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Seventeen compounds, rather selective, direct or indirect inhibitors and activators of PKA, PKG, and PKC, were analysed for effects on vascular CaV1.2 channel current (ICa1.2) by using the patch-clamp technique in single rat tail artery myocytes. The aim was to investigate how PKs regulate ICa1.2 and disclose any unexpected modulation of CaV1.2 channel function by these agents. The cAMP analogues 8-Br-cAMP and 6-Bnz-cAMP partially reduced ICa1.2 in dialysed cells, while weakly increasing it under the perforated configuration. The β-adrenoceptor agonist isoproterenol and the adenylate cyclase activator forskolin concentration-dependently increased ICa1.2; this effect was reversed by PKA inhibitors H-89 and KT5720, but not by PKI 6-22. The cGMP analogue 8-Br-cGMP, similarly to the NO-donor SNP, moderately reduced ICa1.2, this effect being reversed to a slight stimulation under the perforated configuration. Among PKG inhibitors, Rp-8-Br-PET-cGMPS decreased current amplitude in a concentration-dependent manner while Rp-8-Br-cGMPS was ineffective. The non-specific phosphodiesterase inhibitor IBMX increased ICa1.2, while H-89, KT5720, and PKI 6-22 antagonized this effect. The PKC activator PMA, but not the diacylglycerol analogue OAG, stimulated ICa1.2 in a concentration-dependent manner; conversely, the PKCα inhibitor Go6976 markedly reduced basal ICa1.2 and, similarly to the PKCδ (rottlerin) and PKCe translocation inhibitors antagonised PMA-induced current stimulation. The ensemble of findings indicates that the stimulation of cAMP/PKA, in spite of the paradoxical effect of both 8-Br-cAMP and 6-Bnz-cAMP, or PKC pathways enhanced, while that of cGMP/PKG weakly inhibited ICa1.2 in rat tail artery myocytes. Since Rp-8-Br-PET-cGMPS and Go6976 appeared to block directly CaV1.2 channel, their docking to the channel protein was investigated. Both compounds appeared to bind the α1C subunit in a region involved in CaV1.2 channel inactivation, forming an interaction network comparable to that of CaV1.2 channel blockers. Therefore, caution should accompany the use of these agents as pharmacological tools to elucidate the mechanism of action of drugs on vascular preparations.

Vascular smooth muscle cells (VSMCs) that function as synthetic units play important roles in cardiovascular diseases. Extracellular nucleotides, such as ATP, have been shown to act via activation of P2 purinoceptors implicated in various inflammatory diseases, we hypothesized that extracellular nucleotides contribute to vascular diseases via up-regulation of inflammatory proteins, including cyclooxygenase-2 (COX-2) and cytosolic phospholipase A2 (cPLA2) in VSMCs. However, the mechanisms of ATP-induced cPLA2 and COX-2 expression and PGE2 synthesis remain largely unclear. We showed that pretreatment with the inhibitors of STAT3 (CBE), NADPH oxidase [diphenyleneiodonium chloride (DPI) or apocynin (APO)], ROS [N-acetyl-l-cysteine (NAC)], and PKC (Ro-318220, Gö6983, or Rottlerin) or transfection with siRNAs of STAT3 and p47(phox) markedly inhibited ATPγS-induced cPLA2 and COX-2 mRNA/protein expression and promoter activity and PGE2 secretion. ATPγS further stimulated PKC, p47(phox), and STAT3 translocation. Moreover, ATPγS-induced STAT3 phosphorylation and translocation was inhibited by pretreatment with the inhibitors of PKC, NADPH oxidase, and ROS. ATPγS enhanced NADPH oxidase activity and ROS generation in VSMCs, which were reduced by pretreatment with Ro-318220, Gö6983, or Rottlerin. Finally, we found that ATPγS significantly induced cyclin D1 expression and VSMCs proliferation, which were inhibited by pretreatment with NAC, APO, DPI, Ro-318220, Gö6983, Rottlerin, or CBE or transfection with siRNAs of COX-2 and cyclin D1. We also demonstrated that ATPγS induced cyclin D1 expression via a PGE2-dependent pathway. These results suggested that ATPγS-induced cPLA2/COX-2 expression and PGE2 secretion is mediated through a PKC/NADPH oxidase/ROS/STAT3-dependent pathway in VSMCs.

