Norman L. Block

Stromal and Epithelial Expression of Tumor Markers Hyaluronic Acid and HYAL1 Hyaluronidase in Prostate Cancer

Hyaluronic acid (HA), a glycosaminoglycan, regulates cell adhesion and migration. Hyaluronidase (HAase), an endoglycosidase, degrades HA into small angiogenic fragments. Using an enzyme-linked immunosorbent assay-like assay, we found increased HA levels (3–8-fold) in prostate cancer (CaP) tissues when compared with normal (NAP) and benign (BPH) tissues. The majority (∼75–80%) of HA in prostate tissues was found to exist in the free form. Primary CaP fibroblast and epithelial cells secreted 3–8-fold more HA than respective NAP and BPH cultures. Only CaP epithelial cells and established CaP lines secreted HAase and the secretion increased with tumor grade and metastasis. The pH activity profile and optimum (4.2; range 4.0–4.3) of CaP HAase was identical to the HYAL1-type HAase present in human serum and urine. Full-length HYAL1 transcript and splice variants were detected in CaP cells by reverse transcriptase-polymerase chain reaction, cloning, and sequencing. Immunoblotting confirmed secretion of a ∼60-kDa HYAL1-related protein by CaP cells. Immunohistochemistry showed minimal HA and HYAL1 staining in NAP and BPH tissues. However, a stromal and epithelial pattern of HA and HYAL1 expression was observed in CaP tissues. While high HA staining was observed in tumor-associated stroma, HYAL1 staining in tumor cells increased with tumor grade and metastasis. The gel-filtration column profiles of HA species in NAP, BPH, and CaP tissues were different. While the higher molecular mass and intermediate size HA was found in all tissues, the HA fragments were found only in CaP tissues. In particular, the high-grade CaP tissues, which showed both elevated HA and HYAL1 levels, contained angiogenic HA fragments. The stromal-epithelial HA and HYAL1 expression may promote angiogenesis in CaP and may serve as prognostic markers for CaP.

URINARY HYALURONIC ACID AND HYALURONIDASE: MARKERS FOR BLADDER CANCER DETECTION AND EVALUATION OF GRADE

PURPOSE Specific patterns of progression and frequent recurrence of bladder tumors determine the choice of treatment, frequency of surveillance, quality of life, and ultimately, patient prognosis. The prognosis would be improved if an accurate noninvasive test was available for diagnosis. Identification of markers that function in bladder cancer progression would be helpful in designing such diagnostic tests. The glycosaminoglycan, hyaluronic acid (HA), promotes tumor metastasis. Hyaluronidase (HAase), an endoglycosidase, degrades HA into small fragments that promote angiogenesis. We have previously shown that both HA and HAase are associated with bladder cancer and may function in bladder tumor angiogenesis. In this study we examined whether urinary HA and HAase levels serve as bladder cancer markers. MATERIALS AND METHODS Among the 513 urine specimens analyzed, 261 were from transitional cell carcinoma (TCC) patients, 9 from patients with non-TCC tumors, and 243 from controls (normals, patients with other genitourinary (GU) conditions or a history of bladder cancer (HxBCa)). The urinary HA and HAase levels were measured by two ELISA-like assays that utilize a biotinylated HA binding protein for detection. These levels were normalized to total urinary protein and were expressed as ng./mg. (HA test) and mU/mg. (HAase test), respectively. RESULTS The urinary HA levels were elevated (2.5 to 6.5 fold) in bladder cancer patients (1173.7+/-173.4; n = 261) as compared with normals (246.1+/-38.5; n = 41); GU patients (306.6+/-32.2; n = 133), and patients with a HxBCa (351.1+/-49.1; n = 69) (p <0.001). The urinary HAase levels were elevated (3 to 7 fold) in G2/G3 bladder cancer patients (26.2+/-3.2) as compared with normals (4.5+/-0.9) and patients with either GU conditions (5.8+/-1.3), HxBCa (8.2+/-2.6) or G1 tumors (9.7+/-2.5) (p <0.001). The HA test showed 83.1% sensitivity, 90.1% specificity and 86.5% accuracy in detecting bladder cancer, regardless of the tumor grade. The HAase test showed 81.5% sensitivity, 83.8% specificity and 82.9% accuracy to detect G2/G3 patients. Combining the inferences of the HA and HAase tests (HA-HAase test) resulted in detection of bladder cancer, regardless of tumor grade and stage, with higher sensitivity (91.2%) and accuracy (88.3%), and comparable specificity (84.4%). CONCLUSION Our results show that the HA-HAase urine test is a noninvasive, highly sensitive and specific method for detecting bladder cancer and evaluating its grade.

