RY10-4的抗肿瘤血管生成和侵袭研究
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摘要
黄酮(flavonoids)作为一种在植物中广泛存在的化合物，也大量的存在于人们的日常饮食中，其集多种优良的生理、药理，保健活性于一身，默默的维系着人类的身体健康。黄酮类化合物从日常的身体状态的调节到肿瘤以及心血管等重大疾病的预防都发挥着不可忽视的作用。随着研究的深入，在治疗疾病的层面上，近年来，某些黄酮因其低毒、高效、天然的特点而倍受关注。
普通针毛蕨Macrothelypteris torresiana(Gaud.)Ching，为金星蕨科针毛蕨属的植物，在我国长江以南的各省均存在大量分布。民间普遍认为该植物具有清热解毒，软坚散结的功效，因此在传统中医中其常被用于止血及治疗水肿。通过近几年的深入研究发现，世界上多个实验室均发现，普通针毛蕨中所含的特殊黄酮成分原芹菜素（Protoapigenone）在体内和体外的抗肿瘤活性筛选中均有良好的表现。更加令人惊喜的是，原芹菜素抗肿瘤谱较广，对不同来源的肿瘤细胞都表现出明显的细胞毒作用。通过进一步研究发现，以Protoapigenone为代表的一系列的B环为非芳香环的黄酮类化合物，在体内和体外筛选实验中均表现出良好的抗肿瘤活性；而结构相似的传统黄酮的抗肿瘤作用却很微弱。
在前人的研究基础上，本课题以Protoapigenone为先导化合物，采用化学全合成的方法开发出了新化合物RY10-4，其具体命名为2-(1-hydroxy-4-oxo-2,5-cyclohexadien-1-yl)-4H-pyran-4-one，其中包括核心基团1-hydroxycyclohexa-2,5-dien-4-one。在初步的体外筛选中采用肝肿瘤细胞，肺肿瘤细胞，乳腺肿瘤细胞，前列腺肿瘤细胞等对RY10-4进行全面抗肿瘤的体外评价，结果显示人源乳腺肿瘤细胞为该化合物最佳的靶向肿瘤细胞。同时在实验中也观察到，该化合物不但展现出了良好的抗肿瘤作用，且其副作用也比较微弱。这种初步的筛选结果为后续的深入研究提供了基础并指明了研究的方向。通过后续研究发现该化合物可以作用于肿瘤细胞中的多条通路，包括经典的PI3K-AKT和MAPK通路。
在本课题中，针对RY10-4的毒副作用小的特点，采用了抗血管生成和抗细胞迁移两个途径来评价其抗肿瘤的具体作用机制。在抗血管生成的实验中，本实验采用人脐静脉内皮细胞（HUVECs）建立体外模型以模拟RY10-4对血管生长的抑制作用，体外小管形成抑制作用，以及细胞运动能力抑制作用。同时用斑马鱼来模拟细胞的整体的血管生成。通过上述模型，从表观学来评价了RY10-4的抗血管生成的能力。为了探讨具体的药物分子作用机制，实验通过模拟缺氧环境建立AKT-mTOR-HIF-1-VEGF通路评价系统。通过免疫印迹方法在MDA-MB-231和MCF-7两种人源肿瘤细胞中评估RY10-4对该通路的作用。结果发现肿瘤细胞中该通路上关键蛋白磷酸化激活在RY10-4共培养后被弱化，进而使得这条通路被抑制。
由于肿瘤细胞周边的血管生成行为和肿瘤细胞的侵袭与迁移密切相关，实验中采用人源性乳腺癌肿瘤细胞MDA-MB-231，一种易于迁移的乳腺肿瘤细胞作为细胞实验模型。首先通过细胞粘附，和transwell法在体外模拟肿瘤细胞侵袭和迁徙三个过程：肿瘤细胞粘附于相邻组织上，降解细胞外基质，发生迁徙。然后针对细胞的主要结构支撑蛋白即钙粘蛋白环粘蛋白复合体和细胞外基质采用免疫荧光和免疫印迹的方法来检测其表达和分布。结果显示钙粘蛋白以及在胞内与其构成细胞骨架的环粘蛋白在RY10-4作用以后表达量和表达的位置都发生了明显的变化，主要表现在环粘蛋白在胞内的表达增加，而在核内的表达则减少，钙粘蛋白的表达范围更大而且表达量也增加。MMP-2/9作为主要的细胞外基质降解酶，其表达量的多少与细胞外基质的数量息息相关。因此采用免疫印迹的方法对MMP-2/9以及其上游通路的相关蛋白的表达量来进行评价。结果显示MAPK可能是RY10-4作用于肿瘤细胞的另一条通路，MAPK与PI3K-AKT共同影响着MMP-2/9的表达，而这种通路的激活可以被RY10-4通过降低其上关键蛋白的磷酸化的来阻断。
作为一个有开发前景的化合物，本课题建立了RY10-4的HPLC-UV分析方法。并采用该方法建立了大鼠体内的分析模型并初步进行了RY10-4在大鼠体内的药物分布研究。结果显示RY10-4无论是在空白样中还是在动物组织内均可以在HPLC-UV下采用常规的流动相进行高灵敏度的检出。组织分布的初步结果显示RY10-4的在组织中的分布和血液量有正相关，该化合物在血流充盈的器官中的分布情况差别不大，在血液中可以较长时间保持一个相对高的浓度，这对于该化合物的后续的药物开发工作是十分有利的。
Flavones are a series of compounds that widely distributed in plants, and existedin our daily diet in a large amount. They rolled physiological, pharmacological andhealth activities into a one, nourished our body silently. They play an essential rolefrom the daily regulation of the body to the prevention of major diseases such astumors and cardiovascular disease. With further research, flavones have attractedmore and more attention in the aspect of disease treatment due to their characteristicsof natural, high efficiency and low toxicity.
Macrothelypteris torresiana (Gaud.) Ching, widely distributed in the provinces insouthwestern China, is a member of genus Mcrothelypteris in familyThelypteridaceae. The plant has the functions of clearing heat and removing toxicityand softening and resolving hard mass. It was often used in traditional Chinesemedicine to stop the bleeding and the treatment of edema.
