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INTRODUCTION: Protein kinase C (PKC) is a promising drug target for various therapeutic areas. Natural products derived from plants, animals, microorganisms, and marine organisms have been used by humans as medicine from prehistoric times. Recently, several compounds derived from plants have been found to modulate PKC activities through competitive binding with ATP binding site, and other allosteric regions of PKC. As a result fresh race has been started in academia and pharmaceutical companies to develop an effective naturally derived small-molecule inhibitor to target PKC activities. Herein, in this review, we have discussed several natural products and their derivatives, which are reported to have an impact on PKC signaling cascade. METHODS: All information presented in this review article regarding the regulation of PKC by natural products has been acquired by a systematic search of various electronic databases, including ScienceDirect, Scopus, Google Scholar, Web of science, ResearchGate, and PubMed. The keywords PKC, natural products, curcumin, rottlerin, quercetin, ellagic acid, epigallocatechin-3 gallate, ingenol 3 angelate, resveratrol, protocatechuic acid, tannic acid, PKC modulators from marine organism, bryostatin, staurosporine, midostaurin, sangivamycin, and other relevant key words were explored. RESULTS: The natural products and their derivatives including curcumin, rottlerin, quercetin, ellagic acid, epigallocatechin-3 gallate, ingenol 3 angelate, resveratrol, bryostatin, staurosporine, and midostaurin play a major role in the management of PKC activity during various disease progression. CONCLUSION: Based on the comprehensive literature survey, it could be concluded that various natural products can regulate PKC activity during disease progression. However, extensive research is needed to circumvent the challenge of isoform specific regulation of PKC by natural products.

The slow delayed rectifier potassium current (IKs) is formed by the KCNQ1 (Kv7.1) channel, an ion channel of four α‐subunits that modulates KCNE1 β‐subunits. IKs is central to the repolarization of the cardiac action potential. Loss of function mutation reducing ventricular cardiac IKs cause the long‐QT syndrome (LQTS), a disorder that predisposes patients to arrhythmia and sudden death. Current therapy for LQTS is inadequate. Rottlerin, a natural product of the kamala tree, activates IKs and has the potential to provide a new strategy for rational drug therapy. In this study, we show that simple modifications such as penta‐acetylation or penta‐methylation of rottlerin blunts activation activity. Total synthesis was used to prepare side‐chain‐modified derivatives that slowed down KCNQ1/KCNE1 channel deactivation to different degrees. A binding hypothesis of rottlerin is provided that opens the way to improved IKs activators as novel therapeutics for the treatment of LQTS., The polyphenolic natural product rottlerin, isolated from Kamala powder, has been used for centuries in traditional Indian medicine. Among other biological effects, rottlerin has been shown to activate KCNQ1 potassium channels. Rottlerin analogues were synthesized and evaluated electrophysiologically in X. laevis oocytes. A molecular modeling based model was developed to explain the binding‐mode of rottlerin and other KCNQ1 ligands.

3β,7β,25-Trihydroxycucurbita-5,23(E)-dien-19-al (TCD) is a triterpenoid isolated from wild bitter gourd that is a common tropical vegetable with neuroprotective effects. Because excessive glutamate release is a major cause of neuronal damage in various neurological disorders, the aims of this study were to examine the effect of TCD on glutamate release in vitro and to examine the effect of TCD in vivo. In rat cerebrocortical synaptosomes, TCD reduced 4-aminopyridine (4-AP)-stimulated glutamate release and Ca2+ concentration elevation, but had no effect on plasma membrane potential. TCD-mediated inhibition of 4-AP-induced glutamate release was dependent on the presence of extracellular calcium; persisted in the presence of the glutamate transporter inhibitor dl-TBOA, P/Q-type Ca2+ channel blocker ω-agatoxin IVA, and intracellular Ca2+-releasing inhibitors dantrolene and CGP37157; and was blocked by the vesicular transporter inhibitor bafilomycin A1 and the N-type Ca2+ channel blocker ω-conotoxin GVIA. Molecular docking studies have demonstrated that TCD binds to N-type Ca2+ channels. TCD-mediated inhibition of 4-AP-induced glutamate release was abolished by the Ca2+-dependent protein kinase C (PKC) inhibitor Go6976, but was unaffected by the Ca2+-independent PKC inhibitor rottlerin. Furthermore, TCD considerably reduced the phosphorylation of PKC, PKCα, and myristoylated alanine-rich C kinase substrate, a major presynaptic substrate for PKC. In a rat model of kainic acid (KA)-induced excitotoxicity, TCD pretreatment substantially attenuated KA-induced neuronal death in the CA3 hippocampal region. These results suggest that TCD inhibits synaptosomal glutamate release by suppressing N-type Ca2+ channels and PKC activity and exerts protective effects against KA-induced excitotoxicity in vivo.

Understanding the mechanism of SARS‐CoV‐2 infection and identifying potential therapeutics are global imperatives. Using a quantitative systems pharmacology approach, we identified a set of repurposable and investigational drugs as potential therapeutics against COVID‐19. These were deduced from the gene expression signature of SARS‐CoV‐2‐infected A549 cells screened against Connectivity Map and prioritized by network proximity analysis with respect to disease modules in the viral–host interactome. We also identified immuno‐modulating compounds aiming at suppressing hyperinflammatory responses in severe COVID‐19 patients, based on the transcriptome of ACE2‐overexpressing A549 cells. Experiments with Vero‐E6 cells infected by SARS‐CoV‐2, as well as independent syncytia formation assays for probing ACE2/SARS‐CoV‐2 spike protein‐mediated cell fusion using HEK293T and Calu‐3 cells, showed that several predicted compounds had inhibitory activities. Among them, salmeterol, rottlerin, and mTOR inhibitors exhibited antiviral activities in Vero‐E6 cells; imipramine, linsitinib, hexylresorcinol, ezetimibe, and brompheniramine impaired viral entry. These novel findings provide new paths for broadening the repertoire of compounds pursued as therapeutics against COVID‐19., A systems pharmacology approach is proposed to identify small molecules and drugs that target host cells and have anti‐SARS‐CoV‐2 effects. Shortlisted drug candidates are experimentally validated.

International audience; Rabies virus (RABV) is a neurotropic virus that causes fatal encephalitis in humans and animals and still kills up to 59,000 people worldwide every year. To date, only preventive or post-exposure vaccination protects against the disease but therapeutics are missing. After screening a library of 80 kinases inhibitors, we identified two compounds as potent inhibitors of RABV infection: Tyrphostin 9 and Rottlerin. Mechanism of action studies show that both inhibitors interfere with an early step of viral cycle and can prevent viral replication. In presence of Tyrphostin 9, the viral entry through endocytosis is disturbed leading to improper delivery of viral particles in cytoplasm, whereas Rottlerin is inhibiting the transcription, most likely by decreasing intracellular ATP concentrations, and therefore the replication of the viral genome.

Quercetin is a flavonoid polyphenolic compound present in fruits and vegetables that has proven anti-inflammatory activity. The goal of the present investigation was to investigate the effects of quercetin on tumor necrosis factor-α (TNF-α)-induced inflammatory responses via the expression of ICAM-1 and MMP-9 in human retinal pigment epithelial cells (ARPE-19 cells). Real-time PCR, gelatin zymography, and Western blot analysis showed that TNF-α induced the expression of ICAM-1 and MMP-9 protein and mRNA in a time-dependent manner. These effects were attenuated by pretreatment of ARPE-19 cells with quercetin. Quercetin inhibited the TNF-α-induced phosphorylation of PKCδ, JNK1/2, ERK1/2. Quercetin, rottlerin, SP600125 and U0126 attenuated TNF-α-stimulated c-Jun phosphorylation and AP-1-Luc activity. Pretreatment with quercetin, rottlerin, SP600125, or Bay 11-7082 attenuated TNF-α-induced NF-κB (p65) phosphorylation, translocation and RelA/p65-Luc activity. TNF-α significantly increased MMP-9 promoter activity and THP-1 cell adherence, and these effects were attenuated by pretreatment with quercetin, rottlerin, SP600125, U0126, tanshinone IIA or Bay 11-7082. These results suggest that quercetin attenuates TNF-α-induced ICAM-1 and MMP-9 expression in ARPE-19 cells via the MEK1/2-ERK1/2 and PKCδ-JNK1/2-c-Jun or NF-κB pathways.

