Significance of divergent expression of prostaglandin EP4 and EP3 receptors in human prostate cancer.

PGE2 has been implicated in prostate cancer tumorigenesis. We hypothesized that abnormal prostaglandin receptor (EPR) expression may contribute to prostate cancer growth. Twenty-six archived radical prostatectomy specimens were evaluated by immunohistochemistry (IHC) and Western blotting for the expression of EP1, EP2, EP3, and EP4. As a corollary, EPR expression in one normal (PZ-HPV7) and four prostate cancer cell lines (CA-HPV10, LNCaP, PC3, and Du145) were assessed by Western blotting. Prostate cancer and normal cell growth were compared in vitro after EPR blockade, siRNA EPR knockdown, or overexpression. EP1, EP2, EP3, and EP4 receptors were detected by IHC in all areas of benign tissue within the clinical prostate cancer specimens. In areas of prostate cancer, EP4 and EP2 were overexpressed in 85% (22 of 26) and 75% (18 of 24) and EP3 expression was reduced in all (26 of 26, 100%) specimens (P < 0.05 vs. benign tissue). EP1 showed no specific differential expression pattern. Increased EP4 and reduced EP3 was confirmed by Western blotting in fresh clinical specimens and in prostate cancer cell lines (CA-HPV10, LNCaP, PC3, and Du145) compared with the normal prostate cell line (PZ-HPV7). EP2 and EP4 siRNA knockdown resulted in reduced in vitro growth and metastasis-related gene expression (MMP9 and Runx2) of prostate cancer lines, and in vitro migration was inhibited by EP4 antagonists. As a corollary, EP3-overexpressing PC3 cells displayed impaired growth in vitro. Human prostate cancer is associated with EP4 and EP2 overexpression and reduced EP3 expression. These data suggest that targeting specific EPR may represent a novel therapeutic approach for prostate cancer.

Beneficial effects of vitamin E in sperm functions in the rat after spinal cord injury.

UNLABELLED: Male infertility as a result of spinal cord injury (SCI) is associated with abnormal semen qualities including low sperm counts and poor sperm motility and morphology. Clinical studies suggest that reactive oxygen species (ROS)-related events might contribute to abnormal sperm functions after SCI. The current study examined whether impaired sperm functions after SCI can be ameliorated by an antioxidant, vitamin E. Vitamin E feeding of spinal cord transected (SCX) rats during the acute (maintenance) and chronic (restoration) phases of the injury partially preserved sperm viability and mitochondrial potential; similar effects were only seen in spinal cord contused (SCC) rats during the chronic phase. A beneficial effect of vitamin E on sperm motility, however, was only observed in SCX rats during the chronic phase of the injury. These results suggest that ROS-related events might account for some of the effects of cord injury on sperm functions, depending on the extent of injury and time postinjury. Furthermore, we found that sperm heads from SCC and SCX rats were less condensed compared to those from sham control rats. Such effects were attenuated by vitamin E, suggesting that ROS-related events may also contribute to abnormal sperm morphology after SCI. Partial restoration of male accessory gland weights in those rats fed vitamin E further suggests its beneficial effects on the functions of these glands. CONCLUSION: Vitamin E feeding attenuated some of the effects of spinal cord injury on sperm functions and male accessory glands in the rat. These results support a role of ROS-related events in deterioration of semen quality after cord injury. Further understanding of the underlying mechanisms for effects of vitamin E on sperm functions and male accessory glands will provide scientific rationale for the use of vitamin E or other antioxidant as therapeutic means to preserve sperm functions and semen quality in SCI men.

Impaired sperm function after spinal cord injury in the rat is associated with altered cyclic adenosine monophosphate signaling.

