The type III receptor tyrosine kinase fms-like tyrosine kinase 3 (FLT3) is expressed on both normal hematopoietic stem cells and acute myeloid leukemia (AML) cells and regulates their proliferation. Internal tandem duplication (ITD) mutation of FLT3 is present in a third of AML cases, results in constitutive activation and aberrant signaling of FLT3, and is associated with adverse treatment outcomes. While wild-type (WT) FLT3 is predominantly a 150 kDa complex glycosylated cell surface protein, FLT3-ITD is partially retained in the endoplasmic reticulum as a 130 kDa underglycosylated species associated with the chaperones calnexin and heat shock protein (HSP) 90, and mediates aberrant STAT5 signaling, which upregulates the oncogenic serine/threonine kinase Pim-1. FLT3 contains a Pim-1 substrate consensus serine phosphorylation site, and we hypothesized that it might be a Pim-1 substrate. Pim-1 was indeed found to directly interact with and serine-phosphorylate FLT3. Pim-1 inhibition decreased the expression and half-life of 130 kDa FLT3, with partial abrogation by proteasome inhibition, in association with decreased FLT3 binding to calnexin and HSP90, and increased 150 kDa FLT3 expression and half-life, with abrogation by inhibition of glycosylation. These findings were consistent with Pim-1 stabilizing FLT3-ITD as a 130 kDa species associated with calnexin and HSP90 and inhibiting its glycosylation to form the 150 kDa species. Pim-1 knockdown effects were similar. Pim-1 inhibition also decreased phosphorylation of FLT3 at tyrosine 591 and of STAT5, and expression of Pim-1 itself, consistent with inhibition of the FLT3-ITD-STAT5 signaling pathway. Finally, Pim-1 inhibition synergized with FLT3 inhibition in inducing apoptosis of FLT3-ITD cells. This is, to our knowledge, the first demonstration of a role of Pim-1 in a positive feedback loop promoting aberrant signaling in malignant cells. The type III receptor tyrosine kinase fms-like tyrosine kinase 3 (FLT3) is expressed on both normal hematopoietic cells and acute myeloid leukemia (AML) cells and regulates their proliferation proviral insertion site in Moloney murine leukemia virus lymphomagenesis While wild-type FLT3 (FLT3-WT) is synthesized as a 130 kDa underglycosylated, or high-mannose, species, and is then folded in the endoplasmic reticulum (ER) and exported to the Golgi apparatus, where it is glycosylated to form a 150 kDa complex glycosylated species prior to translocation to the cell surface Pim-1 phosphorylates and thereby regulates a number of proteins that are important in key cellular processes, including the pro-apoptotic protein BAD , and this prompted us to test whether Pim-1, expressed downstream of FLT3-ITD FLT3 contains a putative Pim-1 substrate consensus phosphorylation site -ITD FLT3-ITD cell lines included MV4-11 and MOLM-14 . Immunocomplexes were collected using protein A- or protein G-sepharose beads, which were then washed extensively with lysis buffer three times at 4°C.For immunoprecipitation, cells were lysed in buffer  with protease  and phosphatase  inhibitor cocktails. Insoluble material was removed by centrifugation, and antibodies or control immunoglobulins were added to the lysates, followed by incubation at 4°C for 16 hours. Antibodies used for immunoprecipitation included monoclonal anti-Pim-1 (3 µg) and polyclonal anti-FLT3 Cells were lysed in buffer, as above, and immunoblotting was performed as described previously The GST pull-down assay was performed as described previously 50’s of 0.34 µM and 5.5 µM in in vitro kinase and cell-based assays, respectively, while it inhibits Pim-2 kinase with a ten-fold higher IC50 of 3.45 µM Pim-1 kinase was inhibited by incubating cells with the Pim-1-selective kinase inhibitor quercetagetin  at 10 µM in 0.1% DMSO GST-tagged active human Pim-1 protein (Sigma-Aldrich) at 0.250 µg was incubated with 10 µM quercetagetin or DMSO control in 25 µl of 1x kinase buffer  supplemented with 500 µM ATP  at 30°C for 15 minutes. GST-tagged active FLT3 (571-993) protein (Sigma-Aldrich) at 0.5 µg was then added to the reaction mixtures and incubated at 30°C for 30 additional minutes. The reaction was then terminated by adding 9 µL of 4X LDS sample buffer (Invitrogen).5 cells were mixed with lentivirus in a 12-well plate, then cultured for 72 hours after infection. As a control, cells were also infected with equal amounts of plasmid expressing green fluorescent protein (GFP) (Sigma Life Science). Three days after infection with Pim-1 or control shRNA, Pim-1 expression was measured by immunoblotting, with GAPDH as a loading control, to confirm the expected effect or lack of effect on Pim-1 expression. Forty-eight hours after infection, cells were selected for puromycin resistance with 1 µg/ml puromycin for 7 days and then 0.5 µg/ml for 11 days, and stable clones were then isolated and analyzed. On immunoblotting, Pim-1 signal to- noise ratio was significantly low in the three cell lines studied. Pim-1 protein is degraded mainly in the proteasome, with a half-life of approximately 1.7 hours 6 cells of Pim-1 shRNA as well as control shRNA-transduced cells were treated with the proteasome inhibitor MG132 (2 µM) for 3 hours. The MG132-treated samples were then immunoblotted for Pim-1 expression to confirm Pim-1 knockdown.Cells were infected with lentivirus containing Pim-1 small hairpin RNA (shRNA) or non-target control according to the manufacturer's protocol . Briefly, 2.5×106 cells/ml in RPMI 1640 medium with 10% fetal bovine serum (FBS) and 100 µg/ml cycloheximide (CHX) (Sigma-Aldrich) to inhibit new protein synthesis, and residual cellular protein was measured at serial time points by immunoblotting.Cells were cultured at a density of 1×10Mature or complex glycosylation was inhibited by incubating cells with 50 mM 2-deoxy-D-glucose  for 24 hours in the presence and absence of quercetagetin at 10 µM.To evaluate the role of proteasomal degradation in increased FLT3-ITD turnover in the presence of quercetagetin or AR339, cells were incubated with 10 µM quercetagetin or 500 nm AR339 in the presence and absence of the proteasome inhibitor carbobenzoxy-L-leucyl-L-leucyl-L-leucinal  at 1 µM.FLT3-ITD cells were treated with quercetagetin or DMSO control for 3 hours and then lysed in immunoprecipitation buffer as above with 1% SDS. Cell lysates were boiled for 10 minutes. Insoluble material was removed by centrifugation at 23°C and supernatant protein was quantified. To detect ubiquitinated FLT3, FLT3 was immunoprecipitated from the lysate, resolved in a NuPage 4–12% Bis-Tris gel, and immunoblotted with antibody to ubiquitin or FLT3.FLT3-ITD cDNA in pMSCV-puro vector was a kind gift from Dr. Guido Marcucci, the Ohio State University, Columbus, Ohio. To generate the S935D and S935E FLT3-ITD mutants, the serine at position 935 of the FLT3-ITD vector construct was mutated to alanine (A), aspartic acid (D) or glutamic acid (E) using the Site-Directed Mutagenesis kit  as per manufacturer’s instructions. The primers used for site-directed mutagenesis for S935A FLT3-ITD were sense: 5'-tttgactcaaggaaacggccagccttccctaatttg-3' and anti-sense: 5'-caaattagggaaggctggccgtttccttgagtcaaa-3', S935D FLT3-ITD were sense: 5'-tgactcaaggaaacggccagacttccctaatttgacttcg-3' and anti-sense: 5'-cgaagtcaaattagggaagtctggccgtttccttgagtca-3', and those for S935E FLT3-ITD were sense: 5'-tttgactcaaggaaacggccagccttccctaatttg-3' and anti-sense: 5'-caaattagggaaggctggccgtttccttgagtcaaa-3'.PT67 cells were transfected