INTERNATIONAL JOURNAL OF ONCOLOGY 38: 893-902, 2011 Involvement of riboflavin kinase expression in cellular sensitivity against cisplatin GEN HIRANO1,3, Hiroto IZUMI1, YOSHIHIRO Yasuniwa1, SHOHEI SHIMAJIRI2, WANG KE-YONG2, YASUYUKI SASAGIRI2, HitoSHi KuSaBa3, KAORI Matsumoto4, Tetsuya Hasegawa4, Masayuki Akimoto4, KOICHI AKASHI3 and Kimitoshi KOHNO1 Departments of 1Molecular Biology and 2Pathology and Cell Biology, School of Medicine, university of occupational and Environmental Health, Kitakyushu, Fukuoka 807-8555; 3Department of Internal Medicine and Department of Biosystemic Science of Medicine, Graduate School of Medicine, Kyushu university, Fukuoka, Fukuoka 812-8582; 4Faculty of Pharmaceutical Science, Josai International University, Togane, Chiba 283-8555, Japan Received October 27, 2010; Accepted December 23, 2010 DOI: 10.3892/ijo.2011.938 Abstract. Flavin adenine dinucleotide (FAD) is an essential well with Gleason score, known as a good indicator of patient coenzyme for glutathione reductase (GR) which catalyzes the prognosis. The present study suggests that RFK expression is reduction of oxidized glutathione to regenerate the reduced involved not only in cellular protection from oxidative stress form involved in protection against oxidative stress. Riboflavin but also in malignant progression of prostate cancer. kinase (RFK) also known as flavokinase is involved in the first step of bioactivation of riboflavin (RF) to form flavin Introduction mononucleotide (FMN) which can be subsequently converted to FAD in an ATP-dependent reaction catalyzed by FAD Riboflavin, vitamin 2B (RF) is a water-soluble vitamin present synthetase (FADS). We investigated the involvement of RFK in a wide variety of foods. Riboflavin kinase (RFK) also known in cisplatin resistance using human prostate cancer PC3 cells. as flavokinase (1) is required for the conversion of riboflavin RFK overexpression renders cells resistant not only to cisplatin into its active coenzyme derivatives. Riboflavin is phosphorylated but also to hydrogen peroxide (H2O2) and diamide. by RFK to generate flavin mononucleotide (FMN), which is Furthermore, knockdown of RFK expression induced apoptosis. adenylated by FAD synthetase (FADS) to generate flavin We demonstrated that overexpression of RFK increased the adenine dinucleotide (FAD). FAD is a coenzyme for glutathione levels of FAD, FMN and total glutathione and the expression reductase (GR), which mediates the regeneration of reduced of GR and glutathione S-transferase-π (GSTπ). RFK expression glutathione; L-γ-glutamyl-L-cysteinyl-glycine (GSH). Gluta- is up-regulated in cisplatin-resistant P/CDP6 cells in addition thione is the major regulator of the intracellular thiol redox to FAD, total glutathione level, GR and GSTπ. Knockdown of status and effectively scavenges free radicals and other reactive RFK expression also sensitized both PC3 and P/CDP6 cells to oxygen species (ROS). In these reactions, glutathione is cisplatin. Moreover, cellular levels of RFK expression correlate oxidized to form glutathione disulfide (GSSG), which is then reduced to glutathione by the NADPH-dependent GR. If oxidative stress or any other factors limit the GR reaction, then GSSG will accumulate. Glutathione synthesis is primarily regulated by γ-glutamylcysteine synthetase (γ-GCS). Gluta- Correspondence to: Dr Kimitoshi Kohno, Department of Molecular thione-S-transferases (GSTs) catalyze the conjugation of Biology, School of Medicine, university of occupational and glutathione with many endogenous compounds. Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu, The total glutathione level is higher in many multidrug Fukuoka 807-8555, Japan and radiation resistant tumors than in normal tissues (2,3). E-mail: [email protected] Elevated levels of γ-GCS have also been identified in different Abbreviations: 5-FU, 5-fluorouracil; cDNA, complementary DNA; human cancer tissues (2). There are seven known classes of FAD, flavin adenine dinucleotide; FADS, FAD synthetase; FMN, human GSTs. One, GSTπ, is highly expressed in many cancers flavin mononucleotide; GF, galactoflavin; GR, glutathione reductase; and appears to be involved in drug resistance (4-6). However, GSSG, glutathione disulfide; GST, glutathione S-transferase; there are very few studies on the role of RFK expression in H2O2, hydrogen peroxide; RF, riboflavin; RFK, riboflavin kinase; cancer. Cisplatin is widely used at the initial anticancer agent WST-8, water-soluble tetrazolium salt; γ-GCS, γ-glutamylcysteine because of its activity against many human solid tumors (7,8). synthetase Its anticancer effect is obtained by several mechanisms, including formation of DNA adducts and production of reactive oxygen Key words: RFK, B2-vitamers, cisplatin, oxidative