Action of the natural compound esculetin on Ca2+ movement and survival in prostate cancer cells

Esculetin is derived from coumarin and is shown to be the main constituent of the Chinese herb Cortex Fraxini. The molecular paths underlying the action of esculetin are intensively studied. The outcome of esculetin on Ca2+ concentration ([Ca2+]i) in prostate cells is unexplored. Fura-2 was used to detect Ca2+ changes. Death was assessed by using WST-1. At doses of 25-100 mM, esculetin evoked [Ca2+]i raises. This signal was lessened by 15% by exclusion of Ca2+. Esculetin (100 μM) induced Mn2+ entry that implied Ca2+ influx. Esculetin-evoked Ca2+ influx was curbed by 50% by nifedipine (1 mM), econazole (0.5 mM) and SKF96365 (5 mM); phorbol 12-myristate 13 acetate (PMA; 1 nM; a protein kinase C [PKC] activator); and GF109203X (2 mM; a PKC inhibitor. In the absence of Ca2+, pretreatment with the endoplasmic reticulum (ER) Ca2+ pump inhibitor thapsigargin (1 mM) eradicated esculetin-induced [Ca2+]i raises. U73122, a phospholipase C (PLC) suppressor got rid of esculetin-caused [Ca2+]i rises. Esculetin (20-70 mM) evoked death which was not restrained by treatment with the Ca2+ binder BAPTA/AM. In summary, in PC3 cells, esculetin stimulated [Ca2+]i raises by Ca2+ influx through PKC-sensitive store-operated Ca2+ entry and PLC-associated ER Ca2+ discharging. Esculetin provoked Ca2+-independent cell death.

Enhanced neutrophil extracellular trap formation in COVID-19 is inhibited by the PKC inhibitor ruboxistaurin.

Neutrophil extracellular traps (NETs) are web-like DNA and protein lattices which are expelled by neutrophils to trap and kill pathogens, but which cause significant damage to the host tissue. NETs have emerged as critical mediators of lung damage, inflammation and thrombosis in COVID-19 and other diseases, but there are no therapeutics to prevent or reduce NETs that are available to patients. Here, we show that neutrophils isolated from hospitalised patients with COVID-19 produce significantly more NETs in response to LPS compared to cells from healthy control subjects. A subset of patients were captured at follow-up clinics (3-4 month post-infection) and while LPS-induced NET formation is significantly lower at this time point, it remains elevated compared to healthy controls. LPS- and PMA-induced NETs were significantly inhibited by the protein kinase C (PKC) inhibitor ruboxistaurin. Ruboxistaurin-mediated inhibition of NETs in healthy neutrophils reduces NET-induced epithelial cell death. Our findings suggest ruboxistaurin could reduce proinflammatory and tissue-damaging consequences of neutrophils during disease, and since it has completed phase III trials for other indications without safety concerns, it is a promising and novel therapeutic strategy for COVID-19.

Comprehensive Account on the Synthesis of (-)-Balanol and its Analogues

Background: A variety of diseases have been associated with hyperactivation of protein kinase C (PKC) enzymes such as cancer, diabetes, asthma, cardiovascular and central nervous system disorders. There is a dire need to selectively inhibit these enzymes by synthesizing new potent inhibitors. Balanol, a fungal metabolite belonging to the PKC inhibitor family, is especially included in this aspect. Tremendous effort has been put towards the synthesis of balanol by different research groups. Objective: The aim of this review is to provide a detailed description of synthetic approaches adopted for the synthesis of key fragments of balanol (azepane and benzophenone). All the factors that resulted in excellent yield and high enantioselectivity have also been mentioned. Conclusion: It has been shown throughout this review that the synthesis of hexahydroazepine and benzophenone cores of balanol was achieved by employing a variety of important key steps with commercially available starting precursors, which make this total synthesis more valuable. Moreover, this article provides ideas to the synthetic as well as pharmaceutical chemists for the synthesis of (-)-balanol and its analogues.

