Live-Imaging til at studere på mikrotubulus Dynamisk Ustabilitet i taxan-resistent brystkræft
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
Den førende dødsårsag brystkræft er gennem metastase 1, 2. Taxaner, såsom docetaxel og paclitaxel, der i øjeblikket anvendes som førstevalg regimer i behandlingen af metastatisk brystkræft 2, 3, 4, 5, 6. De er en del af en gruppe af mikrotubuli-targeting midler (MTA'er), der forstyrrer mikrotubulusdynamik. Men en af de største udfordringer for anvendelse af taxaner i kurativ terapi er udviklingen af taxan resistens i cancerceller, hvilket fører til recidiv 7. Lægemiddelresistens tegner sig for mere end 90% af alle dødsfald blandt patienter med metastatisk brystkræft 7.
Mikrotubuli dannes ved polymerisation af a- og p-tubulin-heterodimererclass = "xref"> 8, 9. Den præcise regulering af mikrotubulusdynamik er vigtigt for mange cellulære funktioner, herunder celle polarisering, cellecyklusprogression, intracellulær transport, og cellesignalering. Dysregulering af mikrotubuli og deres dynamik vil forstyrre cellefunktion og resultere i celledød 10, 11. Afhængigt af, hvordan de forårsager denne fejlregulering, kan MTA narkotika klassificeres som enten mikrotubuli stabiliserende midler (dvs. taxaner) eller mikrotubuli-destabalizing midler (dvs. vinca alkaloider eller colchicin-site bindemidler) 20. Trods deres modsatte virkninger på mikrotubulus masse, ved en tilstrækkelig dosering, kan begge klasser dræbe kræftceller via deres virkninger på mikrotubulusdynamik 21.
Taxaner fungerer primært ved at stabilisere mikrotubulære spindel 12, der fører tilkromosomal fejljustering. Den efterfølgende perpetual aktivering af spindlen samling kontrolpunkt (SAC) standser cellen i mitose. Langvarig mitosestandsning derefter forårsager apoptose 13, 14. Taxan interagerer med mikrotubuli gennem taxan bindingsstedet på β-tubulin 8, 15, som kun er til stede i samlet tubulin 16.
Flere mekanismer for taxan resistens er blevet foreslået 9, 17. Disse mekanismer omfatter både generel multilægemiddelresistens grund af overekspression af lægemiddel-efflux proteiner og taxan-specifik resistens 5, 9, 18, 19. For eksempel kan taxan-resistente cancerceller har ændret ekspression og funktion af visse β-tubUlin isotyper 5, 9, 19, 20, 21, 22, 23. Ved anvendelse af en in vivo metode til at måle mikrotubulære dynamik ustabilitet, viser vi, at, når sammenlignet med ikke-modstandsdygtige, parentale MCF-7 CC celler 17, de mikrotubulusdynamik af docetaxel-resistente MCF-7 TXT celler er ufølsomme over for docetaxel behandling.
For bedre at forstå funktionen af MTA'erne og den nøjagtige mekanisme af taxan-resistens i kræftceller, er det vigtigt at måle mikrotubulusdynamik. Her rapporterer vi en in vivo fremgangsmåde til at gøre det. Ved at anvende levende billeddannelse i kombination med ekspressionen af GFP-mærkede tubulin i cellerne, kan vi måle mikrotubulusdynamik af MCF-7 TXT og MCF-7-CC-celler med og without docetaxel behandling. Resultaterne kan hjælpe os med at designe mere effektive lægemidler, der kan overvinde taxan modstand.
Protocol
Representative Results
Discussion
Der er to store metoder til at måle mikrotubulære dynamisk ustabilitet: in vitro og in vivo. I in vitro-metode, oprenses tubulin anvendes til at måle mikrotubulus dynamisk ustabilitet med computer-forbedret time-lapse differential interferens-kontrast mikroskopi. I in vivo-metoden, mikroinjiceres fluorescerende tubulin, eller udtrykkes GFP-tubulin, er indarbejdet i mikrotubuli. Dynamikken (vækst og afkortning) af mikrotubuli registreres derefter ved tidsforskudt anvendelse af et f…
Disclosures
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 |
References
- 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).
