板的位置,保持灌胶过程中胶在玻璃板中的位置适当,同时对玻璃板与低端橡胶带的衔接紧密。保持灌胶梳顺利插入到两块玻璃板之间,同时保持灌胶梳位置的正确。将分离胶加入到两块玻璃之间,在加入分离胶的过程中保持操作正确,用吸管将胶液缓慢的加入到两者之间,同时保持加入分离胶的量在合适的位置,加入500ul蒸馏水并放置45分钟,当分离胶凝固后,将蒸馏水用吸水纸洗掉,按照配方配制浓缩胶,将浓缩胶缓缓加入到分离胶上面并放置灌胶梳,待10分钟后将配好的胶移出并放置到电极装置中。
2.2电泳：将凝胶板按照电极装置说明放置,将凝胶板放置妥当后加入1X电泳缓冲液,将内部核心槽放置后加入400ml电泳缓冲液,按照测量的浓度将样品调整浓度,以保持样品蛋白含量相同,对于全细胞蛋白需要50-100ug,而核蛋白需要20-50ug,组蛋白需要50-100ug。根据试验需要将蛋白样品与6xSDS样品缓冲液液或者2xLaemmli混合,蛋白电泳标记物选择全蓝标记分子,需要体积为5u1,将需要电泳的样品在95-100℃中放置3分钟,将样品迅速旋转并加样,一旦胶发生凝固后即从灌胶架上移除并放置到电泳装置中,将电泳装置内室加入1x电泳缓冲液,同时将外室中加入1x电泳缓冲液,移除灌胶梳并加样,并在75v电压下保持1-2小时。
2.3转膜：根据具体实验条件调整,在胶电用完之前,准备好转膜缓冲液,同时在转膜之前5分钟,每块胶准备好2片滤纸和纤维纸,将需纤维素膜提前5分钟浸入到转膜缓冲液中,停止电泳并开始转膜,将胶板从电泳装置中移除,将两块玻璃板分离,将带有胶的玻璃板平稳放置到转膜缓冲液中,慢慢将胶从玻璃板上移除,在转膜缓冲液中放置10分钟将转膜架的黑色面朝下,放置顺序分别为：纤维纸垫、滤纸、胶、膜、滤纸、纤维纸垫。如果装置松散则可以增加滤纸以保持无气泡。将转膜架压紧并放置到转膜槽中,黑对黑,加入冰冻转膜缓冲液,转膜槽加满缓冲液。载电压30V 4℃过夜或者220V室温下1小时,保持整个过程处于冷冻状态。将转膜架打开,将膜平整的放置到干净容皿中,用清水冲洗2-4次,50ml封闭(5%无脂牛奶TBS-T溶液)液室温下封闭1小时,常温下TBST冲洗3次10分钟,加入一抗常温下孵育1小时或者4℃过夜。
2.4检测：常温下TBST冲洗3次10分钟,加入二抗常温下孵育1小时或者4℃过夜,常温下TBST冲洗3次10分钟,将膜上液体吸干放置到保鲜膜上,加入3ml显色剂,并孵育1分钟将显色剂去除,放置到保鲜膜中,随后放置到G:Box装置进行检测,图像获取及数据整理。
3 Spheroid形成试验
将细胞消化计数后按照500细胞/ml的浓度稀释在DMEM/Ham's F-12培养基中,然后根据要求将细胞稀释到Spheroid培养基Complete MammoCult TM Medium中并种植到低粘附培养板中,放置到37℃培养箱中培养,定期观察并拍照,所有Spheroid试验处理过程严格按照实验设计,根据不同的时间点进行图像拍摄,并在实验各个时间点获取Spheroid进行相关的细胞计数以及活性检测试验。
结果：
1药物处理对卵巢癌肿瘤生物学影响
卵巢癌细胞株Hey中无E-cadherin表达而N-cadherin强表达,Trichostatin A、Decitabine及Cisplatin单独处理可以轻度增加N-cadherin的表达,而不能增加E-cadherin的表达。Trichostatin A/Cispaltin组合及Decitabine/Cispaltin组合并不能改变N-cadherin的表达。所有药物处理组合对Pan-AKT及AKT-1无显著影响,表明可能通过其他非AKT信号通路影响细胞,Trichostatin A, Decitabine及Cisplatin单独应用及Trichostatin A/cisplatin联合用药对ABCG2表达无影响。decitabine/Cisplatin可以显著的抑制ABCG2的表达。干细胞标志分子OCT4在Hey中强表达,而NANOG和SOX2中度表达,药物处理无显著影响,凋亡蛋白分析结果显示单独应用Trichostatin A, Decitabine和Cisplatin可以显著提高Bax的表达。SKOV3中强表达E-cadherin,而N-cadherin表达较弱。单独应用Trichostatin A、Decitabine以及Cisplatin对E-cadherin及N-cadherin无显著影响,而联合用药TSA/Cisplatin, Decitabine/Cisplatin可以显著降低E-cadherin及N-cadherin的表达。单独应用药物Decitabine或者Trichostatin A可以激活AKT的表达,联合用药能够明显抑制AKT-1的表达Pan-AKT表达无显著改变。所有处理组均能够抑制ABCG2的表达。NANOG的表达在Decitabine/Cisplatin的作用下受到明显抑制,SOX2受到组合用药Trichostatin A/Cisplatin及Decitabine/Cisplatin抑制作用显著,且表达明显降低,这种现象在OCT4中也观察到。线粒体凋亡信号通路相关蛋白Bcl-2,Bcl-xl以及Bad无显著改变,TSA,Decitabine以及Cisplatin抑制Bax的表达,TSA/CSP和decitabine/CSP对Bax具有完全抑制作用。
2药物处理对卵巢癌细胞表观遗传学影响
DNMT3a和DNMT3b均在Hey细胞中表达。Cisplatin可以升高DNMT3a的表达。DNMT3b表达在处理前后无显著改变。结果显示所有处理对LSD1无影响。组蛋白甲基化进行分析结果显示药物处理对H3K4me2、H3K4me3、H3K9me2、H3K9me3无显著影响。SKOV3细胞中DNMT3a强表达而DNMT3b表达较弱。Trichostatin A, Decitabine以及Cisplatin能够抑制DNMT3a的表达,而Decitabine/Cisplatin的抑制效果更为显著,Trichostatin A/Cisplatin抑制效果处于两者之间。Trichostatin A/Cisplatin, Decitabine/Cisplatin可以明显抑制LSD1的表达,这与Hey细胞存在较大的差异。Trichostatin A/Cisplatin可以显著降低H3K4me2和H3K4me3的水平,Trichostatin A/Ccisplatin可以完全抑制H3K9me3的表达,Decitabine/Cisplatin轻度抑制H3K9me2,药物单独应用呈现出不同的表达方式。Trichostatin A, Decitabine及Cisplatin对H3K4rne2无明显影响,而Trichostatin A和Decitabine对H3K4me3轻度抑制。此外,单独用药及Decitabine/Cisplatin对H3K9me3也有较为明显的抑制作用。
3药物处理对卵巢癌细胞Spheroid形成的影响
Hey形成的Spheroid体积较大并且较为紧密。IOSE形成的Spheroid体积较大。SKOV3形成Spheroid较Hey Spheroid在细胞数目和体积都小,A2780形成的Spheroid不规则并且较为松散。Spheroid对药物的反应具有浓度和时间依赖性,药物组合Trichostatin A/Cisplatin, Decitabine/Cisplatin均能完全抑制Spheroid的形成,Spheroid形成后然后再用药物处理结果显示单药物应用不能抑制Spheroid的生长,而高剂量Cisplatin和Trichostatin A/Cisplatin, Decitabine/C isplatin显著抑制Spheroid的生长。Spheroid形成数目具有药物种类和浓度依赖性。TSA/Cisplatin组合可以获得与高浓度TSA及Cisplatin相同抑制效果。而在SKOV3细胞中,对照组相比,所有组别Spheroid在第5天无显著差异,随着培养天数的增加,TSA, Decitabine及Cisplatin处理组Spheroid生长受到完全抑制,细胞活性检测发现细胞全部发生凋亡。联合用药Trichostatin A/Cisplatin和Decitabine/Cisplatin组与单药处理类似,所有Spheriod形成及细胞活性受到完全抑制。在Spheroid生长抑制实验过程中,研究结果显示TrichostatinA组、Cisplatin5um组以及Trichostatin A/Cisplatin组完全抑制Spheroid的生长及细胞活性,而Decitabine组、Cisplatin低剂量组和Decitabine/Cisplatin组对Spheroid的生长无显著影响。这表明腹水肿瘤细胞团对TSA的反应同高剂量Cisplatin同样敏感。联合用药可以显著减少Spheroid形成数目,且SKOV3细胞Spheroid数目分析发现具有药物种类和浓度特异性。三种药物Trichostatin A, Decitabine和Cisplatin对Spheroid的抑制作用随着药物浓度的增加而增强。与对照组相比,药物组合Trichostatin A/Cisplatin可以使Spheroid形成降低8倍,而Decitabine/Cisplatin组只有5倍。更为有趣的是,Trichostatin A/Cisplatin组合可以获得与高浓度Cisplatin相似效果,这种联合应用的优点同Hey相似。
结论：
Hey细胞强表达N-cadherin且药物处理可以改变N-cadherin的表达。药物处理对ABCG2的影响最为显著,强表达多能性标志分子OCT4/NANOG/SOX2。DNMT及LSD1的表达无显著变化。药物Trichostatin A/CSP组合抑制Spheroid形成及生长能力显著,选择合适的抑制Spheroid形成能力的药物组合可能可以阻止肿瘤的转移而最大保护正常卵巢上皮细胞。与Hey细胞相比,在SKOV3细胞中联合用药较单独用药显著抑制E-cadherin及N-cadherin的表达,所有处理组都可以抑制ABCG2的表达,强表达OCT/NANOG/SOX2,而对线粒体凋亡相关蛋白无显著影响,高表达DNMT3a,而DNMT3b表达较弱,药物处理中联合用药对DNMT3a的效果较单独用药更为显著,联合用药可以显著抑制肿瘤LSD1的表达,而LSD1可以促进组蛋白的去甲基化,这就表明在DNA甲基化、组蛋白修饰以及DNA损伤机制方面存在相互联系,Trichostatin A/Cisplatin联合用药较单独用药可以显著抑制H3K4和H3K9甲基化表达水平,单独药物及联合用药都能完全抑制spheroid的形成,Trichostatin A/CSP单独或者联合用药都能够显著抑制Spheroid的生长。
第二章表观遗传药物联合化疗药物对卵巢癌细胞致瘤能力影响
目的：
依据上述实验结果并通过对临床病例的观察,建立卵巢癌动物模型,通过表观遗传修饰药物、传统化疗药物和联合用药疗法及序贯疗法对卵巢癌细胞肿瘤形成、生长以及腹腔内转移进行的分析,阐述表观遗传药物联合传统化疗药物对卵巢癌的抗肿瘤的效果。
方法：
1肿瘤细胞的准备
将肿瘤细胞hey种植到培养板中,当细胞生长到培养皿面积的70-80%时,用PBS冲洗去2遍,去除漂浮的细胞,加入蛋白酶-EDTA消化,将获取的细胞在1500转/分钟条件下离心2-5分钟并用PBS冲洗两次,随后用计数板对细胞进行计数,用台盼蓝检测细胞活性。将细胞悬浮到培养基中准备接种。
2老鼠准备4-6周龄老鼠,在试验前3天运抵。
3实验过程、后续观察及试验记录
根据实验需要,将10x106肿瘤细胞或者与处理过的肿瘤细胞皮下注射或腹腔注射,注射过程前做好准备工作,包括老鼠状态的观察体重的测量等基本情况记录,在实验过程中注意操作并做好标记,肿瘤种植过程后,密切观察动物反应,待肿瘤接种后第2天再次探望。将动物的状态做好详细记录,每天观察一次,对于状态不佳老鼠增加观察次数,带肿瘤形成后根据要求测量肿瘤大小以及动物荷瘤体重。对于序贯治疗老鼠,根据研究设计进行严格操作,以保证数据的客观性和可靠性。
