Desensitization of the soluble guanylyl cyclase/cGMP pathway by lipopolysaccharide in rat isolated pulmonary artery but not aorta

Background and purpose:

To investigate the function of soluble guanylyl cyclase (sGC)/3′,5′-cyclic guanosine monophosphate (cGMP) pathway in lipopolysaccharide (LPS)-induced changes in vascular reactivity of rat isolated pulmonary artery and aorta.

Experimental approach:

Nitric oxide (NO) production, contraction responses to endothelin-1 (ET-1), relaxation responses to sodium nitroprusside (SNP), 8-pCPT-cGMP, BAY412272 and T-0156, SNP-induced cGMP production and expression of sGC_α1, sGC_β1 and 3′,5′-cyclic nucleotide phosphodiesterase-5 (PDE5) proteins were measured in LPS-treated pulmonary and aortic rings from male Wistar rats.

Key results:

In both vessels, LPS (10 μg mL⁻¹, 20 h) increased NO production, which was inhibited by the selective inducible NOS (iNOS) inhibitor 1400W (1 μM). In the aorta, LPS decreased ET-1-induced contractility and this decrease was inhibited by the selective sGC inhibitor ODQ (10 μM) but not by removal of endothelium, or inhibitors of cyclooxygenase (indomethacin, 10 μM) or iNOS (1400W, 1 μM). Furthermore, aortic relaxation responses to the direct sGC activator BAY412272 were enhanced. In the pulmonary artery, SNP (1 nM to 30 μM)-induced relaxation and cGMP production, BAY412272-induced relaxation and sGC_β1 protein expression were decreased, whereas relaxation responses to the PDE5-specific inhibitor T-0156 (0.1–100 nM) were enhanced. Relaxation responses to the phosphodiesterase-resistant cGMP analogue, 8-pCPT-cGMP, and protein expression levels of sGC_α1 and PDE5 were not altered in either vessel.

Conclusion and implications:

LPS caused a selective hypocontractility of rat aorta to ET-1 mediated mainly through NO-independent sGC activation, whereas in the pulmonary artery, the effect of sGC activation was reduced by a decreased protein expression of sGC_β1 together with increased PDE5 activity.     

Lack of effect of ODQ does not exclude cGMP signalling via NO-sensitive guanylyl cyclase

BACKGROUND AND PURPOSE

Nitric oxide (NO) is known to activate NO-sensitive guanylyl cyclase (NO-GC) and to elicit cGMP production. However, NO has also been proposed to induce cGMP-independent effects. It is accepted practice to use specific NO-GC inhibitors, such as ODQ or NS2028, to assess cGMP-dependent NO effects. Consequently, NO-induced reactions seen in the presence of these inhibitors commonly serve as an affirmation of cGMP independence.

EXPERIMENTAL APPROACH

We evaluated the use of ODQ to discriminate between cGMP-dependent and cGMP-independent NO effects. NO-GC-expressing HEK cells, platelets and tissues from wild type (WT) and NO-GC-deficient mice (GCKO) were used.

KEY RESULTS

NO donors led to accumulation of cGMP in platelets and GC-expressing HEK cells and induced phosphorylation of the vasodilator-stimulated phosphoprotein in platelets; both effects were reduced by ODQ. High concentrations of NO donors, however, overrode the inhibitory effect of ODQ. Correspondingly, ODQ inhibited but did not fully eliminate NO-induced relaxation of aorta and fundus from WT mice. Relaxation induced by endogenously released NO was fully or partially inhibited by ODQ in fundus and aorta, respectively. In aorta and fundus of GCKO mice NO-induced relaxation was absent and served as standard for complete NO-GC inhibition.

CONCLUSIONS AND IMPLICATIONS

High NO concentrations can overcome the inhibitory effect of ODQ on NO-GC. Smooth muscle relaxation induced by NO donors/endogenously released NO in the presence of ODQ in WT was absent in GCKO animals indicating involvement of NO-GC. Accordingly, NO-induced effects in the presence of ODQ do not necessarily prove cGMP independence.     

