Ismail Kaddour-Djebbar

Human ciliary muscle cell responses to FP-class prostaglandin analogs: Phosphoinositide hydrolysis, intracellular Ca2+ mobilization and MAP kinase activation

Phospholipase C induced phosphoinositide (PI) turnover, intracellular Ca(2+) ([Ca(2+)](i)) mobilization and mitogen-activated protein (MAP) kinase activation by FP-class prostaglandin analogs was studied in normal human ciliary muscle (h-CM) cells. Agonist potencies obtained in the PI turnover assays were: travoprost acid ((+)-fluprostenol; EC(50) = 2.6 +/- 0.8 nM) > bimatoprost acid (EC(50) = 3.6 +/- 1.2 nM) > (+/-)-fluprostenol (EC(50) = 4.3 +/- 1.3 nM) >> prostaglandin F(2 alpha) (PGF(2 alpha)) (EC(50) = 134 +/- 17 nM) > latanoprost acid (EC(50) = 198 +/- 83 nM) > S-1033 (EC(50) = 2930 +/- 1420 nM) > unoprostone (EC(50) = 5590 +/- 1490 nM) > bimatoprost (EC(50) = 9600 +/- 1100 nM). Agonist potencies in h-CM cells correlated well with those previously obtained for the cloned human ciliary body-derived FP receptor (r = 0.96, p< 0.001) and that present on h-TM cells (r = 0.94, p< 0.0001). Travoprost acid, PGF(2 alpha) and unoprostone also stimulated [Ca(2+)](i) mobilization in h-CM cells with travoprost acid being the most potent agonist. MAP kinase activity was stimulated in the h-CM cells with the following rank order of activity (at 100 nM): travoprost acid > PGF(2 alpha) > latanoprost acid > PGD(2) > bimatoprost > latanoprost = bimatoprost acid = fluprostenol > PGE(2) = S-1033 > unoprostone > PGI(2). The PI turnover, [Ca(2+)](i) mobilization and MAP kinase activation induced by several of these agonists was blocked by the FP receptor antagonist, AL-8810 (11 beta-fluoro-15-epiindanyl PGF(2 alpha)) (e.g. K(i) = 5.7 microM versus PI turnover). These studies have characterized the biochemical and pharmacological properties of the native FP prostaglandin receptor present on h-CM cells using three signal transduction mechanism assays and a broad panel of FP-class agonist analogs (including free acids of bimatoprost, travoprost and latanoprost) and the FP receptor antagonist, AL-8810.

SAHA-sensitized prostate cancer cells to TNFα-related apoptosis-inducing ligand (TRAIL): Mechanisms leading to synergistic apoptosis

Treatment of cancer cells with histone deacetylase inhibitors (HDACi) such as suberolylanilide hydroxamic acid (SAHA) activates genes that promote apoptosis. To enhance proapoptotic efficiency, SAHA has been used in combination with radiation, kinase inhibitors and cytotoxic drugs. Although several prostate cells respond to TNFα‐Related Apoptosis‐Inducing Ligand (TRAIL), LNCaP are resistant. This model system was utilized to examine the advantages of combined treatment with SAHA and TRAIL. In LNCaP cells, TRAIL induced synergistic apoptosis when combined with even with the lowest dose SAHA. Treatment with caspase inhibitor confirmed that SAHA‐induced apoptosis was mediated through caspases. In addition to induction of apoptosis, SAHA and TRAIL decreased the levels of proapoptotic proteins IKKα, IKKβ and IKKγ, suggesting that SAHA treatment may reduce the activity of NFκB. However, assay for NFκB luciferase reporter activity showed highly significant increase in SAHA‐treated cells, supporting earlier suggestions that HDACi promotes NFκB transcriptional activity. Further analyses to determine the mechanisms by which the combination of SAHA and TRAIL led to synergistic apoptosis indicated that the apoptotic response of LNCaP is due to a complex regulation of death receptor pathway and alterations of NFκB activity at several regulatory steps.

Distinct Nrf2 Signaling Mechanisms of Fumaric Acid Esters and Their Role in Neuroprotection against 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Experimental Parkinson's-Like Disease.

