Lines Established from the Gastrointestinal Tract Antiproliferative Effects of Isoflavones on Human Cancer Cell

Seven isoflavones, biochanin A, daidzein, genistein, genistin, prunectin, puerarin, and pseudobaptigenin were tested for cytostatic and cytotoxic effects on 10 newly established cancer cell lines of the human gastrointestinal origin. Proliferation of HSC-41E6, HSC-45M2, and SH101-P4 stomach cancer cell lines was strongly inhibited by biochanin A and genistein, whereas other stomach, esophageal, and colon cancer lines were moderately suppressed by both compounds. Biochanin A and genistein were cytostatic at low concentrations (<20 pg/ml for biochanin A, <10/tg/ml for genistein) and were cytotoxic at higher concentrations (>40 pg/ml for biochanin A, >20 pg/ml for genistein). DNA fragmentation was observed at cytotoxic doses of both compounds, indicating the apoptotic mode of cell death by the compounds. Chromatin condensation and nuclear fragmentation of each cell line were also observed. The advent of apoptosis was dose dependent for both isoflavones. Biochanin A suppressed tumor growth of HSC-45M2 and HSC-41E6 lines in athymic nude mice. Our results suggest that two of isoflavone derivatives, biochanin A and genistein, inhibit the cell growth of stomach cancer cell lines in vitro through activation of a signal transduction pathway for apoptosis. Moreover, in vivo experiments demonstrate that biochanin A can be used as an anticancer agent. I N T R O D U C T I O N Although the prevalence of human gastric carc inoma is gradually decreasing recently, it still shares a significant portion of cancerrelated deaths in Japan (1). In oriental societies, miso and soy sauce have been traditionally used in a daily diet. Ingestion o f miso diet was shown to reduce the f requency o f s tomach cancers significantly as revealed in epidemiological studies (2). However, there is no experimental study on the mechanism of reduction in s tomach cancer-related death among miso consumers . Miso is made by ferment ing soy bean, rice, and wheat or oat, and contains various biologically active substances such as botanic proteins, vitamins, fats, enzymes, carbohydrates, saponins, isoflavones, phytosterols, and lectins (3, 4). A m o n g these, isoflavones are known to have various biological activities (4). In the present study, we have tested the effect of isoflavones on human gastric tumor cell lines. For this, we have newly established and character ized six cancer cell lines derived f rom the human gastrointestinal tract. In addition, s tomach cancer cell lines we have previously established were also included in the study (5, 6). The effects of seven different isoflavone compounds were examined on cell proliferation o f these cell lines. The results indicated that some isoflavones induced apoptosis of tumor cells. In addition, these isof lavones suppressed tumor growth of the cell lines in a thymic nude mice. Received 6/1/93; accepted 9/27/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by grants from the Ministry of Health and Welfare of Japan, and by a grant from Miso Central Institute, Tokyo, Japan. 2 To whom requests for reprints should be addressed, at Department of Pathology, Research Institute for Nuclear Medicine and Biology, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan. Establishment of Cancer Cell Lines. Tumor tissues were trimmed of fat and necrotic portions and minced with scalpels. The tissue pieces were transferred, together with medium, at 10 to 15 fragments/dish to 60-mm culture dishes (Falcon, Lincoln Park, N J). The patient's ascitic tumor cells were harvested by centrifugation (1000 rpm for 10 min) and plated into 60-mm dishes. Culture dishes initially selectively trypsinized [trypsin, 0.05% (w/v); EDTA, 0.02% (w/v)], to remove overgrowing fibroblasts. In addition, we attempted to remove the fibroblasts mechanically and to transfer the tumor cells selectively. The cells were cultured on dishes at 37~ in a 5% CO2 and 95% air atmosphere. In the early phase of cultivation, the cancer cells were grown in DMEM3/o~-minimal essential medium (1:1). When the cancer cells started to grow and become serially passageable, they were cultivated in DMEM (GIBCO, Grand Island, NY). Both media were supplemented with 10% heat inactivated FCS (Hazleton, Lenexa, KA), penicillin (100 IU/ml), streptomycin (100/xg/ml), 1% (v/v) nonessential amino acids, sodium pyruvate, and L-glutamine (2 mta). All cell lines were frozen in liquid nitrogen at early passage. HSC-45 ceil line was established from stomach cancer of the signet ring cell type from the ascites of a 28-year-old female patient. HSC-41 cell line was established from stomach cancer of the moderately differentiated tubular adenocarcinoma type from freshly resected surgical specimen of the primary tumor of a 45-year-old mate patient. HSC-42 cell line was established from a xenotransplantable human stomach cancer line of the well-differentiated type, H-111 (7). HEC-46 cell line was established from a xenotransplantable human esophageal cancer line of the well-differentiated squamous cell carcinoma type, EH-6(8). HCC-48 and HCC-50 cell lines were established from xenotransplantable human colorectal cancer lines of the well-differentiated adenocarcinoma type, CH-4 and CH-5, respectively (8). HSC-39, HSC-40A, and HSC-43 cell lines were those established previously in our laboratory from signet ring cell gastric carcinomas with a scirrhous stromal reaction (5, 6). SH101 cell line was also reported previously, which was established from a stomach cancer of the well-differentiated adenocarcinoma type (8). ST-Fib and ST-Fib2 cells are normal fibroblasts from nonneoplastic mucosas of the human stomach (5). Subclones were obtained after two cycles of a metal cylinder isolation of colonies and subsequent colony formation in soft agar. Mycoplasma contamination was routinely monitored by a Hoechst 33258 staining kit (Flow Laboratories, McLean, VA). Effect of lsoflavones on Cell Growth. Biochanin A and genistein were purchased from Sigma Chemical Co. (St. Louis, MO). Daidzein, genistin, prunectin, and pseudobaptigenin were obtained from Extrasynthese Laboratories (Geney, France). Puerarin was purchased from Funakoshi Chemicals Co., Ltd. (Tokyo, Japan). Isoflavone was dissolved in DMSO and aliquots were added to culture dishes. Final concentrations of DMSO in the culture medium were kept below 1% (v/v). In order to determine the effect of various compounds on cell proliferation, cells were seeded at 2 • 104 cells with 1 ml of medium/well onto 24-well plates. On the indicated day thereafter, cells were trypsinized and the cell numbers were scored. The extent of growth was also determined by a crystal violet dye elution assay as described previously (9). An equivalent volume of DMSO was added to control cultures which usually had no measurable effects on cell growth. Analysis of Apoptosis. For the detection of apoptosis, cultured cells were processed as described previously (10). Briefly, the cells were seeded onto tissue culture chamber slides (Nunc, Inc., Naperville, IL) and were incubated 3 The abbreviations used are: DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethy! sutfoxide; FCS, fetal calf serum; TK, tyrosine kinase; IDso, drug concentration which inhibited growth by 50%.