Signal transducers and activators of transcription (STATs) were originally discovered as mediators of signal transduction. Persistent aberrant activation of STAT3 is part of the malignant phenotype of hormone-refractory prostate cancer and pancreatic cancer; this is thought to be mediated by homodimers of phosphorylated STAT3, which translocate to the nucleus. One consequence of persistently-activated STAT3 in malignant cells is that they depend upon it for survival. STAT3 is observed to heterodimerize with STAT1 and STAT2; however the contributions of STAT3:STAT1 and STAT3:STAT2 heterodimers to the survival of malignant cells have not been investigated in detail. Previously we reported that single-stranded oligonucleotides containing consensus STAT3 binding sequences (13410 and 13411) were more effective for inducing apoptosis in prostate cancer cells than antisense STAT3 oligonucleotides. Control oligonucleotides (scrambled sequences) had no effect. STAT3-inhibiting oligonucleotide 13410, but not scrambled-sequence oligonucleotides, induced apoptosis in pancreatic cancer cells as well. Here we report that 13410 and derivative olignucleotides induced apoptosis in STAT1-null and STAT2-null fibrosarcoma cell lines U3A and U6A, as well as in the parental fibrosarcoma cell line 2fTGH. The cell lines expressed constitutively-activated STAT3 and depended on its activity for survival. Forty-eight hr after transfection of 13410 or related oligonucleotides, significant apoptosis was observed in 2fTGH, U3A and U6A cells. Scrambled-sequence oligonucleotides had no effect on survival. These data indicate that neither STAT1 nor STAT2 play significant roles in the maintenance of these cells, and by extension that STAT3:STAT1 and STAT3:STAT2 heterodimers regulate a different set of genes from STAT3:STAT3 homodimers.

In 2012, prostate cancer will once again be the second-leading cause of cancer death of American males. Although initially treatable, prostate cancer can recur in a hormone refractory form that is not responsive to current available therapies. The mortality rate associated with hormone refractory prostate cancer is high, and there is an urgent need for new therapeutic agents to treat prostate cancer. A common feature of prostate cancer is the dependence on activated signal transducer and activator of transcription 3 (STAT3), a transcription factor, for survival. More important, inhibition of STAT3 has been shown to induce apoptosis in prostate cancer cells. In recent years, inhibitors of STAT3 have emerged as promising molecular candidates for targeted prostate cancer therapy. The aim of this review is to examine the role of STAT3 in prostate cancer and how inhibitors of STAT3 could advance the quest for treatment of the disease. Janus kinase 2 (JAK2)-targeted therapy appears very promising in the treatment of prostate cancer. It has been shown to decrease symptoms associated with myeloproliferative disorders and increase overall survival of patients compared with the best available therapy. In addition to improved outcome, many JAK2 inhibitors have been found to be tolerable with no adverse impact on quality of life. As such, JAK2 inhibitors may play an important role in the management of patients with prostate cancer. Current studies are evaluating the role of JAK2 inhibitors in solid tumors. Pending clinical trial results will determine the future direction of JAK2 inhibitors in the treatment of patients with prostate cancer.

Background. Eicosanoids are generally recognized to exert potent immunomodulatory properties, including effects on T cell, antigen-presenting cell (APC), and dendritic cell (DC) maturation and function. Since DC maturation and function may also be regulated by store-operated calcium entry (SOCE), we hypothesized that the effects of eicosanoids on DC function may in part be regulated through changes in intracellular calcium.Methods. DC derived from the bone marrow of male Balb/ ByJ mice cultured for 7 d in the presence of granulocyte macrophage colony stimulating factor (GMCSF) and interleukin-4 (IL-4) were used to study the effects of eicosanoids on SOCE and the resulting Ca(2+) mobilization.Results. The 5-lipoxygenase (5-LO) products leukotriene B4 (LTB(4)) and LTD(4), but not LTC(4), depleted Ca(2+) from DC endoplasmic reticulum stores. The specificity of LTB(4) and LTD(4) on Ca(2+) store-depletion was confirmed by the ability of the specific receptor antagonists, LY25583 and MK571, respectively, to abrogate Ca(2+) store depletion. RT-PCR demonstrated DC receptors for LTB(4) (BLT(1) and BLT(2)) and the cysteinyl-LTs (CysLT(1), CysLT(2), and GPR17). We also detected transient receptor potential canonical (TRPC) 1, 2, 4, and 6 and stromal interaction molecule 1 (STIM1) on CD11c D DCs, suggesting these proteins also participate in DC SOCE. In contrast, the cyclooxygenase (CO) metabolite PGE(2) had no effect on DC Ca(2+) mobilization.Conclusions. To our knowledge, these are the first observations of distinct effects of eicosanoids on DC Ca2+ mobilization, which may have important implications for the regulation of DC maturation at sites of immune and non-immune inflammation. Published by Elsevier Inc.

