基于抑制NF-κB的单羰基姜黄素类似物的抗肺癌活性及其机制研究

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题名	基于抑制NF-κB的单羰基姜黄素类似物的抗肺癌活性及其机制研究
其他题名	The studies of MCACs inhibitors targeting NF-κB andtheir pharmacological activities and mechanismagainst lung cancer
作者	翁碧霞
学位类型	硕士
导师	吴晓萍
答辩日期	2016-05-29
学位授予单位	温州医科大学
学位专业	药理学
关键词	MCACs NF-κB信号通路抗肿瘤类似物肺癌
摘要	中文摘要：目的：NF-κB（Nuclear factor κB）是一种普遍存在于哺乳动物细胞核内核转录因子，它的异常激活或过度表达与多种肺癌的发生发展有关。我们前期的研究发现单羰基姜黄素类似物MCACs（Mono-carbonyl Analogues of Curcumin）能够抑制NF-кB信号通路，表现出较好的抗炎作用。为了发现高效低毒的抗肺癌化合物，本文对两类MCACs进行抗肺癌活性筛选，及基于抑制NF-кB信号通路的抗肺癌机制研究，以期能够获得高效低毒的抗肺癌先导化合物。方法：1、姜黄素类似物将Curcumin结构中的β-二酮改构为单羰基得到MCACs，在我们的前期工作中以姜黄素为先导化合物设计合成一系列的MCACs，并通过ESI–MS和1H-NMR鉴定这些化合物结构，保留在化合物库中。我们在筛选抗炎活性的时候发现两类MCACs能够抑制NF-кB的激活：一类是以EF24类MCACs；另一类是多甲氧基取代MCACs。2、抗肺癌活性筛选MTT的实验方法对两种类型的MCACs进行抗肿瘤活性筛选，在不同药物浓度下作用72 h后测定每个化合物对H460、A549、H1975以及正常人肝细胞HL7702的半数抑制浓度（IC50），筛选出抗肺癌效果好且毒性较小的活性化合物。3、体外抗肺癌活性研究用Hoechst 33258染色法和流式细胞仪两种方法检测活性化合物诱导肺癌细胞凋亡的效果以及对细胞周期的抑制。选择出高效低毒的MCACs进行作用机制研究，在A549、H460细胞中Western Blotting方法检测化合物对NF-κB信号通路的抑制，以及NF-κB所调控的下游的信号蛋白IкB、Caspase-3等。4、体内抗肺癌活性研究我们通过细胞层面的实验结果，选出两个具有良好体外抗肺癌活性的化合物在裸鼠体内进行研究，选用Nu/nu BALB/c 裸鼠建立H460异位移植瘤的裸小鼠肿瘤模型，腹腔注射给药，通过计算肿瘤体积、抑瘤率，以及免疫组化的方法来判断化合物的体内抗肿瘤活性。结果：一、EF24类MCACs抑制NF-κB抗H460活性与机制研究1、Ad10和Aeb8在三种肺癌细胞H460、A549和H1975中均表现出良好的抑制增殖作用，尤其对H460细胞的IC50分别达到12.14±0.53 μM和5.01±0.56 μM。同样从受试化合物对正常人肝细胞（HL7702）的IC50中可以看出 Ad10和Aeb8在抑制肺癌细胞的同时对人正常细胞的毒性较小。与阳性对照药EF24和姜黄素相比，Ad10和Aeb8具有相当或者更好的抗肺癌活性以及较低的细胞毒性。2、Ad10和Aeb8在较低的浓度就能有效地抑制H460细胞增殖时间生长曲线，随着药物作用时间的延长，细胞存活率逐渐降低。本文发现Ad10（20 μM）的浓度作用24 h后可以明显地阻滞H460细胞周期的进程，使大多数细胞停留在分裂前期。而Aeb8在1 μM的浓度下即可有效的干扰H460细胞集落形成。3、Ad10和Aeb8在表现出良好抗肺癌活性的同时可以有效地抑制TNF-α诱导的NF-кB通路中IкB的降解，以及其所调控的下游与凋亡相关的蛋白Bcl-2和BAX的表达。4、Ad10和Aeb8在H460肺癌裸鼠模型中表现出与细胞层面相符的抗肺癌活性，它们在抑制裸鼠肿瘤生长的同时能够不同程度地影响NF-кB的信号通路及其所调控的凋亡相关蛋白Bcl-2、BAX以及周期相关蛋白Cyclin D1。二、多甲氧基取代类MCACs抑制NF-κB抗A549活性与机制研究1、通过MTT法测定所有化合物对肺癌细胞H460、A549和H1975的增殖抑制活性，其中大部分化合物都具有较好的抑制肺癌细胞增殖活性（IC50为3.0 μM左右），尤其是3B、11B和12B表现出的抗肺癌活性要比姜黄素更好。2、Hoechst 33258染色法和流式细胞术显示3B、11B和12B能够剂量依赖性的诱导A549细胞发生凋亡，尤其是11B在1 μM的浓度下可以使细胞核发生形变甚至产生碎片。同样的效果从Western Blotting的结果中我们也可以看到：3B、11B和12B能够剂量依赖性的诱导Caspase-3的激活，并抑制Bcl-2的表达。3、3B、11B和12B在A549细胞中能抑制NF-κB信号通路，它们能够剂量依赖性的抑制TNF-α诱导的IKK的磷酸化或IκB的降解，其中12B的效果尤为明显。结论：本文发现一类具有较好研究前景的MCACs类似物，该类化合物普遍具有较好的抗肺癌活性，部分化合物能够抑制NF-κB信号通路、上调其所调控的凋亡蛋白并下调其所调控的抗凋亡蛋白，起到良好的抑制肺癌细胞增殖、诱导凋亡，及抗体内裸鼠肺癌异种移植瘤活性。但是目前对该类化合物作用靶点以及深入的抗肺癌机制还不是十分明确，还有待于进一步探讨。
其他摘要	AbstractPurpose: Nuclear transcription factor NF-κB (Nuclear factor kappa B) is one kind of mammalian nuclei protein family, and whose abnormal activation or excessive expression is associated with the development of a variety of lung cancer. In our previous work, we found that the Mono-carbonyl Analogues of Curcumin (MCACs) expressed preferable anti-inflammation effect by inhibit NF-κB signaling pathway. In order to find high efficiency and low toxicity anti-cancer compounds, on the basis of previous work we screened their antitumor activity and the inhibit effect on NF-кB, in the purpose to find the lead compounds with efficient and low toxicity against lung cancer.Methods:1、Curcumin analoguesWe get the Mono-carbonyl Analogues of Curcumin (MCACs) by altered the structure β-diketone in curcumin to, Mono-carbonyl. So