PKCβ Inhibitor Research Articles

Protein kinase cβ phosphorylates occludin regulating tight junction trafficking in vascular endothelial growth factor-induced permeability in vivo.

Vascular endothelial growth factor (VEGF)–induced breakdown of the blood-retinal barrier requires protein kinase C (PKC)β activation. However, the molecular mechanisms related to this process remain poorly understood. In this study, the role of occludin phosphorylation and ubiquitination downstream of PKCβ activation in tight junction (TJ) trafficking and endothelial permeability was investigated. Treatment of bovine retinal endothelial cells and intravitreal injection of PKCβ inhibitors as well as expression of dominant-negative kinase was used to determine the contribution of PKCβ to endothelial permeability and occludin phosphorylation at Ser490 detected with a site-specific antibody. In vitro kinase assay was used to demonstrate direct occludin phosphorylation by PKCβ. Ubiquitination was measured by immunoblotting after occludin immunoprecipitation. Confocal microscopy revealed organization of TJ proteins. The results reveal that inhibition of VEGF-induced PKCβ activation blocks occludin Ser490 phosphorylation, ubiquitination, and TJ trafficking in retinal vascular endothelial cells both in vitro and in vivo and prevents VEGF-stimulated vascular permeability. Occludin Ser490 is a direct target of PKCβ, and mutating Ser490 to Ala (S490A) blocks permeability downstream of PKCβ. Therefore, PKCβ activation phosphorylates occludin on Ser490, leading to ubiquitination required for VEGF-induced permeability. These data demonstrate a novel mechanism for PKCβ targeted inhibitors in regulating vascular permeability.

PKCβII modulation of myocyte contractile performance

Significant up-regulation of the protein kinase Cβ(II) (PKCβ(II)) develops during heart failure and yet divergent functional outcomes are reported in animal models. The goal here is to investigate PKCβ(II) modulation of contractile function and gain insights into downstream targets in adult cardiac myocytes. Increased PKCβ(II) protein expression and phosphorylation developed after gene transfer into adult myocytes while expression remained undetectable in controls. The PKCβ(II) was distributed in a peri-nuclear pattern and this expression resulted in diminished rates and amplitude of shortening and re-lengthening compared to controls and myocytes expressing dominant negative PKCβ(II) (PKCβDN). Similar decreases were observed in the Ca(2+) transient and the Ca(2+) decay rate slowed in response to caffeine in PKCβ(II)-expressing myocytes. Parallel phosphorylation studies indicated PKCβ(II) targets phosphatase activity to reduce phospholamban (PLB) phosphorylation at residue Thr17 (pThr17-PLB). The PKCβ inhibitor, LY379196 (LY) restored pThr17-PLB to control levels. In contrast, myofilament protein phosphorylation was enhanced by PKCβ(II) expression, and individually, LY and the phosphatase inhibitor, calyculin A each failed to block this response. Further work showed PKCβ(II) increased Ca(2+)-activated, calmodulin-dependent kinase IIδ (CaMKIIδ) expression and enhanced both CaMKIIδ and protein kinase D (PKD) phosphorylation. Phosphorylation of both signaling targets also was resistant to acute inhibition by LY. These later results provide evidence PKCβ(II) modulates contractile function via intermediate downstream pathway(s) in cardiac myocytes.

Role of oxidative stress in methamphetamine-induced dopaminergic toxicity mediated by protein kinase Cδ

This study examined the role of protein kinase C (PKC) isozymes in methamphetamine (MA)-induced dopaminergic toxicity. Multiple-dose administration of MA did not significantly alter PKCα, PKCβI, PKCβII, or PKCζ expression in the striatum, but did significantly increase PKCδ expression. Gö6976 (a co-inhibitor of PKCα and -β), hispidin (PKCβ inhibitor), and PKCζ pseudosubstrate inhibitor (PKCζ inhibitor) did not significantly alter MA-induced behavioral impairments. However, rottlerin (PKCδ inhibitor) significantly attenuated behavioral impairments in a dose-dependent manner. In addition, MA-induced behavioral impairments were not apparent in PKCδ knockout (−/−) mice. MA-induced oxidative stress (i.e., lipid peroxidation and protein oxidation) was significantly attenuated in rottlerin-treated mice and was not apparent in PKCδ (−/−) mice. Consistent with this, MA-induced apoptosis (i.e., terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic cells) was significantly attenuated in rottlerin-treated mice. Furthermore, MA-induced increases in the dopamine (DA) turnover rate and decreases in tyrosine hydroxylase (TH) activity and the expression of TH, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) were not significantly observed in rottlerin-treated or PKCδ (−/−) mice. Our results suggest that PKCδ gene expression is a key mediator of oxidative stress and dopaminergic damage induced by MA. Thus, inhibition of PKCδ may be a useful target for protection against MA-induced neurotoxicity.

