*Article* **Anti-Tumorigenic Effect of a Novel Derivative of 2-Hydroxyoleic Acid and the Endocannabinoid Anandamide on Neuroblastoma Cells**

**Hana Golan 1,2,3, Raphael Mechoulam 4, Reem Smoum 4, Efrat Cohen-Zada 1, Sara Pri-Chen 1, Sapir Wiener 1, Igor Grinberg 1, Dekel D. Bar-Lev 1, Christeeneh G. Haj 4, Tamar Fisher 1,† and Amos Toren 1,2,3,\*,†**


† These authors contributed equally to this work.

**Abstract:** Modulation of the endogenous cannabinoid system has been suggested as a potential anticancer strategy. In the search for novel and less toxic therapeutic options, structural modifications of the endocannabinoid anandamide and the synthetic derivative of oleic acid, Minerval (HU-600), were done to obtain 2-hydroxy oleic acid ethanolamide (HU-585), which is an HU-600 derivative with the anandamide side chain. We showed that treatment of SK-N-SH neuroblastoma cells with HU-585 induced a better anti-tumorigenic effect in comparison to HU-600 as evidenced by 3-[4,5- dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide assay, colony-forming assay, and migration assay. Moreover, HU-585 demonstrated pro-apoptotic properties shown by increased levels of activated caspase-3 following treatment and a better senescence induction effect in comparison to HU-600, as demonstrated by increased activity of lysosomal β-galactosidase. Finally, we observed that combined treatment of HU-585 with the senolytic drugs ABT-263 in vitro, and ABT-737 in vivo resulted in enhanced anti-proliferative effects and reduced neuroblastoma xenograft growth in comparison to treatment with HU-585 alone. Based on these results, we sugges<sup>t</sup> that HU-585 is a pro-apoptotic and senescence-inducing compound, better than HU-600. Hence, it may be a beneficial option for the treatment of resistant neuroblastoma especially when combined with senolytic drugs that enhance its anti-tumorigenic effects.

**Keywords:** 2-hydroxy oleic acid; anandamide; anti Bcl2; endocannabinoid system; membrane lipid therapy; neuroblastoma