In the recent years, several laboratories in the world have found that a particularflavonoid, protoapigenone, which detected in the Macrothelypteris torresiana (Gaud.)Ching have exhibited fine activities both in vivo and in vitro anti-tumor screen. Whatis more commendable to statement is that it has a wide spectrum of anti-tumorsignificant cytotoxicity to several tumor cells from different sources. Protoapigenonerepresent a series of flavonoids with a non aromatic B ring, which can exhibit a goodanti-tumor activity in vivo and in vitro screening experiments are found throughfurther researchs. Meanwhile the traditional flavones with similar structure have littleanti-tumor effects.
Based on the previous studies, used protoapigenone as a model, we adoptedchemical synthesis methods to develop the new compounds RY10-4. It wasspecifically named as2-(1-hydroxy-4-oxo-2,5-cyclohexadien-1-yl)-4H-pyran-4-one,including a core group:1-hydroxycyclohexa-2,5-dien-4-one. In preliminary in vitroscreening, we adopted a liver tumor cells, lung tumor cells, breast tumor cells,prostate tumor cells as target tumor cells, among which breast were most sensitive to RY10-4. Besides the fine anti-tumor activities, no obvious side effects were observed.The results provided the basis for subsequent in-depth study and indicated thedirection of further research. Through a follow-up study we found that the compoundscan act on multiple pathways of tumor cells, such as the classic PI3K-AKT andMAPK pathways.
In the task, taken the characteristics of small side effect of RY10-4into account,anti-angiogenic and anti-cell migration are two promising pathway to evaluate thespecific anti-tumor mechanism. In anti-angiogenesis experimental, firstly humanumbilical vein endothelial cells (HUVECs), which were essential cells in tubeformation, were co-cultured with RY10-4, secondly tube formation inhibition andinhibition of cell motility also accomplished using HUVECs, finally transgeniczebrafish was used as a whole angiogenesis model to assess RY10-4. Through theabove model, we evaluate the ability to anti-angiogenic of RY10-4by superficial test,which gave us a direction for further study. In order to explore the specific mechanismand behavior of RY10-4, we simulated hypoxic environment and established anAKT-mTOR-HIF-1-VEGF pathway evaluation system. We evaluated the role ofRY10-4in the signal pathway in two human source tumor cells lines MDA-MB-231and MCF-7by immunoblotting. The results shows that the phosphorylation of keyprotein of the tumor cells in the pathways was inactivated after the tumor cellsco-cultured with RY10-4, which may be the cause of inhibition of the pathway.