With the introduction of arsenic trioxide and all-trans retinoic acid, the prognosis of acute promyelocytic leukemia has greatly improved. However, all-trans retinoic acid resistance is still unresolved in acute promyelocytic leukemia relapsed patients. In this study, the clinical achievable concentration of 7-hydroxystaurosporine synergized with all-trans retinoic acid to induce terminal differentiation in all-trans retinoic acid resistant acute promyelocytic leukemia cell lines. Though 7-hydroxystaurosporine is a PKC inhibitor, PKC might not be involved in the combination-induced differentiation since other PKC selective inhibitors, Go 6976 and rottlerin failed to cooperate with all-trans retinoic acid to trigger differentiation. The combination significantly enhanced the protein level of CCAAT/enhancer binding protein β and/or PU.1 as well as activated MEK/ERK. U0126 (MEK specific inhibitor) not only suppressed the combination-induced differentiation but also restored the protein level of CCAAT/enhancer binding protein β and/or PU.1. However, RAF-1 inhibitor had no inhibitory effect on MEK activation and the combination-induced differentiation. Therefore, the combination overcame differentiation block via RAF-1 independent MEK/ERK modulation of the protein level of CCAAT/enhancer binding protein β and/or PU.1. These findings may provide a preclinical rationale for the potential role of this combination in the treatment of all-trans retinoic acid resistant acute promyelocytic leukemia patients.

Astaxanthin (AST) is a product made from marine organisms that has been used as an anti-cancer supplement. It reduces pontin expression and induces apoptosis in SKBR3, a breast cancer cell line. Using Western blotting and qRT-PCR analyses, this study revealed that in the T47D and BT20 breast cancer cell lines, AST inhibits expression of pontin and mutp53, as well as the Oct4 and Nanog cancer stem cell (CSC) stemness genes. In addition, we explored the mechanism by which AST eradicates breast cancer cells using pontin siRNAs. Pontin knockdown by pontin siRNA reduced proliferation, Oct4 and Nanog expression, colony and spheroid formation, and migration and invasion abilities in breast cancer cells. In addition, reductions in Oct4, Nanog, and mutp53 expression following rottlerin treatment confirmed the role of pontin in these cells. Therefore, pontin may play a central role in the regulation of CSC properties and in cell proliferation following AST treatment. Taken together, these findings demonstrate that AST can repress CSC stemness genes in breast cancer cells, which implies that AST therapy could be used to improve the efficacy of other anti-cancer therapies against breast cancer cells.

Magnolol is one of the hydroxylated biphenyl compounds from the root and stem bark of Magnolia officinalis, which shown to possess anti-colorectal cancer (CRC) effects. However, the regulatory mechanism of magnolol on apoptosis and NF-&kappa, B signaling in human CRC has not been elucidated. Thus, we investigated the inhibitory mechanism of magnolol on human and mouse CRC (HT-29 and CT-26) in vitro and in vivo. Results from reporter gene assay indicated that both magnolol and rottlerin (PKC&delta, inhibitor) reduced the endogenous NF-&kappa, B activity. In addition, indolactam V (PKC&delta, activator)-induced NF-&kappa, B signaling was significantly suppressed with both magnolol and rottlerin treatment. Results from Western blotting also indicated that phosphorylation of PKC&delta, and NF-&kappa, B -related proteins involved in tumor progression were effectively decreased by magnolol treatment. The invasion capacity of CRC cells was also attenuated by both magnolol and rottlerin. Furthermore, magnolol triggered Fas/Fas-L mediated extrinsic apoptosis and mitochondria mediated intrinsic apoptosis were validated by flow cytometry. Most importantly, tumor growth in both HT-29 and CT-26 bearing mice were suppressed by magnolol, but no pathologic change was detected in mice kidney, spleen, and liver. As confirmed by immunohistochemistry (IHC) staining from tumor tissue, PKC&delta, /NF-&kappa, B signaling and downstream proteins expression were decreased, while apoptotic proteins expression was increased in the magnolol treated group. According to these results, we suggest that the induction of apoptosis through extrinsic/intrinsic pathways and the blockage of PKC&delta, B signaling are associated with the magnolol-inhibited progression of CRC.

Chronic high-glucose exposure results in the production of advanced glycation end-products (AGEs) leading to reactive oxygen species (ROS) generation, which contributes to the development of diabetic cardiomyopathy. PKC&delta, activation leading to ROS production and mitochondrial dysfunction involved in AGE-induced cardiomyocyte apoptosis was reported in our previous study. Diallyl trisulfide (DATS) is a natural cytoprotective compound under various stress conditions. In this study, the cardioprotective effect of DATS against rat streptozotocin (STZ)-induced diabetic mellitus (DM) and AGE-induced H9c2 cardiomyoblast cell/neonatal rat ventricular myocyte (NRVM) damage was assessed. We observed that DATS treatment led to a dose-dependent increase in cell viability and decreased levels of ROS, inhibition of PKC&delta, activation, and recuded apoptosis-related proteins. Most importantly, DATS reduced PKC&delta, mitochondrial translocation induced by AGE. However, apoptosis was not inhibited by DATS in cells transfected with PKC&delta, wild type (WT). Inhibition of PKC&delta, by PKC&delta, kinase-deficient (KD) or rottlerin not only inhibited cardiac PKC&delta, activation but also attenuated cardiac cell apoptosis. Interestingly, overexpression of PKC&delta, WT plasmids reversed the inhibitory effects of DATS on PKC&delta, activation and apoptosis in cardiac cells exposed to AGE, indicating that DATS may inhibit AGE-induced apoptosis by downregulating PKC&delta, activation. Similar results were observed in AGE-induced NRVM cells and STZ-treated DM rats following DATS administration. Taken together, our results suggested that DATS reduced AGE-induced cardiomyocyte apoptosis by eliminating ROS and downstream PKC&delta, signaling, suggesting that DATS has potential in diabetic cardiomyopathy (DCM) treatment.

The present study aimed to analyze the compensatory signaling pathways induced by forkhead domain inhibitor‑6 (FDI‑6), which is a forkhead box protein M1 (FOXM1) inhibitor, in ovarian cancer cells and evaluate the effectiveness of simultaneous inhibition of FOXM1 and the compensatory signaling pathway in decreasing the survival of ovarian cancer cells. The present study identified the proteins involved in the compensatory mechanism activated by FDI‑6 in HeyA8 ovarian cancer cells using western blot analysis and a reverse‑phase protein array. In addition, a cell viability assay was performed to determine the effects of FDI‑6 and the compensatory signaling pathway on cancer cell viability. All experiments were performed in three‑dimensional cell cultures. The present study observed that FDI‑6 stimulated the upregulation of N‑Ras, phosphoprotein kinase Cδ (p‑PKCδ) (S664) and HER3 in HeyA8 cells. Tipifarnib as an N‑Ras inhibitor, rottlerin as a p‑PKCδ (S664) inhibitor and sapitinib as a HER3 inhibitor were selected. The combination of FDI‑6 with tipifarnib attenuated the upregulation of N‑Ras induced by FDI‑6 and the combination of FDI‑6 with sapitinib also attenuated HER3 downstream signaling pathway in HeyA8 cells, as shown by on western blot analysis. Rottlerin downregulated p‑PKCδ (S664) by inhibiting the activity of a Src‑related tyrosine kinase that transfers a phosphate group to PKCδ. Compared with FDI‑6 alone, the addition of tipifarnib or rottlerin to FDI‑6 was significantly more effective in reducing the growth of HeyA8 cells. However, the combination of FDI‑6 and sapitinib did not induce a significant decrease in survival of HeyA8 cells. In conclusion, the addition of tipifarnib or rottlerin to inhibit N‑Ras or p‑PKCδ (S664), respectively, inhibited the compensatory signaling pathway response induced by FDI‑6 in HeyA8 cells. These inhibitors increased the efficacy of FDI‑6, which inhibits FOXM1, in reducing ovarian cancer cell viability.

We previously showed that overexpression of Thy-1 inhibited and knock-down of Thy-1 enhanced endothelial cell migration. Here, we used phorbol-12-myristate-13-acetate (PMA) as an inducer for Thy-1 expression to investigate molecular mechanisms underlying Thy-1 up-regulation. Our data showed that increased levels of Thy-1 mRNA and protein in endothelial cells were observed at 14–18 hours and 20–28 hours after PMA treatment, respectively. Treatment with PMA for 32 hours induced Thy-1 up-regulation and inhibited capillary-like tube formation and endothelial cell migration. These effects were abolished by Röttlerin (a PKC-δ inhibitor), but not Gö6976 (a PKC-α/β inhibitor). Moreover, pre-treatment with Bay 61–3606 (a Syk inhibitor) or Bay 11-7082 (a NF-κB inhibitor) abolished the PMA-induced Thy-1 up-regulation and migration inhibition in endothelial cells. Using the zebrafish model, we showed that PMA up-regulated Thy-1 and inhibited angiogenesis through the PKC-δ-mediated pathway. Surprisingly, we found that short-term (8–10 hours) PMA treatment enhanced endothelial cell migration. However, this effect was not observed in PMA-treated Thy-1-overexpressed endothelial cells. Taken together, our results suggest that PMA initially enhanced endothelial cell migration, subsequently activating the PKC-δ/Syk/NF-κB-mediated pathway to up-regulate Thy-1, which in turn inhibited endothelial cell migration. Our results also suggest that Thy-1 might play a role in termination of angiogenesis.

