SP600125 Research Articles

Function of the transforming growth factor-β1/c-Jun N-terminal kinase signaling pathway in the action of thalidomide on a rat model of pulmonary fibrosis

The aims of this study were to observe the effects of thalidomide on a rat model of pulmonary fibrosis, to determine the protein expression levels of phosphorylated c-Jun N-terminal kinase (p-JNK) and α-smooth muscle actin (α-SMA) and to explore the mechanism underlying the preventive effect of thalidomide on pulmonary fibrosis. Ninety healthy male Wistar rats (200±20 g) were randomly divided into control (N), model (M), SP600125 (SP), thalidomide (T) and SP600125 plus thalidomide (SP + T) groups. Pulmonary fibrosis models were established in groups M, SP, T and SP + T by the intratracheal instillation of bleomycin (BLM). A gavage of thalidomide was administered to the rats in groups T and SP + T once daily, whereas normal saline was administered to the rats in the other groups. The rats in the SP and SP + T groups were injected intraperitoneally with SP600125 following BLM administration, whereas the rats in the other groups received dimethyl sulfoxide. Rats were randomly sacrificed on days 7, 14 and 28. Pathological changes were examined by light microscopy using hematoxylin and eosin staining. Hydroxyproline (HYP) levels in the lung tissues were detected using alkaline hydrolysis. The protein expression levels of p-JNK and α-SMA were measured by immunohistochemical staining and western blot analysis. In group M, alveolitis was most serious on day 7 and then eased on day 14; marked pulmonary fibrosis was observed on day 28. The fibrosis was markedly attenuated in the SP + T group compared with that in group M. The HYP content increased gradually with time after BLM administration and peaked on day 28. On days 14 and 28, the HYP content was lower in groups T and SP than in group M (P<0.05). The expression levels of p-JNK protein and α-SMA were significantly lower in groups SP, T and SP + T than those in group M on day 14 (P<0.05). The expression level of α-SMA was lower in group SP + T than those in groups SP and T on days 14 and 28 (P<0.05). The expression level of p-JNK protein in group T was higher than those in groups SP and SP + T on days 14 and 28 (P<0.05). Thus, thalidomide eased the degree of BLM-induced pulmonary fibrosis in rats by downregulating p-JNK and α-SMA expression.

Modulation of nociceptin and its receptor by phorbol-12-myristate-13-acetate via specific signal transduction kinases

• MM6 cells cultured with different concentrations (10-2-102 ng/ml) of phorbol-12-myristate-13-acetate (PMA) for 48 hrs. • Cells were treated with / without PMA 10 ng/ml for 0, 3, 6, 12, 24, 48 hrs. • mRNA of NOP and the nociceptin precursor prepronociceptin (PNoc) detected by quantitative RT-PCR.2 mRNA expression is presented as ratio of NOP or PNoc / reference gene HPRT (human hypoxanthine phosphoribosyl-transferase) normalized by the ratio of the calibrator (normalized ratio). • Intervention study: MM6 cells pretreated with PD98509 (PD) 30 μM, SP600125 (SP) 10 μM , SB203580 (SB) 10 μM or Bay 117821 (Bay) 3 μM for 1 hr to inhibit specific MAPKK/MEKs, JNK, p38, or NF-κB signaling pathway; followed by a co-culture with PMA 10 ng/ml for 6 or 48 hrs. • Intracellular phosphorylated kinases detected by phospho-flow. • Statistics: Medians with interquartile ranges (IQR); Mann-Whitney U test, subsequent post hoc analysis; level of significance: p<0.05. Figure 1: Time course of NOP and PNoc mRNA expression MM6 cells cultured with / without PMA 10 ng/ml.

SP600125 overcomes antimitotic drug-resistance in cancer cells by increasing apoptosis with independence of P-gp inhibition

The purpose of this study was to identify conditions that increase the sensitivity of resistant cancer cells to antimitotic drugs. Using MTS assays, microscopic observation, assessment of cleaved PARP, FACS analysis, and Hoechst staining, we found that the c-Jun N-terminal kinase (Jnk) inhibitor SP600125 (SP) sensitized the antimitotic drug-resistant KBV20C cancer cell line. The sensitization mechanism was independent of p-glycoprotein (P-gp) inhibition. Interestingly, SP-induced sensitization was greater in resistant KBV20C cancer cells than in KB parent cells. The mechanism of SP-induced sensitization involved G2 arrest. KBV20C cells treated with SP and antimitotic drugs were more sensitized than cells treated with SP alone. This suggests that SP can restore sensitization for antimitotic drugs in resistant cancer cells. Our findings may contribute to the development of SP-based combination therapies for patients receiving anti-cancer agents that target microtubules.