As known that the invasion and migration of tumor cells are closely related to thebehavior of angiogenesis around the tumor cells, so the human resource breast tumorcells MDA-MB-231, which were regarded to prone to migrate were used as cellmodels. Firstly, cell adhesion assay, tumor cell invasion and migration assay by invitro transwell system were used to imitate the three processes of tumorigenesis:adhering to the adjacent tissue, degradation of extracellular matrix, migration.Thenexpression and distribution of the main structural support protein for cells:E-cadherin/β-catenin complex and extracellular matrix were detected by both immunofluorescence and immunoblotting. The results showed expression levels anddistribution of E-cadherin and its corresponding β-catenin that constitute cytoskeletonin intracellular significantly changed after RY10-4treatment. The outcome exhibitedas follows: β-catenin up-regulated in the cytoplasm, while down-regulated in thenucleus. E-cadherin increased in both expression levels and range. MMP-2/9acted asa major extracellular matrix degrading enzyme, their quantity was closely bound up ofthe amount of extracellular matrix. Therefore, we have adopted the Western blotmethod to evaluate MMP-2/9and its related proteins in the upstream pathway. Theresult shows the MAPK may be the other signal pathway RY10-4acts on the tumorcells. MAPK and PI3K-AKT signal pathway contribute to the expression of MMP-2/9,and the activation of the signal pathway can be blocked by reducing thephosphorylation levels of key proteins.
As a compound with the prospects for the development, the subject established aHPLC-UV analysis method for RY10-4. Then we adopted SD rats to analysis thepreliminary pharmacokinetic in vitro of RY10-4.The results show that whether it is inthe blank or the animal tissues, RY10-4can be detected by HPLC-UV using aconventional mobile phase to get a high sensitivity. The tentative results show thatthere is a positive correlation between the tissue distribution and the amount of bloodof RY10-4. The distribution of the compound exhibited only a little differences amongthe organs with plentiful bloods meanwhile it can sustain a relatively highconcentration in the blood in a longer time. This is very beneficial for us to follow-updrug development work.
普通针毛蕨Macrothelypteris torresiana(Gaud.)Ching，为金星蕨科针毛蕨属的植物，在我国长江以南的各省均存在大量分布。民间普遍认为该植物具有清热解毒，软坚散结的功效，因此在传统中医中其常被用于止血及治疗水肿。通过近几年的深入研究发现，世界上多个实验室均发现，普通针毛蕨中所含的特殊黄酮成分原芹菜素（Protoapigenone）在体内和体外的抗肿瘤活性筛选中均有良好的表现。更加令人惊喜的是，原芹菜素抗肿瘤谱较广，对不同来源的肿瘤细胞都表现出明显的细胞毒作用。通过进一步研究发现，以Protoapigenone为代表的一系列的B环为非芳香环的黄酮类化合物，在体内和体外筛选实验中均表现出良好的抗肿瘤活性；而结构相似的传统黄酮的抗肿瘤作用却很微弱。
在前人的研究基础上，本课题以Protoapigenone为先导化合物，采用化学全合成的方法开发出了新化合物RY10-4，其具体命名为2-(1-hydroxy-4-oxo-2,5-cyclohexadien-1-yl)-4H-pyran-4-one，其中包括核心基团1-hydroxycyclohexa-2,5-dien-4-one。在初步的体外筛选中采用肝肿瘤细胞，肺肿瘤细胞，乳腺肿瘤细胞，前列腺肿瘤细胞等对RY10-4进行全面抗肿瘤的体外评价，结果显示人源乳腺肿瘤细胞为该化合物最佳的靶向肿瘤细胞。同时在实验中也观察到，该化合物不但展现出了良好的抗肿瘤作用，且其副作用也比较微弱。这种初步的筛选结果为后续的深入研究提供了基础并指明了研究的方向。通过后续研究发现该化合物可以作用于肿瘤细胞中的多条通路，包括经典的PI3K-AKT和MAPK通路。