with S935 wild type FLT3-ITD and each of the S935 mutated FLT3-ITD plasmid constructs, S935A, S935D and S935E using FuGENE 6 transfection reagent , according to manufacturer instructions. For each construct, stable clones were isolated using the selection antibiotic puromycin (2 µg/ml). The isolated stable clones  were propagated in culture in DMEM medium supplemented with 10% FBS and 2 µg/ml puromycin.Cells were incubated with the FLT3 inhibitors midostaurin  or sorafenib (LC Laboratories) in the presence and absence of quercetagetin or quizartinib  in the presence and absence of AR339. Apoptosis was detected by Annexin V/propidium iodide (PI) labeling , measured by flow cytometry on a FACScan flow cytometer  and analyzed with CellQuest Pro software, version 5.2.1, 2005 .Percentages of apoptotic cells following incubation under different conditions were compared by the Student t-test, two-tailed.in vitro GST pull-down assays were performed. In the co-immunoprecipitation assays, lysates of MV4-11 and MOLM-14 FLT3-ITD cells, and of EOL-1 FLT3-WT cells, all of which express Pim-1, were immunoprecipitated with anti-Pim-1 and immunoblotted with anti-FLT3, and, reciprocally, immunoprecipitated with anti-FLT3 and immunoblotted with anti-Pim-1. Interaction between Pim-1 and FLT3 was detected in MV4-11 and MOLM-14, and also in EOL-1  containing the Pim-1 consensus phosphorylation site at amino acid 935 (RKRPS) as the substrate and immunoprecipitates from MV4-11 cells with anti-Pim-1 antibody or IgG control as the kinase. Direct interaction between Pim-1 and FLT3 resulted in serine phosphorylation of FLT3  to confirm the decreased stability of the 130 kDa FLT3-ITD species observed on Pim-1 inhibition/knockdown. However mutating FLT3-ITD serine 935 to alanine did not mirror the effects of Pim-1 inhibition/knockdown on FLT3-ITD 130 kDa stability. Serines undergo competitive phosphorylation or O-linked glycosylation AML with FLT3-ITD is associated with adverse treatment outcome, and, in particular, short disease-free survival following chemotherapy. FLT3 inhibitors have been incorporated into treatment regimens for AML with FLT3-ITD, and have activity Clinically applicable Pim-1 inhibitors are being developed and are entering clinical trials. Based on our data, combined administration of Pim-1 inhibitors and FLT3 inhibitors should enhance the clinical efficacy of inhibition of the FLT3 pathway in AML with FLT3-ITD. SGI-1776, the first Pim-1 inhibitor to have undergone phase I testing, is also a FLT3 inhibitor Figure S1A.The Pim-1 inhibitor quercetagetin does not inhibit FLT3. In order to facilitate subsequent experiments it was essential to identify a Pim-1 inhibitor that did not also inhibit FLT3. Quercetagetin was known to inhibit Pim-1 kinase B. Quercetagetin does not alter phosphorylation of the PDK1 target Akt or the JNK target c-jun. MV4-11 cells treated with quercetagetin at 10 µM for the indicated time periods were immunoblotted for phospho-c-jun, total jun kinase and the loading control GAPDH, and phospo-Akt, total Akt and GAPDH. Phosphorylation of c-Jun, an indicator of JNK acitivity, and Akt, an indicator of PI3K activity, did not decrease with respect to total c-Jun or Akt expression, indicating absence of effect of quercetagetin on JNK and PI3K in the MV4-11 cell line.(TIF)Click here for additional data file.Figure S2The proteasome inhibitor MG-132 overcomes destabilization of 130 kDa FLT3 by Pim-1 inhibition. In the same experiments as in A) and 500 nM AR339 (B) for the indicated time periods.(TIF)Click here for additional data file.


Reducing the time required for initial integration of bone-contacting implants with host tissues would be of great clinical significance. Changes in osteoblast adhesion formation and reorganization of the F-actin cytoskeleton in response to altered topography are known to be upstream of osteoblast differentiation, and these processes are regulated by the Rho GTPases. Rac and RhoA (through Rho Kinase (ROCK)). Using pharmacological inhibitors, we tested how inhibition of Rac and ROCK influenced osteoblast adhesion, differentiation and mineralization on PT (Pre-treated) and SLA  topographies. Inhibition of ROCK, but not Rac, significantly reduced adhesion number and size on PT, with adhesion size consistent with focal complexes. After 1 day, ROCK, but not Rac inhibition increased osteocalcin mRNA levels on SLA and PT, with levels further increasing at 7 days post seeding. ROCK inhibition also significantly increased bone sialoprotein expression at 7 days, but not BMP-2 levels. Rac inhibition significantly reduced BMP-2 mRNA levels. ROCK inhibition increased nuclear translocation of Runx2 independent of surface roughness. Mineralization of osteoblast cultures was greater on SLA than on PT, but was increased by ROCK inhibition and attenuated by Rac inhibition on both topographies. In conclusion, inhibition of ROCK signalling significantly increases osteoblast differentiation and biomineralization in a topographic dependent manner, and its pharmacological inhibition could represent a new therapeutic to speed bone formation around implanted metals and in regenerative medicine applications. In vivo, titanium forms a direct interface with bone tissue through the process of osseointegration. By modifying the surface topography of the implant surface, formation of bone on titanium surfaces can be further enhanced. It is evident that cellular recognition of surface topography is extremely complex, and remains as of yet, poorly understood. Although many dental implant systems show great clinical success temporally Titanium and its alloys have been used for bone-contacting implants for several decades due to their high biocompatibility, favourable mechanical properties and low corrosion Sand-blasted, Large grit, and Acid etched (SLA) topography produced by grit blasting followed by acid etching . This technique results in a surface with hierarchical levels of roughness; large pores 20–50 μm occur as a result of sand blasting, and smaller micropits 0.5–2 μm in diameter result from HCl/H2SO4 treatment in vitroin vivoMany different topographies are found commercially on dental implants, but moderately roughened topographies, produced through additive techniques such as titanium plasma spraying or subtractive techniques such as grit-blasting, acid etching or anodization are most common In vitro, SLA increases osteoblast attachment, differentiation, and biomineralization, in comparison with smoother topographies, which promote adhesion formation, spreading, F-actin stressfibre formation and proliferation Adhesion formation and F-actin reorganization, whether influenced by topography or not, are regulated by the Rho-family of GTPases Recent studies have shown that actin gene expression is downregulated in mineralizing, but not non-mineralizing osteoblasts All studies involving rats were performed in compliance with the University Council on Animal Care using protocols approved by the Animal Use Subcommittee at the University of Western Ontario specifically for this work.2SO4, and cleaned in dilute nitric acid followed by rinsing with pure water. The three-dimensional surface topography was quantitatively measured using confocal microscopy . Images over an area of 798 μm ×798 μm were obtained with a 20× objective. Three-dimensional roughness parameters Sa (arithmetic mean deviation of the surface), St  and Ssk (skewness of the surface) were calculated using a