【作者】 刘蒙；

【导师】 董卫国；

【作者基本信息】 武汉大学 ， 内科学， 2011， 博士

【摘要】 胃癌是全球常见的消化道恶性肿瘤之一,近年来的发病率呈上升趋势,每年约有9百万人被确诊为胃癌,其中东欧、南美及东亚发病率最高。目前胃癌的治疗方案是以手术切除为主的综合治疗,化学治疗是其重要的辅助治疗手段,但大多数胃癌患者在治疗后容易复发,为了有效防止复发,我们试图寻找一些更有效的、更新机制的医疗手段。那可丁(Noscapine,简称Nos),是一种来自罂粟属鸦片的邻苯二甲酸异喹啉生物碱,在鸦片的生物碱含量中占1%-10%,分子式C22H2307N,相对分子量为413。在过去的75年里,那可丁通常是作为一种没有明显毒副作用的止咳药使用。那可丁的化学结构与已知的微管抑制剂秋水仙碱很相似,其抑制微管的作用已被广泛的认可。目前,那可丁作为一种治疗慢性淋巴细胞性白血病和霍奇金淋巴瘤的药物,已进入Ⅰ、Ⅱ期临床试验阶段。那可丁结合微管蛋白促进微管聚合,从而诱导细胞生长停滞。而且,有报道证实那可丁可通过线粒体途径诱导部分肿瘤细胞调亡。但是,目前尚无文献研究那可丁对胃癌细胞的抗肿瘤作用。本实验提出一个假设：那可丁是否在抗胃癌细胞中扮演重要角色?以及通过何种途径?为了验证这个假设,本研究第一部分体外实验,用不同浓度的那可丁干预胃癌BGC823细胞,观察肿瘤细胞的活力变化和凋亡情况,并初步探讨其发生机制。第二部分体内实验,通过将胃癌BGC823细胞注射在裸鼠皮下,建立人胃癌皮下移植瘤模型,观察不同浓度的那可丁对肿瘤的抑制作用,并初步探讨其发生机制。第三部分在前两部分的基础上,通过RNA干扰构建慢病毒载体并下调CDH17基因,观察CDH17基因联合那可丁对肿瘤的抑制作用。综合整个实验结果,表明低浓度的那可丁即可抑制胃癌BGC823细胞的生长,呈量效关系,凋亡的线粒体通路可能参与其中,并且CHD17基因可以增强那可丁对人胃癌BGC823细胞的杀伤作用,同样,凋亡的线粒体通路可能参与其中,为临床上治疗胃癌找到了一条新的潜在途径。材料和方法1.浓度10μM的那可丁,分别干预四种胃癌细胞(BGC823、SGC7901、MGC803、HGC27)12、24及36 h后,MTT法检测细胞生存率变化,初步筛查敏感细胞。浓度分别为10、100和150μM的那可丁,干预胃癌BGC823细胞12、24及36 h后,MTT法检测细胞生长抑制率变化。不同浓度的那可丁干预胃癌BGC823细胞24 h后,Hoechst33258染色法及流式Annexin V-FITC/PI法检测细胞凋亡情况；Western blotting方法检测procaspase-3和procaspase-9及其激活片段cleaved caspase-3和cleaved caspase-9的表达水平的变化。2.将人胃癌BGC823细胞(5×106,重悬于100μLPBS溶液中)接种于裸鼠右侧背部皮下。当肿瘤体积达100-150 mm3后,那可丁(10mg/kg、20mg/kg、40 mg/kg)每隔3d行瘤内注射,并用游标卡尺测量瘤体长径和宽径,接种后33 d处死裸鼠。观察裸鼠状态和移植瘤的生长状况,绘制生长曲线、称量瘤重、组织病理学观察瘤体形态结构变化及TUNEL方法检测细胞凋亡情况。3.通过构建CDH17-miRNA慢病毒表达载体,下调CDH17基因。设计4条miRNA序列转入CDH17基因。设一个阴性对照组(Lenti-CDH 17-miR-neg)。利用基因转染技术将重组载体转入BGC823细胞(Lenti-CDH 17-miR-B), RT-PCR和Western blotting方法检测BGC823、Lenti-CDH 17-miR-neg和Lenti-CDH 17-miR-B的表达,确定载体是否构建成功。通过Hoechst33258染色法及流式Annexin V-FITC/PI法检测细胞凋亡情况；Western blotting方法检测Bcl-2和Bax的表达水平的变化。建立移植瘤模型观察裸鼠状态和移植瘤的生长状况,绘制生长曲线、称量瘤重、组织病理学观察瘤体形态结构变化及TUNEL方法检测细胞凋亡情况。结果1.那可丁(10μM)干预BGC823、SGC7901、MGC803、HGC27细胞12、24、36h后发现那可丁对BGC823细胞抑制率要明显高于SGC7901、MGC803和HGC27细胞,差异有统计学意义(P<0.05)。浓度为10μM的那可丁干预GBC823细胞12、24及36h后,细胞抑制率分别为(10.26±2.10)%、(21.71±3.51)%及(76.34±6.25)%,并且随着浓度的增加(100和150μM)其抑制作用随之增加(P<0.05)。Hoechst 33258染色法及流式Annexin V-FITC/PI法结果表明那可丁诱导胃癌细胞凋亡且呈量效关系,差异有统计学意义(P<0.05)。以上结果表明那可丁具有杀伤人胃癌细胞的作用。Western blotting法检测结果表明,随着浓度的升高(50、100、150μM),Bcl-2表达明显减弱,而Bax、Cyt-c、cleaved caspase-3和cleaved caspase-9表达明显增强。以上结果表明线粒体通路可能参与了那可丁诱导人胃癌BGC823细胞凋亡的过程。2.不同浓度那可丁干预后,细胞皮下移植瘤生长速度较慢、体积较小,呈量效关系,差异有统计学意义(P<0.05)。瘤体TUNEL检测结果显示,随着药物浓度的增加凋亡细胞明显增多,差异有统计学意义(P<0.05)。3. RT-PCR和Western blotting方法检测证实Lenti-CDH 17-miR-B组的CDH17mRNA和蛋白表达与BGC823组和Lenti-CDH 17-miR-neg组相比明显减少,差异有统计学意义(P<0.05)。而BGC823组和Lenti-CDH 17-miR-neg组之间CDH17表达没有差异(P>0.05)。行体内、外实验后证实那可丁联合下调CDH17基因治疗效果更优于单纯使用那可丁治疗(P<0.05)。结论在体外实验中,那可丁具有杀伤人胃癌BGC823细胞的作用并呈量效关系,线粒体通路可能参与了凋亡过程。在体内实验中,那可丁可以更有效的以凋亡方式抑制人胃癌裸鼠皮下移植瘤的生长,并呈量效关系。下调CDH17基因可以增强那可丁杀伤人胃癌BGC823细胞的作用。基于以上研究结果,我们为临床上治疗胃癌找到了一条新的潜在途径。更多还原

【Abstract】 Gastric cancer remains one of the most common forms of cancer worldwide Around 9 million cases of gastric cancer are diagnosed worldwide each year, with the highest incidence occurring in eastern Asia, Eastern Europe, and the Andean regions of South America Furthermore, most gastric cancer patients have tumor recurrence after treatment In order to prevent recurrence after treatment, it is important to develop medicines which are high efficacy and new mechanisms of action. Noscapine is a phthalide isoquinoline non-narcotic