we synthesized a batch of MCACs according to the leading compound Curcumin, and then identificated their structure through the ESI–MS and 1H-NMR, then retained in compound library. When screening the anti-inflammatory activity of MCACs, we found two class compounds can inhibit the NF-кB, one category is mono-carbonyl curcumin analogues EF24; another kind is multi-methoxyl replaced MCACs.2、Screening the anti-lung cancer activity We used MTT assay to determine the anti-tumor activity of the two kinds compounds, under different concentration gradient of these drugs to determine the half inhibition rate（IC50）against H460, A549, H1975 and HL7702(normal liver cells) . Found active compounds which expressed good effect against lung cancer and lower toxicity.3、The anti-lung cancer activity in vitro At first, Hoechst 33258 and Flow Cytometry methods were used to show that our compounds can significantly induce the lung cancer cells apoptosis or inhibition the cell cycle. Then Western Blotting methods was used to determine the inhibitor ability of NF-κB signaling pathway and their downstream signal protein IкB and Caspase-3 which are regulated by NF-κB.4、The anti-lung cancer activity in vivoAccording to the results of cell experiments, two perfect compounds that expressed preferable antitumor activity in vitro were chose for experiments in vivo, we used Nu/Nu BALB/c nude mice to establish H460 heterotopic transplantation tumor model. Abdominal cavity injection for 30 days, we determined the antitumor activity in vitro through calculate tumor volume, inhibition rate and immunohistochemical.Result: 一、The researches of EF24 MCACs targeting NF-κB and their pharmacological activities and mechanism against H4601. Ad10 and Aeb8 showed good inhibitory effect of proliferation on three types of lung cancer cells A549, H1975 and H460, particularly on H460 cell, with the IC50 of 12.14±0.53 μM and 5.01±0.56 μM respectively. Also we can see from the IC50 of normal liver cells (HL7702), so Ad10 and Aeb8 expressed good inhibition of lung cancer cells and much lower toxicity to normal cells. Compared with the positive control drug EF24 and curcumin, Ad10 and Aeb8 expressed equivalent or better activity agagnist lung cancer and lower cytotoxic. 2. Ad10 and Aeb8 under relatively low concentration can effectively inhibit the growth of H460 cell proliferation time curve, longer duration of drug action, the cell survival rate gradually reduced, the effect is more stable than positive medicine EF24. We also found that Ad10 (20 μM) after 24 h action time can obviously block the H460 cell cycle process, most of cells stay at the early stage of the division. And Aeb8 (1 μM) can interfere H460 cell colony formation effectively. 3. Ad10 and Aeb8 can inhibit the cell proliferation and at the same time can effectively restrain the NF-κB pathway induced by TNF-α such as the degradation of IкB, and their downstream apoptosis related proteins. 4. In nude mice model Ad10 and Aeb8 on H460 expressed the same activity as on the H460 cells, when they inhibited the growth of the nude mouse tumor at the same time can affect the NF-κB signaling pathway and their downstream apoptosis related proteins, such as Bcl-2, BAX and cycle related proteins.