Selective Protein Kinase C Inhibitor Research Articles

Effect of the selective protein kinase C inhibitor, Ro-31-8220, on chemokine-induced Leukocyte recruitment in vivo

The most critical and most important event in acute inflammation is the migration of neutrophils and other inflammatory cells from blood to the site of injury, immune response or infection. Leukocyte recruitment occurs in response to pro-inflammatory mediators such as cytokines and interleukins which are produced at the site of inflammation. Protein kinase C (PKC) is a family of kinases that are involved in the pathophysiology of a variety of inflammatory diseases or disorders such as arthritis, asthma and myocarditis. The effect of Ro-31-8220, the selective PKC inhibitor, on leukocyte transmigration in various inflammatory models is still incompletely understood. The present study explored the effect of the selective and pan inhibitor of PKC, Ro-31-8220, on CXCL1/KC induced leukocyte recruitment especially neutrophils in acute peritonitis model in mice. Ro-31-8220 treatment significantly attenuated the emigration of leukocytes predominately neutrophils in response to CXCL1/KC chemokine. Thereby, Ro-31-8220 treatment ameliorated CXCL1/KC induced acute peritonitis by interfering with emigration of leukocytes. Collectively, our study demonstrates that pharmacological inhibition of PKC in general, may provide the basic key of therapeutic strategy for many inflammatory diseases or immune linked disorders in which PKC was implicated

Impaired Ca2+ handling in resistance arteries from genetically obese Zucker rats: Role of the PI3K, ERK1/2 and PKC signaling pathways

The impact of obesity on vascular smooth muscle (VSM) Ca2+ handling and vasoconstriction, and its regulation by the phosphatidylinositol 3-kinase (PI3K), mitogen activated protein kinase (MAPK) and protein kinase C (PKC) were assessed in mesenteric arteries (MA) from obese Zucker rats (OZR). Simultaneous measurements of intracellular Ca2+ ([Ca2+]i) and tension were performed in MA from OZR and compared to lean Zucker rats (LZR), and the effects of selective inhibitors of PI3K, ERK-MAPK kinase and PKC were assessed on the functional responses of VSM voltage-dependent L-type Ca2+ channels (CaV1.2). Increases in [Ca2+]i induced by α1-adrenoceptor activation and high K+ depolarization were not different in arteries from LZR and OZR although vasoconstriction was enhanced in OZR. Blockade of the ryanodine receptor (RyR) and of Ca2+ release from the sarcoplasmic reticulum (SR) markedly reduced depolarization-induced Ca2+ responses in arteries from lean but not obese rats, suggesting impaired Ca2+-induced Ca2+ release (CICR) from SR in arteries from OZR. Enhanced Ca2+ influx after treatment with ryanodine was abolished by nifedipine and coupled to up-regulation of CaV1.2 channels in arteries from OZR. Increased activation of ERK-MAPK and up-regulation of PI3Kδ, PKCβ and δ isoforms were associated to larger inhibitory effects of PI3K, MAPK and PKC blockers on VSM L-type channel Ca2+ entry in OZR. Changes in arterial Ca2+ handling in obesity involve SR Ca2+ store dysfunction and enhanced VSM Ca2+ entry through L-type channels, linked to a compensatory up-regulation of CaV1.2 proteins and increased activity of the ERK-MAPK, PI3Kδ and PKCβ and δ, signaling pathways.

PKC inhibition of sotrastaurin has antitumor activity in diffuse large B-cell lymphoma via regulating the expression of MCT-1.