alkaloid derived from opium,for more than 75 years, noscapine has traditionally been used as an oral cough suppressant with no know toxic side effects in human. Noscapine’s chemical structure is similar to the known microtubule binding agent, colchicines, and its binding activity to microtubules has been extensively characterized. Noscapine hydrochloride is in phaseⅠ/Ⅱclinical trials for the treatment of chronic lymphocytic leukemia or low grade non Hodgkin’s lymphoma refractory to chemotherapy and hematological malignancies. Noscapine binds stoichiometrically to tubulin and promotes microtubule polymerization, which causes growth arrest of tumor cells during mitosis. Moreover, noscapine anti-cancer activity involve induction of apoptosis via mitochondrial pathways was demonstrated in various cancers. So far, there is no available information about the antitumor effects of noscapine on human gastric cancer cells. In this study, we addressed the hypothesis that noscapine play an important role in mitochondria-mediated apoptosis in gastric cancer cells. To test our hypothesis, we investigated the mechanism of action, by which noscapine induces apoptosis in gastric cancer cell lines. We demonstrated that mitochondrial pathways involved noscapine-mediated apoptosis in gastric cancer cell lines. In vitro study, noscapine was used in four gastric cancer cell lines, BGC823,SGC7901,MGC803 and HGC27, changes and apoptosis of tumor cells was observed, its mechanism was preliminary explored. Secondly, in vivo study, gastric cancer cells xenograft model in nude mice subcutaneously was established, tumor inhibition of noscapine was observed. Finally, we observed tumor inhibition of noscapine combined lock-down CDH17 （Liver-intestine cadherin） gene. Based on the above findings, a potential new way of clinical treatment of gastric cancer was found.Methods1. Gastric cancer cells were treated with noscapine, survival and growth inhibition rate was measured by MTT （3-（4,5-dimethylthiazol-2-yl）-2,5- diphenyltetrazolium bromide） assay; conditions of apoptosis were measured by Hoechst33258 and Annexin V-FITC/PI of Flow Cytomtry; activity of procaspase-3, procaspase-9, cleaved caspase-3 and cleaved caspase-9 were measured by Western blotting.2. Gastric cancer BGC823 cell,5×106, suspended in 100μL PBS, were subcutaneously inoculated into the lower right flank of the nude mice. When the tumors were 100-150 mmJ in size, noscapine （10,20 and 40mg/kg） were administrated via intratumoral injection every 3 days. Tumor growth was monitored using calipers every 3 days. The nude mice were killed at 33 days.Tumor volume （V） was calculated by using the formula:tumor volume V （mm3）=-π/6×length （mm）×width （mm）2. Status of nude mice and inhibitory effect of tumor growth was observed, the tumor volume was calculated, and growth curve was plotted. Representative histological sections of TUNEL （terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling）were taken from mice bearing transplantation tumor were detected.3. Construction of CDH17 miRNA Lentiviruses, A polⅡexpression system plasmid（Invitrogen, Carlsbad, CA, USA）, which is based on the miRNA vector system and includes endogenous murine miR-155 flanking sequences and a spectinomycin