Abstract 9786: Wnt11 Regulates Angiogenesis Through a Non-canonical Wnt-PKC-JNK Pathway

Wnt11 has been implicated in stem cell proliferation and differentiation. However, its role in angiogenesis remains largely unknown. We investigated angiogenic role of Wnt11 on human vascular endothelial cells (HUVECs) and documented its related signaling pathways. HUVECs pre-labeled with PKH26 were seeded in BD Matrigel Matrix (growth factor reduced) at 1.5х10 4 /cm 2 and treated with recombinant Wnt11 proteins at 5μg/ml. Both the length and the number of capillary-like tubes (CLT) were measured after HUVECs were treated for 18 hours. Wnt11 significantly increased the number and length of CLT (Fig. A). The migration of HUVECs assessed with the BD Biocoat™ Angiogenesis System was also promoted by Wnt11 (Fig. B). Immunostaining (Fig. C) and Western blotting (Fig. D) results revealed upregulation of p-pan-PKC and p-JNK in HUVECs transfected with Wnt11 (H Wnt11 ) compared to control cells transduced with empty vector (H GFP ). Moreover, the effect of Wnt11 on CLT formation and HUVECs migration were abrogated when the JNK inhibitor SP600125 (SP) at 5 μM and the PKC inhibitor Calphostin-C (Cal-C) at 0.1 μM were added to the culture system (Fig. E and F). Our data suggested, for the first time, that Wnt11 promotes endothelial cell migration and capillary formation, which is related to the activation of the non-canonical Wnt-PKC-JNK pathways.

Production of reactive oxygen species by withaferin A causes loss of type collagen expression and COX-2 expression through the PI3K/Akt, p38, and JNK pathways in rabbit articular chondrocytes

Withaferin A (WFA) is a major chemical constituent of Withania somnifera, also known as Indian ginseng. Many recent reports have provided evidence of its anti-tumor, anti-inflammation, anti-oxidant, and immune modulatory activities. Although the compound appears to have a large number of effects, its defined mechanisms of action have not yet been determined.We investigated the effects of WFA on loss of type collagen expression and inflammation in rabbit articular chondrocytes. WFA increased the production of reactive oxygen species, suggesting the induction of oxidative stress, in a dose-dependent manner. Also, we confirmed that WFA causes loss of type collagen expression and inflammation as determined by a decrease of type II collagen expression and an increase of cyclooxygenase-2 (COX-2) expression via western blot analysis in a dose- and time- dependent manner. WFA also reduced the synthesis of sulfated proteoglycan via Alcian blue staining and caused the synthesis of prostaglandin E2 (PGE2) via assay kit in dose- and time-dependent manners.Treatment with N-acetyl-l-cysteine (NAC), an antioxidant, inhibited WFA-induced loss of type II collagen expression and increase in COX-2 expression, accompanied by inhibition of reactive oxygen species production. WFA increased phosphorylation of both Akt and p38. Inhibition of PI3K/Akt, p38, and JNK with LY294002 (LY), SB203580 (SB), or SP600125 (SP) in WFA-treated cells rescued the expression of type II collagen and suppressed the expression of COX-2. These results demonstrate that WFA induces loss of type collagen expression and inflammation via PI3K/Akt, p38, and JNK by generating reactive oxygen species in rabbit articular chondrocytes.

Celastrol targets mitochondrial respiratory chain complex I to induce reactive oxygen species-dependent cytotoxicity in tumor cells

