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学科主题: 肿瘤学
题名:
通过联合用药增强胃癌细胞对TRAIL 敏感性的研究
作者: 李琳
答辩日期: 2016-05-25
导师: 季加孚
专业: 肿瘤学
授予单位: 北京大学
授予地点: 北京大学临床肿瘤学院
学位: 博士
关键词: 胃癌 ; TRAIL ; 凋亡 ; 紫杉醇 ; TSA ; 联合用药
其他题名: Potentiation of TRAIL-induced apoptosis in gastric cancer cells by combination therapy
分类号: R735.2
摘要:

第一部分  胃癌中TRAIL受体表达及细胞敏感性分析

 

背景和目的  TRAIL具有选择性诱导肿瘤细胞凋亡而对正常组织细胞无明显毒性的特征,因而在肿瘤治疗中极具应用前景。为评估TRAIL信号在胃癌治疗中的可行性,本研究将检测TRAIL受体在胃癌细胞和组织中的表达情况,并评价重组人源性TRAIL对胃癌细胞的杀伤作用。

方法  研究选取9株胃癌细胞进行实验,采用Cell Counting Kit-8(CCK-8)法检测细胞生长抑制,Annexin V/PI双染色法测定细胞凋亡,Western blot检测Caspase裂解。qRT-PCR和Western blot分别检测胃癌细胞中TRAIL相关受体(DR4、DR5、DcR1、DcR2)mRNA及蛋白的表达,并通过流式细胞仪分析死亡受体在细胞膜表面的表达水平。进一步通过免疫组化方法检测240例胃癌石蜡组织中DR4、DR5的表达情况。

结果  TRAIL对9株胃癌细胞有不同程度的生长抑制作用,这种作用在一定范围内呈时间和剂量依赖性。各株细胞对TRAIL的敏感性不同,其中SNU-16、NUGC3、NCI-N87和SNU-1敏感性最高,低浓度TRAIL(200ng/ml)作用12h能明显抑制胃癌细胞生长,作用24h后50%以上细胞发生凋亡。Caspase-3,-7,-8,-9和PARP的裂解水平与凋亡程度相关。

分析死亡受体在胃癌中的表达情况发现,除SNU-16外,胃癌细胞膜表面普遍高表达DR5,各株细胞DR5的表达量均在90%以上。而DR4的表达量差异很大,TRAIL不敏感的细胞株DR4表达相对较低。免疫组化结果显示,胃癌组织标本中DR4和DR5的表达率分别为56.7%(136/240)和79.2%(190/240)。

结论  TRAIL对胃癌细胞具有不同程度的生长抑制和凋亡诱导作用。其受体DR4和DR5在胃癌组织中高表达。TRAIL药物治疗在胃癌中具有潜在价值。

第二部分  TSA通过抑制ERK/FOXM1通路增强TRAIL诱导的胃癌细胞凋亡

 

背景和目的  前期实验发现TRAIL对胃癌细胞有不同程度的杀伤作用,部分胃癌细胞对TRAIL诱导的凋亡耐受。提高TRAIL敏感性以及寻找敏感性预测标志物是临床应用的关键。本部分研究将分析曲古抑菌素A(Trichostatin A, TSA)对TRAIL介导胃癌细胞凋亡的促进作用,并进一步探讨其协同机制。

方法  研究选取2株非敏感胃癌细胞AGS和SGC-7901进行实验,分别通过CCK-8法测定细胞生长抑制,Annexin V/PI双染色法判定凋亡,Western blot法检测Caspase蛋白裂解。通过流式细胞仪分析细胞膜表面死亡受体表达。qRT-PCR检测TSA处理后FOXM1 mRNA表达。Western blot检测药物作用后FOXM1、死亡受体和抗凋亡蛋白的改变。通过RNA干扰敲低FOXM1、DR5或Bcl-2的表达,分析其对TRAIL敏感性的作用。利用ERK抑制剂U0126验证通路调控关系。

结果  TSA联合TRAIL处理AGS和SGC-7901细胞后,细胞生长抑制明显增强,细胞凋亡量显著增加。与TRAIL单药相比,联合用药组Caspase-3,-7,-8,-9和PARP裂解增强。TSA处理AGS和SGC-7901细胞后,死亡受体DR5表达升高,抗凋亡蛋白XIAP、Mcl-1、Bcl-2和Survivin表达下降。通过RNA干扰敲低DR5或Bcl-2后,都能影响TRAIL介导的细胞毒作用。此外,TSA处理AGS和SGC-7901后明显抑制转录因子FOXM1的表达。值得注意的是,9株胃癌细胞中FOXM1蛋白表达水平和TRAIL敏感性呈负相关趋势。RNA干扰法敲低FOXM1表达后,TRAIL诱导的细胞凋亡量明显增多,并伴随抗凋亡蛋白XIAP和Mcl-1表达降低。进一步分析TSA对FOXM1的调控发现,TSA处理可以降低胃癌细胞中ERK磷酸化水平。pERK和FOXM1的表达在9株胃癌细胞中有正相关趋势。用ERK抑制剂U0126处理AGS和SGC-7901细胞后,FOXM1的表达量随U0126作用时间延长而逐渐降低。联合使用U0126和TRAIL,可以提高TRAIL对胃癌细胞的杀伤作用,起到类似FOXM1敲低的效果。

