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Preliminary Study

Albert Leyva, Ph.D.
Arnold Freeman, M.D.

Cancer Pharmacology Laboratory
Section of Hematology/Oncology
Children's Mercy Hospital
Kansas City, MO 64108

note: this is an abridged version of the original study

A pilot study was conducted to evaluate several plant extracts for anti-cancer potential. Our approach was to test the plant extracts, for cytotoxic effects in a panel of human tumor cell lines and non-tumor fibroblast lines. First, we were looking for any cytotoxic effect, and then examining for differential effects, i.e. more cytotoxic in some cell lines compared to others, including non-tumor cells. This aim is to rule out general poisons. Since the initial experiments proved to be positive, the plant extracts were also tested in tumor cells with known resistance to various conventional anticancer drugs. Even when using crude extracts, it is possible to characterize some chemical properties of the unknown active ingredient by examining its effects in certain cell models and in combination with other agents. This preliminary study of these plant extracts was thus carried out to determine if more extensive investigation is warranted to isolate cytotoxic substances and eventually test them for anitcancer activities in animal models.


Test substances

Eight plant extracts were received from C.E.D. Tech Health Limited and tested for cytotoxic effects in cultured cell lines. These extracts were labeled A,B,C,D,E,F,G,H. All samples were ethanol extracts of the plant leaves with a final alcohol concentration of 16-41% (vol/vol), mean of 24%. All plant extracts were supplied in dark glass bottles and stored at room temperature.

Cell Culture

The following human cancer cell lines were used: leukemia (CEM); colon (HCT8, COLO201, SW837); glioma (U87, U138, U373); lung (SW1573, SW1573-2R160). The cells were obtained commercially from ATCC, except the SW1573 lines that were obtained from Dr. H. Joenge, Free University, Amsterdam, Holland. All cell lines that were maintained in liquid nitrogen sotrage until use. When in use for cytotoxicity testing, cells were maintained in exponetial growth in plastic flasks (Falcon) in RPM1-1640 medium containing 10% feta bovine serum. All cell culture media and reagents were obtained from JRH or GIBCO. Other chemicals were from Sigma. CEM cells grew as stationary suspension (loose) cultures, while the other cell lines grew as adherent monolayers. The adherent cultures were passaged to new flasks and fresh mdium after brief (Leyva, 1990) trypsinization. CEM cells were passaged by dilution in fresh medium. SW1573-2R160 is a variant subline of SW1573 that is known to over express mdr-1 gene coding for P-glycoprotein-170, thus representing classical multi-drug resistance (MDR) (Broxterman, 1989; Deuchars, 1989). This MDR cell line is over 100 times less sensitive to the anticancer drugs doxorubicin, vincristine and etoposide, when compared to the parent cell line SW1573.

Two normal human skin fibroblast lines were used and maintained as with the tumor cell lines, except alpha-MEM medium was used for maintenance culture. CMH-F1 fibroblasts were originated in our laboratory and CCD973 fibroblast line was obtained from ATCC.

Cytotoxicity Assay

Cytotoxicity was deterimied by the MTT assay in multi-well plates (Pieters, 1988; ya, 1990). Cells were plated in 96-well microplates in RPMI-1640 medium plus 10% fetal bovine serum supplement with antibiotics (penicillin, streptomycin, fungizone; not toxic to human cells). The number of cells plated varied for the different cell lines; in most cases 700-2,000 cells per 100 ul per well. Plated cells were then allowed to equilibriate overnight. Plant extracts were diluted in culture medium and 100 ul added to cells. The highest concentration of plant extract tested was always 5%, that resulted in an ethanol concentration of about 1%. Ethanol was tested alone and found to be 30% growth inhibitory at 1% and not inhibitory at 0.5%. Plant exracts were usually tested at several 2-fold sereal dilutions. After cells were incubated with and without added test substances for 3 days under standard culture conditions, the nubmer of metabolically viable cells was determined by the MTT tetrazolium dye assay. Briefly, the old medium was removed and 200 ul of fresh medium with 0.5 mg/ml MTT was added. Cells were then further incubated for 3 hours. The medium was removed and the blue-black MTT formazan produced by the metabolically active cells was dissolved with the 150 ul of dimethyl sulfoxide. Absorbance was determined at 550 nm using the ELISA plate reader (Cambridge Instruments). Absorbance was plotted versus plant extract concentration (%, vol/vol) to obtain dose-response curves. Cytotoxicity was measured as the IC50, the concentration of plant extract or test agent that produces 50% decrease in the number viable cells (MTT absorbance) compared to untreated controls.

Fresh Tumor Specimens

Single-cell suspensions of tumor cells were obtained form surgical specimens of patients with cancer and examined for sensitivity to test substances, as reported elsewhere (Leyva, 1993). Briefly, tissue was minced and then disaggregated enzymatically and mechanically. Viable tumor cells were plated at 50,000/well in 96-well plates. Otherwise, the cytotoxicity assay was the same as for cell lines. An alternative method for small numbers of tumor cells was to plate 5,000 cells in 6-well plates in 1 ml 0.3% agar medium (semi-solid) with and without test agent. After 3 days incubation, MTT is added for overnight to stain viable cells.


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*The name, "Alzium", has been changed to Alsihum due trademark issues. No change has been made to the patented product formula.