N,N-Disubstituted 4-sulfamoylbenzoic Cid Derivatives as Inhibitors of Cytosolic Phospholipase A2α: Synthesis, Aqueous Solubility, and Activity in a Vesicle and a Whole Blood Assay.

BACKGROUND: Cytosolic phospholipase A2α (cPLA2α) is the key enzyme that initiates the arachidonic acid cascade through which proinflammatory lipid mediators can be formed. Therefore, cPLA2α is considered an interesting target for the development of anti-inflammatory drugs. Although several effective inhibitors of the enzyme have been developed, none of them has yet reached clinical application. OBJECTIVE: Recently, we have prepared new 4-sulfamoylbenzoic acid derivatives based on a cPLA2α inhibitor found in a ligand-based virtual screening. The most effective of these compounds were now subjected to further variations in which the substitution pattern on the sulfamoyl nitrogen atom was changed. METHODS: The new compounds were tested in vitro in a vesicle assay for cPLA2α inhibition as well as for their water solubility, metabolic stability, and selectivity towards related enzymes. In addition, they were evaluated ex vivo in a whole blood assay in which metabolites of the arachidonic acid cascade formed after activation of cPLA2α were quantified using a combined online dilution/online solid phase extraction HPLC-MS method. RESULTS: Inhibitors with submicromolar inhibitory in vitro potency were found with favourable water solubility and selectivity. However, their efficacy did not match that of the highly effective, known, structurally related cPLA2a inhibitor giripladib, which was also tested as a reference. One advantage of some of the new compounds compared to giripladib was their significantly improved water solubility. When analyzing the substances in the ex vivo whole blood assay, it was found that the obtained inhibition data correlated better with the in vivo results when the phorbol ester 12-Otetradecanoylphorbol-13-acetate was used for activation of the enzyme in the blood cells instead of the calcium ionophore A23187. CONCLUSION: New compounds with good activity towards cPLA2α and reasonable physicochemical properties were identified. Overall, the results obtained could be helpful in the development of clinically applicable inhibitors of this enzyme.

HIV latency-reversing activity of latex from Euphorbia umbellata (Pax) Bruyns and three diterpenes isolated from this species.

Two unusual phorbol esters, namely 20-deoxyphorbol-3,4,12-triacetate-13-phenylacetate (1) and phorbol-3,4,12,13-tetraacetate-20-phenylacetate (2) plus ingol-3,8,12-triacetate-7-phenylacetate (3) were isolated from the latex of Euphorbia umbellata and identified by HRESIMS and 2D NMR. Compound 1 is herein described for the first time. Assignment of the phenylacetyl group at C-7 in compound 3 was suggested by the HMBC and NOESY spectra obtained in pyridine-d5. In addition to the latex and its distinct terpenoid fractions, the isolated compounds were tested as latent reversal agents against HIV-1-infected J-Lat cells, with reference to phorbol-12-myristate-13-acetate and ingenol-B. Compound 2 reverted 75-80% the viral latency on the GFP-positive cells, resulting EC50 3.70 µg/mL (SI 6.7), while 1 induced 34-40% reactivation at the same concentration range (4-20 µg/mL). The ingol derivative 3 was ineffective. Phorbol esters were confirmed as effective constituents in the latex since the fraction containing them was 2.4-fold more active than the lyophilised latex at the lowest concentration assayed.

In vitro toxicity of latex, its terpenoidal fractions and isolated phorbol esters from Euphorbia umbellata (Pax) Bruyns on monocytic and melanoma cells.