Use of protein kinase C and phospholipase A2 inhibitors in bovine endometrial cells treated with estradiol and calcium ionophore

The release of endometrial prostaglandin-F2α (PGF2α) in bovine females can be induced in vivo by estradiol (E2). However, its role in this mechanism has not been clarified. We hypothesized that E2 stimulates the activity and abundance of protein kinase C (PKC) and phospholipase A2 (PLA2). Our objective in this study was to analyze the effects of PKC and PLA2 inhibitors on PGF2α synthesis induced by E2 and calcium ionophore (CI) in bovine endometrial cells (BEND cells; Experiment 1). Additionally, we evaluated the abundance of PKC and PLA2 in endometrial explants of cows treated or not with E2 17 days after estrus (D17, D0 = estrus; Experiment 2). In Experiment 1, BEND cells were submitted to a PKC inhibitor (10 μM of C25H24N4O2; bisindolylmaleimide I, or BIS I), a PLA2 inhibitor (20 μM of arachydoniltrifluoromethane or AACOCF3), or none. The BEND cells were subsequently treated with E2 and CI, and PGF2α concentrations were measured in the culture medium through radioimmunoassay. For DIF-12 (PGF2α concentration 12 h after treatment subtracted from PGF2α concentration at hour 0), no PKC inhibitor effect was observed (P= 0.2709). However, DIF-12 was lower (P < 0.05) for groups treated with the PLA2 inhibitor and PLA2 inhibitor + CI + E2 groups than the control and CI + E2 groups. Thus, AACOCF3 was an efficient PLA2 inhibitor in the BEND cells culture system, and E2 did not stimulate the synthesis of PKC and PLA2. In Experiment 2, cyclic Nellore heifers received none (n = 5) or 3 mg (n = 6) of 17β-E2 on D17 and were slaughtered 2 h after administration. The abundance of PKC and PLA2 in the endometrial tissue was evaluated using Western blotting analysis. No E2 effect was observed on PKC (P = 0.08) and PLA2 (P = 0.56). We concluded that E2 did not stimulate the activity and abundance of PKC and PLA2.

Effects of Parathyroid Hormone on Osteoporotic Fracture Healing in Mice via Non-Phospholipases C-Dependent Protein Kinase C Signaling Pathway

Objectives: This study was aimed to explore the effects of parathyroid hormone (PTH) on osteoporotic fracture healing in mice and the underlying mechanisms. Methods: Microarray analysis was conducted to analyze the gene expression level in MC3T3-E1 cells. Carboxyfluorescein succinimidyl ester (CFSE) staining and flow cytometry was adopted to analyze the proliferation and apopto-sis of MC3T3-E1 cells. qRT-PCR was used to analyze the mRNA expression level. Fluorescence resonance energy transfer (FRET) assay was conducted to detect PKC activity. The bone mineral density (BMD) and bone volume (BV)/total volume (TV) were determined via enzyme-linked immunosorbent assay (ELISA) and microscopic computed tomography (micro-CT). Results: ERK1/2 was abnormally expressed in MC3T3-E1 cells after GlylArg19hPTH (1-34) + KT5720 treatment. GlylArg19hPTH (1-34)+ KT5720 treatment promoted cell proliferation, inhibited cell apoptosis, and upregulatedthe expression of osteogenesis-related genes (ALP, OPN, Runx2 and OPG) in MC3T3-E1 cells, which were due to the activation of the non-PLC-dependent PKC signaling pathway and can be blocked by PKC inhibitor Go6983 or ERK1/2 inhibitor BVD-523. Moreover, the activity of PKC in MC3T3-E1 cells treated with GlylArg19hPTH (1-34) + KT5720 + Go6983 was alleviated by ERK1/2 inhibitor BVD-523. In vivo, specific activation of the non-PLC-dependent PKC signaling pathway increased the serum levels of APL and OPG in mice with osteoporotic fracture, which were reversed by PKC inhibitor Go6983 and ERK1/2 inhibitor BVD-523. Moreover, PKC inhibitor Go6983 and ERK1/2 inhibitor BVD-523 suppressed the elevation of BV/TV and BMD induced by specific activation of the non-PKC-dependent signaling pathway. Conclusions: Taken together, PTH stimulates osteoporotic fracture healing in mice through the non-PLC-dependent PKC signaling pathway in which ERK1/2 exerts a vital role.

