PAR1-mediated NFkappaB activation promotes survival of prostate cancer cells through a Bcl-xL-dependent mechanism

We have previously reported that protease-activated receptor 1 (PAR1 or thrombin receptor) is over-expressed in metastatic prostate cancer cell lines compared to prostate epithelial cells. In this study, we examined 1,074 prostate biopsies by tissue microarray analysis and demonstrated that PAR1 expression is significantly increased in prostate cancer compared to normal prostate epithelial cells and benign prostatic hyperplasia. We hypothesized that PAR1 activation contributed to prostate cancer cell progression. We demonstrated that stimulation of PAR1 by thrombin or thrombin receptor activating peptide (TRAP6), in androgen-independent DU145 and PC-3 cells resulted in increased DNA binding activity of the NFkappaB p65 subunit. IL-6 and IL-8 levels were also elevated in conditioned media by at least two-fold within 4-6 h of PAR1 activation. This induction of cytokine production was abrogated by pretreatment of cells with the NFkappaB inhibitor caffeic acid phorbol ester. The p38 and ERK1/2 MAPK signaling cascades were also activated by PAR1 stimulation, whereas the SAPK/JNK pathway was unaffected. Inhibition of p38 and ERK1/2 by SB-203589 and PD-098059, respectively, did not abrogate NFkappaB activity, suggesting an independent induction of NFkappaB by PAR1 stimulation. Furthermore, TUNEL assay showed that activation of PAR1 attenuated docetaxel induced apoptosis through the upregulation of the Bcl-2 family protein Bcl-xL. Akt activation was not observed, suggesting that drug resistance induced by PAR1 was independent of PI3K signaling pathway. Because thrombin and PAR1 are over-expressed in prostate cancer patients, targeting the inhibition of their interaction may attenuate NFkappaB signaling transduction resulting in decreased drug resistance and subsequent survival of prostate cancer cells.     

INPP4B suppresses prostate cancer cell invasion

Background

INPP4B and PTEN dual specificity phosphatases are frequently lost during progression of prostate cancer to metastatic disease. We and others have previously shown that loss of INPP4B expression correlates with poor prognosis in multiple malignancies and with metastatic spread in prostate cancer.

Results

We demonstrate that de novo expression of INPP4B in highly invasive human prostate carcinoma PC-3 cells suppresses their invasion both in vitro and in vivo. Using global gene expression analysis, we found that INPP4B regulates a number of genes associated with cell adhesion, the extracellular matrix, and the cytoskeleton. Importantly, de novo expressed INPP4B suppressed the proinflammatory chemokine IL-8 and induced PAK6. These genes were regulated in a reciprocal manner following downregulation of INPP4B in the independently derived INPP4B-positive LNCaP prostate cancer cell line. Inhibition of PI3K/Akt pathway, which is highly active in both PC-3 and LNCaP cells, did not reproduce INPP4B mediated suppression of IL-8 mRNA expression in either cell type. In contrast, inhibition of PKC signaling phenocopied INPP4B-mediated inhibitory effect on IL-8 in either prostate cancer cell line. In PC-3 cells, INPP4B overexpression caused a decline in the level of metastases associated BIRC5 protein, phosphorylation of PKC, and expression of the common PKC and IL-8 downstream target, COX-2. Reciprocally, COX-2 expression was increased in LNCaP cells following depletion of endogenous INPP4B.

Conclusion

Taken together, we discovered that INPP4B is a novel suppressor of oncogenic PKC signaling, further emphasizing the role of INPP4B in maintaining normal physiology of the prostate epithelium and suppressing metastatic potential of prostate tumors.

     

Monocyte surface-bound IL-15 can function as an activating receptor and participate in reverse signaling

IL-15 is a short chain, four-alpha helix cytokine that shares some biological function with IL-2. One striking difference between IL-2 and IL-15 is the ability of monocytes to express IL-15 on their cell surface after activation. In the current study we have investigated the ability of human monocyte cell surface IL-15 to participate in reverse signaling. Cross-linking anti-IL-15 Abs were used as a surrogate ligand for surface IL-15 engagement. Ligation of cell surface-expressed IL-15 induced monocyte adhesion that required the activity of small m.w. GTPases. Reverse signals through surface IL-15 activated the Rho-GTPase Rac3. In addition, engagement of cell surface IL-15 was found to activate a number of signaling pathways, including both extracellular signal-regulated kinase 1/2 and p38, and resulted in the secretion of IL-8. IL-8 production required mitogen-activated protein kinase activity. Thus, the current study has established that cell surface IL-15 is more than just a ligand; it can function as a receptor and participate in reverse signaling that results in cellular adhesion and production of inflammatory cytokines.     