In Brazil, latex from Euphorbia umbellata (African milk tree) has been increasingly used in folk medicine to treat several types of cancer, including melanoma. The effect of lyophilized latex (LL), its hydroethanolic extract (E80), triterpene (F-TRI)- and diterpene (F-DIT)-enriched fractions, along with six isolated phorbol esters from LL and phorbol 12-myristate 13-acetate (PMA) on J774A.1, THP-1, SK-MEL-28, and B16-F10 cell line viability were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. The compounds were identified by 2D-NMR and HRESIMS. The effect of the LL, extract and fractions on cell viability was also assessed through a resazurin reduction assay. At 100 µg/ml, LL, and its fractions moderately inhibited J774A.1 (37.5-59.5%) and THP-1 (12.6-43.6%) metabolism. LL (IC50 70 µg/ml) and F-TRI (IC50 68 µg/ml) were barely more effective against B16-F10 cells, and only F-TRI exerted an inhibitory effect on SK-MEL-28 cells (IC50 66-75 µg/ml). The samples did not effectively inhibit THP-1 growth (IC50 69-87 µg/ml, assessed by MTT). B16-F10 was susceptible to PMA (IC50 53 µM) and two 12-phenylacetate esters (IC50 56-60 µM), while SK-MEL-28 growth was inhibited (IC50 58 µM) by one of these kinds of esters with an additional 4ß-deoxy structure. Synagrantol A (IC50 39 µM) was as effective as PMA (IC50 47 µM) in inhibiting J774A.1 growth in a dose-dependent manner. Furthermore, an in silico study with target receptors indicated a high interaction of the compounds with the PKC proteins. These results provide useful knowledge on the effect of tigliane-type diterpenes on tumor cell from the perspective of medicinal chemistry.

Effect of Kaempferol on Modulation of Vascular Contractility Mainly through PKC and CPI-17 Inactivation.

In this study, we investigated the efficacy of kaempferol (a flavonoid found in plants and plant-derived foods such as kale, beans, tea, spinach and broccoli) on vascular contractibility and aimed to clarify the detailed mechanism underlying the relaxation. Isometric contractions of divested muscles were stored and linked with western blot analysis which was carried out to estimate the phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and phosphorylation-dependent inhibitory protein for myosin phosphatase (CPI-17) and to estimate the effect of kaempferol on the RhoA/ROCK/CPI-17 pathway. Kaempferol conspicuously impeded phorbol ester-, fluoride- and a thromboxane mimetic-derived contractions regardless of endothelial nitric oxide synthesis, indicating its direct effect on smooth muscles. It also conspicuously impeded the fluoride-derived elevation in phospho-MYPT1 rather than phospho-CPI-17 levels and phorbol 12,13-dibutyrate-derived increase in phospho-CPI-17 and phospho-ERK1/2 levels, suggesting the depression of PKC and MEK activities and subsequent phosphorylation of CPI-17 and ERK1/2. Taken together, these outcomes suggest that kaempferol-derived relaxation incorporates myosin phosphatase retrieval and calcium desensitization, which appear to be modulated by CPI-17 dephosphorylation mainly through PKC inactivation.

PKC regulates αKlotho gene expression in MDCK and NRK-52E cells.

Particularly expressed in the kidney, αKlotho is a transmembrane protein that acts together with bone hormone fibroblast growth factor 23 (FGF23) to regulate renal phosphate and vitamin D homeostasis. Soluble Klotho (sKL) is released from the transmembrane form and controls various cellular functions as a paracrine and endocrine factor. αKlotho deficiency accelerates aging, whereas its overexpression favors longevity. Higher αKlotho abundance confers a better prognosis in cardiovascular and renal disease owing to anti-inflammatory, antifibrotic, or antioxidant effects and tumor suppression. Serine/threonine protein kinase C (PKC) is ubiquitously expressed, affects several cellular responses, and is also implicated in heart or kidney disease as well as cancer. We explored whether PKC is a regulator of αKlotho. Experiments were performed in renal MDCK or NRK-52E cells and PKC isoform and αKlotho expression determined by qRT-PCR and Western Blotting. In both cell lines, PKC activation with phorbol ester phorbol-12-myristate-13-acetate (PMA) downregulated, while PKC inhibitor staurosporine enhanced αKlotho mRNA abundance. Further experiments with PKC inhibitor Gö6976 and RNA interference suggested that PKCγ is the major isoform for the regulation of αKlotho gene expression in the two cell lines. In conclusion, PKC is a negative regulator of αKlotho gene expression, an effect which may be relevant for the unfavorable effect of PKC on heart or kidney disease and tumorigenesis.

Biological evaluation of indolactams for in vitro bryostatin 1-like activity.