Goserelin versus orchiectomy in the treatment of advanced prostate cancer: Final results of a randomized trial

OBJECTIVES To compare the efficacy and safety of goserelin and orchiectomy in patients with stage D2 prostate cancer. METHODS A randomized, open, multicenter study was conducted in 283 patients. Patients were allocated to goserelin, 3.6 mg every 28 days or to orchiectomy. Study end points were endocrine response, objective response, time to treatment failure, survival, and tolerability. Objective response was based on modified criteria of the National Prostate Cancer Project. RESULTS Serum testosterone decreased from baseline to castrate levels by week 4 in each group and remained below castrate levels thereafter. Acid phosphatase and alkaline phosphatase concentrations also decreased in each group. The goserelin and orchiectomy groups had similar results for objective response (82% versus 77%) and had similar medial times to treatment failure (52 versus 53 weeks) and survival (119 versus 136 weeks). No significant interactions between treatments and prognostic factors were observed. Adjusting for baseline testosterone concentration had no effect on survival outcome. Race had no influence on outcome or efficacy end points. Common adverse events in both groups were pain, hot flushes, and lower urinary tract symptoms. CONCLUSIONS Goserelin is well tolerated and as effective as orchiectomy in patients with Stage D2 prostate cancer.

Inhibition of cell proliferation, invasion, tumor growth and metastasis by an oral non-antimicrobial tetracycline analog (COL-3) in a metastatic prostate cancer model

Antibiotic forms of tetracycline exhibit antitumor activity in some tumor models. However, their low in vivo efficacy and associated morbidity limit their long‐term application in cancer therapy. This report appraises the efficacy of doxycycline (DC) and non‐antimicrobial, chemically modified tetracyclines (CMTs) against prostate cancer. Both DC and several CMTs inhibited prostate tumor cell proliferation in vitro. Some of the CMTs were significantly more potent than DC. One of the CMTs, 6‐deoxy, 6‐demethyl, 4‐de‐dimethylamino tetracycline (CMT‐3, COL‐3), was the most potent inhibitor (50% inhibition dose [GI50] ≤ 5.0 μg/ml). Exposure of tumor cells to CMT‐3 induced both apoptosis and necrosis. Mitochondrial depolarization and increased levels of reactive hydroxyl radicals were also observed in cells treated with CMT‐3. Cell cycle arrest at the G0/G1 compartment was observed in CMT‐3‐ and DC‐treated cells. DC and CMTs also inhibited the invasive potential of the tumor cells in vitro, from 10% (CMT‐6) to >90% (CMT‐3). CMT‐3 and DC decreased matrix metalloproteinase (MMP)‐2, tissue inhibitor of MMP (TIMP)‐1 and TIMP‐2 secretion in treated cultures and inhibited activity of secreted MMPs, CMT‐3 was a stronger inhibitor. Daily oral gavage of DC and CMT‐3 inhibited tumor growth and metastasis in the Dunning MAT LyLu rat prostate tumor. Decreases in tumor growth (27–35%) and lung metastases were observed (28.9 ± 15.4 sites/animal [CMT‐3‐treated] versus 43.6 ± 18.8 sites/animal [DC‐treated] versus 59.5 ± 13.9 [control]; p < 0.01]. A delay in tumor growth (27 ± 9.3%, p < 0.05), reduction in metastases (58 ± 8%) and decrease in tumor incidences (55 ± 9%, CMT‐3‐treated) were also observed, when rats were predosed for 7 days. No significant drug‐induced morbidity was observed in any of the animals. These results, along with a recently concluded clinical trial, suggest a potential use of CMT‐3 as an oral, nontoxic drug to treat metastatic prostate and other cancers.