Background For improved uptake of oligonucleotide-based therapy, the oligonucleotides often are coupled to peptides that facilitate entry into cells. To this end, novel cell-penetrating peptides (CPPs) were designed for mediating intracellular uptake of oligonucleotide-based therapeutics. The novel peptides were based on taking advantage of the nuclear localization properties of transcription factors in combination with a peptide that would bind putatively to cell surfaces. It was observed that adding a glutamate peptide to the N-terminus of the nuclear localization signal (NLS) of the Oct6 transcription factor resulted in a novel CPP with better uptake and better nuclear colocalization than any other peptide tested. Results Uptake of the novel peptide Glu-Oct6 by cancer cell lines was rapid (in less than 1 hr, more than 60% of DU-145 cells were positive for FITC), complete (by 4 hr, 99% of cells were positive for FITC), concentration-dependent, temperature-dependent, and inhibited by sodium azide (NaN 3 ). Substitution of Phe, Tyr, or Asn moieties for the glutamate portion of the novel peptide resulted in abrogation of novel CPP uptake; however none of the substituted peptides inhibited uptake of the novel CPP when coincubated with cells. Live-cell imaging and analysis by imaging flow cytometry revealed that the novel CPP accumulated in nuclei. Finally, the novel CPP was coupled to a carboxyfluorescein-labeled synthetic oligonucleotide, to see if the peptide could ferry a therapeutic payload into cells. Conclusions These studies document the creation of a novel CPP consisting of a glutamate peptide coupled to the N-terminus of the Oct6 NLS; the novel CPP exhibited nuclear colocalization as well as uptake by prostate and pancreatic cancer cell lines.

Signal transducers and activators of transcription (STAT) were originally discovered as components of signal transduction pathways. Persistent aberrant activation of STAT3 is a feature of many malignancies including prostate cancer and pancreatic cancer. One consequence of persistently activated STAT3 in malignant cells is that they depend on it for survival; thus, STAT3 is an excellent molecular target for therapy. Previously, we reported that single-stranded oligonucleotides containing consensus STAT3 binding sequences (13410 and 13411) were more effective for inducing apoptosis in prostate cancer cells than antisense STAT3 oligonucleotides. Control oligonucleotides (scrambled sequences) had no effect. Here, we report that authentic STAT3 binding sequences, identified from published literature, were more effective for inducing apoptosis in prostate cancer cells and pancreatic cancer cells than was oligonucleotide 13410. Moreover, the authentic STAT3 binding sequences showed differing efficacies in the malignant cell lines depending on whether the canonical STAT3 binding sequence was truncated at the 5′ or the 3′ end. Finally, expression of one STAT3-regulated gene was decreased following treatment, suggesting that STAT3 may regulate the same set of genes in the two types of cancer. We conclude that truncating the 5′ end left intact enough of the canonical STAT3 binding site for effective hybridization to the genome, whereas truncation of the 3′ end, which is outside the canonical binding site, may have affected binding of required cofactors essential for STAT3 activity, thereby reducing the capacity of this modified oligonucleotide to induce apoptosis. Additional experiments to answer this hypothesis are under way. [Mol Cancer Ther 2008;7(6):1543–50]