MCT-1 (multiple copies in T-cell lymphoma-1), a novel oncogene, was originally identified in T-cell lymphoma. A recent study has demonstrated that MCT-1 is highly expressed in 85% of diffuse large B-cell lymphomas (DLBCL). PKC (protein kinase C) plays an essential role in signal transduction for multiple biologically active substances for activating cellular functions and proliferation. In this study, we found that the mRNA and protein expression levels of MCT-1 were visibly decreased after knocking down PKC by siRNA in SUDHL-4 and OCI-LY8 DLBCL cell lines. A selective PKC inhibitor, sotrastaurin, effectively inhibited cell proliferation and induced cell apoptosis in a dose- and time-dependent manner. Meanwhile, we also observed that the cell cycle was arrested in the G1 phase in sotrastaurin-treated cells. In addition, MCT-1 was down-regulated in the sotrastaurin treatment group in vivo. Furthermore, we demonstrated that the PKC inhibitor sotrastaurin induced cell apoptosis and cell cycle arrest in DLBCL cells potentially through regulating the expression of MCT-1. Our data suggest that targeting PKC may be a potential therapeutic approach for lymphomas and related malignancies that exhibit high levels of MCT-1 protein.

Phycocyanin Protects Against UVB-induced Apoptosis Through the PKC α/βII-Nrf-2/HO-1 Dependent Pathway in Human Primary Skin Cells.

Phycocyanin (Pc) is one of the active pigment constituents of Spirulina microalgae. It has been used for its potent antioxidant and anti-inflammatory properties. However, the protective effects of Pc against ultraviolet-B (UVB)-induced primary skin cells damage are still undefined. In the present study, we investigated whether Pc prevented UVB-induced apoptotic cell death in human dermal fibroblasts (HDF) and human epidermal keratinocytes (HEK). Pc induced the transcription of heme oxygenase-1 (HO-1). Furthermore, Pc treatments resulted in a marked increase in nuclear factor erythroid-derived 2 (NF-E2)-like 2 (Nrf-2) nuclear translocation. Also, Pc protected UVB induced apoptosis and reduced the p53 and Bax levels, as well as caspase-3 activation. Pc treatment showed a significantly enhanced effect on the phosphorylation of protein kinase C (PKC) α/β II, but not that of p38 mitogen-activated protein kinase (MAPK) or Akt. Induction of HO-1 induced by Pc was suppressed by Go6976, a selective inhibitor of PKC α/β II. In addition, knockdown of HO-1 by small interfering (siRNA) caused a significant increase in poly (ADP-ribose) polymerase 1 (PARP-1) cleavage and caspase-3 activation after Pc pretreatment. Taken together, our results demonstrate that Pc-induced expression of HO-1 is mediated by the PKC α/β II-Nrf-2/HO-1 pathway, and inhibits UVB-induced apoptotic cell death in primary skin cells.

TSC2/Rheb signaling mediates ERK-dependent regulation of mTORC1 activity in C2C12 myoblasts.

The enhanced rate of protein synthesis in skeletal muscle cells results in a net increase in total protein content that leads to skeletal muscle growth/hypertrophy. The mitogen‐activated protein kinase kinase (MEK)/extracellular signal‐regulated kinase (ERK)‐dependent regulation of the activity of mechanistic target of rapamycin (mTOR) and subsequent protein synthesis has been suggested as a regulatory mechanism; however, the exact molecular processes underlying such a regulation are poorly defined. The purpose of this study was to investigate regulatory mechanisms involved in the MEK/ERK‐dependent pathway leading to mTORC1 activation in skeletal muscle cells. Treatment with phorbol‐12‐myristate‐13‐acetate (PMA), a potent agonist of protein kinase C (PKC) and its downstream effector in the MEK/ERK‐dependent pathway, resulted in the activation of mTORC1 signaling and phosphorylation of the upstream regulator tuberous sclerosis 2 (TSC2) in C2C12 myoblasts. PMA‐induced activation of mTORC1 signaling was partially prevented by treatment with U0126 (a selective inhibitor of MEK1/2) or BIX‐02189 (a selective inhibitor of MEK5) and completely blocked with BIM‐I (a selective inhibitor of upstream PKC). TSC2 phosphorylation at Ser664 (an ERK‐dependent phosphorylation site) was prevented with U0126, and BIM‐I treatment blocked PMA‐induced phosphorylation of TSC2 at multiple residues (Ser664, Ser939, and Thr1462). Overexpression of Ras homolog enriched in brain (Rheb), a downstream target of TSC2, and an mTORC1 activator, was sufficient to activate mTORC1 signaling. We also identified that PMA‐induced activation of mTORC1 signaling was significantly inhibited in the absence of Rheb with siRNA knockdown. These observations demonstrate that the PKC/MEK/ERK‐dependent activation of mTORC1 is mediated through TSC2 phosphorylation and its downstream target Rheb in C2C12 myoblasts.

