摘要
研究背景
癌症严重威胁人类健康,除手术治疗外,化学治疗仍然是肿瘤治疗的主要手段。目前临床广泛应用的化疗药物除传统的细胞毒类药物外,单克隆抗体、小分子化合物抑制剂等靶向药物越来越受到重视,并已经是目前及未来相当长时间内引领化疗领域发展动向的主要研究方向。尽管如此,这些药物仍然存在一定的毒副作用,并产生耐药性,这可能是限制药物成功化疗的主要因素。为克服这些弱点,我们拟对细胞毒类药物吉西他滨进行前体药物修饰,对小分子靶向抑制剂索拉非尼进行结构改造。
吉西他滨以抗癌活性强、抗瘤谱广的特点在化疗药物中占居重要地位,但其在血浆和肝脏中很快被灭活,且严重的毒副作用和耐药性也限制其临床应用。我们认为,对吉西他滨进行前药修饰,在胞啶环的N4位引入具有长侧链酯基酰胺基团,在一定程度上可以增加胃肠道吸收,阻断脱氧胞苷脱氨酶的脱氨作用,增强代谢稳定性,延长半衰期,提高生物利用度,从而达到增加抗癌活性,降低毒副作用的目的。我们从此系列化合物中筛选得到了SL-01(N4-(3-正十二烷氧基甲酰基吡嗪-2-甲酰基)-2’-脱氧-2’,2’-二氟胞苷),发现其具有较高的抗癌活性。本论文第一部分重点评价了化合物SL-01的抗肿瘤活性并对其作用机制进行初步探讨。
首个口服多激酶抑制剂索拉非尼是化疗药物中比较成功的双芳基脲类化合物,但毒副作用以及易产生耐药性也是其临床应用所不可忽视的。根据索拉非尼的结构特点,我们设计合成了一系列基于吲唑或氮杂吲唑的双芳基脲类或硫脲类化合物,初步试验显示出较好的抑制肿瘤细胞增殖的活性。本论文的第二部分对初步筛选得到的15个化合物的体外抗肿瘤活性进行了进一步的筛选,对筛选出的化合物1082-39(N-{4-[1-(4-溴吲唑)]苯基}-N’-[4-氯-3-三氟甲基苯基]脲),探讨其对人黑色素瘤M21体外抗癌活性和作用机制。
第一部分吉西他滨口服衍生物SL-01的抗癌活性研究
实验目的：比较评价化合物SL-01与吉西他滨的抗肿瘤活性,初步探讨SL-01的抗癌作用机制。
实验方法：选用人非小细胞肺癌细胞NCI-H460和人结肠癌细胞HCT-116,以MTT法检测了SL-01对癌细胞增殖的抑制作用,Hochest33258染色法和流式细胞术Annexin V-FITC/PI双染实验测定了SL-01对癌细胞凋亡诱导作用；体内实验,通过建立裸鼠移植NCI-H460和HCT-116肿瘤模型,检测了SL-01对NCI-H460和HCT-116肿瘤细胞生长的抑制作用,进一步以TUNEL法检测了SL-01诱导肿瘤细胞凋亡率,western blotting法检测了SL-01对肿瘤组织caspase-9、caspase3、PARP、 Bax和Bcl-2的调控作用。
实验结果：SL-01(0.156-20μM)与NCI-H460和HCT-116细胞分别孵育24h,48h,72h, SL-01以时间和剂量依赖性方式,显著抑制癌细胞增殖；相同剂量下SL-01与吉西他滨对癌细胞的体外抑制活性相似。以8μM SL-01和吉西他滨分别处理细胞24h,Hoehest33258染色,可见细胞核染色质固缩浓染、或集聚至核膜周边、或出现碎裂呈珠串状等典型凋亡现象。FITC-Annexin V/PI双染结果显示,SL-01明显诱导细胞凋亡,8μM的SL-01作用24h,诱导NCI-H460和HCT-116细胞凋亡率分别为39.6%和41.4%,高于相同剂量下吉西他滨的凋亡活性(NCI-H460:27.8%,HCT-116:24.6%),裸鼠实验表明,口服给予SL-01(10、20、30、μmol/kg,三天一次)三周,可明显抑制NCI-H460和HCT-116肿瘤生长,抑制率分别为31.8%、43.1%、58.0%和36.4%、45.8%、63.1%,且对裸鼠体重影响不甚明显；静脉注射给予同剂量吉西他滨(30μmol/kg,三天一次)对NCI-H460和HCT-116肿瘤生长的抑制率分别是41.5%和46.9%,但动物体重下降较明显。对肿瘤组织进行TUNEL分析,显示SL-01处理组(10、20、30μmol/kg)肿瘤组织中TUNEL阳染率明显增高,NCI-H460和HCT-116组织中TUNEL阳染率分别为25.6%、41.7%、54.3%和20.7%、33.6%、47.7%,吉西他滨处理的NCI-H460和HCT-116组织中阳染率则分别为37.2%和31.2%。Western blotting实验发现,SL-01对肿瘤组织caspase-9、 caspase3和PARP裂解活化程度及对Bax/Bcl-2上调作用均明显高于吉西他滨。
实验结论：SL-01对NCI-H460和HCT-116细胞的体外增殖抑制作用与吉西他滨相似,但对裸鼠接种的人癌细胞生长抑制作用明显优于吉西他滨。SL-01的抗癌活性与其诱导凋亡作用有关,符合caspase依赖性凋亡诱导作用机制。
第二部分吲唑双芳基脲类化合物的抗癌活性研究第一节吲唑双芳基脲类化合物抗癌活性筛选实验(体外实验)
实验目的：对新设计的15个基于吲唑或氮杂吲唑的双芳基脲类或硫脲类化合物筛选,初步评价筛选出的5个化合物对血管生成的抑制作用和对黑色素瘤细胞M21增殖的抑制作用。