Scleral fibroblast activation occurs in glaucomatous and myopic eyes. Here we perform an unbiased screen to identify kinase inhibitors that reduce fibroblast activation to diverse stimuli in vitro and to in vivo intraocular pressure (IOP) elevation. Primary cultures of peripapillary scleral (PPS) fibroblasts from two human donors were screened using a library of 80 kinase inhibitors to identify compounds that inhibit TGFβ-induced extracellular matrix (ECM) synthesis. Inhibition of myofibroblast differentiation was verified by alpha smooth muscle actin (αSMA) immunoblot and collagen contraction assay. Inhibition of IOP-induced scleral fibroblast proliferation was assessed by ELISA assay for proliferating cell nuclear antigen (PCNA). The initial screen identified 7 inhibitors as showing>80% reduction in ECM binding. Three kinase inhibitors were verified to reduce TGFβ-induced αSMA expression and cellular contractility (rottlerin, PP2, tyrphostin 9). The effect of three Src inhibitors, bosutinib, dasatinib, and SU-6656, on myofibroblast differentiation was evaluated, with only dasatinib significantly inhibiting TGFβ-induced ECM synthesis, αSMA expression, and cellular contractility at nanomolar dosages. Subconjunctival injection of dasatinib reduced IOP-induced scleral fibroblast proliferation compared to control (4.9 ± 11.1 ng/sclera with 0.1 μM versus 88.7 ± 38.6 ng/sclera in control, P

Application of high-dosage UVB irradiation in phototherapeutic dermatological treatments present health concerns attributed to UV-exposure. In assessing UV-induced photobiological damage, we investigated dose-dependent effects of UVB irradiation on human keratinocyte cells (HaCaT). Our study implemented survival and apoptosis assays and revealed an unexpected dose response wherein higher UVB-dosage induced higher viability. Established inhibitors, such as AKT- (LY294002), PKC- (Gö6976, and Rottlerin), ERK- (PD98059), P38 MAPK- (SB203580), and JNK- (SP600125), were assessed to investigate UV-induced apoptotic pathways. Despite unobvious contributions of known signaling pathways in dose-response mediation, microarray analysis identified transcriptional expression of UVB-response genes related to the respiratory-chain. Observed correlation of ROS-production with UVB irradiation potentiated ROS as the underlying mechanism for observed dose responses. Inability of established pathways to explain such responses suggests the complex nature underlying UVB-phototherapy response.

Du et al. demonstrate that PKCδ modulates BACE1 expression and amyloidogenic amyloid precursor protein processing. Inhibition of PKCδ markedly reduces BACE1 expression, β-amyloid levels, and amyloid plaque formation and also rescues cognitive deficits in Alzheimer disease mouse models., β-amyloid protein (Aβ) plays a central role in the pathogenesis of Alzheimer disease (AD). Aβ is generated from sequential cleavage of amyloid precursor protein (APP) by β-site APP-cleaving enzyme 1 (BACE1) and the γ-secretase complex. Although activation of some protein kinase C (PKC) isoforms such as PKCα and ε has been shown to regulate nonamyloidogenic pathways and Aβ degradation, it is unclear whether other PKC isoforms are involved in APP processing/AD pathogenesis. In this study, we report that increased PKCδ levels correlate with BACE1 expression in the AD brain. PKCδ knockdown reduces BACE1 expression, BACE1-mediated APP processing, and Aβ production. Conversely, overexpression of PKCδ increases BACE1 expression and Aβ generation. Importantly, inhibition of PKCδ by rottlerin markedly reduces BACE1 expression, Aβ levels, and neuritic plaque formation and rescues cognitive deficits in an APP Swedish mutations K594N/M595L/presenilin-1 with an exon 9 deletion–transgenic AD mouse model. Our study indicates that PKCδ plays an important role in aggravating AD pathogenesis, and PKCδ may be a potential target in AD therapeutics., Graphical Abstract

Abnormal lipid metabolism may contribute to the pathogenesis of non-alcoholic steatohepatitis (NASH). The ATP-binding cassette transporter A1 (ABCA1) protein mediates the transport of cholesterol and phospholipids from cells to apolipoprotein A-I (apoA-I) to generate nascent HDL particles. Previous studies revealed that the overexpression of ABCA1 alleviated hepatic lipid levels by modifying lipid transport. Here, we examined the effect of berberine (BBR) on ABCA1 in QSG-7701 hepatocytes and in mice. BBR decreased hepatic cholesterol and triglyceride levels. It also increased ABCA1 protein levels but not mRNA levels in a time- and dose-dependent manner. The PKCδ inhibitor rottlerin and PKCδ siRNA completely abolished the effect of BBR on ABCA1. BBR also decreased the phosphorylation of ABCA1 serine residues and PKCδ Tyr 311. The inhibition of ABCA1 with its siRNA abolished the reduction in cellular cholesterol levels by BBR. BBR administration to mice fed a methionine choline-deficient diet also significantly increased ABCA1 protein levels and reduced hepatic lipid levels. These results suggest that BBR can reduce steatosis by increasing ABCA1 protein levels through PKCδ to reduce the phosphorylation of serine residues in ABCA1.

Polydopamine (PDA) coating as a bioinspired strategy for nanoparticles (NPs) has been extensively applied in cancer theranostics. However, a cellular-level understanding of nano-biointeraction of these PDA-coated NPs (PDNPs), which drives the fate of them and acts as a critical step to determine their efficacy, still remains unknown. Herein, we utilized the representative mesoporous silica NPs (MSNs) to be coated with PDA and study their nano-bioactivities in cancer cells. HeLa cell line was utilized as a model in this study. The PDNPs were discovered to be internalized through three specific pathways, that is, Caveolae-, Arf6-dependent endocytosis, and Rab34-mediated macropinocytosis (55%, 20% and 37% of uptake inhibition by nystatin, Arf6 knockdown, and rottlerin, respectively). Autophagy-mediated accumulation of PDNPs in lysosomes was observed and the formed PDA shells shedded in the lysosomes. Almost 40% of the NPs were transported out of cells via Rab8/10- and Rab3/26-mediated exocytosis pathways at our tested level. On the basis of these results, a novel combined cancer treatment strategy was further proposed using drug-loaded MSNs-PDA by (i) utilizing naturally intracellular mechanism-controlled PDA shedding for organelle-targeted release of drugs in lysosomes to generate lysosome impairment and (ii) blocking the demonstrated exocytosis pathways for enhanced therapeutic efficacy.

Rottlerin is a cytostatic and cytotoxic drug in a variety of cancer cells. Our previous experience demonstrated that depending upon the genetic/biochemical background of cancer cells, rottlerin is able to induce both apoptotic and autophagic cell death, or dramatically disturb protein homeostasis leading to lethal cellular atrophy. In the current study, we investigated the cytotoxic effects and mechanisms of rottlerin against human amelanotic A375 melanoma cells. In this cell line, rottlerin exhibits its main and newest cytotoxic properties, that is, growth arrest, apoptosis induction, and translation shutoff. In fact, the drug, time-, and dose-dependently, markedly inhibited cell proliferation through cyclin D1 downregulation and induced apoptotic cell death as early as after 18 h treatment. Mechanistically, rottlerin triggered apoptosis by both intrinsic and extrinsic pathways. Both pathways are likely activated by the downregulation of the antiapoptotic B-cell lymphoma 2 (Bcl-2) protein, which simultaneously affects mitochondrial and endoplasmic reticulum (ER) membranes stability. Concomitantly to extrinsic apoptosis induction, the rottlerin-activated ER stress/eukaryotic initiation factor 2 (eIF2) α axis blocked the translational apparatus. The altered proteostasis precluded the complete cells' rescue from death in the presence of apoptosis inhibitors.