二、The researches of multiple methoxy replace MCACs targeting NF-κB and their pharmacological activities and mechanism against A549 1. We used MTT assay to determine the anti-proliferative effect of all the compounds on human lung cancer cells H460, A549and H1975. Most of the compounds showed good activity against lung cancer, and 11 among them have preferable inhibition activity against lung cancer cell proliferation (Mostly of these IC50s are about 3.0 μM), especially the 3B, 11B and 12B. Compared with curcumin, the compounds demonstrated better anti-tumor activity on cancer cells and low toxicity. 2. Hoechst 33258 and Flow Cytometry methods showed that 3B, 11B and 12B can significantly induce the A549 cells apoptosis. Especially about 11B, under the concentration of 1 μM can make the nuclei deformation even fragments occurred. The same result can be found on Western Blotting: 3B, 11B and 12B can induce the activation of Caspase-3 and inhibit the expression of Bcl-2 in a mode of dose dependent. 3. 3 B, 11 B, 12 B can inhibit the NF-κB signaling pathway in a certain degree, They can inhibit TNF-α induced phosphorylation of IKK and promotes the degradation of IκB in dose dependent, especially 12 B.Conclusion:This article found some promising prospects of MCACs analogues. These compounds generally have excellent anti-lung cancer activity. Among them some compounds can inhibit NF-кB signaling pathway, raised the regulation of apoptosis protein and cut its regulation of antiapoptotic proteins. They expressed a good inhibition of lung cancer cell proliferation, inducing apoptosis, and anti-lung cancer in nude mice tumor. But at present the targets and mechanism of this kind compounds were still unclear, so the mechanisms of anti-tumor activity and targets of such compounds need a further and deep study
语种	中文
学号	11005032
发布年限	2010-05-27
毕业论文分类号	0R90
原始专题	药学院
学位论文研究方向	小分子抗肿瘤活性及其机制研究
参考文献	参考文献 [1] HJ Kim , N Hawke3, AS Baldwin. NF-kB and IKK as therapeutic targets in cancer. Cell Death and Differentiation 2013, 13: 738–747. [2] Bark H, Choi CH. PSC833, cyclosporine analogue, downregulates MDR1 expression by activating JNK/c-Jun/AP-1 and suppressing NF-kappaB. Cancer chemotherapy and pharmacology 2010, 65(6):1131-1136. [3] Chen G, Umelo IA, Lv S, et al. miR-146a inhibits cell growth, cell migration and induces apoptosis in non-small cell lung cancer cells. PLoS One 2013, 8(3):e60317. [4] La Motte Rouge T, Galluzzi L, Olaussen KA,et al. A novel epidermal growth factor receptor inhibitor promotes apoptosis in non-small cell lung cancer cells resistant to erlotinib. Cancer Res 2007, 67(13):6253-6262. [5] Leighl NB, Paz-Ares L, Douillard JY,et al. Randomized phase III study of matrix metalloproteinase inhibitor BMS-275291 in combination with paclitaxel and carboplatin in advanced non-small-cell lung cancer: National Cancer Institute of Canada-Clinical Trials Group Study BR.18. Journal of clinical oncology 2005, 23(12):2831-2839. [6] Zhang L, Ge W, Hu K,et al. Endostar down-regulates HIF-1 and VEGF expression and enhances the radioresponse to human lung adenocarcinoma cancer cells. Molecular biology reports 2012, 39(1):89-95. [7] Gitlitz BJ, Moon J, Glisson BS,et al. Sorafenib in platinum-treated patients with extensive stage small cell lung cancer: a Southwest Oncology Group (SWOG 0435) phase II trial. