【作者】 武丹威；

【导师】 张德昌； 叶莱英；

【作者基本信息】 北京协和医学院 ， 药理学， 2013， 博士

【摘要】 研究背景癌症是严重威胁人类健康的疾病之一。因此,寻找高效低毒,作用靶点明确的抗肿瘤药物或者有效的联合用药方案是当前药理学以及临床治疗学共同关注的热点问题。羧胺三唑(Carboxyamidotriazole, CAI)是一种非细胞毒类的抗肿瘤药物,能抑制体内,体外多种肿瘤细胞的增殖,促进肿瘤细胞凋亡。先前的研究认为其作用机制与CAI抑制钙离子跨膜内流有关,但其分子机制尚无定论。我们课题组在深入研究CAI药理作用的过程中,发现CAI不仅具有抗肿瘤作用,还在一系列急、慢性炎症模型中表现出良好的抗炎作用,其作用机制与下调动物炎症部位或巨噬细胞释放炎性细胞因子有关。近年来基础和临床研究表明癌症与慢性炎症之间存在联系。其中一个联系点就是在炎癌发展中均发挥重要作用的巨噬细胞。在肿瘤发展的过程中,肿瘤微环境中的巨噬细胞和相关促炎细胞因子会扮演“帮凶”的角色,促进肿瘤的生长,转移。目前针对肿瘤内巨噬细胞和某些促炎因子治疗癌症已得到广泛认可和接受。综合以上背景,我们提出以下假说：CAI是否是通过作用于在炎、癌过程中均发挥重要作用的巨噬细胞而起抗炎及抗癌作用。CAI除能抑制炎症模型中促炎细胞因子释放,是否也能抑制肿瘤微环境中促炎细胞因子的产生。同时,如果以上假说成立,我们是否能够将CAI与另一种抑制炎性细胞因子的药物合用,以增强CAI的抗肿瘤疗效。在本项课题中,我们将围绕以上工作假说展开研究。研究方法采用酶联免疫吸附法(ELISA)检测CAI对脂多糖(LPS)诱导的RAW264.7细胞及佐剂性关节炎(AA)大鼠腹腔巨噬细胞释放肿瘤坏死因子-α(TNF-a)的影响；采用逆转录-聚合酶链式反应实验(RT-PCR)检测CAI对LPS诱导的RAW264.7细胞TNF-a mRNA表达的影响；通过DNFB诱导的迟发型超敏反应考察CAI对细胞免疫的影响：通过中性红实验考察CAI对非特异性免疫的影响；建立C57BL/6小鼠Lewis肺癌(LLC)移植瘤模型,以肿瘤生长、动物体重、生存期和血管生成等指标评价CAI及COM组(CAI与DEX合用,Combination, COM)对移植瘤模型的疗效；采用免疫组化的方法观察肿瘤组织中TNF-a表达情况；采用ELISA法检测肿瘤组织匀浆中TNF-a的水平；采用免疫荧光法鉴定从肿瘤内提取巨噬细胞,采用RT-PCR法检测肿瘤内巨噬细胞TNF-a mRNA的表达水平。建立A549裸鼠移植瘤模型,以肿瘤生长,生存期,血管生成等指标来评价CAI及COM组对移植瘤模型的疗效；采用ELISA法检测肿瘤组织匀浆和腹腔巨噬细胞中TNF-a, TGF-p的水平。采用ELISA法检测CAI及COM组对肿瘤条件培养基(LCM)诱导的小鼠腹腔巨噬细胞中TNF-α水平的影响；采用CCK-8法检测CAI及COM组对单独培养的LLC细胞增殖的影响；建立小鼠腹腔巨噬细胞与LLC细胞共培养模型,采用CCK-8法测定腹腔巨噬细胞对LLC细胞增殖的作用及药物对此作用的影响。采用ELISA法及RT-PCR法检测CAI及COM组对LCM诱导RAW264.7细胞TNF-a,IL-6水平的影响；建立小鼠巨噬细胞系RAW264.7细胞与LLC细胞共培养模型,采用结晶紫染料测定RAW264.7细胞对LLC细胞侵袭,迁移的作用及药物对此作用的影响。研究结果1. CAI (40能够明显抑制LPS诱导RAW264.7细胞释放TNF-a的水平,抑制率为35.36%(p<0.01)。 CAI (10,20μmol/L)均能够明显抑制RAW264.7细胞TNF-a mRNA表达(p＜0.01)。2. CAI (20mg/kg)对AA大鼠腹腔巨噬细胞释放炎性细胞因子TNF-α有明显抑制作用(p＜0.01)。3. CAI (20mg/kg)对DNFB所诱导的小鼠迟发型超敏反应没有抑制作用。CAI(5、10、20和40μmol/L)对体外培养正常大鼠腹腔巨噬细胞吞噬功能没有影响。4. CAI (20mg/kg)对LLC移植瘤模型中肿瘤的生长,血管生成有一定抑制作用。同时可延长荷瘤动物的生存期。与小剂量DEX (1mg/kg)联合用药后,药物的疗效优于两种药物单独使用时的效果。同时各给药组动物体重之间没有明显差异。5. CAI (20mg/kg)对LLC移植瘤模型中肿瘤组织的TNF-a水平有抑制作用,与小剂量DEX (1mg/kg)联合用药后,抑制作用优于单用药组。6. CAI (20mg/kg)对于LLC移植瘤模型中肿瘤内巨噬细胞TNF-α mRNA表达有明显抑制作用(p＜0.01),与小剂量DEX (1mg/kg)联合用药后,抑制作用优于单用药组(p＜0.01)。7. CAI (20mg/kg)对A549移植瘤模型中肿瘤的生长,血管生成有一定抑制作用。与小剂量DEX联合用药后,药物的疗效优于两种药物单独使用时的效果。8. CAI (20mg/kg)对A549移植瘤模型肿瘤组织匀浆TNF-α和TGF-β水平有一定抑制作用,与小剂量DEX联合用药后,抑制作用优于单用药组。CAI(20mg/kg)对A549移植瘤模型中裸鼠腹腔巨噬细胞TNF-α的释放有明显的抑制作用(p＜0.01),与小剂量DEX联合用药后,抑制作用优于单用药组。9. CAI (5、10、20、40μmol/L)对单独培养的LLC细胞的增殖有明显抑制作用(p＜0.01),并呈时间剂量依赖关系。DEX对LLC细胞的增殖没有影响；CAI与小剂量DEX合用对LLC细胞的增殖抑制作用与单独应用CAI相比,没有增强。10. CAI (30μmol/L)能够抑制LCM诱导小鼠腹腔巨噬细胞释放TNF-a, IL-6的水平,升高IL-10的水平。CAI与DEX (10μmol/L)合用能够明显抑制TNF-α,IL-6含量,其抑制作用优于单用药情况(p＜0.05)。11.将小鼠腹腔巨噬细胞与LLC细胞共培养可促进LLC细胞的增殖。CAI和DEX对LLC细胞的增殖均具有明显抑制作用(p＜0.05)。CAI和DEX合用对LLC细胞的增殖抑制作用优于单用药情况(p＜0.01)。12. CAI (20在一定程度上能够抑制LCM诱导RAW264.7细胞释放TNF-a, IL-6的水平。与DEX (10μmol/L)合用后,其抑制作用优于单用药情况。13. CAI (30能够抑制LCM诱导RAW264.7细胞TNF-a, IL-6mRNA的表达(p＜0.01)。与DEX (10μmol/L)合用,抑制TNF-a, IL-6mRNA表达更明显。