Resveratrol dimers are novel sphingosine kinase 1 inhibitors and affect sphingosine kinase 1 expression and cancer cell growth and survival

BACKGROUND AND PURPOSE

Sphingosine kinase 1 catalyses formation of the bioactive lipid, sphingosine 1-phosphate, which protects cancer cells from apoptosis. Therefore, sphingosine kinase 1 is a novel target for intervention with anti-cancer agents. We have assessed the effect of the anti-cancer agent, resveratrol and its dimers (ampelopsin A and balanocarpol) on sphingosine kinase 1 activity and on survival of MCF-7 breast cancer cells.

EXPERIMENTAL APPROACH

Ampelopsin A and balanocarpol were purified from Hopea dryobalanoides and their effect on sphingosine kinase 1 activity and expression, [³H] thymidine incorporation, ERK-1/2 phosphorylation and PARP activity assessed in MCF-7 cells.

KEY RESULTS

Resveratrol, ampelopsin A and balanocarpol were novel inhibitors of sphingosine kinase 1 activity. Balanocarpol was a mixed inhibitor (with sphingosine) of sphingosine kinase 1 with a K_ic= 90 ± 10 µM and a K_iu of ∼500 µM. Balanocarpol and ampelopsin A also induced down-regulation of sphingosine kinase 1 expression and reduced DNA synthesis, while balanocarpol stimulated PARP cleavage in MCF-7 breast cancer cells. Resveratrol was a competitive inhibitor (with sphingosine) of sphingosine kinase 1 with a K_ic= 160 ± 40 µM, reduced sphingosine kinase 1 expression and induced PARP cleavage in MCF-7 cells.

CONCLUSIONS AND IMPLICATIONS

Each molecule of balanocarpol may bind at least two sphingosine kinase 1 catalytic molecules to reduce the activity of each simultaneously. These findings suggest that resveratrol, ampelopsin A and balanocarpol could perturb sphingosine kinase 1-mediated signalling and this might explain their activity against MCF-7 breast cancer cells.

LINKED ARTICLE

This article is commented on by Hergst and Yun, pp. 1603–1604 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.01898.x     

FTY720 (fingolimod) increases vascular tone and blood pressure in spontaneously hypertensive rats via inhibition of sphingosine kinase

BACKGROUND AND PURPOSE

FTY720 (Fingolimod) is a recently approved orally administered drug for the treatment of multiple sclerosis. Phase II and III clinical trials have demonstrated that this drug modestly increases BP. We previously showed that inhibition of sphingosine kinase increases vascular tone and BP in hypertensive, but not normotensive rats. Since FTY720 is reported to have inhibitory effects on sphingosine kinase, we investigated whether FTY720 increases vascular tone and BP only in hypertensive rats via this mechanism.

EXPERIMENTAL APPROACH

The contractile and BP modulating effects of FTY720 were studied in vivo and ex vivo (wire myography) in age-matched normotensive Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs).

KEY RESULTS

Oral administration of FTY720 induced an increase in mean arterial pressure in SHR, whereas a decrease in BP was observed in WKY rats, as measured 24 h after administration. Similar to the sphingosine kinase inhibitor dimethylsphingosine (DMS), FTY720 induced large contractions in isolated carotid arteries from SHR, but not in those from WKY. In contrast, the phosphorylated form of FTY720 did not induce contractions in isolated carotid arteries from SHR. FTY720-induced contractions were inhibited by endothelium denudation, COX and thromboxane synthase inhibitors, and by thromboxane receptor antagonism, indicating that (like DMS-induced contractions) they were endothelium-dependent and mediated by thromboxane A₂.

CONCLUSIONS AND IMPLICATIONS

These data demonstrate that FTY720 increases vascular tone and BP only in hypertensive rats, most likely as a result of its inhibitory effect on sphingosine kinase.     

The sphingosine kinase inhibitor N,N-dimethylsphingosine inhibits neointimal hyperplasia

Background and purpose:

Sphingosine-1-phosphate and its receptors may be involved in vascular smooth muscle cell (VSMC) proliferation following vascular injury. Here, we evaluate the effect of d-erythro-N,N-dimethylsphingosine (DMS), a sphingosine kinase (SK) inhibitor, on VSMC proliferation, apoptosis and neointimal formation.