moving average Gaussian filter with a cut-off wavelength of 30 μm. Three measurements each were made on three samples per group. The surface topography was qualitatively examined using scanning electron microscopy (Phillips XL30), using a tungsten filament as electron emitter and a secondary electron detector. Images were acquired with an acceleration voltage of 20 kV. The surface topography was qualitatively examined using scanning electron microscopy (Phillips XL30), using a tungsten filament as electron emitter and a secondary electron detector. Images were acquired with an acceleration voltage of 20 kV. To quantify levels of nanoroughness on PT surfaces, Atomic force microscopy was performed using a Digital Instruments Multimode AFM with NanoScope IIIa controller . Measurements were either made in height mode, with a scan direction of 0° , while recording the trace and retrace height information (measured in nm), or in lateral-force mode, with a scan direction of 90°, while recording the trace and retrace lateral force data .SLA and PT Ti discs were prepared as previously described in Miron et al 2 atmosphere with 100% relative humidity at 37°C.Rat Calvarial Osteoblasts (RCOs) were isolated from 0–5 days old Sprague Dawley® rats by enzymatic digestion as previously described 2 tissue culture plastic flasks using a 0.05% trypsin and 0.2 g/L EDTA·4Na solution and counted using a Beckman CoulterTM MultisizerTM 3 Coulter Counter® . RCOs were seeded at 3×104 cells/cm2 for real-time RT-qPCR analysis and at 1×104 cells/cm2 for immunofluorescence. For experiments lasting longer than 24 hours, cells were supplemented at seeding with 50 µg/ml of ascorbic acid and 2 mM β-glycerol phosphate to induce differentiation. Rac activity was inhibited using NSC23766  reconstituted in sterile water and used at a final concentration of 100 mM. ROCK signaling was inhibited using Y-27632 . Y-27632 was reconstituted in dimethyl sulfoxide (DMSO) and used at a final concentration of 10 μM. The NSC23766 and Y-27632 inhibitors were added at the time of cell seeding and media was changed daily to ensure continuous presence and activity of the inhibitors. To assess bone nodule formation, tetracycline (Sigma-Aldrich) was added to the media at 5 μg/ml. Images were imported into imageJ, and the intensity of staining and the numbers of nodules per surface counted calculated.For experiments, cells were released from 75 cmTo assess the efficacy of NCS23766 and Y27632 on inhibition of Rac1 and ROCK respectively, osteoblasts were cultured on tissue culture plastic to ensure sufficient protein could be extracted for the assays. Cells were grown in the presence of the inhibitors for 1 and 3 weeks, with osteogenic media serving as a control. Rac1 activity was assayed using G-LISA™ Rac1 assay biochem kit™  according to manufacturers instructions. ROCK activity was quantified using the Rho-associated kinase activity assay (Millipore). Activity was normalized to GAPDH to ensure equal protein loading.Cells designated for immunofluorecent labeling were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) and permeabilized with 0.1% Triton-X 100 in PBS for five minutes. Non-specific antibody interactions were blocked using 3% bovine serum albumin (BSA) in PBS for 30 minutes prior to the addition of the primary antibody. Samples were incubated with primary antibodies to vinculin , osteocalcin , and bone sialoprotein (Gift from Dr Harvey Goldberg) Runx2  at a 1∶100 dilution. All antibodies were diluted in 0.5% BSA in PBS. After a 1 hour incubation with the primary antibody, the surfaces were rinsed three times and then incubated with a 1∶200 dilution of Alexa Fluor® 488 goat anti-mouse IgG  for visualization of the primary antibody and a 1∶100 dilution of Rhodamine phalloidin  for visualization of filamentous actin. Nuclei were counterstained using 4’,6 diamidino-2-phenylindole (DAPI) using Vectashield Mounting Medium . To visualize bone nodule formation by tetracycline incorporation, an illumination wavelength of 488 nm was used as previously described Bglap, Rn01455285_g1), bone sialoprotein , bone morphogenetic protein-2  Runx2  and ATF-4  were obtained from Applied BiosystemsTM . RT-PCR was run on the extracted RNA using a final reaction volume of 15 μl per well. TaqMan® One-Step RT-PCR Master Mix Reagents was added to 50 ng of RNA per well in order to obtain the final reaction volume. Each well contained RNA from one titanium surface. All experiments were completed in quadruplicate and repeated three independent times. Gene expression levels were reported relative to 18S levels and normalized to day 1 PT control cells.Total RNA was extracted using TRIZOL® reagent at 1 and 7 days according to the manufacturer’s recommendations. Probes for osteocalcin . p<0.05 was considered significant.a of the SLA topography was 1.435 µm in comparison with the PT, which was 0.331 µm (t) (defined as the height difference between the highest and lowest points) for PT was 2.3 µm and for SLA was 8.95 µm. As expected from the SEM micrographs, the Ssk of PT was negative indicating a predominance of smooth valleys, while SLA had a positive skew, which is characteristic of surfaces with peaks. AFM analysis of PT surfaces revealed that the surfaces have significant nanoroughness Topographical-induced adhesion and morphological changes influence osteoblast migration, differentiation, and mineralization on titanium To investigate the roles of Rac and ROCK signaling in osteoblast response to PT and SLA topographies, we utilized the well-characterized pharmacological inhibitors NSC23766 (Rac) and Y27632 (RhoA-ROCK). NSC23766 is a cell-permeable inhibitor of Rac activation through inhibition of the Rac-specific guanine exchange factors TrioN and Tiam 1, both of which catalyze GDP/GTP exchange. With regards to specificity, NSC23766 likely also inhibits Rac2 and Rac3, but Rac2 is mainly expressed in hematopoietic lineages and Rac3 is associated with neuronal development. NSC23766 has no effect on cdc42 or RhoA activation. To inhibit ROCK indirectly, we used Y27632, which is a non-isoform-selective inhibitor that targets the ATP-dependent kinase domains of both ROCK1 and ROCK2. Essentially Y27632 works by competing with ATP for binding to the kinases domains of the ROCKs. Based on previous studies The most prominent finding from our study is that inhibition of ROCK signaling in osteoblasts significantly increases osteocalcin (PT and SLA) and bone sialoprotein (SLA) expression at 1 week, and biomineralization at 4 weeks. Recent evidence suggests that ROCK inhibition results in an increase in Wnt signalling in MC3T3 osteoblasts cultured on titanium The measured changes in gene expression in osteoblasts induced by ROCK inhibitors were mirrored by increases in biomineralization at 4 weeks on both PT and SLA topographies, assessed through tetracycline incorporation. Inhibition of Rac activity reduced nuclear translocation while ROCK inhibition increased nuclear translocation of the osteocalcin transcription factors Runx2. Interestingly, Runx2 gene expression levels did not change under Rac or ROCK inhibition (data not shown), suggesting that upregulation of the gene is not required and it is nuclear accumulation of the protein that is altered. ROCK inhibition has been previously demonstrated to increase osteocalcin synthesis and biomineralization of mouse calvarial osteoblasts Inhibition of ROCK signalling resulted in a significant reduction in FA formation