Our previous observations of changes in the expression of cAMP-dependent genes and the cAMP-responsive element modulator (CREM) in rat testicular cells after spinal cord injury (SCI) implied abnormal cAMP signaling as one of the mechanisms underlying the effects of SCI on spermatogenesis. It was postulated that such effects might contribute to abnormal sperm function after SCI. In this study, we examined this possibility. In spinal cord-contused (SCC) and -transected (SCX) rats, impaired sperm motility was accompanied by an increase in sperm cAMP content. Treatment of SCX rats with exogenous testosterone or follicle-stimulating hormone resulted in a further decrease in sperm motility, whereas sperm cAMP either increased or remained unchanged. These effects differed from those in sham control rats that received identical treatments. Results of these experiments also demonstrated that impaired sperm motility in SCC and SCX rats was accompanied by decreases in sperm viability and mitochondrial potential, thus suggesting a possible link between these changes. We concluded that impaired sperm motility after SCI was associated with decreases in sperm viability and mitochondrial potential. These effects occurred in the face of elevated sperm cAMP content and changes in its regulation, suggesting that altered cAMP signaling events might contribute to impairment of sperm motility and perhaps other sperm functions after SCI.

Effects of exogenous testosterone on testicular function during the chronic phase of spinal cord injury: dose effects on spermatogenesis and Sertoli cell and sperm function.

INTRODUCTION: Exogenous testosterone has been shown to attenuate spinal cord injury (SCI)-related regression of spermatogenesis in the rat. The current experiment investigated the effects of exogenous testosterone in testicular and sperm functions in the rat during the chronic phase of SCI. METHODS: Chronic SCI rats were given subcutaneous implants of testosterone-filled silastic capsules (TC). Northern blot cDNA hybridization was used to measure testicular levels of Sertoli cell- and germ cell-specific transcripts. Western blot and immunohistochemistry were used to determine protein level and cellular localization, respectively, of cyclic adenosine monophosphate-responsive element modulator (CREM) in the testes. Flow cytometry was used to determine sperm viability and mitochondrial potential. RESULTS: Spontaneous restoration of spermatogenesis occurred in 7 of the 8 untreated SCI rats. Although exogenous testosterone restored complete spermatogenesis in all SCI rats, regressed seminiferous epithelium remained in 30% to 70% of tubular cross sections in these rats. These effects were associated with altered responses of germ cell-specific mRNA transcripts to exogenous testosterone, and abnormal cellular distribution of CREM. Sperm of untreated SCI rats exhibited lowered motility, viability, and mitochondrial potential. Implantation of 10 cm of TC worsened sperm motility in sham control and SCI rats, but restored sperm viability and mitochondrial potential in SCI rats. CONCLUSION: Administration of exogenous testosterone to SCI rats during the chronic phase of injury failed to facilitate spermatogenic restoration over that achieved in untreated SCI rats. Abnormalities in postmeiotic spermatogenic differentiation could contribute to these effects, and perhaps the production of sperm with abnormal morphology and/or functions during the chronic phase of SCI.

Signal transducer and activator of transcription 3 (STAT3) activation in prostate cancer: Direct STAT3 inhibition induces apoptosis in prostate cancer lines.

Signal transducers and activators of transcription (STAT) were originally discovered as components of cytokine signal transduction pathways. Persistent activation of one STAT, STAT3, is a common feature of prostate cancer. Activated STAT3 was found in pathology specimens obtained from prostatectomy in the cancerous areas but not in the normal margins. Because the activation of STAT3 is mediated by the action of an upstream Janus kinase (JAK) kinase, usually JAK1 or JAK2, the activation step for STAT3 might itself be a target for therapy in prostate cancer. However, the redundancy of upstream kinases may make this strategy unreliable for therapy. To develop molecular targets for prostate cancer treatment, JAK kinase and STAT3 inhibition of two prostate cancer lines were compared. DU145 and NRP-154 cells were treated with JAK kinase inhibitors, analyzed for onset of apoptosis, and measured by annexin V binding and propidium iodide uptake. Activation of caspases in the cells was determined by measuring cleaved caspase-3 following treatment. For determining the effect on mitochondrial membrane depolarization that accompanies apoptosis, the fluorescent dye JC-1 was used. STAT3 was specifically inhibited by transfecting either a dominant-negative (DN) STAT3 plasmid or antisense STAT3 oligonucleotides into the cells. To look for reduction in STAT3 levels within cells, fixed and permeabilized prostate cancer cells were stained with antibody to STAT3. We found that more than one JAK kinase is involved in STAT3 activation in prostate cancer lines. AG490 (JAK2 specific) induced apoptosis in DU145 but not in NRP-154 prostate cancer lines, whereas piceatannol (JAK1 specific) induced apoptosis in NRP-154 but not in DU145 cells. Next, we demonstrated efficacy of specific STAT3 inhibitors in prostate cancer lines. Both induction of apoptosis and reduction in intracellular STAT3 protein were observed following treatment with antisense STAT3 oligonucleotides, while transfection of a DN-STAT3 plasmid into both prostate cancer cell lines resulted in loss of viability and onset of apoptosis. We conclude that STAT3-specific inhibitors, rather than JAK kinase-specific inhibitors, should be more useful therapeutically in treating androgen-resistant prostate cancer and that STAT3 is an appropriate target in the treatment of prostate cancer.