4实验结束时,麻醉处死、记录观察、测量数据、分析结果。
5肿瘤体积计算公式=宽度2x长度x0.52。
结果：
1药物处理对卵巢癌细胞Hey成瘤能力的影响
药物组合Trichostatin A/Cisplatin组动物肿瘤体积较对照组减少约4倍。TSA0.3um组也有明显的肿瘤抑制效果且Decitabine 10um组和Cisplatin 1um组明显,Decitabine/Cisplatin抗肿瘤效果介于Decitabine 10um组和Cisplatin 1um组之间。从体积角度来看肿瘤的抑制能力顺序分别为：Trichostatin A/Cisplatin>Trichostatin A>Decitabine/ Cisplatin>Decitabine>control,从肿瘤重量角度来看肿瘤抑制能力顺序分别为：Trichostatin A/Cisplatin>Trichostatin A>Decitabine/Cisplatin>Decitabine>Cisplatin>control。肿瘤生长曲线提示Trichostatin A/Cisplatin联合用药较其他用药方案更能抑制肿瘤生长,并使肿瘤生长维持在较低的水平。
2药物处理对卵巢癌细胞Hey种植及转移能力的影响
根据对照组动物模型转移特点我们选取了十个部位作为参考,包括肝脏、脾、腹膜、肠系膜、大网膜、胃、盆腔壁、横隔、膀胱以及卵巢。实验结果显示TSA 0.3umm组和Cisplatin 1um组无肿瘤转移灶形成。而与此相对,Decitabine10um组,Trichostatin A/Cisplatin组及TSA/Decitabine组存在不同程度的转移灶形成。Decitabine 10um组主要转移部位有肝脏、脾、腹膜、肠系膜、横隔、膀胱以及卵巢。Trichostatin A/Cisplatin主要转移部位有肝脏、脾、大网膜以及膀胱,decitabine/Cisplatin主要转移部位有肝脏、脾、大网膜及卵巢。我们还观察了药物对种植后细胞转移能力的影响,结果显示所有组别的肿瘤均出现了转移。
3药物处理对卵巢癌细胞Hey肿瘤生长能力的影响
P53表达情况进行检测,结果显示CisplatinlowTrichostatin Ahigh、CisplatinloWDecitabinehigh、Trichostatin AlowCisplatinlow、DecitabinelowCisplatinlow可以导致P53升高。将卵巢癌细胞株种植到老鼠皮下待肿瘤形成后进行腹腔内药物治疗以观察药物对肿瘤生长能力的影响,结果发现肿瘤抑制能力的顺序分别：CisplatinlowDecitabinehigh>DecitabinelowCisplatinlow>CisplatinlowTrichostatin Ahigh>Control>Trichostatin AlowCisplatinlow>Trichostatin AhighDecitabinehigh。其中CisplatinlowDecitabinehigh抗肿瘤生长能力最强。
结论：
药物组合Trichostatin A/Cisplatin较单独用药更能抑制肿瘤生长和降低肿瘤负荷,并使肿瘤生长维持在较低的水平。Trichostatin A及Cisplatin处理过的细胞不能够在腹腔内形成肿瘤种植,其他药物组和联合用药具有肿瘤种植的器官特异性,序贯疗法Cisplatinlowdecitabinehigh抗肿瘤生长能力最强,DecitabinelowCisplatinlow治疗策略也具有较好的抗肿瘤效果。
Ovarian cancer is the fifth lethal cancer of female reproduction systerm malignancies in America, the high mortility dues to the late stage diagnosis when the cancer cells spread outside of the ovaries. Although good prognosis of the surgery following by chemotherapy could be achieved, but just 30% of the patients who could survive 5 years after the diagnosis, the challenage of the ovarian cancer is the lack of early detection methods and the metastasis and chemoresistance of late stage ovarian cancer cells, Detection in early stage and comphrehensive, safety and effective treatment strategies of ovarian cancer are two of the most important issues should be investigated in future.
Genetics and epigenetics work together to determine the fate and progression of human cancer, the epigenetics is different from genetics, it is heritable and reverseble and could change the statue of the cell from malignancy to normal and vice versa. The disturbance and ablation of the epigenetic regulation involve in the mechanism of most human disease, With the development of the research of epigenetics of human cancer, especially the DNA methylation, histone methylation and histone acetylation and other modifications, the epigenetic realated enzymes and proteins would become the target of the cancer treatment.
The disorder of the epigenetic statue could increase the instability of the genome and eventually cause cancer, So if the combination of the epigenetic modifiers and conventional chemotherapeutic reagents could make the cancer cell resensitizing to conventional chemotherapeutic reagents in ovarian cancer is unknown, but the application of the inhibitors of DNA methyltransferase, histone methyltransferase and histone deacetylase are becoming more and more in preclinical and clinical trials.
We assume that the combination and sequential treatment strategies could affect human ovarian cancer in aspect of molecular biology, cellular biology, morphology and tumorgenicity and would provide us a promising treatment strategy for ovarian cancer patients.
Chapter 1 Effect of combination of epigenetic modifiers and conventional chemotherapeutic reagent on human ovarian cancer cell line
OBJECTIVE:
To investigate the effect of the single or combination of epigenetic modifiers and conventional chemotherapeutic reagents on the tumor biology, epigenetic regulation, spheroid formation and growth of Hey cells, and to find the detailed mechanism of the response of Hey to epigenetic modification and try to figure out and improve the the management of metastasis and chemoresistance of the ovarain cancer.
METHODS:
1 Cell preparation and Isolation of protein
1.1 Cell preparation:Cells (treated or untreated) are growing to 80-90% confluence and trypsinized after removing growth medium. Cells are washed once with PBS and pelleted by centrifugation for protein isolation.
Total isolation of protein:Prepare 1X extraction buffer by adding 1 ml of 5X extraction buffer to 4 ml of distilled water. Add DTT solution and PIC to 1X extraction buffer at the 1:1000 ratios. Re-suspend cell pellet in 100μl of ice cold 1X extraction buffer per 106 adherent cells or 2 x 106 non-adherent cells. Transfer the cell solution to a microcentrifuge vial. Incubate on ice for 15 min with vigorous vortex 5 sec/5 min. Centrifuge the cell solution for 10 min at 14,000 rpm at 4℃and transfer the supernatant into a new microcentrifuge vial. Measure the protein concentration of cell extract. Use immediately or aliquot and freeze supernatant at -80℃until further use. Avoid repeated freezing/thawing.
