PO.ET06.05 · 实验与分子治疗
用印楝（Azadirachta indica）柠檬苦素类化合物Nimbolide靶向治疗犬膀胱癌
Targeting canine bladder cancer with Nimbolide, a Neem ( Azadirachta indica ) limonoid
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：伴远处转移的膀胱癌（BlCa）患者5年相对生存率约为9%。因此，亟需针对转移性BlCa的新型创新疗法。研究发现，犬会自发发生肌层浸润性膀胱癌（MIBC），且10%在诊断时进展为远处转移。目前大多数患有MIBC的犬采用非甾体抗炎药（NSAIDs）治疗；然而，反应率<25%。我们的目标是寻找可用于犬的低成本、低毒性天然化合物。Nimbolide是从印楝叶中提取的一种柠檬苦素类化合物，已发现其对人BlCa细胞系具有抗肿瘤作用。因此，我们检验了该天然产物是否对犬BlCa细胞系有效。
方法：我们从普渡大学的Deborah Knapp博士处获得了两种犬BlCa细胞系——K9TCC-PU-AXC（AXC，能在裸鼠中形成肿瘤）和K9TCC-PU-Pu（PuPu，不能形成肿瘤）。通过MTT法及3D打印球体上的活/死细胞染色评估细胞活力。采用流式细胞术评估凋亡率。通过LC3B和p62的表达评估自噬，通过波形蛋白（vimentin）、Snail-1、Snail-2的表达评估EMT，并使用Cytation 5平台通过划痕实验评估细胞迁移。
结果：Nimbolide对PuPu细胞（IC50 = 0.656 μM）抑制增殖的效果强于AXC细胞（IC50 = 1.2 μM）。流式细胞术分析显示，AXC对nimbolide不发生凋亡，而PuPu则发生凋亡；然而，nimbolide在两种细胞中均引起自噬。另一方面，nimbolide阻止了AXC细胞的上皮-间质转化（EMT）和迁移，但对PuPu细胞无此作用。在人真皮成纤维细胞中未观察到nimbolide的这些作用，突显了nimbolide的特异性和选择性。
结论：基于这些结果，我们计划在未来研究中将nimbolide开发为治疗犬BlCa的治疗工具。
致谢：本文内容完全由作者负责，不代表美国国立卫生研究院、退伍军人事务部或美国政府的官方观点。我们感谢加州大学综合癌症中心（UCDCCC）支持基金（P30CA093373）为本项目提供的慷慨资助。
查看英文原文 English abstract
Background: The 5-year relative survival rate for Bladder Cancer (BlCa) patients with distant metastases is about 9%. Hence, novel and innovative therapies for metastatic BlCa are required. Dogs were found to develop muscle-invasive BlCa (MIBC) spontaneously and 10% progress to distant metastases at the time of diagnoses. Currently most dogs with MIBC are treated with non-steroidal anti-inflammatory drugs (NSAIDs); however, <25% respond to them. Our quest was to identify low-cost natural compounds of low toxicity that can be used in dogs. Nimbolide, a limonoid extracted from Neem leaves, was found to have anti-tumor effects in human BlCa cell lines. Hence, we tested whether this natural product will be effective in canine BlCa cell lines.
Methods: We obtained two canine BlCa cell lines - K9TCC-PU-AXC (AXC), that formed tumors in nude mice and K9TCC-PU-Pu (PuPu), which did not, from Dr. Deborah Knapp, Purdue University. Cell viability was estimated by MTT assay and by live/dead cell staining on 3D-printed spheroids. Flow cytometry was used to estimate the rate of apoptosis. Autophagy was estimated by the expression of LC3B and p62 whereas EMT was estimated by the expression of vimentin, Snail-1, Snail-2, and cell migration was estimated by scratch assay using a Cytation 5 platform.
Results: Nimbolide was more effective in suppressing proliferation in PuPu cells (IC 50 = 0.656 µM) compared to AXC cells (IC 50 = 1.2 µM). Flow cytometric analysis showed that AXC did not undergo apoptosis in response to nimbolide whereas PuPu did; however, nimbolide caused autophagy in both cells. On the other hand, nimbolide prevented epithelial mesenchymal transition (EMT) and migration in AXC, but not in PuPu cells. These effects of nimbolide were not seen in human dermal fibroblasts, underlining the specificity and selectivity of nimbolide.
Conclusions: Based on these results, we intend to develop nimbolide as a therapeutic tool in the treatment of canine BlCa in future studies.
Acknowledgements: The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health, the Department of Veterans Affairs or the United States Government. We are grateful for a generous award from the University of California Comprehensive Cancer Center (UCDCCC) Support Grant (P30CA093373) for this project.
利益披露 Disclosure
C. Suen, None..
E. Sharma, None..
G. Gilchrist, None..
N. Batra, None..
C. A. Lucchesi, None..
K. A. Iczkowski, None..
R. B. Rebhun, None..
P. M. Ghosh, None.