Zosuquidar 3HCl

LY335979 competitively inhibits equilibrium binding of Pgp by blocking [³H]azidopine photoaffinity labeling of the Pgp in CEM/VLB₁₀₀ plasma membranes. [1] LY335979 alone shows the cytotoxicity to drug-sensitive and MDR cell lines with IC50 ranging from 6 μM-16 μM and produces its ability to completely reverse the resistance of the oncolytics to the MDR cell lines P388/ADR, MCF7/ADR, 2780AD, or UCLA-P3.003VLB at concentration of 0.1 and 0.5 μM. [1] LY335979 significantly restores drug sensitivity in P-gp-expressing leukemia cell lines including K562/HHT40, K562/HHT90, K562/DOX and HL60/DNR, and enhances the cytotoxicity of anthracyclines and ozogamicin (Mylotarg) in primary AML blasts with active P-gp. [2] A latest paper indicates that LY335979 completely inhibits apically directed transport of (Z)-endoxifen in the ABCB1-transduced cells. [3]

• ATPase-Assay

  Die P-Glycoprotein-ATPase-Aktivität wird durch die Freisetzung von anorganischem Phosphat aus ATP gemessen. Der Assay wird in einer 96-Well-Platte für 90 Minuten bei 37 °C durchgeführt. Membranen (8 μg-10 μg Protein) werden in einem Gesamtvolumen von 100 μL Puffer A inkubiert, der 5 mM Natriumazid, 1 mM Ouabain, 1 mM EGTA, 3 mM ATP, ein ATP-regenerierendes System bestehend aus 5 mM Phosphoenolpyruvat und 3,6 Einheiten/mL Pyruvatkinase in Anwesenheit und Abwesenheit von 1 mM Natriumvanadat enthält. Die Pgp-ATPase-Aktivität ist als der Vanadat-sensitive Anteil der gesamten ATPase-Aktivität definiert. Die Platten werden 3 Minuten nach Zugabe der Detektionslösung abgelesen. Die Absorption wird bei 690 nm mit einem Mikrotiterplattenleser gemessen. Eine Phosphat-Standardkurve wird verwendet, um die gebildete μmol Phosphat zu berechnen. Proben werden in dreifacher Ausführung gemessen.

• Zelllinien

  CEM/VLB₁₀₀, P388/ADR, MCF7/ADR, 2780AD, and UCLA-P3.OO3VLB cells

• Konzentrationen

  0.05 μM to 5 μM

• Inkubationszeit

  72 hours

• Methode

  Cell viability is determined using a modified the MTT dye reduction method. Cells are harvested during logarithmic growth phase, and seeded in 96-well plates. The cells are then cultured for 72 hours in the presence of oncolytics with or without modulators. MCF-7 and MCF-7/ADR cells are incubated 24 hours before the addition of the drug with and without the LY335979. LY335979 is prepared as 2 mM DMSO stocks and added to wells to give final concentrations ranging from 0.05 to 5 μM. After 72 hours, 20 μL of freshly prepared MTT(5 mg/mL in Dulbecco's PBS) is added to each well and incubated for 4 hours in a 37 °C incubator containing 5% CO₂. Cells are pelleted in a Sorvall RT6000B centrifuge, 70 μL of medium is carefully removed from each well, and 100 μL of 2-propanol/0.04 N HC1 is added. Cells are resuspended 5-10 times with a Multipipettor or until no particulate matter is visible. Plates are immediately read on a Titertek Multiskan MCC/340 microplate reader Flow Laboratories with a test wavelength of 570 nm and a reference wavelength of 630 nm. Controls are measured in quadruplicate and modulators are measured in duplicate. Cytotoxicity analyses are also performed using the CeliTiter 96 AQueous assay kit.

• Tiermodelle

  P388 or P388/ADR cells are implanted by i.p. injection into female BDF₁ mice.

• Dosierungen

  ≤30 mg/kg

• Verabreichung

  Administered via i.p. and i.v.