Multiple lines of evidence support the idea that autophagy plays an essential role in the development of drug resistance, self-renewal, differentiation, and tumorigenic potentials of cancer stem cells (CSCs). Rottlerin (ROT) is widely used as a protein kinase C-delta (PKC-δ) inhibitor. Recent studies revealed that ROT induces apoptosis through engagement of mitochondria. However, it is not known whether ROT-induced apoptosis is associated with other mechanisms such as autophagy. Here we found that ROT induced autophagy followed by induction of apoptosis via inhibition of PI3K/Akt/mTOR pathway and activation of caspase cascade in human pancreatic CSCs. ROT induced a dose- and time-dependent inhibition of cell survival and induction of cytoplasmic vacuolations. The conversion of microtubule-associated protein LC3-I to LC3-II, and increased accumulations of Atg7 and Beclin-1 were also observed in CSCs treated with ROT. Prolonged exposure of CSCs to ROT eventually caused apoptosis which was associated with the suppression of phosphorylated Akt (Ser473) and mTOR (Ser2448), downregulation of XIAP, cIAP-1, Bcl-2 and Bcl-X(L), induction of Bax, activation of caspase-3 and -9, and concomitant degradation of PARP. ROT-induced apoptosis was enhanced by dominant negative AKT, Akt1/2 inhibitor, and rapamycin. Our study also demonstrates that gene silencing of Atg7 and Beclin1, or cotreatment of the autophagosome inhibitor, 3-methyladenine, inhibited ROT-induced autophagy and accelerated ROT-induced apoptosis. The knockdown of PKC-δ did not block ROT-induced autophagy and cell death, suggesting these effects of ROT were exerted through PKC-δ-independent pathway. In summary, our data demonstrate that ROT can induce autophagy which leads to cell death in pancreatic CSCs.

Combination therapy, which can optimize killing activity to cancers and minimize drug resistance, is a mainstream therapy against hormone-refractory prostate cancers (HRPCs). Rottlerin, a natural polyphenolic component, synergistically increased PC-3 (a HRPC cell line) apoptosis induced by camptothecin (a topoisomerase I inhibitor). Using siRNA technique to knockdown protein kinase C-δ (PKCδ), the data showed that rottlerin-mediated synergistic effect was PKCδ-independent, although rottlerin has been used as a PKCδ inhibitor. Rottlerin potentiated camptothecin-induced DNA fragmentation at S phase and ATM phosphorylation at Ser1981. The effect was correlated to apoptosis (r2 = 0.9). To detect upstream signals, the data showed that camptothecin acted on and stabilized topoisomerase I-DNA complex, leading to the formation of camptothecin-trapped cleavage complexes (TOP1cc). The effect was potentiated by rottlerin. To determine DNA repair capability, the time-related γH2A.X formation was examined after camptothecin removal. Consequently, rottlerin significantly inhibited camptothecin removal-mediated decline of γH2A.X formation at S phase, indicating the impairment of DNA repair activity in the presence of rottlerin. The combinatory treatment of camptothecin and rottlerin induced conformational change and activation of Bax and formation of truncated Bad, suggesting the contribution of mitochondria stress to apoptosis. In summary, the data suggest that rottlerin-mediated camptothecin sensitization is through the augmented stabilization of TOP1cc, leading to an increase of DNA damage stress and, possibly, an impairment of DNA repair capability. Subsequently, mitochondria-involved apoptosis is triggered through Bax activation and truncated Bad formation. The novel discovery may provide an anticancer approach of combinatory use between rottlerin and camptothecin for the treatment of HRPCs.

Patients with rheumatoid arthritis (RA) are at increased risk of developing infections and appear to be particularly susceptible to septic arthritis. Lipoteichoic acid (LTA), a cell wall component of Gram-positive bacteria is an amphiphilic, negatively charged glycolipid. However, the effects of LTA on human synovial fibroblasts are largely unknown. We investigated the signaling pathway involved in IL-6 production stimulated by LTA in rheumatoid arthritis synovial fibroblasts (RASF). LTA caused concentration- and time-dependent increases in IL-6 production. LTA-mediated IL-6 production was attenuated by Toll-like receptor 2 (TLR2) monoclonal antibody or siRNA. Pretreatment with PKCdelta inhibitor (rottlerin), c-Src inhibitor (PP2), AP-1 inhibitor (tanshinone IIA) and NF-kappaB inhibitor (PDTC and TPCK) also inhibited the potentiating action of LTA. However, focal adhesion kinase (FAK) mutant and siRNA did not affect LTA-mediated IL-6 production. Stimulation of cells with LTA increased the PKCdelta and c-Src phosphorylation and kinase activity. LTA increased the accumulation of p-c-Jun and p-p65 in the nucleus, as well as AP-1 and NF-kappaB luciferase activity. LTA-mediated increase of AP-1 and NF-kappaB luciferase activity was inhibited by rottlerin and PP2 or TLR2 and PKCdelta siRNA or c-Src mutant. Our results suggest that LTA-increased IL-6 production in human synovial fibroblasts via the TLR2 receptor, PKCdelta, c-Src, AP-1 and NF-kappaB signaling pathways.