1.2 Isolation of histone:Harvest cells and pellet the cells by centrifugation at 1000 rpm for 5 min at 4℃. Dilute 10X Pre-Lysis Buffer into 1X Pre-Lysis Buffer with distilled water at a 1:10 ratio. Re-suspend cells in the Diluted 1X Pre-Lysis Buffer at 106cells/ml and lyse cells on ice for 10min with gentle stirring. Centrifuge at 3000 rpm for 5 min at 4℃. If cell lysates are prepared in a 1.5ml to 2ml size vial, centrifuge at 10,000 rpm for lmin at 4℃, Remove supernatant. Re-suspend cell/tissue pellet in 3 volumes of Lysis Buffer and incubate on ice for 30 min. Centrifuge at 12,000 rpm for 5 min at 4℃and transfer the supernatant fraction (containing acid-soluble proteins) into a new vial. Prepare Balance-DTT Buffer by adding DTT Solution to Balance Buffer at a 1:500 ratio. Add 0.3 volumes of the Balance-DTT Buffer to the supernatant immediately. Quantify the protein concentration with an OD reader. BSA can be used as a standard. Aliquot and store the extract at -20℃for several days, or -80℃for long-term storage. Avoid repeated freezing/thawing.
1.3 Nuclear extraction:Growing cells to 70-80% confluence on a culture plate or flask. Remove the growth medium and wash cells with PBS twice and then remove PBS. Add lml of fresh PBS per 20 cm2 area, and scrape cells into a 15ml conical tube. (Alternative option:detach cells with trypsin-EDTA and collect cells into a 15ml conical tube. Count cells in a hemacytometer). Centrifuge the cells for 5min at 1000 rpm and discard the supernatant. Dilute NE1 with distilled water at a 1:10 ratio. Add DTT solution and PIC to ice cold diluted NE1 (IX) at a 1:1000 ratio. Re-suspend cell pellet in 100μl of diluted NE1 per 106 cells and transfer to a micro centrifuge vial. Incubate on ice for 10min. Vortex vigorously for 10 sec and centrifuge the preparation for 1 min at 12,000 rpm. Carefully remove the cytoplasmic extract from the nuclear pellet. Add DTT solution and PIC to NE2 at a 1:1000 ratio. Add 2 volumes of NE2 containing DTT and PIC to nuclear pellet (about 10μl per 106 cells or per 2 mg tissues). Incubate the extract on ice for 15 min with vortex 5sec/ 3min. The extract (especially tissue extract) can be further sonicated for 3 X 10 sec to increase nuclear protein extraction. Centrifuge the suspension for 10 min at 14,000 rpm at 4℃and transfer the supernatant into a new microcentrifuge vial. Measure the protein concentration of the nuclear extract. Use immediately or aliquot and freeze the supernatant at -80℃until further use. Avoid repeated freezing/thawing.
2 Western Blotting Protocol
2.1 Making gel:Place a short plate on top of the spacer plate, Slide the two plates into the casting frame, keeping the short plate facing front. Insure both plates are flush at the bottom on a level surface. Lock the pressure cams to secure the glass plates. Engage the spring loaded lever and place the gel cassette assembly on the gray casting stand gasket. Insure the horizontal ribs on the back of the casting frame are flush against the face of the casting stand and the glass plates are perpendicular to the level surface. The lever pushes the Spacer Plate down against the gray rubber gaskets. Place comb in between glass plates. Only put teeth in between glass. Mark 1 cm below where teeth are on outside of glass plate. Pour separating gel into 50 ml beaker:see recipes below, Take up 7 ml of gel using 5 ml pipet. Put pipet on edge of glass and hold at a 90 degree angle and begin filling to mark made on glass plate. Add 500ul of H2O. Let sit for 45 minutes. When gel is polymerized, invert apparatus and empty H2O. Pour in stacking gel. Place comb into gel. Let sit for 45 minutes. Remove the gel cassette sandwich from the casting frame and place it into the electrode assembly with the short plate facing inward. Slide the gel cassette sandwich and electrode assembly into the clamping frame. Press down the electrode assembly while closing the two cam levers of the clamping frame. Lower into the mini tank and add 400mls of lx Running Buffer.
2.2 Sample preparation:Dilute sample if necessary in dilution buffer below (or RIP A),For whole cell preps, typically use 50-100ug of protein per lane (20-50ug for nuclear,50-100 for histone), For each sample, add desired amount of protein to a microcentrifuge tube followed by 6x SDS Sample buffer or 2x Laemmli. Note:BME should be added fresh to the loading buffer, For the protein markers, add 5ul of markers to tube and lul of 6x sample buffer or 5ul of 2x Laemmli buffer. (Note:if you want to be able to see the marker while the gel is running, load 7-8ul instead of 5ul. Incubate at 95-100℃for 3 minutes. Quick spin the sample and load on gel.
2.3 Running gel:Once gel is polymerized, remove gel sandwich from casting stand and insert into inner cooling core of running rig. Fill inner core with 1X running buffer. Fill outer chamber with 1X running buffer until half of the screws are covered. (make 400ml for mini rig,500ml/gel for Criterion), Blow out wells. Load samples. Run at 75 volts for 1-2 hours or longer depending on the amount of separation. Before gel is done running, get out 2-3 pyrex dishes and fill with transfer buffer. Soak 2 filter papers and 2 fiber pads per gel for at least 5 minutes. Soak membrane in transfer buffer for at least 5 minutes. Stop electrophoresis.
2.4 Membrane transfer:Remove gel. Separate plates. Use spacers to separate. For Criterion rig, use lid to help pop open the cassette. Cut off stacking gel/wells with razor or spacers. Put plates into transfer buffer and nudge gel off plate with spatula into the transfer buffer, Soak gel for 10 minutes. Open sandwich with black side down and stack in this order:fiber pad, filter paper, gel, membrane, filter paper, fiber pad. Make sure to roll out bubbles after gel, membrane and second fiter paper. Close and put into transfer chamber. Black to back. Put in frozen block. Fill transfer chamber with transfer buffer. Transfer at 30V overnight in the cold room or at 100V for 1-2 hours on bench, changing ice pack half way through if needed.
2.5 Washes and detection:Open sandwich. Put membrane in clean container. Rinse membrane with dH2O 2-4x. Block with 50mls 1 hr RT (Blocking buffer:5% non-fat dry milk in TBS-T), Wash with TBS-T 3×10min at RT, Add primary antibody and incubate for 1 hour at RT or overnight at 4C. Wash in TBS-T 3×10 minutes at RT. Add secondary antibody at 1:1000 in TBST with 5% milk and incubate for 1 hour at RT. Wash in TBS-T 3×10min at RT. Drip dry the membrane onto a paper towel and lie flat on a piece of Saran Wrap with the protein side facing up. Mix 3 mls of chemiluminescent substrate Reagent 1:1 and pour onto the membrane. Let incubate for 1 minute. Dump solution off of membrane and drip dry on paper towel. Cover membrane with saran wrap. Expose on G:box for 1 minute to 5 minute depending on the strength of signal.
3 Spheroid formation assay
The cells are cultured in 6/12/24/96 wells ultra-low attachment plates at a concentration of 500 cells/ml in 2 ml/well Complete MammoCultTM Medium in a humidified incubator. A minority population of cells will began to grow as multi-cellular spheres, as opposed to the majority of cells which will not survive. Cells are examined microscopically on a daily basis and growth rates recorded. All the treatment proceure are accroding to the experiment schematic representation and all the spheroid would be taken pictures and assayed in aspect of size, cell number, spheroid number and cell viability.
RESULTS:
1 The effect of the treatment on the tumor biology of Hey cells
Our data indicated that no E-cadherin has been detected in Hey cells, but strongly expression of N-cadherin. Trichostatin A, Decitabine and Cisplatin alone could slightly increased the expression of E-cadherin, but not E-cadherin. The combination of Trichostatin A/Cisplatin and Decitabine/Cisplatin could not change the E-cadherin expression. There is no significanlty change of Pan-AKT and AKT-1 in presence of drugs, The combination of Decitabine/Cisplatin could significantly suppress the ABCG2, but not found in other treatment group. The pluripotency markers OCT4 strongly expression in Hey cells, but moderate expression of NANOG and SOX2, No effect of treatment on the expression of these pluripotency markers. Mitochondrial apoptosis pathway protein Bax has been increased after the single drug treatment. Our data indicated that strongly E-cadherin has been detected in Hey cells, but weak expression of N-cadherin. Trichostatin A, Decitabine and Cisplatin alone could not increase the expression of E-cadherin, but the combination of Trichostatin A/Cisplatin and Decitabine/Cisplatin could significantly change the E-cadherin and N-cadherin expression, Trichostatin A and decitabine could increase AKT1, but the combination of decitabine/Cisplatin could suppress the AKT1,All the treatment significantly suppress the ABCG2, Decitabine/Cisplatin suppress the expression of NANOG, but SOX2 and OCT4could be affected by both combiantion, Bax could be suppressed by Trichostatin A/Cisplatin and Decitabine/Cisplatin.
