Dietary Supplement 4-Methylumbelliferone: An Effective Chemopreventive and Therapeutic Agent for Prostate Cancer

Effective control of localized prostate cancer (PCa) and of its metastatic spread by consumption of a nontoxic dietary supplement can potentially delay/avoid treatment of low-risk localized PCa and halt progression in patients with advanced disease. 4-methylumbelliferone (4-MU; 7-hydroxy–4-methylcoumarin or hymecromone) is a dietary supplement consumed in Europe and Asia for improving liver health. 4-MU is known to inhibit synthesis of hyaluronic acid (HA), a nonsulfated glycosaminoglycan. HA and HA family members—HA synthases (ie, HAS1, HAS2, HAS3), HA receptors (ie, cluster of differentiation antigen 44 [CD44], hyaluronan-mediated motility receptor [RHAMM]) and hyaluronidases (mainly HYAL-1)—promote tumor growth and progression. HA family members are potential diagnostic and prognostic markers for several cancers (1–7). For example, HA expression is elevated in PCa tissues, and together with HYAL-1 expression it predicts biochemical recurrence (8,9). In tumor tissues, HA is contributed by both tumor cells and associated stroma and is a mixture of large polymers and HA fragments, some of which are angiogenic (2). Interaction between pericellular HA and CD44/RHAMM promotes cell survival, cancer stemness, motility, and invasion by activating intracellular signaling (10–15). In preclinical models, while HA synthases and/or HYAL-1 enhance tumor growth, metastasis, and angiogenesis, their knockdown inhibits tumor cell functions (16–22). Because cancer cells express more than one HA synthase, targeting HA synthesis by small molecule inhibitors may be effective in cancer chemoprevention and treatment (12). Inhibition of HA synthesis by 4-MU has been examined in a few model systems (23–27). Mammalian cells synthesize HA using UDP-glucuronic acid (UGA) and UDP-N-acetyl-D-glucosamine. UGA is a substrate for UDP-glucuronosyltransferases (28–30). In cells treated with 4-MU, UDP-glucuronosyltransferase transfers glucuronic acid onto 4-MU. This depletes the intracellular pool of UGA, leading to blockade of HA synthesis. The Km values of UDP-glucuronosyltransferases and HA synthases range between 100 and 900 µM; consequently, the IC50 of 4-MU to inhibit HA synthesis is approximately 0.4mM (~70 µg/mL; [28–30]). 4-MU also downregulates the expression of HAS2, HAS3, and UDP-dehydrogenase; these are key enzymes in glycosaminoglycan synthesis (31,32). Because of its fluorescence, 4-MU is widely used as a fluorescent indicator in enzyme assays. In small clinical trials, 4-MU has shown choleretic and antispasmodic properties with improvement in liver and gallbladder functions (14,33–35). Although a coumarin-derivative, 4-MU lacks antisperminogenic and anti-aromatase activities of coumarin, and unlike Coumadin it has no anticoagulant activity (36–39). The maximum tolerated dose of 4-MU in mice is 2.8 to 7.3g/kg [National Institute for Occupational Safety and Health (NIOSH) registry: registry of toxic effects of chemical substances (RTECs) number GN7000000]. We have previously reported that, at the IC50 for HA synthesis, 4-MU inhibits proliferation, invasion, and motility of PCa cells in vitro and subcutaneous growth of PC3-ML-xenografts (39). 4-MU has also shown antitumor activity in a few tumor models at doses of 1 to 3g/day; however, 4-MU has not been evaluated for its chemopreventive activity and/or therapeutic efficacy in transgenic models, at various stages of cancer progression, or at doses comparable with those in human use [23-25,27). We evaluated chemopreventive and therapeutic efficacy of 4-MU in three PCa mouse models—TRAMP, PC3-ML/Luc+ skeletal metastasis, and DU145 subcutaneous implantation. We also investigated the mechanism of action of 4-MU.