20-HEDE (WIT 002)

Cell experiment:

Human NSCLC cell lines (e.g A549) are seeded onto the upper well of the chamber. Subsequently, serum-free medium with HET0016 (10 μM), WIT002 (10 μM), WIT003 (0.01, 0.1, 1 μM), or ethanol as a control is added to the upper chamber, while the lower well is filled to the top (500 μL) with RPMI-1640 containing 5% fetal calf serum (FCS) as a chemoattractant. Cells are allowed to migrate for 5 h. Cells that have invaded to the bottom surface of the filter are counted with an ocular micrometer in a blinded manner, counting a minimum of 10 high-powered fields (HPF) [2]. Human renal cell adenocarcinoma lines (RPTC) 786-O or 769-P cells are plated and next day (0 hrs) transferred to serum-free medium containing either EGF or ET-1. Cells are exposed either to 10 μM WIT002 or vehicle. Cell counting is performed at the day of transfer to serum free medium (0 hrs) and 24, 48 and 72 hrs thereafter. Medium is changed to fresh containing mitogens and drugs every 24 hrs. Data presented are characteristic experiment from at least two separate experiments, each performed in triplicate[3].

Animal experiment:

Mice[3]Experiments are carried out on 6-week immunodefficient athymic nude mice weighing 20-26 g. Animals are acclimated for 1 week prior to injection with renal cell carcinoma. Immediately before each implantation, the cells are trypsinized, counted and resuspended in 10% serum containing RPMI media. The concentration of cells is adjusted to 40 millions/mL and 4 million cells/animal are injected subcutaneously. The cells are allowed to grow for 7-15 days until the size of the tumors reach approximately 0.1 cm3. The mice then receive daily subcutaneous injection (s.c.) injections of WIT 002 (10 mg/kg/day in 200 μL) in an isotonic NaPO4 buffer (pH 9.0) or vehicle (0.1 M NaPO4 solution pH 9.0). The diameter of the tumor is measured on every 3-4 days for 2 weeks using precision calibers. At the end of the experiment the mice are euthanized with CO2 and the tumors were excised to confirm the diameter measurements[3].

References:

[1]. Ming Yua . et al. Effects of a 20-HETE antagonist and agonists on cerebral vascular tone. Eur J Pharmacol. 2004 Feb 23;486(3):297-306.
[2]. Wei Yu, et al. Cytochrome P450 ω-hydroxylase promotes angiogenesis and metastasis by upregulation of VEGF and MMP-9 in non-small cell lung cancer. Cancer Chemother Pharmacol. 2011 Sep; 68(3): 619-29.
[3]. Anna Alexanian, et al. Down-regulation of 20-HETE Synthesis and Signaling Inhibits Renal Adenocarcinoma Cell Proliferation and Tumor Growth. Anticancer Res. 2009 October ; 29(10): 3819-3824.