VCOM Louisiana Research Day Program

Pharmacology

Hassan Y. Ebrahim 1 , PhD; Abu Bakar Siddique 1 , PhD; Zakaria Y. Abd Elmageed 2 , PhD; and Khalid A. El Sayed 1 *, PhD 1 School of Basic Pharmaceutical and Toxicological Sciences, College of Pharmacy, University of Louisiana Monroe, Monroe, Louisiana; 2 Department of Pharmacology, Edward Via College of Osteopathic Medicine, Monroe, Louisiana. 52 THE EXTRA-VIRGIN OLIVE OIL (S)-(-)-OLEOCANTHAL: A PROMISING NUTRACEUTICAL FOR THE CONTROL OF PROSTATE CANCER

Context: Prostate cancer (PC) ranks the second most diagnosed malignancy and fifth leading cause of men cancer death. Androgen deprivation therapy (ADT) is the standard treatment for management of the hormone naïve PC phenotype. Despite positive initial response, PC acquires resistance to ADT and progress to aggressive metastatic castration resistance (mCRPC) with limited therapeutic options and high mortality. (-)-Oleocanthal (OC) is a unique extra-virgin olive oil (EVOO) phenolic with documented health benefits including anticancer. Objectives: Current study aimed at uncovering OC efficacy against different CRPC cells proliferation, colonization, migration, and invasion in vitro . The study validated SET- and MYND-containing protein 2 (SMYD2) as OC potential molecular target mediating its anti PC effects. Research aimed to evaluate OC oral efficacy in relevant in vivo progression and recurrence PC models. Methods: OC was extracted from EVOO using our patented liquid-liquid extraction methodology. Formazan-based cell viability was used to access effect of OC treatments on the proliferation of PC-3, PC-3M, DU145, CWR-R1ca PC cells, while the colony formation

assay used to gauge its effect on PC single-cell colonization. Transwell invasion and migration kits used to assess the anti-invasive and antimigratory effects of OC. Western blot (WB) analyses and CRISPER-Cas9 were implemented to validate OC mechanistic molecular mode of action. In vivo efficacy of OC was assessed in tumor progression and recurrence models in nude mice engrafted with mCRPC luciferase tagged CWR-R1ca (CWR-R1ca-Luc) cells. Immunofluorescence staining used to evaluate OC effect on progression and vascular markers in tumor samples. Results: OC exhibited a significant dose and time-dependent proliferation and colonization inhibition against all tested PC cells, with IC 50 values ranged in nM-low µM levels. OC showed indiscernible toxicity on immortalized/non tumorigenic RWPE-1 prostatic epithelial cells at higher dose level. OC showed remarkable antimigratory and anti-invasive activities against PC cells. The Eli Lilly Open Innovation Drug Discovery Program showed that OC significantly inhibited SMYD2-mediated methylation of p53 using the scintillation proximity assay. WB analyses of collected CWR-R1ca-Luc tumor samples treated with OC showed significant downregulation of SMYD2 expression and its downstream effectors EZH2 and p56, which

correlated with tumor progression suppression. In addition, the expression of proliferation and vascular markers, Ki67 and CD31 respectively, were suppressed in OC-treated tumor samples, indicating the significant OC antitumor effect. Daily OC 10 mg/kg treatments of nude mice for 30 days after primary tumor surgical excision noticeably prevented locoregional and distant recurrences, evidenced by IVIS bioluminescence live animal imaging. Conclusions: The study proved the in vivo remarkable SMYD2-mediated progression and recurrence inhibitory effects of the EVOO phenolic OC in mCRPC. OC is effective anti-PC lead feasible for development as a nutraceutical for use as adjuvant therapy for mCRPC progression and recurrence in patients and survivors.

68