Small molecule activators of protein kinase C (PKC) have traditionally been classified as either tumor promoters or suppressors. Although bryostatin 1 has well established anti-cancer activity, most natural products that target the PKC regulator domain exhibit tumor promotion properties. In this study, we examine a focused library of indolactam analogues in cell-based assays to establish the structural features of the scaffold that enhance bryostatin 1-like activity. These systematic biological assessments identified specific indole substitution patterns that impart diminished tumor promotion behavior in vitro for indolactam analogues, while still maintaining nanomolar potency for PKC.

Biphasic alcoholysis coupled with high-speed countercurrent chromatography for high performance on separating phorbol from Croton tiglium Linn extracts.

Phorbol is a tetracyclic diterpenoid found in Euphorbia tirucalli, Croton tiglium, and Rehmannia glutinosa, and is nuclear of various phorbol esters. The rapid obtaining of phorbol with high purity highly contributes to its application, such as synthesizing phorbol esters with designable side chains and particular therapeutic efficacy. This study introduced a biphasic alcoholysis method for obtaining phorbol from croton oil by using polarity imparity organic solvents in both phases and established a high-speed countercurrent chromatography method for simultaneous separation and purification of phorbol. The optimized operation conditions of biphasic alcoholysis were a reaction time of 91 min, a temperature of 14°C, and a croton oil-methanol ratio of 1:30 (g:ml). The phorbol during the biphasic alcoholysis was 3.2-fold higher in content than that obtained in conventional monophasic alcoholysis. The optimized high-speed countercurrent chromatography method was using the ethyl acetate/n-butyl alcohol/water at 4.7:0.3:5 (v:v:v) with Na2 SO4 at 0.36 g/10 ml as the solvent system, using the mobile phase flow rate of 2 ml/min, the revolution of 800 r/min, under which the retention of the stationary phase was achieved at 72.83%. The crystallized phorbol following high-speed countercurrent chromatography was obtained as high purity of 94%.

Comparison of the ligand binding site of C1 domains: a molecular dynamics simulation study of the C1 domain-phorbol 13-acetate-membrane system.

C1 domains are lipid-binding structural units of about 50 residues. Typical C1 domains associate with the plasma membrane and bind to diacylglycerol/phorbol ester during the activation of the proteins containing these domains. Although the overall structure of the C1 domains are similar, there are differences in their primary sequence and in the orientation of the ligand/lipid binding residues. To gain structural insights into the ligand/lipid binding, we performed molecular docking of phorbol 13-acetate into the C1 domain and 1.0 µs molecular dynamics simulation on the C1 domain-ligand-lipid ternary system for PKCθ C1A, PKCδ C1B, PKCßII C1B, PKCθ C1B, Munc13-1 C1, and ßII-Chimaerin C1. We divided these C1 domains into three types based on the orientations of Gln-27 and Trp/Tyr-22. In type 1, Trp/Tyr-22 is outside and Gln-27 is inside the ligand binding pocket. In type 2, both Trp/Tyr-22 and Gln-27 are outside the ligand binding pocket, and in type 3, Trp/Tyr-22 is inside and Gln-27 is outside the pocket. The type 1 C1 domains showed higher ligand binding and higher membrane binding with a shorter distance between the C1 domain and the membrane than the type 2 and type 3. For ligand binding, Pro-11 plays a major role in the type 1 and 2, and Gly-23 in the type 1 and type 3 C1 domains. This study elucidates the role of Gln-27, Trp-22, Pro-11 and Gly-23 in ligand/lipid binding in typical C1 domains and bears significance in developing selective modulators of C1 domain-containing proteins.Communicated by Ramaswamy H. Sarma.

The Effect of Luteolin on the Modulation of Vascular Contractility via ROCK and CPI-17 Inactivation.

In this investigation, we made a study of the efficacy of luteolin (a flavonoid found in plants such as vegetables, herbs and fruits) on vascular contractibility and to elucidate the mechanism underlying the relaxation. Isometric contractions of denuded muscles were stored and combined with western blot analysis which was conducted to assess the phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and phosphorylation-dependent inhibitory protein for myosin phosphatase (CPI-17) and to examine the effect of luteolin on the RhoA/ROCK/CPI-17 pathway. Luteolin significantly alleviated phorbol ester-, fluoride- and thromboxane mimetic-elicited contractions regardless of endothelial nitric oxide synthesis, implying its direct effect on smooth muscle. It also significantly alleviated the fluoride-elicited elevation in pCPI-17 and pMYPT1 levels and phorbol 12,13-dibutyrate-elicited increase in pERK1/2 level, suggesting depression of ROCK and PKC/MEK activity and ensuing phosphorylation of MYPT1, CPI-17 and ERK1/2. Taken together, these results suggest that luteolin-elicited relaxation includes myosin phosphatase reactivation and calcium desensitization, which seems to be arbitrated by CPI-17 dephosphorylation via ROCK/PKC inhibition.

