Potent growth suppressive activity of curcumin in human breast cancer cells: Modulation of Wnt/β-catenin signaling

Abnormal activation of the Wnt/β-catenin signaling pathway and subsequent upregulation of β-catenin driven downstream targets—c-Myc and cyclin D1 is associated with development of breast cancer. The objective of our study was to determine if curcumin could modulate the key elements of Wnt pathway in breast cancer cells; an effect that might underscore its usefulness for chemoprevention/treatment of this malignancy. Curcumin showed a cytotoxic effect on MCF-7 cells with 50% inhibitory concentration (IC₅₀) of 35 μM; while IC₅₀ for MDA-MB-231 cells was 30 μM. Treatment with low cytostatic dose of 20 μM curcumin showed G₂/M arrest in both breast cancer cells. The effect of curcumin (20 μM) treatment on expression of Wnt/β-catenin pathway components in breast cancer cells (MCF-7 and MDA-MB-231) was analyzed by immunofluorescence and Western blotting. Curcumin was found to effectively inhibit the expression of several Wnt/β-catenin pathway components—disheveled, β-catenin, cyclin D1 and slug in both MCF-7 and MDA-MB-231. Immunofluorescence analysis showed that curcumin markedly reduced the nuclear expression of disheveled and β-catenin proteins. Further, the protein levels of the positively regulated β-catenin targets—cyclin D1 and slug, were downregulated by curcumin treatment. The expression levels of two integral proteins of Wnt signaling, GSK3β and E-cadherin were also altered by curcumin treatment. In conclusion, our data demonstrated that the efficacy of curcumin in inhibition of cell proliferation and induction of apoptosis might occur through modulation of β-catenin pathway in human breast cancer cells.     

Synergistic anti-proliferative effects of vitamin D derivatives and 9-cis retinoic acid in SH-SY5Y human neuroblastoma cells

This study examines the effect of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃], two vitamin D analogues (KH 1060 and EB 1089, which are 20-epi-22-oxa and 22,24-diene-analogues, respectively), 9-cis retinoic acid and all-trans retinoic acid on proliferation of SH-SY5Y human neuroblastoma cells, after treatment for 7 days. Cell number did not change when the cells were incubated with 1, 10 or 100 nM 1,25(OH)₂D₃ or its derivatives, but significantly decreased in the presence of the two retinoids (0.001–10 μM final concentration). A synergistic inhibition was observed, when SH-SY5Y cells were treated combining 0.1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060, and 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM EB 1089. Acetylcholinesterase activity showed a significant increase, in comparison with controls, after treatment of the cells for 7 days with 0.1 or 1 μM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060 or 10 nM EB 1089. This increase was synergistic, combining 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or EB 1089. The levels of the c-myc encoded protein remarkably decreased after treatment of SH-SY5Y cells for 1, 3, 7 days with 0.1 and 1 μM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060 or 10 nM EB 1089. In particular, the association of 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM EB 1089 resulted in a synergistic c-myc inhibition, in comparison with that obtained in the presence of the retinoid alone. These findings may have therapeutic implications in human neuroblastoma.     

Effect of modulators of cytoskeletal function on desensitization and recovery of PGE2-responsive ovarian adenylate cyclase

Exposure of cultured Graafian follicles to PGE₂ for 20 h resulted in a loss of the cyclic AMP response to fresh hormone. This desensitization was prevented by addition to the medium of D₂O (25–50%) or Li⁺ (0.6 – 6 mM), agents believed to stabilize microtubules, as well as by phalloidin (1.0 – 10 μM), believed to stabilize the polymerized state of actin, in a dose-dependent manner.The spontaneous recovery of responsiveness to PGE₂ upon incubation of refractory follicles for 6 h in hormone-free medium was prevented by addition to the medium of cytochalasin B (CB; 3 μg/ml) or of the actin-binding myosin subfragment HMM S-1 (80 μg/ml) or of anti-actin serum; viz. by agents likely to interfere with microfilament function. D₂O (50%) caused morphological damage to the inner layer of the membrana granulosa and severe depression of protein synthesis. The other drugs used (phalloidin, LiCl and cytochalasin B) had no such effects. Resensitization of refractory follicles was also prevented by cycloheximide (10 μg/ml) and by actinomycin D (10 μg/ml). It is speculated that the recovery process may involve the insertion of a newly synthesized protein, such as PG-receptor, into the membrane by a mechanism dependent on microfilament action.     