单用CAI (20μmol/L)也能够抑制LCM诱导RAW264.7细胞IL-1mRNA表达(p＜0.01)。CAI和DEX (10μmol/L)合用抑制IL-1mRNA表达,与单用药相比,具有极显著性差异(p＜0.01)。14.侵袭,迁移实验结果显示：CAI (30μmol/L)和DEX (10μmol/L)均能够抑制RAW264.7细胞对LLC细胞迁移和侵袭的促进作用。尤其是CAI和DEX合用对LLC细胞的增殖迁移和侵袭作用,与单用药相比,具有极显著性差异(p<0.05)。结论1.CAI不仅具有抗肿瘤作用,而且还通过抑制炎性细胞因子的释放而发挥抗炎作用。2.CAI的抗肿瘤作用机制与抑制肿瘤微环境中细胞因子的产生有关,从而间接影响肿瘤增殖、迁移、侵袭和肿瘤组织内的血管新生。3.CAI与小剂量DEX联合用药通过进一步抑制炎性细胞因子的释放增强CAI的抗肿瘤作用,降低不良反应。打破CAI单独使用抗肿瘤作用较弱的瓶颈。4.CAI兼具抗肿瘤及抗炎作用,因此可作为探索炎～癌关系的工具药物。同时通过对CAI作用机制的研究,可能能够发现炎～癌转化过程中其它关键物质,对于明确炎～癌转化的分子网络,具有重要意义。更多还原

【Abstract】 BackgroundCarboxyamidotriazole (CAI) is a non-cytotoxic anti-cancer drug, which can inhibit proliferation and induce apoptosis of several tumor cell lines. Previous study shows that the mechanism of action is the inhibition of transmembrane calcium influx, but the molecular mechanism has not been fully understood. The study of pharmacological functions of CAI by our research group shows that CAI not only had favourable anti-cancer activities, but also had anti-inflammation action in a variety of animal models of acute and chronic inflammation via inhibiting the expression of several cytokines (such as tumor necrosis factor-alpha) at the site of inflammation and macrophages.The macrophages play a critical role in the relationship between cancer and inflammation. During the development of tumorgenesis, the macrophages and pro-inflammatory cytokines act as accomplices for the proliferation, migration, invasion and metastasis of neoplastic in the tumor microenvironment. There is also a consensus about the treatment of cancer through macrophages and pro-inflammatory cytokines.As mentioned above, we hypothesize that the anti-cancer and anti-inflammation effects of CAI may function via the macrophages. CAI may not only inhibit the release of pro-inflammatory cytokines of animal inflammation models, but also can inhibit the product of pro-inflammatory cytokines in the tumor microenvironment. Ifthe presumptions above are correct, we could combine a drug which can suppress pro-inflammatory cytokines with CAI in vivo and in vitro, thus increasing the usage and power of C Al.MethodsAdjuvant-Induced Arthritis (AA) Model and LPS induced RAW264.7macrophages inflammation model was established. We analyzed the effect of CAI on TNF-a production in peritoneal macrophages isolated from AArats and LPS induced RAW264.7macrophages using enzyme-linked immuno-sorbent assay (ELISA). TNF-a mRNA expression level was analyzed by RT-PCR assay. Neutral red experiment is used to examine the impact of CAI on the non-specific immune function. DNFB-induced delayed-type hypersensitivity is used to examine the impact of CAI on the cellular immunity. Lewis Lung Carcinoma xenograft Model was established and validated. The effect of