Angiotensin II (AngII) and the mineralocorticoid receptor (MR) ligand aldosterone both contribute to cardiovascular disorders, including hypertension and adverse vascular remodeling. We previously demonstrated that AngII activates MR-mediated gene transcription in human vascular smooth muscle cells (SMCs), yet the mechanism and the impact on SMC function are unknown. Using an MR-responsive element-driven transcriptional reporter assay, we confirm that AngII induces MR transcriptional activity in vascular SMCs and endothelial cells, but not in Cos1 or human embryonic kidney-293 cells. AngII activation of MR was blocked by the MR antagonist spironolactone or eplerenone and the protein kinase C-δ (PKCδ) inhibitor rottlerin, implicating both in the mechanism. Similarly, small interfering RNA knockdown of PKCδ in SMCs prevented AngII-mediated MR activation, whereas knocking down of MR blocked both aldosterone- and AngII-induced MR function. Coimmunoprecipitation studies reveal that endogenous MR and PKCδ form a complex in SMCs that is enhanced by AngII treatment in association with increased serine phosphorylation of the MR N terminus. AngII increased mRNA expression of the SMC-MR target gene, FKBP51, via an MR-responsive element in intron 5 of the FKBP51 gene. The impact of AngII on FKBP51 reporter activity and gene expression in SMCs was inhibited by spironolactone and rottlerin. Finally, the AngII-induced increase in SMC number was also blocked by the MR antagonist spironolactone and the PKCδ inhibitor rottlerin. These data demonstrate that AngII activates MR transcriptional regulatory activity, target gene regulation, and SMC proliferation in a PKCδ-dependent manner. This new mechanism may contribute to synergy between MR and AngII in driving SMC dysfunction and to the cardiovascular benefits of MR and AngII receptor blockade in humans.

Hypoxia-inducible factor 1 (HIF-1) plays important roles in cancer cell biology. HIF-1&alpha, is reportedly activated by several factors, including protein kinase C (PKC), in addition to hypoxia. We investigated the role of PKC isoforms and the effects of vitamin K2 (VK2) in the activation process of HIF-1&alpha, Human hepatocellular carcinoma (HCC)-derived Huh7 cells were cultured under normoxic and hypoxic (1% O2) conditions with or without the PKC stimulator TPA. The expression, transcriptional activity and nuclear translocation of HIF-1&alpha, were examined under treatment with PKC inhibitors, siRNAs against each PKC isoform and VK2. Hypoxia increased the expression and activity of HIF-1&alpha, TPA increased the HIF-1&alpha, activity several times under both normoxic and hypoxic conditions. PKC-&delta, siRNA-mediated knockdown, PKC-&delta, inhibitor (rottlerin) and pan-PKC inhibitor (Ro-31-8425) suppressed the expression and transcriptional activity of HIF-1&alpha, VK2 significantly inhibited the TPA-induced HIF-1&alpha, transcriptional activity and suppressed the expression and nuclear translocation of HIF-1&alpha, induced by TPA without altering the HIF-1&alpha, mRNA levels. These data indicate that PKC-&delta, enhances the HIF-1&alpha, transcriptional activity by increasing the nuclear translocation, and that VK2 might suppress the HIF-1&alpha, activation through the inhibition of PKC in HCC cells.

Exposure to UVR stimulates the cAMP signaling pathway, which leads to melanin deposits in skin tissues. Although melanogenesis can be beneficial by protecting skin from UVR-induced damage, excessive or uneven deposits of melanin can cause various skin hyperpigmentation disorders. Because cAMP-responsive element binding protein (CREB) and CREB-regulated transcription coactivators (CRTC) play a major role in conveying cAMP signals that induce transcription of microphthalmia-associated transcription factor and melanin production, we screened for a CREB or CRTC inhibitor and identified rottlerin (Rot) as a potent inhibitor of its transcriptional activity. Rot suppressed melanin production in both basal and cAMP-stimulated cultured melanocytes by downregulating melanogenic gene expression. In addition, topical application of Rot on the tails of mice decreased melanin accumulation. Mechanistically, we showed that Rot decreased the mitochondrial membrane potential, which then activated AMPK, leading to the phosphorylation and nuclear exclusion of CRTC3 and suppressing the expression of CREB target genes, including MITF. Our study demonstrates that Rot is an active antimelanogenic agent and suggests that screening for an inhibitor of CREB or CRTC transcriptional activity is a promising strategy by which to discover better drugs to treat skin hyperpigmentary disorders.

We previously reported that oral formulations containing indomethacin nanoparticles (IND-NPs) showed high bioavailability, and, consequently, improved therapeutic effects and reduced injury to the small intestine. However, the pathway for the transintestinal penetration of nanoparticles remained unclear. Thus, in this study, we investigated whether endocytosis was related to the penetration of IND-NPs (72.1 nm) using a transcell set with Caco-2 cells or rat intestine. Four inhibitors of various endocytosis pathways were used [nystatin, caveolae-dependent endocytosis (CavME), dynasore, clathrin-dependent endocytosis (CME), rottlerin, macropinocytosis, and cytochalasin D, phagocytosis inhibitor], and all energy-dependent endocytosis was inhibited at temperatures under 4 &deg, C in this study. Although IND-NPs showed high transintestinal penetration, no particles were detected in the basolateral side. IND-NPs penetration was strongly prevented at temperatures under 4 &deg, C. In experiments using pharmacological inhibitors, only CME inhibited penetration in the jejunum, while in the ileum, both CavME and CME significantly attenuated penetration. In conclusion, we found a novel pathway for the transintestinal penetration of drug nanoparticles. Our hypothesis was that nanoparticles would be taken up into the intestinal epithelium by endocytosis (CME in jejunum, CavME and CME in ileum), and dissolved and diffused in the intestine. Our findings are likely to be of significant use for the development of nanomedicines.

Objective: Intervertebral disc (IVD) degeneration and disc herniation are major causes of lower back pain, which involve the presence of inflammatory mediators and tissue invasion by immune cells. Intercellular adhesion molecule 1 (ICAM1, also termed CD54) is an adhesion molecule that mediates cell-cell interactions, particularly between immune cells and target tissue. The aim of this study was to examine the intracellular signaling pathways involved in inflammatory stimuli-induced ICAM1 expression in human anulus fibrosus (AF) cells. Methods: Quantitative reverse transcription-polymerase chain reaction (qPCR), western blotting, and flow cytometry were performed to dissect the roles of different signaling pathways in inflammatory stimuli-mediated ICAM1 expression. Results: Using qPCR and western blot analyses, a significant increase in ICAM1 expression was observed in AF cells after stimulation of lipopolysaccharide (LPS) plus interferon-gamma (IFN&gamma, ) in a time-dependent manner. Flow cytometry revealed ICAM1 upregulation on the surface of AF cells. Importantly, LPS plus IFN&gamma, treatment also significantly promoted Chemokine ligand (CCL)2 expression, but not CCL3. The enhanced ICAM1 expression was abolished after incubation with antibody against CCL2. In AF cells, treatment with LPS plus IFN&gamma, activated the FAK/ERK/GSK3 signaling pathways, promoted a time-dependent increase in PKC&delta, phosphorylation, and promoted PKC&delta, translocation to the nucleus. Treatment with the pharmacological PKC&delta, inhibitor, rottlerin, effectively blocked the enhanced productions of ICAM1 and CCL2. Conclusions: Inflammatory stimuli in AF cells are part of a specific pathophysiology in IVD degeneration and disc herniation that modulates CCL2/ICAM1 activation through the FAK/ERK/GSK3 and PKC&delta, signaling pathways in AF cells.

Dysregulation of neutrophil apoptosis causes pathogenesis and aggravation of allergy. S100A9 exists as one of the proteins in the neutrophils, triggering inflammatory responses by activating the immune cells. In this study, we investigated whether S100A9 affects constitutive neutrophil apoptosis by activating the monocytes in normal and allergic subjects. Supernatant from human monocytic THP-1 cells after treatment with S100A9 suppressed normal neutrophil apoptosis by inhibiting the activations of caspase 9 and caspase 3. S100A9 upregulated the release of MCP-1, IL-6, and IL-8 in THP-1 cells. An increase in cytokine was suppressed by CLI-095, a Toll-like receptor (TLR) 4 inhibitor, PP2, a Src inhibitor, rottlerin, a PKCδ inhibitor, MAP kinase inhibitors, including PD98059, SB202190, and SP600125, and BAY-11-7085, an NF-κB inhibitor. Src, PKCδ, ERK1/2, p38 MAPK, and JNK were phosphorylated by S100A9. The phosphorylation of Src and PKCδ was suppressed by CLI-095, and the activation of ERK1/2, p38 MAPK, and JNK was inhibited by CLI-095, PP2, and rottlerin. S100A9 induced NF-κB activity, and the activation was suppressed by CLI-095, PP2, rottlerin, and MAPK kinase inhibitors. In normal and allergic subjects, supernatant from normal and allergic monocytes after stimulation with S100A9 suppressed normal and allergic neutrophil apoptosis, respectively; MCP-1, IL-6, and IL-8 in the supernatant was increased by S100A9. The cytokine secretion induced by S100A9 is related to TLR4, Src, PKCδ, ERK1/2, p38 MAPK, JNK, and NF-κB. Taken together, S100A9 induces anti-apoptotic effect on normal and allergic neutrophils by increasing cytokine secretion of monocytes. These findings may help us to better understand neutrophil apoptosis regulated by S100A9 and pathogenesis of allergic diseases.