Preservation of spermatogenesis in spinal cord injured rats with exogenous testosterone. Relationship with serum testosterone levels and cellular localization of cAMP responsive element modulator.

UNLABELLED: The current experiment examined the effects of exogenous testosterone (T) on spermatogenesis in rats with spinal cord injury (SCI) and their relationship with the cellular distribution of a cyclic AMP-responsive element modulator (CREM) in testicular cells. Implantation of T-filled Silastic capsules (TCs, 1-20 cm) resulted in dose-dependent, biphasic changes in testicular T levels and spermatogenesis in SCI rats. However, dose responsiveness of spermatogenesis to exogenous T in SCI rats differed from that in sham control rats. Specifically, implantation of 2-cm TCs enhanced the effects of SCI on spermatogenesis, resulting in total regression of the seminiferous epithelium. Although 3-cm TCs maintained complete spermatogenesis in sham control rats, this regimen failed to support complete spermatogenesis in SCI rats. Although complete spermatogenesis was maintained in SCI rats given 5-20-cm TC implants, various abnormalities persisted. Cellular distribution of CREM remained normal in SCI rats but was altered in those SCI rats that received 3- or 5-cm TC implants. Such effects were associated with reduced CREM proteins in testicular tissues. These results were consistent with altered cAMP signaling and its regulation in testicular cells after SCI and provided possible mechanistic explanations for the effects of SCI on spermatogenesis. CONCLUSION: SCI resulted in changes in the responsiveness of spermatogenesis to exogenous T. These effects were associated with altered cAMP/CREM signaling in testicular cells. Further studies, including a study of the relationship between serum T levels and normalcy of sperm functions and the role of neural-endocrine interactions in mediating the effects of SCI on spermatogenesis and sperm function, are needed so that therapeutic regimens can be designed for clinical use.

Alteration of cyclic adenosine 3',5'-monophosphate signaling in rat testicular cells after spinal cord injury.

INTRODUCTION: Earlier studies demonstrated that the effects of spinal cord injury (SCI) on spermatogenesis were associated with altered Sertoli cell responses to treatment with follicle-stimulating hormone (FSH) and/or testosterone (T). Because of the importance of the cyclic adenosine 3',5'-monophosphate (cAMP) signal pathway in hormonal actions on Sertoli cells and spermatogenesis, the purpose of this study was to determine whether cAMP signaling in testicular cells is altered after SCI. METHODS: Rats with SCI were treated with FSH, T, or FSH + T for 7 or 14 days. Northern blot cDNA hybridization was used to measure testicular levels of Sertoli and germ cell-specific transcripts encoded by genes that contain cAMP responsive element (CRE) and/or steroid hormone responsive element (HRE). Cellular distribution of CRE modulator (CREM) was determined by immunohistochemistry. RESULTS: Treatment of sham control rats with FSH or T + FSH for 2 weeks resulted in decreases in mRNAs for CREM and CRE binding protein (CREB). Concomitantly, levels of mRNA for Sertoli cell inhibin alpha and germ cell-specific protamine 1 (Pm-1), transition protein 2 (TP-2), and lactate dehydrogenase C (LDHC) were all reduced. In contrast, identical FSH and/or T treatments resulted in increases in levels of CREM and CREB mRNAs in the testes of SCI rats; these effects were associated with similar changes in mRNAs for inhibin alpha, Pm-1, TP-2, and LDHC. The effects of SCI on CREM expression were corroborated by similar changes in its distribution in testicular cells. CONCLUSION: SCI is associated with changes in FSH and/or T regulation of cAMP/CRE and HRE signaling in testicular cells. These effects may mediate the effects of SCI on spermatogenesis.