Tyrosine Kinase Inhibitors Inhibit Multiple Steps of the Cell Cycle of Vascular Smooth Muscle Cells

Protein tyrosine kinase (PTK) inhibitors have been reported to inhibit proliferation of vascular smooth muscle cells (SMC). To elucidate the made of this inhibition, the effects on the cell cycle of cultured vascular SMC of three PTK inhibitors with different modes of action (methyl 2,5-dihydroxyeinnamate, genistein, and herbimycin A) were studied. Rat aortic SMC were synchronized to the G0 phase of the cell cycle and then released to proceed through the cell cycle by the addition of platelet-derived growth factor (PDGF), and [³H]thymidine incorporation into DNA was measured. The three PTK inhibitors all inhibited PDGF-induced DNA synthesis in a dose-dependent fashion, with IC₅₀ values of 4.7 ± 1.4 μM for methyl 2,5.dihydroxycinnamate, 6.7 ± 2.5 μM for genistein, and 0.17 ± 0.07 μM for herbimycin A. Time course studies suggested that the agents inhibited early G1 phase but not the G0-G1 transition. The lack of effect on the G0-G1 transition was also supported by the finding that the agents did not inhibit the ligand-induced autophosphorylation of PDGF receptor nor the induction of c-fos mRNA at concentrations which were sufficient to inhibit DNA synthesis. PTK inhibitors inhibited progression of the S phase when they were added to SMC that had been arrested at the G1-S border with hydroxyurea. Methyl 2,5-dihydroxyeinnamate also blocked the M phase when it was added to SMC cultured in the presence of 10% fetal calf serum, while genistein and herbimycin A did not inhibit the M phase under the same experimental conditions. In accordance with our previous observation, methyl 2,5-dihydroxycinnamate impaired microtubule networks and formation of the mitotic spindle during the M phase. Our findings indicated that PTK inhibitors inhibit multiple steps of the vascular SMC cell cycle.     

Novel oxindole/benzofuran hybrids as potential dual CDK2/GSK-3β inhibitors targeting breast cancer: design,synthesis, biological evaluation,and in silico studies

The serine/threonine protein kinases CDK2 and GSK-3β are key oncotargets in breast cancer cell lines, therefore, in the present study three series of oxindole-benzofuran hybrids were designed and synthesised as dual CDK2/GSK-3β inhibitors targeting breast cancer (5a–g, 7a–h, and 13a–b). The N¹-unsubstituted oxindole derivatives, series 5, showed moderate to potent activity on both MCF-7 and T-47D breast cancer cell lines. Compounds 5d–f showed the most potent cytotoxic activity with IC₅₀ of 3.41, 3.45 and 2.27 μM, respectively, on MCF-7 and of 3.82, 4.53 and 7.80 μM, respectively, on T-47D cell lines, in comparison to the used reference standard (staurosporine) IC₅₀ of 4.81 and 4.34 μM, respectively. On the other hand, the N¹-substituted oxindole derivatives, series 7 and 13, showed moderate to weak cytotoxic activity on both breast cancer cell lines. CDK2 and GSK-3β enzyme inhibition assay of series 5 revealed that compounds 5d and 5f are showing potent dual CDK2/GSK-3β inhibitory activity with IC₅₀ of 37.77 and 52.75 nM, respectively, on CDK2 and 32.09 and 40.13 nM, respectively, on GSK-3β. The most potent compounds 5d–f caused cell cycle arrest in the G2/M phase in MCF-7 cells inducing cell apoptosis because of the CDK2/GSK-3β inhibition. Molecular docking studies showed that the newly synthesised N¹-unsubstituted oxindole hybrids have comparable binding patterns in both CDK2 and GSK-3β. The oxindole ring is accommodated in the hinge region interacting through hydrogen bonding with the backbone CO and NH of the key amino acids Glu81 and Leu83, respectively, in CDK2 and Asp133 and Val135, respectively, in GSK-3β. Whereas, in series 7 and 13, the N¹-substitutions on the oxindole nucleus hinder the compounds from achieving these key interactions with hinge region amino acids what rationalises their moderate to low anti-proliferative activity.     

