Narciclasine

Kinase experiment:

The RhoA G-LISA assay is used. The assay uses a 96-well plate coated with the Rho binding domain of Rho family effector proteins. The active GTP-bound form of the Rho family protein but not the inactive GDP-bound form from biological samples will bind to the plate. Bound active Rho family protein is then detected by incubation with a specific primary antibody followed by a secondary antibody conjugated to horseradish peroxidase. The signal is then developed using OPD. U373 and GL19 glioblastoma multiforme cells are serum starved for 24 h and left untreated or treated with Narciclasine (100 nM) for 3 min, 5 min, 30 min, 1 h, and 2 h or treated with 2.0 μg/mL of the cell-permeable Rho inhibitor for 4 h in serum-free medium at 37°C with and without subsequent Narciclasine treatment. This product consists of highly purified C3 transferase covalently linked to a proprietary cell-penetrating moiety via a disulfide bond. The cell-penetrating moiety allows rapid and efficient transport through the plasma membrane. Once in the cytosol, the cell-penetrating moiety is released, thereby allowing C3 transferase to freely diffuse intracellularly and inactive RhoA, RhoB, and RhoC but not related GTPases such as Cdc42 or Rac1. C3 transferase inhibits Rho proteins by ADP ribosylation on Asn41 in the effector binding domain of the GTPase. Cells are then lysed and RhoA activity is measured by the RhoA G-LISA Activation Assay or alternatively cells are stained for F-actin[1].

Cell experiment:

Cell[1]The Narciclasine IC50 concentration, the Narciclasine concentration that decreased by 50% the global growth rate of a given cell population, is assessed with the MTT assay. The cells are incubated for 72 h in the presence and absence of the Narciclasine (with concentrations ranging between 10-9 and 10-5 M concentrate) for the determination of Narciclasine IC50 values[1].

Animal experiment:

Mice[1] The Hs683 cell line and GL19 primoculture grafted into the brains of nude immunodeficient mice both produced invasive brain tumors. Xenograft-bearing mice receive vehicle alone, oral temozolomide at 40 mg/kg (5 administrations per week for 5 consecutive weeks), or Narciclasine at 1 mg/kg either oral (once per week for 5 weeks) or i.v. (twice per week for 5 weeks). Drug administration is initiated respectively on days 5 and 7 post-tumor grafting for the Hs683 and GL19 models. The temozolomide dose and treatment schedule are selected based on previous optimized regimens. Narciclasine dose and treatment schedule are selected based on Narciclasine toxicity study in rats after oral administration and pharmacokinetic study that we have recently published. In toxicity study, Narciclasine (25, 10, or 1 mg/kg) is administered five times a week for 3 weeks and the no adverse effect level dose is defined to be 1 mg/kg/d p.o., with minimal acanthosis reactive changes and minor variations in some biochemistry parameters observed at this dose level considered to be nonadverse.

References:

[1]. Lefranc F, et al. Narciclasine, a plant growth modulator, activates Rho and stress fibers in glioblastoma cells. Mol Cancer Ther. 2009 Jul;8(7):1739-50.
[2]. Wahyuni DS, et al. The use of bio-guided fractionation to explore the use of leftover biomass in Dutch flower bulb production as allelochemicals against weeds. Molecules. 2013 Apr 17;18(4):4510-25.