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 kinase C (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 protein β (C/EBPβ) and C/EBPe 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.

Rottlerin, isolated from a medicinal plant Mallotus phillippinensis, has been demonstrated to inhibit cellular growth and induce cytoxicity in glioblastoma cell lines through inhibition of calmodulin-dependent protein kinase III. Emerging evidence suggests that rottlerin exerts its antitumor activity as a protein kinase C inhibitor. Although further studies revealed that rottlerin regulated multiple signaling pathways to suppress tumor cell growth, the exact molecular insight on rottlerin-mediated tumor inhibition is not fully elucidated. In the current study, we determine the function of rottlerin on glioma cell growth, apoptosis, cell cycle, migration and invasion. We found that rottlerin inhibited cell growth, migration, invasion, but induced apoptosis and cell cycle arrest. Mechanistically, the expression of Cdc20 oncoprotein was measured by the RT-PCR and Western blot analysis in glioma cells treated with rottlerin. We observed that rottlerin significantly inhibited the expression of Cdc20 in glioma cells, implying that Cdc20 could be a novel target of rottlerin. In line with this, over-expression of Cdc20 decreased rottlerin-induced cell growth inhibition and apoptosis, whereas down-regulation of Cdc20 by its shRNA promotes rottlerin-induced anti-tumor activity. Our findings indicted that rottlerin could exert its tumor suppressive function by inhibiting Cdc20 pathway which is constitutively active in glioma cells. Therefore, down-regulation of Cdc20 by rottlerin could be a promising therapeutic strategy for the treatment of glioma.

The molecular bases of disease provide exceptional prospect to translate research findings into new drugs. Nevertheless, to develop new and novel chemical entities takes huge amount of time and efforts, mainly due to the stringent processes. Therefore, drug repurposing is one of such strategies which is being used in recent times to identify new pharmacophores. The essential first step in discovery of the specific inhibitor with low toxicity is the identification and elucidation of pathways exclusive to target pathogen. One such target is the shikimate pathway, which is essential for algae, higher plants, bacteria and fungi. Since, this enzyme system is absent in higher eukaryotes and in mammals, the enzymes involved in the pathway provide an attractive target for the development of potentially selective and non toxic antimicrobial agents. Since, so far there is no specific inhibitor which is able to restrain mycobacterial shikimate pathway; we expanded the use of a known kinase inhibitor; Rottlerin, in order to predict the prototype in discovering the specific molecules against this enzyme. For the first time we have shown that Rottlerin inhibits extracellular mycobacteria by affecting Shikimate Kinase (SK) and this effect is further enhanced during the intracellular infection due to the added effect of PKC- δ down-regulation. The molecular docking of Rottlerin with both the mycobacterial SKs, corroborated the inhibition data, and revealed that the effects of SK, in slow and in fast grower mycobacteria are due to the changes in affinity of binding with the drug.

// Xuyuan Yin 1, * , Yu Zhang 2, * , Jingna Su 1, * , Yingying Hou 1 , Lixia Wang 1 , Xiantao Ye 1 , Zhe Zhao 1 , Xiuxia Zhou 1 , Yali Li 3 , Zhiwei Wang 1, 4 1 The Cyrus Tang Hematology Center and Collaborative Innovation Center of Hematology, Jiangsu Institute of Hematology, The First Affiliated Hospital, Soochow University, Suzhou, China 2 Department of Oncology, Guizhou People’s Hospital, Guizhou, China 3 Department of Anesthesiology, Shenzhen People’s Hospital, Shenzhen Anesthesiology Engineering Center, The Second Clinical Medical College, Jinan University, Guangdong, China 4 Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Cambridge, MA, USA * These authors contributed equally to this work Correspondence to: Yali Li, email: lyl00111@163.com Zhiwei Wang, email: zwang6@bidmc.harvard.edu Keywords: rottlerin, breast cancer, Skp2, invasion, apoptosis Received: June 06, 2016 Accepted: August 15, 2016 Published: August 25, 2016 ABSTRACT Studies have investigated the tumor suppressive role of rottlerin in carcinogenesis. However, the molecular mechanisms of rottlerin-induced anti-tumor activity are largely unclear. Skp2 (S-phase kinase associated protein 2) has been validated to play an oncogenic role in a variety of human malignancies. Therefore, inactivation of Skp2 could be helpful for the treatment of human cancers. In the current study, we explore whether rottlerin could inhibit Skp2 expression, leading to inhibition of cell growth, migration and invasion in breast cancer cells. We found that rottlerin treatment inhibited cell growth, induced apoptosis and cell cycle arrest. We also revealed that rottlerin suppressed cell migration and invasion in breast cancer cells. Mechanically, we observed that rottlerin significantly down-regulated the expression of Skp2 in breast cancer cells. Importantly, overexpression of Skp2 abrogated rottlerin-mediated tumor suppressive activity, whereas down-regulation of Skp2 enhanced rottlerin-triggered anti-tumor function. Strikingly, we identified that rottlerin exhibited its anti-tumor potential partly through inactivation of Skp2 in breast cancer. Our findings indicate that rottlerin could be a potential safe agent for the treatment of breast cancer.

Adiponectin, an adipokine, accumulated in lung system via T-cadherin after allergens/ozone challenge. However, the roles of adiponectin on lung pathologies were controversial. Here we reported that adiponectin stimulated expression of inflammatory proteins, cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2 (COX-2), and production of reactive oxygen species (ROS) in human alveolar type II A549 cells. AdipoR1/2 involved in adiponectin-activated NADPH oxidase and mitochondria, which further promoted intracellular ROS accumulation. Protein kinase C (PKC) may involve an adiponectin-activated NADPH oxidase. Similarly, p300 phosphorylation and histone H4 acetylation occurred in adiponectin-challenged A549 cells. Moreover, adiponectin-upregulated cPLA2 and COX-2 expression was significantly abrogated by ROS scavenger ( N-acetylcysteine) or the inhibitors of NADPH oxidase (apocynin), mitochondrial complex I (rotenone), PKC (Ro31-8220, Gö-6976, and rottlerin), and p300 (garcinol). Briefly, we reported that adiponectin stimulated cPLA2 and COX-2 expression via AdipoR1/2-dependent activation of PKC/NADPH oxidase/mitochondria resulting in ROS accumulation, p300 phosphorylation, and histone H4 acetylation. These results suggested that adiponectin promoted lung inflammation, resulting in exacerbation of pulmonary diseases via upregulating cPLA2 and COX-2 expression together with intracellular ROS production. Understanding the adiponectin signaling pathways on regulating cPLA2 and COX-2 may help develop therapeutic strategies on pulmonary diseases.

Proteinase-activated receptor 2 (PAR-2) and toll-like receptor 4 (TLR4) are involved in innate immune responses and signaling cross-talk between these receptor molecules has the potential to augment an ongoing inflammatory response. The aim of this study was to evaluate the possible cooperative influence of PAR-2 and TLR4 on IL-12p40 production by macrophages after stimulation with lipopolysaccharide (LPS). During culture, GM-CSF upregulated PAR-2 expression by macrophages in a time-dependent manner. Stimulation with LPS enhanced IL-12p40 production by macrophages in a concentration-dependent manner. While human neutrophil elastase (HNE) did not induce IL-12p40 production, pretreatment of macrophages with HNE synergistically increased the IL-12p40 protein level after LPS exposure. Silencing of TLR4 with small interfering RNA blunted the synergistic enhancement of IL-12p40 by HNE combined with LPS. Silencing of β-arrestin 2, p22phox, or ERK1/2 also inhibited an increase of IL-12p40. Interestingly, transfection of macrophages with small interfering RNA duplexes for DUOX-2, EGFR, TLR4, or TRAF6 significantly blunted the increase of IL-12p40 in response to treatment with HNE plus LPS. U73122 and Rottlerin also inhibited the increased production of IL-12p40. In conclusion, HNE is involved in transactivation of TLR4 through activation of DUOX-2/EGFR and synergistically enhances IL-12p40 production by macrophages stimulated with LPS.