Discovery of orally active chalcones as histone lysine specific demethylase 1 inhibitors for the treatment of leukaemia

Histone lysine specific demethylase 1 (LSD1) has emerged as an attractive molecule target for the discovery of potently anticancer drugs to treat leukaemia. In this study, a series of novel chalcone derivatives were designed, synthesised and evaluated for their inhibitory activities against LSD1 in vitro. Among all these compounds, D6 displayed the best LSD1 inhibitory activity with an IC₅₀ value of 0.14 μM. In the cellular level, compound D6 can induce the accumulation of H3K9me1/2 and inhibit cell proliferation by inactivating LSD1. It exhibited the potent antiproliferative activity with IC₅₀ values of 1.10 μM, 3.64 μM, 3.85 μM, 1.87 μM, 0.87 μM and 2.73 μM against HAL-01, KE-37, P30-OHK, SUP-B15, MOLT-4 and LC4-1 cells, respectively. Importantly, compound D6 significantly suppressed MOLT-4 xenograft tumour growth in vivo, indicating its great potential as an orally bioavailable candidate for leukaemia therapy.     

On the regulation of tyrosine transaminase, glutamatic dehydrogenase and aspartic transaminase in Tetrahymena

Aspartic transaminase, tyrosine transaminase, lactic dehydrogenase, and glutamic dehydrogenase were studied in Tetrahymena pyriformis in order to gain a better understanding of the control of the entrance and exit of metabolic intermediates to and from the major carbohydrate pathways. Glucose decreased the activity of aspartic transaminase, tyrosine transaminase and glutamic dehydrogenase but not lactic dehydrogenase. Actinomycin D (6 and 12 μg/ml) blocked the decrease in glutamic dehydrogenase and aspartic transaminase activity caused by glucose; 12 μg/ml partially prevented the decrease in tyrosine transaminase activity. Actinomycin D alone had little effect on enzyme activity. Uracil incorporation into RNA was doubled by 6 μg/ml actinomycin D, a concentration which did not alter the RNA content of the cells. At 12 μg/ml this drug caused a small decrease in RNA spec. act. Cycloheximide at 10 μg/ml, a concentration which inhibited protein synthesis by 70%, caused a three-fold increase in aspartic transaminase and a two-fold increase in glutamic dehydrogenase. In the presence of both cycloheximide and glucose, the drug effect predominated. Thus both actinomycin D and cycloheximide blocked the glucose-induced decrease in enzyme activity. These results suggest that the levels of aspartic transaminase, glutamic dehydrogenase, and probably tyrosine transaminase are regulated at least in part by a degradative control system.     