Protein kinase C-δ signaling regulates glucagon secretion from pancreatic islets.

Accumulating evidence supports the "glucagonocentric hypothesis", in which antecedent α-cell failure and inhibition of glucagon secretion are responsible for diabetes progression. Protein kinase C (PKC) is involved in glucagon secretion from α-cells, although which PKC isozyme is involved and the mechanism underlying this PKC-regulated glucagon secretion remains unknown. Here, the involvement of PKCδ in the onset and progression of diabetes was elucidated. Immunofluorescence studies revealed that PKCδ was expressed and activated in α-cells of STZ-induced diabetic model mice. Phorbol 12-myristate 13-acetate (PMA) stimulation significantly augmented glucagon secretion from isolated islets. Pre-treatment with quercetin and rottlerin, PKCδ signaling inhibitors, significantly suppressed the PMA-induced elevation of glucagon secretion. While Go6976, a Ca2+-dependent PKC selective inhibitor did not suppress glucagon secretion. Quercetin suppressed PMA-induced phosphorylation of Tyr311 of PKCδ in isolated islets. However, quercetin itself had no effect on either glucagon secretion or glucagon mRNA expression. Our data suggest that PKCδ signaling inhibitors suppressed glucagon secretion. Elucidation of detailed signaling pathways causing PKCδ activation in the onset and progression of diabetes followed by the augmentation of glucagon secretion could lead to the identification of novel therapeutic target molecules and the development of novel therapeutic drugs for diabetes. J. Med. Invest. 64: 122-128, February, 2017.

Stress Signals, Mediated by Membranous Glucocorticoid Receptor, Activate PLC/PKC/GSK-3β/β-catenin Pathway to Inhibit Wound Closure

Glucocorticoids (GCs), key mediators of stress signals, are also potent wound healing inhibitors. To understand how stress signals inhibit wound healing, we investigated the role of membranous glucocorticoid receptor (mbGR) by using cell-impermeable BSA-conjugated dexamethasone. We found that mbGR inhibits keratinocyte migration and wound closure by activating a Wnt-like phospholipase (PLC)/ protein kinase C (PKC) signaling cascade. Rapid activation of mbGR/PLC/PKC further leads to activation of known biomarkers of nonhealing found in patients, β-catenin and c-myc. Conversely, a selective inhibitor of PKC, calphostin C, blocks mbGR/PKC pathway, and rescues GC-mediated inhibition of keratinocyte migration invitro and accelerates wound epithelialization of human wounds exvivo. This novel signaling mechanism may have a major impact on understanding how stress response via GC signaling regulates homeostasis and its role in development and treatments of skin diseases, including wound healing. To test tissue specificity of this nongenomic signaling mechanism, we tested retinal and bronchial human epithelial cells and fibroblasts. We found that mbGR/PLC/PKC signaling cascade exists in all cell types tested, suggesting a more general role. The discovery of this nongenomic signaling pathway, in which glucocorticoids activate Wnt pathway via mbGR, provides new insights into how stress-mediated signals may activate growth signals in various epithelial and mesenchymal tissues.

Staurosporine enhances ATRA-induced granulocytic differentiation in human leukemia U937 cells via the MEK/ERK signaling pathway.