2 Effect of the treatment on epigenetic regulation of Hey cell line
DNMT3a and DNMT3b could be detected in Hey cells and Cisplatin could slightly increase the expression of DNMT3a, but not of DNMT3b, No effect on LSD1 and histone methylation markers. DNMT3a is strongly expression in SKOV3 cells but slightly expression of DNMT3b, Trichostatin A, Decitabine and Cisplatin could decrease the expression of DNMT3a, but Decitabine/Cisplatin has more suppress effect on DNMT3a, Interestingly, Trichostatin A/Cisplatin and Decitabine/Cisplatin could totally suppress LSD1. The effect of drugs on the histone methylation is diversity and complexity.
3 Effect of the treatment on spheroid formation
The Hey spheroid is big and compact in compare with the IOSE, SKOV3 and A2780. Interestingly, the A2780 spheroid is loose and out of shape, the formation of the spheroid has the features of time and dose response in presence of the drugs, and the combination of Trichostatin/Cisplatin and Decitabine/Cisplatin could totally suppress the shperoid formation. The combination strategies also could suppress the spheroid growth and the number of spheroid formation. The Spheroid formation of SKOV3 in presence of different treatment strategies has time and dose dependent effect and all the strategies significantly suppress the formation of Spheroid, in the spheroid growth assay. Trichostatin A, Cisplatin 5um and Trichostatin A/Cisplatin totally suppressed the Spheroid growth and Spheroid cell viability, but Decitabine,Cisplatin lum and Decitabine/Cisplatin has no significant effect, The combination of Trichostatin A/Cisplatin could suppress the Spheroid number in 8-fold in compare with control group. So the combination could achieve great Spheroid inhibition effect.
CONCLUSION:
High expression of N-cadherin could be detected in Hey cell line and the it could be changed in presence of drugs alone or combination, the ABCG2 has been significantly suppressed by the decitabine/CSP combination and pluripotent markers OCT4/NANOG/SOX2 highly express. No significantly inhibition of DNMT and LSD1 and even the histone methylation marks, TSA/CSP blcoked the formation and growth of spheroid, and the optimal strategy could not only kill the cancer cells but also have the protection effect of normal epithelial surficial ovarain cells. The combination of Trichostatin, Decitabine and Cisplatin could significantly supppress the expression of E-cadherin and N-cadherin in SKOV3 cells, The ABCG2 could be totally suppressed by all treatment. Strongly expression of pluripotency markers OCT/NANOG/SOX2 could be found, No effect has been detected on the mitochondrial apoptosis pathway, DNMT 3a was highly expressed but not of DNMT3a, Interestingly, The Trichostatin A/Cisplatin and Decitabine/Cisplatin could significantly suppress the LSD1. But the expression of histone methylation markers showed more diversity and complexity, Methylation of H3k4 and H3K9 reduced in presence of Trichostatin A/Cisplatin. Moreover, Drug alone or combination could totally suppress the formation and growth of spheroid of SKOV3.
Chapter 2 Effect of combination of epigenetic modifiers and conventional chemotherapeutic reagent on human ovarian cancer cell line Hey tumorigenicity in mouse model
OBJECTIVE:
To establish the animal model of ovarian tumor formation, growth, abdominal metastasis and the effect of treatment of combination and sequential on tummorgenecity and behavior of ovarain cancer, try to find a promising strategy to cure human ovarain cancer.
METHODS
1 Preparation of tumor cells
Growing cells in complete medium and when cells reach 70-80% confluent, replace medium with fresh medium to remove dead and detached cells when 3-4 hrs before harvesting. Remove medium and wash cells with could PBS. Add a minimum amount of trypsin-EDTA. Disperse cells and add complete medium (10:1 to 5:1). Centrifuge immediately at 1500 rpm for 2-5 min and wash twice with PBS and store cells. Count cells using a hemocytometer. Using typan blue staining to exclude dead cells. Mix cells 1:1 with typan blue solution. Cells should be suspended in a volume of 300μl contains required number of cells per injection.10×106 cells are needed per injection per mouse.
2 Preparation of mice. Mice should be 4-6 weeks old. Allow 3-5days acclimatization period after mice have arrived.
3 Preparation of injection Clean and sterilize the inoculation area of the mice with ethanol and/or iodine solutions. Use lml syringe and a 27- or 30-gauge needle. Mix cells and draw the cells into a syringe without a needle. Inject cells(10×106) subcutaneously (s.c.) or Intraperitoneal injection into the lower flank of the mice or abdominal. The animal tumor assay 1 and 2 should be terminated after 2wks and the assay 3 therapy should start after 1 weeks when the tumors reach an average volume of 0.5cm×0.5cm. Tumor diameters are measured by digital calipers, and the tumor volume is calculated by the formula:Volume= width×2×length×0.52.
RESULTS:
1 Effect of the treatment on tumorgenicity of Hey cells in mice
In compare with control, The Trichostatin A/Cisplatin treated cells could suppress the tumor size 4-fold and Trichostatin A 0.3um group also has notable suppression effect, the sequence of the tumor suppression is Trichostatin A/Cisplatin>Trichostatin/Cisplatin>Decitabine/Cisplatin>decitabine>control, from weight aspect:Trichostatin A/Cisplatin>Trichostatin >Decitabine/Cisplatin>Decitabine>Cisplatin>control, the tumor growth curve also showed the same effect.
2 Effect of the treatment on metastasis of Hey cells in mice
We establised the animal model and selected ten sites as indicator to analysis the effect of the treatment on the metastasis and implantation of ovarain cancer cell Hey in mouse metastasis model. These sites are liver, spleen, omentum, mesenchy, diaphage, stomach, bladder, abdominal wall, pelvic cavity and ovaries, we found that Trichostatin A 0.3um and Cisplatin lum totally suppress the metastasis, and Decitabine, Trichostatain A/Cisplatin and Decitabine/Cisplatin have organ specific patterns.
3 Effect of the sequential treatment on the tumor growth inhibition
According to the expression of P53, we selected CSPlowdecitabinehigh, DecitabinelowCisplatinlow, CisplatinlowTrichostatinAhigh, TrichostatinAlowCisplatinlow as sequential treatment to look at if these strategies could suppress the growth of tumor, The results indicated that CisplatinlowDecitabinehigh has the most tumor growth inhibition effect.
CONCLUSION:
Trichostatin A/Cisplatin has the most tumor growth suppression and tumor burden effect and maintain the tumor growth in a low level in a mice tumorgenicity model, Trichostatin A and Cisplatin alone could totally suppress the metastasis in the tumor metastasis model, but this assay need double confirm in the future and try to figure out the detailed mechanism. The sequential treatment of CisplatinlowDecitabinehigh has the most tumor growth inhibition effect, DecitabinelowCisplatinlow also could suppress the growth, it indicated that the combinationof Decitabine and Cisplatin has the synergistic effect.