实验方法：采用MTT法,检测了1121-39等15个双芳基脲类化合物对人肿瘤细胞MDA-MB-231、PLC/PRF/5、HCT-116、786-0、NCI-H460生长的抑制作用;化合物1121-39、1122-10、1122-38、1122-37和1082-39对人黑色素瘤细胞M21及人脐静脉内皮细胞HUVEC和EA.hy926生长抑制作用；利用划痕损伤法,检测化合物1121-39、1122-38和1082-39对HUVEC细胞迁移能力的影响；以斑马鱼胚胎模型法,检测化合物1121-39、1122-10、1122-38、1122-37和1082-39对血管生成的影响。
实验结果：结果显示,除化合物1121-43、1122-26、1121-40外,新合成的其他化合物与MDA-MB-231、PLC/PRF/5、HCT-116和NCI-H460细胞分别孵育72h,其IC50值多低于索拉非尼,而对于786-0细胞,只有1122-38、1082-39、1122-37、1104-40和1106-78的IC50值小于索拉非尼。筛选出的化合物1121-39、1122-10、1122-38、1122-37和1082-39与EA-hy-926细胞作用72h,其IC50值远低于索拉非尼;而对于HUVEC细胞,只有化合物1121-39的IC50值为4.81±1.97μM；对于M21细胞,化合物1122-38和1082-39作用72h的IC5o值远低于索拉非尼,1122-10和1122-37的IC50值与索拉非尼相当,而1121-39对M21细胞增殖无明显抑制作用。划痕实验显示,化合物1121-39、1122-38和1082-39能抑制HUVEC细胞迁移作用,以5μM作用24h,对HUVEC的迁移抑制率分别为23.16%、41.32%和45.63%,明显低于相同剂量下的索拉非尼的抑制率71.85%。斑马鱼胚胎模型显示,阳性对照药物索拉非尼显著抑制斑马鱼胚胎体节间血管形成,致血流停滞和不同程度心囊水肿,循环系统障碍,而化合物1121-39、1122-10、1122-38、1122-37和1082-39对斑马鱼胚胎体节间的血管生成无明显影响,未见血流异常等现象。实验结论：筛选获得到的吲唑双芳基脲类化合物1121-39、1122-10、1122-38、1122-37和1082-39,体外对多种人癌细胞的增殖有明显抑制作用,且活性优于索拉非尼,但对斑马鱼胚胎血管生成没有影响。
第二节化合物1082-39体外抗肿瘤活性研究及其机制探讨
实验目的：比较评价化合物1082-39和索拉非尼对人黑色素瘤M21的抗癌活性,探讨1082-39的抗癌作用机制。
实验方法：采用MTT法,测定了化合物1082-39和索拉非尼对人黑色素瘤细胞M21作用24、48、72h的半数抑制率。以流式细胞术Annexin V-FITC/PI双染法检测了1082-39与M21细胞孵育48h后的凋亡诱导作用。采用JC-1染色法,观察了不同浓度1082-39作用48h,对M21线粒体膜电位的影响。提取M21细胞总蛋白,以western-blotting法检测了M21细胞受体c-Kit表达水平,检测了1082-39作用48h对M21细胞增殖核抗原PCNA,caspase-9、caspase3、PARP、Bax、Bcl-2、 Mc1-1等凋亡相关蛋白、Akt及相关信号NF-κB、mTOR、GSK3β、c-Myc、survivin、 PI3K、EGFR、Wnt2表达水平的影响；分别分离提取线粒体蛋白及核蛋白,采用western-blotting法检测了1082-39对M21细胞色素c的重新分布及P-catenin入核的影响。
实验结果：化合物1082-39与M21细胞分别孵育24、48、72h,其IC50值均明显低于索拉非尼,以72h最为明显。流式细胞术显示,1082-39明显引起M21细胞凋亡,作用48h后,其5μM组凋亡率40.73%,高于同剂量索拉非尼的凋亡率25.26%。以0、0.625、1.25、2.5、5μM的1082-39处理细胞48h,JC-1染色为红色荧光逐渐减弱,绿色荧光逐渐增强,绿色与红色荧光光密度比值增加,最高组为2.71±0.2,高于索拉非尼组的光密度值之比(2.13±0.6)。Western blotting结果显示,与细胞孵育48h,1082-39比索拉非尼明显下调M21细胞中PCNA表达量；1082-39显著上调Bax表达,下调Mcl-1表达,而索拉非尼则引起Mcl-1表达量明显下调,但对Bax和Bcl-2表达影响较弱,二者均能上调Bax/Bcl-2,Bax/Mcl-1值,引起细胞色素c由线粒体到胞浆重新分布；对于凋亡执行蛋白,1082-39可以明显促进casepase-9和casepase-3裂解活化,PARP裂解增加,而索拉非尼对casepase-9和casepase-3活化作用较弱；Western blotting结果显示,M21细胞中无受体c-Kit表达,但1082-39与索拉非尼均能抑制Akt磷酸化活化,并抑制其上游蛋白PI3K、EGFR、Wnt2及下游蛋白NF-κB、mTOR、GSK3β、c-Myc、survivin表达或活化,且以1082-39作用更为明显。
实验结论：1082-39具有比索拉非尼更好的抑制M21细胞增殖活性,诱导M21凋亡作用,其机制可能与调节Akt信号通路有关。