Signal transducer and activator of transcription (STAT) proteins are latent cytoplasmic proteins that transmit cell-surface signals generated by ligand-receptor interactions to the nucleus. They are activated mainly by phosphorylation. Of the STAT proteins, STAT3 and to a lesser extent STAT5, is associated with transformed cells, particularly in tumors arising from oncogenes that activate tyrosine kinase signaling pathways. In benign cells, STAT3 like other STAT proteins, is transiently activated then deactivated by multiple regulatory proteins. However, in many malignant cells, the activation of STAT3 is constitutive; this persistent and aberrant signaling by STAT3 results in the continued expression of anti-apoptotic genes of the bcl family and of survivin, cell-cycling genes, angiogenic factors such as VEGF, and metastasis-promoting factors such as matrix metalloproteases. The activity of some oncogenic viral proteins, notably v-src, is due to persistent activation of STAT3. When malignant cells express constitutive STAT3 activation, their very survival depends upon STAT3's continued activation or expression or DNA-binding activity inhibiting STAT3 at any of these steps results in cancer cell apoptosis, whereas benign cells survive the same treatment. This review will discuss the critical role of persistent STAT3 in the maintenance of the malignant state, and the efforts published to date for inhibiting STAT3 as a future therapeutic approach for cancer treatment; limitations of each inhibitory strategy will be included in so far as information is available.

Dendritic cells (DCs) control the segue from innate to adaptive immunity. Moreover, depending upon their milieu, DCs can either induce or inhibit immune responses. Whether DCs are immune stimulatory or tolerogenic apparently rests with whether or not the DCs express activated signal transducer and activator of transcription-3 (STAT3), the transcription factor induced by IL-6-like cytokines and IL-10. DCs expressing activated STAT3 produce less IL-12, which results in less effector T cell development. Moreover, DCs expressing activated STAT3 also express the tryptophan-catabolising enzyme indoleamine 2,3-dioxygenase. The kynurenine products of tryptophan catabolism induce T cell apoptosis; this area is of major interest to researchers working on tolerogenic DCs. In various disease models ranging from tumours to autoimmune diseases, administration of STAT3-activating cytokines resulted in attenuation of immune responses. Other corroborating evidence was obtained using conditional STAT3-deficient mice, or mice defective in cytokine signalling. Thus, persistently activating STAT3 in DCs may be a feasible strategy for controlling allograft rejection.

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.

Interleukin-6 (IL-6) is a pleiomorphic cytokine whose growth factor properties play an important role in the development and progression of many types of cancer. IL-6 is produced in response to a variety of stimuli, and is required for the development of T and B lymphocytes to effector cells. In certain neoplasias, such as multiple myeloma, IL-6 is both produced and required for survival by the cancer cell itself. In other neoplasias, IL-6 may come from tissue surrounding the tumour. Thus, therapeutic strategies aimed at inhibiting the production, expression or action of IL-6 would be quite beneficial in the treatment of cancer. Moreover, IL-6 is a pathophysiological factor in several hyperproliferative diseases and the paraneoplastic syndromes that often accompany cancer, such as cachexia and osteoporosis; thus, anti-IL-6 therapy would be useful in treating these entities as well. This expert opinion acquaints the reader with IL-6, its physiological responses, the cancer types with which it is associated, and discusses the current state of therapy aimed at inhibiting it.

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.

Signal transducers and activators of transcription (STATs) are involved in growth regulation of cells. They are usually activated by phosphorylation at specific tyrosine residues. In neoplastic cells, constitutive activation of STATs accompanies growth dysregulation and resistance to apoptosis through changes in gene expression, such as enhanced anti-apoptotic gene expression or reduced pro-apoptotic gene expression. Activated STAT3 is thought to play an important role in prostate cancer (PCA) progression. Because we are interested in how persistently-activated STAT3 changes the cellular phenotype to a malignant one in prostate cancer, we used expression vectors containing a gene for constitutively-activated STAT3, called S3c, into NRP-152 rat and BPH-1 human benign prostatic epithelial cells.