Antagonism by ETYA of the effects of leukotrienes on ileum and lung parenchymal strips independent of effects on arachidonic acid metabolism

5,8,11,14-Eicosatetraynoic acid (ETYA), a compound which inhibits both the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism, antagonized the contraction of segments of guinea-pig ileal longitudinal muscle produced by SRS-A (IC₅₀ = 2.73 μM). This activity was unaffected by pretreatment of the tissues with 10 μM indomethacin. Phenidone, another mixed cyclooxgenese-lipoxygenese inhibitor, was inactive. FPL-55712, an SRS-A antagonist, was a very potent inhibitor (IC₅₀ = 0.011 μM).BW755C and NDGA nonselectively inhibited the contractions of the guinea-pig ileal longitudinal muscle induced by SRS-A or histamine.ETYA antagonized the contraction of the guinea-pig ileal strip produced by 6 nM synthetic LTC₄ (IC₅₀ = 9.3 μM). FPL-55712 demonstrated an IC₅₀ of 0.3 μM in a similar series of experiments. ETYA, 1, 3 or 10 μM did not inhibit the contractions elicited by 0.5 μM of histamine.This was not a tissue-selective effect since 100 μM ETYA antagonized the LTC₄-induced contraction of the guinea-pig lung parenchymal strip preparation.These data demonstrate that ETYA antagonized the contractile effect of the leukotrienes on tissues from the gastrointestinal tract and lung. Furthermore, the inability of indomethacin or phenidone to inhibit the contractile response suggests that antagonism by ETYA may occur by a mechanism independent of cyclooxygenase and lipoxygenase enzymes.     

Contractile effects of prostaglandin E2 in rat rectum: Sensitivity to the prostaglandin antagonists diphloretin phosphate and SC 19220

Prostaglandin E₂ (PGE₂) applied cumulatively (1 nM − 1 μM) induced concentration-dependent tonic contractions in the longitudinal muscle of isolated rat rectum. The PGE₂ effects were not altered by guanethidine (50 μM), whereas atropine (3 μM), guanethidine plus atropine or tetrodotoxin (0.1 μM) reduced them to an almost equal extent and increased the EC₅₀ values for PGE₂. The after-contractions following electrical stimulation were enhanced by PGE₂ (10 nM) and inhibited by atropine. Diphloretin phosphate (DPP, 100 μM) shifted the regression lines for PGE₂ to the right in both untreated and tetrodotoxin-treated preparations, and thereby increased the EC₅₀ values. Slopes of the concentration-effect lines for PGE₂ before and after DPP differed in the presence of tetrodotoxin. The regression line for PGE₂ with SC 19220 (100 μM) in tetrodotoxin-treated preparations was shifted to the right in a parallel fashion. It is concluded that PGE₂ exerted both a neural (cholinergic) and a smooth muscle effect. There may be a competitive antagonism between SC 19220 and PGE₂ but the block by DPP may be nonselective.     

Benzoxazole derivatives as new VEGFR-2 inhibitors and apoptosis inducers: design,synthesis, in silico studies,and antiproliferative evaluation

In this study, a set of novel benzoxazole derivatives were designed, synthesised, and biologically evaluated as potential VEGFR-2 inhibitors. Five compounds (12d, 12f, 12i, 12l, and 13a) displayed high growth inhibitory activities against HepG2 and MCF-7 cell lines and were further investigated for their VEGFR-2 inhibitory activities. The most potent anti-proliferative member 12 l (IC₅₀ = 10.50 μM and 15.21 μM against HepG2 and MCF-7, respectively) had the most promising VEGFR-2 inhibitory activity (IC₅₀ = 97.38 nM). A further biological evaluation revealed that compound 12l could arrest the HepG2 cell growth mainly at the Pre-G1 and G1 phases. Furthermore, compound 12l could induce apoptosis in HepG2 cells by 35.13%. likely, compound 12l exhibited a significant elevation in caspase-3 level (2.98-fold) and BAX (3.40-fold), and a significant reduction in Bcl-2 level (2.12-fold). Finally, docking studies indicated that 12l exhibited interactions with the key amino acids in a similar way to sorafenib.     