Although all-trans retinoic acid (ATRA) is regarded as a prominent example of differentiation therapy, it is not effective for the treatment of other subtypes of acute myeloid leukemia(AML) beyond acute promyelocytic leukemia (APL). Therefore, new strategies need to be explored to extend the efficacy of ATRA-based therapy to non-APL AML patients. In the present study, staurosporine, a protein kinaseC(PKC) pan-inhibitor, exhibited synergism with ATRA to promote granulocytic differentiation in poorly ATRA-sensitive U937 cells but not in ATRA unresponsive K562 and Kasumi cells. Staurosporine or the combined treatment did not affect PKC activity in U937 cells. Moreover, other selective PKC inhibitors, UCN-01, Go6976 or rottlerin failed to enhance ATRA‑induced granulocytic differentiation in U937 cells. Therefore, staurosporine-enhanced ATRA-induced granulocytic differentiation in U937 cells may be independent of PKC. Staurosporine activated mitogen‑activated protein kinase kinase(MEK) and extracellular signal‑regulated kinase(ERK). Meanwhile, staurosporine also enhanced ATRA-promoted upregulation of the protein level of CCAAT/enhancer‑binding proteinβ(C/EBPβ) and C/EBPε in U937 cells. Furthermore, blockade of MEK activation suppressed staurosporine‑enhanced differentiation as well as the elevated protein level of C/EBPs. Taken together, we concluded that staurosporine enhanced ATRA‑induced granulocytic differentiation in U937 cells via MEK/ERK-mediated modulation of the protein level of C/EBPs.

Mechanisms of connecting tubule glomerular feedback enhancement by aldosterone.

Connecting tubule glomerular feedback (CTGF) is a mechanism where an increase in sodium (Na) concentration in the connecting tubule (CNT) causes the afferent arteriole (Af-Art) to dilate. We recently reported that aldosterone within the CNT lumen enhances CTGF via a nongenomic effect involving GPR30 receptors and sodium/hydrogen exchanger (NHE), but the signaling pathways of this mechanism are unknown. We hypothesize that aldosterone enhances CTGF via cAMP/protein kinase A (PKA) pathway that activates protein kinase C (PKC) and stimulates superoxide (O2-) production. Rabbit Af-Arts and their adherent CNTs were microdissected and simultaneously perfused. Two consecutive CTGF curves were elicited by increasing the CNT luminal NaCl. We found that the main effect of aldosterone was to sensitize CTGF and we analyzed data by comparing NaCl concentration in the CNT perfusate needed to achieve half of the maximal response (EC50). During the control period, the NaCl concentration that elicited a half-maximal response (EC50) was 37.0 ± 2.0 mmol/l; addition of aldosterone (10-8 mol/l) to the CNT lumen decreased EC50 to 19.3 ± 1.3 mmol/l (P ≤ 0.001 vs. Control). The specific adenylyl cyclase inhibitor 2',3'-dideoxyadenosine (ddA; 2 × 10-4 mol/l) and the PKA inhibitor H-89 dihydrochloride hydrate (H-89; 2 × 10-6 mol/l) prevented the aldosterone effect. The selective PKC inhibitor GF109203X (10-8 mol/l) also prevented EC50 reduction caused by aldosterone. CNT intraluminal addition of O2- scavenger tempol (10-4 mol/l) blocked the aldosterone effect. We conclude that aldosterone inside the CNT lumen enhances CTGF via a cAMP/PKA/PKC pathway and stimulates O2- generation and this process may contribute to renal damage by increasing glomerular capillary pressure.

An Evolutionarily Conserved PLC-PKD-TFEB Pathway for Host Defense.

The mechanisms that tightly control the transcriptionof host defense genes have not been fully elucidated. We previously identified TFEB as a transcription factor important for host defense, but the mechanisms that regulate TFEB during infection remained unknown. Here, we used C.elegans to discover a pathway that activates TFEB during infection. Gene dkf-1, which encodes a homolog of protein kinase D (PKD), was required for TFEB activation in nematodes infected with Staphylococcus aureus. Conversely, pharmacological activation of PKD wassufficient to activate TFEB. Furthermore, phospholipase C (PLC) gene plc-1 was also required for TFEB activation, downstream of Gαq homolog egl-30 and upstream of dkf-1. Using reverse and chemical genetics, we discovered a similar PLC-PKD-TFEB axis in Salmonella-infected mouse macrophages. In addition, PKCα was required in macrophages. These observations reveal a previously unknown host defense signaling pathway, which has been conserved across one billion years of evolution.

Protein kinase C-mediated mu-opioid receptor phosphorylation and desensitization in rats, and its prevention during early diabetes.

