实时成像来研究抗紫杉烷乳腺癌微管动态失稳
Summary
In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells.
Abstract
Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane resistance in cancerous cells drastically reduces the effectiveness of the drugs’ long-term usage. Accumulated evidence suggests that the mechanisms underlying taxane resistance include both general mechanisms, such as the development of multidrug resistance due to the overexpression of drug-efflux proteins, and taxane-specific mechanisms, such as those that involve microtubule dynamics.
Because taxanes target cell microtubules, measuring microtubule dynamic instability is an important step in determining the mechanisms of taxane resistance and provides insight into how to overcome this resistance. In the experiment, an in vivo method was used to measure microtubule dynamic instability. GFP-tagged α-tubulin was expressed and incorporated into microtubules in MCF-7 cells, allowing for the recording of the microtubule dynamics by time lapse using a sensitive camera. The results showed that, as opposed to the non-resistant parental MCF-7CC cells, the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitive to docetaxel treatment, which causes the resistance to docetaxel-induced mitotic arrest and apoptosis. This paper will outline this in vivo method of measuring microtubule dynamic instability.
Introduction
乳腺癌死亡率的主要原因就是通过转移1，2。紫杉烷类，如多西他赛和紫杉醇，目前用作一线转移性乳腺癌2，3，4，5，6的治疗方案。他们是一群扰乱微管动力学微管靶向代理（MTA）的一部分。然而，在治疗性治疗使用紫杉烷类的最大挑战之一是在癌细胞中紫杉烷抗性，从而导致疾病复发7的发展。耐药占转移性乳腺癌7之间所有死亡的90％以上。
微管是由α-和β微管蛋白异源二聚体的聚合形成类=“外部参照”> 8，9。微管动力学的精确调节是许多细胞功能，包括细胞极化，细胞周期进程，细胞内运输和细胞信号传导的重要。微管和它们的动态的失调会扰乱细胞的功能，导致细胞死亡10,11。取决于它们怎样导致此失调，MTA药物可归类为微管稳定剂（即紫杉烷）或微管destabalizing剂（ 即，长春花生物碱或秋水仙碱点结合剂）20。尽管微管的质量的相反的效果，以足够的剂量，这两个类可以通过其对微管动力学21作用杀死癌细胞。
紫杉烷类通过稳定微管主轴12主要作用，导致染色体错位。在纺锤体装配检验点（SAC）的后续永久激活逮捕的有丝分裂细胞。延长的有丝分裂停滞然后导致凋亡13,14。紫杉烷与通过对β微管蛋白8，15，这是只有在组装的微管蛋白16存在的紫杉烷的结合位点的微管相互作用。
对紫杉烷抗性多种机制已经提出9,17。这些机制包括由于药物外排蛋白与紫杉类药物特异性抵抗力5，9，18，19的表达了一般多药耐药性。例如，耐紫杉类癌细胞可能已经改变了某些β桶的表达和功能同型球蛋白5，9，19，20，21，22，23。通过使用体内方法测量微管动态不稳定，我们表明，相对于非抗性，父母的MCF-7 的CC单元17时，耐多西紫杉醇的MCF-7 的TXT细胞的微管动力学是不敏感的多西他赛治疗。
为了更好地了解的MTA的功能，并在癌细胞紫杉烷抗性的确切机制，重要的是测量微管动力学。这里，我们报告这样的体内方法。通过使用实时成像结合GFP标记的微管蛋白在细胞中的表达，我们可以测量的MCF-7 的TXT和MCF-7 的CC细胞和Wi的微管动力学thout多西紫杉醇治疗。研究结果可以帮助我们设计出能够克服阻力紫杉更加有效的药物。
Protocol
Representative Results
Discussion
有两种主要的方法来测量微管动态不稳定性： 在体外和体内。在体外方法中，纯化的微管蛋白是用来测量与计算机增强的时间推移微分干涉反差显微镜微管动态不稳定性。在体内方法中，显微注射荧光微管蛋白，或表达GFP的微管蛋白，掺入微管。微管的动力学（生长和缩短）然后通过时间推移在间期细胞10，20，25</…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This research is supported by funding from CBCF (to ZW).