and disruption of F-actin stressfibres, particularly in cells on PT. Rac1 and Cdc42 stimulate the formation of focal contacts, with ROCK regulating the subsequent formation of actin stressfibres bundles and the development of mature FAs. On SLA surfaces, osteoblasts form adhesions consistent in size with focal complexes, larger mature FAs being relatively rare Although the current SLA topography has been a very effective topography for promoting osseointegration clinically in vitroin vitroin vitro. One of the major issues facing patient-specific tissue regeneration is the amount of time required for the patient’s cells to migrate into, proliferate, differentiate, and produce matrix and mineral within a scaffold ex vivo. The use of a ROCK inhibitor could accelerate osteoblast infiltration, differentiation, and mineralization in both ex vivo (addition to media in bioreactors) and in vivo (systemic administration) seeded scaffolds for bone tissue regeneration. This would ultimately reduce the amount of time required to produce patient-specific tissues suitable for implantation. However, if mesenchymal stem cells were used as a cell source, it is likely that ROCK inhibitors may only function in temporal manner beyond initial scaffold colonization/cell proliferation and MSC commitment to the osteogenic lineage.The effectiveness of ROCK inhibition on accelerating osteoblast differentiation and mineralization has huge implications not only for enhancing implant integration, but also for bone tissue engineering applications. In 1986, Bellows et al. demonstrated the necessity of ascorbic acid (AA) and beta-glycerol phosphate (BGP) for matrix secretion and mineralization of osteoblasts respectively We have demonstrated differences in the impact of cytoskeletal-regulating proteins on osteoblast adhesion formation, differentiation, and mineralization in response to substratum topography. While topographical manipulation osteoblasts can significantly affect differentiation, we demonstrate that pharmaceutical inhibition of ROCK activity acts synergistically with osteoconductive SLA to effectively speed osteoblast maturation, differentiation and mineralization. Further elucidation of the pathways through which adhesion and cytoskeletal changes regulate gene expression in osteoblasts will be instrumental in developing scaffolds and bioactive materials for bone tissue regeneration in future dental and orthopaedic applications.


The riluzole-induced Smad linker phosphorylation is mechanistically different from the Smad linker phosphorylation induced by TGFβ. We also demonstrate that riluzole-induced Smad linker phosphorylation is independent of the expression of the metabotropic glutamate receptor 1 (GRM1), which is one of the glutamate receptors whose involvement in human melanoma has been documented. We further show that riluzole upregulates the expression of INHBB and PLAU, two genes associated with the TGFβ signaling pathway. The non-canonical increase in Smad linker phosphorylation induced by riluzole could contribute to the modulation of the pro-oncogenic functions of Smads in late stage melanomas.Riluzole, an inhibitor of glutamate release, has shown the ability to inhibit melanoma cell xenograft growth. A phase 0 clinical trial of riluzole as a single agent in patients with melanoma resulted in involution of tumors associated with inhibition of both the mitogen-activated protein kinase (MAPK) and phophoinositide-3-kinase/AKT (PI3K/AKT) pathways in 34% of patients. In the present study, we demonstrate that riluzole inhibits AKT-mediated glycogen synthase kinase 3 (GSK3) phosphorylation in melanoma cell lines. Because we have demonstrated that GSK3 is involved in the phosphorylation of two downstream effectors of transforming growth factor beta (TGFβ), Smad2 and Smad3, at their linker domain, our aim was to determine whether riluzole could induce GSK3β-mediated linker phosphorylation of Smad2 and Smad3. We present evidence that riluzole increases Smad2 and Smad3 linker phosphorylation at the cluster of serines 245/250/255 and serine 204 respectively. Using GSK3 inhibitors and siRNA knock-down, we demonstrate that the mechanism of riluzole-induced Smad phosphorylation involved GSK3β. In addition, GSK3β could phosphorylate the same linker sites  Transforming growth factor-beta (TGFβ) plays a dual role in melanoma, mediating tumor suppressive activities at early stages and prooncogenic activities at later stages of tumor progression From studies on epithelial cells, carcinomas, gliomas and melanomas, it appears that Smads, through their linker domain, are at the point of convergence of major cellular signaling pathways, involving ERK, JNK, p38, CDK, GSK3β. GSK3β activity is negatively regulated upon AKT phosphorylation on serine 9 One drug that can inhibit AKT activity is riluzole, an inhibitor of glutamate release and a FDA approved therapeutic agent for the treatment of amyotrophic lateral sclerosis The present study was designed to determine whether riluzole treatment could result in GSK3β-mediated linker phosphorylation of Smad2 and Smad3, through the inhibition of AKT activity. We present evidence that riluzole inhibits AKT-mediated GSK3β phosphorylation, and increases Smad2 and Smad3 linker phosphorylation by a mechanism involving GSK3β. We also show that riluzole upregulates the expression of two genes associated with the TGFβ signaling pathway, INHBB (coding for Inhibin beta B) and PLAU (coding for the urokinase plasminogen activator).WM793, WM278, and 1205LU were kindly provided by Dr. M. Herlyn  and SB431542 were purchased from Sigma-Aldrich. CHIR-99021 (CT99021) was purchased from Selleck Chemicals . Recombinant human TGFβ1 was purchased from R&D systems, Inc .5/dish or well). The following day, cells were serum-starved for about 16 hours before being incubated with 50 mM Nacl or 50 mM LiCl for 2 and 5 hours. For CT99021 experiment, serum-starved cells were incubated in the presence of DMSO or 2 µM CT99021 for two hours. For riluzole treatment, serum-starved cells were incubated with or without 25 µM riluzole for 9 hours in the absence or presence of either GSK3 inhibitor: LiCl (50 mM) or CT99021 (2 µM). For the TGFβ treatment, serum-starved cells were incubated in the absence or presence of 200 pM TGFβ for 1 or 2 hours.Cells were seeded in 35 mm dishes or 6-well plates , WM793 cells were transfected with 50 nM non targeting control siRNA or GSK3α/β siRNA . The cells were then resuspended in warm media and split into two wells of a 6 well tissue culture plate and placed in the 37°C incubator overnight. The following day, the medium was replaced with fresh culture medium for recovery. After overnight serum starvation, cells were incubated with or without 25 µM riluzole for 9 hours before protein extraction.The WM793 melanoma cell line was grown to 80% confluency. 