Crotofolane-Type Diterpenoids: Crotocascarins R-V, Rearranged Trinorcrotofolane: Crotocascarin δ and a Phorbol Derivative from the Leaves of Croton cascarilloides.

Five new crotofolanes, named crotocascarins R-V (1-5), one rearranged trinorcrotofolane, crotocascarin δ, and one phorbol derivative were isolated from the EtOAc-soluble fraction of the MeOH extract of the leaves of Croton cascarilloides. Crotocascarins R (1), T (3), and U (4) possessed isobutyric acid as an acyl moiety and crotocascarin B (2) an acetyl group, whereas crotocascarin V (5) was elucidated to be a hydroxylated compound of crotocascarin K at the 9-position. Crotocascarin δ (6) was a trinor rearranged crotofolane with a tertiary hemiketal functional group at the 8-position. The absolute configuration of the 8-position was determined by the comparison of the experimental electronic circular dichroism (ECD) spectrum and calculated ECD spectra. Compound 7 was a phorbol ester derivative with a peroxide functional group. The fatty acid attached at the 12-position was found to be a single species-i.e., lauric acid (C-12)-from the evidence of the mass spectral data.

Study of TPA on enhancing the anti-tumor effects of cisplatin and reducing its renal toxicity / 中国临床药理学与治疗学

AIM: To investigate the effects of phorbol ester (TPA) on the anti-tumor effect and renal toxicity of cisplatin (CP). METHODS: MTT assay was used to examine the effect of TPA on the proliferation inhibition of CP in A549 and SPC-A-1 lung cancer cells. Also the effect of TPA on acute toxicity of CP was observed by once injection of high dose CP through caudal vein; The tumor-bearing mice model was explored to investigate the effect of TPA on tumor inhibition ratio and renal toxicity of CP in vivo. And the effect of TPA on renal oxidative stress induced by CP was detected. RESULTS: 1 ng/mL TPA could significantly enhance the inhibitory effect of CP on cell proliferation. In acute toxicity test, TPA could significantly reduce the toxicity of CP and prolong the survival time of animals. And the tumor weight (P<0.05), serum creatinine (P<0.05) and urea nitrogen levels (P<0.01) in TPA combined with CP group were significantly lower than those in CP group. Meanwhile, the results of HE staining showed that the renal tissue damage was significantly reduced in the combined group compared with CP group. The contents of MDA in renal tissue were decreased (P<0.01). However, the contents of GSH and the activity of SOD were increased (P<0.05) in TPA and CP combined group. CONCLUSION: TPA can enhance the inhibitory effect of CP on cell proliferation and inhibit tumor growth in tumor-bearing mice. At the same time, TPA can reduce the renal toxicity of CP, which may be related to the inhibition of renal oxidative stress induced by CP.

Molecular dynamics simulation studies on binding of activator and inhibitor to Munc13-1 C1 in the presence of membrane.