Backgrounds
Cancer remains a serious threat to mankind. Besides surgery, chemotherapy is still the major treatment for cancer. Currently, cytotoxic drugs are widely used chemotherapy drugs in clinic. In addition, targeted drugs such as small molecule inhibitors have been focused in recent years. This will lead the development trends of chemotherapy currently and in future for a long time. Nonetheless, toxicity and drug resistance are considered to be the main obstacles for successful chemotherapy. In order to overcome these disadvantages, we developed prodrugs for cytotoxic agent gemcitabine and made structure modification for small molecule targeted inhibitor sorafenib.
Gemcitabine, showing effective against a variety of cancers, has played a prominent role in cancer treatment. However, gemcitabine is rapidly inactivated in plasma and liver. Serious toxcities and drug resistance also limited its use in clinic. The introduction of prodrug moieties at the N4-position of the cytidine ring to form a hydrolysable amide linkage would improve its oral bioavailability, decrease its susceptibility to deamination, increase metabolism stability, maintain the active concentration for a longer time, and reduce the toxicity. SL-01, dodecyl-3-((1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl) carbamoyl) pyrazine-2-carboxylate, was selected from these compounds. Our goal in this study in Part1was to evaluate the efficacy of SL-01on the growth of human cancers.
Sorafenib, the first oral multi-kinase inhibitor, is a successful example of diarylurea derivatives. However, concomitant toxicities and drug resistance cannot be ignored in clinic. A new series of diarylurea compounds containing indazole or azaindazole moiety based on the structural features of sorafenib was synthesized in our group. These compounds showed good activiy in preliminary test. In Part2,15 diarylurea compounds were identified for their anticancer activity. The anticancer activity of compound1082-39(N-(4-(1-(4-bromoindazole))phenyl)-N'-(4-Chloro-3-trifluoromethylphenyl)urea), one of indazole diarylurea compounds, was evaluated in human melanoma cell M21and its mechanism underlying were also investigated.