Granulocyte-macrophage colony stimulating factor (GM-CSF) induces procoagulant activity of macrophages. Tissue factor (TF) is a membrane-bound glycoprotein and substance P (SP) is a pro-inflammatory neuropeptide involved in the formation of membrane blebs. This study investigated the role of SP in TF release by GM-CSF-dependent macrophages. SP significantly decreased TF levels in whole-cell lysates of GM-CSF-dependent macrophages. TF was detected in the culture supernatant by enzyme-linked immunosorbent assay after stimulation of macrophages by SP. Aprepitant (an SP/neurokinin 1 receptor antagonist) reduced TF release from macrophages stimulated with SP. Pretreatment of macrophages with a radical scavenger(pyrrolidinedithiocarbamate) also limited the decrease of TF in whole-cell lysates after stimulation with SP. A protein kinase C inhibitor (rottlerin) partially blocked this macrophage response to SP, while it was significantly inhibited by a ROCK inhibitor (Y-27632) or a dynamin inhibitor (dinasore). An Akt inhibitor (perifosine) also partially blocked this response. Furthermore, siRNA targeting p22phox, β-arrestin 2, or Rho A, blunted the release of TF from macrophages stimulated with SP. In other experiments, visceral adipocytes derived from cryopreserved preadipocytes were found to produce SP. In conclusion, SP enhances the release of TF from macrophages via the p22phox/β-arrestin 2/Rho A signaling pathway.

Fascin-1, an actin-bundling protein, plays an important role in cancer cell migration and invasion; however, the underlying mechanism remains unclear. On the basis of a 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced cell migration model, it was shown that TPA increased fascin-1 mRNA and protein expression and fascin-1-dependent cell migration. TPA dose- and time-dependently increased PKCδ and STAT3α activation and GSK3β phosphorylation; up-regulated Wnt-1, β-catenin, and STAT3α expression; and increased the nuclear translocation of β-catenin and STAT3α. Rottlerin, a PKCδ inhibitor, abrogated the increases in STAT3α activation and β-catenin and fascin-1 expression. WP1066, a STAT3 inhibitor, suppressed TPA-induced STAT3α DNA binding activity and β-catenin expression. Knockdown of β-catenin attenuated TPA-induced fascin-1 and STAT3α expression as well as cell migration. In addition to MCF-7, migration of Hs578T breast cancer cells was inhibited by silencing fascin-1, β-catenin, and STAT3α expression as well. TPA also induced Wnt-1 expression and secretion, and blocking Wnt-1 signaling abrogated β-catenin induction. DHA pretreatment attenuated TPA-induced cell migration, PKCδ and STAT3α activation, GSK3β phosphorylation, and Wnt-1, β-catenin, STAT3α, and fascin-1 expression. Our results demonstrated that TPA-induced migration is likely associated with the PKCδ and Wnt-1 pathways, which lead to STAT3α activation, GSK3β inactivation, and β-catenin increase and up-regulation of fascin-1 expression. Moreover, the anti-metastatic potential of DHA is partly attributed to its suppression of TPA-activated PKCδ and Wnt-1 signaling.

Background/Aims: The phytochemical polyphenol rottlerin is a potent activator of diverse Ca2+ -sensitive K+ channels. Those channels play a decisive role in the execution of eryptosis, the suicidal death of erythrocytes, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the stimulation of eryptosis includes increase of cytosolic Ca2+ activity ([Ca2+]i) and ceramide. The present study explored, whether rottlerin induces eryptosis and, if so, to test for the involvement of Ca2+ entry and ceramide. Methods: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified by determination of haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to rottlerin (1 - 5 µM) significantly increased the percentage of annexin-V-binding cells, an effect paralleled by significant decrease of forward scatter. Up to 5 µM rottlerin failed to significantly increase average Fluo3-fluorescence. Rottlerin (5 µM) did, however, significantly increase the ceramide abundance. Rottlerin (5 µM) further significantly increased hemolysis. The effect of rottlerin (5 µM) on annexin-V-binding was virtually abolished by removal of extracellular Ca2+. Conclusions: Rottlerin stimulates eryptosis with erythrocyte shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by and at least in part due to Ca2+ entry and ceramide.

The MTT assay (to a less degree MTS, XTT or WST) is a widely exploited approach for measuring cell viability/drug cytotoxicity. MTT reduction occurs throughout a cell and can be significantly affected by a number of factors, including metabolic and energy perturbations, changes in the activity of oxidoreductases, endo-/exocytosis and intracellular trafficking. Over/underestimation of cell viability by the MTT assay may be due to both adaptive metabolic and mitochondrial reprogramming of cells subjected to drug treatment-mediated stress and inhibitor off-target effects. Previously, imatinib, rottlerin, ursolic acid, verapamil, resveratrol, genistein nanoparticles and some polypeptides were shown to interfere with MTT reduction rate resulting in inconsistent results between the MTT assay and alternative assays. Here, to test the under/overestimation of viability by the MTT assay, we compared results derived from the MTT assay with the trypan blue exclusion assay after treatment of glioblastoma U251, T98G and C6 cells with three widely used inhibitors with the known direct and side effects on energy and metabolic homeostasis - temozolomide (TMZ), a DNA-methylating agent, temsirolimus (TEM), an inhibitor of mTOR kinase, and U0126, an inhibitor of MEK1/2 kinases. Inhibitors were applied shortly as in IC50 evaluating studies or long as in studies focusing on drug resistance acquisition. We showed that over/underestimation of cell viability by the MTT assay and its significance depends on a cell line, a time point of viability measurement and other experimental parameters. Furthermore, we provided a comprehensive survey of factors that should be accounted in the MTT assay. To avoid result misinterpretation, supplementation of the tetrazolium salt-based assays with other non-metabolic assays is recommended.

Primary open-angle glaucoma (POAG) is a leading cause of irreversible blindness worldwide, and elevated intraocular pressure (IOP) is a major risk factor. While IOP is mainly controlled by adjusting the outflow resistance in the trabecular meshwork (TM), drugs that act directly on the TM are rare. In this study, we discovered a novel compound and pathway that acts on the TM and decreases IOP by genomic, proteomic, and bioinformatic analyses of POAG-derived TMs and experimental validation. Overlapping differentially expressed genes of the TM between patients with POAG and normal controls from two independent gene expression profiles in public databases were analyzed and matched by using the Connectivity Map (CMap). Rottlerin was identified as a potential compound. Subsequent experiments confirmed that rottlerin reversed POAG phenotypes in vitro and that it decreased IOP and actin/extracellular matrix accumulation in vivo with no detectable ocular side effects. SwissTargetPrediction in combination with pathway analysis predicted that the effects of rottlerin may be mediated by activation of the Rap1 pathway. Finally, we confirmed that rottlerin upregulated Rap1 and the downstream PI3K/AKT pathway independent of the MAPK/ERK pathway in a dexamethasone-induced POAG cell model.

Rottlerin as a natural agent, which is isolated from Mallotus philippinensis, has been identified to play a critical role in tumor inhibition. However, the molecular mechanism of rottlerin-mediated anti-tumor activity is still ambiguous. It has been reported that EZH2 exhibits oncogenic functions in a variety of human cancers. Therefore, inhibition of EZH2 could be a promising strategy for the treatment of human cancers. In this study, we aim to explore whether rottlerin could inhibit tumorigenesis via suppression of EZH2 in prostate cancer cells. Multiple approaches such as FACS, Transwell invasion assay, RT-PCR, Western blotting, and transfection were performed to determine our aim. We found that rottlerin treatment led to inhibition of cell growth, migration and invasion, but induction of apoptosis in prostate cancer cells. Importantly, we defined that rottlerin decreased the expression of EZH2 and H3K27me3 in prostate cancer cells. Moreover, overexpression of EZH2 abrogated the rottlerin-induced inhibition of cell growth, migration, and invasion in prostate cancer cells. Consistently, down-regulation of EZH2 enhanced rottlerin-triggered anti-tumor function. Collectively, our work demonstrated that rottlerin exerted its tumor suppressive function via inhibition of EZH2 expression in prostate cancer cells. Our findings indicated that rottlerin might be a potential therapeutic compound for treating patients with prostate cancer.

Autophagy, as a highly conserved cellular degradation and recycling process, plays an important part in maintaining cellular homeostasis. PKC signaling is involved in multiple pathways including cell cycle progression, tumorigenesis, migration and autophagy. Literatures about PKC and autophagy from PubMed databases were reviewed in this study. Studies regarding the association of PKC and autophagy remain debatable. Different duration of the stimulation of autophagy and distinct cell contexts result in different function of PKC in regulating autophagy. The subcellular localization of PKCs and their downstream regulators may influence the autophagy regulation as well. As important intracellular components, the mitochondria play an important role in regulating autophagy, by metabolic modulation and structural derangement. Phase II studies regarding PKC-β inhibitor, enzastaurin, showed promising results in MCL, DLBCL and recurrent high-grade gliomas. However, the detailed mechanism is still in need. The mechanism of PKC-β in mediating autophagy in lymphoma and high-grade gliomas remains elusive as well. Moreover, several studies were in agreement that rottlerin enhanced autophagy in breast cancer cells, which warrants further clinical studies to verify PKC-δ as a therapeutic target. Thus, identifying the function of PKC in modulating autophagy and conducting related clinical studies help find novel target for chemotherapy.