Munc13-1 is a presynaptic active zone protein that plays a critical role in priming the synaptic vesicle and releasing neurotransmitters in the brain. Munc13-1 acts as a scaffold and is activated when diacylglycerol (DAG)/phorbol ester binds to its C1 domain in the plasma membrane. Our previous studies showed that bryostatin 1 activated the Munc13-1, but resveratrol inhibited the phorbol ester-induced Munc13-1 activity. To gain structural insights into the binding of the ligand into Munc13-1 C1 in the membrane, we conducted 1.0 µs molecular dynamics (MD) simulation on Munc13-1 C1-ligand-lipid ternary system using phorbol 13-acetate, bryostatin 1 and resveratrol as ligands. Munc13-1 C1 shows higher conformational stability and less mobility along membrane with phorbol 13-acetate and bryostatin 1 than with resveratrol. Bryostatin 1 and phorbol ester remained in the protein active site, but resveratrol moved out of Munc13-1 C1 during the MD simulation. While bryostatin 1-bound Munc13-1 C1 showed two different positioning in the membrane, phorbol 13-acetate and resveratrol-bound Munc13-1 C1 only showed one positioning. Phorbol 13-acetate formed hydrogen bond with Ala-574 and Gly-589. Bryostatin 1 had more hydrogen bonds with Trp-588 and Arg-592 than with other residues. Resveratrol formed hydrogen bond with Ile-590. This study suggests that different ligands control Munc13-1 C1's mobility and positioning in the membrane differently. Ligand also has a critical role in the interaction between Munc13-1 C1 and lipid membrane. Our results provide structural basis of the pharmacological activity of the ligands and highlight the importance of membrane in Munc13-1 activity.Communicated by Ramaswamy H. Sarma.

The Effect of Galangin on the Regulation of Vascular Contractility via the Holoenzyme Reactivation Suppressing ROCK/CPI-17 rather than PKC/CPI-17.

In this study, we investigated the influence of galangin on vascular contractibility and to determine the mechanism underlying the relaxation. Isometric contractions of denuded aortic muscles were recorded and combined with western blot analysis which was performed to measure the phosphorylation of phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17) and myosin phosphatase targeting subunit 1 (MYPT1) and to evaluate the effect of galangin on the RhoA/ROCK/CPI-17 pathway. Galangin significantly inhibited phorbol ester-, fluoride- and thromboxane mimetic-induced vasoconstrictions regardless of endothelial nitric oxide synthesis, suggesting its direct effect on vascular smooth muscle. Galangin significantly inhibited the fluoride-dependent increase in pMYPT1 and pCPI-17 levels and phorbol 12,13-dibutyrate-dependent increase in pERK1/2 level, suggesting repression of ROCK and MEK activity and subsequent phosphorylation of MYPT1, CPI-17 and ERK1/2. Taken together, these results suggest that galangin-induced relaxation involves myosin phosphatase reactivation and calcium desensitization, which appears to be mediated by CPI-17 dephosphorylation via not PKC but ROCK inactivation.

DNA-PKcs kinase activity stabilizes the transcription factor Egr1 in activated immune cells.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is known primarily for its function in DNA double-stranded break repair and nonhomologous end joining (NHEJ). However, DNA-PKcs also has a critical yet undefined role in immunity impacting both myeloid and lymphoid cell lineages spurring interest in targeting DNA-PKcs for therapeutic strategies in immune-related diseases. To gain insight into the function of DNA-PKcs within immune cells, we performed a quantitative phosphoproteomic screen in T cells to identify phosphorylation targets of DNA-PKcs. Our results indicate that DNA-PKcs phosphorylates the transcription factor Egr1 (early growth response protein 1) at serine 301. Expression of Egr1 is induced early upon T cell activation and dictates T cell response by modulating expression of cytokines and key costimulatory molecules such as IL (interleukin) 2, IL6, IFNγ, and NFκB. Inhibition of DNA-PKcs by treatment with a DNA-PKcs specific inhibitor NU7441 or shRNA knockdown increased proteasomal degradation of Egr1. Mutation of serine 301 to alanine via CRISPR-Cas9 reduced EGR1 protein expression and decreased Egr1-dependent transcription of IL2 in activated T cells. Our findings identify DNA-PKcs as a critical intermediary link between T cell activation and T cell fate and a novel phosphosite involved in regulating Egr1 activity.

Endogenous neurosteroids pregnanolone and pregnanolone sulfate potentiate presynaptic glutamate release through distinct mechanisms.

