(CANCER RESEARCH 52, 5707-5712, October 15, 1992] Carotenoids Up-Regulate Connexin43 Gene Expression Independent of Their Provitamin A or Antioxidant Properties1 Li-Xin Zhang, Robert V. Cooney, and John S. Bertram2 Molecular Oncology Unit, Cancer Research Center of Hawaii. University of Hawaii, Honolulu, Hawaii 96813 ABSTRACT their antioxidant capabilities were not. Neither activity was associated with the provitamin status of the various carotenoids Epidemiological evidence and studies in whole animals and cell cul tested (16). ture have indicated that carotenoids have cancer chemopreventive ac Here, we explore the mechanism by which various caro tion. In mouse C3H10T1/2 cells, this activity is highly correlated with the ability of carotenoids to up-regulate gap junctional intercellular tenoids elevate junctional communication. Gap junctions are communication. Here, we report that in mouse cells, carotenoids in composed of transmembrane proteins called connexins, which crease the expression of connexM.ì,a gene that encodes a major gap form a family of proteins (17). One member of this family, junction protein. This effect appears unrelated to their provitamin A Cx43,3 is a major gap junctional protein in various mammalian or antioxidant properties, since carotenoids with and without provitamin cells including 10T1/2 cells (18). The data indicate that caro A activity increased levels of connexin43 mRNA and protein, whereas tenoids enhance gap junctional communication by increasing the antioxidants methyl-bixin and a-tocopherol were inactive. More the levels of Cx43 mRNA and protein. This effect appears over, the active carotenoid canthaxanthin did not induce the vitamin unrelated to their provitamin A activity or antioxidant proper A-inducible gene retinoic acid receptor-0. Connexln4i is the first caro- ties. tenoid-inducible gene described in mammals. By indicating an addi tional pathway through which carotenoids function, these data provide a mechanistic basis for cancer chemoprevention by carotenoids and may MATERIALS AND METHODS lead to a re-evaluation of carotenoid physiology. Chemicals. Lycopene and fi-carotene were obtained from Sigma Chemical Co. (St. Louis, MO) and were 90 and 80% pure, respectively, INTRODUCTION as determined by high-performance liquid chromatography. Canthax anthin and /rans-methyl-bixin were gifts from Hoffmann-LaRoche Carotenoids, yellow and red pigments found in vegetables (Nutley, NJ), and were >99% and 90% pure, respectively. Because of its and fruits, are common components of the human diet. Accu mulating evidence suggests that carotenoids, particularly 0-car- purity and greater chemical stability, canthaxanthin was used for many of the molecular studies. The synthetic benzoidal derivative of retinoic otene, have diverse beneficial roles in animals and humans. acid, TTNPB, was a kind gift from Hoffmann-LaRoche. a-Tocopherol Among these are: provitamin A activity (1), cancer chemopre was purchased from Sigma. vention (2-4), stimulation of immune responses (5), suppres Cell Culture. Mouse embryonic fibroblast C3H/10T1/2 cells were sion of atherosclerosis (6), and prevention of cataract formation cultured in Eagle's basal medium with Earle's salts (GIBCO), supple (7). High dose ^-carotene is clinically approved for use in mented with 5% fetal bovine serum (Hyclone Laboratories) and 25 jig/ml gentamicin sulfate, and were incubated at 37°Cin5% COi in air patients with erythropoietic protoporphyria (8), is currently undergoing clinical trial as a cancer preventive agent (9), and, as described previously (15,19). Unless otherwise mentioned, cells were seeded in plastic culture dishes (Falcon). When confluent, 1 week post- together with canthaxanthin, is a widely used, United States Department of Agriculture-approved, food coloring agent (10). seeding, the medium was changed to 3% fetal bovine serum. Drug treatment started with the first medium change unless otherwise indi Until recently, biological activity of carotenoids in mammals cated. Cell number was counted by Coulter counter. The murine em was considered limited to those with provitamin A activity. bryonal teratocarcinoma F9 cell line was kindly supplied by Dr. Lor However, since Peto étal.