Transplantation of human islets without immunosuppression

Significance Diabetes mellitus type 1 is an autoimmune disease that results in irreversible destruction of insulin-producing beta cells. Substantial advances have been made in beta cell replacement therapies over the last decades. However, lack of eligible donor organs and the need for chronic immunosuppression to prevent rejection critically limit a widespread application of these strategies. In this paper we present the clinical success of using a bioartificial pancreas for the transplantation of insulin-producing islets without affecting the immune system. In a patient with long-standing type-1 diabetes we could demonstrate persistent graft function and regulated insulin secretion without the need for immune-modulating medication. This strategy opens up avenues for more widespread and safe application of various cell-based therapies. Transplantation of pancreatic islets is emerging as a successful treatment for type-1 diabetes. Its current stringent restriction to patients with critical metabolic lability is justified by the long-term need for immunosuppression and a persistent shortage of donor organs. We developed an oxygenated chamber system composed of immune-isolating alginate and polymembrane covers that allows for survival and function of islets without immunosuppression. A patient with type-1 diabetes received a transplanted chamber and was followed for 10 mo. Persistent graft function in this chamber system was demonstrated, with regulated insulin secretion and preservation of islet morphology and function without any immunosuppressive therapy. This approach may allow for future widespread application of cell-based therapies.

Clinical benefits of bicalutamide compared with flutamide in combined androgen blockade for patients with advanced prostatic carcinoma: Final report of a double-blind, randomized, multicenter trial

OBJECTIVES To compare the efficacy and tolerability of bicalutamide and flutamide, each combined with luteinizing hormone-releasing hormone analogue (LHRH-A) therapy, in patients with metastatic (Stage D2) prostate cancer. METHODS This was a randomized, double-blind (for antiandrogen therapy), multicenter study with a two-by-two factorial design. Eight hundred thirteen patients were allocated 1:1 to bicalutamide (50 mg once daily) and flutamide (250 mg three times daily) and 2:1 to goserelin acetate (3.6 mg every 28 days) and leuprolide acetate (7.5 mg every 28 days). RESULTS The median times to progression and death were 97 and 180 weeks for the bicalutamide plus LHRH-A group compared with 77 and 148 weeks for the flutamide plus LHRH-A group. The hazard ratio for time to progression for bicalutamide plus LHRH-A to flutamide plus LHRH-A was 0.93 (95% confidence interval [CI] 0.79 to 1.10, P = 0.41) and that for survival time was 0.87 (95% CI 0.72 to 1.05, P = 0.15). The therapies were generally well tolerated. The most common adverse event in the two groups was hot flashes. The incidence of hematuria was significantly higher for the bicalutamide plus LHRH-A group than for the flutamide plus LHRH-A group (12% versus 6%, P = 0.007), but no patient withdrew from therapy because of hematuria. There was a significantly (26% versus 12%, P < 0.001) higher incidence of diarrhea and more withdrawals for diarrhea (25 patients versus 2) for the flutamide plus LHRH-A group relative to the bicalutamide plus LHRH-A group. CONCLUSIONS With a median follow-up time of 160 weeks, the combination of bicalutamide plus LHRH-A was well tolerated and had equivalent time to progression and survival compared with flutamide plus LHRH-A. Treatment with bicalutamide plus LHRH-A resulted in longer median survival than treatment with flutamide plus LHRH-A.

Improvement of islet function in a bioartificial pancreas by enhanced oxygen supply and growth hormone releasing hormone agonist

Islet transplantation is a feasible therapeutic alternative for metabolically labile patients with type 1 diabetes. The primary therapeutic target is stable glycemic control and prevention of complications associated with diabetes by reconstitution of endogenous insulin secretion. However, critical shortage of donor organs, gradual loss in graft function over time, and chronic need for immunosuppression limit the indication for islet transplantation to a small group of patients. Here we present a promising approach to address these limitations by utilization of a macrochamber specially engineered for islet transplantation. The s.c. implantable device allows for controlled and adequate oxygen supply and provides immunological protection of donor islets against the host immune system. The minimally invasive implantable chamber normalized blood glucose in streptozotocin-induced diabetic rodents for up to 3 mo. Sufficient graft function depended on oxygen supply. Pretreatment with the growth hormone-releasing hormone (GHRH) agonist, JI-36, significantly enhanced graft function by improving glucose tolerance and increasing β-cell insulin reserve in rats thereby allowing for a reduction of the islet mass required for metabolic control. As a result of hypervascularization of the tissue surrounding the device, no relevant delay in insulin response to glucose changes has been observed. Consequently, this system opens up a fundamental strategy for therapy of diabetes and may provide a promising avenue for future approaches to xenotransplantation.

Nitrosamine Formation in Bladder Infections and its Role in the Etiology of Bladder Cancer

Dimethylnitrosamine, a powerful carcinogen, is produced in the urine of patients with urinary tract infections of Proteus mirabilis and Escherichia coli when nitrite is present. Treatment with tetracycline antibiotics does not enhance the concentration of this carcinogen but, rather, sharply reduces it. The results emphasize the importance of proper antibiotic therapy for the infections to reduce the possibility of the subsequent development of bladder cancer.