A monoterpene,unique component of thyme honeys,induces apoptosis in prostate cancer cells via inhibition of NF-κB activity and IL-6 secretion

We have previously demonstrated that Greek thyme honey inhibits significantly the cell viability of human prostate cancer cells. Herein, 15 thyme honey samples from several regions of Greece were submitted to phytochemical analysis for the isolation, identification and determination (through modern spectral means) of the unique thyme honey monoterpene, the compound trihydroxy ketone E-4-(1,2,4-trihydroxy-2,6,6-trimethylcyclohexyl)-but-3-en-2-one.We investigated the anti-growth and apoptotic effects of the trihydroxy ketone on PC-3 human androgen independent prostate cancer cells using MTT assay and Annexin V-FITC respectively. The molecular pathways involved to such effects were further examined by evaluating its ability to inhibit (a) the NF-κB phosphorylation (S536), (b) JNK and Akt phosphorylation (Thr183/Tyr185 and S473 respectively) and (c) IL-6 production, using ELISA method. The anti-microbial effects of the trihydroxy ketone against a panel of nine pathogenic bacteria and three fungi were also assessed.The trihydroxy ketone exerted significant apoptotic activity in PC-3 prostate cancer cells at 100 μM, while it inhibited NF-κB phosphorylation and IL-6 secretion at a concentration range 10⁻⁶–10⁻⁴ M. Akt and JNK signaling were not found to participate in this process. The trihydroxy ketone exerted significant anti-microbial profile against many human pathogenic bacteria and fungi (MIC values ranged from 0.04 to 0.57 mg/ml). Conclusively, the Greek thyme honey-derived monoterpene exerted significant apoptotic activity in PC-3 cells, mediated, at least in part, through reduction of NF-κB activity and IL-6 secretion and may play a key role in the anti-growth effect of thyme honey on prostate cancer cells.     

Monocytes conditioned media stimulate fibronectin expression and spreading of inflammatory breast cancer cells in three-dimensional culture: A mechanism mediated by IL-8 signaling pathway

Background

Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer characterized by invasion of carcinoma cells into dermal lymphatic vessels where they form tumor emboli over expressing adhesion molecule E-cadherin. Although invasion and metastasis are dynamic processes controlled by complex interaction between tumor cells and microenvironment the mechanisms by which soluble mediators may regulate motility and invasion of IBC cells are poorly understood. The present study investigated the effect of media conditioned by human monocytes U937 secreted cytokines, chemokines and growth factors on the expression of adhesion molecules E-cadherin and fibronectin of human IBC cell line SUM149. Furthermore, cytokines signaling pathway involved were also identified.

Results

U937 secreted cytokines, chemokines and growth factors were characterized by cytokine antibody array. The major U937 secreted cytokines/chemokines were interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1/CCL2). When SUM149 cells were seeded in three dimensional (3D) models with media conditioned by U937 secreted cytokines, chemokines and growth factors; results showed: 1) changes in the morphology of IBC cells from epithelial to migratory spindle shape branched like structures; 2) Over-expression of adhesion molecule fibronectin and not E-cadherin. Further analysis revealed that over-expression of fibronectin may be mediated by IL-8 via PI3K/Akt signaling pathway.

Conclusion

The present results suggested that cytokines secreted by human monocytes may promote chemotactic migration and spreading of IBC cell lines. Results also indicated that IL-8 the major secreted cytokine by U937 cells may play essential role in fibronectin expression by SUM149 cells via interaction with IL-8 specific receptors and stimulation of PI3K/Akt signaling pathway.     