(\ 1) in 1981 first raised the question, raine J. Gudas (Harvard) and cultured as described (20). Briefly, the "Can dietary 0-carotene materially reduce human cancer cells were cultured in Dulbecco's modified Eagle's medium (Flow) con rates?", the focus of carotenoid research has shifted to include taining 10% fetal calf serum (Hyclone Laboratories) and 25 *¿g/ml studies of their properties as antioxidants (12, 13). At present, gentamicin sulfate and were incubated at 37°Cin 5% CO: ¡na¡r-Cells therefore, 2 competing, but not mutually exclusive, hypotheses were seeded in 150-mm plastic culture dishes. Since this transformed exist to explain carotenoid action in mammals: (a) conversion cell line rapidly lifts off from the culture dish after attaining confluence, to retinoids, vitamin A-like compounds possessing multiple it was treated on the second day after seeding. Treatment. All carotenoids tested were delivered to cells using tet- physiological properties; and (b) activity of the intact molecule as a lipid-phase antioxidant. In previous studies using carcino rahydrofuran as solvent (99.9% with 0.025% butylated hydroxytoluene; purchased from Aldrich Chemical Co.) as described previously (15). gen-treated C3H10T1/2 cells, we have confirmed that caro TTNPB and a-tocopherol were dissolved in acetone unless indicated. tenoids can function as cancer preventive agents (14, 15) and The final concentration of acetone or tetrahydrofuran in the culture have demonstrated a novel action of carotenoids: that of up- medium was 0.5%. regulating gap junctional communication (16). The ability of Gap Junctional Communication Assay. Cell cultures were grown in carotenoids to enhance communication was highly correlated 60-mm dishes and treated with drugs after confluence. Gap junctional with their ability to inhibit neoplastic transformation, however, communication was measured by microinjection of the fluorescent tracer dye Lucifer yellow CH (Sigma) as a 10% solution in 0.33 MLiCI Received 4/23/92; accepted 8/3/92. as described previously (21). Micropipettes were prepared from glass The costs of publication of this article were defrayed in part by the payment of capillary tubes with the aid of a Flaming-Brown micropipette puller page charges. This article must therefore be hereby marked advertisement in accord (Sutler Instrument Co., Novata, CA), and Lucifer yellow was microin- ance with 18 U.S.C. Section 1734 solely to indicate this fact. jected into a single cell with an Eppendorf microinjector. The total 1This research was supported by Grant BC686 from the American Cancer Society. 2 To whom requests for reprints should be addressed, at University of Hawaii at 3 The abbreviations used are: Cx43, connexin43; TTNPB. tetrahydrotetramethyl Manoa, Cancer Research Center, Department of Molecular Oncology. 1236 Lau- naphthalenyl propenylben/oic acid: SDS. sodium dodecyl sulfate: RAR, retinoic hala Street, Honolulu, HI 96813. acid receptor. 5707 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1992 American Association for Cancer Research. UP-REGULATION OF CONNEXIN43 GENE EXPRESSION BY CAROTENOIDS number of fluorescent neighboring cells was counted 10 min after mi- croinjection and served as an index of gap junctional communication. The communicating networks were digitized using a video camera and stored for later analysis. Lycopene Isolation of Protein. Cell cultures were grown in 150-mm dishes and were treated after reaching confluence. The cells were washed with Mx^vA^v^v^v^^v^kx V^ ice-cold phosphate-buffered saline containing 1 ITIMNaF and 1 imi CA ' ' phenylmethylsulfonyl fluoride. After centrifugation at 3,000 rpm for ^^ ß-Carotene 10 min, cell pellets were either frozen at -70°C for future analysis or lysed in 30-50 ¡i\oflysis buffer (1% Nonidet P-40, 0.05 M iodoaceta- mide, 10 mM phenylmethylsulfonyl fluoride. 1 m\i EDTA, 1 MMleu- peptin. 2 ¿ig/mlaprotinin, 0.7 ¿tg/mlpepstatin in borate buffer, pH 8.0) for 1-2 h at 4°C.Lysates were then centrifuged at 10,000 x g for 15 min, and the supernatant was used as the source of Cx43. Protein content was measured with a commercial Protein Assay Reagent kit (Pierce Chemical Co., Rockford, IL) according to the manufacture's instructions. Methyl-Bixin Protein Electrophoresis and \Yestern Blotting. Cell lysates contain ing an equal amount of protein (usually 35-50 Mg/lane)were mixed with Fig. 1. Structures of carotenoids tested. 2x SDS sample buffer containing 20% 0-mercaptoethanol. After at least 15 min at room temperature, the samples were electrophoresed on 123456 78 a 10% SDS-polyacrylamide mini-gel (Bio-Rad, Richmond, CA) at 200 V. A prestained protein standard was also loaded for indication of both molecular weight and transference efficiency. After electrophoresis. Western blots were performed as previously described using a rabbit Cx43 mm«.an polyclonal antibody raised against a 15-mer synthetic polypeptide lo cated in the C-terminal domain of rat heart Cx43 (22). Immunofluorescent Detection of Gap Junctional Plaques. This anal B 012347 ysis was essentially performed as previously described (22) using the same polyclonal anti-Cx43 antibody as described above. Isolation of Total RNA and Poly(A)' RNA. After treatment, cells Cx43 were harvested with cold phosphate-buffered saline containing