The anticancer agent PB-100, selectively active on malignant cells, inhibits multiplication of sixteen malignant cell lines, even multidrug resistant

THE ANTICANCER AGENT PB-100, SELECTIVELY ACTIVE ON MALIGNANT CELLS, INHIBITS MULTIPLICATION OF SIXTEEN MALIGNANT CELL LINES, EVEN MULTIDRUG RESISTANT   Mirko Beljanski† Centre Oncologique et Biologique de Recherches Appliquées, Domaine de la Source, St. Prim 38370, France. Send correspondence to Mrs. Mirko Beljanski, c/o Natural Source 214 East 52nd street, 3rd floor, New York, NY 10022, USA.     ABSTRACT The plant-derived anticancer agent PB-100 selectively destroys cancer cells, even when multidrug resistant; yet, it does not inhibit normal (non-malignant) cell multiplication. Testing of PB-100 on sixteen malignant cell lines, several multidrug resistant, as well as on five normal cell lines, confirmed our previous results. Flavopereirine and dihydroflavopereirine, the active principles of PB-100, were chemically synthesized and displayed the same selectivity for tumor cells as the purified plant extract, being active at even lower concentrations.     INTRODUCTION From its very beginnings almost half a century ago, cancer chemotherapy has faced dramatic problems. Lack of selectivity of conventional anticancer agents, which damage not only malignant but also normal cells, in particular blood cells, has made the scientific community aware of the need for more specifically selective drugs (Pisha et al., 1995). Another drawback, which arose just after cancer chemotherapy was started, was the appearance of drug-resistant cancer cells (Moscow and Cowan, 1988; Israel, 1989; Gottesman, 1993). Several resistance mechanisms have been identified, which researchers are actively trying to overcome; however, few satisfactory solutions have been proposed; moreover, trying to suppress or bypass drug resistance generally means adding a new drug to the anticancer regimen, thus increasing the risk of adverse side effects. We demonstrated experimentally that our anticancer agent PB-100 selectively destroys cancer cells, but has no effect on their normal (non-malignant) counterparts (Beljanski and Bugiel, 1978; Beljanski, 1979, 1983; Beljanski and Beljanski, 1982, 1984, 1986). Moreover, it has proven to be active, in vitro, on the highly malignant U251 glioblastoma cells, which are both radio- and BCNU-resistant (Beljanski et al., 1993; Beljanski and Crochet, 1994). We have now tested the activity of PB-100 in vitro on sixteen different malignant cell lines, some of which are resistant to conventional therapy, as well as on five normal cell lines. Results presented here confirm previous ones, by showing destruction of over 90% of cancer cells (74 to 98%, depending on cell line) using maximal product concentrations of 100 µg/ml; normal cells remained unaffected. PB-100 is the plant-derived alkaloid flavopereirine, utilized in the form of a purified plant extract containing both flavopereirine and dihydroflavopereirine. Pure synthetic flavopereirine and dihydroflavopereirine proved to be more active on U251 cancer cells than the natural product; like the latter, these compounds spared normal astrocytes.   MATERIAL AND METHODS Chemicals Trypsin, RPMI 1640 culture medium, fetal calf serum, glutamine: Gibco, Grand Island, NY, USA. Anticancer agents: PB-100 was purified in our laboratory and checked for purity using HPLC, UV absorbance spectra and thin layer chromatography. Pure, synthetic flavopereirine and dihydroflavopereirine were a gift from Dr. A. Fürstner, Max-Planck Institute, Muhlheim, Germany. Cell lines and culture techniques Brain cell lines: U251 human 1,3-Bis (2-chloroethyl)-1 nitrosourea (BCNU)- and radiation-resistant glioblastoma; CCF-STTG-l and SW 1088 human astrocytomas; C6 rat glial tumor; CRL 1656 Mpf normal astrocytes. Ovarian cell lines: ES2 human carcinoma, exhibiting low to moderate multidrug resistance (MDR) (doxorubicin, cisplatin, carmustine, etoposide, cyanomorpholine doxorubicin...); SW 626, human adenocarcinoma. Breast cancer cell lines: MCF-7 and ZR-75-1 human carcinoma. Prostate cell line: cisplatin-, adriamycin- and etoposide-resistant PC3 human carcinoma. Colon cell lines: LoVo human adenocarcinoma; chemoresistant CaCo-2 human adenocarcinoma; CCD-18 Co human normal colon cells. Thyroid cell line: TT human medullary carcinoma. Pancreatic cell line: MIA PaCa-2 human carcinoma. Hepatic cell lines: Sk-Hep 1 human adenocarcinoma; clone 9 normal rat liver cells. Kidney cell lines: A 498 human carcinoma; NRK-49F normal rat kidney fibroblasts. Skin cell lines: G-361 human malignant melanoma; CCD-974Sk normal human fibroblasts. All cell lines were obtained from ATCC, Rockville, MD, USA, except U251, obtained from SCC, Uppsala, Sweden. Cell culture After checking for the absence of mycoplasms and bacteria, cells were grown at 37°C as monolayer confluent cells in RPMI 1640 medium supplemented with 10% calf serum. To avoid cell membrane sensitization, no antibiotics were used. Stock cultures were duplicated weekly after addition of trypsin (+ 0.005% EDTA), to disperse cells for inoculation. All cell lines were cultured in several 6-well tissue culture plates (9.8 cm2 wells), starting from a 4 x l04-inoculum, and grown for 48 h (doubling time: 20-24 h), prior to assays. Cell growth inhibition The antiproliferative activity of PB-100 was determined following addition of increasing concentrations of the agent to culture media containing the cell inoculum. Dead cells were found in suspension; remaining viable cells were trypsinized and counted with a Coulter counter.   RESULTS Assays using the plant-derived purified extract After each cell line had been grown in vitro for 48 h either in the absence or in the presence of concentrations ranging from 10 to 100 µg/ml of PB-100, the number of surviving cells was estimated as the percentage of cells growing in the absence of PB-100 (100%). Anticancer agent activity was dose dependent (Beljanski et al., 1993); l00 µg/ml was found to be the most active concentration (Table I). Typically, from 80 to 98% of malignant cells, depending on the cell line, were killed by this concentration of PB-100, which left over 95% of normal cells viable. Multiplication of normal astrocytes was actually increased, perhaps due to the presence of ATPase in the culture medium (Beljanski et al., 1993).     Table I indicates that PB-100 is as active on drug-resistant tumor lines as on other malignant lines. Examples found in Table I are: U251 BCNU- and radiation-resistant human glioblastoma cells, ES2 multidrug-resistant human ovarian carcinoma cells, and PC-3 multidrug-resistant human prostate carcinoma cells. According to our 20 years of experience, PB-100 itself never induced cancer cell resistance either in vitro or in vivo. Assays using pure flavopereirine and dihydroflavopereirine Using U251 BCNU-resistant glioblastoma cells and normal CRL 1656 astrocytes as controls, we compared the effect of pure synthetic (...truncated)