Experimental approach:

Growth responses in vitro to fetal calf serum (FCS) were measured by [³H]-thymidine incorporation and extracellular signal-regulated kinase-1/2 (ERK-1/2) activation in quiescent primary cultures of porcine VSMC in the presence and absence of various concentrations of the SK inhibitor DMS. In vivo treatment with DMS was delivered with a local endoluminal catheter, following balloon injury of coronary arteries. The artery intimal formation was investigated by angiography, myography and histomorphometry.

Key results:

In vitro experiments indicated that DMS induced a dose-dependent reduction in [³H]-thymidine incorporation and ERK-1/2 activation via a protein kinase C (PKC) independent mechanism with an IC₅₀ value of 12 ± 6 and 15 ± 10 µM respectively. DMS also reduced Akt signalling. Four weeks following in vivo delivery of DMS, complete functional endothelial regeneration was observed in all treatment groups, with significant reduction of intimal formation (vehicle 23.7 ± 4.6% vs. DMS infusion 8.92 ± 2.9%, P < 0.05).

Conclusions and implications:

Taken together, these results demonstrate that local administration of the SK inhibitor, DMS, reduced neointimal formation, and this effect could involve inhibition of ERK-1/2 and Akt signalling, and modulation of smooth muscle growth.     

Sphingosine kinase 1 is critically involved in nitric oxide-mediated human endothelial cell migration and tube formation

Background and purpose:

Sphingosine kinases (SKs) convert sphingosine to sphingosine 1-phosphate (S1P), which is a bioactive lipid that regulates a variety of cellular processes including proliferation, differentiation and migration.

Experimental approach:

We used the human endothelial cell line EA.hy926 to investigate the effect of nitric oxide (NO) donors on SK-1 expression, and on cell migration and tube formation.

Key results:

We showed that exposure of EA.hy926 cells to Deta-NO (125–1000 µM) resulted in a time- and concentration-dependent up-regulation of SK-1 mRNA and protein expression, and activity with a first significant effect at 250 µM of Deta-NO. The increased SK-1 mRNA expression resulted from an enhanced SK-1 promoter activity. A similar effect was also seen with various other NO donors. In mechanistic terms, the NO-triggered effect occurred independently of cGMP, but involved the classical mitogen-activated protein kinase cascade because the MEK inhibitor U0126 abolished the NO-induced SK-1 expression. The effect of NO was also markedly reduced by the thiol-reducing agent N-acetylcysteine, suggesting a redox-dependent mechanism. Functionally, Deta-NO triggered an increase in the migration of endothelial cells in an adapted Boyden chamber assay, and also increased endothelial tube formation in a Matrigel assay. These responses were both abolished in cells depleted of SK-1.

Conclusions and implications:

These data show that NO donors up-regulate specifically SK-1 expression and activity in human endothelial cells, and SK-1 in turn critically contributes to the migratory capability and tube formation of endothelial cells. Thus, SK-1 may be considered an attractive novel target to interfere with pathological processes involving angiogenesis.     

2-Phenyl-5-(pyrrolidin-1-yl)-1-(3,4,5-trimethoxybenzyl)-1H-benzimidazole, a benzimidazole derivative, inhibits growth of human prostate cancer cells by affecting tubulin and c-Jun N-terminal kinase

BACKGROUND AND PURPOSE

The c-Jun N-terminal kinase (JNK) and tubulin are, frequently, targets for developing anti-cancer drugs. A major obstacle to successful development is P-glycoprotein (P-gp)-mediated resistance. Here, we have assessed a compound that inhibited growth of cancer cells, for effects on JNK and tubulin and as a substrate for P-gp.

EXPERIMENTAL APPROACH

Several pharmacological and biochemical assays were used to characterize signalling pathways of 2-phenyl-5-(pyrrolidin-1-yl)-1-(3,4,5-trimethoxybenzyl)-1H-benzimidazole (PPTMB), a benzimidazole analogue, in prostate cancer cells.