Activation of RET tyrosine kinase regulates interleukin-8 production by multiple signaling pathways

Interleukin-8 (IL-8) is known to contribute to human cancer progression through its potential function as a mitogenic, angiogenic, or motogenic factor. We found a high level of IL-8 production in SK-N-MC human primitive neuroectodermal tumor cells transfected with the human RET gene (SK-N-MC (RET) cells) in response to glial cell line-derived neurotrophic factor (GDNF) stimulation. IL-8 was also produced at high levels in TT human medullary thyroid carcinoma and TPC-1 human papillary thyroid carcinoma cell lines both of which express activated RET tyrosine kinase. To investigate which signaling pathways are responsible for IL-8 expression, we treated SK-N-MC (RET) cells with several kinase inhibitors before GDNF stimulation. The results showed that a MEK1 inhibitor, PD98059, a p38MAPK inhibitor, SB202190, and a protein kinase C (PKC) inhibitor, Calphostin C, markedly decreased the IL-8 secretion from SK-N-MC (RET) cells at 24 h after GDNF stimulation. In contrast, a phosphatidylinositol 3-kinase (PI3-K) inhibitor, LY294002, increased its secretion. These results thus suggested that IL-8 production by RET tyrosine kinase is regulated by multiple signaling pathways.     

B cell-derived IL-15 enhances CD8 T cell cytotoxicity and is increased in multiple sclerosis patients

Multiple lines of evidence suggest that CD8 T cells contribute to the pathogenesis of multiple sclerosis (MS). However, the sources and involvement of cytokines such as IL-15 in activating these cells is still unresolved. To investigate the role of IL-15 in enhancing the activation of CD8 T cells in the context of MS, we determined cell types expressing the bioactive surface IL-15 in the peripheral blood of patients and evaluated the impact of this cytokine on CD8 T cell cytotoxicity and migration. Flow cytometric analysis showed a significantly greater proportion of B cells and monocytes from MS patients expressing IL-15 relative to controls. We established that CD40L activation of B cells from healthy donors increased their IL-15 levels, reaching those of MS patients. We also demonstrated an enhanced cytotoxic profile in CD8 T cells from MS patients upon stimulation with IL-15. Furthermore, we showed that IL-15 expressed by B cells and monocytes is sufficient and functional, enhancing granzyme B production by CD8 T cells upon coculture. Exposure of CD8 T cells to this cytokine enhanced their ability to kill glial cells as well as to migrate across an in vitro inflamed human blood-brain barrier. The elevated levels of IL-15 in patients relative to controls, the greater susceptibility of CD8 T cells from patients to IL-15, in addition to the enhanced cytotoxic responses by IL-15-exposed CD8 T cells, stresses the potential of therapeutic strategies to reduce peripheral sources of IL-15 in MS.     

The vanilloid capsaicin induces IL-6 secretion in prostate PC-3 cancer cells

Capsaicin (8-methyl-N-vanillyl-6-nonenamide), a constituent of green and red peppers, has been linked with suppression of tumorigenesis through a mechanism that is not well understood. In the present study, we examined the effects of capsaicin on the production of the cytokine interleukin (IL)-6 by PC-3 cells at both protein and mRNA levels which were evaluated by ELISA and real-time PCR, respectively. Capsaicin-treated PC-3 cells increased the synthesis and secretion of IL-6 which was abrogated by the transient receptor potential vanilloid receptor subtype 1 (TRPV1) antagonist capsazepine, as well as by inhibitors of PKC-α, phosphoinositol-3 phosphate kinase (PI-3K), Akt and extracellular signal-regulated protein kinase (ERK). We analyzed the role of capsaicin in the tumor necrosis factor (TNF)-α secretion by PC-3 cells which was increased at shorter times than IL-6 production. Furthermore, incubation of PC-3 cells with an anti-TNF-α antibody blocked the capsaicin-induced IL-6 secretion. These results raise the possibility that capsaicin-mediated IL-6 increase in prostate cancer PC-3 cells is regulated at least in part by TNF-α secretion and signaling pathway involving Akt, ERK and PKC-α activation.     