Materials
| Dulbecco's Modified Eagle's Medium (DMEM) | Sigma-Aldrich | D5796 | |
| Non-essential amino acids | Life Technologies, Invitrogen | 11140-050 | |
| FBS | Gibco, Invitrogen | 12483 | |
| Anti-Anti (100x) | Life Technologies, Invitrogen | 15240-062 | |
| docetaxel | Sigma-Aldrich | 01885-5mg-F | |
| DMEM phenol red-free | Gibco, Invitrogen | 21063 | |
| CellLight Reagent *BacMam 2.0* GFP-tubulin | ThermoFisher Scientific | C10613 | Key reagent for expressing GFP tubulin in cells |
| CellLight Reagent *BacMam 2.0* GFP | ThermoFisher Scientific | B10383 | Control |
| Dimethyl Sulfoxide (DMSO) | Sigma-Aldrich+B9:AA9 | 472301 | for dissoving decetaxel |
| 22-mm glass coveslip | Fisher Scientifics | 12-545-101 | |
| 6-well culture plate | Greiner Bio-One International | 6 Well Celi Culture Plate | |
| DeltaVision Microscopy Imaging Systems | GE Health | This system is equipped with weather station for controlling temperature and CO2. It also equipped with Worx Software for deconvolution and time lapse control. | |
| Trypsin-EDTA (0.25%), phenol red | ThermoFisher Scientific | 25200056 | |
| Bright-Line Hemacytometer Set, Hausser Scientific | Hausser Scientific, Distributed by VWR | Supplier No.: 1492 VWR No.:15170-172 |
Riferimenti
- Kamangar, F., Dores, G. M., Anderson, W. F. Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic regions of the world. J Clin Oncol. 24 (14), 2137-2150 (2006).
- Yardley, D. A. Drug resistance and the role of combination chemotherapy in improving patient outcomes. Int J Breast Cancer. 2013, 137414 (2013).
- Jassem, J., et al. Doxorubicin and paclitaxel versus fluorouracil, doxorubicin, and cyclophosphamide as first-line therapy for women with metastatic breast cancer: final results of a randomized phase III multicenter trial. J Clin Oncol. 19 (6), 1707-1715 (2001).
- Nabholtz, J. M., et al. Docetaxel and doxorubicin compared with doxorubicin and cyclophosphamide as first-line chemotherapy for metastatic breast cancer: results of a randomized, multicenter, phase III trial. J Clin Oncol. 21 (6), 968-975 (2003).
- Zelnak, A. Overcoming taxane and anthracycline resistance. Breast J. 16 (3), 309-312 (2010).
- Rivera, E. Implications of anthracycline-resistant and taxane-resistant metastatic breast cancer and new therapeutic options. Breast J. 16 (3), 252-263 (2010).
- Longley, D. B., Johnston, P. G. Molecular mechanisms of drug resistance. J Pathol. 205 (2), 275-292 (2005).
- Downing, K. H., Nogales, E. Crystallographic structure of tubulin: implications for dynamics and drug binding. Cell Struct.Funct. 24 (5), 269-275 (1999).
- McGrogan, B. T., Gilmartin, B., Carney, D. N., McCann, A. Taxanes, microtubules and chemoresistant breast cancer. Biochim.Biophys.Acta. 1785 (2), 96-132 (2008).
- Kamath, K., Oroudjev, E., Jordan, M. A. Determination of microtubule dynamic instability in living cells. Methods Cell Biol. 97, 1-14 (2010).
- Dumontet, C., Jordan, M. A. Microtubule-binding agents: a dynamic field of cancer therapeutics. Nat Rev Drug Discov. 9 (10), 790-803 (2010).