A promising approach to neurotherapeutics involves activating the nuclear-factor-E2-related factor 2 (Nrf2)/antioxidant response element signaling, which regulates expression of antioxidant, anti-inflammatory, and cytoprotective genes. Tecfidera, a putative Nrf2 activator, is an oral formulation of dimethylfumarate (DMF) used to treat multiple sclerosis. We compared the effects of DMF and its bioactive metabolite monomethylfumarate (MMF) on Nrf2 signaling and their ability to block 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced experimental Parkinsons disease (PD). We show that in vitro DMF and MMF activate the Nrf2 pathway via S-alkylation of the Nrf2 inhibitor Keap1 and by causing nuclear exit of the Nrf2 repressor Bach1. Nrf2 activation by DMF but not MMF was associated with depletion of glutathione, decreased cell viability, and inhibition of mitochondrial oxygen consumption and glycolysis rates in a dose-dependent manner, whereas MMF increased these activities in vitro. However, both DMF and MMF upregulated mitochondrial biogenesis in vitro in an Nrf2-dependent manner. Despite the in vitro differences, both DMF and MMF exerted similar neuroprotective effects and blocked MPTP neurotoxicity in wild-type but not in Nrf2 null mice. Our data suggest that DMF and MMF exhibit neuroprotective effects against MPTP neurotoxicity because of their distinct Nrf2-mediated antioxidant, anti-inflammatory, and mitochondrial functional/biogenetic effects, but MMF does so without depleting glutathione and inhibiting mitochondrial and glycolytic functions. Given that oxidative damage, neuroinflammation, and mitochondrial dysfunction are all implicated in PD pathogenesis, our results provide preclinical evidence for the development of MMF rather than DMF as a novel PD therapeutic. SIGNIFICANCE STATEMENT Almost two centuries since its first description by James Parkinson, Parkinsons disease (PD) remains an incurable disease with limited symptomatic treatment. The current study provides preclinical evidence that a Food and Drug Administration-approved drug, dimethylfumarate (DMF), and its metabolite monomethylfumarate (MMF) can block nigrostriatal dopaminergic neurodegeneration in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of PD. We elucidated mechanisms by which DMF and its active metabolite MMF activates the redox-sensitive transcription factor nuclear-factor-E2-related factor 2 (Nrf2) to upregulate antioxidant, anti-inflammatory, mitochondrial biosynthetic and cytoprotective genes to render neuroprotection via distinct S-alkylating properties and depletion of glutathione. Our data suggest that targeting Nrf2-mediated gene transcription using MMF rather than DMF is a promising approach to block oxidative stress, neuroinflammation, and mitochondrial dysfunction for therapeutic intervention in PD while minimizing side effects.

Cat iris sphincter smooth-muscle contraction: comparison of FP-class prostaglandin analog agonist activities.

The pharmacologic characteristics of a number of FP-class prostaglandin (PG) analogs were determined by using the cat iris sphincter smooth-muscle-contraction assay. Cumulative concentration-response curves were generated for each compound. The relative agonist potencies (EC(50)) of the compounds were: cloprostenol (0.0012 +/- 0.0004 nM) >> travoprost acid (0.46 +/- 0.13 nM) = bimatoprost acid (0.99 +/- 0.19 nM) > (+/-)-fluprostenol (15.8 +/- 2.6 nM) = PGF(2alpha) (18.6 +/- 1.8 nM) > latanoprost acid (29.9 +/- 1.6 nM) > bimatoprost (140 +/- 45 nM) > S-1033 (588 +/- 39 nM) > unoprostone (UF-021; 1280 +/- 50 nM; n = 4-14). The maximum response induced by travoprost acid (122% +/- 2.3% maximum response relative to PGF(2alpha)) was significantly greater than that induced by all the other PG compounds (P < 0.001 - P < 0.02). Interestingly, the FP-receptor antagonist, AL-8810, behaved as a moderate efficacy partial agonist (EC(50) = 2140 +/- 190 nM; 63 +/- 4.3% maximum response relative to PGF(2alpha)), indicating that the cat iris contains an extremely well-coupled FP-receptor population, and/or the tissue contains an extremely high density of the FP-receptor and/or spare receptors. The cat iris contraction data were well correlated with other FP-receptor-mediated signal-transduction processes, including FP-receptor binding in bovine corpus luteum (r = 0.86), FP-receptor binding in human iris (r = 0.61), phosphoinositide (PI) hydrolysis in human ciliary muscle and trabecular meshwork cells (r = 0.77 - 0.86), PI turnover in rat and mouse cells (r = 0.73 - 0.76) and via cloned human FP-receptor (r = 0.9), and rat uterus contraction (r = 0.84). These data confirm the presence of functional FP-receptors in the cat iris sphincter, which are exquisitely well coupled and which respond to a variety of FP-class PG analogs with differing potencies.