Although fluorescent dyes combined with flow cytometry have been used to confirm the viability of sperm in the past, methods to detect damage to spermatozoa following injury have been limited to use of dyes, which are often difficult to adequately compensate for in a single laser system.

Signal transducers and activators of transcription (STAT) were originally discovered as components of cytokine signal transduction pathways. Persistent activation of one of these transcription factors, STAT3, is a feature of many malignancies, including hormone-resistant prostate cancer. In this regard, malignant cells expressing persistently activated STAT3 become dependent on it for survival, thus rendering STAT3 a potential molecular target for therapy of hormone-resistant prostate cancer. Previously, we reported that antisense oligonucleotides specific for STAT3 were better at inducing apoptosis than inhibitors of JAK1 or JAK2, the upstream activating kinases of STAT3. Here, we report that novel single-stranded oligonucleotides, which putatively block STAT3-DNA binding, were better at inducing hormone-resistant prostate cancer apoptosis than antisense STAT3 oligonucleotides. We observed that the novel STAT3-inhibiting oligonucleotides induced apoptosis by a mitochondrial-dependent pathway involving the activation of caspase-3. Prostate cell lines not expressing persistently activated STAT3 did not become apoptotic after treatment with these same oligonucleotides. Scrambled-sequence control oligonucleotides had none of the effects of the active sequence oligonucleotides on any variable measured. Furthermore, the novel STAT3-inhibiting oligonucleotides, but not scrambled-sequence control oligonucleotide, significantly reduced the volume of s.c. DU145 tumors in vivo. Histologic examination of the tumors revealed no infiltrate of mononuclear or granulocytic cells, which would be indicative of evocation of a nonspecific immune response by the oligonucleotides. We conclude that single-stranded oligonucleotides based on the binding sequences of STAT3 are an additional strategy to design inhibitors for this molecular target and that these inhibitors should be useful as experimental therapeutics for hormone-resistant prostate cancer.

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.

The effects of kindled seizures elicited from sites in the left and right temporal lobes on mitogen-induced proliferation (LPS, Con A, PHA) and induction of representative TH1 (IFN-γ) and TH2 (IL-10, IL-4) cytokines were determined in activated rat splenocytes. With reference to cell proliferation, the changes depended on the hemispheric side and location of kindling. Kindling of the left side mediated significant increase in cell proliferation by LPS. Left side kindling resulted in decreased cell proliferation by PHA. Although right side kindling showed no change when taken together, further analysis showed that the reduced proliferation byPHAwas mediated when the pyriform cortex was kindled with no change from amygdaloid nuclei. Similar hemispheric polarization was observed in the production of IL-10 and IFN-γ by Con A-stimulated splenocytes in left side kindled rats. Hence, kindled temporal lobe seizures induced changes in specific immune functions. These effects are not only lateralized but are also specific with respect to the particular region kindled. Since epileptic patients have altered immune functions, this report contributes to our understanding of this complex immune-brain cross-talk in epilepsy.

An estimated 30% of cancer deaths are attributed to cachexia and its consequences. Cachexia (wasting syndrome) is the hypercatabolism of the body's carbon sources, proteins and lipids, for conversion into energy. It is induced by a variety of pathological conditions, including cancer. Among the inflammatory responses to cancer is the synthesis of cytokines, including IL-6 and related cytokines. These cytokines have been found to induce cachexia by altering metabolism of lipids and proteins. IL-6-like cytokines have been found to inhibit lipid biosynthesis by adipocytes, which increased the rate of lipid catabolism. Others have described the atrophy and increased catabolism of muscle protein due to IL-6. A cytokine closely-related to IL-6 is leptin, which plays a major role in lipid metabolism under normal conditions. The role of leptin in pathological conditions such as cancer cachexia has not yet been fully elucidated. Detailed mechanistic information about the induction of cancer cachexia by IL-6-like cytokines requires more research.