Low-Concentration Arsenic Trioxide Inhibits Skeletal Myoblast Cell Proliferation via a Reactive Oxygen Species-Independent Pathway

Myoblast proliferation and differentiation are essential for skeletal muscle regeneration. Myoblast proliferation is a critical step in the growth and maintenance of skeletal muscle. The precise action of inorganic arsenic on myoblast growth has not been investigated. Here, we investigated the in vitro effect of inorganic arsenic trioxide (As₂O₃) on the growth of C2C12 myoblasts. As₂O₃ decreased myoblast growth at submicromolar concentrations (0.25–1 μM) after 72 h of treatment. Submicromolar concentrations of As₂O₃ did not induce the myoblast apoptosis. Low-concentration As₂O₃ (0.5 and 1 μM) significantly suppressed the myoblast cell proliferative activity, which was accompanied by a small proportion of bromodeoxyuridine (BrdU) incorporation and decreased proliferating cell nuclear antigen (PCNA) protein expression. As₂O₃ (0.5 and 1 μM) increased the intracellular arsenic content but did not affect the reactive oxygen species (ROS) levels in the myoblasts. Cell cycle analysis indicated that low-concentrations of As₂O₃ inhibited cell proliferation via cell cycle arrest in the G1 and G2/M phases. As₂O₃ also decreased the protein expressions of cyclin D1, cyclin E, cyclin B1, cyclin-dependent kinase (CDK) 2, and CDK4, but did not affect the protein expressions of p21 and p27. Furthermore, As₂O₃ inhibited the phosphorylation of Akt. Insulin-like growth factor-1 significantly reversed the inhibitory effect of As₂O₃ on Akt phosphorylation and cell proliferation in the myoblasts. These results suggest that submicromolar concentrations of As₂O₃ alter cell cycle progression and reduce myoblast proliferation, at least in part, through a ROS-independent Akt inhibition pathway.     

Purine receptors are required for DHA-mediated neuroprotection against oxygen and glucose deprivation in hippocampal slices

Docosahexaenoic acid (DHA) is important for central nervous system function during pathological states such as ischemia. DHA reduces neuronal injury in experimental brain ischemia; however, the underlying mechanisms are not well understood. In the present study, we investigated the effects of DHA on acute hippocampal slices subjected to experimental ischemia by transient oxygen and glucose deprivation (OGD) and re-oxygenation and the possible involvement of purinergic receptors as the mechanism underlying DHA-mediated neuroprotection. We observed that cellular viability reduction induced by experimental ischemia as well as cell damage and thiobarbituric acid reactive substances (TBARS) production induced by glutamate (10 mM) were prevented by hippocampal slices pretreated with DHA (5 μM). However, glutamate uptake reduction induced by OGD and re-oxygenation was not prevented by DHA. The beneficial effect of DHA against cellular viability reduction induced by OGD and re-oxygenation was blocked with PPADS (3 μM), a nonselective P2X_1–5 receptor antagonist as well as with a combination of TNP-APT (100 nM) plus brilliant blue (100 nM), which blocked P2X₁, P2X₃, P2X_2/3, and P2X₇ receptors, respectively. Moreover, adenosine receptors blockade with A₁ receptor antagonist DPCPX (100 nM) or with A_2B receptor antagonist alloxazine (100 nM) inhibited DHA-mediated neuroprotection. The addition of an A_2A receptor antagonist ZM241385 (50 nM), or A₃ receptor antagonist VUF5574 (1 μM) was ineffective. Taken together, our results indicated that neuroprotective actions of DHA may depend on P2X, A₁, and A_2B purinergic receptors activation. Our results reinforce the notion that dietary DHA may act as a local purinergic modulator in order to prevent neurodegenerative diseases.     