Novel Role of Androgens in Mitochondrial Fission and Apoptosis

Androgen and androgen receptors (AR) play critical roles in the proliferation of prostate cancer through transcriptional regulation of target genes. Here, we found that androgens upregulated the expression of dynamin-related protein 1 (Drp1), which is involved in the induction of mitochondrial fission, a common event in mitosis and apoptosis. Clinical tissue samples and various prostate cancer cell lines revealed a positive correlation between Drp1 and AR levels. Treatment of androgen-sensitive cells with an AR agonist, R1881, and antagonist, bicalutamide, showed that Drp1 is transcriptionally regulated by androgens, as confirmed by an AR ChIP-seq assay. Live imaging experiments using pAcGFP1-Mito stably transfected LNCaP (mito-green) cells revealed that androgen did not induce significant mitochondrial fission by itself, although Drp1 was upregulated. However, when treated with CGP37157 (CGP), an inhibitor of mitochondrial Ca2+ efflux, these cells exhibited mitochondrial fission, which was further enhanced by pretreatment with R1881, suggesting that androgen-induced Drp1 expression facilitated CGP-induced mitochondrial fission. This enhanced mitochondrial fission was correlated with increased apoptosis. Transfection with dominant-negative (DN-Drp1, K38A) rescued cells from increased apoptosis, confirming the role of androgen-induced Drp1 in the observed apoptosis with combination treatment. Furthermore, we found that CGP reduced the expression of Mfn1, a protein that promotes mitochondrial fusion, a process which opposes fission. We suggest that androgen-increased Drp1 enhanced mitochondrial fission leading to apoptosis. The present study shows a novel role for androgens in the regulation of mitochondrial morphology that could potentially be utilized in prostate cancer therapy. Mol Cancer Res; 9(8); 1067–77. ©2011 AACR.

Effects of prostaglandin F2α and latanoprost on phosphoinositide turnover, myosin light chain phosphorylation and contraction in cat iris sphincter

The effects of the ocular hypotensive agents prostaglandin F(2alpha) (PGF(2alpha)) and its analog latanoprost on intraocular pressure (IOP) in both animals and human have been investigated extensively in the last two decades. While there is general agreement that application of these PGs to the eye alters IOP by altering the aqueous humor outflow of the eye via the uveoscleral and trabecular meshwork pathways, the mechanism of action of these agents on IOP lowering remains unclear. There is evidence which suggests that myosin light kinase (MLC kinase) may be involved in the IOP-lowering effects of these agents. Thus, the purpose of the present work was to investigate in cat iris sphincter the effects of these PGs on the MLC kinase signaling pathway, inositol phosphates production, MLC phosphorylation and contraction, in order to gain more information about the mechanism through which these agents modulate smooth muscle function and lower IOP. [(3)H]myo-inositol phosphates production was measured by ion-exchange chromatography, MLC kinase activity was measured by incorporation of (32)Pi into MLC, and changes in muscle tension were recorded isometrically. PGF(2alpha) and latanoprost induced contraction in a concentration-dependent manner with EC(50) values of 18.6 and 29.9 nM, respectively, and increased inositol phosphates production in a concentration-dependent manner. At 1 microM, PGF(2alpha) and latanoprost increased inositol phosphates formation by 125 and 102% over basal, respectively. PGF(2alpha) and latanoprost increased MLC phosphorylation in a concentration- and time-dependent manner, at 1 microM and 5 min incubation, the PGs increased the MLC response by 181 and 176% over basal, respectively. In general, PGF(2alpha) was slightly more potent in inducing the biochemical and pharmacological responses. Wortmannin, ML-7 and ML-9, selective inhibitors of MLC kinase, inhibited significantly PGF(2alpha)- and latanoprost-induced MLC phosphorylation and contraction. These results demonstrate for the first time involvement of the MLC kinase pathway in the FP receptor function of this ocular tissue. Contraction-relaxation of smooth muscle alters the shape and stiffness of smooth muscle cells and MLC kinase, through myosin phosphorylation and dephosphorylation, has been shown to be involved in cytoskeletal remodeling, cytoskeletal alterations, and IOP lowering. In light of these reports and the findings presented here we suggest that alterations in the MLC kinase signaling pathway and its derived second messengers, which leads to changes in contraction-relaxation of the smooth muscles of the anterior segment, could facilitate aqueous humor outflow and thus contribute to the IOP-lowering effects of the FP-class PGs.