CAI on anti-tumor was evaluated through measuring tumor growth, animal body weight, survival, angiogenesis level. Tumor tissues were removed for immunohistochemical experiment. The tumor homogenate was determined by ELISA assay. The macrophages extracted from tumor tissues were stained and indentified by F4/80immuno-fluorescent test. TNF-a mRNA expression level was analyzed by RT-PCR.A549xenograft model in nude mice was established and validated. The effect of CAI, DEX, and their combination on anti-tumor was evaluated using tumor growth and angiogenesis. The specimens of tumor homogenate or peritoneal macrophages were subjected to ELISA for cytokines including:TNF-α and transforming growth factor-beta (TGF-p). CAI and co-administered group (COM Group, CAI and DEX combination group) used with ELISA to detect TNF-a level in tumor conditioned medium induced murine peritoneal macrophages. Using CCK-8assay on Lewis Lung Carcinoma cells (LLC) cultured alone or a co-culture model of mouse peritoneal macrophages and LLC cells to detect how the drug affects LLC cells proliferation. CAI and co-administered group used with ELISA to detect TNF-a level in tumor conditioned medium induced RAW264.7macrophages.The TNF-a and Interleukin-6(IL-6) expression levels in RAW264.7macrophages were tested by ELISA and RT-PCR assay. The migration and invasion of the LLC cells cultured alone or with RAW264.7macrophages were measured with crystal violet agent.Results1. CAI (40umol/L) inhibited the releases of TNF-a from LPS-induced RAW264.7cells (p<0.01). The inhibition rate was35.36%(p<0.01). CAI (10,20μmol/L)suppressed the expression of mRNA for TNF-a.(p<0.01).2. CAI (20mg/kg) suppressed TNF-a production in peritoneal macrophages isolated from rats of adjuvant-induced arthritis (p<0.01)3. CAI at a dose of20mg/kg was proven to be neutral in DNFB-induced contact hypersensitivity assay. CAI (5、10、20and40μmol/L) had little influence of on phagocytosis of rat peritoneal macrophages in vitro.4. CAI (20mg/kg) in Lewis Lung Carcinoma (LLC) xenograft model inhibit the growth of mouse transplanted tumor, blood vessels growth and prolong the survival time. The anti-tumor effect of CAI combined with low dose DEX (1mg/kg) was more than that of single drug respectively. The addition of DEX did not influence the body weights.5. In20mg/kg CAI group, the TNF-a level in tumor was decreased. The inhibitory effect of CAI combined with low dose DEX on TNF-a level was more than that of single drug respectively.6. LLC tumor tissue macrophages were identified by staining F4/80. The result of realtime RT-PCR showed that, compared with PEG group, mRNA from LLC tumor tissue macrophages for TNF-a in CAI, DEX and combination group was decreased by75%,88%and91%, respectively.7. In