Part1Anti-tumor activity of SL-01, an oral derivative of gemcitabine
Objective:Our goal in this study was to evaluate the efficacy of SL-01on the growth of human cancers and mechnasim underlying with gemcitabine as control.
Methods:Experiments were performed on human non-small cell lung cancer NCI-H460and colon cancer HCT-116both in vitro and in vivo. In vitro assays, the anti-proliferative activity was determined by the MTT assay and the apoptosis induction was estimated with Hoechst33258staining and Annexin V-FITC and propidium iodide (PI) staining analysis by using flow cytometry. In vivo experiments, the anti-tumor effect of SL01was examined by NCI-H460and HCT-116xenografts bearing in mice. The TUNEL assay was used to evaluate the efficacy of apoptosis induction in tumor tissues. Further examination with western blotting analysis was employed to determine the levels of apoptosis related proteins including caspase-9, caspase3, PARP, Bax and Bcl-2.
Results:After incubation for24,48and72h, SL-01(0.156-20μM) significantly inhibited the growth of NCI-H460and HCT-116cells in a dose-and time-dependent manner. The inhibitory potency is similar to that of gemcitabine. Both SL-01and gemcitabine (8μM) were found to potently induce NCI-H460and HCT-116cells to apoptosis, showing cell shrinkage and membrane integrity loss or deformation, nuclear fragmentation and chromatin compaction determined by Hochest33258staining. Annexin V-FITC/PI double staining showed that the apoptosis rates of NCI-H460and HCT-116were39.6%and41.4%after treatment with8μM SL-01for24h, which were higher than that of gemcitabine at the same dose (27.8%for NCI-H460and24.6%for HCT-116). In vivo, oral administration with SL-01once every three days for3weeks effectively delayed the growth of NCI-H460and HCT-116without significant loss of body weight in mice. At dosages of10,20and30μmol/kg of SL-01, the inhibition rates on growth of NCI-H460and HCT-116xenografts were31.8%,43.1%,58.0%and36.4%,45.8%,63.1%, respectively. In comparison, injection30μmol/kg gemcitabine once every three days inhibited the growth of NCI-H460and HCT-116xenografts by41.5%and46.9%, with a significant reduction in body weight. TUNEL staining showed that positive staining cells were increased in xenografts treated with SL-01. The percentages of apoptotic cells induced by10,20and30μmol/kg of SL-01were25.6%,41.7%,54.3%for NCI-H460and20.7%,33.6%.47.7%for HCT-116, respectively. While, gemcitabine (30μmol/kg) induced TUNEL staining positive cells in NCI-H460and HCT-116xenografts by37.2%and31.2%, respectively. Western blotting assay showed that SL-01induced activation of caspase-9, caspase-3and cleaved PARP, as well as increased Bax/Bcl-2ratio in cancer cells. These biological activities of SL-01were more potential than that of gemcitabine.
Conclusion:SL-01showed similar in vitro but superior in vivo inhibitory effect to gemcitabine. The higher potency of SL-01in vivo was associated with its better ability in induction of caspase dependent apoptosis.
Part2Anti-tumor activity study on indazole based diarylurea derivatives
Chapter1Identification of indazole based diarylurea derivatives for their in vitro anti-tumor activity
Objective:15indazole diarylurea compounds were identified for their anticancer activity. The effects of the selected5diarylurea compounds on antiangiogenesis and anti-proliferation against human melanoma cell M21were further investigated.
Methods:The antiproliferative activities of15indazole diarylurea compounds against tumor cells MDA-MB-231, PLC/PRF/5, HCT-116,786-0, NCI-H460, and compounds1121-39,1122-10,1122-38,1122-37,1082-39against M21cells and human umbilical vein endothelial cells HUVEC and EA.hy926cells, were determined by the MTT assay. The scratch assay was employed to investigate the effects of1121-39,1122-38and1082-39on the migaration ability of HUVECs. The effects of1121-39,1122-10,1122-38,1122-37and1082-39on angiogenesis were evaluated by using the model of zebrafish embryo.