Activation of the α2A adrenoceptor in microglia promotes LPS-induced TNF-α production and cognitive impairment in mice

Background: a2A adrenoceptor receptor (a2A-AR) plays an important role in inflammatory response in Kupffer cells in sepsis. Blockage of a2A-AR inhibits lipopolysaccharide (LPS)-induced tumor necrosis factor a (TNF-a) production and protects the target organ functions in sepsis animal models; however, its expression and function in microglia have remained obscure. This study aimed to determine whether a2A-AR was expressed in microglia and whether its activation would exacerbate microglial inflammation and sepsis-related neurological dysfunction. Methods: Western blotting and immunofluorescence were used to detect a2A-AR expression in BV-2 microglia. Enzyme-linked immunosorbent assay (ELISA) was used to assess the TNF-a production in the supernatant after LPS induced BV-2 cells were pretreated with a2A-AR agonist BHT933, and/or a2A-AR antagonist BRL44408, and also in the supernatants derived from BV-2 cells treated with BHT933 and/or PKC inhibitor. Signaling pathways including JNK，P38，ERK，IκBa, CREB and PCK were detected by western blotting. a2A-AR gene knock-out (KO) and wild type (WT) mice were prepared by intraperitoneal injection of LPS. Lectin /TNF-a labeled microglia and synaptophysin/NeuN expression in the hippocampus were localized by immunofluorescence. Morris water maze test, Rotating-stick test, Elevated plus maze test and Open-field test were conducted over 4 weeks.Results: a2A-AR was constitutively expressed in BV-2 microglia, which was enhanced by LPS. Pretreatment with BHT933 promoted LPS-induced IκB and JNK phosphorylation, and TNF-a secretion in BV-2 microglia which were abrogated by BRL44408. Activation of a2A-AR by BHT933 also increased PKC phosphorylation in LPS-treated BV-2 microglia. PKC inhibitor significantly reversed the promoting effects of BHT933 on IκB and JNK phosphorylation as well as TNF-a secretion in LPS-treated BV-2 microglia. Furthermore, LPS treatment significantly increased hippocampal microglia activation and TNF-a expression, decreased hippocampal synaptophysin expression, and impaired cognitive and motor functions in WT mice, all of which were markedly reversed by a2A-AR gene knockout. Conclusion: a2A-AR activation promotes LPS-induced IκB and JNK phosphorylation as well as TNF-a production in microglia through the PKC signaling pathway. Knockout of a2A-AR gene significantly improves LPS-induced cognitive and motor impairments in mice, indicating that a2A-AR is a potential therapeutic target for sepsis-associated encephalopathy.

Embelin as a Novel Inhibitor of PKC in the Prevention of Platelet Activation and Thrombus Formation

Embelin is a quinone derivative and found in the fruits of Embelia ribes Burm.f. Embelin has been identified as a small molecular inhibitor of X-chromosome-linked inhibitor of apoptosis proteins, and has multiple biological activities, including antioxidation, anti-inflammation, and antitumor effects. However, the effect of embelin in platelets remains unclear. Thus, this study investigated the antiplatelet mechanism of embelin. Our data revealed that embelin could inhibit platelet aggregation induced by various agonists, including the protein kinase C (PKC) activator phorbol 12,13-dibutyrate (PDBu). Embelin, as well as the PKC inhibitor Ro 31-8220, markedly reduced PDBu-mediated phosphorylation of the PKC substrate, suggesting that embelin may be a PKC inhibitor for platelets. Embelin could block PKC downstream signaling and events, including the inhibition of protein kinase B and mitogen-activated protein kinase activation, granule release, and glycoprotein IIbIIIa activation. Moreover, embelin could delay thrombus formation in the mesenteric microvessels of mice, but did not significantly affect the tail bleeding time. In conclusion, we demonstrated that embelin is a PKC inhibitor and possesses antiplatelet and antithrombotic effects. The further analysis is necessary to more accurately determine clinical therapeutic potential of embelin in all clinical thromboembolic events with disturbance of thrombocyte function.