BackgroundCelastrol is an active ingredient of the traditional Chinese medicinal plant Tripterygium Wilfordii, which exhibits significant antitumor activity in different cancer models in vitro and in vivo; however, the lack of information on the target and mechanism of action of this compound have impeded its clinical application. In this study, we sought to determine the mode of action of celastrol by focusing on the processes that mediate its anticancer activity.MethodsThe downregulation of heat shock protein 90 (HSP90) client proteins, phosphorylation of c-Jun NH2-terminal kinase (JNK), and cleavage of PARP, caspase 9 and caspase 3 were detected by western blotting. The accumulation of reactive oxygen species (ROS) was analyzed by flow cytometry and fluorescence microscopy. Cell cycle progression, mitochondrial membrane potential (MMP) and apoptosis were determined by flow cytometry. Absorption spectroscopy was used to determine the activity of mitochondrial respiratory chain (MRC) complexes.ResultsCelastrol induced ROS accumulation, G2-M phase blockage, apoptosis and necrosis in H1299 and HepG2 cells in a dose-dependent manner. N-acetylcysteine (NAC), an antioxidative agent, inhibited celastrol-induced ROS accumulation and cytotoxicity. JNK phosphorylation induced by celastrol was suppressed by NAC and JNK inhibitor SP600125 (SP). Moreover, SP significantly inhibited celastrol-induced loss of MMP, cleavage of PARP, caspase 9 and caspase 3, mitochondrial translocation of Bad, cytoplasmic release of cytochrome c, and cell death. However, SP did not inhibit celastrol-induced ROS accumulation. Celastrol downregulated HSP90 client proteins but did not disrupt the interaction between HSP90 and cdc37. NAC completely inhibited celastrol-induced decrease of HSP90 client proteins, catalase and thioredoxin. The activity of MRC complex I was completely inhibited in H1299 cells treated with 6 μM celastrol in the absence and presence of NAC. Moreover, the inhibition of MRC complex I activity preceded ROS accumulation in H1299 cells after celastrol treatment.ConclusionWe identified ROS as the key intermediate for celastrol-induced cytotoxicity. JNK was activated by celastrol-induced ROS accumulation and then initiated mitochondrial-mediated apoptosis. Celastrol induced the downregulation of HSP90 client proteins through ROS accumulation and facilitated ROS accumulation by inhibiting MRC complex I activity. These results identify a novel target for celastrol-induced anticancer activity and define its mode of action.

Cytotoxic effects of metal protoporphyrins in glioblastoma cells: Roles of albumin, reactive oxygen species, and heme oxygenase-1

We investigate the cytotoxic effect of metal protoporphyrins including ferric protoporphyrin (FePP; hemin), cobalt protoporphyrin (CoPP), and tin protoporphyrin (SnPP) in glioblastoma cells C6 and GBM8401. Data of MTT assay show that FePP and CoPP, but not SnPP, significantly reduce the viability of glioma cells C6 and GBM8401 in the absence of serum. In the condition with fetal bovine serum (FBS) or bovine serum albumin (BSA), the cytotoxic effect of FePP and CoPP was completely inhibited. Binding of FePP, CoPP, and SnPP with BSA was examined via spectrophotometer analysis, and the protective effect of serum against FePP and CoPP-induced cell death was abolished by BSA depletion. A loss in the integrity of DNA with an occurrence of apoptotic events including DNA ladders, caspase 3 and PARP protein cleavage, and chromatin-condensed cells is observed in FePP-treated or CoPP-treated C6 cells. An increase in intracellular peroxide level was examined in FePP, but not CoPP, -treated C6 cells, and N-acetyl- l-cysteine (NAC) addition significantly protected C6 cells from FePP, but not CoPP, -induced cell death with reducing FePP-stimulated reactive oxygen species (ROS) production. Activation of extracellular regulated kinases (ERKs) and c-Jun-N-terminal kinases (JNKs) with an increase in the heme oxygenase-1 (HO-1) protein was observed in FePP-treated or CoPP-treated C6 cells in the absence of FBS or BSA, and adding JNKs inhibitor SP600125 (SP), but not ERKs inhibitor PD98059 (PD), significantly attenuated FePP-induced or CoPP-induced HO-1 protein expression in accordance with reducing JNKs protein phosphorylation. However, PD98059, SP600125, or transfection of C6 cells with antisense HO-1 oligonucleotides show no effect on the cytotoxicity elicited by FePP and CoPP in C6 cells. Effect of serum and BSA on the cytotoxicity of metal protoporphyrins in glioma cells is first demonstrated in the present study, and the roles of ROS, MAPKs, and HO-1 were elucidated.