Niclosamide suppresses cancer cell growth by inducing Wnt co-receptor LRP6 degradation and inhibiting the Wnt/β-catenin pathway

The Wnt/β-catenin signaling pathway is important for tumor initiation and progression. The low density lipoprotein receptor-related protein-6 (LRP6) is an essential Wnt co-receptor for Wnt/β-catenin signaling and represents a promising anticancer target. Recently, the antihelminthic drug, niclosamide was found to inhibit Wnt/β-catenin signaling, although the mechanism was not well defined. We found that niclosamide was able to suppress LRP6 expression and phosphorylation, block Wnt3A-induced β-catenin accumulation, and inhibit Wnt/β-catenin signaling in HEK293 cells. Furthermore, the inhibitory effects of niclosamide on LRP6 expression/phosphorylation and Wnt/β-catenin signaling were conformed in human prostate PC-3 and DU145 and breast MDA-MB-231 and T-47D cancer cells. Moreover, we showed that the mechanism by which niclosamide suppressed LRP6 resulted from increased degradation as evident by a shorter half-life. Finally, we demonstrated that niclosamide was able to induce cancer cell apoptosis, and displayed excellent anticancer activity with IC(50) values less than 1 μM for prostate PC-3 and DU145 and breast MDA-MB-231 and T-47D cancer cells. The IC(50) values are comparable to those shown to suppress the activities of Wnt/β-catenin signaling in prostate and breast cancer cells. Our data indicate that niclosamide is a unique small molecule Wnt/β-catenin signaling inhibitor targeting the Wnt co-receptor LRP6 on the cell surface, and that niclosamide has a potential to be developed a novel chemopreventive or therapeutic agent for human prostate and breast cancer.     

Adrenomedullin inhibits prostate cancer cell proliferation through a cAMP-independent autocrine mechanism

Adrenomedullin (AM) is a multifunctional peptide expressed in the normal and malignant prostate, and in prostate cancer cells. To elucidate the potential role of AM in prostate cancer, we have transfected the human AM gene into PC-3, DU 145, and LNCaP prostate cancer cells. Northern blot, Western blot, and radioimmunoassay techniques confirmed an increase in the synthesis and secretion of the 6kDa mature peptide, in the AM-transfected clones. Proliferation and cell cycle assays demonstrated that AM overexpression inhibited cell proliferation in PC-3 and LNCaP cells through a G0/G1 cell cycle arrest, but not in DU 145 cells. In vivo growth assays also confirmed that, at least in PC-3, AM produced a very significant reduction of tumor volume. In addition, the three cell lines expressed the CL/RCP/RAMP-2 receptor complex by RT-PCR, which suggests that AM peptide acts through an autocrine loop in prostate cancer cells. Although cAMP elevation is the most common pathway involved in AM signalling, stimulation of PC-3, DU 145, and LNCaP with synthetic AM did not increase intracellular cAMP. However, short-term stimulation of PC-3 cells with synthetic AM increased ERK1/2 activation. On the contrary, long-term stimulation, or AM overexpression, caused a reduction in the basal activation of ERK1/2. In summary, our results demonstrate that AM (either overexpressed or exogenously added) causes an inhibition of prostate cancer cell growth. This inhibition does not depend on changes in intracellular cAMP levels, but may be related to ERK1/2 activation.     

Multipathways for transdifferentiation of human prostate cancer cells into neuroendocrine-like phenotype

The neuroendocrine (NE) cell is a minor cell population in normal human prostate glands. The number of NE cells is increased in advanced hormone-refractory prostate carcinomas (PCA). The mechanism of increased NE cell population in these advanced tumors is poorly understood. We examined molecular mechanisms which may be involved in the regulation of the transdifferentiation process of human PCA cells leading to a NE phenotype. We compared PCA cell lines LNCaP and PC-3 in the following medium conditions: steroid-reduced (SR), interleukin-6 (IL-6)-supplemented, or dibutyrate cAMP (db-cAMP)-supplemented. We found that androgen-responsive C-33 LNCaP cells responded to all treatments, having a neuronal-like morphology. In contrast, C-81 LNCaP cells, having a decreased androgen responsiveness, had a less pronounced effect although followed a similar trend. Androgen-unresponsive PC-3 cells showed little change in their morphology. Grown in the SR condition, the level of neuron-specific enolase (NSE), a marker of neuronal cells, was upregulated in C-33 LNCaP cells, while to a lesser degree in the presence of IL-6. In the presence of db-cAMP, the NSE level in C-33 cells was decreased, lower than that in control cells. An opposite effect was observed for C-81 LNCaP cells. Nevertheless, the NSE level was only elevated in db-cAMP-treated PC-3 cells, but no change was found in PC-3 cells grown in the SR- or IL-6-supplemented medium. Thus, a similar gross phenotypic change may correlate with differential molecular expressions. We also analyzed the expression of protein tyrosine phosphatase alpha (RPTPalpha) since it plays a critical role in normal neuronal differentiation and signaling. Our results showed that the expression of RPTPalpha correlates with the NE phenotypic change of LNCaP cells in the SR condition. In summary, our data clearly show that the molecular process by which cultured human prostate cancer cells undergo a transdifferentiation process to a NE cell-like phenotype is accompanied by differential expressions of different markers, and a gross NE cell-like phenotype can occur by exposing PCA cells to different pharmacological agents.     