Ultraviolet B irradiation and activation of protein kinase D in primary mouse epidermal keratinocytes

Our previous studies demonstrated that protein kinase D (PKD), a serine/threonine kinase implicated in various cell processes, is upregulated in basal cell carcinoma (BCC), supporting a possible tumorigenic role for PKD in skin. As the greatest risk factor for BCC is sun exposure, the ability of ultraviolet B (UVB) irradiation to activate PKD in primary mouse keratinocytes was investigated. Using western analysis with two autophosphorylation-specific antibodies, we show for the first time that UVB activated PKD in a time- and dose-dependent manner. UVB-induced PKD activation was verified using an in vitro kinase assay. Furthermore, activation was reduced by antioxidant pretreatment, suggesting a link with oxidative stress. UVB-induced PKD activation was mediated primarily by Src family tyrosine kinases rather than protein kinase C (PKC), and in fact, UVB did not alter PKC-mediated transphosphorylation. UVB induced apoptosis dose dependently, and this death could be prevented by overexpression of wild-type PKD, but not mutant PKD or the empty adenovirus. Indeed, a mutant that cannot be phosphorylated by Src kinases exacerbated UVB-elicited apoptosis. Thus, our data indicate that UVB irradiation of keratinocytes induces Src-mediated activation of PKD, which protects cells from UVB-stimulated apoptosis, providing a possible explanation for the observed upregulation of PKD in BCC.

Therapeutic advantage of combining calcium channel blockers and TRAIL in prostate cancer

Disruption of intracellular calcium initiates multiple cell-damaging processes, such as apoptosis. In normal cells, the levels of Ca2+ are low in the mitochondria, whereas in apoptotic cells, Ca2+ increases. Mitochondria uptake Ca2+ via an inner membrane channel called the uniporter and extrude it into the cytoplasm through a Na+/Ca2+ exchanger. Overload of Ca2+ in the mitochondria in CGP-treated cells leads to its damage, thus affecting cellular function and survival. The goal of these experiments was to determine the importance of mitochondrial calcium ([Ca2+]m) in apoptosis of prostate cancer cells. Furthermore, we have examined the advantages of increasing the [Ca2+]m and treating the cells with tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), a potent apoptotic agent. Our results show that, under these treatment conditions, inhibiting the Na+/Ca2+ exchanger using benzothiazepin CGP-37157 (CGP) did not induce apoptosis. However, combination of CGP and TRAIL increased the apoptotic response ∼25-fold compared with control. Increase in apoptosis followed enhanced levels of [Ca2+]m and was accompanied by pronounced mitochondrial changes characteristic of mitochondria-mediated apoptosis. Experiments with calcium ionophores showed that mere increase in cytosolic and/or mitochondrial Ca2+ was not sufficient to induce apoptosis. These results have therapeutic implications as inhibitors of Na+/Ca2+ exchanger are being used for treating some neurologic and cardiologic ailments, and TRAIL induces apoptosis preferentially in cancer cells. Furthermore, this system provides an excellent model to investigate the role of [Ca2+]m in apoptosis. [Mol Cancer Ther 2006;5(8):1958–66]