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.     

Effect of oral organic nitrates on expression and activity of vascular soluble guanylyl cyclase

Background and purpose:

The regulation of vascular soluble guanylyl cyclase (sGC) expression by nitric oxide (NO) is still under discussion. In vitro, NO has been shown to downregulate the expression of sGC but it is unclear if this mechanism is operative in vivo and occurs during nitrate treatment.

Experimental approach:

We investigated whether high dose isosorbide mononitrate (ISMN) or pentaerythrityl tetranitrate (PETN) treatment changes vascular sGC expression and activity in vivo. New Zealand White rabbits received a standard diet, 2 or 200 mg ISMN kg⁻¹ d⁻¹ for 16 weeks, and C57BL/6 mice received a standard diet, 6, 60 or 300 mg PETN kg⁻¹ d⁻¹ for four weeks. Absorption was checked by measuring the plasma levels of the drug/metabolite.

Key results:

Western blots of rabbit aortic rings showed similar protein levels of sGC α1- (P=0.2790) and β1-subunits (P=0.6900) in all groups. Likewise, ANOVA showed that there was no difference in the expression of sGC in lungs of PETN-treated mice (P=0.0961 for α1 and P=0.3709 for β1). The activities of isolated sGC in response to SNAP (1 μM–1 mM) were identical in aortae of ISMN-treated rabbits (P=0.0775) and lungs of PETN-treated mice (P=0.6348). The aortic relaxation response to SNAP slightly decreased at high ISMN but not at high PETN.

Conclusions and implications:

These data refute the hypothesis that therapeutic treatment with long acting NO donors has a significant impact on the regulation of vascular sGC expression and activity in vivo.     

Caffeine inhibits the viability and osteogenic differentiation of rat bone marrow-derived mesenchymal stromal cells

Background and purpose:

Soluble guanylate cyclase (sGC) is the signal transduction enzyme most responsible for mediating the effects of nitric oxide (NO). Recently, NO-independent small molecule activators of sGC have been developed that have promising clinical activities. We have shown that the secreted matrix protein thrombospondin-1 (TSP-1) binds to CD47 and potently inhibits NO stimulation of sGC in endothelial and vascular smooth muscle cells (VSMCs) and platelets. Here we show that TSP-1 signalling via CD47 inhibits sGC activation by NO-independent sGC activating small molecules.

Experimental approach:

Vascular smooth muscle cells and washed human platelets were pretreated with TSP-1 (2.2 nM) in the presence of haeme-dependent sGC activators (YC-1, BAY 41-2272), and a haeme-independent activator (meso-porphyrin IX), and cGMP levels were measured. The effect of sGC activators on platelet aggregation and contraction of VSMC embedded in collagen gels was also assayed in the presence and absence of TSP-1.

Key results:

Thrombospondin-1 inhibited sGC activator-dependent increase in cGMP in VSMC and platelets. TSP-1 pretreatment also inhibited the ability of these agents to delay thrombin-induced platelet aggregation. TSP-1 pretreatment reduced the ability of sGC activating agents to abrogate VSMC contraction in vitro.

Conclusions and implications:

This work demonstrates that TSP-1 is a universal inhibitor of sGC, blocking both haeme-dependent and haeme-independent activation. These data coupled with the reported increases in TSP-1 with age, diabetes, ischaemia/reperfusion, and atherosclerosis implies that the therapeutic potential of all drugs that activate sGC could be compromised in disease states where TSP-1/CD47 signalling is elevated.     

BAY 41-2272, a soluble guanylate cyclase agonist, activates human mononuclear phagocytes

BACKGROUND AND PURPOSE

Phagocyte function is critical for host defense against infections. Defects in phagocytic function lead to several primary immunodeficiencies characterized by early onset of recurrent and severe infections. In this work, we further investigated the effects of BAY 41-2272, a soluble guanylate cyclase (sGC) agonist, on the activation of human peripheral blood monocytes (PBM) and THP-1 cells.