3.10Recombinant GST Smad2 and GST Smad3 fusion proteins (3 µg) and 1 µl of GSK3β (New England Biolabs) were incubated at 30°C for 1 hour in a 30 µl reaction containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 5 mM DTT and 0.5 mM ATP. After 1 hour incubation, SDS sample buffer was added to terminate the kinase reactions. The reaction products were then analyzed by immunoblotting using the pSmad2 (S245/250/255) and pSmad3 (S204) phosphopeptide antibodies.Cells were harvested, washed with phosphate-buffered saline, and extracted in the presence of protease and phosphatase inhibitors (Roche) as previously described UACC903 cells were stably transfected with the pcDNA6-GRM1 construct  by following the lipofectin  transfection manual provided. Stable clones UACC903-G2 (abbreviated as G2) and UACC903-G4 (abbreviated as G4) over-expressing GRM1 were propagated under selection of 10 ug/ml Blasticidin in RPMI 1640 containing 10% FBS. Control cell line UACC903-V1 (abbreviated as V1) was derived from UACC903 transfected with pcDNA6/V5-HisA empty vector.2 first strand kit  for the TFGβ/BMP signaling pathway PCR arrays. For the TFGβ/BMP signaling pathway PCR array, the cDNAs were added to the RT2 qPCR master mix, and the mixture was aliquoted across the PCR array, according to the manufacturer recommendations. The qPCR was performed in One Step Plus qPCR instrument . All primers for SYBR qRT-PCR were purchased from Qiagen . Changes in gene expression were calculated using the delta delta Ct method. All experiments were independently replicated 3 times.Total RNA was isolated using Trizol  and Direct-zol RNA miniprep kit  following manufacturer’s instruction. One microgram of total RNA was used for cDNA synthesis using SuperScript II cDNA synthesis kit  for standart qPCR and RTWe previously demonstrated that the two pan-CDK/GSK3 inhibitors, flavopiridol We previously reported that riluzole inhibited phosphorylation of AKT, suggesting that this agent could negatively affect AKT activity in vitro kinase assay  as a key player in melanoma development We previously mentioned that preclinical studies 2 Profiler™ PCR Arrays to identify known TGFβ target genes whose expression could be modulated by riluzole. For this purpose, RNAs were isolated from WM793 melanoma cells incubated in the absence or presence of riluzole. cDNAs converted from these RNAs were processed for real-time PCR using one PCR array for the untreated cells-derived cDNA and one array for the riluzole-treated cells-derived cDNA. By comparing the two, we identified four genes whose expression was increased or decreased in the presence of riluzole as compared with untreated WM793 melanoma cells (The positive effect of riluzole on Smad2 and Smad3 linker phosphorylation  promptedma cells . By qPCRma cells . The INHin vitro kinase assays confirmed that GSK3β could phosphorylate Smad2 and Smad3 at the cluster of serines 245/250/255 and serine 204 respectively.In the present study, we described a cross-talk between three pathways involved in melanoma biology, the TGFβ signaling pathway Interestingly, we also demonstrate riluzole induced Smad linker phosphorylation in the GRM1 negative UACC930 melanoma cell line TGFβ-induced Smad linker phosphorylation has been described in a wide variety of cellular systems, including melanoma cells. The different kinases involved in each of these studies include JNK, CDKs, GSK3, depending on the phosphorylation site and the cellular context The effect of riluzole on the linker phosphorylation of Smad2 and Smad3, downstream effectors of TGFβ, will likely modulate TGFβ signaling and the expression of TGFβ target genes. Our previous report suggested that Smad3 linker phosphorylation might contribute to the resistance to TGFβ-mediated cell growth inhibition in melanoma, by inhibiting the expression of p15 and p21. However, Smad3 linker phosphorylation did not inhibit the expression of PAI-1, involved in TGFβ pro-oncogenic effects. Therefore, Smad3 activity would be inhibited on promoters involved in cell growth inhibition, such as p15 and p21, but fully competent for regulating some of the genes involved in TGFβ prooncogenic effects We initiated the characterization of genes associated with the TGFβ signaling pathway and whose expression was modulated by riluzole. Our goal was to define possible mediators of riluzole action downstream of the TGFβ signaling pathway. Since TGFβ exerts pro-oncogenic activities at late stages of melanoma development, it is important to determine whether riluzole can have a negative effect on the expression of genes involved in TGFβ pro-oncogenic activities. This could explain the inhibition of melanoma cell growth in mice and the involution of some of the tumors in patients treated with riluzole. Alternatively, a positive regulation of genes involved in TGFβ pro-oncogenic activities by riluzole could explain the mixed responses to riluzole and the failure of some patients to respond to riluzole in clinical trials At this point of the study, we cannot directly link the riluzole-induced linker phosphorylation of Smad2 and Smad3 to the upregulation of INHBB and PLAU expression. This would require an extensive analysis using stable transfectants expressing wild-type and Smad mutant forms unable to be phosphorylated in the specific GSK3 sites. However, our study revealed an important cross-talk between three melanoma signaling pathways, the glutamate signaling, the PI3K/AKT pathway and the TGFβ signaling pathway. We then focused on the TGFβ signaling pathway as a possible mediator of riluzole actions. We then identified two genes whose upregulation by riluzole might be detrimental to a more complete response to this agent in clinical trials. The identification of cross-talks such as those described in this study could be instrumental in predicting responses to riluzole-based therapy.


N-acetyl-p-aminophenol  reduces the paracellular transport activities of itself and co-administered substances. The aim of this study was to find out how APAP reduces the bioavailability of small molecules which pass paracellularly, and which tight junction proteins are involved in the regulation of paracellular transport using a Caco-2 barrier model.Drugs and diet interact with each other in mutually influencing their bioavailability. In our previous work we have shown that in vitro model is widely used by the pharmaceutical industry to predict the absorption of orally administered drugs. To construct a Caco-2 barrier model, the Caco-2 cell line, a continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells, was seeded onto inserts  for 21 days. After differentiation, the cells were treated topically with 10 mM APAP for 24 h. The cell transepithelial electrical resistance (TER) and capacitance (Ccl) were determined by two different impedance-measuring systems. The membrane permeability was tested by differently sized molecules: Lucifer Yellow (520 Da), 3–5 kDa and 40 kDa Fluorescein thiocarbamoyl (FITC)-dextran. The tight junction proteins  were investigated using Western blot analysis and immunofluorescence staining.The Caco-2 i.e. membrane integrity, which correlated significantly with the decrease of permeability of the small molecules Lucifer Yellow (ca. 520 Da) and FITC-dextran (3–5 kDa) but not of large molecules (40 kDa FITC-dextran). The tight junction protein ZO-1 and its tyrosine phosphorylation were up-regulated after 24 h treatment with APAP.APAP increased the TER value, Tight junction proteins play an important role in maintaining the intestinal barrier function. We assume that APAP affects the paracellular transport pathway through disassembly of the tight junction. We suppose that APAP leads to remodelling of the tight junction protein ZO-1 through changes in the level of protein expression and in tyrosine phosphorylation. APAP may reduce the bioavailability of co-administered substances through remodelling the tight junction by regulating ZO-1 and its tyrosine phosphorylation. As a result, the paracellular transport activity for small molecules is decreased as is net intestinal absorption.