The Antipsoriatic Agent Monomethylfumarate Has Antiproliferative, Prodifferentiative, and Anti-Inflammatory Effects on Keratinocytes

Monomethylfumarate (MMF) is thought to be the bioactive ingredient of the drug Fumaderm (Biogen Idec, Cambridge, MA), licensed in Germany since 1994 for the treatment of moderate-to-severe psoriasis. Psoriasis is a common inflammatory hyperproliferative skin disorder that involves cross-talk between different cell types, including immune cells and keratinocytes. Psoriatic lesions are characterized by hyperproliferation, aberrant differentiation, and inflammation, with the psoriatic cytokine network maintained by communication between immune cells and keratinocytes. Recently, there is increasing evidence regarding the pivotal role of keratinocytes in mediating the disease process, and these cells can be regarded as safe therapeutic targets. From the data available on human subjects treated with Fumaderm, MMF is an effective antipsoriatic agent with known effects on immune cells. However, little is known about its direct effects on keratinocytes. We hypothesized that MMF has direct antiproliferative, prodifferentiative, and anti-inflammatory effects on keratinocytes. Indeed, MMF dose-dependently inhibited [3H]thymidine incorporation into DNA, indicating a direct antiproliferative action on keratinocytes. MMF significantly increased the protein level of keratin 10, the early keratinocyte differentiation marker, and the activity of transglutaminase, a late differentiation marker. These results are consistent with an ability of MMF to promote keratinocyte differentiation and inhibit proliferation, thereby improving psoriatic lesions. In 12-O-tetradecanoylphorbol-13-acetate (TPA)–induced keratinocytes, MMF significantly inhibited the expression of the proinflammatory cytokines, tumor necrosis factor-α (TNFα), interleukin-6, and interleukin-1α as well as the production of TNFα. Our results support the notion that MMF has direct antiproliferative, prodifferentiative, and anti-inflammatory effects on keratinocytes, highlighting its potential use as a multifactorial antipsoriatic agent.

Mitofusin 1 degradation is induced by a disruptor of mitochondrial calcium homeostasis, CGP37157: a role in apoptosis in prostate cancer cells.

Mitochondria constantly divide (mitochondrial fission) and fuse (mitochondrial fusion) in a normal cell. Disturbances in the balance between these two physiological processes may lead to cell dysfunction or to cell death. Induction of cell death is the prime goal of prostate cancer chemotherapy. Our previous study demonstrated that androgens increase the expression of a mitochondrial protein involved in fission and facilitate an apoptotic response to CGP37157 (CGP), an inhibitor of mitochondrial calcium efflux, in prostate cancer cells. However, the regulation and role of mitochondrial fusion proteins in the death of these cells have not been examined. Therefore, our objective was to investigate the effect of CGP on a key mitochondrial fusion protein, mitofusin 1 (Mfn1), and the role of Mfn1 in prostate cancer cell apoptosis. We used various prostate cancer cell lines and western blot analysis, qRT-PCR, siRNA, M30 apoptosis assay and immunoprecipitation techniques to determine mechanisms regulating Mfn1. Treatment of prostate cancer cells with CGP resulted in selective degradation of Mfn1. Mfn1 ubiquitination was detected following immunoprecipitation of overexpressed Myc-tagged Mfn1 protein from CGP-treated cells, and treatment with the proteasomal inhibitor lactacystin, as well as siRNA-mediated knockdown of the E3 ubiquitin ligase March5, protected Mfn1 from CGP-induced degradation. These data indicate the involvement of the ubiquitin-proteasome pathway in CGP-induced degradation of Mfn1. We also demonstrated that downregulation of Mfn1 by siRNA enhanced the apoptotic response of LNCaP cells to CGP, suggesting a likely pro-survival role for Mfn1 in these cells. Our results suggest that manipulation of mitofusins may provide a novel therapeutic advantage in treating prostate cancer.