BACKGROUND AND PURPOSE: Neurosteroids influence neuronal function and have multiple promising clinical applications. Direct modulation of postsynaptic neurotransmitter receptors by neurosteroids is well characterized, but presynaptic effects remain poorly understood. Here, we report presynaptic glutamate release potentiation by neurosteroids pregnanolone and pregnanolone sulfate and compare their mechanisms of action to phorbol 12,13-dibutyrate (PDBu), a mimic of the second messenger DAG. EXPERIMENTAL APPROACH: We use whole-cell patch-clamp electrophysiology and pharmacology in rat hippocampal microisland cultures and total internal reflection fluorescence (TIRF) microscopy in HEK293 cells expressing GFP-tagged vesicle priming protein Munc13-1, to explore the mechanisms of neurosteroid presynaptic modulation. KEY RESULTS: Pregnanolone sulfate and pregnanolone potentiate glutamate release downstream of presynaptic Ca2+ influx, resembling the action of a phorbol ester PDBu. PDBu partially occludes the effect of pregnanolone, but not of pregnanolone sulfate. Calphostin C, an inhibitor that disrupts DAG binding to its targets, reduces the effect PDBu and pregnanolone, but not of pregnanolone sulfate, suggesting that pregnanolone might interact with a well-known DAG/phorbol ester target Munc13-1. However, TIRF microscopy experiments found no evidence of pregnanolone-induced membrane translocation of GFP-tagged Munc13-1, suggesting that pregnanolone may regulate Munc13-1 indirectly or interact with other DAG targets. CONCLUSION AND IMPLICATIONS: We describe a novel presynaptic effect of neurosteroids pregnanolone and pregnanolone sulfate to potentiate glutamate release downstream of presynaptic Ca2+ influx. The mechanism of action of pregnanolone, but not of pregnanolone sulfate, partly overlaps with that of PDBu. Presynaptic effects of neurosteroids may contribute to their therapeutic potential in the treatment of disorders of the glutamate system.

PKCε regulates Rho GTPases and actin cytoskeleton reorganization in non-small cell lung cancer cells.

Oncogenic protein kinase C epsilon (PKCε) promotes the formation of membrane ruffles and motility in non-small cell lung cancer (NSCLC) cells. We found that PKCε is down-regulated when NSCLC cells undergo epithelial-to-mesenchymal transition (EMT) in response to TGF-ß, thus becoming dispensable for migration and invasion in the mesenchymal state. PKCε silencing or inhibition leads to stress fibre formation, suggesting that this kinase negatively regulates RhoA activity. Ruffle formation induced by PKCε activation in the epithelial state is dependent on PI3K, but does not involve the PI3K-dependent Rac-GEFs Ect2, Trio, Vav2 or Tiam1, suggesting alternative Rac-GEFs as mediators of this response. In the proposed model, PKCε acts as a rheostat for Rho GTPases that differs in the epithelial and mesenchymal states.

Influence of serum concentration in retinoic acid and phorbol ester induced differentiation of SH-SY5Y human neuroblastoma cell line.

Numerous protocols to establish dopaminergic phenotype in SH-SY5Y cells have been reported. In most of these protocols there are variations in concentration of serum used. In this paper, we compared the effects of high (10%), low (3%) and descending (2.5%/1%) serum concentration in differentiation medium containing different proportion of retinoic acid (RA) and 12-O-Tetradecanoylphorbol-13-acetate (TPA) or RA-only on the undifferentiated SH-SY5Y cells with regards to cell morphology, biochemical and gene expression alterations. Cells differentiated in culture medium containing low and descending serum concentrations showed increased number of neurite projections and reduced proliferation rates when compared to undifferentiated cells. The SH-SY5Y cells differentiated in culture medium containing 3% RA and low serum or descending (2.5%/1% RA/TPA) were found to be more susceptible to 6-hydroxydopamine (6-OHDA) induced cytotoxicity. Cells differentiated with RA/TPA or RA differentiated showed increased production of the α-synuclein (SNCA) neuroprotein and dopamine neurotransmitter compared to undifferentiated cells, regardless serum concentrations used. There was no significant difference in the expression of tyrosine hydroxylase (TH) gene between undifferentiated and differentiated SH-SY5Y cells. However, the expression of dopamine receptor D2 (DRD2) gene was markedly increased (p<0.05) in differentiated cells with 3% serum and RA only when compared to undifferentiated cells. In conclusion, to terminally differentiate SH-SY5Y cells to be used as a cell-based model to study Parkinson's disease (PD) to investigate molecular mechanisms and drug discovery, the optimal differentiation medium should contain 3% serum in RA-only.