Painful diabetic neuropathy is associated with impaired opioid analgesia; however, the precise mechanism in sensory neurons remains unclear. This study aimed to identify putative mechanisms involved in modified opioid responsiveness during early streptozotocin-induced diabetes in rats. In this study, we demonstrate that in diabetic animals, impaired peripheral opioid analgesia is associated with a reduction in functional mu-opioid receptor (MOR) G protein coupling. Mu-opioid receptor immunoreactive neurons colocalized with activated forms of protein kinase C (PKC) and with the receptor for advanced glycation end products (RAGE) during streptozotocin-induced diabetes. Moreover, MOR phosphorylation at Thr370 in sensory neurons of diabetic rats, and thus desensitization, was due to RAGE-dependent PKC activation. Importantly, blocking PKC activation using PKC selective inhibitor, silencing RAGE with intrathecal RAGE siRNA, or inhibiting advanced glycation end product (AGE) formation prevented sensory neuron MOR phosphorylation and, consequently, restored MOR G protein coupling and analgesic efficacy. Thus, our findings give the first in vivo evidence of a RAGE-dependent PKC-mediated heterologous MOR phosphorylation and desensitization in sensory neurons under pathological conditions such as diabetic neuropathy. This may unravel putative mechanisms and suggest possible prevention strategies of impaired opioid responsiveness.

Impact Of Hypoxia In The Expression And Regulation Of The TASK‐1 Potassium Channel In Cardiac Myocytes

TASK‐1 is a member of the two‐pore domain potassium channel family and strongly expressed in the heart. However, little is known about the roles and regulation of TASK‐1 channels in cardiac hypoxia. The present study was designed to investigate whether hypoxia modulated the expression and function of TASK‐1 and whether protein kinase C (PKC) was critical in hypoxia‐mediated processes in adult rat cardiac myocytes. The effects of acute hypoxia on the TASK‐1 channel function were chemically induced by sodium cyanide (NaCN). Activity of TASK‐1 in cardiomyocytes was investigated by the whole‐cell patch‐clamp technique. The current flowing through TASK‐1 channels in cardiac myocytes was quantified by analyzing the effects of extracellular pH or the selective TASK‐1 inhibitor methanandamide on the steady‐state current‐voltage relation. The voltage protocol, which included voltage ramps from +40 to −100 mV preceded by a test pulse from 0 to +40 mV, was designed to inactivate voltage‐dependent ion channels. In addition, a blocker cocktail, that had no effect on TASK‐1 channels expressed in HEK293T cells, was used to eliminate a number of ion channels in cardiac myocytes. Our data show that exposure of cardiac myocytes to NaCN for 5 minutes reduced the steady‐state outward current at +40 mV, an effect that was abolished by pretreatment with methanandamide. Either methanandamide or extracellular acidification (from pH 7.4 to 6.4) decreased the outward current, identifying TASK‐1 as the channel mediating this effect. Interestingly, the NaCN‐induced reduction in the outward current was largely prevented by pretreatment with the PKC selective inhibitor bisindolylmaleimide. To assess the expression and protective role of TASK‐1 in hypoxia, the prolonged hypoxia (4 hours) was employed by incubating cells in an airtight chamber in which O2 was replaced by N2 with glucose‐free Tyrode's solution. While 4 hours of hypoxia caused a significant reduction in the viability of cardiac myocytes, the inhibition of cardiac TASK‐1 channels by methanandamide further increased hypoxia‐induced cell death. Western blot analysis showed that 4 hours of hypoxia led to a decrease in the protein expression of TASK‐1 in cardiac myocytes. In summary, we provided the evidence that TASK‐1 channels in cardiomyocytes is regulated by hypoxia‐induced PKC signaling in cardiomyocytes. Our data are also supportive for a possible role of TASK‐1 channels in the cardioprotection against hypoxia.

