RK-33

Cell experiment:

Briefly, 8×102 MCF-7 cells are seeded in a 96-well plate, and treated with different concentrations of RK-33 loaded nanoparticles and unloaded nanoparticles next day. After 72-h incubation, MTS reagent is added to the cells, and the absorbance is measured at 490 nm after 2-h incubation with MTS reagent. The experiment is repeated three independent times.

Animal experiment:

Mice are randomLy redistributed into four groups of eight according to their tumor growth, which results in an approximately equal distribution of tumor size at the beginning of radiation and RK-33 drug treatment. The four groups of mice are blindly chosen for four different experimental procedures, including control (injection of DMSO only), RK-33 treatment (injection of RK-33 only 50 mg/kg), radiation (one-time radiation of 5 Gy), or radiation and RK-33 treatment (combination of radiation of 5 Gy and RK-33 injection). RK-33 and DMSO are injected intraperitoneally thrice weekly for two weeks. Radiation is performed at the beginning of drug injection using the Small Animal Radiation Research Platform (SARRP) with a circular beam of 1 cm diameter, focusing on the tumor site. Mice of each group are euthanized 0 h and 24 h after radiation and tumors are extracted for γH2AX, cleaved Caspase 3, and Ki67 staining. The remaining mice of each group are imaged with a Xenogen IVIS Spectrum, with injection of D-luciferin 5 minutes before imaging. Mice are euthanized after six weeks of imaging and tumors are extracted for H&E staining, cleaved Caspase 3, and i67 staining. Morphology of the tumors after RK-33 and radiation treatment is assessed by a veterinary pathologist on hematoxylin and eosin stained sections.

References:

[1]. Xie M,et al. RK-33 radiosensitizes prostate cancer cells by blocking the RNA helicase DDX3. Cancer Res. 2016 Sep 12.
[2]. Bol GM, e tal. PLGA nanoparticle formulation of RK-33: an RNA helicase inhibitor against DDX3. Cancer Chemother Pharmacol. 2015 Oct;76(4):821-7.