Live-Imaging om te studeren microtubuli Dynamic Instabiliteit in taxaan-resistente borstkankers
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
De belangrijkste oorzaak van de sterfte aan borstkanker is door middel van metastase 1, 2. Taxanen zoals paclitaxel en docetaxel, worden momenteel gebruikt als eerstelijnsbehandeling regimes voor de behandeling van metastatische borstkanker 2, 3, 4, 5, 6. Ze behoren tot een groep van microtubulus richtende middelen (MTA) die dynamiek van microtubuli verstoren. Een van de grootste uitdagingen voor gebruik taxanen curatieve therapie is de ontwikkeling van resistentie taxaan in kankercellen, wat leidt tot terugkeer van de ziekte 7. Geneesmiddelresistentie goed voor meer dan 90% van alle sterfgevallen bij patiënten met metastatische borstkanker 7.
Microtubuli worden gevormd door de polymerisatie van α- en β-tubuline heterodimerenclass = "xref"> 8, 9. De precieze regeling van de dynamiek van microtubuli is van belang voor vele cellulaire functies, zoals mobiele polarisatie, voortgang van de celcyclus, intracellulair transport en cell signaling. Ontregeling van microtubuli en hun dynamiek celfunctie verstoren en leiden tot celdood 10, 11. Afhankelijk van hoe zij veroorzaken deze ontregeling, kan MTA drugs worden geclassificeerd als microtubule stabiliserende middelen (dat wil zeggen, taxanen) of microtubule-destabalizing middelen (dat wil zeggen, vinca alkaloïden of colchicine plaatse bindmiddelen) 20. Ondanks hun tegengestelde effecten op microtubuli massa, op een voldoende dosering, beide klassen kunnen kankercellen te doden door hun effecten op de dynamiek van microtubuli 21.
Taxanen werken voornamelijk door het stabiliseren van de microtubule as 12, waardoorchromosomale foutieve uitlijning. De daaropvolgende voortdurende activering van de spindel montage checkpoint (SAC) arresteert de cel in mitose. Langdurige mitose veroorzaakt apoptose dan 13, 14. Taxaan reageert met microtubuli door het taxaan bindingsplaats op β-tubuline 8, 15, die alleen aanwezig in gemonteerde tubuline 16.
Meerdere mechanismen voor taxaan weerstand zijn voorgesteld 9, 17. Deze mechanismen omvatten zowel algemene multidrug resistentie door de overexpressie van drug-efflux eiwitten en taxaan-specifieke weerstand 5, 9, 18, 19. Bijvoorbeeld kan taxaan-resistente kankercellen veranderde expressie en functie van bepaalde β-badUlin isotypen 5, 9, 19, 20, 21, 22, 23. Door toepassing van een in vivo methode om microtubule dynamische instabiliteit meten, laten we zien dat, in vergelijking met niet-resistente, ouderlijke MCF-7 cellen 17 CC, de microtubule dynamiek van docetaxel-resistente MCF-7 TXT cellen zijn ongevoelig voor behandeling met docetaxel.
Om een beter begrip van de functie van MTA en het exacte mechanisme van taxaan-resistentie in kankercellen, is het essentieel om microtubule dynamiek meten. Hier rapporteren we een in vivo werkwijze voor het doen. Via levende beeldvorming in combinatie met de expressie van GFP-gelabeld tubuline in cellen, kunnen we de microtubule dynamiek van MCF-7 TXT en MCF-7 cellen met CC en wi metenthout de behandeling met docetaxel. Het resultaat kan ons helpen het ontwerpen van meer doeltreffende geneesmiddelen die taxaan weerstand kan overwinnen.
Protocol
Representative Results
Discussion
Er zijn twee belangrijke methoden microtubule dynamische instabiliteit meten: in vitro en in vivo. In de in vitro werkwijze wordt gezuiverd tubuline gebruikt microtubule dynamische instabiliteit-computer verbeterde time-lapse differentiële interferentie contrast microscopie meten. In de in vivo werkwijze gemicroinjecteerd fluorescerende tubuline of expressie GFP-tubuline, wordt opgenomen in microtubules. De dynamiek (groei en verkorting) van de microtubuli wordt vervolgens geregistre…
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 |
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