KEY RESULTS

PPTMB inhibited proliferation of several human prostate cancer cell lines. It displayed similar activity against a P-gp-rich cell line, indicating that PPTMB was not a substrate for P-gp. PPTMB induced G2/M arrest of the cell cycle and subsequent apoptosis, using flow cytometry. Tubulin polymerization assays and Western blot analysis showed that PPTMB directly acted on tubulin and caused disruption of microtubule dynamics, inducing mitotic arrest and sustained high levels of cyclin B1 expression and Cdk1 activation. Subsequently, mitochondria-related apoptotic cascades were induced, including Bcl-2 and Bcl-xL phosphorylation, Mcl-1 down-regulation, truncated Bad formation and activation of caspase-9 and -3. PPTMB stimulated JNK phosphorylation at Thr¹⁸³/Tyr¹⁸⁵. SP600125, a specific JNK inhibitor, significantly inhibited apoptotic signalling, indicating that JNK plays a key role in PPTMB action. PPTMB showed a 10-fold higher potency against prostate cancer cells than normal prostate cells.

CONCLUSIONS AND IMPLICATIONS

PPTMB is an effective anti-cancer agent. It disrupted microtubule dynamics, leading to mitotic arrest of the cell cycle and JNK activation, which in turn stimulated the mitochondria-related apoptotic cascades in prostate cancer cells.     

Loss of sphingosine kinase-1 in carcinoma cells increases formation of reactive oxygen species and sensitivity to doxorubicin-induced DNA damage

BACKGROUND AND PURPOSE

Sphingosine kinases (SK) catalyse the formation of sphingosine 1-phosphate, which is a key lipid mediator regulating cell responses such as proliferation, survival and migration. Here we have investigated the effect of targeted inhibition of SK-1 on cell damage and elucidated the mechanisms involved.

EXPERIMENTAL APPROACH

Three human carcinoma cell lines (colon HCT-116, breast MDA-MB-231, lung NCI-H358) were used, which were either transduced with shRNA constructs to deplete SK-1, or treated with a SK-1 inhibitor. Cell growth and viability were assayed by [³H]thymidine incorporation and colony formation. Reactive oxygen species (ROS) were measured by fluorescence and apoptosis by annexin V with flow cytometry. Proteins were analysed by Western blotting. DNA damage was induced by doxorubicin.

KEY RESULTS

Knock-down of SK-1 by shRNA strongly inhibited DNA synthesis and colony formation of carcinoma cells. SK-1 knock-down (SK-1kd) cells revealed dysfunctional extracellular signal-regulated protein kinase and PKB/Akt cascades, and contained increased levels of ROS. After SK-1kd, treatment with doxorubicin increased DNA damage, measured by histone-2AX phosphorylation. Similar effects were found in cells with a SK-1 inhibitor and doxorubicin. The increased damage response in SK-1kd cells was accompanied by greater reduction of DNA synthesis and colony formation, and by more pronounced apoptosis. Addition of a NADPH oxidase inhibitor reduced the increased apoptosis in doxorubicin-treated SK-1kd cells.

CONCLUSIONS AND IMPLICATIONS

SK-1kd in carcinoma cells triggered oxidative stress by increasing intracellular Ros production. Targeted inhibition of SK-1 represents a promising approach to sensitize cells to DNA damage and facilitate apoptosis upon doxorubicin treatment.     

A combinatorial in silico and cellular approach to identify a new class of compounds that target VEGFR2 receptor tyrosine kinase activity and angiogenesis

BACKGROUND AND PURPOSE

Vascular endothelial growth factor receptor 2 (VEGFR2) is an attractive therapeutic target for the treatment of diseases such as cancer. Small-molecule VEGFR2 inhibitors of a variety of chemical classes are currently under development or in clinical use. In this study, we describe the de novo design of a new generation pyrazole-based molecule (JK-P3) that targets VEGFR2 kinase activity and angiogenesis.

EXPERIMENTAL APPROACH

JK-P compound series were designed using de novo structure-based identification methods. Compounds were tested in an in vitro VEGFR2 kinase assay. Using primary endothelial cells, JK-P compounds were assessed for their ability to inhibit VEGF-A-stimulated VEGFR2 activation and intracellular signalling. We tested these compounds in cell migration, proliferation and angiogenesis assays.