Internalization of Staphylococcus aureus by Bovine Endothelial Cells is Associated with the Activity State of NF‐κB and Modulated by the Pro‐inflammatory Cytokines TNF‐α and IL‐1β

Bacterial internalization is an important process in the pathogenesis of infectious diseases in which nuclear factor kappaB (NF-kappaB) plays a prominent role. We present pharmacological evidence indicating that in bovine endothelial cells (BEC) the internalization of Staphylococcus aureus, a pathogenic bacterium that causes mastitis in bovine cattle, was associated with the activation of NF-kappaB. The internalization of S. aureus increased when BEC were stimulated with alpha-tumour necrosis factor (TNF-alpha) or beta-interleukin 1 (IL-1beta) which are known activators of NF-kappaB. SN50 (an inhibitor peptide of NF-kappaB nuclear translocation) and BAY 11-7083 (a chemical that inhibits the IkappaBalpha phosphorylation) caused significant reduction in S. aureus intracellular number, indicating that its internalization was associated with the NF-kappaB activity. Furthermore, specific inhibition of c-Jun N-terminal kinase with SP600125 (SP) or p-38 with SB203580 (SB) did not cause any change in the S. aureus intracellular number compared with the untreated control. Finally, TNF-alpha treatment of BEC after the addition of both SP and SB, induced a significant increase in S. aureus internalization above the control value. These data indicate that NF-kappaB activity is associated with S. aureus internalization and suggest that this transcription factor may play a role in the pathophysiology of bovine mastitis caused by this bacterium.

Activated c-Jun N-Terminal Kinase Is a Therapeutic Target in Advanced Stage ALK-Negative Anaplastic Large Cell Lymphoma.

Anaplastic large cell lymphoma kinase (ALK)-negative anaplastic large cell lymphoma (ALCL) is an aggressive T cell lymphoma. To find a novel therapeutic target for aggressive ALK-negative ALCL, we studied two ALK-negative CD30-positive ALCL cell lines, N1 and N2, which were previously reported. In vitro culture, N1 and N2 represent early and advanced stage lymphoma phenotype, respectively. Increased tumorigenicity of N2 was confirmed by its xenotransplantability in SCID mice, whereas N1 was not transplantable either in SCID or in NOD/SCID mice. Subcutaneous tumors formed in the flank of SCID mice inoculated with N2 consisted of large basophilic lymphoma cells with prominent nucleoli, strongly positive for CD2 and CD30. Subcutaneous tumor showed TCR γ rearrangement identical with those in N2, and expressed aberrant CD30 protein with molecular weight 100 Kd, which was expressed only in N2, but not in N1. Those suggested subcutaneous tumors had characteristics of advanced stage ALCL. We hypothesized that constitutively activated c-Jun N-terminal kinase (JNK) is a therapeutic target in advanced stage ALK-negative ALCL, because, (1) JNKs were highly phosphorylated in N2 compared to in N1 studied by immunoblotting using phospho-specific antibodies against JNKs (N1=1.2±0.1 and N2=2.9±0.3, p=0.01, signal intensity); (2)specific JNK inhibitor, SP600125(SP) significantly inhibited cell growth of N2 compared to those of N1in culture for three days(N1=95±1.8 and N2=58±1.6 at 5μM SP, p<0.05; N1=89±0.1 and N2=29±2.4 at 50μM SP, p<0.01, % of viable cell No. of control). Incubation with SP restored the expression of co-stimulatory molecule, CD86 protein, and putative tumor suppressor gene, JunB protein, both of which were strongly downregulated in N2 compared with in N1. Despite of significant growth inhibition, constitutive activation of AP-1 was still demonstrated in N2 incubated with 50μM SP, whereas it was completely inhibited in N1 in electro mobility shift assay using AP-1 DNA consensus sequence(N1=0.2±0.1 and N2=8.3±0.2, p<0.01, arbitrary density units). It suggested that SP exhibited its effect in pathway(s) other than transcriptional regulation through AP-1 complex in ALCL cell lines, N2. In vivo model, intraperitoneal injection of SP (30mg/weight) alternate days for 22days significantly suppressed the growth of subcutaneous tumors (DMSO=1.4±0.5 and SP=0.2±0.1, p<0.05, tumor weight[g] at day26). Tumors in SP-treated mice were exclusively localized in subcutaneous region, in contrast, tumors infiltrated and produced signs of hindlimb paralysis in two of three DMSO-treated mice. Histopathologically tumors in SP-treated showed increase of cells with nuclear condensation, suggesting apoptotic cell death. Furthermore, in addition to partial inhibition of phosphorylation of JNK, tumors in SP-treated mice showed significant decrease of levels of both mRNA (64±13%, p<0.05, real-time PCR) and protein (62±2%, p=0.02, signal intensity by immunoblotting) of BCL2, compared with those in DMSO-treated mice. Whereas no change was found in Bcl-XL expression. No change was found in CD30 protein expression. Taken together, the present study showed that aberrantly activated JNK(s) is a therapeutic target in some types of advanced stage ALK-negative ALCL, although details of signaling pathways triggered by JNK(s) in ALCL cells remained to be elucidated.