A role for PKCβ in altering calcium homeostasis under hyperglycemic conditions in human endothelial cells

Cardiovascular diseases (CVD) are leading causes of morbidity and mortality for diabetic patients. Endothelial cell dysfunction is a hallmark of CVD. Intracellular Ca2+ concentration ([Ca2+]i) may regulate endothelial cell function. Here, we investigated the effects of hyperglycemia on [Ca2+]i and a role for protein kinase C (PKC) in regulating [Ca2+]i in the human endothelial cell line, EA.hy926. Cells were cultured in normal (5.5 mM, NG) or high (25 mM, HG) glucose media then treated with either a) vehicle (0.1% DMSO), b) LY341684 (selective PKCβ inhibitor, 100 nM, 24h), or c) phorbol 12‐myristate 13‐acetate (PMA, PKC activator, 200 nM, 4h). Using a spectrofluorometer, [Ca2+]i in cells loaded with Fura 2‐AM was monitored in the absence of extracellular Ca2+. Thapsigargin (SERCA inhibitor, 1 μM,) induced a transient increase in [Ca2+]i which was similar among the experimental groups of cells. However, when 1.5 mM Ca2+ was added, Ca2+ entry was significantly increased in cells cultured in HG compared to NG. This elevated Ca2+ entry in HG‐treated cells was attenuated by treatment with LY341684. Cells treated with PMA in NG showed an increased Ca2+ entry similar to cells cultured in HG. Our results suggest that hyperglycemia enhanced Ca2+ entry in endothelial cells is PKCβ dependent. Thus, PKCβ inhibitors may restore normal endothelial cell function under hyperglycemic conditions. Supported by NIH/NIDCR.

Role of mitochondrial reactive oxygen species and actin polymerization in PKC‐dependent constriction of small pulmonary but not mesenteric arteries

PKC plays a central role in the regulation of vascular smooth muscle (VSM) tone. However, little is known regarding mechanisms by which PKC mediates contraction in pulmonary VSM. We hypothesized that PKCβ elicits pulmonary vasoconstriction through a unique signaling axis involving VSM mitochondrial reactive oxygen species (ROS) and actin polymerization not observed in systemic arteries. The PKC activator phorbol 12‐myristate 13‐acetate (PMA) caused constriction in both endothelium‐disrupted, pressurized rat pulmonary (~150 μm diameter) and mesenteric (~165 μm diameter) arterioles that was independent of an increase in VSM Ca2+ (measured by fura‐2 fluorescence). Consistent with our hypothesis, the PKCβ inhibitor LY‐333,531 (10 nM), the O2− scavenger tiron (10 mM), the mitochondrial‐targeted antioxidant Mito‐CP (0.5 μM), and the actin polymerization inhibitor cytochalasin B (10 μM), blunted reactivity to PMA in pulmonary arteries while having no effect in mesenteric arterioles. Furthermore, PMA increased mitochondrial ROS (measured by Mito‐SOX fluorescence) and caused actin polymerization (measured by Alexa Fluor 488 phalloidin/Alexa Fluor 594 DNase staining) in cultured human pulmonary artery smooth muscle cells. We conclude that PKCβ uniquely enhances mitochondrial ROS production and actin polymerization in the pulmonary circulation to mediate vasoconstriction.