It is widely accepted that apoptosis is closely associated with cancer cell death. However, whether autophagy induces tumor cell death has not been fully elucidated. Various studies have discussed the antitumor properties of rottlerin in human malignancies. The current study aimed to investigate the effects of rottlerin, a natural product isolated from the kamala tree (Mallotus philipensis), on growth inhibition and autophagy in gastric cancer (GC) cell lines in vitro. The results of the present study demonstrated that rottlerin suppressed cell growth, induced autophagy and apoptosis, and reduced migration and invasion in the SGC-7901 and MGC-803 GC cell lines. Furthermore, rottlerin led to microtubule-associated protein 1 light chain 3β-II augmentation and the enrichment of autophagosomes. In addition, the protein expression levels of mechanistic target of rapamycin kinase and S-phase kinase-associated protein 2 were downregulated in GC cells following rottlerin treatment, which is associated with autophagy. The protein levels of caspase-3, cleaved-caspase-3, total poly (ADP-ribose) polymerase (PARP) and cleaved-PARP exhibited no marked alterations in the GC cells following rottlerin treatment, indicating that caspases were likely not involved in rottlerin-induced GC apoptosis. In summary, the results of the present study indicate that rottlerin may inhibit invasion and promote apoptosis in GC cells, which may be mediated by the activation of autophagy. Therefore, rottlerin may be of value in the treatment of GC.

Flavonoids have been chronicles of the history of a long way journey in the cure of physiological or pathophysiological conditions in various diseases including cancer. Our previous findings suggest the extensive mechanism of quercetin (QUE) mediated regression of cell survival, cell proliferation, oxidative stress, inflammation, and angiogenesis via modulating PI3K and PKC signaling in lymphoma as well as hepatocellular carcinoma. PI3K-PKC pathway is a key monitor of mammalian cells regulated by its different isoenzymes, which may exert similar or opposite cellular effects by differential coupling of signaling pathways. Put forward the invention of selective inhibitors against various isoenzymes is beneficial to reduce the burden of inclusive deleterious effects of drug for normal physiological process. Therefore, we hypothesized the improved anticancer efficacy of QUE in combination with isoenzyme inhibitors—rottlerin (ROT-PKCδ inhibitor), G0 6983 (PKCα inhibitor), and PI-103 (p110α-class I PI3K inhibitor) in MCF-7 and RAW 264.7 cells. QUE significantly improves the cytotoxicity of ROT + G0 6983 ranged 30–55% and PI-103 ranged 24–63% after 24–48 h against MCF-7 cells. Additionally in the presence of QUE, the improved cytotoxicity of ROT + G0 6983 is observed to range 69–75% and PI-103 ranged 45–88% after 24–48 h in RAW 264.7 cells. This increment in cell deaths are positively correlated with enhanced morphological alteration observed in MCF-7 cells. Further, QUE significantly increases the attenuation of PKCα level approximately by 50% in combination with PI-103. Overall results of the current study suggested that QUE improves the synergistic anticancer efficacy in combination with PI-103, ROT, and G0 6983 in MCF-7 and RAW 264.7 cells.

High circulating androgen in women with polycystic ovary syndrome (PCOS) may increase the risk of cardiovascular disease in offspring. The aim of the present study is to investigate whether maternal androgen excess in the rat PCOS model would lead to cardiac hypertrophy in offspring. Maternal testosterone propionate (maternal-TP)-treated adult female offspring displayed cardiac hypertrophy associated with local high cardiac dihydrotestosterone (DHT). The molecular markers of cardiac hypertrophy along with androgen receptor (AR) and PKCδ, were increased in the Maternal-TP group. Treatment of primary neonatal rat ventricular cardiomyocytes (NRCMs) and H9c2 cells with DHT significantly increased cell size and upregulated PKCδ expression, which could be attenuated by AR antagonist. Treatment with phorbol 12-myristate 13-acetate (PMA), a PKC activator, significantly increased cell size and upregulated myh7 level. Rottlerin, that may inhibit PKCδ, significantly reduced the hypertrophic effect of DHT and PMA on NRCMs and H9c2 cells. Chromatin immunoprecipitation revealed that AR could bind to Pkcδ promoter. Our results indicate that prenatal exposure to testosterone may induce cardiac hypertrophy in adult female rats through enhanced Pkcδ expression in cardiac myocytes.

In the past few years, we focused the interest on rottlerin, an old/new natural substance that, over the time, has revealed a number of cellular and molecular targets, all potentially implicated in the fight against cancer. Past and recent literature well demonstrated that rottlerin is an inhibitor of enzymes, transcription factors and signaling molecules that control cancer cell life and death. Although the rottlerin anticancer activity has been mainly ascribed to apoptosis and/or autophagy induction, recent findings unveiled the existence of additional mechanisms of toxicity. The major novelties highlighted in this mini review are the ability to bind and inhibit key molecules, such as ERK and mTOR, directly, thus independently of upstream signaling cascades, and to cause a profound dysregulation of cap-dependent protein translation through the mTORC1/4EBP1/eIF4E axis and by inhibition of eIF2, an initiation factor of translation that is negatively regulated by endoplasmic reticulum (ER) stress. These last mechanisms, proved to be lethal in cancer cell lines derived from breast and skin, strongly enforce the potential of rottlerin as a promising natural lead compound for the development of novel therapeutic approaches.

Thrombin has been known to activate inflammatory genes including matrix metalloproteinases (MMPs). The elevated expression of MMP-9 has been observed in patients with neuroinflammatory diseases and may contribute to the pathology of brain diseases. However, the mechanisms underlying thrombin-induced MMP-9 expression in SK-N-SH cells remain unknown. The effects of thrombin on MMP-9 expression were examined in SK-N-SH cells by gelatin zymography, Western blot, real-time PCR, promoter activity assay, and cell migration assay. The detailed mechanisms were analyzed by using pharmacological inhibitors and small intefering RNA (siRNA) transfection. Here, we demonstrated that thrombin induced the expression of proform MMP-9 and migration of SK-N-SH cells, which were attenuated by pretreatment with the inhibitor of thrombin (PPACK), Gq (GPA2A), PC-PLC (D609), PI-PLC (ET-18-OCH3), nonselective protien kinase C (PKC, GF109203X), PKCα/βII (Go6983), PKCδ (Rottlerin), p38 mitogen-activated protein kinases (MAPK) (SB202190), JNK1/2 (SP600125), or NF-κB (Bay11-7082 or Helenalin) and transfection with siRNA of Gq, PKCα, PKCβ, PKCδ, p38, JNK1/2, IKKα, IKKβ, or p65. Moreover, thrombin-stimulated PKCα/βII, PKCδ, p38 MAPK, JNK1/2, or p65 phosphorylation was abrogated by their respective inhibitor of PPACK, GPA2A, D609, ET-18-OCH3, Go6983, Rottlerin, SB202190, SP600125, Bay11-7082, or Helenalin. Pretreatment with these inhibitors or transfection with MMP-9 siRNA also blocked thrombin-induced SK-N-SH cell migration. Our results show that thrombin stimulates a Gq/PLC/PKCs/p38 MAPK and JNK1/2 cascade, which in turn triggers NF-κB activation and ultimately induces MMP-9 expression and cell migration in SK-N-SH cells.

The first total synthesis of rottlerin is described. The methodology allows the development of potential novel protein kinase C δ (PKCδ) analogues for better treatment of various diseases. Kamalachalcone A and dimeric rottlerin were synthesized in a very practical and economical way using FeCl3 as a catalyst.

Treatment of patients with B-NHL with rituximab and CHOP has resulted in significant clinical responses. However, a subset of patients develops resistance to further treatments. The mechanism of unresponsiveness in vivo is not known. We have reported the development of rituximabresistant clones derived from B-NHL cell lines as models to investigate the mechanism of resistance. The resistant clones exhibit hyper-activated survival/anti-apoptotic pathways and no longer respond to a combination of rituximab and drugs. Recent studies reported the therapeutic efficacy in mice bearing B-cell lymphoma xenografts following treatment with the anti-CD20-hIFNα fusion protein. We hypothesized that the fusion protein may bypass rituximab resistance and inhibit survival signaling pathways. Treatment of the rituximab- resistant clones with anti-CD20-hIFNα, but not with rituximab, IFNα, or rituximab+IFNα resulted in significant inhibition of cell proliferation and induction of cell death. Treatment with anti-CD20-hIFNα sensitized the cells to apoptosis by CDDP, doxorubicin and Treanda. Treatment with anti-CD20-hIFNα inhibited the NF-κB and p38 MAPK activities and induced the activation of PKC-σ and Stat-1. These effects were corroborated by the use of the inhibitors SB203580 (p38 MAPK) and Rottlerin (PKC-σ). Treatment with SB203580 enhanced the sensitization of the resistant clone by anti-CD20-hIFNα to CDDP apoptosis. In contrast, treatment with Rotterin inhibited significantly the sensitization induced by anti-CD20-hIFNα. Overall, the findings demonstrate that treatment with anti-CD20-hIFNα reverses resistance of B-NHL. These findings suggest the potential application of anti-CD20-hIFNα in combination with drugs in patients unresponsive to rituximab.containing regimens.