KEY RESULTS

JK-P3 and JK-P5 were predicted to bind the VEGFR2 kinase domain with high affinity, and both compounds showed pronounced inhibition of endogenous VEGFR2 kinase activity in primary human endothelial cells. Only JK-P3 inhibited VEGF-A-stimulated VEGFR2 activation and intracellular signalling. Interestingly, JK-P3 inhibited endothelial monolayer wound closure and angiogenesis but not endothelial cell proliferation. Both compounds inhibited fibroblast growth factor receptor kinase activity in vitro, but not basic fibroblast growth factor-mediated signalling in endothelial cells.

CONCLUSIONS AND IMPLICATIONS

This is the first report that describes an anti-angiogenic inhibitor based on such a pyrazole core. Using a de novo structure-based identification approach is an attractive method to aid such drug discovery. These results thus provide an important basis for the development of multi-tyrosine kinase inhibitors for clinical use in the near future.     

Development of β-amino-carbonyl compounds as androgen receptor antagonists

Aim： Androgen receptor （AR） antagonists have proven to be useful in the early control of prostate cancer. The aim of this study was to identify and characterize a novel β-amino-carbonyl-based androgen receptor antagonist. Methods Different isomers of the β-amino-carbonyl compounds were obtained by chiral separation. The bioactivities of the isomers were evaluated by AR nuclear translocation, mammalian two-hybrid, competitive receptor binding and cell proliferation assays. The expression of genes downstream of AR was analyzed with real-time PCR. The therapeutic effects on tumor growth in vivo were observed in male SClD mice bearing LNCaP xenografts. Results： Compound 21 was previously identified as an AR modulator by the high-throughput screening of a diverse compound library. In the present study, the two isomers of compound 21, termed compounds 21-1 and 2.1.-2, were characterized as partial AR agonists in terms of androgen-induced AR nuclear translocation, prostate-specific antigen expression and cell proliferation. Further structural modifications led to the discovery of a androgen receptor antagonist （compound 6012）, which blocked androgen receptor nuclear translocation, androgen-responsive gene expression and androgen-dependent LNCaP cell proliferation. Four stereoisomers of compound 6012 were isolated, and their bioactivities were assessed. The pharmacological effects of 6012, including AR binding, androgen-induced AR translocation, NH2- and COOH-terminal interaction, growth inhibition of LNCaP cells in vitro and LNCaP xenograft growth in nude mice, were mainly restricted to isomer 6012-4 （1R, 3S）. Conclusion： Compound 6012-4 was determined to be a novel androgen receptor antagonist with prostate cancer inhibitory activities comparable to bicalutamide both in vitro and in vivo.     

Nucleotide oligomerization domain 1 is a dominant pathway for NOS2 induction in vascular smooth muscle cells: comparison with Toll-like receptor 4 responses in macrophages

Background and purpose:

Gram-negative bacteria contain ligands for Toll-like receptor (TLR) 4 and nucleotide oligomerization domain (NOD) 1 receptors. Lipopolysaccharide (LPS) activates TLR4, while peptidoglycan products activate NOD1. Activation of NOD1 by the specific agonist FK565 results in a profound vascular dysfunction and experimental shock in vivo.

Experimental approach:

Here, we have analysed a number of pharmacological inhibitors to characterize the role of key signalling pathways in the induction of NOS2 following TLR4 or NOD1 activation.

Key results:

Vascular smooth muscle (VSM) cells expressed NOD1 mRNA and protein, and, after challenge with Escherichia coli or FK565, NOS2 protein and activity were induced. Macrophages had negligible levels of NOD1 and were unaffected by FK565, but responded to E. coli and LPS by releasing increased NO and expression of NOS2 protein. Classic pharmacological inhibitors for NF-κB (SC-514) and mitogen-activated protein kinase (SB203580, PD98059) signalling pathways inhibited responses in both cell types regardless of agonist. While TLR4-mediated responses in macrophages were specifically inhibited by the pan-caspase inhibitor z-VAD-fmk and the PKC inhibitor Gö6976, NOD1-mediated responses in VSM cells were inhibited by the Rip2 inhibitor PP2.