Inhibition of crosstalk between Bcr-Abl and PKC signaling by PEITC, augments imatinib sensitivity in chronic myelogenous leukemia cells

Chronic myelogenous leukemia (CML), a clonal hyperproliferation of immature blood cells accounts for 20% of adult leukemia cases. Reciprocal translocation of chromosomes 9 and 22, results into Bcr-Abl fusion and is responsible for expression of a tyrosine kinase protein p210bcr/abl, which mediates several survival pathways and confer therapeutic resistance. Protein kinase C (PKC), a family of serine threonine kinases play an important role in the process of leukemogenesis. A crosstalk between Bcr-Abl and PKC signaling has been documented. Therefore, targeting p210bcr/abl and its associated signaling proteins using non-toxic natural means will be an effective strategy for antileukemic therapy. Aim of the present study is to investigate whether PEITC, a natural isothiocyanate in combination with imatinib mesylate (IM), a tyrosine kinase inhibitor could increase the therapeutic efficacy of IM by modulating the expression of p210bcr/abl. Enhanced cytotoxic efficacy of IM by PEITC was further validated using another myelogenous leukemia cell line, KU812. It was observed that PEITC in combination with IM efficiently downregulated the expression of p210bcr/abl in chronic myelogenous leukemia cell lines (K-562). PEITC inhibited the expressions of PKCα, PKCβII and PKCζ (both phosphorylated and total form). Expression of Raf1 and ERK1/2, two important target proteins in PKC signaling cascade was diminished. The result indicated that PEITC ultimately reduced expression of Raf1 and ERK1/2 through Bcr-Abl and PKC inhibition. This result was further confirmed by UCN-01, a selective PKC inhibitor and IM; indicating an association between p210bcr/abl and PKC with Raf1 and ERK1/2. PEITC thus may have enormous potential in synergistic therapy of leukemia by enhancing drug efficacy.

Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca2+ signalling in human platelets

Rises in cytosolic Ca2+ concentration ([Ca2+]cyt) are central in platelet activation, yet many aspects of the underlying mechanisms are poorly understood. Most studies examine how experimental manipulations affect agonist-evoked rises in [Ca2+]cyt, but these only monitor the net effect of manipulations on the processes controlling [Ca2+]cyt (Ca2+ buffering, sequestration, release, entry and removal), and cannot resolve the source of the Ca2+ or the transporters or channels affected. To investigate the effects of protein kinase C (PKC) on platelet Ca2+ signalling, we here monitor Ca2+ flux around the platelet by measuring net Ca2+ fluxes to or from the extracellular space and the intracellular Ca2+ stores, which act as the major sources and sinks for Ca2+ influx into and efflux from the cytosol, as well as monitoring the cytosolic Na+ concentration ([Na+]cyt), which influences platelet Ca2+ fluxes via Na+/Ca2+ exchange. The intracellular store Ca2+ concentration ([Ca2+]st) was monitored using Fluo-5N, the extracellular Ca2+ concentration ([Ca2+]ext) was monitored using Fluo-4 whilst [Ca2+]cyt and [Na+]cyt were monitored using Fura-2 and SFBI, respectively. PKC inhibition using Ro-31-8220 or bisindolylmaleimide I potentiated ADP- and thrombin-evoked rises in [Ca2+]cyt in the absence of extracellular Ca2+. PKC inhibition potentiated ADP-evoked but reduced thrombin-evoked intracellular Ca2+ release and Ca2+ removal into the extracellular medium. SERCA inhibition using thapsigargin and 2,5-di(tert-butyl) l,4-benzohydroquinone abolished the effect of PKC inhibitors on ADP-evoked changes in [Ca2+]cyt but only reduced the effect on thrombin-evoked responses. Thrombin evokes substantial rises in [Na+]cyt which would be expected to reduce Ca2+ removal via the Na+/Ca2+ exchanger (NCX). Thrombin-evoked rises in [Na+]cyt were potentiated by PKC inhibition, an effect which was not due to altered changes in non-selective cation permeability of the plasma membrane as assessed by Mn2+ quench of Fura-2 fluorescence. PKC inhibition was without effect on thrombin-evoked rises in [Ca2+]cyt following SERCA inhibition and either removal of extracellular Na+ or inhibition of Na+/K+-ATPase activity by removal of extracellular K+ or treatment with digoxin. These data suggest that PKC limits ADP-evoked rises in [Ca2+]cyt by acceleration of SERCA activity, whilst rises in [Ca2+]cyt evoked by the stronger platelet activator thrombin are limited by PKC through acceleration of both SERCA and Na+/K+-ATPase activity, with the latter limiting the effect of thrombin on rises in [Na+]cyt and so forward mode NCX activity. The use of selective PKC inhibitors indicated that conventional and not novel PKC isoforms are responsible for the inhibition of agonist-evoked Ca2+ signalling.