肿瘤的发生及发展由遗传学和表观遗传学共同决定。与遗传学不同的是,表观遗传学具有可逆性并能使细胞转换到正常状态,表观遗传调控网络的破坏可能是导致疾病的重要机制,包括染色体不稳定性引起的相关综合征以及精神发育迟缓等。通过对表观遗传学相关酶类位点药物研究发现,在表观遗传调控中,将表观遗传修饰改变作为治疗方法将和化疗一样成为肿瘤防治的重要手段。目前新的检测方法的发展对解释表观遗传改变引起的疾病提供了重要依据。随着表观遗传药物在治疗疾病过程中所发挥的作用,原来越多的表观修饰抑制剂的作用机理得到深入研究,特别是DNA甲基化转移酶抑制剂和组蛋白去乙酰化酶以及组蛋白去甲基化酶抑制剂。也有研究发现表观遗传药物对恶性肿瘤具有较好的抗肿瘤作用,
我们推测传统化疗药物联合表观遗传药物可以改善卵巢癌细胞的生物学行为,我们希望通过对卵巢癌细胞表观遗传修饰表达方式的研究,探讨联合用药和序贯用药的方法是否对卵巢癌分子生物学、细胞生物学、形态学及致瘤能力产生影响,以期为寻求新的卵巢癌治疗策略提供依据,从而达到有效抑制肿瘤生长的目的。
第一章表观遗传药物联合化疗药物对卵巢癌细胞株影响
目的：
通过表观遗传修饰药物及传统化疗药物对卵巢癌转移灶及腹水肿瘤细胞株生物学功能、表观遗传修饰、Spheroid形成的分析,试图寻找卵巢癌转移部位肿瘤对表观遗传修饰药物的反应,通过对表观遗传修饰规律的发现,为进一步改善传统化疗药物治疗效果,尤其是为有效腹水肿瘤及转移部位肿瘤的治疗提供依据。
方法：
1细胞准备及蛋白提取
1.1药物处理的细胞蛋白酶消化后,细胞转移到15ml离心管,将细胞用PBS冲洗一遍在1000转/分钟条件下离心5分钟,待提蛋白。
1.2总蛋白提取方案：准备1x提取液并将1ml提取液加入到4ml蒸馏水中,加入DTT溶液,并使DTT与提取液的比例为1：1000,将106粘附细胞重悬浮的细胞放入到100ul的冰冷1x提取液中,将含有细胞的溶液转移到1ml离心管中,冰上放置15分钟,并且每5分钟摇晃5秒,4-C条件下14000转/分钟离心细胞溶液10分钟,并将上清液转移到新的1ml离心管中,细胞蛋白提取液定量,将提取的细胞裂解液放置到-80℃冰箱备用。
1.3组蛋白提取方案：将细胞收取并在1000转/分钟4℃条件下离心5分钟,用蒸馏水将10x预裂解液稀释成1x,将106细胞重新悬浮在裂解液中放置在冰上10分钟,该过程中轻轻摇晃,然后在3000转4℃条件下离心5分钟,如果细胞裂解液的体积为1.5-2ml则需要在10,000转/分钟4℃条件下离心1分钟并丢弃上清液。将200ul的裂解液加入到含有离心沉淀的离心管中并在冰上放置30分钟,将离心管在12,000转/分钟4℃条件下放置5分钟,并将上清液转移到新的离心管中,准备DTT平衡溶液,将DTT溶液以1：500的比例配制(0.3mlDTT平衡液加入到1ml上清液中),用OD值进行蛋白定量,BSA作为标准对照,将获取的组蛋白提取液放置到-20℃中保存数天或者在-80℃中长期保存,避免反复冻融和冷冻。
1.4核蛋白提取方案：细胞消化待提取,按照1：20(例如3mlPBS加入到100mm培养板中)的比例将新鲜PBS加入到培养板中,将细胞刮掉并放置到15ml离心管中,在1000转/分钟条件下离心5分钟并丢弃上清液,将NE1溶液用水按照1：10比例稀释,将DTT和PIC以1：1000的比例加入到冰冷稀释1XNE1液中,将细胞按照106每100ul稀释NE1液的比例混合并转移到新的1ml离心管中,将离心管在冰上放置10分钟,每2分钟震荡10秒,随后在12,000转/分钟下离心1分钟,将细胞浆小心与细胞核沉淀分离,将DTT和PIC以1：1000的比例与NE2溶液混合,将2体积的含有DTT和PIC的NE2溶液加入到沉淀物中,将提取物在冰上放置15分钟并每3分钟震荡5秒,细胞提取物随后超声3x10秒为了增加核蛋白的提取效率,将提取液在14,000转4℃条件下离心10分钟,并将上清液转移到新的离心管中,对核蛋白进行定量,将提取液迅速的冷冻到-80℃冰箱中,避免反复冻融。
2 Western Blotting
2.1配胶：将灌胶架放置到试验台中,保持水平,将两块玻璃板放置到灌胶架中,保持短板面朝外,保证两块玻璃板在同一水平位置,将两块玻璃压紧,以保持密闭性,调整灌胶架以及玻璃板的位置,保持灌胶过程中胶在玻璃板中的位置适当,同时对玻璃板与低端橡胶带的衔接紧密。保持灌胶梳顺利插入到两块玻璃板之间,同时保持灌胶梳位置的正确。将分离胶加入到两块玻璃之间,在加入分离胶的过程中保持操作正确,用吸管将胶液缓慢的加入到两者之间,同时保持加入分离胶的量在合适的位置,加入500ul蒸馏水并放置45分钟,当分离胶凝固后,将蒸馏水用吸水纸洗掉,按照配方配制浓缩胶,将浓缩胶缓缓加入到分离胶上面并放置灌胶梳,待10分钟后将配好的胶移出并放置到电极装置中。
2.2电泳：将凝胶板按照电极装置说明放置,将凝胶板放置妥当后加入1X电泳缓冲液,将内部核心槽放置后加入400ml电泳缓冲液,按照测量的浓度将样品调整浓度,以保持样品蛋白含量相同,对于全细胞蛋白需要50-100ug,而核蛋白需要20-50ug,组蛋白需要50-100ug。根据试验需要将蛋白样品与6xSDS样品缓冲液液或者2xLaemmli混合,蛋白电泳标记物选择全蓝标记分子,需要体积为5u1,将需要电泳的样品在95-100℃中放置3分钟,将样品迅速旋转并加样,一旦胶发生凝固后即从灌胶架上移除并放置到电泳装置中,将电泳装置内室加入1x电泳缓冲液,同时将外室中加入1x电泳缓冲液,移除灌胶梳并加样,并在75v电压下保持1-2小时。
2.3转膜：根据具体实验条件调整,在胶电用完之前,准备好转膜缓冲液,同时在转膜之前5分钟,每块胶准备好2片滤纸和纤维纸,将需纤维素膜提前5分钟浸入到转膜缓冲液中,停止电泳并开始转膜,将胶板从电泳装置中移除,将两块玻璃板分离,将带有胶的玻璃板平稳放置到转膜缓冲液中,慢慢将胶从玻璃板上移除,在转膜缓冲液中放置10分钟将转膜架的黑色面朝下,放置顺序分别为：纤维纸垫、滤纸、胶、膜、滤纸、纤维纸垫。如果装置松散则可以增加滤纸以保持无气泡。将转膜架压紧并放置到转膜槽中,黑对黑,加入冰冻转膜缓冲液,转膜槽加满缓冲液。载电压30V 4℃过夜或者220V室温下1小时,保持整个过程处于冷冻状态。将转膜架打开,将膜平整的放置到干净容皿中,用清水冲洗2-4次,50ml封闭(5%无脂牛奶TBS-T溶液)液室温下封闭1小时,常温下TBST冲洗3次10分钟,加入一抗常温下孵育1小时或者4℃过夜。
2.4检测：常温下TBST冲洗3次10分钟,加入二抗常温下孵育1小时或者4℃过夜,常温下TBST冲洗3次10分钟,将膜上液体吸干放置到保鲜膜上,加入3ml显色剂,并孵育1分钟将显色剂去除,放置到保鲜膜中,随后放置到G:Box装置进行检测,图像获取及数据整理。
3 Spheroid形成试验
将细胞消化计数后按照500细胞/ml的浓度稀释在DMEM/Ham's F-12培养基中,然后根据要求将细胞稀释到Spheroid培养基Complete MammoCult TM Medium中并种植到低粘附培养板中,放置到37℃培养箱中培养,定期观察并拍照,所有Spheroid试验处理过程严格按照实验设计,根据不同的时间点进行图像拍摄,并在实验各个时间点获取Spheroid进行相关的细胞计数以及活性检测试验。
结果：
1药物处理对卵巢癌肿瘤生物学影响
卵巢癌细胞株Hey中无E-cadherin表达而N-cadherin强表达,Trichostatin A、Decitabine及Cisplatin单独处理可以轻度增加N-cadherin的表达,而不能增加E-cadherin的表达。Trichostatin A/Cispaltin组合及Decitabine/Cispaltin组合并不能改变N-cadherin的表达。所有药物处理组合对Pan-AKT及AKT-1无显著影响,表明可能通过其他非AKT信号通路影响细胞,Trichostatin A, Decitabine及Cisplatin单独应用及Trichostatin A/cisplatin联合用药对ABCG2表达无影响。decitabine/Cisplatin可以显著的抑制ABCG2的表达。干细胞标志分子OCT4在Hey中强表达,而NANOG和SOX2中度表达,药物处理无显著影响,凋亡蛋白分析结果显示单独应用Trichostatin A, Decitabine和Cisplatin可以显著提高Bax的表达。SKOV3中强表达E-cadherin,而N-cadherin表达较弱。单独应用Trichostatin A、Decitabine以及Cisplatin对E-cadherin及N-cadherin无显著影响,而联合用药TSA/Cisplatin, Decitabine/Cisplatin可以显著降低E-cadherin及N-cadherin的表达。单独应用药物Decitabine或者Trichostatin A可以激活AKT的表达,联合用药能够明显抑制AKT-1的表达Pan-AKT表达无显著改变。所有处理组均能够抑制ABCG2的表达。NANOG的表达在Decitabine/Cisplatin的作用下受到明显抑制,SOX2受到组合用药Trichostatin A/Cisplatin及Decitabine/Cisplatin抑制作用显著,且表达明显降低,这种现象在OCT4中也观察到。线粒体凋亡信号通路相关蛋白Bcl-2,Bcl-xl以及Bad无显著改变,TSA,Decitabine以及Cisplatin抑制Bax的表达,TSA/CSP和decitabine/CSP对Bax具有完全抑制作用。
2药物处理对卵巢癌细胞表观遗传学影响
DNMT3a和DNMT3b均在Hey细胞中表达。Cisplatin可以升高DNMT3a的表达。DNMT3b表达在处理前后无显著改变。结果显示所有处理对LSD1无影响。组蛋白甲基化进行分析结果显示药物处理对H3K4me2、H3K4me3、H3K9me2、H3K9me3无显著影响。SKOV3细胞中DNMT3a强表达而DNMT3b表达较弱。Trichostatin A, Decitabine以及Cisplatin能够抑制DNMT3a的表达,而Decitabine/Cisplatin的抑制效果更为显著,Trichostatin A/Cisplatin抑制效果处于两者之间。Trichostatin A/Cisplatin, Decitabine/Cisplatin可以明显抑制LSD1的表达,这与Hey细胞存在较大的差异。Trichostatin A/Cisplatin可以显著降低H3K4me2和H3K4me3的水平,Trichostatin A/Ccisplatin可以完全抑制H3K9me3的表达,Decitabine/Cisplatin轻度抑制H3K9me2,药物单独应用呈现出不同的表达方式。Trichostatin A, Decitabine及Cisplatin对H3K4rne2无明显影响,而Trichostatin A和Decitabine对H3K4me3轻度抑制。此外,单独用药及Decitabine/Cisplatin对H3K9me3也有较为明显的抑制作用。
