Hyaluronidase is a hyaluronic acid-degrading endoglycosidase that is present in many toxins and the levels of which are elevated in cancer. Hyaluronidases (HAases)1 are a family of enzymes that are crucial for the spread of bacterial infections, toxins present in various venoms, and possibly, cancer progression (1-6). In humans, six HAase genes have been identified. These genes occur in clusters of three at two chromosomal locations (Ref. 7 and human genome blast search). HYAL1, HYAL2, and HYAL3 occur on chromosome 3p21.3, and PH20, HYAL4, and HYALP1 occur on chromosome 7q31.3. With the possible exception of HYAL4 and HYALP1, all other HAases degrade hyaluronic acid (HA) (7).HA is a nonsulfated glycosaminoglycan made up of repeating disaccharide units, D-glucuronic acid, and N-acetyl-D-glucosamine. HA is present in body fluids, tissues, and the extracellular matrix (8 -10). It keeps tissues hydrated and maintains osmotic balance and cartilage integrity (8, 10). HA also actively regulates cell adhesion, migration, and proliferation by interacting with specific cell surface receptors such as CD44 and RHAMM (11). The concentrations of HA are elevated in several inflammatory diseases and various carcinomas (e.g. bladder, prostate, breast, lung, colon, and so forth; Refs. 9 and 12-19). For example, we have shown that urinary HA concentration is a highly sensitive and specific marker for detecting bladder cancer, regardless of its grade (18,19). In tumor tissues, HA may promote tumor growth and metastasis probably by actively supporting tumor cell migration and offering protection against immune surveillance (20 -22). Small fragments of HA, generated by HAases, stimulate angiogenesis (23-25). We recently showed that HA fragments of ϳ10 -15 disaccharide units stimulate endothelial cell proliferation by acting through cell surface HA receptor, RHAMM, and activating the mitogenactivated protein kinase pathway (26). We have also shown that elevated levels of HYAL1-type HAase coincide with the presence of angiogenic HA fragments in prostate tumor tissues and in the urine of bladder cancer patients (17,27).Among the six HAases, HYAL1, HYAL2, and PH20 are well characterized. HYAL1 type HAase was originally purified from human plasma and urine (28, 29). However, we have shown that HYAL1 is the major tumor-derived HAase expressed in bladder and prostate cancer tissues (17,30). It has a optimum pH range of 4.0 -4.3, and the enzyme is 50 -80% active at pH 4.5 (17). have shown that a lack of functional HYAL1 results in a disorder called mucopolysaccharidosis IX. In this study the authors identified that aa Glu 268 is crucial for HYAL1 activity. HYAL2 was originally designated as the lysosomal HAase, and it cleaves high molecular mass HA into ϳ20-kDa HA fragments (32). It has a pH optimum of ϳ4.0 and is possibly less active than other HAases. HYAL-2 may also be exposed to the cell surface through a GPI anchor (32). The third HAase gene in the 3p21.3 locus is HYAL3. Although
In this paper we report the syntheses of the 39-peptide C(Acm)LNELDADEQADL-CESLHDHADE-LYRSCLARFGDDGENL, 1, and of the 25-peptide C(Acm)LNELDADEQADL-CLARFGDDGENL, 2, by means of thiol capture ligations using precursor peptides bearing b l o c b g groups only on cysteine residues. The ligations were made in each case at the underlined cysteine, cleanly and in high yield. For each of the above syntheses, an acidolytically deblocked 13-peptide dibenzofuranyl ester, 6-[C(Acm)LNELDADEQADLeucinyloxy]-4-mercaptodibenzofuran, 3, was prepared in pure form in 52% overall yield through three stages: (1) stepwise synthesis on a solid-phase resin loaded with the dibenzofuran template, (2) acidolytic removal of the tert-butyl esters of the resin-bound peptide, and (3) preparative cleavage of the deblocked peptidyloxydibenzofuran ester from the resin. In the case of both the 39-peptide and the 25-peptide, significant rate enhancements were seen for the 08-acyl-transfer step of the thiol capture sequence when both the N-terminal and C-terminal fragments had been previously side-chain deblocked, in comparison with the cases when only the C-terminal fragment had been side-chain deblocked. In the 13-peptide + 12-peptide ligation to form the 25-peptide 2, a t1p = 5 min was seen for the leucine-cysteine amide bond-forming reaction. A model leucine-cysteine 08-acyl transfer as well as leucinecysteine OJV-acyl transfers between protected peptide fragments, however, showed the expected t1p = 4 h. Rationalization of this observed 40-fold rate enhancement is offered that identifies the aspartic acid side chain carboxylate, 12 residues in sequence from the N-terminus and penultimate to the amide ligation site, as apossible intramolecular general base catalyst for the proton-transfer step during the OJV-acyl transfer.
Many tumors constitutively express high levels of the inducible form of proinflammatory enzyme, cyclooxygenase-2 (COX-2). Increased COX-2 expression is associated with tumor cell resistance to many cytotoxic chemotherapy drugs. Furthermore, increased resistance to cytotoxic antitumor drugs is also known to be dependent on associated stromal cells in many tumors. We investigated whether prostate tumor-associated stromal cells, marrow-derived osteoblasts, affect cytotoxicity of 2 antitumor drugs, COL-3 and docetaxel (TXTR), and whether it is dependent on COX-2 activity. We further examined whether inhibiting the activity of COX-2 negate the stroma-induced decrease in drug sensitivity in tumor cells. COX-2-specific inhibitor celecoxib (CXB) was used to inhibit COX-2 activity and associated alteration in cell death signaling was investigated. Coculturing PC-3ML cells with osteoblasts decreased the cytotoxicity of the tested antitumor drugs and was associated with increased COX-2 activity in PC-3ML cells. A significant decrease in drug-induced PGE 2 increase and an increase in cytotoxicity were observed when cells were treated with COL-3 or TXTR combined with CXB. Cytotoxicity of single or combination treatment increased apoptosis, which was associated with caspase-3 and -9 activation, PARP cleavage, increased BAD protein, but decreased protein levels of XIAP and BCL-xL . Oral administration of CXB (40 mg/kg) to mice with PC-3ML tumors for 42 days increased tumor latency, decreased tumor growth and enhanced tumor control with COL-3 or TXTR. Overall, a synergistic enhancement of antitumor activity in combination treatment was observed in vitro and an additive effect in vivo. These observations suggest a potential clinical use of combined dosing of COX-2 inhibitors and cytotoxic drugs at lower, nontoxic dose than currently used to treat advanced prostate cancer. ' 2005 Wiley-Liss, Inc.
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