Inhibition of protein kinase C by isojacareubin suppresses hepatocellular carcinoma metastasis and induces apoptosis in vitro and in vivo.

Targeted inhibition of protein kinase C (PKC) inhibits hepatocellular carcinoma (HCC) proliferation and metastasis. We previously reported the cytotoxicity of a series of synthetic phenyl-substituted polyoxygenated xanthone derivatives against human HCC. In the current study, the most potent natural product, isojacareubin (ISJ), was synthesized, and its cellular-level antihepatoma activities were evaluated. ISJ significantly inhibited cell proliferation and was highly selective for HCC cells in comparison to nonmalignant QSG-7701 hepatocytes. Moreover, ISJ exhibited pro-apoptotic effects on HepG2 hepatoma cells, as well as impaired HepG2 cell migration and invasion. Furthermore, ISJ was a potent inhibitor of PKC, with differential actions against various PKC isotypes. ISJ selectively inhibited the expression of aPKC (PKCζ) in the cytosol and the translocation of cytosolic PKCζ to membrane site. ISJ also directly interacted with cPKC (PKCα) and nPKC (PKCδ, PKCε and PKCμ) and thereby inhibited the early response of major MAPK phosphorylation and the late response of HCC cell invasion and proliferation. In a hepatoma xenograft model, ISJ pretreatment resulted in significant antihepatoma activity in vivo. These findings identify ISJ as a promising lead compound for the development of new antihepatoma agents and may guide the search for additional selective PKC inhibitors.

ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells.

During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y1 receptor agonist, induced a large signal while UTPyS (P2Y2 agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y1. Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC.

Cantharidin Induces Apoptosis Through the Calcium/PKC-Regulated Endoplasmic Reticulum Stress Pathway in Human Bladder Cancer Cells.

Bladder cancer is a common malignancy worldwide. However, there is still no effective therapy for bladder cancer. In this study, we investigated the cytotoxic effects of cantharidin [a natural toxin produced (pure compound) from Chinese blister beetles (Mylabrisphalerata or Mylabriscichorii) and Spanish flies (Cantharis vesicatoria)] in human bladder cancer cell lines (including: T24 and RT4 cells). Treatment of human bladder cancer cells with cantharidin significantly decreased cell viability. The increase in the expressions of caspase-3 activity and cleaved form of caspase-9/-7/-3 were also increased in cantharidin-treated T24 cells. Furthermore, cantharidin increased the levels of phospho-eIF2α and Grp78 and decreased the protein expression of procaspase-12, which was accompanied by the increase in calpain activity in T24 cells. Cantharidin was capable of increasing the intracellular Ca (2+) and the phosphorylation of protein kinase C (PKC) in T24 cells. The addition of BAPTA/AM (a Ca (2+) chelator) and RO320432 (a selective cell-permeable PKC inhibitor) effectively reversed the increase in caspase-3 and calpain activity, the phosphorylation levels of PKC and eIF2α and Grp78 protein expression, and the decrease in procaspase-12 expression induced by cantharidin. Importantly, cantharidin significantly decreased the tumor volume (a dramatic 71% reduction after 21 days of treatment) in nude mice xenografted with T24 cells. Taken together, these results indicate cantharidin induced human bladder cancer cell apoptosis through a calcium/PKC-regulated ER stress pathway. These findings suggest that cantharidin may be a novel and potential anticancer agent targeting on bladder cancer cells.