Dibenzoylmethane (DB), a minor constituent of the root extract of licorice, belongs to the flavonoid family. Hydroxydibenzoylmethane (HDB) and hydroxymethyldibenzoylmethane (HMDB) have an identical structure to DB, but also possess a hydroxyl group and a hydroxyl and methyl group bonded to aromatic rings, respectively. They inhibit cellular proliferation and induce apoptosis in a variety of types of cancer cell, however, the antimetastatic effects of DB, HDB and HMDB on human breast carcinoma cells remain to be elucidated. The present study aimed to clarify the molecular mechanisms underlying the effects of DB and its analogues on phorbol‑12‑myristate 13‑acetate (PMA)‑induced MCF‑7 cell metastasis. The results revealed that DB, HDB and HMDB inhibited cell migration and invasion. In addition, PMA‑mediated MCF‑7 cell invasion was inhibited by DB, HDB and HMDB by inhibiting the expression of matrix metalloproteinase (MMP)‑9. Rottlerin, a protein kinase C (PKC)δ inhibitor and LY294002, a phosphatidylinositide 3‑kinase (PI3K) inhibitor, reduced the PMA‑mediated expression of MMP‑9 and cell invasion. Furthermore, DB, HDB and HMDB prevented the activation of PKCδ and PI3K by inhibiting their phosphorylation. The present study was the first, to the best of our knowledge, to demonstrate the antimetastatic potential of DB, HDB and HDMB, which decreased cancer cell invasion through the PI3K/PKCδ‑mediated MMP‑9 pathway.

Protein kinase C (PKC)-δ is a member of the PKC family. It has been implicated in tumor suppression as well as survival of various cancers. The aggressive malignancy of T lymphocytes known as adult T-cell leukemia (ATL) is associated with human T-cell leukemia virus type 1 (HTLV-1) infection. In this study, we show that HTLV-1-infected T cells are characterized by phosphorylation and nuclear translocation of PKC-δ. Expression of HTLV-1 regulatory protein Tax increased PKC-δ phosphorylation. Blockade of PKC-δ by rottlerin suppressed PKC-δ phosphorylation and inhibited cell viability in HTLV-1-infected T-cell lines and primary ATL cells. Rottlerin induced cell cycle arrest at the G1 phase and caspase-mediated apoptosis of HTLV-1-infected T cells. Rottlerin downregulated the expression of proteins involved in G1/S cell cycle transition, cyclin D2, CDK4 and 6, and c-Myc, resulting in dephosphorylation of retinoblastoma protein (pRb). Furthermore, rottlerin reduced the expression of important anti-apoptotic proteins (e.g., survivin, XIAP, Bcl-xL and c-FLIP) and Bcl-2 phosphorylation, and activated the pro-apoptotic protein Bax. Our results showed that permanent activation of nuclear factor-κB (NF-κB) by HTLV-1 Tax allows infected cells to escape cell cycle arrest and apoptosis and that PKC-δ mediates Tax-induced activation of NF-κB. Based on these findings, new therapies designed to target PKC-δ could be potentially useful in the treatment of ATL.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin best characterized for its survival and differentiative effects on neurons. Recent studies demonstrated that BDNF and its receptors are also expressed in the peripheral vasculature, where it stimulates angiogenesis and promotes the survival of endothelial cells. This study was designed to investigate the angiogenic effects of BDNF and its expressional regulation by tumor necrosis factor (TNF-α) and protein kinase C (PKC) in endothelial cells. In the Matrigel angiogenesis assay, BDNF-stimulated vascular tube formation of human umbilical vein endothelial cells (HUVEC) was completely blocked by an inhibition of the TrkB receptor, but only partially inhibited by the inhibition of the p75NTR signaling. Treatment of HUVEC and HUVEC-derived EA.hy 926 cells with TNF-α resulted in a downregulation of BDNF expression, which could be prevented by the TNFR1 antagonist WP9QY. BDNF downregulation by TNF-α was associated with decreased angiogenic activity of HUVEC. The effect of TNF-α on BDNF expression could not be abolished by the inhibition of PKC. Treatment of HUVEC and EA.hy 926 cells with PKC-activating phorbol esters (phorbol-12-myristate-13-acetate, PMA or phorbol-12,13-dibutyrate) resulted in a downregulation of BDNF expression, whereas the inactive 4α-phorbol-12,13-didecanoate was without effect. PMA had no significant effect on BDNF mRNA stability and the downregulation of BDNF mRNA expression by PKC activation was likely a transcriptional event. BDNF downregulation by PMA could be prevented by PKC inhibitors Gö 6983 and rottlerin, but not by Gö 6976. Thus, a Gö 6983/rottlerin-sensitive PKC isoform is likely to be responsible for PMA-induced BDNF downregulation.

There are two established depigmenting agent assays currently in use. However, these methods are unreliable and time-consuming. Therefore, it will be valuable to establish a better assay system for depigmenting agent analysis. In this study, we established a melanogenesis regulation assay system using a fluorescent protein reporter combined with the promoters for the microphthalmia-associated transcription factor (MITF), tyrosinase (Tyr) and dopachrome tautomerase (Dct) genes in MeWo human melanoma cells. We used several melanogenesis regulators, including theophylline, hesperetin, arbutin and rottlerin, to confirm the function of this assay system. The established MeWo/pMITF-EGFP, MeWo/pTyr-EGFP and MeWo/pDct-EGFP stable cells integrated the pMITF-EGFP, pTyr-EGFP and pDct-EGFP plasmids into their genomic DNA. These stably transfected cells were used to examine alterations in the expression of the MITF, Tyr and Dct genes. All of the tested compounds, including theophylline, hesperetin, arbutin and rottlerin, could be analyzed in the stable cells, producing reliable results. Therefore, we believe that this melanogenesis regulation assay system can be used as a rapid and reliable assay system to analyze the regulation of melanogenesis by many known or unknown compounds.

Dysregulated release of neutrophil reactive oxygen species and proteolytic enzymes contributes to both acute and chronic inflammatory diseases. Therefore, molecular regulators of these processes are potential targets for new anti-inflammatory therapies. We have shown previously that myristoylated alanine-rich C-kinase substrate (MARCKS), a well-known actin binding protein and protein kinase C (PKC) substrate, is a key regulator of neutrophil functions. In the current study, we investigate the role of PKC-mediated MARCKS phosphorylation in neutrophil migration and adhesion in vitro. We report that treatment of human neutrophils with the δ-PKC inhibitor rottlerin significantly attenuates f-Met-Leu-Phe (fMLF)-induced MARCKS phosphorylation (IC50=5.709 μM), adhesion (IC50=8.4 μM), and migration (IC50=6.7 μM), while α-, β-, and ζ-PKC inhibitors had no significant effect. We conclude that δ-PKC-mediated MARCKS phosphorylation is essential for human neutrophil migration and adhesion in vitro. These results implicate δ-PKC-mediated MARCKS phosphorylation as a key step in the inflammatory response of neutrophils.

Rottlerin has been reported to exert its anti-tumor activity in various types of human cancers. However, the underlying molecular mechanism has not been fully elucidated. In the current study, we explored whether rottlerin exhibits its tumor suppressive function in hepatocellular carcinoma cells. Our MTT assay results showed that rottlerin inhibited cell growth in hepatocellular carcinoma cells. Moreover, we found that rottlerin induced cell apoptosis and caused cell cycle arrest at G1 phase. Furthermore, our wound healing assay result demonstrated that rottlerin retarded cell migration in hepatocellular carcinoma cells. Additionally, rottlerin suppressed cell migration and invasion. Notably, we found that rottlerin upregulated DDX3 expression and subsequently downregulated Cyclin D1 expression and increased p21 level. Importantly, down-regulation of DDX3 abrogated the rottlerin-mediated tumor suppressive function, whereas overexpression of DDX3 promoted the anti-tumor activity of rottlerin. Our study suggests that rottlerin exhibits its anti-cancer activity partly due to upregulation of DDX3 in hepatocellular carcinoma cells.