resistance gene, was used for the construction of human CDH17 miRNA expression plasmids. Four precursor miRNA sequences （SRI-4） targeting to CDH17 （GenBank accession number NM₀04063）were designed using an Internet application system （Invitrogen, Carlsbad, USA）. And a randomized sequence was construct as a negative-control. These double-stranded oligonucleotides were inserted into pcDNATM6.2-GW/EmGFP-miR expression vector （Invitrogen, Carlsbad, USA） and then transient transfected into BGC823 cells according to the operating manual of Lipofectamine 2000 （Invitrogen, Carlsbad, USA）, then DNA sequencing were performed for confirming successful construction. RT-PCR and western blot showed that the SR4 yielded the best suppression efficiency （data not shown）, therefore, The pDONRTM221 vector was used as an intermediate to transfer the pre-miRNA expression cassette into the lentiviral expression plasmid （pLenti6/V5-DEST） using Gateway Technology （Invitrogen, Carlsbad, USA）. The new miRNA expression vectors （plenti-CDH17-miR） and packaging vectors were cotransfected into 293 FT cells with Lipofectamine2000 according to the manufacturer’s instructions （Invitrogen） the viral supernatant was harvested 48 hours after transfection.BGC823 cells were transduced with specific （Lenti-CDH17-miR-B） or negative control lentiviral vectors （Lenti-CDH17-miR-neg, Mock） and selected for stable integrants by culturing a complete medium containing blasticidin for 2 weeks.Results1. After treatment of the four gastric cancer cell lines （BGC823, SGC7901, MGC803, HGC27） with 10μM noscapine, cell viability was determined by MTT asssy. noscapine significantly inhibited the proliferation of all cell lines tested. Especially, noscapine at 12 h,24 h and 36 h inhibited BGC823 cells viability by （10.26±2.10）% （21.71±3.51）%and （76.34±6.25）%of control group respectively. Furthermore, the number of BGC823 cells was greatly decreased by incubation 100μM and 150μM noscapine, while a minimal decrease was shown in 10μM noscapine after treatment for 24h.These data indicated that noscapine has a potential not only to prevent gastric cancer cell growth, but also to reduce the number of gastric cancer cells. In addition, the morphological changes of the apoptotic BGC823 cells were detected by Hoechst 33258 staining. Chromatin condensation and nuclear fragmentation are the classic characteristics of apoptotic cells. In the control cells, the nuclei were stained a weak homogeneous blue, while in treatment groups, bright chromatin condensation and nuclear fragmentation could be found. Apoptosis is characterized by distinct morphological features such as nuclear fragmentation and chromatin condensation. Noscapine has been reported to have an anti-tumor effect by apoptosis induction in various solid tumors. Thus, we examined here if it resulted in gastric cancer cell death via apoptosis. BGC823 cells were treated with noscapine （50-150μM） for 24h followed by annexin V/PI staining to examine the proportion of apoptotic cells. It revealed that noscapine caused cell apoptosis in a dose-dependent manner. The apoptosis is initiated by a caspase cascade. We therefore addressed whether noscapine induced apoptosis by caspase signaling. BGC823 cells were treated with 