Beverly E. Barton

IL-6-like cytokines and cancer cachexia: consequences of chronic inflammation.

An estimated 30% of cancer deaths are attributed to cachexia and its consequences. Cachexia (wasting syndrome) is the hypercatabolism of the bodys 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 cach exia 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.

Interleukin-6 and new strategies for the treatment of cancer, hyperproliferative diseases and paraneoplastic syndromes

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.

Stable expression of constitutively-activated STAT3 in benign prostatic epithelial cells changes their phenotype to that resembling malignant cells

BackgroundSignal 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.ResultsWe observed that prostatic cell lines stably expressing S3c required STAT3 expression for survival, because they became sensitive to antisense oligonucleotide for STAT3. However, S3c-transfected cells were not sensitive to the effects of JAK inhibitors, meaning that STAT3 was constitutively-activated in these transfected cell lines. NRP-152 prostatic epithelial cells lost the requirement for exogenous growth factors. Furthermore, we observed that NRP-152 expressing S3c had enhanced mRNA levels of retinoic acid receptor (RAR)-α, reduced mRNA levels of RAR-β and -γ, while BPH-1 cells transfected with S3c became insensitive to the effects of androgen, and also to the effects of a testosterone antagonist. Both S3c-transfected cell lines grew in soft agar after stable transfection with S3c, however neither S3c-transfected cell line was tumorigenic in severe-combined immunodeficient mice.ConclusionsWe conclude, based on our findings, that persistently-activated STAT3 is an important molecular marker of prostate cancer, which develops in formerly benign prostate cells and changes their phenotype to one more closely resembling transformed prostate cells. That the S3c-transfected cell lines require the continued expression of S3c demonstrates that a significant phenotypic change occurred in the cells. These conclusions are based on our data with respect to loss of growth factor requirement, loss of androgen response, gain of growth in soft agar, and changes in RAR subunit expression, all of which are consistent with a malignant phenotype in prostate cancer. However, an additional genetic change may be required for S3c-transfected prostate cells to become tumorigenic.

STAT3: a potential therapeutic target in dendritic cells for the induction of transplant tolerance

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.

IL-6 signaling by STAT3 participates in the change from hyperplasia to neoplasia in NRP-152 and NRP-154 rat prostatic epithelial cells

BackgroundSTAT3 phosphorylation is associated with the neoplastic state in many types of cancer, including prostate cancer. We investigated the role of IL-6 signaling and phosphorylation of STAT3 in 2 rat prostatic epithelial lines. NRP-152 and NRP-154 cells were derived from the same rat prostate, yet the NRP-152 cells are not tumorigenic while the NRP-154 cells are tumorigenic. These lines are believed to represent 2 of the stages in the development of prostate cancer, hyperplasia and neoplasia. Differences in signaling pathways should play a role in the 2 phenotypes, hyperplastic and neoplastic.MethodsWe looked at the phosphorylation state of STAT3 by intracellular flow cytometry, using phospho-specific antibodies to STAT3. We used the same method to examine IL-6 production by the cell lines. We also measured apoptosis by binding of fluorescent annexin V to the cells.ResultsAlthough both cells lines made IL-6 constitutively, phosphorylated-STAT3 was present in untreated NRP-154 cells, but not in NRP-152 cells. Treatment with dexamethasone inhibited the IL-6 production of NRP-152 cells, but enhanced that of NRP-154 cells. Treatment with the JAK2 inhibitor AG490 induced apoptosis in NRP-152, but not NRP-154 cells.ConclusionsWe conclude from these experiments that STAT3 activity plays a role in the phenotype of NRP-154 cell, but not NRP-152 cells. The significance of alternative IL-6 signaling pathways in the different phenotypes of the 2 cell lines is discussed.

STAT3 Inhibition in Prostate and Pancreatic Cancer Lines by STAT3 Binding Sequence Oligonucleotides: Differential Activity Between 5′ and 3′ Ends

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]

Effects of hemispheric lateralization and site specificity on immune alterations induced by kindled temporal lobe seizures

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-gamma) 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 by PHA was 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-gamma 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.

Potential use of STAT3 inhibitors in targeted prostate cancer therapy: future prospects

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.

Eicosanoid-Induced Store-Operated Calcium Entry in Dendritic Cells

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 (GM-CSF) 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 B(4) (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(+) 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 Ca(2+) mobilization, which may have important implications for the regulation of DC maturation at sites of immune and non-immune inflammation.

Creation of a novel peptide with enhanced nuclear localization in prostate and pancreatic cancer cell lines

BackgroundFor 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.ResultsUptake 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 (NaN3). 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.ConclusionsThese 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.