3药物处理对卵巢癌细胞Spheroid形成的影响
Hey形成的Spheroid体积较大并且较为紧密。IOSE形成的Spheroid体积较大。SKOV3形成Spheroid较Hey Spheroid在细胞数目和体积都小,A2780形成的Spheroid不规则并且较为松散。Spheroid对药物的反应具有浓度和时间依赖性,药物组合Trichostatin A/Cisplatin, Decitabine/Cisplatin均能完全抑制Spheroid的形成,Spheroid形成后然后再用药物处理结果显示单药物应用不能抑制Spheroid的生长,而高剂量Cisplatin和Trichostatin A/Cisplatin, Decitabine/C isplatin显著抑制Spheroid的生长。Spheroid形成数目具有药物种类和浓度依赖性。TSA/Cisplatin组合可以获得与高浓度TSA及Cisplatin相同抑制效果。而在SKOV3细胞中,对照组相比,所有组别Spheroid在第5天无显著差异,随着培养天数的增加,TSA, Decitabine及Cisplatin处理组Spheroid生长受到完全抑制,细胞活性检测发现细胞全部发生凋亡。联合用药Trichostatin A/Cisplatin和Decitabine/Cisplatin组与单药处理类似,所有Spheriod形成及细胞活性受到完全抑制。在Spheroid生长抑制实验过程中,研究结果显示TrichostatinA组、Cisplatin5um组以及Trichostatin A/Cisplatin组完全抑制Spheroid的生长及细胞活性,而Decitabine组、Cisplatin低剂量组和Decitabine/Cisplatin组对Spheroid的生长无显著影响。这表明腹水肿瘤细胞团对TSA的反应同高剂量Cisplatin同样敏感。联合用药可以显著减少Spheroid形成数目,且SKOV3细胞Spheroid数目分析发现具有药物种类和浓度特异性。三种药物Trichostatin A, Decitabine和Cisplatin对Spheroid的抑制作用随着药物浓度的增加而增强。与对照组相比,药物组合Trichostatin A/Cisplatin可以使Spheroid形成降低8倍,而Decitabine/Cisplatin组只有5倍。更为有趣的是,Trichostatin A/Cisplatin组合可以获得与高浓度Cisplatin相似效果,这种联合应用的优点同Hey相似。
结论：
Hey细胞强表达N-cadherin且药物处理可以改变N-cadherin的表达。药物处理对ABCG2的影响最为显著,强表达多能性标志分子OCT4/NANOG/SOX2。DNMT及LSD1的表达无显著变化。药物Trichostatin A/CSP组合抑制Spheroid形成及生长能力显著,选择合适的抑制Spheroid形成能力的药物组合可能可以阻止肿瘤的转移而最大保护正常卵巢上皮细胞。与Hey细胞相比,在SKOV3细胞中联合用药较单独用药显著抑制E-cadherin及N-cadherin的表达,所有处理组都可以抑制ABCG2的表达,强表达OCT/NANOG/SOX2,而对线粒体凋亡相关蛋白无显著影响,高表达DNMT3a,而DNMT3b表达较弱,药物处理中联合用药对DNMT3a的效果较单独用药更为显著,联合用药可以显著抑制肿瘤LSD1的表达,而LSD1可以促进组蛋白的去甲基化,这就表明在DNA甲基化、组蛋白修饰以及DNA损伤机制方面存在相互联系,Trichostatin A/Cisplatin联合用药较单独用药可以显著抑制H3K4和H3K9甲基化表达水平,单独药物及联合用药都能完全抑制spheroid的形成,Trichostatin A/CSP单独或者联合用药都能够显著抑制Spheroid的生长。
第二章表观遗传药物联合化疗药物对卵巢癌细胞致瘤能力影响
目的：
依据上述实验结果并通过对临床病例的观察,建立卵巢癌动物模型,通过表观遗传修饰药物、传统化疗药物和联合用药疗法及序贯疗法对卵巢癌细胞肿瘤形成、生长以及腹腔内转移进行的分析,阐述表观遗传药物联合传统化疗药物对卵巢癌的抗肿瘤的效果。
方法：
1肿瘤细胞的准备
将肿瘤细胞hey种植到培养板中,当细胞生长到培养皿面积的70-80%时,用PBS冲洗去2遍,去除漂浮的细胞,加入蛋白酶-EDTA消化,将获取的细胞在1500转/分钟条件下离心2-5分钟并用PBS冲洗两次,随后用计数板对细胞进行计数,用台盼蓝检测细胞活性。将细胞悬浮到培养基中准备接种。
2老鼠准备4-6周龄老鼠,在试验前3天运抵。
3实验过程、后续观察及试验记录
根据实验需要,将10x106肿瘤细胞或者与处理过的肿瘤细胞皮下注射或腹腔注射,注射过程前做好准备工作,包括老鼠状态的观察体重的测量等基本情况记录,在实验过程中注意操作并做好标记,肿瘤种植过程后,密切观察动物反应,待肿瘤接种后第2天再次探望。将动物的状态做好详细记录,每天观察一次,对于状态不佳老鼠增加观察次数,带肿瘤形成后根据要求测量肿瘤大小以及动物荷瘤体重。对于序贯治疗老鼠,根据研究设计进行严格操作,以保证数据的客观性和可靠性。
4实验结束时,麻醉处死、记录观察、测量数据、分析结果。
5肿瘤体积计算公式=宽度2x长度x0.52。
结果：
1药物处理对卵巢癌细胞Hey成瘤能力的影响
药物组合Trichostatin A/Cisplatin组动物肿瘤体积较对照组减少约4倍。TSA0.3um组也有明显的肿瘤抑制效果且Decitabine 10um组和Cisplatin 1um组明显,Decitabine/Cisplatin抗肿瘤效果介于Decitabine 10um组和Cisplatin 1um组之间。从体积角度来看肿瘤的抑制能力顺序分别为：Trichostatin A/Cisplatin>Trichostatin A>Decitabine/ Cisplatin>Decitabine>control,从肿瘤重量角度来看肿瘤抑制能力顺序分别为：Trichostatin A/Cisplatin>Trichostatin A>Decitabine/Cisplatin>Decitabine>Cisplatin>control。肿瘤生长曲线提示Trichostatin A/Cisplatin联合用药较其他用药方案更能抑制肿瘤生长,并使肿瘤生长维持在较低的水平。
2药物处理对卵巢癌细胞Hey种植及转移能力的影响
根据对照组动物模型转移特点我们选取了十个部位作为参考,包括肝脏、脾、腹膜、肠系膜、大网膜、胃、盆腔壁、横隔、膀胱以及卵巢。实验结果显示TSA 0.3umm组和Cisplatin 1um组无肿瘤转移灶形成。而与此相对,Decitabine10um组,Trichostatin A/Cisplatin组及TSA/Decitabine组存在不同程度的转移灶形成。Decitabine 10um组主要转移部位有肝脏、脾、腹膜、肠系膜、横隔、膀胱以及卵巢。Trichostatin A/Cisplatin主要转移部位有肝脏、脾、大网膜以及膀胱,decitabine/Cisplatin主要转移部位有肝脏、脾、大网膜及卵巢。我们还观察了药物对种植后细胞转移能力的影响,结果显示所有组别的肿瘤均出现了转移。
3药物处理对卵巢癌细胞Hey肿瘤生长能力的影响
P53表达情况进行检测,结果显示CisplatinlowTrichostatin Ahigh、CisplatinloWDecitabinehigh、Trichostatin AlowCisplatinlow、DecitabinelowCisplatinlow可以导致P53升高。将卵巢癌细胞株种植到老鼠皮下待肿瘤形成后进行腹腔内药物治疗以观察药物对肿瘤生长能力的影响,结果发现肿瘤抑制能力的顺序分别：CisplatinlowDecitabinehigh>DecitabinelowCisplatinlow>CisplatinlowTrichostatin Ahigh>Control>Trichostatin AlowCisplatinlow>Trichostatin AhighDecitabinehigh。其中CisplatinlowDecitabinehigh抗肿瘤生长能力最强。
结论：
药物组合Trichostatin A/Cisplatin较单独用药更能抑制肿瘤生长和降低肿瘤负荷,并使肿瘤生长维持在较低的水平。Trichostatin A及Cisplatin处理过的细胞不能够在腹腔内形成肿瘤种植,其他药物组和联合用药具有肿瘤种植的器官特异性,序贯疗法Cisplatinlowdecitabinehigh抗肿瘤生长能力最强,DecitabinelowCisplatinlow治疗策略也具有较好的抗肿瘤效果。
Ovarian cancer is the fifth lethal cancer of female reproduction systerm malignancies in America, the high mortility dues to the late stage diagnosis when the cancer cells spread outside of the ovaries. Although good prognosis of the surgery following by chemotherapy could be achieved, but just 30% of the patients who could survive 5 years after the diagnosis, the challenage of the ovarian cancer is the lack of early detection methods and the metastasis and chemoresistance of late stage ovarian cancer cells, Detection in early stage and comphrehensive, safety and effective treatment strategies of ovarian cancer are two of the most important issues should be investigated in future.
