增强细胞凋亡和自噬的诱导合成一种新型的C-1 7-deoxypancratistatin的类似物在人类乳癌细胞和神经母细胞瘤与他莫昔芬
Summary
我们已合成了新型防癌抗癌作为本地pancratistatin活动相媲美的pancratistatin模拟;有趣的是，与他莫昔芬治疗组合的产生在细胞凋亡和自噬的诱导线粒体与非癌成纤维细胞的影响最小的目标大幅提高。因此，与他莫昔芬结合JCTH-4可以提供一个安全的抗癌疗法。
Abstract
乳腺癌是最常见的癌症之一，除在北美的妇女。目前许多抗癌药物，包括电离辐射，通过DNA损伤诱导细胞凋亡。不幸的是，这种治疗方法有非选择性的肿瘤细胞，在正常细胞产生类似的毒性。我们已经报道（PST），通过天然化合物pancratistatin的选择性诱导肿瘤细胞凋亡。最近，一种新的PST模拟，7 deoxypancratistatin（JCTH-4）的C-1乙酰衍生物，从头合成产生，它在多种癌细胞株展品媲美选择性诱导凋亡活性。最近，自噬已被牵连在恶性肿瘤亲生存和死亡机制在化疗反应。三苯氧胺（TAM）总是表现出亲生存在许多癌症细胞自噬的诱导。在这项研究中，JCTH-4单独和谭相结合的疗效，诱导细胞死亡在人类乳腺癌canceR（MCF7细胞）和神经母细胞瘤细胞（SH-SY5Y细胞）进行了评估。谭单独诱导细胞自噬，但单独JCTH-4微不足道的细胞死亡而造成的一些诱导的自噬诱导细胞凋亡的重要。有趣的是，组合的治疗取得了大幅增加在细胞凋亡和自噬的诱导。我们监测的组合使用治疗时间推移显微镜MCF7细胞的形态学变化时间依赖性接受TAM诱导细胞自噬，JCTH-4诱导的细胞凋亡和自噬，并加速细胞死亡。我们已经证明了这些化合物诱导细胞凋亡/自噬在这些癌细胞的线粒体定位。重要的是，这些治疗并不影响非癌的人成纤维细胞的生存。因此，这些结果表明，JCTH-4在与谭相结合，可以作为一个安全和非常强大的抗癌疗法对乳腺癌和神经母细胞瘤细胞。
Protocol
介绍细胞凋亡，或I型程序性细胞死亡，是一种生理过程，可以运行外在，通过死亡配体结合死亡受体，或本质。细胞凋亡的内在途径是由细胞内的压力，如DNA损伤和线粒体功能障碍，最终导致线粒体，线粒体膜电位的耗散（MMP）的释放凋亡因子从线粒体间隙的通透性，和随后的执行细胞凋亡1。 自噬是一个过程，在其中一个单元休息，降解，回收自…
Discussion
PST和类似的化合物已被证实具有抗癌特性11-15,21。我们以前曾报道自然的PST线粒体选择性破坏肿瘤细胞，从而诱导细胞凋亡12,14凋亡因子的释放。这是最有可能的，JCTH-4的行为，通过相同的机制;造成JCTH-4 TMRM染色（ 图5a），并增加从SH-SY5Y细胞中分离出线粒体ROS生成在MCF7细胞基质金属蛋白酶的崩溃（ 图5B），线粒体功能障碍的指标。因此，诱导细胞凋亡由J…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作已经得到了哥伦布章9671（安大略省温莎市），以及CIHR弗雷德里克班廷和查尔斯·加拿大研究生奖学金颁发给丹尼斯·马骑士。感谢他们的协助，你罗伯特·霍奇和伊丽莎白菲达尔戈达席尔瓦随着时间推移显微镜。谢谢凯蒂Facecchia时间推移显微镜视频编辑。我们也想感谢这个手稿的严格审查Sudipa六月查特吉和Phillip特伦布莱。这项工作是致力于记忆凯文Couvillon谁失去了对抗癌症的斗争，他在2010年。
Materials
| Material Name | Company | Catalogue number |
| SH-SY5Y cell line | ATCC | CRL-2266 |
| Dulbecco’s Modified Eagles Medium F-12 HAM | Sigma-Aldrich | 51448C |
| Fetal bovine serum | Gibco BRL | 16000-044 |
| MCF7 cell line | ATCC | HTB-22 |
| RPMI-1640 medium | Sigma-Aldrich | R 0883 |
| Apparently normal human fetal fibroblast cell line (NFF) | Coriell Institute for Medical Research | AG04431B |
| Dulbecco’s Modified Eagle’s Medium, High Glucose medium | Thermo Scientific | SH30022.01 |
| Tamoxifen citrate salt | Sigma-Aldrich | T9262 |
| 35 mm glass bottom culture dishes | MatTek | P35G-014-C |
| Leica DMI6000 B inverted microscope | Leica Microsystems | N/A |
| Hoechst 33342 dye | Molecular Probes | H3570 |
| Leica DM IRB inverted fluorescence microscope | Leica Microsystems | N/A |
| Annexin V AlexaFluor-488 | Invitrogen | A13201 |
| Trypan Blue solution | Sigma-Aldrich | T8154-20ML |
| Haemocytometer | Fisher Scientific | 267110 |
| WST-1 reagent | Roche Applied Science | 11644807001 |
| Wallac Victor3 1420 Multilabel Counter | PerkinElmer | 1420-011 |
| Tetramethylrhodamine methyl ester (TMRM) | Gibco BRL | T-668 |
| Glass tissue grinder | Fisher Scientific | K8885300-0002 |
| BioRad protein assay | Bio-Rad Laboratories | 500-0001Bottom of Form |
| Amplex Red | Invitrogen | A12222 |
| Horseradish peroxidase (HRP) | Sigma-Aldrich | P8125 |
| SpectraMax Gemini XPS | Molecular Devices | 3126666 |
| Anti-LC3 antibody raised in rabbit | Novus Biologicals | NB100-2220 |
| Anti-mouse HRP-conjugated secondary antibody | Abcam | ab6728 |
| Anti-rabbit HRP-conjugated secondary antibody | Abcam | ab6802 |
| Chemiluminescence peroxidase substrate | Sigma-Aldrich | CPS160 |
| Monodansylcadaverine | Sigma-Aldrich | 30432 |
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Tags
Apoptotic InductionAutophagic InductionSynthetic C-1 Analogue7-deoxypancratistatinBreast AdenocarcinomaNeuroblastoma CellsTamoxifenAnti-cancer TreatmentIonizing RadiationDNA DamageSelective ApoptosisPancratistatinJCTH-4AutophagyMalignanciesPro-survival MechanismPro-death MechanismChemotherapyCell DeathMCF7 CellsSH-SY5Y Cells
Cite This Article
Ma, D., Collins, J., Hudlicky, T., Pandey, S. Enhancement of Apoptotic and Autophagic Induction by a Novel Synthetic C-1 Analogue of 7-deoxypancratistatin in Human Breast Adenocarcinoma and Neuroblastoma Cells with Tamoxifen. J. Vis. Exp. (63), e3586, doi:10.3791/3586 (2012).