Inhibition of Protein Kinase β2 Attenuates NOS Uncoupling and Myocardial Dysfunction in Streptozotocin‐Induced Diabetic Rats

Hyperglycemia induced protein kinase C (PKC) β activation is implicated in diabetic cardiomyopathy, which is also associated with impaired function of endothelial nitric oxide (NO) synthase (eNOS). We postulated that PKCβ2 inhibition may attenuate myocardial dysfunction by rescuing eNOS function and reducing eNOS uncoupling in streptozotocin (STZ)‐induced diabetes. Control (C) or STZ‐induced diabetic (D) rats were treated or untreated with PKCβ inhibitor LY333531 (LY, 1 mg/kg/day) for four weeks. The ratio of peak velocity of early and late diastolic filling (E/A) measured by echocardiography was decreased, while left ventricular isovolumic relaxation time (IVRT) and the heart to body weight ratio were increased in D rats. All of these changes were attenuated by LY (P<0.05 D vs. C). Cardiac O2− and nitrotyrosine production were increased, whereas NO were decreased in D rats. Diabetes induced increase of O2− levels was blocked by the NOS inhibitor L‐NAME, indicating “NOS uncoupling”. In addition, cardiac iNOS expression was increased, while levels of eNOS, p‐eNOS (Ser 1177) and p‐Akt (Ser 473) expression were decreased in D rats. All the alterations were attenuated or reversed by LY. We conclude that PKCβ2 inhibition may attenuate myocardrial dysfunction in diabetes by restoring Akt‐dependent eNOS/NO signaling and maintaining eNOS coupling.Supported by RGC/GRF (782910, 766709)

Gender differences in aortic endothelial function of streptozotocin‐induced diabetic rats: possible involvement of protein kinase C beta and nitric oxide production

Little is known of the interaction between diabetes and gender in the vasculature. The objective of this study was to investigate whether there are gender differences in rat aortic endothelial function in streptozotocin (STZ, 60mg/kg, iv)‐induced diabetes and the roles of protein kinase C beta (PKCβ) and nitric oxide (NO). Endothelium‐dependent vasodilatation (EDV) to acetylcholine (ACh; 10−8 to 10−5M) was measured in aortic rings precontracted with phenylephrine (PE; 2μM) before and after pretreatment with LY341684 (1μM), a selective PKCβ inhibitor. Constrictor response curves to PE (10−8 to 10−5M) were also generated before and after incubation with L‐NAME (200μM), an endothelial nitric oxide synthase inhibitor. STZ‐induced diabetes impaired aortic EDV to ACh only in females. Inhibition of PKCβ increased the sensitivity to ACh in both control and diabetic male rats. However, PKCβ inhibition enhanced the sensitivity to ACh only in aorta taken from diabetic female rats. Addition of L‐NAME resulted in a significant potentiation of the contractile responses to PE in all groups. However, aorta from control females had a greater maximal potentiation of the PE responses than others. These data suggest that the predisposition of female rat aorta to vascular injury in diabetes is possibly due to differences in enhanced PKCβ activation and decreased basal NO production (supported by NIH/NIDCR).

Chemotherapy-Associated Angiogenesis in Neuroblastoma Tumors

The influences of cytotoxic drugs on endothelial cells remain incompletely understood. Herein, we examined the effects of chemotherapeutic agents in experimental angiogenesis models and analyzed vessel densities in clinical neuroblastoma tumor samples. Cisplatin (20 to 500 ng/mL), doxorubicin (4 to 100 ng/mL), and vincristine (0.5 to 4 ng/mL), drugs commonly involved in neuroblastoma therapy protocols, induced pro-angiogenic effects in different angiogenesis models. They enhanced endothelial cell tube formation, endothelial cell sprouting from spheroids, formation of tip cells in the sprouting assay, expression of αvβ3 integrin, and vitronectin binding. All three drugs increased global cellular kinase phosphorylation levels, including the angiogenesis-relevant molecules protein kinase Cβ and Akt. Pharmacological inhibition of protein kinase Cβ or Akt upstream of phosphatidylinositol 3-kinase reduced chemotherapy-induced endothelial cell tube formation. Moreover, the investigated chemotherapeutics dose dependently induced vessel formation in the chick chorioallantoic membrane assay. Tumor samples from seven high-risk patients with neuroblastoma were analyzed for vessel density by IHC. Results revealed that neuroblastoma samples taken after chemotherapy consistently showed an enhanced microvessel density compared with the corresponding samples taken before chemotherapy. In conclusion, our data show that chemotherapy can activate endothelial cells by inducing multiple pro-angiogenic signaling pathways and exert pro-angiogenic effects in vitro and in vivo. Moreover, we report a previously unrecognized clinical phenomenon that might, in part, be explained by our experimental observations: chemotherapy-associated enhanced vessel formation in tumors from patients with neuroblastoma.