Delayed inhibition of DNA synthesis in mouse jejunum by low doses of actinomycin D

Actinomycin D (0.008 μg/gm of body weight) injected intraperitoneally every two hours, produced a prompt 50% inhibition of RNA synthesis in the jejunum of mice, and a delayed inhibition of DNA synthesis, that reached its maximum inhibition (68% of control values) 4.5 hours after the first injection of actinomycin D. Autoradiographic studies indicated that this low level of actinomycin D inhibited a step in the G₁ phase of the cell cycle, preventing the initiation, but not affecting the continuation, of DNA biosynthesis. The activity of DNA polymerase was not affected under these conditions. The results are substantially similar to those previously obtained with Ehrlich ascites cells growing in the peritoneal cavity of mice and can be interpreted as indicating that in the G₁ phase of dividing cells there is an actinomycin sensitive step whose inhibition prevents the entrance of cells into the DNA-synthesis phase.     

Synthesis and biological evaluation of 3,4-diaryl-5-aminoisoxazole derivatives

A series of cis-restricted 3,4-diaryl-5-aminoisoxazoles have been synthesized and evaluated for their biological activities. Among them, compound 11a and 13a displayed potent cytotoxic activities in vitro against five human cancer cell lines with IC₅₀ values in the low micromolar range and two compounds inhibited tubulin polymerization with IC₅₀ value of 1.8, and 2.1 μM, respectively, similar to that of CA-4. Compound 13a could arrest at the G2/M phase of the cell cycle at the concentration of 0.1 and 1.0 μM and induce apoptosis at 0.1–1.0 μM.     

Inhibition of Excessive Cell Proliferation by Calcilytics in Idiopathic Pulmonary Arterial Hypertension

Idiopathic pulmonary arterial hypertension (IPAH) is a rare and progressive disease of unknown pathogenesis. Vascular remodeling due to excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs) is a critical pathogenic event that leads to early morbidity and mortality. The excessive cell proliferation is closely linked to the augmented Ca²⁺ signaling in PASMCs. More recently, we have shown by an siRNA knockdown method that the Ca²⁺-sensing receptor (CaSR) is upregulated in PASMCs from IPAH patients, involved in the enhanced Ca²⁺ response and subsequent excessive cell proliferation. In this study, we examined whether pharmacological blockade of CaSR attenuated the excessive proliferation of PASMCs from IPAH patients by MTT assay. The proliferation rate of PASMCs from IPAH patients was much higher (~1.5-fold) than that of PASMCs from normal subjects and patients with chronic thromboembolic pulmonary hypertension (CTEPH). Treatment with NPS2143, an antagonist of CaSR or calcilytic, clearly suppressed the cell proliferation in a concentration-dependent manner (IC₅₀ = 2.64 μM) in IPAH-PASMCs, but not in normal and CTEPH PASMCs. Another calcilytic, Calhex 231, which is structurally unrelated to NPS2143, also concentration-dependently inhibited the excessive proliferation of IPAH-PASMCs (IC₅₀ = 1.89 μM). In contrast, R568, an activator of CaSR or calcimimetic, significantly facilitated the proliferation of IPAH-PASMCs (EC₅₀ = 0.33 μM). Similar results were obtained by BrdU incorporation assay. These results reveal that the excessive PASMC proliferation was modulated by pharmacological tools of CaSR, showing us that calcilytics are useful for a novel therapeutic approach for pulmonary arterial hypertension.     

Prostaglandins E1 and E2 stimulate the proliferation of pulmonary artery smooth muscle cells

Prostaglandins E₁ and E₂ are thought to be inhibitors of the growth of systemic vascular smooth muscle cells (SMC). However, their effect on the proliferation of SMC from the pulmonary artery (PA) has not been described and was the subject of this investigation. Cultures of bovine PA SMC were exposed to PGE₁ and PGE₂ under various conditions and their growth was assessed. PGE₁ and PGE₂ did not inhibit the growth of PA SMC in 10% fetal calf serum (FCS), but instead caused a dose dependent (10 ng - 1 μg/ml) increase in [³H]-thymidine incorporation when added to cultures containing 0.5% FCS; the highest doses resulted in 95% and 75% increases in [³H]-thymidine uptake at 24 hours with PGE₁ and PGE₂ respectively. This was accompanied by a modest increase in actual cell numbers (e.g., 20% with 1 μg/ml PGE₁). Furthermore, PGE₁ could mimic insulin-like growth factor (IGF-1) by potentiating the stimulation of SMC growth by fibroblast growth factor, suggesting that PGE₁ may act as a progression factor in the growth cycle of these cells. There was, however, no effect of PGE₁ on the proliferation of bovine aortic SMC. We conclude that, contrary to most reported effects on systemic SMC, PGE₁ and PGE₂ do not inhibit the proliferation of PA SMC but rather stimulate it.     