50μM, 100μM and 150μM noscapine for 24h, followed by Western blot to assess the caspase activation. Cleaved forms of caspase-3 and caspase-9 were detected, indicating that noscapine induced apoptosis via caspase cascade. Bax protein was upregulation and Bcl-2 protein was downregulation after noscapine treatment. It is established that Bcl-2 inhibits Bax activity, which reduces mitochondrial membrane potential, leading to cell apoptosis.2. Following the investigation of apoptosis induction in BGC823 cell in vitro, the anti-tumor effect of noscapine was evaluated. The in vivo study was investigated in xenograft model. After growing for 6 days, the tumor xenografts reached a mean size of 100 mm3. We chose 18 mice with tumor xenografts of around 100 mm3 in size and randomly divided them into three groups. There were no statistical differences among the sizes of all the groups. The nude mice were given different treatments. Noscapine cause the inhibitory effects on the growth of tumor in vivo. Control group, in which tumors grew progressively and reached 1000 mm3 within 18 days. However, the treatment groups were significantly suppressed. Treatment with noscapine reduced tumor growth in a dose-dependent manner. At the end of 33 days, noscapine at 10 mg/kg,20 mg/kg and 40mg/kg inhibited tumor growth by 45%,60%and 81%of control group （2178 mm） respectively （Fig.5 A）. Compare with control group, tumor weight was only 0.206g in high dose group at the end of the experiment. TUNEL assay of the subcutaneous tumor sections demonstrated noscapine caused obvious cell death in tumor mass via apoptosis, whereas less apoptosis was found in control group （p< 0.05）. These results proved that noscapine has significant anti-tumoral potential in vivo.3. The lentiviral vector expression cassette allowed for the permanent expression of GFP and CDH17 miRNA in transduced cells. To test the knockdown efficiency, we examined the expression of CDH17 on both mRNA and protein levels. Data showed that after being transfected with a MOI of 10 for 72 hours, the mRNA and protein levels of CDH17 were downregulated in lenti-CDH17-miR-B cells compared with BGC823 and lenti-CDH17-miR-neg cells. Downexpression of CHD17 gene can enhance apoptosis-inducing effects of noscapine on human gastric cancer BGC823 cells. Apoptosis was detected using flow cytometry and Hoechst33258 staining. The treatment with noscapine upregulated Bax protein, downregulated Bcl-2 and Bcl-xL protein. In addition, Cyt-c protein was upregulated, suggesting that the apoptosis is Bcl-dependent pathway. Moreover, in xenograft tumor mouse model, downexpression of CHD17 increased the efficacy of cisplatin-induced inhibition of tumor growth in nude mice via apoptosis induction which was demonstrated by TUNEL assay.ConclusionsThese data of the study suggest that noscapine induces apoptosis in gastric cancer cells via mitochondrial pathways in vitro and in vivo. Downexpression of extrinsic CHD17 gene can conspicuously ameliorate apoptosis-inducing effects of cisplatin on human gastric cancer BGC823 cells, which is a novel strategy to improve chemotherapeutic effects on gastric cancer. Based on the above findings, a potential new way of clinical treatment of colon cancer was found.更多还原