Several reports have implicated reactive oxygen and nitrogen metabolites (RONS) in the initiation and/or progression of inflammatory bowel diseases (IBDs). We have investigated the role of three key RONS-metabolizing enzymes (inducible nitric oxide synthase [iNOS], superoxide dismutase [SOD], nicotinamide adenine dinucleotide phosphate [NADPH] oxidase) in a murine model of IBD. Mice genetically deficient (−/−) in either iNOS or the p47phox subunit of NADPH oxidase, transgenic (Tg) mice that overexpress SOD, and their respective wild-type (WT) littermates were fed dextran sulfate sodium (DSS) in drinking water for 7 days to induce colitis. In addition, the specific iNOS inhibitor 1400W was used in DSS-treated WT and p47phox−/− mice. WT mice responded to DSS feeding with progressive weight loss, bloody stools, elevated serum NOX and colonic mucosal injury with neutrophil infiltration. Both the onset and severity of colitis were significantly attenuated in iNOS−/− and 1400W-treated WT mice. While the responses to DSS did not differ between WT and p47phox−/− mice, enhanced protection was noted in 1400W-treated p47phox−/− mice. Interestingly, SODTg mice exhibited more severe colitis than their WT littermates. These findings reveal divergent roles for superoxide and iNOS-derived NO in intestinal inflammation.

Hyaluronic acid and HYAL1-type hyaluronidase show high accuracy in detecting bladder cancer and evaluating its grade, respectively. Hyaluronic acid promotes tumor progression; however, the functions of hyaluronidase in cancer are largely unknown. In this study, we stably transfected HT1376 bladder cancer cells with HYAL1-sense (HYAL1-S), HYAL1-antisense (HYAL1-AS), or vector cDNA constructs. Whereas HYAL1-S transfectants produced 3-fold more HYAL1 than vector transfectants, HYAL1-AS transfectants showed f f 90% reduction in HYAL1 production. HYAL1-AS transfectants grew four times slower than vector and HYAL1-S transfectants and were blocked in the G 2 -M phase of the cell cycle. The expression of cdc25c and cyclin B1 and cdc2/p34-associated H1 histone kinase activity also decreased in HYAL1-AS transfectants. HYAL1-S transfectants were 30% to 44% more invasive, and HYAL1-AS transfectants were f f 50% less invasive than the vector transfectants in vitro. In xenografts, there was a 4-to 5-fold delay in the generation of palpable HYAL1-AS tumors, and the weight of HYAL1-AS tumors was 9-to 17-fold less than vector and HYAL1-S tumors, respectively (P < 0.001). Whereas HYAL1-S and vector tumors infiltrated skeletal muscle and blood vessels, HYAL1-AS tumors resembled benign neoplasia. HYAL1-S and vector tumors expressed significantly higher amounts of HYAL1 (in tumor cells) and hyaluronic acid (in tumor-associated stroma) than HYAL1-AS tumors. Microvessel density in HYAL1-S tumors was 3.8-and 9.5-fold higher than that in vector and HYAL1-AS tumors, respectively. These results show that HYAL1 expression in bladder cancer cells regulates tumor growth and progression and therefore serves as a marker for high-grade bladder cancer. (Cancer Res 2005; 65(6): 2243-50)

Hyaluronidases degrade hyaluronic acid, which promotes metastasis. HYAL1 type hyaluronidase is an independent prognostic indicator of prostate cancer progression and a biomarker for bladder cancer. However, it is controversial whether hyaluronidase (e.g., HYAL1) functions as a tumor promoter or as a suppressor. We stably transfected prostate cancer cells, DU145 and PC-3 ML, with HYAL1-sense (HYAL1-S), HYAL1-antisense (HYAL1-AS), or vector DNA. HYAL1-AS transfectants were not generated for PC-3 ML because it expresses little HYAL1. HYAL1-S transfectants produced V42 milliunits (moderate overproducers) or z80 milliunits hyaluronidase activity (high producers). HYAL1-AS transfectants produced <10% hyaluronidase activity when compared with vector transfectants (18-24 milliunits). Both blocking HYAL1 expression and high HYAL1 production resulted in a 4-to 5-fold decrease in prostate cancer cell proliferation. HYAL1-AS transfectants had a G 2 -M block due to decreased cyclin B1, cdc25c, and cdc2/p34 expression and cdc2/p34 kinase activity. High HYAL1 producers had a 3-fold increase in apoptotic activity and mitochondrial depolarization when compared with vector transfectants and expressed activated proapoptotic protein WOX1. Blocking HYAL1 expression inhibited tumor growth by 4-to 7-fold, whereas high HYAL1 producing transfectants either did not form tumors (DU145) or grew 3.5-fold slower (PC-3 ML). Whereas vector and moderate HYAL1 producers generated muscle and blood vessel infiltrating tumors, HYAL1-AS tumors were benign and contained smaller capillaries. Specimens of high HYAL1 producers were 99% free of tumor cells. This study shows that, depending on the concentration, HYAL1 functions as a tumor promoter and as a suppressor and provides a basis for anti-hyaluronidase and high-hyaluronidase treatments for cancer. (Cancer Res 2005; 65(17): 7782-9)

The tumor cell-derived hyaluronidase HYAL-1 degrades hyaluronic acid (HA) into pro-angiogenic fragments that support tumor progression. Although HYAL-1 is a critical determinant of tumor progression and a marker for cancer diagnosis and metastasis prediction, it has not been evaluated as a target for cancer therapy. Similarly, sulfated hyaluronic acid (sHA) has not been evaluated for biological activity, although it is a HAase inhibitor. In this study we show that sHA is a potent inhibitor of prostate cancer. sHA blocked the proliferation, motility and invasion of LNCaP, LNCaP-AI, DU145 and LAPC-4 prostate cancer cells, also inducing caspase 8-dependent apoptosis associated with downregulation of Bcl-2 and phospho-Bad. sHA inhibited Akt signaling including androgen receptor (AR) phosphorylation, AR-activity, NFkb activation and VEGF expression. These effects were traced to a blockade in complex formation between PI3K and HA receptors and to a transcriptional downregulation of HA receptors, CD44 and RHAMM, along with PI3K inhibition. Angiogenic HA fragments or overexpression of myristoylated-Akt or HA receptors blunted these effects of sHA, implicating a feedback loop between HA receptors and PI3K/Akt signaling in the mechanism of action. In an animal model, sHA strongly inhibited LNCaP-AI prostate tumor growth without causing weight loss or apparent serum-organ toxicity. Inhibition of tumor growth was accompanied by a significant decrease in tumor angiogenesis and an increase in apoptosis index. Taken together, our findings offer mechanistic insights into the tumor-associated HA-HAase system and a preclinical proof-of-concept of the safety and efficacy of sHA to control prostate cancer growth and progression.