ATP also blocked inositol phosphates  generation and [Ca2+]i mobilization induced by thrombin. Thrombin reduced cAMP levels which were restored in the presence of ATP. SQ-22536, an adenylate cyclase (AC) inhibitor, partially reduced the inhibition exerted by ATP on TIPA. 12-lipoxygenase (12-LO) inhibitors, nordihidroguaretic acid (NDGA) and 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15(S)-HETE), strongly prevented ATP-mediated TIPA inhibition. Additionally, ATP inhibited the increase of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE) induced by thrombin. Pretreatment with both SQ-22536 and NDGA almost completely abolished ATP-mediated TIPA inhibition. Our results describe for the first time that ATP implicates both AC and 12-LO pathways in the inhibition of human platelets aggregation in response to agonists.In this study we have investigated the role of extracellular ATP on thrombin induced-platelet aggregation (TIPA) in washed human platelets. ATP inhibited TIPA in a dose-dependent manner and this inhibition was abolished by apyrase but not by adenosine deaminase (ADA) and it was reversed by extracellular magnesium. Antagonists of P2Y Several agonists including ADP, thrombin, and thromboxane A1 and P2Y12, involved in the ADP-induced platelet responses have been cloned. The P2Y1 receptor mediates PLC activation via a Gαq subunit and subsequently regulates intracellular calcium ([Ca2+]i) mobilization and platelet shape changes 12 receptor, on the other hand, is coupled to the Gαi subunit, which prevents the activation of AC, whereupon the intracellular cAMP concentration decreases. P2Y12 receptor behaves as a negative regulator of platelet activation 12-dependent Gαi activation also potentiates the release of granule contents IIbβ3 integrin via phosphoinositide-3 kinase ADP is released during platelet activation, becoming a critical molecule in hemostasis. ADP also cooperates with other molecules, including thrombin, to potentiate many platelet responses 1 and P2Y12 receptors αq and Gαi pathways 12 activation to stimulate Gαi and activate PAR1 via Gαq but also, at high concentrations, it can regulate PAR4 pathway 12 antagonists) selectively block the ADP component in the thrombin response resulting in a potent inhibition of platelet activation whereas they are ineffective for P2Y1ADP-induced platelet aggregation requires coactivation of P2Y1 and P2Y12 receptors 1 receptor in human platelets and increases the intracellular calcium concentration without generating platelet aggregation 1 receptors play an important role in platelet activation, particularly under conditions of shear stress and thus during arterial thrombosis ATP and ADP are present in platelets at approximately equimolar concentrations 2+]i mobilization, this suggests that an extracellular P2X-like site could be responsible for the effects of these nucleotides , extracellular ATP, GTP and AMP increased the 12(S)-HETE production. ATP activated 12-LO by an unknown mechanism and increased by 3-fold the 12(S)-HETE formation. A purinergic binding site is proposed to activate this pathway ATP and other nucleotides such as, GTP, GDP or GDP-β-S inhibit both thrombin- and ADP-mediated platelet activation 12 receptor and the inhibition of the intracellular levels of 12(S)-HETE.The aim of this work was to examine the interaction between extracellular ATP and platelets exposed to thrombin. Our results suggest that AC and the 12-LO pathways are implicated in the inhibition of TIPA mediated by ATP. This physiological inhibition of human platelets in response to strong agonists is mediated by a combined action between the P2Y3P5P), fibrinogen, acid citrate dextrose, ADA, ADP, ATP, α,β-methylene ATP, βγ-methylene ATP, benzoyl ATP, 2 methylthio ATP, apyrase, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), N-[2-hydroxyethyl] piperazine-N′-[2-ethanesulfonic acid] (HEPES), 15(S)-HETE, 3- isobutyl-1-methylxanthine (IBMX), milrinone, dipyridamole, NDGA, protein kinase, 3′, 5′-cyclic AMP dependent (PKA), polyethylenimine, sodium nitroprusside (SNP) and thrombin were obtained from Sigma-Aldrich Chemical . Fura-2 AM was from Molecular Probes . Myo-[2-3H]inositol was from GE Healthcare . Dowex AG1-X8 column was from Bio-Rad Laboratories . Trichloroacetic acid (TCA) was purchased from Panreac . 1H-Adenosine 3′, 5′ biphosphate  and stirred before treatment. Aggregation was measured as the percentage of the maximum change in light transmission against a buffer blank for 10 min. All assays were performed in the presence of 1 mM CaCl2.Platelets were acquired from the Blood Bank at the Galdakao Hospital in Spain. Platelet aggregation was carried out as described previously 2+]i levels were monitored and calculated as described 8/ml in a nominally Ca2+-free standard medium  were loaded with 1 µM Fura-2 AM and 0.018 U/ml apyrase grade V at 37°C for 45 min. Subsequently, acid citrate dextrose was added , and cells were washed by centrifugation . Finally, cells were resuspended in fresh standard medium; apyrase-free at 2×108 cells/ml. Cell aliquots (0.5 ml) were transferred to a cuvette and prewarmed at 37°C for 5 min.[Ca2 or without external Ca2+ but with 2 mM EGTA-K2-H2.Fluorescence was measured with a spectrofluorimeter  equipped with a thermostated cell holder and a magnetic stirrer. Fura-2 fluorescence was monitored continuously using monochromator settings of 340 and 380 nm (excitation) and 505 nm (emission). These experiments were performed either in the presence of 1 mM CaCl3H]inositol phosphates was carried out as described 3H]inositol at 37°C for 3 hours. Subsequently, cells were washed and resuspended in HEPES buffer (pH 7.4) at 6–8×108 cells/ml and then allowed to rest for ≥25 min before experimentation. Platelet suspensions (0.5 ml) were prewarmed at 37°C for 5 min followed by placing them into the aggregometer (37°C) and stirred. After stimulation, reaction was stopped by adding 0.5 ml ice-cold 10% (vol/vol) perchloric acid. Samples were centrifuged at 1,000×g for 5 min at 4°C, and the supernatants were neutralized with 1.5 M KOH/75 mM HEPES and recentrifuged at 1,500×g for 5 min at 4°C. The supernatant was diluted with 10 mM HEPES (pH 7.4)/2 mM EDTA. The [3H]inositol phosphates were separated by anion-exchange chromatography on a Dowex AG1-X8 column as described Measurement of i. In order to investigate whether ATP blocked PLC activation mediated by thrombin, we examined inositol phosphate generation in platelets. As shown in 1), bisphosphate (IP2) and trisphosphate (IP3) generation induced by all thrombin concentrations tested (from 0.025 U/ml to 0.5 U/ml). Moreover, maximal concentration of ATP (3 mM) inhibited also basal levels of IP1, IP2, IP3 by 39, 17 and 11%, respectively. Taken together these results indicate that ATP blocks platelet aggregation induced by thrombin and this process should require PLC activation to induce inositol phosphate generation and calcium mobilization.A key element in early signalling is the PLC, which mediates inositol phosphate break-down to generate IP3, DAG and i induced by thrombin being this inhibition independent of the extracellular