Conclusions and implications:

Our findings suggest a selective role for NOD1 in VSM cells, and highlight NOD1 as a potential novel therapeutic target for the treatment of vascular inflammation.     

Oxidized LDL-induced angiogenesis involves sphingosine 1-phosphate: prevention by anti-S1P antibody

BACKGROUND AND PURPOSE

Neovascularization occurring in atherosclerotic lesions may promote plaque expansion, intraplaque haemorrhage and rupture. Oxidized LDL (oxLDL) are atherogenic, but their angiogenic effect is controversial; both angiogenic and anti-angiogenic effects have been reported. The angiogenic mechanism of oxLDL is partly understood, but the role of the angiogenic sphingolipid, sphingosine 1-phosphate (S1P), in this process is not known. Thus, we investigated whether S1P is involved in the oxLDL-induced angiogenesis and whether an anti-S1P monoclonal antibody can prevent this effect.

EXPERIMENTAL APPROACH

Angiogenesis was assessed by capillary tube formation by human microvascular endothelial cells (HMEC-1) cultured on Matrigel and in vivo by the Matrigel plug assay in C57BL/6 mice.

KEY RESULTS

Human oxLDL exhibited a biphasic angiogenic effect on HMEC-1; low concentrations were angiogenic, higher concentrations were cytotoxic. The angiogenic response to oxLDL was blocked by the sphingosine kinase (SPHK) inhibitor, dimethylsphingosine, by SPHK1-siRNA and by an anti-S1P monoclonal antibody. Moreover, inhibition of oxLDL uptake and subsequent redox signalling by anti-CD36 and anti-LOX-1 receptor antibodies and by N-acetylcysteine, respectively, blocked SPHK1 activation and tube formation. In vivo, in the Matrigel plug assay, low concentrations of human oxLDL or murine oxVLDL also triggered angiogenesis, which was prevented by i.p. injection of the anti-S1P antibody.

CONCLUSION AND IMPLICATIONS

These data highlight the role of S1P in angiogenesis induced by oxLDL both in HMEC-1 cultured on Matrigel and in vivo in the Matrigel plug model in mice, and demonstrate that the anti-S1P antibody effectively blocks the angiogenic effect of oxLDL.     

3��,4��-Dihydroxyflavonol down-regulates monocyte chemoattractant protein-1 in smooth muscle: role of focal adhesion kinase and PDGF receptor signalling

Background and purpose:

We investigated the effects of a synthetic flavonol, 3′,4′-dihydroxyflavonol (DiOHF) on the expression of monocyte chemoattractant protein-1 (MCP-1) in rat vascular smooth muscle cells.

Experimental approach:

MCP-1 expression was assessed by quantitative real-time PCR and protein phosphorylation by immunoprecipitation and Western blots.

Key results:

DiOHF (1–30 µmol·L⁻¹) concentration-dependently reduced MCP-1 expression in both quiescent cells and cells stimulated with platelet-derived growth factor (PDGF) or interleukin 1-β. The effect of DiOHF was associated with a suppression of focal adhesion kinase (FAK)-mediated signalling. In vitro kinase assays demonstrated that DiOHF is a potent inhibitor of FAK kinase activity (EC₅₀= 2.4 µmol·L⁻¹). Expression of FAK-related non-kinase reduced basal MCP-1 expression, but not that induced by PDGF or interleukin 1-β. DiOHF also inhibited autophosphorylation of PDGF receptors. The PDGF receptor inhibitor AG-1296 potently suppressed basal and PDGF-induced MCP-1 expression. Inhibition of extracellular signal-regulated kinase activation by DiOHF, either directly or indirectly, may also be involved in its effects on MCP-1 expression. DiOHF had no inhibitory effect on either p38 or nuclear factor-κB activation. Moreover, DiOHF inhibited smooth muscle cell spreading (a FAK-mediated response) and proliferation.