stress, prostate species (9,10). Multiple mechanisms for cisplatin resistance cancer have been proposed such as reduced drug accumulation and 894 HIRANO et al: RiBoFlaviN KiNaSe and CisplatiN ReSiStance transport (11-13) and enhanced DNA repair (14,15). Another indicates the start codon and stop codon): 5'-ATGCCCCGAGCG mechanism for cisplatin resistance is the up-regulation of GACTGCATTATG-3' and 5'-TCAGTGGCCATTCATTATTT the detoxification system via glutathione biosynthesis and TGCTTTTAG-3'. This PCR product was cloned into the conjugation (1,3,16,17). In fact, once inside the cell, cisplatin pGEM-T easy vector (Promega, Madison, WI, USA). To has to be activated through aquation reactions before being construct a plasmid expressing Flag-tagged RFK, N-terminal able to interact with DNA. The formation of aquated cisplatin Flag-tagged RFK cDNA were ligated into pcDNA3 vector is rate limited by its interaction with many endogenous (Invitrogen). detoxification molecules, among which glutathione is the most important and effective (5,18). On the other hand, it is Cloning of stable transfectants. The cloning of stable transfectant unknown whether the glutathione recycling system is involved has been described (21). Briefly, PC3 cells were transfected in cisplatin resistance. with pcDNA3-Flag RFK with Superfect reagent and cultured The present studies were designed to determine whether with 500 µg/ml neomycin for 15-20 days. The resulting colonies RFK is involved in the glutathione-dependent antioxidant were isolated and the cellular expression level of Flag-RFK in system. These initial studies indicate that RFK expression is each clone was investigated by Western blot analysis with involved in the cellular sensitivity against cisplatin or oxidative anti-Flag (M2) antibody. stress and malignant progression of cancer. Western blot analysis. Whole-cell lysates were prepared as Materials and methods previously described (22,23). The indicated amounts of whole-cell lysates were separated by sodium dodecyl sulfate- Cell culture. Human epidermoid cancer HeLa cells and prostate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred cancer PC3 cells were cultured in Eagle's minimal essential to polyvinylidene difluoride (PvDF) microporous membranes medium. Human lung cancer A549 and H1299 cells, liver (Millipore, Billerica, Ma, uSa) using a semi-dry blotter. the cancer HepG2 cells and colon cancer DLD1 and CaCo-2 cells blotted membranes were treated with 5% (w/v) skimmed milk were cultured in RPMI-1640 medium. Human pancreatic in 10 mM Tris, 150 mM NaCl and 0.2% (v/v) Tween-20, and cancer MIA PaCa and Panc2 cells were cultured in Dulbecco's incubated for 1 h at room temperature with the primary antibody. modified eagle's medium. Media were purchased from Nissui The following antibodies and dilutions were used: 1:1,000 Seiyaku (Tokyo, Japan) and contained 10% fetal bovine serum. dilution of anti-RFK, 1:1,000 dilution of anti-GSTπ, 1:1,000 The cisplatin-resistant P/CDP6 cells were derived from PC3 dilution of anti-γ-GCSc, 1:1,000 dilution of anti-glutathione cells as described previously (19,20). All cell lines were reductase, 1:1,000 dilution of anti-caspase 3, 1:10,000 dilution maintained in a 5% CO2 atmosphere at 37˚C. of anti-Flag, and 1:5,000 dilution of anti-β-actin. The membranes were then incubated for 45 min at room temperature with a Antibodies and drugs. To generate the polyclonal anti-RFK peroxidase-conjugated secondary antibody and were developed antibody, the synthetic peptide CNFFQvSKSKiMNGH was using chemiluminescence (Amersham, Piscataway, NJ, USA). used as an immunogen and the antisera were obtained from The images were obtained by image analyzer (LAS-4000 mini, New Zealand white rabbits after multiple immunization. Fujifilm,t okyo, Japan). to induce cleaved caspase 3 expression, Anti-GSTπ antibody (610719) was purchased from BD PC3 cells were incubated with 1 µM vP16 for 24 h. Bioscience (San Jose, Ca, uSa). antibodies against γ-GCSc (sc-22755) and glutathione reductase (sc-32886) were purchased Knockdown analysis using siRNAs. The following double- from Santa Cruz Biotechnology (Santa Cruz, Ca, uSa). anti- stranded RNA 25-bp oligonucleotides were commercially caspase-3 (no. 9662) antibody was purchased from Cell generated (Invitrogen): 5'-AAGUAAGGCAGGUGCCUCA Signaling Technology (Danvers, MA, USA). Anti-β-actin UAAUGC-3' (sense) and 5'-GCAUUAUGAGGCACCUGCCU (AC-15) and anti-Flag (M2) (F3165) antibodies were purchased UACUU-3' (antisense); RFK siRNA #1, 5'-AAGAUUAUCUAC from Sigma (St. Louis, MO, USA). Cisplatin, paclitaxel, CACUUGCUCAGGA-3' (sense) and 5'-UCCUGAGCAAGUG 5-fluorouracil (5-Fu) and etoposide (vP16) were purchased GUAGAUAAUCUU-3' (antisense); RFK siRNA no. 2, 5'-UUC from Sigma. Adriamycin