- Jordan, M. A., Wilson, L. Microtubules as a target for anticancer drugs. Nat.Rev.Cancer. 4 (4), 253-265 (2004).
- Gascoigne, K. E., Taylor, S. S. How do anti-mitotic drugs kill cancer cells?. J.Cell Sci. 122 (15), 2579-2585 (2009).
- Kavallaris, M. Microtubules and resistance to tubulin-binding agents. Nat.Rev.Cancer. 10 (3), 194-204 (2010).
- Diaz, J. F., Valpuesta, J. M., Chacon, P., Diakun, G., Andreu, J. M. Changes in microtubule protofilament number induced by Taxol binding to an easily accessible site. Internal microtubule dynamics. J.Biol.Chem. 273 (50), 33803-33810 (1998).
- Abal, M., Andreu, J. M., Barasoain, I. Taxanes: microtubule and centrosome targets, and cell cycle dependent mechanisms of action. Curr Cancer Drug Targets. 3 (3), 193-203 (2003).
- Wang, H., et al. Multiple mechanisms underlying acquired resistance to taxanes in selected docetaxel-resistant MCF-7 breast cancer cells. BMC Cancer. 14 (37), (2014).
- Lal, S., Mahajan, A., Chen, W. N., Chowbay, B. Pharmacogenetics of target genes across doxorubicin disposition pathway: a review. Curr. Drug Metab. 11 (1), 115-128 (2010).
- Murray, S., Briasoulis, E., Linardou, H., Bafaloukos, D., Papadimitriou, C. Taxane resistance in breast cancer: mechanisms, predictive biomarkers and circumvention strategies. Cancer Treat.Rev. 38 (7), 890-903 (2012).
- Kamath, K., Wilson, L., Cabral, F., Jordan, M. A. BetaIII-tubulin induces paclitaxel resistance in association with reduced effects on microtubule dynamic instability. J.Biol.Chem. 280 (13), 12902-12907 (2005).
- Banerjee, A. Increased levels of tyrosinated alpha-, beta(III)-, and beta(IV)-tubulin isotypes in paclitaxel-resistant MCF-7 breast cancer cells. Biochem.Biophys.Res.Commun. 293 (1), 598-601 (2002).
- Wiesen, K. M., Xia, S., Yang, C. P., Horwitz, S. B. Wild-type class I beta-tubulin sensitizes Taxol-resistant breast adenocarcinoma cells harboring a beta-tubulin mutation. Cancer Lett. 257 (2), 227-235 (2007).
- Iseri, O. D., Kars, M. D., Arpaci, F., Gunduz, U. Gene expression analysis of drug-resistant MCF-7 cells: implications for relation to extracellular matrix proteins. Cancer Chemother.Pharmacol. 65 (3), 447-455 (2010).
- Hembruff, S. L., et al. Role of drug transporters and drug accumulation in the temporal acquisition of drug resistance. BMC.Cancer. 8, 318 (2008).
- Yenjerla, M., Lopus, M., Wilson, L. Analysis of dynamic instability of steady-state microtubules in vitro by video-enhanced differential interference contrast microscopy with an appendix by Emin Oroudjev. Methods Cell Biol. 95, 189-206 (2010).
- Sammak, P. J., Gorbsky, G. J., Borisy, G. G. Microtubule dynamics in vivo: a test of mechanisms of turnover. J Cell Biol. 104 (3), 395-405 (1987).
- Walker, R. A., et al. Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies. J Cell Biol. 107 (4), 1437-1448 (1988).
- Desai, A., Mitchison, T. J. Microtubule polymerization dynamics. Annu Rev Cell Dev Biol. 13, 83-117 (1997).
- Walczak, C. E. Microtubule dynamics and tubulin interacting proteins. Curr Opin Cell Biol. 12 (1), 52-56 (2000).
