1

Natural Products as a Source of Anticancer Drugs Drs. Cesar M Compadre and Russell B. Melchert

I. Introduction In the 1950s, scientists discovered two plant-derived antileukemic agents, vinblastine and vincristine, and isolated podophyllotoxin (later modified by a pharmaceutical company to the clinically useful anticancer agents, etoposide and teniposide). These discoveries prompted the National Cancer Institute (NCI) and the US Department of Agriculture to start the systematic collection and screening of plants for antitumor activity in 1960. During the next 22 years, over 35,000 plant samples were collected, and researchers tested over 114,000 plant extracts. In addition, over 18,000 extracts of marine organisms were tested between 1975 and 1982. The NCI also collaborated closely with the pharmaceutical industry in the testing of over 180,000 microbial extracts. The NCI developed a new natural products acquisition program in 1986 by awarding contracts for the collection of plants in tropical and subtropical regions worldwide, and for marine organisms in the Indo-Pacific region. One area of the world that has been relatively less studied as a source of anticancer drugs is Mexico and Central America. In collaboration with scientists from the National University of Mexico, our approach began with a detailed analysis of the historical and current sources on the use of medicinal plants in Mexico. We then targeted Cactuses from the Tehuacan Valley (Figure 1) in the Central part of Mexico. In the past year we have collected several plant species reputed to have anticancer activity. For example one of the species is Stenocereus weberii, shown in Figure 2, this specimen is calculated to over 200 years old. Extracts of these species were prepared and transported back to Arkansas. In previous years, we have discovered potential lead extracts from these plants that have some anticancer activity. Among the critical questions remaining is whether these lead extracts are cytotoxic to normal, non-transformed cell lines. The goal of this project is to determine whether extracts of Stenocereus weberii exert cytotoxic effects in a normal, non-transformed proliferating cell line (cardiac fibroblasts), and if so, how the toxicity profile compares to activity in transformed proliferating cell line (MCF-7 breast cancer cell line).

Figure 1. Plant collections are being conducted in the Tehuacan Valley in Central Mexico

Figure 2. UAMS Pharmacy student Jonathan Goldman and Professor Raul Enriquez from the National University of Mexico collecting a sample of Stenocereus weberii in Central Mexico, in June 2004

2

II. Methods for In Vitro Cytotoxicity Assessment A. Student Responsibilities. Using “high-throughput” techniques, the student will be responsible for growing the nontransformed fibroblast cell line, exposing the cells to various extract preparations, and comparing the cytotoxic potential of the extracts to a standard cytotoxic agent (vinblastine). If certain extracts are found to exert significant cytotoxic activity, then further tests will compare the cytotoxicity profile (e.g. LD50) with that of the cytotoxic activity in MCF-7 breast cancer cells. The student will present their research findings at a local, regional, and/or national scientific meeting. B. Cell Line. Cardiac fibroblasts were obtained in our laboratory through primary isolation from adult rat myocardium. The cells grow well in Minimum Essential Medium (Eagle’s Modification) supplemented with 10% fetal calf serum (FCS) up through passage 2. These cells are currently stored in liquid nitrogen in our laboratory. The cancer cell line (MCF-7 breast cancer) was obtained from the American Type Culture Collection (ATCC). The MCF-7 cell line is grown in Minimum Essential Medium (Eagle’s Modification) supplemented with 10% fetal calf serum (FCS) and appropriate antibiotics (penicillin G, streptomycin, and amphotericin B) to prevent contamination. The cell line is stored in liquid nitrogen and thawed prior to use. Then the cells are grown to near confluency (~70-80%), harvested from the tissue culture flasks with trypsin-EDTA and reseeded at a split ratio of 1:3 in the experimental substrate (multi-well tissue culture plates). Cells require “refeeding” every 2-3 days until experimentation. Once the cells reach near confluency, they will be utilized for cytotoxicity testing. C. Cytotoxicity Testing.

PI Cytotoxicity Test. Propidium iodide is excluded from cells with intact cell membranes; however, when the membrane is damaged, PI enters the cell and stains nucleic acids. An increase in fluorescence staining indicates increased plasma membrane damage. Quantitative measurement of propidium iodide (PI) uptake in cell lines exposed to various extracts or vinblastine will be performed. A BioTek FL500 fluorescence multi-plate reader is used with excitation wavelength set at 520 nm (25 nm half bandwidth) and emission intensity monitored at wavelength 645 nm (40 nm half bandwidth). Total cellular PI uptake will be determined in cultures that are lysed (observed by phase contrast microscopy) by addition of Triton X 100 to a final concentration of 0.2% (v/v). Uptake of PI (cell death) will be expressed as a percent of total cellular PI uptake.

MTT Viability Test. Conversion of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; a yellow water-soluble tetrazolium dye) to MTT formazan (a blue water-insoluble enzymatic product) will be measured to determine cell viability. Viable cells with active mitochondria (and consequently, active mitochondrial dehydrogenases) reduce MTT to MTT formazan, and MTT formazan production is directly proportional to cell viability. Following extract or vinblastine exposure, MTT (0.5 mg/mL) will be added and the cells allowed to incubate for 3 hr. Following incubation, MTT formazan will be extracted with 1.0 N HCl in isopropyl alcohol (1:24). A Packard SpectraCount microplate reader will be used to determine MTT formazan production by measuring absorbance at 550 nm. MTT formazan production (cell viability) will be expressed as a percent of control cells. D. Laboratory Safety. Although the cell lines have been tested by ATCC and are negative for HIV and Hepatitis B, there is a high likelihood that the cell lines would test positive for CMV. In either case, the MCF-7 and PC-3 cell lines are of human origin and thus will be handled as if they are HIV, Hepatitis B, and CMV positive. In addition, the student will be working with a known cytotoxic agent (vinblastine), potential mutagen/carcinogen (propidium iodide), and unknown cytotoxic potential of compounds present in the extracts. The student will be educated and trained for appropriate handling, use, and disposal of all cell lines and chemicals.