Genetics and epigenetics work together to determine the fate and progression of human cancer, the epigenetics is different from genetics, it is heritable and reverseble and could change the statue of the cell from malignancy to normal and vice versa. The disturbance and ablation of the epigenetic regulation involve in the mechanism of most human disease, With the development of the research of epigenetics of human cancer, especially the DNA methylation, histone methylation and histone acetylation and other modifications, the epigenetic realated enzymes and proteins would become the target of the cancer treatment.
The disorder of the epigenetic statue could increase the instability of the genome and eventually cause cancer, So if the combination of the epigenetic modifiers and conventional chemotherapeutic reagents could make the cancer cell resensitizing to conventional chemotherapeutic reagents in ovarian cancer is unknown, but the application of the inhibitors of DNA methyltransferase, histone methyltransferase and histone deacetylase are becoming more and more in preclinical and clinical trials.
We assume that the combination and sequential treatment strategies could affect human ovarian cancer in aspect of molecular biology, cellular biology, morphology and tumorgenicity and would provide us a promising treatment strategy for ovarian cancer patients.
Chapter 1 Effect of combination of epigenetic modifiers and conventional chemotherapeutic reagent on human ovarian cancer cell line
OBJECTIVE:
To investigate the effect of the single or combination of epigenetic modifiers and conventional chemotherapeutic reagents on the tumor biology, epigenetic regulation, spheroid formation and growth of Hey cells, and to find the detailed mechanism of the response of Hey to epigenetic modification and try to figure out and improve the the management of metastasis and chemoresistance of the ovarain cancer.
METHODS:
1 Cell preparation and Isolation of protein
1.1 Cell preparation:Cells (treated or untreated) are growing to 80-90% confluence and trypsinized after removing growth medium. Cells are washed once with PBS and pelleted by centrifugation for protein isolation.
Total isolation of protein:Prepare 1X extraction buffer by adding 1 ml of 5X extraction buffer to 4 ml of distilled water. Add DTT solution and PIC to 1X extraction buffer at the 1:1000 ratios. Re-suspend cell pellet in 100μl of ice cold 1X extraction buffer per 106 adherent cells or 2 x 106 non-adherent cells. Transfer the cell solution to a microcentrifuge vial. Incubate on ice for 15 min with vigorous vortex 5 sec/5 min. Centrifuge the cell solution for 10 min at 14,000 rpm at 4℃and transfer the supernatant into a new microcentrifuge vial. Measure the protein concentration of cell extract. Use immediately or aliquot and freeze supernatant at -80℃until further use. Avoid repeated freezing/thawing.
1.2 Isolation of histone:Harvest cells and pellet the cells by centrifugation at 1000 rpm for 5 min at 4℃. Dilute 10X Pre-Lysis Buffer into 1X Pre-Lysis Buffer with distilled water at a 1:10 ratio. Re-suspend cells in the Diluted 1X Pre-Lysis Buffer at 106cells/ml and lyse cells on ice for 10min with gentle stirring. Centrifuge at 3000 rpm for 5 min at 4℃. If cell lysates are prepared in a 1.5ml to 2ml size vial, centrifuge at 10,000 rpm for lmin at 4℃, Remove supernatant. Re-suspend cell/tissue pellet in 3 volumes of Lysis Buffer and incubate on ice for 30 min. Centrifuge at 12,000 rpm for 5 min at 4℃and transfer the supernatant fraction (containing acid-soluble proteins) into a new vial. Prepare Balance-DTT Buffer by adding DTT Solution to Balance Buffer at a 1:500 ratio. Add 0.3 volumes of the Balance-DTT Buffer to the supernatant immediately. Quantify the protein concentration with an OD reader. BSA can be used as a standard. Aliquot and store the extract at -20℃for several days, or -80℃for long-term storage. Avoid repeated freezing/thawing.
1.3 Nuclear extraction:Growing cells to 70-80% confluence on a culture plate or flask. Remove the growth medium and wash cells with PBS twice and then remove PBS. Add lml of fresh PBS per 20 cm2 area, and scrape cells into a 15ml conical tube. (Alternative option:detach cells with trypsin-EDTA and collect cells into a 15ml conical tube. Count cells in a hemacytometer). Centrifuge the cells for 5min at 1000 rpm and discard the supernatant. Dilute NE1 with distilled water at a 1:10 ratio. Add DTT solution and PIC to ice cold diluted NE1 (IX) at a 1:1000 ratio. Re-suspend cell pellet in 100μl of diluted NE1 per 106 cells and transfer to a micro centrifuge vial. Incubate on ice for 10min. Vortex vigorously for 10 sec and centrifuge the preparation for 1 min at 12,000 rpm. Carefully remove the cytoplasmic extract from the nuclear pellet. Add DTT solution and PIC to NE2 at a 1:1000 ratio. Add 2 volumes of NE2 containing DTT and PIC to nuclear pellet (about 10μl per 106 cells or per 2 mg tissues). Incubate the extract on ice for 15 min with vortex 5sec/ 3min. The extract (especially tissue extract) can be further sonicated for 3 X 10 sec to increase nuclear protein extraction. Centrifuge the suspension for 10 min at 14,000 rpm at 4℃and transfer the supernatant into a new microcentrifuge vial. Measure the protein concentration of the nuclear extract. Use immediately or aliquot and freeze the supernatant at -80℃until further use. Avoid repeated freezing/thawing.
2 Western Blotting Protocol
2.1 Making gel:Place a short plate on top of the spacer plate, Slide the two plates into the casting frame, keeping the short plate facing front. Insure both plates are flush at the bottom on a level surface. Lock the pressure cams to secure the glass plates. Engage the spring loaded lever and place the gel cassette assembly on the gray casting stand gasket. Insure the horizontal ribs on the back of the casting frame are flush against the face of the casting stand and the glass plates are perpendicular to the level surface. The lever pushes the Spacer Plate down against the gray rubber gaskets. Place comb in between glass plates. Only put teeth in between glass. Mark 1 cm below where teeth are on outside of glass plate. Pour separating gel into 50 ml beaker:see recipes below, Take up 7 ml of gel using 5 ml pipet. Put pipet on edge of glass and hold at a 90 degree angle and begin filling to mark made on glass plate. Add 500ul of H2O. Let sit for 45 minutes. When gel is polymerized, invert apparatus and empty H2O. Pour in stacking gel. Place comb into gel. Let sit for 45 minutes. Remove the gel cassette sandwich from the casting frame and place it into the electrode assembly with the short plate facing inward. Slide the gel cassette sandwich and electrode assembly into the clamping frame. Press down the electrode assembly while closing the two cam levers of the clamping frame. Lower into the mini tank and add 400mls of lx Running Buffer.
2.2 Sample preparation:Dilute sample if necessary in dilution buffer below (or RIP A),For whole cell preps, typically use 50-100ug of protein per lane (20-50ug for nuclear,50-100 for histone), For each sample, add desired amount of protein to a microcentrifuge tube followed by 6x SDS Sample buffer or 2x Laemmli. Note:BME should be added fresh to the loading buffer, For the protein markers, add 5ul of markers to tube and lul of 6x sample buffer or 5ul of 2x Laemmli buffer. (Note:if you want to be able to see the marker while the gel is running, load 7-8ul instead of 5ul. Incubate at 95-100℃for 3 minutes. Quick spin the sample and load on gel.
2.3 Running gel:Once gel is polymerized, remove gel sandwich from casting stand and insert into inner cooling core of running rig. Fill inner core with 1X running buffer. Fill outer chamber with 1X running buffer until half of the screws are covered. (make 400ml for mini rig,500ml/gel for Criterion), Blow out wells. Load samples. Run at 75 volts for 1-2 hours or longer depending on the amount of separation. Before gel is done running, get out 2-3 pyrex dishes and fill with transfer buffer. Soak 2 filter papers and 2 fiber pads per gel for at least 5 minutes. Soak membrane in transfer buffer for at least 5 minutes. Stop electrophoresis.
2.4 Membrane transfer:Remove gel. Separate plates. Use spacers to separate. For Criterion rig, use lid to help pop open the cassette. Cut off stacking gel/wells with razor or spacers. Put plates into transfer buffer and nudge gel off plate with spatula into the transfer buffer, Soak gel for 10 minutes. Open sandwich with black side down and stack in this order:fiber pad, filter paper, gel, membrane, filter paper, fiber pad. Make sure to roll out bubbles after gel, membrane and second fiter paper. Close and put into transfer chamber. Black to back. Put in frozen block. Fill transfer chamber with transfer buffer. Transfer at 30V overnight in the cold room or at 100V for 1-2 hours on bench, changing ice pack half way through if needed.
2.5 Washes and detection:Open sandwich. Put membrane in clean container. Rinse membrane with dH2O 2-4x. Block with 50mls 1 hr RT (Blocking buffer:5% non-fat dry milk in TBS-T), Wash with TBS-T 3×10min at RT, Add primary antibody and incubate for 1 hour at RT or overnight at 4C. Wash in TBS-T 3×10 minutes at RT. Add secondary antibody at 1:1000 in TBST with 5% milk and incubate for 1 hour at RT. Wash in TBS-T 3×10min at RT. Drip dry the membrane onto a paper towel and lie flat on a piece of Saran Wrap with the protein side facing up. Mix 3 mls of chemiluminescent substrate Reagent 1:1 and pour onto the membrane. Let incubate for 1 minute. Dump solution off of membrane and drip dry on paper towel. Cover membrane with saran wrap. Expose on G:box for 1 minute to 5 minute depending on the strength of signal.