calcium, moreover, ATP also inhibited phosphatidyl inositol break-down at all concentrations of thrombin tested. These results suggest that PLC activity is affected by ATP. In agreement with that, Soslau et al. reported that extracellular ATP inhibited the calcium mobilization from intracellular pools by various agonists like collagen, thrombin or an analogue to thromboxane A21 receptors 1-receptor desensitization, platelet preparations were treated with apyrase 1 receptor would not be involved in this early signalling due to this receptor desensitizes in milliseconds without the presence of apyrase Besides aggregation, we found that ATP also inhibited the increase of the [CaMany compounds exert their inhibition on TIPA through an increase in basal levels of cyclic nucleotides such as prostaglandins and NO donors The levels of cAMP in platelets play a key role in the control of the activation/aggregation by thrombin. Eigenthaler et al. estimated that intracellular cAMP concentration in unstimulated washed platelets was 4.4 µM, similar to the concentration of cAMP binding sites of PKA 12 receptors, AR-C67085 also increased in a dose-dependent manner the cAMP levels with a maximum increase at 10 µM. No potentiation or additive effect was observed when AR-C67085 was added to a maximal concentration of ATP, suggesting that ATP was acting on P2Y12 receptors with low affinity Thrombin reduces cAMP concentrations below the basal levels through the ADP secreted from dense granules in thrombin activated platelets 1 receptors with the antagonist A3P5P or N6-methyl-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2179) had no effect on TIPA and it had no additional effects in combination with AR-C69931, an antagonist of P2Y121 receptor-null mice, in which the absence of expression of these receptors did not affect TIPA 12 receptors with AR-C67085 did not inhibit thrombin-induced irreversible platelet aggregation; it slightly inhibited both phases of platelet aggregation, the primary response-rate of aggregation and the final response-maximal aggregation in response to 0.025 U/ml thrombin. Inhibition of maximal aggregation was reached at 1 µM, the contribution of this receptor to platelet aggregation was estimated about ∼12% in our conditions. Similar results were obtained in studies made on whole blood aggregation 12 receptor activation mediated by ADP and inhibiting platelet activation/aggregation induced by thrombin.It has been reported that ADP contributes to platelet aggregation in response to low concentrations of thrombin 2 is a substrate of 12-LO for the production of 12(S)-HETE in platelets 2 activity (not shown), furthermore thrombin increases the 12(S)-HETE levels Extracellular nucleotides such as ATP, GTP and AMP are involved in the activation of cyclooxygenase and 12-LO in washed human platelets When both AC and 12-LO were simultaneously inactivated by SQ-22536 and NDGA respectively, ATP-mediated TIPA inhibition was almost completely abolished. This effect was additive not synergistic, suggesting the presence of two different and independent mechanisms whereby ATP exerted its inhibition. According to with this, platelet incubated either with NDGA or 15(S)-HETE did not lead to any significant change in cAMP levels (data not shown), indicating that 12-LO inhibition did not interfere with the effect of ATP on AC. Conversely the inhibition of AC with SQ-22536 did not affect thrombin stimulated 12(S)-HETE formation or ATP-mediated inhibition of this hydroxylipid (data not shown).12-receptor pathways led by thrombin. In addition, we postulate that the rapid effect of ATP on TIPA inhibition could also be originated by a direct allosteric interaction between the ATP and a P2 receptor different to P2Y12. Although the molecular mechanism remains to be elucidated; 2) through another unknown molecular mechanism that involves to the 12-LO pathway. Future studies will focus on the P2 receptor identification as well as the characterization of the molecular events leading to the cooperation between cAMP and 12-LO pathways to inhibit the platelet aggregation induced by thrombin.In conclusion, we have shown that the inhibition of either AC or 12-LO pathway reverses ATP-mediated inhibition of TIPA. Furthermore, ATP exerts its inhibitory effect over TIPA at least through two different and independent signalling routes: 1) by regulating AC activity; the intracellular cAMP levels negatively affects the intracellular signalling controlled by thrombin, and also interferes with the ADP/P2Y


Modulation of DNA base excision repair (BER) has the potential to enhance response to chemotherapy and improve outcomes in tumours such as melanoma and glioma. APE1, a critical protein in BER that processes potentially cytotoxic abasic sites (AP sites), is a promising new target in cancer. In the current study, we aimed to develop small molecule inhibitors of APE1 for cancer therapy.in vitro analyses using fluorescence-based APE1 cleavage assays and counter screened using endonuclease IV cleavage assays, fluorescence quenching assays and radiolabelled oligonucleotide assays. Biochemical APE1 inhibitors were then subjected to detailed cytotoxicity analyses.An industry-standard high throughput virtual screening strategy was adopted. The Sybyl8.0  molecular modelling software suite was used to build inhibitor templates. Similarity searching strategies were then applied using ROCS 2.3  to extract pharmacophorically related subsets of compounds from a chemically diverse database of 2.6 million compounds. The compounds in these subsets were subjected to docking against the active site of the APE1 model, using the genetic algorithm-based programme GOLD2.7 . Predicted ligand poses were ranked on the basis of several scoring functions. The top virtual hits with promising pharmaceutical properties underwent detailed 50 for APE1 inhibition ranged between 30 nM and 50 μM. We demonstrated that APE1 inhibitors lead to accumulation of AP sites in genomic DNA and potentiated the cytotoxicity of alkylating agents in melanoma and glioma cell lines.Several specific APE1 inhibitors were isolated by this approach. The ICOur study provides evidence that APE1 is an emerging drug target and could have therapeutic application in patients with melanoma and glioma. Monofunctional alkylating agents are routinely used for the treatment of patients with advanced melanoma and glioma. However, the response rate to chemotherapy is modest and the overall prognosis is poor. The cytotoxicity of alkylating agents is directly related to their propensity to induce genomic DNA damage. However, the ability of cancer cells to recognize this damage and initiate DNA repair is an important mechanism for therapeutic resistance that negatively impacts upon therapeutic efficacy. Pharmacological inhibition of DNA repair, therefore, has the potential to enhance the cytotoxicity of alkylating agents and improve patient outcomes .N-glycosidic bond, leaving a potentially cytotoxic apurinic/apyrimidinic (AP) site intermediate (E. coli homologue of APE1 (E. coli endonuclease IV (The DNA base excision repair (BER) pathway is critically