Apoptotic Anticancer Effect

Two anthracenone C-glycosides, alvaradoins E and F, isolated from the leaves of Alvaradoa haitiensis Urb. (Simaroubaceae), were found to have potent inhibitory activities with cultured cancer cells. Using the in vivo hollow fiber model, these compounds demonstrated significant growth inhibition at the i.p. site when tested with KB, LNCaP, and Col2 cells. To determine if these anthracenone C-glycosides mediated anticancer activity through an apoptotic pathway, a series of assays were performed with the 10S isomeric compound, alvaradoin E. With a DAPI assay, treatment of LNCaP cells with alvaradoin E at concentrations of 0.4, 2, 10, or 50 ÌM for 24 or 48 h showed chromatin condensation, a morphological characteristic of apoptosis. Mitochondrial membrane potential, analyzed with a DiOC6 uptake assay, showed that treatment of LNCaP cells with 0.07, 0.14, 0.28, 0.56, 0.86, and 1.12 ÌM alvaradoin E for 12 h caused dose-dependent membrane depolarization, another indication of early apoptosis. Also, with an annexin V-FITC assay system, treatment of HL-60 cells with 0.07 ÌM alvaradoin E for 24 h increased annexin V-FITC binding from 3 to 25.9% (8.6-fold). Finally, with the TUNEL assay system, treatment of HL-60 cells with 1.12 ÌM alvaradoin E for 32 h increased FITC-dUTP binding from 1.2 to 12.1% (10-fold). These data suggest alvaradoin E is an effective anticancer agent that induces apoptosis. Additional studies to establish clinical utility should be of interest. In a search for potential anticancer agents of plant origin conducted through a National Cooperative Drug Discovery Group program (1), a crude chloroform extract from the leaves of Alvaradoa haitiensis Urb. (Simaroubaceae) was found to have significant inhibitory activity with cultured KB cells. Through bioassay-guided fractionation, two new isomeric anthracenone C-glycosides, alvaradoins E (10S) and F (10R), along with several structural analogs were isolated (2). Alvaradoin E exhibited activity in the in vivo P388 mouse leukemia model (2). Traditional antitumor research has been based on the ability of antitumor agents to cause drug-induced cellular lesions through general cytotoxic mechanisms. However, a growing body of evidence strongly suggests that some antitumor agents can trigger apoptosis, an irreversible process leading to rapid 779 Abbreviations: DAPI, 6,4'-diamidino-2-phenylindole; DiOC6, 3',3'dihexyloxacarbocyanine iodide; EDTA, ethylenediaminetetraacetic acid; MMP/¢æm, mitochondrial membrane potential; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NCDDG, National Cooperative Drug Discovery Group; PBS, phosphatebuffered saline; PI, propidium iodide; PS, phosphatidylserine; PVP, polyvinylpyrrolidone; TdT, terminal deoxynucleotidyl transferase; TUNEL, terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling. *Current address: College of Pharmacy, The Ohio State University, 500 W. 12th Avenue, Columbus, OH 43210. *Current address: Purdue University, Heine Pharmacy Building, Room 104, 575 Stadium Mall Drive, West Lafayette, IN 47907. **Deceased July 6, 2002. Correspondence to: Dr. A. Douglas Kinghorn, Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, 500 West 12th Avenue, Columbus, OH 43210-1291, U.S.A. Tel: (614) 247-8094, Fax: (614) 247-8642, email: kinghorn.4@osu.edu