在本课题中，针对RY10-4的毒副作用小的特点，采用了抗血管生成和抗细胞迁移两个途径来评价其抗肿瘤的具体作用机制。在抗血管生成的实验中，本实验采用人脐静脉内皮细胞（HUVECs）建立体外模型以模拟RY10-4对血管生长的抑制作用，体外小管形成抑制作用，以及细胞运动能力抑制作用。同时用斑马鱼来模拟细胞的整体的血管生成。通过上述模型，从表观学来评价了RY10-4的抗血管生成的能力。为了探讨具体的药物分子作用机制，实验通过模拟缺氧环境建立AKT-mTOR-HIF-1-VEGF通路评价系统。通过免疫印迹方法在MDA-MB-231和MCF-7两种人源肿瘤细胞中评估RY10-4对该通路的作用。结果发现肿瘤细胞中该通路上关键蛋白磷酸化激活在RY10-4共培养后被弱化，进而使得这条通路被抑制。
由于肿瘤细胞周边的血管生成行为和肿瘤细胞的侵袭与迁移密切相关，实验中采用人源性乳腺癌肿瘤细胞MDA-MB-231，一种易于迁移的乳腺肿瘤细胞作为细胞实验模型。首先通过细胞粘附，和transwell法在体外模拟肿瘤细胞侵袭和迁徙三个过程：肿瘤细胞粘附于相邻组织上，降解细胞外基质，发生迁徙。然后针对细胞的主要结构支撑蛋白即钙粘蛋白环粘蛋白复合体和细胞外基质采用免疫荧光和免疫印迹的方法来检测其表达和分布。结果显示钙粘蛋白以及在胞内与其构成细胞骨架的环粘蛋白在RY10-4作用以后表达量和表达的位置都发生了明显的变化，主要表现在环粘蛋白在胞内的表达增加，而在核内的表达则减少，钙粘蛋白的表达范围更大而且表达量也增加。MMP-2/9作为主要的细胞外基质降解酶，其表达量的多少与细胞外基质的数量息息相关。因此采用免疫印迹的方法对MMP-2/9以及其上游通路的相关蛋白的表达量来进行评价。结果显示MAPK可能是RY10-4作用于肿瘤细胞的另一条通路，MAPK与PI3K-AKT共同影响着MMP-2/9的表达，而这种通路的激活可以被RY10-4通过降低其上关键蛋白的磷酸化的来阻断。
作为一个有开发前景的化合物，本课题建立了RY10-4的HPLC-UV分析方法。并采用该方法建立了大鼠体内的分析模型并初步进行了RY10-4在大鼠体内的药物分布研究。结果显示RY10-4无论是在空白样中还是在动物组织内均可以在HPLC-UV下采用常规的流动相进行高灵敏度的检出。组织分布的初步结果显示RY10-4的在组织中的分布和血液量有正相关，该化合物在血流充盈的器官中的分布情况差别不大，在血液中可以较长时间保持一个相对高的浓度，这对于该化合物的后续的药物开发工作是十分有利的。
Flavones are a series of compounds that widely distributed in plants, and existedin our daily diet in a large amount. They rolled physiological, pharmacological andhealth activities into a one, nourished our body silently. They play an essential rolefrom the daily regulation of the body to the prevention of major diseases such astumors and cardiovascular disease. With further research, flavones have attractedmore and more attention in the aspect of disease treatment due to their characteristicsof natural, high efficiency and low toxicity.
Macrothelypteris torresiana (Gaud.) Ching, widely distributed in the provinces insouthwestern China, is a member of genus Mcrothelypteris in familyThelypteridaceae. The plant has the functions of clearing heat and removing toxicityand softening and resolving hard mass. It was often used in traditional Chinesemedicine to stop the bleeding and the treatment of edema.
In the recent years, several laboratories in the world have found that a particularflavonoid, protoapigenone, which detected in the Macrothelypteris torresiana (Gaud.)Ching have exhibited fine activities both in vivo and in vitro anti-tumor screen. Whatis more commendable to statement is that it has a wide spectrum of anti-tumorsignificant cytotoxicity to several tumor cells from different sources. Protoapigenonerepresent a series of flavonoids with a non aromatic B ring, which can exhibit a goodanti-tumor activity in vivo and in vitro screening experiments are found throughfurther researchs. Meanwhile the traditional flavones with similar structure have littleanti-tumor effects.