EXPERIMENTAL APPROACH

THP-1 cells and PBM viability was evaluated by methylthiazoletetrazolium assay; reactive oxygen species production by lucigenin chemiluminescence; gene and protein expression of NAPDH oxidase components by qRT-PCR and Western blot analysis, respectively; phagocytosis and microbicidal activity by co-incubation, respectively, with zymosan and Escherichia coli; and cytokine release by elisa.

KEY RESULTS

BAY 41-2272, compared with the untreated group, increased spreading of monocytes by at least 35%, superoxide production by at least 50%, and gp91^PHOX and p67^PHOX gene expression 20 to 40 times, in both PBM and THP-1 cells. BAY 41-2272 also augmented phagocytosis of zymosan particles threefold compared with control, doubled microbicidal activity against E. coli and enhanced the release of TNF-α and IL-12p70 by both PBM and THP-1 cells. Finally, by inhibiting sGC with ODQ, we showed that BAY 41-2272-induced superoxide production and phagocytosis is not dependent exclusively on sGC activation.

CONCLUSIONS AND IMPLICATIONS

In addition to its ability to induce vasorelaxation and its potential application for therapy of vascular diseases, BAY 41-2272 was shown to activate human mononuclear phagocytes. Hence, it is a novel pro-inflammatory drug that may be useful for controlling infections in the immunocompromised host.     

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.     

Cyclic bisbibenzyls induce growth arrest and apoptosis of human prostate cancer PC3 cells

Aim:

To investigate the cytotoxic effects of four cyclic bisbibenzyls, Riccardin C (Ric), Pakyonol (Pak), Marchantin M (Mar), and Plagiochin E (Pla) against chemoresistant prostate cancer PC3 cells.

Methods:

Cell growth was assayed by MTT method, and apoptotic related protein Bcl-2 and Bax, poly(ADP-ribose) polymerase (PARP) were examined by Western blotting. Cell cycle and apoptosis of PC3 cells were evaluated with flow cytometry and morphologic examinations.

Results:

The four compounds inhibited proliferation and elicited cell death in a dose- and time-dependent manner with IC₅₀ values of 3.22 μmol/L for Ric, 7.98 μmol/L for Pak, 5.45 μmol/L for Mar, and 5.99 μmol/L for Pla, respectively. Furthermore, exposed to these chemicals caused a decrease in the antiapoptotic protein Bcl-2 and an increase in proapoptotic Bax expression. PARP cleavage and caspase-3 activity were also observed.

Conclusion:

The results suggest that cyclic bisbibenzyls could be used for the development of novel therapeutic chemicals against prostate cancer.     

Thalidomide attenuates nitric oxide-driven angiogenesis by interacting with soluble guanylyl cyclase

Background and purpose:

Nitric oxide (NO) promotes angiogenesis by activating endothelial cells. Thalidomide arrests angiogenesis by interacting with the NO pathway, but its putative targets are not known. Here, we have attempted to identify these targets.

Experimental approach:

Cell-based angiogenesis assays (wound healing of monolayers and tube formation in ECV304, EAhy926 and bovine arterial endothelial cells), along with ex vivo and in vivo angiogenesis assays, were used to explore interactions between thalidomide and NO. We also carried out in silico homology modelling and docking studies to elucidate possible molecular interactions of thalidomide and soluble guanylyl cyclase (sGC).

Key results:

Thalidomide inhibited pro-angiogenic functions in endothelial cell cultures, whereas 8-bromo-cGMP, sildenafil (a phosphodiesterase inhibitor) or a NO donor [sodium nitroprusside (SNP)] increased these functions. The inhibitory effects of thalidomide were reversed by adding 8-bromo-cGMP or sildenafil, but not by SNP. Immunoassays showed a concentration-dependent decrease of cGMP in endothelial cells with thalidomide, without affecting the expression level of sGC protein. These results suggested that thalidomide inhibited the activity of sGC. Molecular modelling and docking experiments revealed that thalidomide could interact with the catalytic domain of sGC, which would explain the inhibitory effects of thalidomide on NO-dependent angiogenesis.