PKCβ inhibition with ruboxistaurin reduces oxidative stress and attenuates left ventricular hypertrophy and dysfuntion in rats with streptozotocin-induced diabetes

Oxidative stress plays critical roles in the development of diabetic cardiovascular complications, including myocardial hypertrophy. The β isoform of PKC (protein kinase C) is preferentially overexpressed in the myocardium of diabetic subjects accompanied with increased activation of the pro-oxidant enzyme NADPH oxidase, which may exacerbate oxidative stress. We hypothesized that myocardial PKCβ is a major upstream mediator of oxidative stress in diabetes and that PKCβ inhibition can attenuate myocardial hypertrophy and dysfunction. Control or streptozotocin-induced diabetic rats were treated with the selective PKCβ inhibitor RBX (ruboxistaurin; 1mg/kg of body weight per day) or the antioxidant NAC (N-acetylcysteine) for 4weeks. LV (left ventricular) dimensions and functions were detected by echocardiography. 15-F2t-isoprostane (a specific index of oxidative stress) and myocardial activities of superoxide dismutase as well as protein levels of NADPH oxidase were assessed by immunoassay or Western blotting. Echocardiography revealed that the LV mass/body weight ratio was significantly increased in diabetic rats (P<0.01 compared with the control group) in parallel with the impaired LV relaxation. A significant increase in cardiomyocyte cross-sectional area was observed in diabetic rats accompanied by an increased production of O2- (superoxide anion) and 15-F2t-isoprostane (all P<0.05 compared with the control group). RBX normalized these changes with concomitant inhibition of PKCβ2 activation and prevention of NADPH oxidase subunit p67phox membrane translocation and p22phox overexpression. The effects of RBX were comparable with that of NAC, except that NAC was inferior to RBX in attenuating cardiac dysfunction. It is concluded that RBX can ameliorate myocardial hypertrophy and dysfunction in diabetes, which may represent a novel therapy in the prevention of diabetic cardiovascular complications.

Differential activations of PKC/PKA related to microvasculopathy in diabetic GK rats

Microvasculopathy is the most serious and predictable threat to the health of diabetic patients, which often results in end-stage renal disease, blindness, and limb amputations. Up to the present, the underlying mechanisms have remained elusive. Here, it was found that the differential activations of PKC/PKA were involved in diabetic microvasculopathy in diabetic GK rats. By real-time PCR, Western blot, immunohistochemistry, and enzyme activity assay, upregulation of PKC was prominent in kidney but was not significant in liver and brain. The expression and activity of PKA were lowered in kidney but comparable in brain and liver during diabetic nephropathy. Furthermore, the generation of reactive oxygen species, production of nitric oxide, and expression of inducible nitric oxide synthase induced by advanced glycation end products were inhibited by PKCβ inhibitor LY-333531 or a PKA agonist in rat glomerular microvascular endothelial cells. Finally, albuminuria was significantly lowered by a PKA agonist and boosted by a PKA antagonist. It suggested that the differential activations of PKC/PKA related to microvasculopathy in diabetes and that activation of PKA may protect the diabetic microvasculature.

Phosphorylation of JAK2 by serotonin 5-HT2A receptor activates both STAT3 and ERK1/2 pathways and increases growth of JEG-3 human placental choriocarcinoma cell

Serotonin 5-HT2A receptor activation improves viability, increases DNA synthesis and activates JAK2-STAT3 and MEK1/2-ERK1/2 signalling pathways in JEG-3 human trophoblast choriocarcinoma cells. The goal of this study was to characterize the signal transduction cascade involved in 5-HT2A receptor-induced growth of JEG-3 cells. Selective 5-HT2A receptor agonist, DOI, induced JEG-3 cell growth was inhibited by the inhibitor of JAK2 (AG490), MEK1/2 (U0126), phospholipase C-β (PLC-β; U73122) and protein kinase C-β (PKC-β; Gö6976)), whereas the selective phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002) had no effect. Specific inhibitors of PLC-β, PKC-β and Ras (farnesylthiosalicylic acid) inhibit activation of ERK1/2, whereas the PKC-ζ inhibitor GF109203X had no effect. Interestingly, inhibition of JAK2 prevented DOI-induced phosphorylation of ERK1/2 whereas inhibition of ERK1/2 pathway had no effect on DOI-induced activation of STAT3. Taken together, our results demonstrate that both the JAK2-STAT3 and PLC-β-PKC-β-Ras-ERK1/2 signalling pathways are involved in the stimulation of JEG-3 cell growth mediated by DOI. Moreover, this study shows that activation of JAK2 by the 5-HT2A receptor is essential to activate both STAT3 and ERK1/2 signalling pathways as well as to increase JEG-3 choriocarcinoma cell growth and survival.