Spinal cord contusion impairs sperm motility in the rat without disrupting spermatogenesis.

Our previous studies demonstrated various abnormalities in spermatogenesis after spinal cord injury (SCI) in cord-transected rats. In this study, we examined whether abnormalities in spermatogenesis in SCI rats were related to the degree of SCI. We used spinal cord-contused (SCC) rats as a model. Adult male Sprague-Dawley rats were subjected to various degrees of cord contusion caused by the weight of a rod dropped from different heights (12.5, 25, 50, and 75 mm) using a New York University IMPACTOR. Testicular histology revealed persistent complete spermatogenesis in all SCC rats 4, 8, or 14 weeks after cord contusion regardless of the extent of SCI. Northern blot complementary DNA (cDNA) hybridization revealed transient but significant decreases in the levels of Sertoli cell-specific transcripts in SCC rats. In addition, levels of messenger RNA (mRNA) transcripts for germ cell-specific transition protein-2 and protamine-1 were consistently decreased in these rats. Such effects were related to the height of the weight drop and were associated with reduced levels of mRNA for cyclic adenosine monophosphate (cAMP) responsive element modulator (CREM). These results demonstrated specific effects of SCI on spermiogenesis and were consistent with altered cAMP signaling in testicular cells after SCI. Sperm motility was also significantly decreased in SCC rats and was related to the height of weight drop. Normal sperm motility recovered only in those rats injured by weight drop from 12.5- and 25-mm heights. In summary, current results demonstrate persistent abnormalities in spermiogenesis and sperm motility in rats that suffered spinal cord contusion by weight drop. Such effects were related to the height of the weight drop and thus to the extent of SCI.

Immunohistochemical localization of the retinoic Acid receptors in human prostate.

Retinoic acid receptors (RARs) are nuclear transcription factors that mediate the effects of retinoids. Aberrant expression and regulation of RARs have been linked to various malignancies, including steroid-related breast and cervical cancers. Our previous results also suggest that prostate cancer is associated with altered RAR signaling. To understand the relationship between RAR signaling and prostate cancer, the current study examined the cellular distribution of RAR-alpha, -beta, and -gamma in human prostate tissues exhibiting different pathologic conditions. In histologically normal epithelium, both RAR-alpha and -gamma were present throughout the epithelium with minimal nuclear accumulation. RAR-beta was present only in basal epithelial nuclei. On the contrary, RAR-alpha was significantly increased in the nuclei of luminal epithelial cells, and both RAR-beta and -gamma were increased in basal and luminal epithelial nuclei in glands exhibiting benign prostatic hyperplasia (BPH). RAR-alpha was also increased in luminal epithelial nuclei in glands exhibiting prostatic intra-epithelial neoplasia (PIN). In these glands, RAR-beta was persisting in basal epithelial nuclei that were also RAR-gamma positive. In low- and intermediate-grade cancerous glands, RAR-alpha was also significantly increased in luminal epithelial nuclei, and a strong RAR-gamma signal was seen in some cells. RAR-beta was absent in these glands. Both RAR-alpha and -gamma were also increased in high-grade cancer cells. In conclusion, current results demonstrated changes in cellular distribution of RAR-alpha and -gamma in human prostate tissues exhibiting different pathologies. These results suggest links between altered RAR signaling and deregulated cell growth and/or tumorigenic transformation of prostate epithelial cells.