Conclusion and implications:

Our results showed that thalidomide interacted with sGC, suppressing cGMP levels in endothelial cells, thus exerting its anti-angiogenic effects. These results could lead to the formulation of thalidomide-based drugs to curb angiogenesis by targeting sGC.     

Distinct molecular requirements for activation or stabilization of soluble guanylyl cyclase upon haem oxidation-induced degradation

Background and purpose:

In endothelial dysfunction, signalling by nitric oxide (NO) is impaired because of the oxidation and subsequent loss of the soluble guanylyl cyclase (sGC) haem. The sGC activator 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino)methyl[benzoic]acid (BAY 58-2667) is a haem-mimetic able to bind with high affinity to sGC when the native haem (the NO binding site) is removed and it also protects sGC from ubiquitin-triggered degradation. Here we investigate whether this protection is a unique feature of BAY 58-2667 or a general characteristic of haem-site ligands such as the haem-independent sGC activator 5-chloro-2-(5-chloro-thiophene-2-sulphonylamino-N-(4-(morpholine-4-sulphonyl)-phenyl)-benzamide sodium salt (HMR 1766), the haem-mimetic Zn-protoporphyrin IX (Zn-PPIX) or the haem-dependent sGC stimulator 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272).

Experimental approach:

The sGC inhibitor 1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) was used to induce oxidation-induced degradation of sGC. Activity and protein levels of sGC were measured in a Chinese hamster ovary cell line as well as in primary porcine endothelial cells. Cells expressing mutant sGC were used to elucidate the molecular mechanism underlying the effects observed.

Key results:

Oxidation-induced sGC degradation was prevented by BAY 58-2667 and Zn-PPIX in both cell types. In contrast, the structurally unrelated sGC activator, HMR 1766, and the sGC stimulator, BAY 41-2272, did not protect. Similarly, the constitutively haem-free sGC mutant β₁H105F was stabilized by BAY 58-2667 and Zn-PPIX.

Conclusions:

The ability of BAY 58-2667 not only to activate but also to stabilize oxidized/haem-free sGC represents a unique example of bimodal target interaction and distinguishes this structural class from non-stabilizing sGC activators and sGC stimulators such as HMR 1766 and BAY 41-2272, respectively.     

Inhibition of nitric oxide-activated guanylyl cyclase by calmodulin antagonists

Background and purpose:

Nitric oxide (NO) controls numerous physiological processes by activation of its receptor, guanylyl cyclase (sGC), leading to the accumulation of 3′-5′ cyclic guanosine monophosphate (cGMP). Ca²⁺-calmodulin (CaM) regulates both NO synthesis by NO synthase and cGMP hydrolysis by phosphodiesterase-1. We report that, unexpectedly, the CaM antagonists, calmidazolium, phenoxybenzamine and trifluoperazine, also inhibited cGMP accumulation in cerebellar cells evoked by an exogenous NO donor, with IC₅₀ values of 11, 80 and 180 µM respectively. Here we sought to elucidate the underlying mechanism(s).

Experimental approach:

We used cerebellar cell suspensions to determine the influence of CaM antagonists on all steps of the NO-cGMP pathway. Homogenized tissue and purified enzyme were used to test effects of calmidazolium on sGC activity.

Key results:

Inhibition of cGMP accumulation in the cells did not depend on changes in intracellular Ca²⁺ concentration. Degradation of cGMP and inactivation of NO were both inhibited by the CaM antagonists, ruling out increased loss of cGMP or NO as explanations. Instead, calmidazolium directly inhibited purified sGC (IC₅₀= 10 µM). The inhibition was not in competition with NO, nor did it arise from displacement of the haem moiety from sGC. Calmidazolium decreased enzyme V_max and K_m, indicating that it acts in an uncompetitive manner.