12-Deoxyphorbol Esters Induce Growth Arrest and Apoptosis in Human Lung Cancer A549 Cells Via Activation of PKC-δ/PKD/ERK Signaling Pathway.

Prostratin, a non-tumor promoting 12-deoxyphorbol ester, has been reported as a protein kinase C (PKC) activator and is shown to have anti-proliferative activity in certain cancer cell types. Here we show that GRC-2, a prostratin analogue isolated from Euphorbia grandicornis, is ten-fold more potent than prostratin for inhibiting the growth of human non-small cell lung cancer (NSCLC) A549 cells. Flow cytometry assay revealed that GRC-2 and prostratin inhibited cell cycle progression at the G2/M phase and induced apoptosis. The cytotoxic effect of GRC-2 and prostratin was accompanied by activation and nuclear translocation of PKC-δ and PKD as well as hyperactivation of extracellular signal-related kinase (ERK). Knockdown of either PKC-δ, PKD or ERK significantly protected A549 cancer cells from GRC-2- and prostratin-induced growth arrest as well as apoptosis. Taken together, our results have shown that prostratin and a more potent analogue GRC-2 reduce cell viability in NSCLC A549 cells, at least in part, through activation of the PKC-δ/PKD/ERK pathway, suggesting the potential of prostratin and GRC-2 as anticancer agents.

Phorbol ester activates human mesenchymal stem cells to inhibit B cells and ameliorate lupus symptoms in MRL.Faslpr mice.

Rationale: Systemic lupus erythematosus (SLE) is a multi-organ autoimmune disease characterized by autoantibody production by hyper-activated B cells. Although mesenchymal stem cells (MSCs) ameliorate lupus symptoms by inhibiting T cells, whether they inhibit B cells has been controversial. Here we address this issue and reveal how to prime MSCs to inhibit B cells and improve the efficacy of MSCs in SLE. Methods: We examined the effect of MSCs on purified B cells in vitro and the therapeutic efficacy of MSCs in lupus-prone MRL.Faslpr mice. We screened chemicals for their ability to activate MSCs to inhibit B cells. Results: Mouse bone marrow-derived MSCs inhibited mouse B cells in a CXCL12-dependent manner, whereas human bone marrow-derived MSCs (hMSCs) did not inhibit human B (hB) cells. We used a chemical approach to overcome this hurdle and found that phorbol myristate acetate (PMA), phorbol 12,13-dibutyrate, and ingenol-3-angelate rendered hMSCs capable of inhibiting IgM production by hB cells. As to the mechanism, PMA-primed hMSCs attracted hB cells in a CXCL10-dependent manner and induced hB cell apoptosis in a PD-L1-dependent manner. Finally, we showed that PMA-primed hMSCs were better than naïve hMSCs at ameliorating SLE progression in MRL.Faslpr mice. Conclusion: Taken together, our data demonstrate that phorbol esters might be good tool compounds to activate MSCs to inhibit B cells and suggest that our chemical approach might allow for improvements in the therapeutic efficacy of hMSCs in SLE.

Post-tetanic potentiation lowers the energy barrier for synaptic vesicle fusion independently of Synaptotagmin-1.

Previously, we showed that modulation of the energy barrier for synaptic vesicle fusion boosts release rates supralinearly (Schotten, 2015). Here we show that mouse hippocampal synapses employ this principle to trigger Ca2+-dependent vesicle release and post-tetanic potentiation (PTP). We assess energy barrier changes by fitting release kinetics in response to hypertonic sucrose. Mimicking activation of the C2A domain of the Ca2+-sensor Synaptotagmin-1 (Syt1), by adding a positive charge (Syt1D232N) or increasing its hydrophobicity (Syt14W), lowers the energy barrier. Removing Syt1 or impairing its release inhibitory function (Syt19Pro) increases spontaneous release without affecting the fusion barrier. Both phorbol esters and tetanic stimulation potentiate synaptic strength, and lower the energy barrier equally well in the presence and absence of Syt1. We propose a model where tetanic stimulation activates Syt1-independent mechanisms that lower the energy barrier and act additively with Syt1-dependent mechanisms to produce PTP by exerting multiplicative effects on release rates.