Activation of STAT3 by Gαs Distinctively Requires Protein Kinase A, JNK, and Phosphatidylinositol 3-Kinase

Signal transducer and activator of transcription 3 (STAT3) can be stimulated by several G(s)-coupled receptors, but the precise mechanism of action has not yet been elucidated. We therefore examined the ability of Galpha(s)Q226L (Galpha(s)QL), a constitutively active mutant of Galpha(s), to stimulate STAT3 Tyr705 and Ser727 phosphorylations in human embryonic kidney 293 cells. Apart from Galpha(s)QL, the stimulation of Galpha(s) by cholera toxin or beta2-adrenergic receptor and the activation of adenylyl cyclase by forskolin, (Sp)-cAMP, or dibutyryl-cAMP all promoted both STAT3 Tyr705 and Ser727 phosphorylations. Moreover, the removal of Galpha(s) by RNA interference significantly reduced the beta2-adrenergic receptor-mediated STAT3 phosphorylations, denoting its capacity to regulate STAT3 activation by a G protein-coupled receptor. The possible downstream signaling molecules involved were assessed by using specific inhibitors and dominant negative mutants. Induction of STAT3 Tyr705 and Ser727 phosphorylations by Galpha(s)QL was suppressed by inhibition of protein kinase A, Janus kinase 2/3, Rac1, c-Jun N-terminal kinase (JNK), or phosphatidylinositol 3-kinase, and a similar profile was observed in response to beta2-adrenergic receptor stimulation. In contrast to the Galpha16-mediated regulation of STAT3 in HEK 293 cells (Lo, R. K., Cheung, H., and Wong, Y. H. (2003) J. Biol. Chem. 278, 52154-52165), the Galpha(s)-mediated responses, including STAT3-driven luciferase activation, were resistant to inhibition of phospholipase Cbeta. Surprisingly, Galpha(s)-mediated phosphorylation at Tyr705, but not at Ser727, was resistant to inhibition of c-Src, Raf-1, and MEK1/2 as well as to the expression of dominant negative Ras. Therefore, as with other Galpha-mediated activations of STAT3, the stimulatory signal arising from Galpha(s) is transduced via multiple signaling pathways. However, unlike the mechanisms employed by Galpha(i) and Galpha(14/16), Galpha(s) distinctively requires protein kinase A, JNK, and phosphatidylinositol 3-kinase for STAT3 activation.

E47 phosphorylation by p38 MAPK promotes MyoD/E47 association and muscle-specific gene transcription

Selective recognition of the E-box sequences on muscle gene promoters by heterodimers of myogenic basic helix-loop-helix (bHLH) transcription factors, such as MyoD, with the ubiquitous bHLH proteins E12 and E47 is a key event in skeletal myogenesis. However, homodimers of MyoD or E47 are unable of binding to and activating muscle chromatin targets, suggesting that formation of functional MyoD/E47 heterodimers is pivotal in controlling muscle transcription. Here we show that p38 MAPK, whose activity is essential for myogenesis, regulates MyoD/E47 heterodimerization. Phosphorylation of E47 at Ser140 by p38 induces MyoD/E47 association and activation of muscle-specific transcription, while the nonphosphorylatable E47 mutant Ser140Ala fails to heterodimerize with MyoD and displays impaired myogenic potential. Moreover, inhibition of p38 activity in myocytes precludes E47 phosphorylation at Ser140, which results in reduced MyoD/E47 heterodimerization and inefficient muscle differentiation, as a consequence of the impaired binding of the transcription factors to the E regulatory regions of muscle genes. These findings identify a novel pro-myogenic role of p38 in regulating the formation of functional MyoD/E47 heterodimers that are essential for myogenesis.

A novel selective inhibitor of jnk suppresses alloreactivity and prolongs cardiac allograft survival

Activation of activator protein-1 (AP-1) is crucial for T cell activation, and is regulated by JNK (Jun N-terminal kinase). SP600125 (SP) is a recently discovered selective inhibitor of JNK that suppresses inflammatory cytokine synthesis in vitro. Aim of this study is to demonstrate in vivo immunosuppressive action of this compound.