Conclusions and implications:

The disruption of every stage of NO signal transduction by common CaM antagonists, unrelated to CaM antagonism, cautions against their utility as pharmacological tools. More positively, the compounds exemplify a novel class of sGC inhibitors that, with improved selectivity, may be therapeutically valuable.     

Role of connexin 43 in the maintenance of spontaneous activity in the guinea pig prostate gland

BACKGROUND AND PURPOSE

To investigate the role of connexin 43 in the maintenance of spontaneous activity in prostate tissue from young and old guinea pigs.

EXPERIMENTAL APPROACH

Conventional intracellular microelectrode and tension recording techniques, coupled with Western blot analysis and immunohistochemistry for connexin 43 (CX43) were used. The effects of three gap junction uncouplers, 18β glycyrrhetinic acid (10 µM, 40 µM), carbenoxolone (10 µM, 50 µM) and octanol (0.5 mM, 1 mM), were studied in cells displaying slow wave activity and on spontaneously contracting tissue from prostate glands of young (2–5 months) and old (9–16 months) guinea pigs.

KEY RESULTS

18β Glycyrrhetinic acid (40 µM), carbenoxolone (50 µM) or octanol (0.5 mM) abolished slow wave activity in prostate tissue from young and old guinea pigs and depolarized membrane potential by approximately 5 mV. These treatments also abolished all contractions in both sets of prostate tissue. These effects were reversed upon washout. Western blot analysis and CX43 immunohistochemistry showed that there was no age-related difference in the expression and distribution of CX43 in prostate tissues.

CONCLUSION AND IMPLICATIONS

When gap junctional communication via CX43 was disrupted, spontaneous activity was abolished at a cellular and whole tissue level; CX43 is therefore essential for the maintenance of spontaneous slow wave activity and subsequent contractile activity in the guinea pig prostate gland.     

The concomitant coronary vasodilator and positive inotropic actions of the nitroxyl donor Angeli's salt in the intact rat heart: contribution of soluble guanylyl cyclase-dependent and -independent mechanisms

Background and Purpose

The NO redox sibling nitroxyl (HNO) elicits soluble guanylyl cyclase (sGC)-dependent vasodilatation. HNO has high reactivity with thiols, which is attributed with HNO-enhanced left ventricular (LV) function. Here, we tested the hypothesis that the concomitant vasodilatation and inotropic actions induced by a HNO donor, Angeli''s salt (sodium trioxodinitrate), were sGC-dependent and sGC-independent respectively.

Experimental Approach

Haemodynamic responses to Angeli''s salt (10 pmol–10 μmol), alone and in the presence of scavengers of HNO (L-cysteine, 4 mM) or of NO [hydroxocobalamin (HXC), 100 μM] or a selective inhibitor of sGC [1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), 10 μM], a CGRP receptor antagonist (CGRP_8–37, 0.1 μM) or a blocker of voltage-dependent potassium channels [4-aminopyridine (4-AP), 1 mM] were determined in isolated hearts from male rats.

Key Results

Angeli''s salt elicited concomitant, dose-dependent increases in coronary flow and LV systolic and diastolic function. Both L-cysteine and ODQ shifted (but did not abolish) the dose–response curve of each of these effects to the right, implying contributions from HNO and sGC in both the vasodilator and inotropic actions. In contrast, neither HXC, CGRP_8–37 nor 4-AP affected these actions.

Conclusions and Implications

Both vasodilator and inotropic actions of the HNO donor Angeli''s salt were mediated in part by sGC-dependent mechanisms, representing the first evidence that sGC contributes to the inotropic and lusitropic action of HNO in the intact heart. Thus, HNO acutely enhances LV contraction and relaxation, while concomitantly unloading the heart, potentially beneficial actions in failing hearts.     

The xanthine derivative KMUP-1 inhibits models of pulmonary artery hypertension via increased NO and cGMP-dependent inhibition of RhoA/Rho kinase

Background and purpose:

KMUP-1 is known to increase cGMP, enhance endothelial nitric oxide synthase (eNOS) and suppress Rho kinase (ROCK) expression in smooth muscle. Here, we investigated the mechanism of action of KMUP-1 on acute and chronic pulmonary artery hypertension (PAH) in rats.

