Levende Imaging å studere microtubule Dynamic Ustabilitet i taxan-resistent brystkreft
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 største årsaken til brystkreft dødelighet er gjennom metastase 1, 2. Taxaner slik som docetaxel og paclitaxel, er for tiden brukes som førstelinjebehandling i behandling av metastatisk brystkreft 2, 3, 4, 5, 6. De er en del av en gruppe mikrotubulidynamikk målretting midler (MTAS) som forstyrrer mikrotubulidynamikk. Imidlertid er en av de største utfordringene for ved hjelp av taksaner i kurativ terapi er utviklingen av taksan resistens i cancerceller, noe som fører til tilbakefall av sykdommen 7. Drug motstand står for mer enn 90% av alle dødsfall blant pasienter med metastatisk brystkreft 7.
Mikrotubuli er dannet ved polymerisering av a- og p-tubulin heterodimererclass = "xref"> 8, 9. Den nøyaktige regulering av mikrotubulidynamikk er viktig for mange cellulære funksjoner, inkludert celle polarisering, cellesyklusprogresjon, intracellulær transport, og cellesignalisering. Dysregulering av mikrotubuler og deres dynamikk vil forstyrre cellefunksjon og føre til celledød 10, 11. Avhengig av hvordan de forårsaker denne feilregulering, kan MTA legemidler klassifiseres som enten mikrotubulidynamikk stabiliserende midler (dvs. taxaner) eller microtubule-destabalizing midler (dvs. vincaalkaloider eller kolkisin stedet bindemidler) 20. Til tross for deres motsatte effekter på microtubule masse, på en tilstrekkelig dose, kan begge klasser drepe kreftceller gjennom sine effekter på mikrotubulidynamikk 21.
Taxaner funksjon først og fremst ved å stabilisere microtubule spindelen 12, som fører tilkromosom forskyvning. Den påfølgende evigvarende aktivering av spindelenheten sjekkpunkt (SAC) arrestasjoner cellen i mitose. Langvarig mitotisk arrest deretter forårsaker apoptose 13, 14. Taxan samvirker med mikrotubuli gjennom taksan bindingssetet på β-tubulin 8, 15, som bare er til stede i montert tubulin 16.
Flere mekanismer for taxan motstand har vært foreslått 9, 17. Disse mekanismene innbefatter både generell multimedikamentresistens på grunn av overekspresjon av stoff-effluks proteiner og taxan-spesifikke motstand 5, 9, 18, 19. For eksempel kan taxan-resistente kreftcellene har forandret ekspresjon og funksjon av visse β-karSienna isotyper 5, 9, 19, 20, 21, 22, 23. Ved hjelp av en in vivo-metode for å måle mikrotubulus dynamisk ustabilitet viser vi at, sammenlignet med ikke-resistente, foreldre MCF-7-celler CC-17, de mikrotubul-dynamikk av docetaxel-resistent MCF-7-celler TXT er ufølsomme for docetaxel behandling.
For bedre å forstå funksjonen av MTA-er, og den eksakte mekanismen for taxan-resistens i kreftceller, er det viktig å måle mikrotubul-dynamikk. Her rapporterer vi en in vivo metode for å gjøre det. Ved å bruke levende avbildning i kombinasjon med uttrykket av GFP-merket tubulin i cellene, kan vi måle mikrotubulidynamikk av MCF-7 TXT og MCF-7 CC celler med og without docetaxel behandling. Resultatene kan hjelpe oss å designe mer effektive legemidler som kan overvinne taxan motstand.
Protocol
Representative Results
Discussion
Det er to hovedfremgangsmåter for å måle mikrotubul dynamisk ustabilitet: in vitro og in vivo. Ved in vitro-metoden, ble renset tubulin brukes til å måle mikrotubul dynamisk ustabilitet med datamaskinen forbedret time-lapse differensialinterferenskontrastmikroskopi. I in vivo-metode, mikroinjisert fluorescerende tubulin, eller uttrykt GFP-tubulin, er innlemmet i mikrotubuli. Dynamikken (vekst og forkorting) av mikrotubuli blir deretter registrert av time-lapse ved hjelp av et fø…
Declarações
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 |
Referências
- 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).