Potentiation of anti-aggregating activity of PGI2 by 7-ethoxycarbonyl-6,8-dimethyl-4-hydroxymethyl-1(2H)-phthalazinone (EG-626) in rabbit platelets in vitro

Anti-aggregating activity of 7-ethoxycarbonyl-6,8-dimethyl-4-hydroxymethyl-1(2H)-phthalazinone (EG-626) was tested using rabbit platelets in vitro. EG-626 alone, when added before, prevented platelet aggregation induced by ADP, as did PGI₂, papaverine and dipyridamole. Spontaneous disaggregation was also accelerated when EG-626 was added after the maximal aggregation induced by ADP. EG-626 alone also inhibited platelet aggregation induced by collagen and arachidonic acid. ID₅₀s of these agents in ADP-induced aggregation were 7–9 nM for PGI₂, 223 μM for EG-626, 266 μM for papaverine and 957 μM for dipyridamole. When EG-626 was used in combination with PGI₂, a threshold dose (50 μM) of EG-626 potentiated the anti-aggregating effect of subthreshold dose (3 nM) of PGI₂ upto 100% inhibition in collagen-induced platelet aggregation. The marked potentiating effect of EG-626 was accompanied by an accumulation of cyclic AMP in the platelets. These effects might be due to inhibition of phosphodiesterase. Papaverine and dipyridamole, other phosphodiesterase inhibitors, also potentiated the anti-aggregating activity of PGI₂. The activity of papaverine, however, was one eighth of EG-626 and that of dipyridamole was much less. The most effective combination of PGI₂ and EG-626 to induce 50% inhibition was obtained with 20% of ID₅₀ of each agent, whereas that of PGI₂ and papaverine or dipyridamole was 39 or 41%, respectively.     

Synergistic regulation of endogenous TRPM2 channels by adenine dinucleotides in primary human neutrophils

The Ca²⁺-permeable TRPM2 channel is a dual function protein that is activated by intracellular ADPR through its enzymatic pyrophosphatase domain with Ca²⁺ acting as a co-factor. Other TRPM2 regulators include cADPR, NAADP and H₂O₂, which synergize with ADPR to potentiate TRPM2 activation. Although TRPM2 has been thoroughly characterized in overexpression or cell-line systems, little is known about the features of TRPM2 in primary cells. We here characterize the regulation of TRPM2 activation in human neutrophils and report that ADPR activates TRPM2 with an effective half-maximal concentration (EC₅₀) of 1 μM. Potentiation by Ca²⁺ is dose-dependent with an EC₅₀ of 300 nM. Both cADPR and NAADP activate TRPM2, albeit with lower efficacy than in the presence of subthreshold levels of ADPR (100 nM), which significantly shifts the EC₅₀ for cADPR from 44 to 3 μM and for NAADP from 95 to 1 μM. TRPM2 activation by ADPR can be suppressed by AMP with an IC₅₀ of 10 μM and cADPR-induced activation can be blocked by 8-Bromo-cADPR. We further show that 100 μM H₂O₂ enables subthreshold concentrations of ADPR (100 nM) to activate TRPM2. We conclude that agonistic and antagonistic characteristics of TRPM2 as seen in overexpression systems are largely compatible with the functional properties of TRPM2 currents measured in human neutrophils, but the potencies of agonists in primary cells are significantly higher.