Conclusions and implications:

This is the first report on a flavonoid compound (DiOHF) that is a potent FAK inhibitor. DiOHF also inhibits PDGF receptor autophosphorylation. These effects underlie the inhibitory action of DiOHF on MCP-1 expression in smooth muscle cells. Our results suggest that DiOHF might be a useful tool for dissection of the (patho)physiological roles of FAK signalling.British Journal of Pharmacology (2009) 157, 597–606; doi:10.1111/j.1476-5381.2009.00199.x; published online 9 April 2009     

The serine-threonine kinase p90RSK is a new target of enzastaurin in follicular lymphoma cells

BACKGROUND AND PURPOSE

Follicular lymphoma is the second most common non-Hodgkin''s lymphoma and, despite the introduction of rituximab for its treatment, this disease is still considered incurable. Besides genetic alterations involving Bcl-2, Bcl-6 or c-Myc, follicular lymphoma cells often display altered B-cell receptor signalling pathways including overactive PKC and PI3K/Akt systems.

EXPERIMENTAL APPROACH

The effect of enzastaurin, an inhibitor of PKC, was evaluated both in vitro on follicular lymphoma cell lines and in vivo on a xenograft murine model. Using pharmacological inhibitors and siRNA transfection, we determined the different signalling pathways after enzastaurin treatment.

KEY RESULTS

Enzastaurin inhibited the serine-threonine kinase p90RSK which has downstream effects on GSK3β. Bad and p70S6K. These signalling proteins control follicular lymphoma cell survival and apoptosis; which accounted for the inhibition by enzastaurin of cell survival and its induction of apoptosis of follicular lymphoma cell lines in vitro. Importantly, these results were replicated in vivo where enzastaurin inhibited the growth of follicular lymphoma xenografts in mice.

CONCLUSIONS AND IMPLICATIONS

The targeting of p90RSK by enzastaurin represents a new therapeutic option for the treatment of follicular lymphoma.     

Sphingosine and FTY720 are potent inhibitors of the transient receptor potential melastatin 7 (TRPM7) channels

Background and Purpose

Transient receptor potential melastatin 7 (TRPM7) is a unique channel kinase which is crucial for various physiological functions. However, the mechanism by which TRPM7 is gated and modulated is not fully understood. To better understand how modulation of TRPM7 may impact biological processes, we investigated if TRPM7 can be regulated by the phospholipids sphingosine (SPH) and sphingosine-1-phosphate (S1P), two potent bioactive sphingolipids that mediate a variety of physiological functions. Moreover, we also tested the effects of the structural analogues of SPH, N,N-dimethyl-D-erythro-sphingosine (DMS), ceramides and FTY720 on TRPM7.

Experimental Approach

HEK293 cells stably expressing TRPM7 were used for whole-cell, single-channel and macropatch current recordings. Cardiac fibroblasts were used for native TRPM7 current recording.

Key Results

SPH potently inhibited TRPM7 in a concentration-dependent manner, whereas S1P and other ceramides did not produce noticeable effects. DMS also markedly inhibited TRPM7. Moreover, FTY720, an immunosuppressant and the first oral drug for treatment of multiple sclerosis, inhibited TRPM7 with a similar potency to that of SPH. In contrast, FTY720-P has no effect on TRPM7. It appears that SPH and FTY720 inhibit TRPM7 by reducing channel open probability. Furthermore, endogenous TRPM7 in cardiac fibroblasts was markedly inhibited by SPH, DMS and FTY720.

Conclusions and Implications

This is the first study demonstrating that SPH and FTY720 are potent inhibitors of TRPM7. Our results not only provide a new modulation mechanism of TRPM7, but also suggest that TRPM7 may serve as a direct target of SPH and FTY720, thereby mediating S1P-independent physiological/pathological functions of SPH and FTY720.

Linked Article

This article is commented on by Rohacs, pp. 1291–1293 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12070     

Sesquiterpenoids from myrrh inhibit androgen receptor expression and function in human prostate cancer cells

Aim:

To examine whether two naturally occurring sesquiterpenoids (ST1 and ST2) with anti-proliferative activity in prostate cancer cells inhibit androgen receptor (AR) signaling.

Methods:

Human prostate cancer cell lines LNCaP and PC3 were used. The expression of AR, AR translocation into the nucleus, and expression levels of AR coactivators ARA70 and steroid receptor coactivator-1 (SRC-1) in LNCaP cells were examined using real-time PCR and Western blot. Changes in prostate-specific antigen (PSA) protein levels, PSA promoter activity, and androgen response element (ARE)-mediated reporter gene activity were examined using enzyme-linked immunoabsorbent assay (ELISA) and transient transfection assays. Co-immunoprecipitation was performed to analyze the interaction between AR and the AR coactivators in ST1- and ST2-treated cells.

