Tasquinimod prevents the angiogenic rebound induced by fractionated radiation resulting in an enhanced therapeutic response of prostate cancer xenografts.

BACKGROUND: Tasquinimod is a novel inhibitor of tumor angiogenesis which enhances therapeutic efficacy when combined with androgen ablation and/or taxane-based chemotherapies in pre-clinical prostate cancer models. It has entered registration Phase III evaluation for the treatment of castration resistant prostate cancer. Since tasquinimod suppresses the angiogenic switch induced by tumor hypoxia as prostate cancers outgrow their blood supply, this raises the issue of whether tasquinimod also suppresses the angiogenic rebound induced by fractionated radiation thereby enhancing therapeutic response to fractionated radiation. METHODS: Human endothelial and prostate cancer cells in culture and human prostate cancer xenografts growing in castrated male nude mice were evaluated for their response to radiation alone and in combination with tasquinimod. RESULTS: At clinically relevant drug levels, tasquinimod significantly (P < 0.05) enhances anti-cancer efficacy of fractionated radiation with optimal timing for initiating daily tasquinimod treatment being after completion of the fractionated radiation. CONCLUSIONS: Based upon cell culture studies and tumor tissue oxygenation (i.e., pO(2)), tumor vascular volume, and tumor blood vessel density measurements, the mechanism for such enhancement and optimal timing involves tasquinimod's ability to prevent the angiogenic rebound induced by fractionated radiation.

Pharmacologic basis for the enhanced efficacy of dutasteride against prostatic cancers.

PURPOSE: Prostatic dihydrotestosterone (DHT) concentration is regulated by precursors from systemic circulation and prostatic enzymes of androgen metabolism, particularly 5alpha-reductases (i.e., SRD5A1 and SRD5A2). Therefore, the levels of expression SRD5A1 and SRD5A2 and the antiprostatic cancer growth response to finasteride, a selective SRD5A2 inhibitor, versus the dual SRD5A1 and SRD5A2 inhibitor, dutasteride, were compared. EXPERIMENTAL DESIGN: Real-time PCR and enzymatic assays were used to determine the levels of SRD5A1 and SRD5A2 in normal versus malignant rat and human prostatic tissues. Rats bearing the Dunning R-3327H rat prostate cancer and nude mice bearing LNCaP or PC-3 human prostate cancer xenografts were used as model systems. Tissue levels of testosterone and DHT were determined using liquid chromatography-mass spectrometry. RESULTS: Prostate cancer cells express undetectable to low levels of SRD5A2 but elevated levels of SRD5A1 activity compared with nonmalignant prostatic tissue. Daily oral treatment of rats with the SRD5A2 selective inhibitor, finasteride, reduces prostate weight and DHT content but did not inhibit R-3327H rat prostate cancer growth or DHT content in intact (i.e., noncastrated) male rats. In contrast, daily oral treatment with even a low 1 mg/kg/d dose of the dual SRD5A1 and SRD5A2 inhibitor, dutasteride, reduces both normal prostate and H tumor DHT content and weight in intact rats while elevating tissue testosterone. Daily oral treatment with finasteride significantly (P < 0.05) inhibits growth of LNCaP human prostate cancer xenografts in intact male nude mice, but this inhibition is not as great as that by equimolar oral dosing with dutasteride. This anticancer efficacy is not equivalent, however, to that produced by castration. Only combination of dutasteride and castration produces a greater tumor inhibition (P < 0.05) than castration monotherapy against androgen-responsive LNCaP cancers. In contrast, no response was induced by dutasteride in nude mice bearing androgen-independent PC-3 human prostatic cancer xenografts. CONCLUSIONS: These results document that testosterone is not as potent as DHT but does stimulate prostate cancer growth, thus combining castration with dutasteride enhances therapeutic efficacy.

In vivo analysis of chronic phosphodiesterase-5 inhibition with sildenafil in penile erectile tissues: no tachyphylaxis effect.

