Abstract. We have demonstrated that blue light has anti-cancer effects in cultured cancer cells and tumor-bearinganimals. Based on our experimental findings, in addition tocytostatic activity that suppresses the proliferation of B16melanoma cells, blue light may exert cytocidal activity throughinteraction with vitamin(s) contained in the culture medium.The present study was undertaken to identify the specificvitamins with which blue light interacts and to investigate thefactors responsible for its cytocidal activity. B16 melanomacells were incubated in media supplemented with variousvitamins and exposed to blue light for 10 min. Cell necrosiswas observed only in media containing riboflavin (0.4 mg/l).The effects of other components of visible light on riboflavinwere also studied. Riboflavin-containing media were exposedto light of each of the three primary colors (red, green andblue) and the effects on the colony-forming capacity of B16melanoma cells were evaluated. Cell necrosis was inducedonly in media exposed to blue light. The effects of riboflavinincreased in a concentration-dependent manner in the rangefrom 0.3 to 1.0 mg/l in blue-light-exposed media and wereantagonized by the presence of catalase (200 U/ml). Thesefindings suggest that cell necrosis is probably induced byactive oxygen species such as hydrogen peroxide formed bythe reaction of riboflavin with blue light.IntroductionAlthough numerous studies have been carried out to investigatethe relationships between light and disease, with specialattention paid to the application of radiation and ultravioletrays (UV) to cancer treatment (1-5) and the evaluation ofcarcinogenic (6-10) and immunosuppressive (11) effects, theeffects of visible light are still not well understood in themedical field. We examined the effects of components ofvisible light on the growth of cancer cells using light-emittingdiodes (LEDs), which have selective light-emission spectra,and found that blue light suppressed the growth of B16melanoma cells in a time-dependent manner (12). The effectsof blue light appeared to be attributable to the inhibition ofDNA synthesis and cell division. When the blood of rats with1-ethyl-1-nitrosourea (ENU)-induced leukemia was exposedto blue light for 3 h during extracorporeal circulation, thegrowth of leukemic cells was suppressed, with no significantdifference in the growth of normal lymphocytes, compared tothe non-exposure control group (13). When B16 melanomacells exposed to blue light and incubated for 7 days wereinjected intravenously into mice, the metastasis of B16melanoma cells to the lung was suppressed (14). The inductionof skin tumors by 12- O-tetradecanoylphorbol-13-acetate inthe v-Ha-ras transgenic mouse was also suppressed by bluelight exposure (15). An interesting phenomenon was notedin the course of these studies: when the culture medium forthe B16 melanoma cells was switched from Eagle's MEM(Nihon Pharmaceutical Co., Ltd., Tokyo, Japan; hereinafterreferred to simply as ‘MEM’) to Dulbecco's MEM (D5796,Sigma Aldrich, St. Louis, MO; hereinafter referred to as‘D-MEM’), the effects of exposure to blue light were markedlyincreased, and powerful cytocidal activity, which was not seenin the experiments using MEM, was observed. Preliminaryinvestigations revealed that some vitamin(s) contained inD-MEM might be related to these effects (unpublished data).We therefore conducted additional studies in order to identifythe vitamin(s) reacting with the blue light as well as the cyto-cidal factor(s) formed in the cell culture medium followingexposure to blue light.Materials and methodsLED device. The apparatus was constructed using red, greenand blue LEDs (Nichia Corp., Tokushima, Japan) as reportedpreviously (12). Each LED was driven by direct current froma standard power supply (S82K-10024, Omron, Tokyo,Japan). The blue LEDs used for evaluation of the effects ofexposure to blue light had the following specifications: current30.1 mA, wavelength 470 nm and irradiance 5.7 mW/cm2. TheLEDs used for comparison of the effects of light of the threeprimary colors had the following specifications: red = current32.0 mA, wavelength 634 nm and irradiance 2.27 mW/cm2;green = 30.5 mA, 518 nm and 2.26 mW/cm2; and blue =8.0 mA, 470 nm and 2.24 mW/cm2.Cell culture. B16 melanoma cells (JCRB0202, Human ScienceResearch Resources Bank, Osaka, Japan) cultured in MEMINTERNATIONAL JOURNAL OF ONCOLOGY 22: 1291-1295, 2003Augmentation of the inhibitory effect of blue light on the growth of B16 melanoma cells by riboflavinMASAYUKI OHARA, TATSUO FUJIKURA and HIROSHI FUJIWARANutrition Research Institute, Otsuka Pharmaceutical Factory, Muya-cho, Naruto, Tokushima 772-8601, JapanReceived January 15, 2003; Accepted March 3, 2003_________________________________________Correspondence to: Dr Masayuki Ohara, Otsuka PharmaceuticalFactory, Inc., Muya-cho, Naruto, Tokushima 772-8601, JapanE-mail: [email protected] words: blue light, LED, B16 melanoma cell, riboflavinwith 10% fetal bovine serum (FBS, Life Technologies,Inc., Grand Island, NY) were used in the experiments whentheir growth rates had stabilized after cultivation for threegenerations. Cells were incubated at 37˚C in a 5% CO2/95%air atmosphere.Culture media. Eagle's MEM (MEM) was purchased fromNihon Pharmaceutical Co., Ltd. (Tokyo, Japan). Dulbecco'sMEM (D5796, D-MEM) was purchased from Sigma Aldrich(St. Louis, MO).Effects of culture medium on the growth of B16 melanomacells. A cell suspension (2 ml) in MEM or D-MEM (sup-plemented with 10% FBS) containing 5x103cells/ml wasinoculated into 6-well microplates (Becton Dickinson,Franklin Lakes, NJ). After incubation for 24 h, the cultureswere exposed to blue light for 20 min and then incubatedfor an additional 4 days. Another set of 6-well microplatescontaining MEM or D-MEM (2 ml/well) was exposed to bluelight for 20 min. Immediately after blue light exposure, eachmedium was used to replace 1.8 ml of the supernatant of thecell suspensions (2 ml) in MEM or D-MEM (supplementedwith 10% FBS) containing 5x103cells/ml. The cell cultureswere then incubated for 4 days. The number of viable cellswas then counted by trypan blue staining.Culture medium with vitamins. MEM was supplementedwith 10% FBS and amino acids (MEM amino acid solution,Life Technologies, Inc.) to adjust the concentration of eachamino acid to closely match that of D-MEM, the amino acidconcentration of which is about twice that of MEM. Theresultant solution was