Rosiglitazone attenuates insulin-like growth factor 1 receptor survival signaling in PC-3 cells

PPARgamma, a member of the peroxisome proliferator-activated receptor family, is overexpressed in prostate cancer. Natural and synthetic ligands of PPARgamma via genomic and nongenomic actions promote cell cycle arrest and apoptosis of several prostate cancer cells, in vitro. Insulin-like growth factor 1 (IGF-1) inhibits the adriamycin-induced apoptosis of PC-3 human prostate cancer cells. Therefore, we have analyzed the ability of two PPARgamma ligands,15dPGJ2 and rosiglitazone, a natural and a synthetic PPARgamma ligand, respectively, to increase the adriamycin-induced cytotoxicity of PC-3 cells and to suppress the IGF-1 survival effect on adriamycin-induced apoptosis of PC-3 cells. Our data revealed that both the PPARgamma ligands increased the adriamycin-induced cytostasis of PC-3 cells, however, only rosiglitazone added to the adriamycin-induced apoptosis of PC-3 cells. In addition, rosiglitazone attenuated the type I IGF receptor (IGF-1R) survival signaling on adriamycin-induced apoptosis of PC-3 cells via its nongenomic action on ERK1/2 and AKT phosphorylation. Because the IGF-1R signaling is probably the most important host tissue (bone) metastasis microenvironment-related survival signaling for prostate cancer cells, we conclude that rosiglitazone effects on IGF-1R-mediated activation of ERK1/2 and AKT could have clinical implications for the management of androgen ablation-refractory and chemotherapy-resistant advanced prostate cancer with bone metastasis.     

CpG-A oligonucleotides induce a monocyte-derived dendritic cell-like phenotype that preferentially activates CD8 T cells

Human B cells and plasmacytoid dendritic cells recognize CpG motifs within microbial DNA via Toll-like receptor 9. Two functionally distinct types of CpG motif containing oligonucleotides (CpG ODN) have been described, CpG-A and CpG-B. In contrast to CpG-B, CpG-A induces high amounts of type I IFN (IFN-alpha and IFN-beta) in plasmacytoid dendritic cells. In the present study, we examined the effects of CpG-A on human primary monocytes. In PBMC stimulated with CpG-A and GM-CSF, monocytes showed excellent survival, increased in size and granularity, and within 3 days developed a dendritic cell-like phenotype that was characterized by down-regulation of CD14, partial up-regulation of CCR7, and an increased surface expression of costimulatory and Ag-presenting molecules. This effect could be inhibited by a combination of blocking Abs to type I IFN, and no such CpG-A-induced changes were observed in purified monocytes. Although IL-12 production by this dendritic cell-like phenotype required additional stimulation with CD40 ligand, this cell type spontaneously up-regulated IL-15 expression. Consistent with the known effect of IL-15 on effector and memory CD8 T cells, the frequency of CCR7(-)/CD45RA(-) CD8 T cells was selectively increased in allogeneic T cell assays. Furthermore, this dendritic cell type was more potent to support both the generation and the IFN-gamma production of autologous influenza matrix peptide-specific memory CD8 T cells as compared with dendritic cells generated in the presence of GM-CSF and IL-4. In conclusion, monocytes exposed to the cytokine milieu provided by CpG-A rapidly develop a dendritic cell-like phenotype that is well equipped to support CD8 T cell responses.