结论  TSA可以增强TRAIL对胃癌细胞的生长抑制和凋亡诱导作用。其机制与TSA促进死亡受体表达,下调抗凋亡蛋白表达,以及抑制ERK/FOXM1通路有关。另外,我们的研究结果提示FOXM1有望成为预测胃癌细胞中TRAIL敏感性的标志物。

第三部分 胃癌中紫杉醇与TRAIL协同作用研究

 

背景和目的  如何提高敏感性是TRAIL药物治疗遇到的主要问题,而联合用药是解决此问题的有效方式。紫杉醇是胃癌临床治疗的常用药物,本项目研究紫杉醇对TRAIL诱导胃癌细胞凋亡的影响,并探讨紫杉醇增强TRAIL抗肿瘤活性的机制。

方法  研究选取TRAIL耐药细胞株SGC-7901和MGC-803进行实验,分别通过CCK-8法测定细胞生长抑制,Annexin V/PI双染色法定量凋亡,Western blot检测Caspase截断。通过流式细胞仪分析细胞膜表面死亡受体的表达。Western blot检测药物作用后死亡受体和抗凋亡蛋白的改变。通过siRNA干扰技术,研究c-IAP1和TRAIL敏感性的关系。利用ERK,JNK和p38的小分子抑制剂U0126,SP600125和SB202190分析MAPK通路对TRAIL介导胃癌细胞凋亡的影响。构建裸鼠胃癌皮下成瘤模型,观察紫杉醇/TRAIL联合使用对肿瘤生长的抑制作用。

结果  紫杉醇和TRAIL联合作用于SGC-7901和MGC-803细胞,与单独用药组相比能显著提高细胞的生长抑制率和凋亡率。与TRAIL单药相比,联合用药组Caspase-3,-7,-8,-9和PARP裂解增强。动物实验发现,贯续使用紫杉醇和TRAIL能有效抑制胃癌移植瘤生长(P<0.05)。 紫杉醇处理SGC-7901和MGC-803细胞后,死亡受体DR4、DR5的表达升高,且DR4在细胞膜表面的表达增强。紫杉醇单独使用或联合TRAIL使用,细胞中抗凋亡蛋白c-IAP1,c-IAP2,Livin和Mcl-1的表达都显著降低。此外,紫杉醇处理SGC-7901和MGC-803细胞后,ERK、JNK和p38的磷酸化水平降低。分别使用它们的抑制剂U0126,SP600125和SB202190处理细胞,可以增强TRAIL诱导的细胞凋亡和生长抑制作用。

结论  紫杉醇通过上调死亡受体,下调抗凋亡蛋白,以及抑制MAPKs激酶活性增强TRAIL的肿瘤杀伤作用。

英文摘要:

Section one  Susceptibility of gastric cancer cells to TRAIL and

expression of Death receptors

 

Objectives: TRAIL holds promise for cancer therapy as it has unique capacity to ively trigger apoptosis in cancer cells. In this study, we assessed TRAIL sensitivity and receptor expression in gastric cancer (GC).

Methods: We analyzed the cytotoxic and apoptotic inducing effect of TRAIL in 9 GC cell lines. After cells were treated with TRAIL, cell viability, apoptosis rate and caspase activation were examined by CCK-8 assay, Annexin V/PI staining and Western blot, respectively. DR4/DR5 expression in cell lines was evaluated by qRT-PCR, Western blot and flow cytometry. The expression of DR4/DR5 in tumor tissues was assessed by immunohistochemistry (IHC) in patients bearing gastric carcinoma (n=240).

Results: GC cells had varying degrees of sensitivity to TRAIL. Results showed TRAIL inhibited cell growth in both time- and dose-dependent manners. SNU-16, NUGC3, NCI-N87 and SNU-1 were defined as sensitive cells, in which over 50% growth inhibition was achieved when treated with low dose of TRAIL (200ng/ml) for 24h. Cleavage of caspase proteins showed the activation of apoptosis pathway and revealed that both extrinsic and mitochondrial pathway were involved in TRAIL-induced apoptosis. Flow cytometry analysis revealed a predominant cell surface expression of DR5 in all these cells except SNU-16. DR4, however, exhibited diverse levels in 9 cell lines and the top three sensitive cells expressed the highest levels. Upregulation of both DR4 (56.7%, 136/240) and DR5 (79.2%, 190/240) was also found in GC tissue compaired with gastric mucosa using IHC.

Conclusions: TRAIL exerts cytotoxic and apoptotic activity on GC cells. DR4 and DR5 highly expressed in GC tissue suggesting therapeutic potential of TRAIL.