Experimental approach:

We measured pulmonary vascular contractility, wall thickening, eNOS immunostaining, expressions of ROCK II, RhoA activation, myosin phosphatase target subunit 1 (MYPT1) phosphorylation, eNOS, soluble guanylyl cyclase (sGC), protein kinase G (PKG) and phosphodiesterase 5A (PDE-5A), blood oxygenation and cGMP/cAMP, and right ventricular hypertrophy (RVH) in rats.

Key results:

In rings of intact pulmonary artery (PA), KMUP-1 relaxed the vasoconstriction induced by phenylephrine (10 µM) or the thromboxane A₂-mimetic U46619 (0.5 µM). In endothelium-denuded PA rings, this relaxation was reduced. In acute PAH induced by U46619 (2.5 µg·kg⁻¹·min⁻¹, 30 min), KMUP-1 relaxed vasoconstriction by enhancing levels of eNOS, sGC and PKG, suppressing those of PDE-5A, RhoA/ROCK II activation and MYPT1 phosphorylation, and restoring oxygenation in blood and cGMP/cAMP in plasma. Incubating smooth muscle cells from PA (PASMCs) with KMUP-1 inhibited thapsigargin-induced Ca²⁺ efflux and angiotensin II-induced Ca²⁺ influx. In chronic PAH model induced by monocrotaline, KMUP-1 increased eNOS and reduced RhoA/ROCK II activation/expression, PA wall thickening, eNOS immunostaining and RVH. KMUP-1 and sildenafil did not inhibit monocrotaline-induced PDE-5A expression.

Conclusion and implications:

KMUP-1 decreased PAH by enhancing NO synthesis by eNOS, with consequent cGMP-dependent inhibition of RhoA/ROCK II and Ca²⁺ desensitization in PASMCs. KMUP-1 has the potential to reduce vascular resistance, remodelling and RVH in PAH.     

Non-THC cannabinoids inhibit prostate carcinoma growth in vitro and in vivo: pro-apoptotic effects and underlying mechanisms

BACKGROUND AND PURPOSE

Cannabinoid receptor activation induces prostate carcinoma cell (PCC) apoptosis, but cannabinoids other than Δ⁹-tetrahydrocannabinol (THC), which lack potency at cannabinoid receptors, have not been investigated. Some of these compounds antagonize transient receptor potential melastatin type-8 (TRPM8) channels, the expression of which is necessary for androgen receptor (AR)-dependent PCC survival.

EXPERIMENTAL APPROACH

We tested pure cannabinoids and extracts from Cannabis strains enriched in particular cannabinoids (BDS), on AR-positive (LNCaP and 22RV1) and -negative (DU-145 and PC-3) cells, by evaluating cell viability (MTT test), cell cycle arrest and apoptosis induction, by FACS scans, caspase 3/7 assays, DNA fragmentation and TUNEL, and size of xenograft tumours induced by LNCaP and DU-145 cells.

KEY RESULTS

Cannabidiol (CBD) significantly inhibited cell viability. Other compounds became effective in cells deprived of serum for 24 h. Several BDS were more potent than the pure compounds in the presence of serum. CBD-BDS (i.p.) potentiated the effects of bicalutamide and docetaxel against LNCaP and DU-145 xenograft tumours and, given alone, reduced LNCaP xenograft size. CBD (1–10 µM) induced apoptosis and induced markers of intrinsic apoptotic pathways (PUMA and CHOP expression and intracellular Ca²⁺). In LNCaP cells, the pro-apoptotic effect of CBD was only partly due to TRPM8 antagonism and was accompanied by down-regulation of AR, p53 activation and elevation of reactive oxygen species. LNCaP cells differentiated to androgen-insensitive neuroendocrine-like cells were more sensitive to CBD-induced apoptosis.

CONCLUSIONS AND IMPLICATIONS

These data support the clinical testing of CBD against prostate carcinoma.