Results:MTT assay showed that most of these diarylureas derivatives, except1121-43,1122-26and1121-40, showed lower IC50s than sorafenib in MDA-MB-231, PLC/PRF/5, HCT-116, and NCI-H460cells. But in786-0cells, only IC50s for1122-38,1082-39,1122-37,1104-40and1106-78were lower than that of sorafenib. IC50s for these5selected compounds1121-39,1122-10,1122-38,1122-37and1082-39against EA.hy926cells were lower than that of sorafenib. Only1121-39significantly inhibited the proliferation of HUVEC cells with IC50at4.81±1.97μM. In M21cell, IC50s forl122-38and1082-39were lower than that of sorafenib.1122-10and1122-37exihibited similar IC50S to sorafenib.1121-39showed no effect on proliferation of M21. Exposure at5μM for24h,1121-39,1122-38and1082-39inhibited the migration of HUVEC by23.16%,41.32%and45.63%, respectively, as estimated by scratch assay. Their effects were much lower than sorafenib with the rate of71.85%. Results from the model of Zebrafish embryo showed that these compounds markedly inhibited the formation of intersegmental blood vessel in Zebrafish embryo, leading to blakage of blood flow and pericardium edema. However, compounds1121-39,1122-10,1122-38,1122-37and1082-39showed no effect on the formation of intersegmental blood vessel in zebrafish embryo.
Conclusion:Indazole diarylurea compounds1121-39,1122-10,1122-38,1122-37and1082-39showed more potential anti-proliferative activity than sorafenib against most tumor cell lines in vitro. These compounds exhibited no effect on the angiogenesis of Zebrafish embryo.
Chapter2Anti-tumor activity in vitro and mechanism study of1082-39
Objective:To evaluate the efficacy of1082-39on proliferation of human melanoma cell M21and mechnasim underlying with sorafenib as control.
Methods:After treatment with1082-39or sorafenib for24,48,72h, the IC50s were determined by MTT assay. Annexin V-FITC and PI staining was exploited to assess the apoptosis rates induced by1082-39in M21cell. After JC-1staining, the change of M21cell mitochondria membrane potential was observed under fluorescence microscope. The expression of c-Kit in M21cells; the effect of1082-39on the expressions of proliferating cell nuclear antigen PCNA; apoptosis related proteins including caspase-9, caspase3, PARP, Bax, Bcl-2, Mcl-1; Akt and Akt signaling pathway related proteins such as NF-κB, mTOR, GSK3β, c-Myc, surviving, PI3K, EGFR, Wnt2, as well as the redistribution of cytochrome c and the nucleal translocation of β-catenin were evaluated by using western blotting assay.
Results:MTT showed that IC50S for1082-39against M21cells were lower than that of sorafenib at indicated exposure time, especially72h. Annexin V-FITC and PI staining indicated that1082-39effectively induced M21cells to apoptosis. The rate of apoptosis caused by48h treatment with5μM of1082-39was40.7%, which was higher than that of the same dose of sorafenib with the rate at25.3%. After treatment with0,0.625,1.25,2.5,5μM of1082-39for48h, the red fluorescence of JC-1was gradually decreased and the green fluorescence was correspondingly increased. The ratios of green to red fluorescence were increased in a dose-dependent manner to the maximum at2.71±0.2, which was a little higher than that of sorafenib (2.13±0.6).1082-39was found to down-regulate the expression of PCNA in M21cells more potential than sorafenib as determine by western blotting assay.1082-39significantly increased the expression of Bax and slightly decreased the expression of Mcl-1. Sorafenib markedly decreased the expression of Mcl-1with weak effect on Bax and Bcl-2. Both1082-39and sorafenib could increase the ratio of Bax/Bcl-2and Bax/Mcl-1, leading to redistribution of cytochrome c.1082-39significantly induced the activation of caspase-9, caspase-3and cleaved PARR However, sorafenib showed less effct on these proteins. Western blotting results also showed that there was no expression of c-Kit in M21cells. But the phosphorylation of Akt, and the expression or activation of its upstream proteins PI3K, EGFR, Wnt2. as well as downstream proteins NF-κB, mTOR, GSK3β,c-Myc, survivin, were inhibited by1082-39and sorafenib. These biological activities of1082-39were more potential than that of sorafenib.
Conclusion:1082-39showed better anti-proliferative and pro-apoptotic activiy than sorafenib in M21cells. The mechnasim underlying was associated with its activity in regulation of Akt signaling pathway.
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