Single-agent therapy with bicalutamide: A comparison with medical or surgical castration in the treatment of advanced prostate carcinoma

OBJECTIVES Single-agent therapy with bicalutamide, a nonsteroidal antiandrogen, was compared with castration, either surgical or medical, in patients with untreated Stage D2 prostate cancer. METHODS In an open, randomized, multicenter trial, patients were randomized to treatment with 50 mg bicalutamide (n = 243) once daily or to castration (n = 243), either orchiectomy or depot injection of goserelin acetate every 28 days. Primary efficacy endpoints were times to treatment failure and objective disease progression and survival. Assessments included review of measurable metastases, prostate dimensions, Eastern Cooperative Oncology Group performance status, pain, analgesic requirements, and quality of life responses. RESULTS The median duration of therapy was 39 weeks for bicalutamide-treated patients and 42 weeks for castrated patients; treatment failure occurred in 53% and 42% and disease progression in 43% and 33%, respectively. Treatment effects favored castration for both endpoints (P < or = 0.002), with hazard ratios (bicalutamide:castration) of 1.54 (95% confidence interval [CI], 1.18 to 2.00) for time to treatment failure and 1.6 (95% CI, 1.19 to 2.15) for time to disease progression. From the 1-year survival analysis, the hazard ratio for probability of death was 1.29 (95% CI, 0.96 to 1.72). Thus far, with a median follow-up of 86 weeks, median survival has not been reached in either group. Changes from baseline in several quality of life variables were significantly different (P < or = 0.01) between treatment groups periodically from months 1 to 6, and all favored bicalutamide. Overall, the antiandrogen was well tolerated compared with castration; with bicalutamide, hot flushes occurred less often and breast tenderness and gynecomastia more often. CONCLUSIONS Although a dosage of 50 mg of bicalutamide once daily was not as effective as castration, the favorable quality of life outcomes and the low incidence of nonhormonal adverse events provide reasons to evaluate bicalutamide, as a single therapeutic agent, at higher doses.

Blocking CD40-TRAF6 signaling is a therapeutic target in obesity-associated insulin resistance.

Significance Inflammation is a critical contributor to the pathogenesis of metabolic disorders associated with obesity. A group of molecules crucial in regulating the immune system are costimulatory molecules, including CD40. Our current study shows that CD40 acts as a double-edged sword in the metabolic syndrome through the initiation of differential signaling cascades. The CD40-TNF receptor-associated factor (TRAF) 2/3/5 signaling pathway protects against metabolic dysfunction and inflammation associated with obesity; conversely, the CD40-TRAF6 pathway contributes to the detrimental consequences of obesity. In the present study, we therefore designed, validated, and used a small-molecule inhibitor that blocks CD40-TRAF6 interactions. The improvement of insulin resistance by this specific CD40-TRAF6 inhibitor could represent a therapeutic breakthrough in the field of immunometabolism. The immune system plays an instrumental role in obesity and insulin resistance. Here, we unravel the role of the costimulatory molecule CD40 and its signaling intermediates, TNF receptor-associated factors (TRAFs), in diet-induced obesity (DIO). Although not exhibiting increased weight gain, male CD40−/− mice in DIO displayed worsened insulin resistance, compared with wild-type mice. This worsening was associated with excessive inflammation of adipose tissue (AT), characterized by increased accumulation of CD8+ T cells and M1 macrophages, and enhanced hepatosteatosis. Mice with deficient CD40-TRAF2/3/5 signaling in MHCII+ cells exhibited a similar phenotype in DIO as CD40−/− mice. In contrast, mice with deficient CD40-TRAF6 signaling in MHCII+ cells displayed no insulin resistance and showed a reduction in both AT inflammation and hepatosteatosis in DIO. To prove the therapeutic potential of inhibition of CD40-TRAF6 in obesity, DIO mice were treated with a small-molecule inhibitor that we designed to specifically block CD40-TRAF6 interactions; this compound improved insulin sensitivity, reduced AT inflammation, and decreased hepatosteatosis. Our study reveals that the CD40-TRAF2/3/5 signaling pathway in MHCII+ cells protects against AT inflammation and metabolic complications associated with obesity whereas CD40-TRAF6 interactions in MHCII+ cells aggravate these complications. Inhibition of CD40-TRAF6 signaling by our compound may provide a therapeutic option in obesity-associated insulin resistance.