Name: live imaging to study microtubule dynamic instability in taxane-resistant breast cancers
Uploaded: 2017-02-20T13:00:00
Description: Watch this Scientific Journal Video about Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers at JoVE.com

This research is supported by funding from CBCF (to ZW).

1. Vorbereitung der Zellen für Live-Imaging <ol><li> Zellkultur und Seeding <ol><li> Verwenden MCF-7 Brustkrebszellen , ausgewählt für die Resistenz gegen Docetaxel (MCF-7 TXT) und deren nicht-resistenten parentalen Zelllinie (MCF-7 CC). Die detaillierten Auswahlprozess und die Charakterisierung dieser ausgewählten Zelllinien wurden zuvor 24 beschrieben. </li><li> Wachsen alle Zellen in 10 cm-Kulturschalen bei 37 ° C in einem Medium, 90% Dulbecco best...

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Es gibt zwei Hauptmethoden Mikrotubuli dynamische Instabilität zu messen: in vitro und in vivo. In dem in vitro - Verfahren wird gereinigtes Tubulin verwendet Mikrotubuli dynamische Instabilität mit Computer-enhanced Zeitrafferdifferentialinterferenzkontrastmikroskopie zu messen. In dem in vivo - Verfahren, mikroinjiziert Fluoreszenz Tubulin oder Tubulin-GFP exprimiert, in Mikrotubuli eingebaut. Die Dynamik (Wachstum und Verkürzung) der Mikrotubuli wird dann durch Zeitraffer unter ...

<html> Die Haupttodesursache Brustkrebs ist durch Metastase 1, 2. Taxane wie Docetaxel und Paclitaxel, werden derzeit als First-Line - Therapien bei der Behandlung von metastasierendem Brustkrebs 2, 3, 4, 5, 6 verwendet. Sie sind Teil einer Gruppe von Mikrotubuli-Targeting-Mittel (MTAs), die die Dynamik der Mikrotubuli stören. Allerdings ist eine der größten Herausforderungen für die Taxane in kurativen Therapie ist die Entwicklung von Taxan - Resistenz von Krebszellen unter Verwendung, die 7 bis Wiederauftreten der Krankheit führt. Arzneimittelresistenz ist für mehr als 90% aller Todesfälle bei Patienten mit metastasiertem Brustkrebs , 7. Mikrotubuli sind durch die Polymerisation von α- und β-Tubulin-Heterodimeren gebildetclass = &quot;xref&quot;&gt; 8, 9. Die genaue Regelung der Dynamik der Mikrotubuli ist wichtig für viele zelluläre Funktionen, einschließlich der Zellpolarisation, Zellzyklus, den intrazellulären Transport und Zellsignalisierung. Dysregulation von Mikrotubuli und deren Dynamik werden die Zellfunktion stören und führen in den Zelltod 10, 11. Je nachdem , wie sie diese Dysregulation verursachen kann MTA Medikamente entweder als Mikrotubuli stabilisierende Mittel eingestuft werden (dh Taxane) oder Mikrotubuli-destabalizing Mittel (dh, Vinca - Alkaloiden oder Colchicin-Bindungsstelle Mittel) 20. Trotz ihrer gegenläufigen Effekte auf Mikrotubuli Masse, bei einer ausreichenden Dosierung, beide Klassen können 21 Krebszellen durch ihre Auswirkungen auf die Dynamik der Mikrotubuli töten. Taxane funktionieren in erster Linie durch die Mikrotubuli Spindel 12 zu stabilisieren, was zuchromosomale Fehlausrichtung. Die anschließende unaufhörlichen Aktivierung der Spindelanordnung checkpoint (SAC) hemmt die Zelle in die Mitose. Längerer mitotischen Arrest bewirkt dann , dass die Apoptose 13, 14. Taxan interagiert mit Mikrotubuli durch die Taxan - Bindungsstelle auf β-Tubulin - 8, 15, die im zusammengebauten Tubulin 16 nur vorhanden ist. Mehrere Mechanismen für Taxan Widerstand haben 9 vorgeschlagen, 17. Diese Mechanismen umfassen sowohl allgemeine Multidrug - Resistenz durch die Überexpression von arzneimittel Efflux Proteine und Taxan-spezifischen Widerstand 5, 9, 18, 19. Beispielsweise Taxan-resistenten Krebszellen Expression und Funktion bestimmter β-Tub möglicherweise verändertulin Isotypen 5, 9, 19, 20, 21, 22, 23. Durch die Verwendung eines in vivo - Verfahren Mikrotubuli dynamische Instabilität zu messen, zeigen wir , dass, wenn im Vergleich zu nicht-resistenten, elterliche MCF-7 - Zellen CC 17 die Mikrotubuli - Dynamik von Docetaxel-resistenten MCF-7 - Zellen zu TXT Docetaxel Behandlung unempfindlich sind. Zum besseren Verständnis der Funktion von MTAs und der genaue Mechanismus der Taxan-Resistenz in Krebszellen zu verstehen, ist es wichtig, die Dynamik der Mikrotubuli zu messen. Hier berichten wir über eine in vivo - Methode, dies zu tun. Durch die Verwendung von Echtzeit- Bildgebung in Kombination mit der Expression von GFP-markierten Tubulin in Zellen, können wir die Dynamik der Mikrotubuli von MCF-7 TXT und MCF-7 CC - Zellen mit und wi messenthout Behandlung mit Docetaxel. Die Ergebnisse können uns wirksamer Medikamente helfen entwerfen, die Taxan Widerstand überwinden kann.