Results:

In LNCaP cells, ST1 and ST2 (40 μmol/L) led to a significant decrease in the expression of AR as well as a reduction of AR translocation into the nucleus, but had no effect on AR protein translation. ST1 and ST2 treatment also resulted in a significant decrease in the level of PSA protein secreted into the medium and was able to suppress PSA promoter-dependent and ARE-dependent luciferase activity. Furthermore, decreased expression of ARA70 and SRC-1 was observed when LNCaP cells were exposed to ST1 and ST2, which interfered with their ability to interact with AR.

Conclusion:

The observations suggest that suppression of AR transactivation by ST1 and ST2 may be mediated, in part, by inhibiting AR nuclear translocation and/or interfering with the interaction between AR and its coactivators ARA70 and SRC-1. Therefore, sesquiterpenoids could be developed as novel therapeutic agents for treating prostate cancer.     

Pathway specific modulation of S1P1 receptor signalling in rat and human astrocytes

Background and Purpose

The sphingosine 1-phosphate receptor subtype 1 (S1P1R) is modulated by phosphorylated FTY720 (pFTY720), which causes S1P1R internalization preventing lymphocyte migration thus limiting autoimmune response. Studies indicate that internalized S1P1Rs continue to signal, maintaining an inhibition of cAMP, thus raising question whether the effects of pFTY720 are due to transient initial agonism, functional antagonism and/or continued signalling. To further investigate this, the current study first determined if continued S1P1R activation is pathway specific.

Experimental Approach

Using human and rat astrocyte cultures, the effects of S1P1R activation on cAMP, pERK and Ca²⁺ signalling was investigated. In addition, to examine the role of S1P1R redistribution on these events, a novel biologic (MNP301) that prevented pFTY720-mediated S1P1R redistribution was engineered.

Key Results

The data showed that pFTY720 induced long-lasting S1P1R redistribution and continued cAMP signalling in rat astrocytes. In contrast, pFTY720 induced a transient increase of Ca²⁺ in astrocytes and subsequent antagonism of Ca²⁺ signalling. Notably, while leaving pFTY720-induced cAMP signalling intact, the novel MNP301 peptide attenuated S1P1R-mediated Ca²⁺ and pERK signalling in cultured rat astrocytes.

Conclusions and Implications

These findings suggested that pFTY720 causes continued cAMP signalling that is not dependent on S1P1R redistribution and induces functional antagonism of Ca²⁺ signalling after transient stimulation. To our knowledge, this is the first report demonstrating that pFTY720 causes continued signalling in one pathway (cAMP) versus functional antagonism of another pathway (Ca²⁺) and which also suggests that redistributed S1P1Rs may have differing signalling properties from those expressed at the surface.     

Targeting V1A-vasopressin receptors with [Arg6, D-Trp7,9, NmePhe8]-substance P (6-11) identifies a strategy to develop novel anti-cancer therapies

Background and purpose

The anti-cancer agent [Arg⁶, D-Trp^7,9, N^mePhe⁸]-substance P (6-11) (SP-G) modulates gastrin releasing peptide (GRP) and arginine vasopressin signalling in small cell lung cancer cells leading to growth arrest and apoptosis. We have shown that SP-G acts as a biased agonist at GRP receptors. This work examines the hypothesis that SP-G acts as a biased agonist at the V_1A vasopressin receptor.

Experimental approach

The human V_1A receptor was expressed in CHO-K1 cells. Extracellular regulated kinase (ERK) activation and intracellular Ca²⁺ were measured using activation state-specific antibodies and Fura-2-AM respectively. The effect of SP-G on tumourigenicity was assessed by colony assay.