PKCs: Pernicious kinase culprits in acetaminophen pathogenesis

PKCs: Pernicious kinase culprits in acetaminophen pathogenesis

Aortic Superoxide Production at the Early Hyperglycemic Stage in a Rat Type 2 Diabetes Model and the Effects of Pravastatin

Endothelium-derived superoxide induces vascular dysfunctions. The aim of this study was to examine the activity of protein kinase C (PKC) isoforms and endothelial nitric oxide synthase (eNOS), which leads to vascular superoxide production in type 2 diabetes, in addition to the effects of pravastatin. We studied these mechanisms in Otsuka Long-Evans Tokushima Fatty (OLETF) rats (type 2 diabetes model) at the early hyperglycemic stage (vs. non-diabetic Long-Evans Tokushima Otsuka [LETO] rats). Superoxide production and catalase activity were measured in aortas, as were the protein expressions of PKCδ and phospho-Ser(1177) eNOS. Superoxide production was increased in OLETF rats, and this increase was inhibited by the selective conventional PKC (cPKC) inhibitor Gö6976 and by the non-selective cPKC and novel PKC inhibitor GF109203X. Phospho-Ser(1177) eNOS was significantly increased in OLETF rats, whereas the protein expressions of PKCδ and phosopho-Thr(505) PKCδ and catalase activity were all greatly reduced. Pravastatin administration to OLETF rats in vivo had normalizing effects on all of these variables. The increment in superoxide production seen in OLETF rats (but not the production in pravastatin-treated OLETF rats) was abolished by high concentration of N(ω)-nitro-L-arginine methyl ester (electron transport inhibitor of eNOS), by sepiapterin (precursor of tetrahydrobiopterin), and by LY294002 (phosphatidylinositol 3-kinase [PI3-kinase] inhibitor). In OLETF rats at the early hyperglycemic stage, aortic superoxide production is increased owing to activation of uncoupled eNOS through phosphorylation by PI3-kinase/Akt. This may be related to the observed reduction in PKCδ/catalase activities. Pravastatin inhibited endothelial superoxide production via normalization of PKCδ/catalase activities.

Abstract B248: Antitumor activity mediated by N-myristoylated cationic peptides (myrCP).

Abstract Although new chemotherapeutic agents are improving the survival rate of cancer patients, all of them have serious toxic side effects. Thus, it is still necessary to search for new agents with minimal side effects or to enhance the effectiveness of known cytotoxic agents at a reduced dose. The modification of peptides by adding myristol group is commonly used to facilitate intracellular delivery. It has been shown recently that the combination of myristoylation and the cationic charge of peptide confer new biological activity to the peptide. We examined the possibility of using such peptides for the treatment of cancer. In present studies, we evaluated the effect of two N-myristoylated cationic peptides (myrCP) on several human cancer cell lines: H1299 (lung), MCF7 (breast), and PC3 (prostate). The first peptide mimics the endogenous Protein kinase C (PKC) pseudosubstrate region and it is considered as a selective inhibitor of PKC (myr-SIYRRGARRWRKL-OH). This peptide has a dual effect on cancer cells as it prevents proliferation by inhibiting PKC and also serves as a cytotoxic agent. The second peptide that is a scrambled version of the first peptide (myr-RLYRKRIWRSAGR-OH) was used as a control to ensure that cytotoxic effect is not only dependent on PKC inhibition. We found that both myrCP had superior cytotoxic effect on cancer cells. Moreover, they dramatically enhanced the activity of commonly used chemotherapeutic agents such as mitoxantrone and cisplatin which were active in combination with peptides at significantly lower concentration. Since the lipophilic nature of myristoylation facilitates penetration of these peptides across cell membranes, we analyzed what lipids can be involved in such interaction. Membrane lipid arrays showed that myrCP preferably binds to PtdIns (3, 4, 5) P3, phosphatidic acid and at less extent to PI (4, 5) P2. Our results suggested that lipid membrane composition of cells could play an essential role in efficacy of myrCP. In summary, the potential outcome of the current studies will be the development of a useful therapeutic tool for efficient killing of tumor cells. Considering that cytotoxic effects of myrCP are non-specific to the type of cancer, a similar strategy can be applied for many different malignancies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B248. Citation Format: Karina Krotova, George Aslanidi. Antitumor activity mediated by N-myristoylated cationic peptides (myrCP). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B248.