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer 2010, 5(11):1835-1840. [8] Fuchs O.Targeting of NF-kappaB signaling pathway, other signaling pathways and epigenetics in therapy of multiple myeloma. Cardiovascular & hematological disorders drug targets 2013,13(1):16-34. [9] Khan N, Afaq F, Kweon MH,et al. Oral consumption of pomegranate fruit extract inhibits growth and progression of primary lung tumors in mice. Cancer Res 2007, 67(7):3475-3482. [10] Gamble C, McIntosh K, Scott R, Ho KH, Plevin R, Paul A: Inhibitory kappa B Kinases as targets for pharmacological regulation. British journal of pharmacology 2012, 165(4):802-819. [11] Stathopoulos GT, Zhu Z, Everhart MB, Kalomenidis I, Lawson WE, Bilaceroglu S, Peterson TE, Mitchell D, Yull FE, Light RW et al: Nuclear factor-kappaB affects tumor progression in a mouse model of malignant pleural effusion. American journal of respiratory cell and molecular biology 2006, 34(2):142-150. [12] Gupta SC, Sundaram C, Reuter S, Aggarwal BB: Inhibiting NF-kappaB activation by small molecules as a therapeutic strategy. Biochim Biophys Acta, 1799(10-12):775-787. [13] Tang X, Liu D, Shishodia S,et al. Nuclear factor-kappaB (NF-kappaB) is frequently expressed in lung cancer and preneoplastic lesions. Cancer 2006, 107(11):2637-2646. [14] Anto RJ, Mukhopadhyay A, Shishodia S, et al. Cigarette smoke condensate activates nuclear transcription factor-kappaB through phosphorylation and degradation of IkappaB(alpha): correlation with induction of cyclooxygenase-2. Carcinogenesis 2002, 23(9):1511-1518. [15] Aggarwal S, Ichikawa H, Takada Y,et al. Curcumin (diferuloylmethane) down-regulates expression of cell proliferation and antiapoptotic and metastatic gene products through suppression of IkappaBalpha kinase and Akt activation. Mol Pharmacol 2006, 69(1):195-206. [16] Chearwae W, Shukla S, Limtrakul P,et al. Modulation of the function of the multidrug resistance-linked ATP-binding cassette transporter ABCG2 by the cancer chemopreventive agent curcumin. Mol Cancer Ther 2006, 5(8):1995-2006. [17] Ikeda R, Vermeulen LC, Lau E,et al. Establishment and characterization of irinotecan-resistant human non-small cell lung cancer A549 cells. Molecular medicine reports 2010, 3(6):1031-1034. [18] Ye MX, Zhao YL, Li Y,et al. Curcumin reverses cis-platin resistance and promotes human lung adenocarcinoma A549/DDP cell apoptosis through HIF-1alpha and caspase-3 mechanisms. Phytomedicine 2012,19(8-9):779-787. [19] Pan Y, Wang Y, Cai L,et al. Inhibition of high glucose-induced inflammatory response and macrophage infiltration by a novel curcumin derivative prevents renal injury in diabetic rats. British journal of pharmacology 2012, 166(3):1169-1182. [20] Kasinski AL, Du Y, Thomas SL,et al. Inhibition of IkappaB kinase-nuclear factor-kappaB signaling pathway by 3,5-bis(2-flurobenzylidene)piperidin-4-one (EF24), a novel