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	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Sigma-Aldrich</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acids</td>
			<td>Life Technologies, Invitrogen</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco, Invitrogen</td>
			<td>12483</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Anti (100x)</td>
			<td>Life Technologies, Invitrogen</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>docetaxel</td>
			<td>Sigma-Aldrich</td>
			<td>01885-5mg-F</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM phenol red-free</td>
			<td>Gibco, Invitrogen</td>
			<td>21063</td>
			<td></td>
		</tr>
		<tr>
			<td>CellLight Reagent *BacMam 2.0* GFP-tubulin</td>
			<td>ThermoFisher Scientific</td>
			<td>C10613</td>
			<td>Key reagent for expressing GFP tubulin in cells</td>
		</tr>
		<tr>
			<td>CellLight Reagent *BacMam 2.0* GFP</td>
			<td>ThermoFisher Scientific</td>
			<td>B10383</td>
			<td>Control</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich+B9:AA9</td>
			<td>472301</td>
			<td>for dissoving decetaxel</td>
		</tr>
		<tr>
			<td>22-mm glass coveslip</td>
			<td>Fisher Scientifics</td>
			<td>12-545-101</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plate</td>
			<td>Greiner Bio-One International</td>
			<td>6 Well Celi Culture Plate</td>
			<td></td>
		</tr>
		<tr>
			<td>DeltaVision Microscopy Imaging Systems</td>
			<td>GE Health</td>
			<td></td>
			<td>This system is equipped with weather station for controlling temperature and CO2. It also equipped with Worx Software for deconvolution and time lapse control.</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>ThermoFisher Scientific</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Bright-Line Hemacytometer Set, Hausser Scientific</td>
			<td>Hausser Scientific, Distributed by VWR</td>
			<td>Supplier No.: 1492 VWR No.:15170-172</td>
			<td></td>
		</tr>
	</tbody>
</table>

live imaging to study microtubule dynamic instability in taxane-resistant breast cancers

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 &#945;-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.

Mit dem Protokoll hier vorgestellten, untersuchten wir die Wirkung von Docetaxel auf die Mikrotubuli - Dynamik der normalen (MCF-7 CC) und Docetaxel-resistenten (MCF-7 TXT) Brustkrebszellen. Zwei Sätze von Aufnahmen zeigen die Wirkung von Docetaxel (0,5 uM) auf Mikrotubuli Wachstum und Verkürzung in MCF-7 CC und MCF-7 - TXT - Zellen (1A). Wir auch die Geschwindigke...

Watch this Scientific Journal Video about Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers at JoVE.com

Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

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.

Live-Imaging zur Untersuchung Mikrotubuli dynamische Instabilität in Taxan-resistenten Mammakarzinome

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane ...

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Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Kombinierte Verwendung von Tail Vein Metastasis Assays und Real-Time In Vivo Imaging zur Quantifizierung der metastasierenden Kolonisation und Belastung von Brustkrebs in der Lunge

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

Verwendung computerbasierter Bildanalyse zur Verbesserung der Quantifizierung von Lungenmetastasen im 4T1-Brustkrebsmodell

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. ...