1 Graviola ( Annona muricata ) Exerts Antiproliferative , 2 Anti-clonogenic and Pro-apoptotic Effects in Human 3 Non-Melanoma Skin Cancer UW-BCC 1 and A 431 4 Cells In Vitro : Involvement of Hedgehog Signaling 5 6

Non-melanoma skin cancers (NMSCs) are the leading cause of skin cancer-related 35 morbidity and mortality. Effective strategies are needed to control NMSC occurrence and 36 progression. Non-toxic, plant-derived extracts have been shown to exert multiple anti-cancer effects. 37 Graviola (Annona muricata), a tropical fruit-bearing plant, has been used in traditional medicine 38 against multiple human diseases including cancer. The current study investigated the effects of 39 graviola leaf and stem extract (GLSE) and its solvent-extracted fractions on two human NMSC cell 40 lines, UW-BCC1 and A431. GLSE was found to: i) dose-dependently suppress UW-BCC1 and A431 41 cell growth, motility, wound closure, and clonogenicity; ii) induce G0/G1 cell cycle arrest by 42 downregulating cyclin/cdk factors while upregulating cdk inhibitors, and (iv) induce apoptosis as 43 evidenced by cleavage of caspases-3, -8 and PARP. Further, GLSE suppressed levels of activated 44 hedgehog (Hh) pathway components Smo, Gli 1/2, and Shh while inducing SuFu. GLSE also 45 decreased the expression of pro-apoptotic protein Bax while decreasing the expression of the anti46 apoptotic protein Bcl-2. We determined that these activities were concentrated in an 47 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 1 June 2018 doi:10.20944/preprints201806.0020.v1 © 2018 by the author(s). Distributed under a Creative Commons CC BY license. acetogenin/alkaloid-rich dichloromethane subfraction of GLSE. Our data identify graviola extracts 48 and their constituents as promising sources for new chemopreventive and therapeutic agent(s) to 49 be further developed for the control of NMSCs. 50