PKCβ-dependent phosphorylation of the glycine transporter 1

The extracellular levels of the neurotransmitter glycine in the brain are tightly regulated by the glycine transporter 1 (GlyT1) and the clearance rate for glycine depends on its rate of transport and the levels of cell surface GlyT1. Over the years, it has been shown that PKC tightly regulates the activity of several neurotransmitter transporters. In the present work, by stably expressing three N-terminus GlyT1 isoforms in porcine aortic endothelial cells and assaying for [32P]-orthophosphate metabolic labeling, we demonstrated that the isoforms GlyT1a, GlyT1b, and GlyT1c were constitutively phosphorylated, and that phosphorylation was dramatically enhanced, in a time dependent fashion, after PKC activation by phorbol ester. The phosphorylation was PKC-dependent, since pre-incubation of the cells with bisindolylmaleimide I, a selective PKC inhibitor, abolished the phorbol ester-induced phosphorylation. Blotting with specific anti-phospho-tyrosine antibodies did not yield any signal that could correspond to GlyT1 tyrosine phosphorylation, suggesting that the phosphorylation occurs at serine and/or threonine residues. In addition, a 23–40%-inhibition on Vmax was obtained by incubation with phorbol ester without a significant change on the apparent Km value. Furthermore, pre-incubation of the cells with the selective PKCα/β inhibitor Gö6976 abolished the downregulation effect of phorbol ester on uptake and phosphorylation, whereas the selective PKCβ inhibitors (PKCβ inhibitor or LY333531) prevented the phosphorylation without affecting glycine uptake, defining a specific role of classical PKC on GlyT1 uptake and phosphorylation. Taken together, these data suggest that conventional PKCα/β regulates the uptake of glycine, whereas PKCβ is responsible for GlyT1 phosphorylation.

Epithelial–mesenchymal transdifferentiation of renal tubular epithelial cells induced by urinary proteins requires the activation of PKC-α and βI isozymes

Proteinuria is a common feature for almost all glomerular diseases and reflects the severity of the glomerular lesion. The presence of a large amount of proteins in tubular fluid, however, may also contribute to the development of RIF (renal interstitial fibrosis). Endocytosis of albumin in proximal tubular cells triggers PKC (protein kinase C)-dependent generation of reactive oxygen species and secretion of chemokines. As a family including 12 isozymes, which PKC isozymes participate in RIF is still unclear. EMT (epithelial-mesenchymal transdifferentiation) of RTECs (renal tubular epithelial cells) plays a crucial role in the progress of RIF induced by proteinuria. In the present study, we investigated the role of classical PKC isozymes in the proteinuria-induced EMT of RTECs. Employing immunochemical staining, we found that PKC-α, -βI and -βII were expressed in glomerulus and in RTECs in both normal and diseased renal tissues, while PKC-γ was only expressed in podocytes in the glomerulus. Treatment of HK-2 cells with extracted urinary proteins resulted in EMT, as evidenced by morphological changes, decreased E-cadherin expression, increased α-SMA (α-smooth muscle actin) expression, as well as production of type I collagen and fibronectin. Western blot analysis of PKC isozymes in the cytosolic compared with membrane fraction revealed translocation of PKC-α and -βI, but not PKC-βII, in HK-2 cells undergoing EMT. Pretreatment with selective PKC-α inhibitor G-6976 or PKC-β inhibitor significantly attenuated EMT induced by urinary proteins. In summary, the present study suggested that PKC-α and -βI play critical roles in the EMT of RTECs in response to urinary proteins.