3 Spheroid formation assay
The cells are cultured in 6/12/24/96 wells ultra-low attachment plates at a concentration of 500 cells/ml in 2 ml/well Complete MammoCultTM Medium in a humidified incubator. A minority population of cells will began to grow as multi-cellular spheres, as opposed to the majority of cells which will not survive. Cells are examined microscopically on a daily basis and growth rates recorded. All the treatment proceure are accroding to the experiment schematic representation and all the spheroid would be taken pictures and assayed in aspect of size, cell number, spheroid number and cell viability.
RESULTS:
1 The effect of the treatment on the tumor biology of Hey cells
Our data indicated that no E-cadherin has been detected in Hey cells, but strongly expression of N-cadherin. Trichostatin A, Decitabine and Cisplatin alone could slightly increased the expression of E-cadherin, but not E-cadherin. The combination of Trichostatin A/Cisplatin and Decitabine/Cisplatin could not change the E-cadherin expression. There is no significanlty change of Pan-AKT and AKT-1 in presence of drugs, The combination of Decitabine/Cisplatin could significantly suppress the ABCG2, but not found in other treatment group. The pluripotency markers OCT4 strongly expression in Hey cells, but moderate expression of NANOG and SOX2, No effect of treatment on the expression of these pluripotency markers. Mitochondrial apoptosis pathway protein Bax has been increased after the single drug treatment. Our data indicated that strongly E-cadherin has been detected in Hey cells, but weak expression of N-cadherin. Trichostatin A, Decitabine and Cisplatin alone could not increase the expression of E-cadherin, but the combination of Trichostatin A/Cisplatin and Decitabine/Cisplatin could significantly change the E-cadherin and N-cadherin expression, Trichostatin A and decitabine could increase AKT1, but the combination of decitabine/Cisplatin could suppress the AKT1,All the treatment significantly suppress the ABCG2, Decitabine/Cisplatin suppress the expression of NANOG, but SOX2 and OCT4could be affected by both combiantion, Bax could be suppressed by Trichostatin A/Cisplatin and Decitabine/Cisplatin.
2 Effect of the treatment on epigenetic regulation of Hey cell line
DNMT3a and DNMT3b could be detected in Hey cells and Cisplatin could slightly increase the expression of DNMT3a, but not of DNMT3b, No effect on LSD1 and histone methylation markers. DNMT3a is strongly expression in SKOV3 cells but slightly expression of DNMT3b, Trichostatin A, Decitabine and Cisplatin could decrease the expression of DNMT3a, but Decitabine/Cisplatin has more suppress effect on DNMT3a, Interestingly, Trichostatin A/Cisplatin and Decitabine/Cisplatin could totally suppress LSD1. The effect of drugs on the histone methylation is diversity and complexity.
3 Effect of the treatment on spheroid formation
The Hey spheroid is big and compact in compare with the IOSE, SKOV3 and A2780. Interestingly, the A2780 spheroid is loose and out of shape, the formation of the spheroid has the features of time and dose response in presence of the drugs, and the combination of Trichostatin/Cisplatin and Decitabine/Cisplatin could totally suppress the shperoid formation. The combination strategies also could suppress the spheroid growth and the number of spheroid formation. The Spheroid formation of SKOV3 in presence of different treatment strategies has time and dose dependent effect and all the strategies significantly suppress the formation of Spheroid, in the spheroid growth assay. Trichostatin A, Cisplatin 5um and Trichostatin A/Cisplatin totally suppressed the Spheroid growth and Spheroid cell viability, but Decitabine,Cisplatin lum and Decitabine/Cisplatin has no significant effect, The combination of Trichostatin A/Cisplatin could suppress the Spheroid number in 8-fold in compare with control group. So the combination could achieve great Spheroid inhibition effect.
CONCLUSION:
High expression of N-cadherin could be detected in Hey cell line and the it could be changed in presence of drugs alone or combination, the ABCG2 has been significantly suppressed by the decitabine/CSP combination and pluripotent markers OCT4/NANOG/SOX2 highly express. No significantly inhibition of DNMT and LSD1 and even the histone methylation marks, TSA/CSP blcoked the formation and growth of spheroid, and the optimal strategy could not only kill the cancer cells but also have the protection effect of normal epithelial surficial ovarain cells. The combination of Trichostatin, Decitabine and Cisplatin could significantly supppress the expression of E-cadherin and N-cadherin in SKOV3 cells, The ABCG2 could be totally suppressed by all treatment. Strongly expression of pluripotency markers OCT/NANOG/SOX2 could be found, No effect has been detected on the mitochondrial apoptosis pathway, DNMT 3a was highly expressed but not of DNMT3a, Interestingly, The Trichostatin A/Cisplatin and Decitabine/Cisplatin could significantly suppress the LSD1. But the expression of histone methylation markers showed more diversity and complexity, Methylation of H3k4 and H3K9 reduced in presence of Trichostatin A/Cisplatin. Moreover, Drug alone or combination could totally suppress the formation and growth of spheroid of SKOV3.
Chapter 2 Effect of combination of epigenetic modifiers and conventional chemotherapeutic reagent on human ovarian cancer cell line Hey tumorigenicity in mouse model
OBJECTIVE:
To establish the animal model of ovarian tumor formation, growth, abdominal metastasis and the effect of treatment of combination and sequential on tummorgenecity and behavior of ovarain cancer, try to find a promising strategy to cure human ovarain cancer.
METHODS
1 Preparation of tumor cells
Growing cells in complete medium and when cells reach 70-80% confluent, replace medium with fresh medium to remove dead and detached cells when 3-4 hrs before harvesting. Remove medium and wash cells with could PBS. Add a minimum amount of trypsin-EDTA. Disperse cells and add complete medium (10:1 to 5:1). Centrifuge immediately at 1500 rpm for 2-5 min and wash twice with PBS and store cells. Count cells using a hemocytometer. Using typan blue staining to exclude dead cells. Mix cells 1:1 with typan blue solution. Cells should be suspended in a volume of 300μl contains required number of cells per injection.10×106 cells are needed per injection per mouse.
2 Preparation of mice. Mice should be 4-6 weeks old. Allow 3-5days acclimatization period after mice have arrived.
3 Preparation of injection Clean and sterilize the inoculation area of the mice with ethanol and/or iodine solutions. Use lml syringe and a 27- or 30-gauge needle. Mix cells and draw the cells into a syringe without a needle. Inject cells(10×106) subcutaneously (s.c.) or Intraperitoneal injection into the lower flank of the mice or abdominal. The animal tumor assay 1 and 2 should be terminated after 2wks and the assay 3 therapy should start after 1 weeks when the tumors reach an average volume of 0.5cm×0.5cm. Tumor diameters are measured by digital calipers, and the tumor volume is calculated by the formula:Volume= width×2×length×0.52.
RESULTS:
1 Effect of the treatment on tumorgenicity of Hey cells in mice
In compare with control, The Trichostatin A/Cisplatin treated cells could suppress the tumor size 4-fold and Trichostatin A 0.3um group also has notable suppression effect, the sequence of the tumor suppression is Trichostatin A/Cisplatin>Trichostatin/Cisplatin>Decitabine/Cisplatin>decitabine>control, from weight aspect:Trichostatin A/Cisplatin>Trichostatin >Decitabine/Cisplatin>Decitabine>Cisplatin>control, the tumor growth curve also showed the same effect.
2 Effect of the treatment on metastasis of Hey cells in mice
We establised the animal model and selected ten sites as indicator to analysis the effect of the treatment on the metastasis and implantation of ovarain cancer cell Hey in mouse metastasis model. These sites are liver, spleen, omentum, mesenchy, diaphage, stomach, bladder, abdominal wall, pelvic cavity and ovaries, we found that Trichostatin A 0.3um and Cisplatin lum totally suppress the metastasis, and Decitabine, Trichostatain A/Cisplatin and Decitabine/Cisplatin have organ specific patterns.
3 Effect of the sequential treatment on the tumor growth inhibition
According to the expression of P53, we selected CSPlowdecitabinehigh, DecitabinelowCisplatinlow, CisplatinlowTrichostatinAhigh, TrichostatinAlowCisplatinlow as sequential treatment to look at if these strategies could suppress the growth of tumor, The results indicated that CisplatinlowDecitabinehigh has the most tumor growth inhibition effect.
CONCLUSION:
Trichostatin A/Cisplatin has the most tumor growth suppression and tumor burden effect and maintain the tumor growth in a low level in a mice tumorgenicity model, Trichostatin A and Cisplatin alone could totally suppress the metastasis in the tumor metastasis model, but this assay need double confirm in the future and try to figure out the detailed mechanism. The sequential treatment of CisplatinlowDecitabinehigh has the most tumor growth inhibition effect, DecitabinelowCisplatinlow also could suppress the growth, it indicated that the combinationof Decitabine and Cisplatin has the synergistic effect.
引文
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