 

 

Section 2  Trichostatin A potentiates TRAIL-induced antitumor effects via inhibition of ERK/FOXM1 pathway in gastric cancer

 

Objectives: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an ideal apoptosis inducer in GC, yet part of cancer cells exhibit resistance to TRAIL-mediated apoptosis. This necessitates the exploration of strategy that re-sensitizes cancer cells to TRAIL and elucidation of molecular mechanism responsible for TRAIL resistance. In this part of study, we investigate the sensitizing effects of Trichostatin A (TSA), an HDAC inhibitor, on TRAIL resistance in GC.

Methods: We analyzed the synergistic effect of TSA and TRAIL in AGS and SGC-7901 cells. Cell viability, apoptosis rate and caspase activation were examined by CCK-8 assay, Annexin V/PI staining and Western blot, respectively. Cell surface expression of DRs was evaluated by flow cytometry. FOXM1 mRNA expression after TSA exposure was evaluated by qRT-PCR. Western blot was used to detect FOXM1, DRs and apoptosis related molecular. After silencing FOXM1, DR5 or Bcl-2 by siRNA, susceptibility of TRAIL was evaluated by cell viability and apoptosis assay. ERK inhibitor U0126 was used to verify the signal regulation.

Results: We found that TSA augmented TRAIL-induced cytotoxicity and apoptosis in GC cells in AGS and SGC-7901 cells. Treatment of cells with both TSA and TRAIL resulted in increased activation of caspase-3, -7,-8,-9 and PARP. Besides, upregulation of DR5 and downregulation of anti-apoptotic proteins including XIAP, Mcl-1, Bcl-2 and Survivin were detected after TSA exposure. Deletion of DR5 and Bcl-2 by siRNA affected TRAIL-induced cytotoxicity. Noticeably, TSA treatment inhibited FOXM1, which expression level showed negative correlation with TRAIL sensitivity. Similarly, silencing of FOXM1 by siRNA resensitized cancer cells to TRAIL and strengthened the TRAIL-augmenting effect of TSA. In addition, TSA treatment resulted in an effectively inactivation of pERK. A positive correlation trend was found between FOXM1 and p-ERK expression in 9 GC cell lines. U0126 (ERK specific inhibitor) treatment resulted in decrease of FOXM1. Co-application of U0126 facilitated TRAIL-mediated cytotoxicity.

Conclusions: TSA-mediated sensitization of GC cells to TRAIL was closely correlated with up-regulation of DR5, down-regulation of anti-apoptotic proteins, and inhibition of FOXM1 via ERK dephosphorylation. This study provided novel evidences that FOXM1 might serve as a biomarker for predicting sensitivity of GC to TRAIL.

 

 

 

Section 3  Paclitaxel enhances tumoricidal potential of TRAIL

 in gastric cancer

 

Objectives: TRAIL therapy is greatly hampered by its resistance. A preclinical successful strategy is to identify combination treatments that sensitizes resistant cancers to TRAIL. The aim of this study was to investigate the clinical potential of TRAIL in conjunction with chemotherapeutic drugs paclitaxel (PTX).

 Methods: We analyzed the synergistic effect of PTX and TRAIL in SGC-7901 and MGC-803 cells. Cell viability, apoptosis rate and caspase activation were examined by CCK-8 assay, Annexin V/PI staining and Western blot, respectively. Cell surface DRs expression was evaluated by flow cytometry. DRs and apoptosis related molecular was detected by western blot after drug exposure. RNA interference was used to evaluate c-IAP1 effect on TRAIL susceptibility. Pharmacological inhibitor U0126, SP600125 and SB202190 were used to verify MAPKs effects on TRAIL signaling. The growth inhibition of tumors was evaluated in terms of volume in a SGC-7901-implanted nude mice model.

Results: We found combined administration of PTX markedly enhanced TRAIL-induced apoptosis in resistant cancer cells both in vitro and in vivo. The sensitization to TRAIL was accompanied by activation of mitochondrial apoptotic pathway, upregulation of TRAIL receptors and downregulation of anti-apoptotic proteins including c-IAP1, c-IAP2, Livin and Mcl-1. Noticeably, PTX could suppress the activation of mitogen-activated protein kinases (MAPKs). Inhibition of MAPKs using specific inhibitors (ERK inhibitor U0126, JNK inhibitor SP600125 and P38 inhibitor SB202190) facilitated TRAIL-mediated apoptosis and cytotoxicity. Additionally, SP600125 upregulated TRAL receptors as well as downregulated c-IAP2 and Mcl-1.

Conclusions: Our comprehensive analyses gave new insight into the role of PTX on enhancing TRAIL sensitivity, and provided theoretical references on the development of combination treatment in TRAIL-resistant gastric cancer.

语种: 中文
相关网址: 查看原文
内容类型: 学位论文
URI标识: http://ir.bjmu.edu.cn/handle/400002259/125691
北京大学临床肿瘤学院_学位论文

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作者单位: 北京大学临床肿瘤学院

Recommended Citation:
李琳. 通过联合用药增强胃癌细胞对TRAIL 敏感性的研究[D]. 北京大学临床肿瘤学院. 北京大学. 2016.
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