PURPOSE: Conflicting information exists regarding the long-term efficacy of phosphodiesterase-5 (PDE5) inhibitor therapy for erectile dysfunction, particularly in regard to whether therapeutic resistance occurs. We investigated the erectile response, and cavernous PDE5 expression and activity after continuous long-term administration of sildenafil at verified therapeutic plasma concentrations, applying an in vivo rat model of age related erectile dysfunction. MATERIALS AND METHODS: Male Fisher 344 young (4 months old) and aged (19 months old) rats (National Institute of Aging, Bethesda, Maryland) were injected with sildenafil mesylate (20 mg/kg) or saline subcutaneously every 8 hours for 3 weeks. After a 10 to 18-hour washout period electrical stimulation of the cavernous nerve was performed to assess penile erection. Penes were excised to measure PDE5 protein expression and activity, and blood was collected for sildenafil measurement. Responses were compared with those determined 30 minutes after a single sildenafil injection. RESULTS: Chronic sildenafil treatment increased the detumescence phase in young and aged rats (p <0.05), although aged rats showed a greater increase than young rats. Baseline cavernous PDE5 expression and activity were greater in aged vs young rats (p <0.05). After chronic sildenafil treatment cavernous PDE5 expression was increased in young (p <0.05) but not in aged rats. Chronic and acute sildenafil treatment similarly inhibited PDE5 activity in the penis of young and aged rats (p <0.05), coincident with its free plasma concentrations equivalent to clinically therapeutic ranges. CONCLUSIONS: Pharmacological PDE5 inhibitor therapy with sildenafil chronically does not result in treatment resistance. Rather, therapeutic efficacy is maintained and apparently more pronounced with erectile impairment than with normal erectile ability.

Inactivation of phosphorylated endothelial nitric oxide synthase (Ser-1177) by O-GlcNAc in diabetes-associated erectile dysfunction.

Impaired endothelial nitric oxide synthase (eNOS) function is associated with erectile dysfunction in diabetes mellitus, but the exact molecular basis for the eNOS defect in the diabetic penis remains unclear. We investigated whether hyperglycemia increases O-GlcNAc modification of eNOS in the penis, preventing phosphorylation at the primary positive regulatory site on the enzyme and hampering mechanisms of the erectile response. Type I diabetes mellitus was induced in male rats by alloxan (140 mg/kg, i.p.). After 5 wk, the diabetic rat penis exhibited increased O-GlcNAc modification of eNOS and decreased eNOS phosphorylation at Ser-1177 at baseline compared with the control rat penis; eNOS phosphorylation at Thr-495, Ser-615, and Ser-633 was not affected. In addition, eNOS phosphorylation at Ser-1177 was impaired in the diabetic rat penis in response to penile blood flow (shear stress) elicited by electrical stimulation of the cavernous nerve (ES) and to recombinant human VEGF165. Phosphorylation of Akt, a mediator of shear stress-induced eNOS phosphorylation at Ser-1177, was decreased in the diabetic penis at baseline, but it was restored by ES. Erectile response to shear stress elicited by ES and to VEGF was decreased in diabetic compared with control rats. This work demonstrates that eNOS inactivation occurs in the diabetic penis by a glycosylation mechanism specifically at Ser-1177, by which the enzyme is rendered incapable of activation by fluid shear stress stimuli and VEGF signaling. In vivo penile erection paradigm supports the physiologic relevance of O-GlcNAc modification in vascular disorders associated with diabetes.

Erection capability is potentiated by long-term sildenafil treatment: role of blood flow-induced endothelial nitric-oxide synthase phosphorylation.

Despite demonstrated clinical efficacy of sildenafil for the temporary treatment of erectile dysfunction, the possibility that sildenafil used long-term durably augments erectile ability remains unclear. We investigated whether continuous long-term administration of sildenafil at clinically relevant levels to aged rats "primes" the penis for improved erectile ability and involves nitric oxide (NO) or RhoA/Rho-kinase signaling pathways. In aged, but not young rats, sildenafil prolonged erection and increased the protein expressions of phosphorylated endothelial NO synthase (eNOS) at serine-1177 and phosphorylated Akt at serine-473 in penes. Only in the young rat penis, protein expressions of phosphodiesterase-5 and phosphomyosin phosphatase target subunit 1, a marker of Rho-kinase activity, were increased by sildenafil. Sildenafil inhibited phosphodiesterase-5 activity in penes of young and aged rats coincident with assayed free plasma levels of the drug equivalent to clinically therapeutic measurements. We conclude that erectile ability can be enhanced under preconditions of erectile impairment by long-term inhibition of phosphodiesterase-5 and that the effect is mediated by Akt-dependent eNOS phosphorylation. The lack of erectile ability enhancement in young rats by long-term phosphodiesterase-5 inhibition may relate to restrained NO signaling by phosphodiesterase-5 up-regulation, lack of incremental Akt and eNOS phosphorylation, and heightened Rho-kinase signaling in the penis.

Age-related changes in phosphorylation of endothelial nitric oxide synthase in the rat penis.