HDAC inhibitors potentiate the apoptotic effect of enzastaurin in lymphoma cells

Enzastaurin is an investigational PKCβ inhibitor that has growth inhibitory and pro-apoptotic effects in both B and T-cell lymphomas. We investigated the cytotoxicity and mechanisms of cell death of the combination of enzastaurin and low concentrations of histone deacetylase (HDAC) inhibitors in B-cell and T-cell lymphoma cell lines and primary lymphoma/leukemia cells. Combined enzastaurin/suberoylanilide hydroxamic acid treatment synergistically induced apoptosis in diffuse large B-cell lymphoma and T-cell lymphoma cell lines, and primary lymphoma/leukemia samples. Similarly, combined treatment of B-cell-like lymphoma cells with enzastaurin and two different HDAC inhibitors, valproic acid and (2E,4E)-6-(4-chlorophenylsulfanyl)-2,4-hexadienoic acid hydroxyamide synergistically induced apoptosis, suggesting the synergy is generalizable to other HDAC inhibitors. Our data indicate that enzastaurin/HDAC inhibitors therapy can synergistically inhibit growth and induce apoptosis in lymphoid malignancies and may be an effective therapeutic strategy. Potential mechanisms including enzastaurin mediated inhibition of HDAC inhibitor-induced compensatory survival pathways are discussed.

Enzastaurin hydrochloride for lymphoma: reassessing the results of clinical trials in light of recent advances in the biology of B-cell malignancies

Introduction: The B-cell receptor (BCR) is critical for the development and persistence of B-cell non-Hodgkin lymphoma (B-NHL). Protein kinase C-beta (PKC-β) has been identified as one of the key signaling hubs downstream of the BCR and constitutes a valuable target in B-NHL. As a potent PKC-β inhibitor, enzastaurin is currently being tested in Phase II/III trials.Areas covered: This review summarizes the latest results and ongoing clinical trials with enzastaurin in light of basic scientific advances in the understanding of various lymphoid cancers, including diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL) and Waldenström's macroglobulinemia (WM).Expert opinion: While its continued clinical development is uncertain, enzastaurin should be regarded as a stepping stone for the development of future therapies; indeed, the recent research has provided valuable insight into the possible molecular mechanisms that explain its limited clinical activity especially in the treatment of DLBCL and MCL. It should be noted that there is still some interest in enzastaurin, in combination, for the treatment of WM.

Submaximal Inhibition of Protein Kinase C Restores ADP-induced Dense Granule Secretion in Platelets in the Presence of Ca2+

Protein kinase C (PKC) is a family of serine/threonine kinases that play isoform-specific inhibitory and stimulatory roles in platelet activation. We show here that the pan-PKC inhibitor Ro31-8220 can be used to dissect these events following platelet activation by ADP. Submaximal concentrations of Ro31-8220 potentiated aggregation and dense granule secretion to ADP in plasma anticoagulated with citrate, in d-Phe-Pro-Arg-chloromethyl ketone-anticoagulated plasma, which has physiological levels of Ca2+, and in washed platelets. Potentiation was retained on inhibition of cyclooxygenase and was associated with an increase in intracellular Ca2+. Potentiation of aggregation and secretion was abolished by a maximally effective concentration of Ro31-8220, consistent with a critical role of PKC in secretion. ADP-induced secretion was potentiated in the presence of an inhibitor of PKCβ but not in the presence of available inhibitors of other PKC isoforms in human and mouse platelets. ADP-induced secretion was also potentiated in mouse platelets deficient in PKCϵ but not PKCθ. These results demonstrate that partial blockade of PKC potentiates aggregation and dense granule secretion by ADP in association with increased Ca2+. This provides a molecular explanation for the inability of ADP to induce secretion in plasma in the presence of physiological Ca2+ concentrations, and it reveals a novel role for PKC in inhibiting platelet activation by ADP in vivo. These results also demonstrate isoform-specific inhibitory effects of PKC in platelets.

A novel bisindolymaleimide derivative (WK234) inhibits proliferation and induces apoptosis through the protein kinase Cβ pathway, in chronic myelogenous leukemia K562 cells

WK234, a novel bisindolymaleimide derivative, was designed as a protein kinase Cβ (PKCβ) inhibitor. The objective of this study was to evaluate the anti-tumor activity of WK234 in the human chronic myelogenous leukemia (CML) K562 cell line and to investigate possible mechanisms of its action. The results show that WK234 inhibited K562 cell proliferation in a time- and dose-dependent manner. WK234 increased cytochrome C release and caspase-3 cleavage, which indicates that it induced apoptosis via mitochondria- and caspase-mediated pathways. Western blotting showed that PKCβ1, PKCβ2, and their phosphorylation levels were effectively decreased after 2–4 h of WK234 treatment. Meanwhile the phosphorylation status of PKCβ downstream proteins, glycogen synthase kinase 3α/β (GSK3α/β) and extracellular signal-regulated kinase (ERK), were inhibited. WK234 blocked phorbol myristate acetate (PMA)-induced Ser660 phosphorylation of PKCβ2 located at the cell membrane, and increased Ser660 PKCβ2 expression within the cytoplasm and the nucleus. These results indicate that WK234 inhibited cell proliferation and induced apoptosis through suppressing the PKCβ signal pathway. WK234 might be a promising candidate for the treatment of CML.