Recently, it was shown that rottlerin inhibits insulin-stimulated glucose uptake and reduces intracellular adenosine triphosphate (ATP) levels in 3T3-L1 adipocytes, suggesting that these two events are causally linked. However, several other reports show that ATP-depletion induces glucose uptake in both muscle cells and adipocytes. In the present study, the mechanism of inhibition by rottlerin was studied in detail, in order to resolve this apparent discrepancy. It was found that rottlerin strongly reduces insulin-stimulated 2-deoxyglucose (2-DOG) uptake in 3T3-L1 adipocytes by a partial inhibition of the translocation of the insulin-responsive GLUT4 glucose transporter towards the plasma membrane (PM). Whereas the insulin-induced phosphatidyl-inositol-3' (PI-3') kinase signaling pathway is unaffected by rottlerin, Cbl tyrosine phosphorylation, which provides an essential, PI-3' kinase-independent signal towards GLUT4 translocation, is markedly attenuated. Furthermore, we also observed a direct inhibitory effect of rottlerin on insulin-induced glucose uptake in 3T3-L1 adipocytes. The direct inhibition of insulin-stimulated 2-DOG uptake by rottlerin displayed characteristics of uncompetitive inhibition: with the K(m(app)) of glucose uptake reduced from 1.6 to 0.9 mM and the V(max(app)) reduced from 5.2 to 1.0 nmol/minmg in the presence of rottlerin. In conclusion, rottlerin inhibits multiple steps involved in insulin-stimulated 2-DOG uptake in 3T3-L1 adipocytes. The observed reduction in GLUT4 translocation towards the PM and the uncompetitive inhibition of the glucose transport process provide alternative explanations for the inhibitory effects of rottlerin aside from the effects of rottlerin on intracellular levels of ATP.

Polysialylation of the neural cell adhesion molecule (NCAM PSA) is necessary for the consolidation processes of hippocampus-based learning. Previously, we have found inhibition of protein kinase C delta (PKCdelta) to be associated with increased polysialyltransferase (PST) activity, suggesting inhibitors of this kinase might ameliorate cognitive deficits. Using a rottlerin template, a drug previously considered an inhibitor of PKCdelta, we searched the Compounds Available for Purchase (CAP) database with the Accelrys((R)) Catalyst programme for structurally similar molecules and, using the available crystal structure of the phorbol-binding domain of PKCdelta, found that diferuloylmethane (curcumin) docked effectively into the phorbol site. Curcumin increased NCAM PSA expression in cultured neuro-2A neuroblastoma cells and this was inversely related to PKCdelta protein expression. Curcumin did not directly inhibit PKCdelta activity but formed a tight complex with the enzyme. With increasing doses of curcumin, the Tyr(131) residue of PKCdelta, which is known to direct its degradation, became progressively phosphorylated and this was associated with numerous Tyr(131)-phospho-PKCdelta fragments. Chronic administration of curcumin in vivo also increased the frequency of polysialylated cells in the dentate infragranular zone and significantly improved the acquisition and consolidation of a water maze spatial learning paradigm in both adult and aged cohorts of Wistar rats. These results further confirm the role of PKCdelta in regulating PST and NCAM PSA expression and provide evidence that drug modulation of this system enhances the process of memory consolidation.

Rottlerin is a pharmacological inhibitor of protein kinase C (PKC) theta, a novel PKC selectively expressed in T lymphocytes. PKC theta is known to regulate T cell receptor (TCR)/CD28 signalling pathways in T lymphocytes, but the impact of PKC theta inhibition on human T cell responses remains undefined. In this work, we describe the effects of rottlerin on the responses of CD4+ and CD8+ human T lymphocytes upon polyclonal activation. We observed a dose-dependent inhibition of CD4+ and CD8+ T cell proliferation in response to anti-CD3/anti-CD28 antibodies stimulation in the presence of rottlerin. This inhibition was associated with impaired CD25 expression and decreased interleukin (IL)-2 production in activated T cells. In contrast, rottlerin did not alter IL-2-induced T cell proliferation. Furthermore, we demonstrated that rottlerin blocked interferon (IFN) gamma, IL-10 and IL-13 mRNA expression in TCR/CD28 activated CD4+ T cells. These findings place rottlerin as a potent immunosuppressive agent for the development of novel therapies in T cell mediated immune disorders.