AIM: Aging is associated with erectile dysfunction (ED) attributed to reduced nitric oxide synthase (NOS) activity and nitric oxide bioavailability. However, the mechanism for this effect has not been fully investigated. We evaluated (i) whether age-related ED involves dysregulation of endothelial NOS (eNOS) phosphorylation; and (ii) whether vascular endothelial growth factor (VEGF) exerts erectile effects and operates via eNOS phosphorylation in aged rats. METHODS: Male Fischer 344 "young" (4-month-old) and "aged" (19-month-old) rats were used. Electrical stimulation of the cavernous nerve (CNS) was performed to generate penile erection. Erectile response in the presence of rhVEGF165 was evaluated by intracavernosal pressure monitoring 25 minutes after intracavernosal injection of VEGF. Penes were excised at baseline, with or without rhVEGF treatment, and after CNS for Western immunoblot of phospho-eNOS (Ser-1177 and Thr-495), phospho-Akt, and eNOS. RESULTS: Erectile response was significantly reduced in aged rats compared with young rats. Phospho-eNOS (Ser-1177) and phospho-Akt were significantly reduced, while phospho-eNOS (Thr-495) was significantly increased, in the aged penis at baseline and after CNS. rhVEGF significantly improved erection and reversed downregulated Ser-1177, but not upregulated Thr-495 phosphorylation, on eNOS in aged penes. eNOS protein was significantly increased in aged penes. CONCLUSIONS: Age-related ED is associated with eNOS inactivation through a decrease in phosphorylation of its positive regulatory site (Ser-1177) and an increase in phosphorylation of its negative regulatory site (Thr-495) in the penis. Altered phosphorylation/constitutive activation of eNOS by fluid shear stress may be a major determinant of compromised vascular homeostasis of the aged penis. The finding that VEGF rapidly induces erection and partly corrects alterations in eNOS phosphorylation in the aged rat penis suggests impaired eNOS activation by deficient endogenous VEGF and supports the potential for growth factor therapy in the treatment of age-related ED.

Immunophilin ligands promote penile neurogenesis and erection recovery after cavernous nerve injury.

PURPOSE: We investigated the long-term effect of immunophilin ligands on erection physiology and cavernous tissue histology using rat models of unilateral and bilateral cavernous nerve (CN) injury. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were administered the immunophilin ligand FK506 (5 mg/kg subcutaneously daily for 5 days), the nonimmunosuppressant FK506 derivative GPI1046 (3 to 3-pyridyl)-1-propyl(2 seconds)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidine carboxylate) (40 mg/kg subcutaneously daily for 5 or 28 days) or saline immediately upon induction of 2 paradigms of cavernous nerve injury, namely focal transection of the CN unilaterally, and a combination of focal transection of the CN unilaterally and excision of a 5 mm segment of contralateral CN (BIL treated). At 28 days following CN injury electrical stimulation of the transected CN and penile erection monitoring were performed. Whole penes were removed and evaluated immunohistochemically for the nitric oxide generator neuronal nitric oxide synthase, the neuronal marker synaptophysin, the endothelial marker CD31 and the smooth muscle marker alpha-actin. RESULTS: In unilaterally treated groups erection recovery was significantly greater in FK506 treated than in saline treated animals ((83.5% +/- 9.2% vs 24.3% +/- 8.1%, p <0.001) and similarly greater in GPI1046 treated animals than in the respective saline treated controls for this group (57.9% +/- 12.6% vs 23.8% +/- 7.0%, p <0.05). In BIL treated groups erection recovery for FK506 and GPI1046 treated rats exceeded saline treated values by approximately 70% (p <0.05) and 40% (p = 0.14), respectively. After 28 days of continuous treatment in BIL treated groups erection recovery for GPI1046 treated rats exceeded saline treated values by 140% (p <0.05). Neuronal and endothelial staining was preserved after immunophilin ligand treatment. CONCLUSIONS: Immunophilin ligands exert neurotrophic effects on the penile innervation that preserve cavernous tissue structure and promote erectile function recovery in rats after extensive CN injury.

Akt-dependent phosphorylation of endothelial nitric-oxide synthase mediates penile erection.

In the penis, nitric oxide (NO) can be formed by both neuronal NO synthase and endothelial NOS (eNOS). eNOS is activated by viscous drag/shear stress in blood vessels to produce NO continuously, a process mediated by the phosphatidylinositol 3-kinase (PI3kinase)/Akt pathway. Here we show that PI3-kinase/Akt physiologically mediates erection. Both electrical stimulation of the cavernous nerve and direct intracavernosal injection of the vasorelaxant drug papaverine cause rapid increases in phosphorylated (activated) Akt and eNOS. Phosphorylation is diminished by wortmannin and LY294002, inhibitors of PI3-kinase, the upstream activator of Akt. The two drugs also reduce erection. Penile erection elicited by papaverine is reduced profoundly in mice with targeted deletion of eNOS. Our findings support a model in which rapid, brief activation of neuronal NOS initiates the erectile process, whereas PI3-kinase/Akt-dependent phosphorylation and activation of eNOS leads to sustained NO production and maximal erection.