LINKED ARTICLE

This article is commented on by Pacher et al., pp. 76–78 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.02121.x     

A nitric oxide/Ca(2+)/calmodulin/ERK1/2 mitogen-activated protein kinase pathway is involved in the mitogenic effect of IL-1beta in human astrocytoma cells

Background and purpose:

Evidence is accumulating to support a role for interleukin-1β (IL-1β) in astrocyte proliferation. However, the mechanism by which this cytokine modulates this process is not fully elucidated.

Experimental approach:

In this study we used human astrocytoma U-373MG cells to investigate the role of nitric oxide (NO), intracellular Ca²⁺ concentration ([Ca²⁺]_i), and extracellular signal-regulated protein kinase (ERK) in the signalling pathway mediating IL-1β-induced astrocyte proliferation.

Key results:

Low IL-1β concentrations induced dose-dependent ERK activation which paralleled upregulation of cell division, whereas higher concentrations gradually reversed both these responses by promoting apoptosis. Pretreatment with the nonspecific NOS inhibitor, N-ω-nitro-l-arginine methyl ester (L-NAME) or the selective iNOS inhibitor, N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide dihydrochloride (1400W), antagonized ERK activation and cell proliferation induced by IL-1β. Inhibition of cGMP formation by the guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), partially inhibited ERK activation and cell division. Functionally blocking Ca²⁺ release from endoplasmic reticulum with ryanodine or 2-aminoethoxydiphenylborane (2-APB), inhibiting calmodulin (CaM) activity with N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide hydrochloride (W7) or MAPK kinase activity with 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthiol]butadiene (U0126) downregulated IL-1β-induced ERK activation as well as cell proliferation. The cytokine induced a transient and time-dependent increase in intracellular NO levels which preceded elevation in [Ca²⁺]_i.

Conclusions and implications:

These data identified the NO/Ca²⁺/CaM/ERK signalling pathway as a novel mechanism mediating the mitogenic effect of IL-1β in human astrocytes. As astrocyte proliferation is a hallmark of reactive astrogliosis, our results reveal a new potential target for therapeutic intervention in neuroinflammatory disorders.     

Retigeric acid B-induced mitophagy by oxidative stress attenuates cell death against prostate cancer cells in vitro

Aim:

Retigeric acid B (RAB), a pentacyclic triterpenic acid from Lobaria kurokawae Yoshim, has been found to induce apoptosis in prostate cancer cells. The aim of this study was to investigate the roles of mitochondrial damage-caused mitophagy in RAB-induced prostate cancer cell death in vitro.

Methods:

Human prostate cancer PC3 and LNCaP cells were tested. Cell viability was analyzed with MTT assay. Cell apoptosis, ROS level and mitochondrial transmembrane potential (mtΔψ) were measured with flow cytometry. Autophagy- and apoptosis-related proteins were studied using Western blotting. GFP-LC3B puncta, mitochondrial swelling and mitophagy were examined morphologically. Quantitative RT-PCR was used to measure LC3B mRNA level, and siRNA was used to knock down LC3BII.

Results:

In both PC3 and LNCaP cells, RAB (15 μmol/L) increased ROS accumulation and decreased mtΔψ in a time-dependent manner. Furthermore, RAB induced mitochondrial swelling and mitophagy, significantly increased LC3B expression and conversion of LC3BI to LC3BII, and the elimination of mitochondria by LC3BII-containing autophagolysosomes. In addition, RAB suppressed the PI3K/Akt/mTOR pathway activation. Pretreatment of PC3 cells with autophagy inhibitor 3-MA (5 mmol/L) or the lysosomal protease inhibitor CQ (10 μmol/L) significantly increased RAB-induced apoptosis. Similar results were obtained in RAB-treated PC3 cells with LC3B knocked down.

Conclusion:

RAB induces mitochondrial damage and mitophagy that attenuates RAB-induced prostate cancer cell death. Thus, suppression of mitophagy might be a potential strategy for improving the chemotherapeutic effects of RAB.