Based on the previous studies, used protoapigenone as a model, we adoptedchemical synthesis methods to develop the new compounds RY10-4. It wasspecifically named as2-(1-hydroxy-4-oxo-2,5-cyclohexadien-1-yl)-4H-pyran-4-one,including a core group:1-hydroxycyclohexa-2,5-dien-4-one. In preliminary in vitroscreening, we adopted a liver tumor cells, lung tumor cells, breast tumor cells,prostate tumor cells as target tumor cells, among which breast were most sensitive to RY10-4. Besides the fine anti-tumor activities, no obvious side effects were observed.The results provided the basis for subsequent in-depth study and indicated thedirection of further research. Through a follow-up study we found that the compoundscan act on multiple pathways of tumor cells, such as the classic PI3K-AKT andMAPK pathways.
In the task, taken the characteristics of small side effect of RY10-4into account,anti-angiogenic and anti-cell migration are two promising pathway to evaluate thespecific anti-tumor mechanism. In anti-angiogenesis experimental, firstly humanumbilical vein endothelial cells (HUVECs), which were essential cells in tubeformation, were co-cultured with RY10-4, secondly tube formation inhibition andinhibition of cell motility also accomplished using HUVECs, finally transgeniczebrafish was used as a whole angiogenesis model to assess RY10-4. Through theabove model, we evaluate the ability to anti-angiogenic of RY10-4by superficial test,which gave us a direction for further study. In order to explore the specific mechanismand behavior of RY10-4, we simulated hypoxic environment and established anAKT-mTOR-HIF-1-VEGF pathway evaluation system. We evaluated the role ofRY10-4in the signal pathway in two human source tumor cells lines MDA-MB-231and MCF-7by immunoblotting. The results shows that the phosphorylation of keyprotein of the tumor cells in the pathways was inactivated after the tumor cellsco-cultured with RY10-4, which may be the cause of inhibition of the pathway.
As known that the invasion and migration of tumor cells are closely related to thebehavior of angiogenesis around the tumor cells, so the human resource breast tumorcells MDA-MB-231, which were regarded to prone to migrate were used as cellmodels. Firstly, cell adhesion assay, tumor cell invasion and migration assay by invitro transwell system were used to imitate the three processes of tumorigenesis:adhering to the adjacent tissue, degradation of extracellular matrix, migration.Thenexpression and distribution of the main structural support protein for cells:E-cadherin/β-catenin complex and extracellular matrix were detected by both immunofluorescence and immunoblotting. The results showed expression levels anddistribution of E-cadherin and its corresponding β-catenin that constitute cytoskeletonin intracellular significantly changed after RY10-4treatment. The outcome exhibitedas follows: β-catenin up-regulated in the cytoplasm, while down-regulated in thenucleus. E-cadherin increased in both expression levels and range. MMP-2/9acted asa major extracellular matrix degrading enzyme, their quantity was closely bound up ofthe amount of extracellular matrix. Therefore, we have adopted the Western blotmethod to evaluate MMP-2/9and its related proteins in the upstream pathway. Theresult shows the MAPK may be the other signal pathway RY10-4acts on the tumorcells. MAPK and PI3K-AKT signal pathway contribute to the expression of MMP-2/9,and the activation of the signal pathway can be blocked by reducing thephosphorylation levels of key proteins.
As a compound with the prospects for the development, the subject established aHPLC-UV analysis method for RY10-4. Then we adopted SD rats to analysis thepreliminary pharmacokinetic in vitro of RY10-4.The results show that whether it is inthe blank or the animal tissues, RY10-4can be detected by HPLC-UV using aconventional mobile phase to get a high sensitivity. The tentative results show thatthere is a positive correlation between the tissue distribution and the amount of bloodof RY10-4. The distribution of the compound exhibited only a little differences amongthe organs with plentiful bloods meanwhile it can sustain a relatively highconcentration in the blood in a longer time. This is very beneficial for us to follow-updrug development work.
引文
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