Hyaluronan (HA) is a glycosaminoglycan polymer that often accumulates in malignancy. Megadalton complexes of HA with proteoglycans create a hydrated connective tissue matrix, which may play an important role in tumor stroma formation. Through its colloid osmotic effects, HA complexes contribute to tumor interstitial fluid pressure, limiting the effect of therapeutic molecules on malignant cells. The therapeutic potential of enzymatic remodeling of the tumor microenvironment through HA depletion was initially investigated using a recombinant human HA-degrading enzyme, rHuPH20, which removed HA-dependent tumor cell extracellular matrices in vitro. However, rHuPH20 showed a short serum half-life (t 1/2 < 3 minutes), making depletion of tumor HA in vivo impractical. A pegylated variant of rHuPH20, PEGPH20, was therefore evaluated. Pegylation improved serum half-life (t 1/2 = 10.3 hours), making it feasible to probe the effects of sustained HA depletion on tumor physiology. In high-HA prostate PC3 tumors, i.v. administration of PEGPH20 depleted tumor HA, decreased tumor interstitial fluid pressure by 84%, decreased water content by 7%, decompressed tumor vessels, and increased tumor vascular area >3-fold. Following repeat PEGPH20 administration, tumor growth was significantly inhibited (tumor growth inhibition, 70%). Furthermore, PEGPH20 enhanced both docetaxel and liposomal doxorubicin activity in PC3 tumors (P < 0.05) but did not significantly improve the activity of docetaxel in low-HA prostate DU145 tumors. The ability of PEGPH20 to enhance chemotherapy efficacy is likely due to increased drug perfusion combined with other tumor structural changes. These results support enzymatic remodeling of the tumor stroma with PEGPH20 to treat tumors characterized by the accumulation of HA. Mol Cancer Ther; 9(11); 3052-64. ©2010 AACR.
Increased deposition of specific extracellular matrix (ECM) components is a characteristic of insulin-resistant skeletal muscle. Hyaluronan (HA) is a major constituent of the ECM. The hypotheses that 1) HA content is increased in the ECM of insulin-resistant skeletal muscle and 2) reduction of HA in the muscle ECM by long-acting pegylated human recombinant PH20 hyaluronidase (PEGPH20) reverses high-fat (HF) diet–induced muscle insulin resistance were tested. We show that muscle HA was increased in HF diet–induced obese (DIO) mice and that treatment of PEGPH20, which dose-dependently reduced HA in muscle ECM, decreased fat mass, adipocyte size, and hepatic and muscle insulin resistance in DIO mice at 10 mg/kg. Reduced muscle insulin resistance was associated with increased insulin signaling, muscle vascularization, and percent cardiac output to muscle rather than insulin sensitization of muscle per se. Dose-response studies revealed that PEGPH20 dose-dependently increased insulin sensitivity in DIO mice with a minimally effective dose of 0.01 mg/kg. PEGPH20 at doses of 0.1 and 1 mg/kg reduced muscle HA to levels seen in chow-fed mice, decreased fat mass, and increased muscle glucose uptake. These findings suggest that ECM HA is a target for treatment of insulin resistance.
Abstract. Calmodulin (CAM) affinity chromatography of a detergent extract of sea urchin sperm yielded ,°20 major proteins. One of these proteins, of Mr 190,000, was purified and used to immunize rabbits. After absorption with living sperm, the serum reacted monospecifically on one-and two-dimensional Western immunoblots with the Mr 190,000 protein. The anti-190-kD serum inhibited 94% of the adenylate cyclase (AC) activity of the CaM eluate. An immunoaffinity column removed 95 % of the AC activity, and the purified (but inactive) Mr 190,000 protein was eluted from the column. The antiserum also inhibited 28 % of the activity of bovine brain CaM-sensitive AC and 90% of the activity of horse sperm CaM-sensitive AC. These data support the hypothesis that the Mr 190,000 protein is sea urchin sperm AC. Although this AC bound to CaM, it was not possible to demonstrate directly a Ca 2÷ or CaM sensitivity. However, two CaM antagonists, calmidazolium and chlorpromazine, both inhibited AC activity, and the inhibition was released by added CaM, suggesting the possibility of regulation of this AC by CaM. Indirect immunofluorescence showed the Mr 190,000 protein to be highly concentrated on only the proximal half of the sea urchin sperm flagellum. This asymmetric localization of AC may be important to its function in flagellar motility. This is the first report of the identification of an AC from animal spermatozoa.IMAL spermatozoa undergo profound physiological changes between the time of their release from the male reproductive tract and their fusion with eggs. Some of these changes, or "activations" are the initiation of motility, capacitation, and the acrosome reaction. A large literature exists on the regulation of the initiation of sperm flagellar motility by Ca 2÷, cAMP, and cAMP-dependent protein kinase (A-kinase; for reviews, see references 1,3,8, 11,21,22). In summary, these data support the hypothesis that increases in cellular Ca 2÷ activate sperm adenylate cyclase, resulting in increased concentrations of cAMP. This in turn activates A-kinase, which phosphorylates axonemal proteins. These phosphorylation events are necessary for the initiation and maintenance of flagellar motility. Sea urchin sperm are a model cell for studying the mechanism of flagellar motility. It is important to the ultimate elucidation of the underlying mechanism to identify, localize, and characterize the enzymes and their protein substrates that function in the process.Previous work (reviewed in reference 3) has shown that sea urchin spermatozoa contain considerable adenylate cyclase (AC) ~ activity. This activity requires Mn-ATP and exhibits a 50-fold activation when living sperm are treated with solu-L. H. Bookbinders present address is Department of Pharmacology, S J-30,
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