Protein kinase C ß inhibition ameliorates experimental mesangial proliferative glomerulonephritis

Activation of protein kinase C (PKC) has been implicated in the pathogenesis of diabetic nephropathy where therapy targeting the β isoform of this enzyme has been examined. However, PKC-β is also increased in various forms of human glomerulonephritis, including IgA nephropathy. Accordingly, we sought to examine the effects of PKC-β inhibition in the Thy1.1 model of mesangial proliferative glomerulonephritis. Following administration of monoclonal OX-7, anti-rat Thy-1.1 antibody, Male Wistar rats were randomized to receive either the PKC-β inhibitor, ruboxistaurin (10 mg/kg per day in chow) or vehicle. Animals were then examined 6 days later. PKC-β inhibition was associated with reductions in mesangial cellularity and extracellular matrix deposition. Proteinuria was, however, unaffected. In vitro, PKC-β inhibition showed modest, dose-dependent reductions in mesangial cell (3) H-thymidine and (3) H-proline incorporations, indices of cell proliferation and collagen synthesis, respectively. The amelioration of the pathological findings of experimental mesangial proliferative glomerulonephritis by PKC-β inhibition suggests the potential clinical utility of this approach as a therapeutic strategy in non-diabetic glomerular disease.

Targeting PKC-β II and PKB Connection: Design of Dual Inhibitors.

Protein kinase C (PKC) has been the center of many cell signaling pathways. PKC isoforms, specifically PKC-β II is linked to both diabetic complications as well as in promotion of angiogenesis and regulation of cancers. PKC-β II activates the PKB/Akt pathway. Enzastaurin, a selective PKC-β II inhibitor has been found to inhibit PKB/Akt by suppressing the regulation of various cancerous pathways. In the present work, we carried out an in depth study on the binding mode of inhibitors of PKC-β II, enzastaurin and ruboxistaurin with the active site residues of PKB and PKC-β II. A ligand based approach has been further used to determine the pharmacophoric features and spatial arrangement of molecules, having common properties necessary for appropriate binding to the active site of both targets. Virtual screening of the respective pharmacophores of both proteins led to identification of hits which may be useful for treatment of diabetic complications and cancer. The study has highlighted important features that may be considered in the future for designing novel inhibitors.

A Gynecologic Oncology Group phase II trial of the protein kinase C-beta inhibitor, enzastaurin and evaluation of markers with potential predictive and prognostic value in persistent or recurrent epithelial ovarian and primary peritoneal malignancies

Objectives Protein kinase C (PKC) activation contributes to proliferation and angiogenesis in epithelial ovarian or primary peritoneal carcinoma (EOC/PPC). A multi-institutional phase II trial was conducted to evaluate the efficacy and safety of PKCβ inhibitor enzastaurin in persistent or recurrent EOC/PPC and to explore potential prognostic and predictive biomarkers. Methods Eligible women with measurable platinum-sensitive and resistant EOC/PPC were treated with continuous administration of oral enzastaurin until disease progression or unacceptable toxicity. A two-stage sequential design was used to evaluate progression-free survival (PFS) ≥ 6-months, tumor response, and toxicity. Translational studies included sequencing of the TP53, PTEN, PIK3CA and PKCβII genes for somatic mutations, quantitative PCR assays for AKT2 and PTEN copy number alterations, and measurement of circulating VEGF-A plasma levels. Results Among 27 eligible and evaluable patients, 3 women with PFS ≥ 6-months (11%) and 2 women with partial responses (7%) were observed. One of them achieved a durable response and remains on the study. No grade 4 adverse events were observed. Most common grade 3 adverse events were constitutional (4) and gastrointestinal (3). Mutations in the TP53 gene and abnormal copy number in the PTEN gene were common (56% and 48% of cases, respectively). Conclusions Enzastaurin was tolerable but had insufficient activity to proceed with the second stage of accrual. However, 1 patient has been progression-free for 44 months. No association between a biomarker and response to enzastaurin has been found. Exploratory analysis suggested an association between survival and PTEN copy number losses.