monoketone analog of curcumin. Mol Pharmacol 2008, 74(3):654-661. [21] Zhu S, Moore TW, Lin X,et al. Synthetic curcumin analog EF31 inhibits the growth of head and neck squamous cell carcinoma xenografts. Integrative biology : quantitative biosciences from nano to macro 2012, 4(6):633-640. [22] Kasinski AL, Du Y, Thomas SL,et al. Inhibition of IkappaB kinase-nuclear factor-kappaB signaling pathway by 3,5-bis(2-flurobenzylidene)piperidin-4-one (EF24), a novel monoketone analog of curcumin. Mol Pharmacol 2008, 74(3):654-661. [23] Amalia H, Sasaki R, Suzuki Y,et al. Vitamin K2-derived compounds induce growth inhibition in radioresistant cancer cells. The Kobe journal of medical sciences 2010, 56(2):E38-49. [24] Davis M, Hatzubai A, Andersen JS,et al Pseudosubstrate regulation of the SCF(beta-TrCP) ubiquitin ligase by hnRNP-U. Genes & development 2002, 16(4):439-451. [25] Wu J, Zhang Y, Cai Y, et al. Discovery and evaluation of piperid-4-one-containing mono-carbonyl analogs of curcumin as anti-inflammatory agents. Bioorganic & medicinal chemistry 2013, 21(11):3058-3065. [26] Kasinski AL, Du Y, Thomas SL,et al. Inhibition of IkappaB kinase-nuclear factor-kappaB signaling pathway by 3,5-bis(2-flurobenzylidene)piperidin-4-one(EF24), a novel monoketone analog of curcumin. Mol Pharmacol 2008, 74(3):654-661. [27] Amalia H, Sasaki R, Suzuki Y,et al. Vitamin K2-derived compounds induce growth inhibition in radioresistant cancer cells. The Kobe journal of medical sciences 2010,56(2):E38-49. [28] Davis M, Hatzubai A, Andersen JS, et al. Pseudosubstrate regulation of the SCF(beta-TrCP)ubiquitin ligase by hnRNP-U. Genes & development 2002, 16(4):439-451. [29] Lee CY, Sher HF, Chen HW, et al. Anticancer effects of tanshinone I in human non-small cell lung cancer. Mol Cancer Ther 2008, 7(11):3527-3538. [30] Olivera A, Moore TW, Hu F, et al. Inhibition of the NF-kappaB signaling pathway by the curcumin analog, 3,5-Bis(2-pyridinylmethylidene)-4-piperidone (EF31): anti-inflammatory and anti-cancer properties. International immunopharmacology 2012, 12(2):368-377. [31] Royt M, Mukherjee S, Sarkar R, et al. Curcumin sensitizes chemotherapeutic drugs via modulation of PKC, telomerase, NF-kappaB and HDAC in breast cancer. Therapeutic delivery 2011, 2(10):1275-1293. [32] Sandur SK, Deorukhkar A, Pandey MK, et al. Curcumin modulates the radiosensitivity of colorectal cancer cells by suppressing constitutive and inducible NF-kappaB activity. International journal of radiation oncology, biology,physics 2009, 75(2):534-542. [33] Shakibaei M, Mobasheri A, Lueders C, et al. Curcumin enhances the effect of chemotherapy against colorectal cancer cells by inhibition of NF-kappaB and Src protein kinase signaling pathways. PLoS One 2013, 8(2):e57218. [34] Wei X, Du ZY, Cui XX, et al. Effects of cyclohexanone analogues of curcumin on growth, apoptosis and NF-kappaB activity in PC-3 human prostate cancer cells. Oncology letters 2012, 4(2):279-284
全文文件名	11005032翁碧霞2014药理学.pdf\|11005032翁碧霞2014药理学.pdf
文献类型	学位论文
条目标识符	https://kms.wmu.edu.cn/handle/3ETUA0LF/115185
专题	温州医科大学
作者单位	溫州医科大学药学院
推荐引用方式 GB/T 7714	翁碧霞. 基于抑制NF-κB的单羰基姜黄素类似物的抗肺癌活性及其机制研究[D]. 温州医科大学,2016.