the A549xenograft model, it is observed that combination of CAI and DEX significantly inhibited the tumor growth and blood vessels growth in the tumor tissues.8. The concentration of TNF-α and TGF-β in tumor homogenate and TNF-a production in peritoneal macrophages of A549tumor bearing mice were all decreased by CAI and/or DEX.9. CCK-8tests showed that CAI (5、10、20and40μmol/L) inhibited LLC cells proliferation in a dose and time dependent manner (p<0.01). DEX could hardly affect the proliferation of LLC cells, and the addition of DEX could not enhance the inhibition of CAI.10. CAI (30μmol/L) could suppress TNF-a, IL-6production and increase IL-10production in the macrophages cultured with LLC cells conditioned medium(LCM), and the addition of DEX (10μmol/L) could decreased TNF-α, IL-6to a dramatic low level (p<0.05)11. The proliferation of LLC cells were significantly enhanced while co-cultured with macrophages. Both CAI (30μmol/L) and DEX (10μmol/L) had the ability to inhibit the LLC cells proliferation in the co-culture system (p<0.05). The promoting action of macrophages vanished while treated with both CAI and DEX (p<0.01)12. ELISA assays showed that both CAI (20μmol/L) and DEX (10μmol/L) could decrease the level of TNF-a secreted from LCM-induced macrophages. The combination could decrease it to a lower level. 13. The result of real time RT-PCR showed that the TNF-α,IL-6and IL-1mRNA level in RAW264.7cell treated with CAI, DEX or the combination were much lower than the control group (p<0.01)14. The migration and invasion of LLC cells were all significantly enhanced while co-cultured with RAW264.7cell. Both CAI(30μmol/L) and DEX (10μmol/L) had the ability to inhibit the LLC cells migration and invasion in the co-culture system. The promoting action of macrophages vanished while treated with both CAI and DEX (p<0.05)ConclusionsCAI, not only does it have anti-tumor effects, it also has the ability to anti-inflammation via suppressing pro-inflammatory expression such as TNF-α.CAI can inhibit the production of cytokines in the tumor micro-environment.This explains how CAI indirectly inhibits tumor proliferation, migration, invasion and tumor blood vessel growth.CAI combined with low doses of dexamethasone can enhance the anti-tumor effect, and further reduce the adverse reactions.This way it can strengthen the weakness of CAI when used on its own, because as a stand alone drug it’s not very effective against tumor cells.CAI has both anti-cancer and anti-inflammatory effects and may become a tool to explore the relationship between inflammation and cancer. Through CAI mechanism, we could find the other key substances in the inflammation related cancer, which is significant for basic and clinical research.更多还原