4-Methylumbelliferone leads to growth arrest and apoptosis in canine mammary tumor cells

Abstract

Abstract. Hyaluronan (HA), a major component of the extracellular matrix (ECM), is synthesized by HA synthase (HAS) 1, HAS2 and HAS3 and is intricately involved in cell growth and metastasis. The HA synthesis inhibitor 4-methylumbelliferone (4-MU) has been reported to exhibit anticancer properties in various types of malignant tumors. However, the underlying mechanisms at the molecular and cellular levels remain unclear. In this study, to establish an animal model for studying the function of HA in human breast cancer, we investigated the antitumor effects of 4-MU using canine mammary tumor (CF33) cells. First, we investigated the effects of 4-MU on HA production in CF33 cells. Quantitative analysis of HA in culture media showed that 4-MU inhibited HA synthesis, accompanied by downregulation of HAS2 mRNA levels, in a dose-dependent manner at 24-72 h. Additionally, we observed a 4-MU-mediated decrease in the extent of the cell-associated HA matrix. We examined the effect of 4-MU on cell growth and apoptosis in CF33 cells. 4-MU markedly inhibited cell proliferation and induced apoptosis in CF33 cells. In particular, our experiments showed that the mechanism of 4-MU-induced apoptosis in CF33 cells involved increased levels of expression of pro-apoptotic BAX mRNA and protein molecules. These data suggest that 4-MU may be a candidate therapeutic agent for the treatment of canine mammary tumors. Furthermore, this study provides the first indication that the canine mammary tumor may be a suitable model for comparative study of the function of HA in human breast cancer. IntroductionMammary tumors in dogs occur at a frequency of 3 times the incidence of mammary tumors in humans and have recorded a higher incidence rate than other livestock (1). Mammary tumors are the most frequent cutaneous neoplasms in female dogs, and approximately 50% are diagnosed as malignant tumors (2). Following surgical excision, approximately 48% of the affected dogs die or are euthanized within 1 year due to tumor recurrence or metastasis (3). Canine mammary tumors are heterogeneous, and the different clinical and biological features (4) make it difficult to determine a prognosis and treatment for affected dogs, as is often the case in humans. Additional and reliable tools for effective therapy are required in veterinary medicine. Furthermore, a recent study revealed clear evidence of a similarity between human and dog tumors in regard to the deregulation of several cancer-related genes, including PI3K/Akt, PTEN and Wnt-β catenin and MAPK signaling (5). Accordingly, canine mammary tumors can also provide a suitable natural model for the comparative study of human breast cancer (5-7).Hyaluronan (HA) is a non-sulfated linear glycosaminoglycan that consists of repeating disaccharide subunits of glucuronic acid and N-acetylglucosamine (8,9). It is ubiquitously distributed in the extra-and pericellular spaces of most animal tissues (8,9). HA plays a critical role in regulating matrix assembly, cell migration, differentiation and proliferation (9-11)...

Anti-tumor and anti-invasion effects of a combination of 4-methylumbelliferone and ionizing radiation in human fibrosarcoma cells

“…Furthermore, it has been demonstrated that 1.0 mM MU exerts its anti-tumor effect on ovarian cancer by suppressing thymidine phosphorylase expression (11). In another study, Saito et al (12) indicated that MU leads to growth arrest and apoptosis mediated by BAX expression and a reduction in HA synthesis. In the present study, the induction of apoptosis by MU in HT1080 cells was observed, suggesting that the intracellular signaling pathway of apoptosis affected by 100 µM MU and is not dependent on the type of tumor.…”

Increased Hyaluronan Levels in HABP1/p32/gC1qR Overexpressing HepG2 Cells Inhibit Autophagic Vacuolation Regulating Tumor Potency

“…4-methyl umbelliferone (4-MU) has been reported to lead to HA synthesis by depleting the cellular UDP-glucoronic acid and downregulating HAS2 and HAS3 [5]. HepG2 and the higher HA containing HepR21 cells, were grown and exposed to different concentrations of 4-MU (0.25, 0.50, 1 and 2 mM) for either 6 or 12 hours as per the experiment along with a set of untreated cells acting as control.…”

“…Altered HA synthesis in tumor cells by HAS activity accelerates tumor growth through the recruitment of HA rich stromal cells and vasculature aided by factors secreted by tumor cells themselves [3], [4]. Consequently, HA synthase inhibitor, 4-MU has been reported to act as anti-tumor agent leading to decreased HA level, growth arrest and apoptosis [5], [6]. Although high molecular weight HA has been observed to enhance cellular proliferation, HA oligosaccharides inhibit anchorage independent growth in tumor cells by suppressing the PI3 Kinase/Akt survival pathway by stimulating expression of the tumor suppressor PTEN [7].…”

Targeting Hyaluronic Acid Family for Cancer Chemoprevention and Therapy

“…4-Methylumbelliferone (4-MU) or 7-hydroxy-4-methylcoumarin is a well-studied inhibitor of HA synthesis (Clarkin, Allen, Wheeler-Jones, Bastow, & Pitsillides, 2011; Kakizaki et al, 2004, 2002; Morohashi et al, 2006; Nakamura et al, 1997; Saito et al, 2013). Mammalian cells synthesize HA, using two building blocks—UDP-glucuronic acid (UGA) and UDP- N -acetyl-D-glucosamine.…”

“…In several studies using tumor cell lines, the IC 50 of 4-MU for inhibiting HA synthesis is in the range of 400 μM (Kakizaki et al, 2004, 2002; Lokeshwar et al, 2010; Nakamura et al, 1997). Recently, it has also been shown that 4-MU downregulates HAS2 and HAS3 expression (Saito et al, 2013). The study showed that 4-MU indeed inhibits both HA and sulfated glycosaminoglycan synthesis in the chick limb bud micromass culture (Clarkin et al, 2011).…”