Key results

In V_1A receptor expressing cells, SP-G caused a sustained activation of ERK via a stimulation of V_1A receptor coupling to G_i. Inhibition of G_i with Pertussis toxin attenuated the inhibition by SP-G of the growth of CHO-K1 cells stably expressing the V_1A receptor. Chimeric V_1A receptors containing the second or third intracellular loop of the V₂ receptor were capable of binding vasopressin and SP-G but had altered ability to activate phospholipase C (PLC) and ERK. The second intracellular loop of the V_1A receptor was essential for vasopressin-stimulated PLC and ERK activation but not for SP-G-induced ERK activation.

Conclusions and implications

This work provides mechanistic insight, for biased agonists at V_1A receptors and highlights a potential role for such agents as anti-cancer agents.     

Natural and synthetic modulators of SK (K(ca)2) potassium channels inhibit magnesium-dependent activity of the kinase-coupled cation channel TRPM7

BACKGROUND AND PURPOSE

Transient receptor potential cation channel subfamily M member 7 (TRPM7) is a bifunctional protein comprising a TRP ion channel segment linked to an α-type protein kinase domain. TRPM7 is essential for proliferation and cell growth. Up-regulation of TRPM7 function is involved in anoxic neuronal death, cardiac fibrosis and tumour cell proliferation. The goal of this work was to identify non-toxic inhibitors of the TRPM7 channel and to assess the effect of blocking endogenous TRPM7 currents on the phenotype of living cells.

EXPERIMENTAL APPROACH

We developed an aequorin bioluminescence-based assay of TRPM7 channel activity and performed a hypothesis-driven screen for inhibitors of the channel. The candidates identified were further assessed electrophysiologically and in cell biological experiments.

KEY RESULTS

TRPM7 currents were inhibited by modulators of small conductance Ca²⁺-activated K⁺ channels (K_Ca2.1–2.3; SK) channels, including the antimalarial plant alkaloid quinine, CyPPA, dequalinium, NS8593, SKA31 and UCL 1684. The most potent compound NS8593 (IC₅₀ 1.6 µM) specifically targeted TRPM7 as compared with other TRP channels, interfered with Mg²⁺-dependent regulation of TRPM7 channel and inhibited the motility of cultured cells. NS8593 exhibited full and reversible block of native TRPM7-like currents in HEK 293 cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes.

CONCLUSIONS AND IMPLICATIONS

This study reveals a tight overlap in the pharmacological profiles of TRPM7 and K_Ca2.1–2.3 channels. NS8593 acts as a negative gating modulator of TRPM7 and is well-suited to study functional features and cellular roles of endogenous TRPM7.     

Advanced glycation end products (AGEs) activate mast cells

BACKGROUND AND PURPOSE

Advanced glycation endproducts (AGEs) represent one of the many types of chemical modifications that occur with age in long-lived proteins. AGEs also accumulate in pathologies such as diabetes, cardiovascular diseases, neurodegeneration and cancer. Mast cells are major effectors of acute inflammatory responses that also contribute to the progression of chronic diseases. Here we investigated interactions between AGEs and mast cells.

EXPERIMENTAL APPROACHES

Histamine secretion from AGEs-stimulated mast cells was measured. Involvement of a receptor for AGEs, RAGE, was assessed by PCR, immunostaining and use of inhibitors of RAGE. Production of reactive oxygen species (ROS) and cytokines was measured.

KEY RESULTS

Advanced glycation endproducts dose-dependently induced mast cell exocytosis with maximal effects being obtained within 20 s. RAGE mRNA was detected and intact cells were immunostained by a specific anti-RAGE monoclonal antibody. AGEs-induced exocytosis was inhibited by an anti-RAGE antibody and by low molecular weight heparin, a known RAGE antagonist. RAGE expression levels were unaltered after 3 h treatment with AGEs. AGE-RAGE signalling in mast cells involves Pertussis toxin-sensitive G_i-proteins and intracellular Ca²⁺ increases as pretreatment with Pertussis toxin, caffeine, 2-APB and BAPTA-AM inhibited AGE-induced exocytosis. AGEs also rapidly stimulated ROS production. After 6 h treatment with AGEs, the pattern of cytokine secretion was unaltered compared with controls.

CONCLUSIONS AND IMPLICATIONS

Advanced glycation endproducts activated mast cells and may contribute to a vicious cycle involving generation of ROS, increased formation of AGEs, activation of RAGE and to the increased low-grade inflammation typical of chronic diseases.