involved in the repair of bases that have been damaged by alkylating agents such as temozolomide and dacarbazine . Althoug of APE1 . The endlease IV , is struUsing either antisense oligonucleotides or RNA interference approaches, several groups have reported that depletion of intracellular APE1 sensitizes mammalian cells to a variety of DNA damaging agents . In heaH-indole-2-carboxylic acid), the first small molecule inhibitor of APE1 that potentiated the cytotoxicity of alkylating agents such as temozolomide .Human APE1, uracil-DNA glycosylase and The oligonucleotides; 5- F-GCCCCCXGGGGACGTACGATATCCCGCTCC-3′ and 3′-Q-CGGGGGCCCCCTGCATGCTATAGGGCGAGG-5′ -terahydrofuran ) , Life Chemicals , Enamine Ltd. , Specs Chemicals , ChemDiv Inc. , Ukrorgsynthesis Ltd  and Sigma-Aldrich.The monofunctional alkylating agent methyl methane sulphonate (MMS) was purchased from Sigma-Aldrich  and dissolved in phosphate-buffered saline. Stock solutions of test compounds were dissolved in DMSO. Temozolomide (TMZ) was a gift from Dr Tracey Bradshaw, School of Pharmacy, University of Nottingham, UK. Potential APE1 inhibitors were purchased from Maybridge Chemicals , ChemBridge Corporation , ASINEX intelligent chemistry  based on the structural features of the APE1 active site (see results and discussion). Using these templates, ROCS 2.3  . Sybyl8.0 was used to build inhibitor templates based on the previously reported APE1 inhibitor (ompounds). The 167M) (New England Biolabs) was incubated in a buffer system consisting of 50 mM Tris-HCl, pH 8.0, 1 mM MgCl2, 50 mM NaCl, 2 mM DTT at 37°C for 10 min. 5′-F-GCCCCCXGGGGACGTACGATATCCCGCTCC-3′ and its complementary Q-labelled oligonucleotide (see above) were annealed in a buffer containing 100 μM Tris-HCl, 50 mM NaCl and 1 μM EDTA. AP-site cleavage was initiated by addition of the annealed substrate (25 nM) to the reaction mix. Fluorescence readings were taken at 5 min intervals during 30 min incubation at 37°C using an Envision Multilabel reader from Perkins Elmer  with a 495 nM excitation and a 512 nM emission filter. If the DNA is cleaved at the abasic site at position 7 from the 5′-end by APE1, the 6-mer fluorescein-containing product will dissociate from its complement by thermal melting. As a result, the quenching effect of the 3′ dabcyl (which absorbs fluorescein fluorescence when in close proximity) is lost, and APE1 activity is measured indirectly as an increase in fluorescence signal . Similar assays were developed for monitoring the AP endonuclease activity of endonuclease IV using a buffering system containing 10 mM HEPES-KOH, pH 7.4, 100 mM KCl and 60 ng of endonuclease IV . The final DMSO concentration was maintained at 1.2% in all assays.A fluorescence-based AP site cleavage assay was performed as described previously with slight modifications , and 50 nμM  before initiating the AP site cleavage assay described in the previous section. Those candidates that showed >90% inhibition of APE1 activity were subjected to serial dilution experiments for IC50 calculations. In addition, screening of potential inhibitors for their specificity (at 100 μM concentration) was performed using endonuclease IV cleavage assays.APE1 was incubated with the candidate inhibitors at 100 50 for APE1 inhibition, the ability of the compounds to inhibit APE1 at a range of concentrations (10 nM–100 μM) were evaluated in black 384-well plates. The reactions were set up as before and fluorescence intensity was measured every 5 min for 30 min following reaction initiation. Using the initial rate values from the assay, percent activity was calculated for each sample relative to a negative DMSO only control. The data was fitted to a sigmoidal dose-response model using Graphpad Prism 3.0  and IC50 values were determined using the formula: % Activity=100/(1+10(log[I]−log IC 50)).To estimate ICM) was incubated with 100 μM of potential APE1 inhibitor in a buffer consisting of 50 μM Tris-HCl, pH 8.0, 1 mM MgCl2, 50 mM NaCl and 2 mM DTT at 37°C for 30 min. Fluorescence intensity was measured every 5 min. Any hits that showed a decrease of more than 50% in the fluorescence intensity were considered as quenchers and discarded from further analyses.To investigate the possibility that compounds might possess intrinsic quenching activity, fluorescence quenching assays were performed. Briefly, the oligonucleotides 5′-F-oligonucleotide (see above) and 3-CGGGGGCCCCCTGCATGCTATAGGGCGAGG-5′ were annealed as described previously. The double stranded oligonucleotide  and the radiolabelled substrate and reaction products were visualised using a phosphorImager .This basic assay was performed as described previously , incubated on ice for 30 min, and clarified by centrifugation at 12 000 × g for 15 min at 4°C , followed by denaturation of samples at 95°C for 10 min. The radiolabeled substrate and product were separated on a standard polyacrylamide-denaturing gel and quantified by phosphorimager analysis.HeLa cells – maintained in DMEM with 10% fetal bovine serum and 1% penicillin–Streptomycin – were harvested, washed with 1 × PBS, and the pellet was resuspended in cold 222 mM KCl plus protease inhibitors (0.5 mμM). Fluorescent DNA substrate was then added to a final concentration of 100, 200 and 500 nM , and enzyme activity was allowed to proceed for 30 min at 37°C. The percentage APE1 cleavage activity was plotted. Lineweaver–Burk plots and kinetic parameters (kcat and KM) were determined from eight independent data points.APE1 protein (80 ng) was incubated at room temperature for 30 min without or with APE1 inhibitor . U89 MG and SNB-19 glioma cell lines were grown in DMEM . HUVEC endothelial cells were grown in a special media (199 Media+HAMS F12), and supplemented with 20% heat-inactivated foetal calf serum, 1% Hepes, 1% Glutamine, 12.5 μg Human EGF, 625 ng of Human bFGF, 3750 units of Heparin, penicillin 0.06 g l−1, and streptomycin 0.1 g l−1) Only cultures with a plating efficiency of over 70% were used for analyses.MeWo, SKMel and MM418 melanoma cancer cell line were grown in RPMI culture medium  containing protease inhibitor  and phosphatase inhibitor cocktail 1 and 2 (Sigma). The protein content of cleared lysates was quantified using the Bradford assay. Proteins (20 μg) were separated by a 10% SDS–PAGE gel using a Tris:Glycine buffer. Following electrophoresis, proteins were transferred onto a nitrocellulose membrane and blocked by incubation with PBST  containing BSA/milk. Membranes were incubated with primary antibodies  1 : 5000 dilution) and infrared dye-labelled secondary antibodies  (IRDye 800CW Donkey Anti-Rabbit IgG (H+L)) and IRDye 680CW Donkey Anti-Mouse IgG (H+L) in the dilution of 1 : 15000 for 60 min. Protein expression was determined by scanning the membranes on Licor–Odyssey's Scanner at the predefined intensity fluorescence channel (700 and 800 nm).Protein samples were prepared by lysing SKMel30, Mewo, MM418 and U89MG cells in RIPA buffer  was added to the pellet and the cell lysate was gently passed several times through a pipette. The resultant viscous solution was centrifuged at 10 000 g for 10 min at 25°C. DNA was precipitated from the supernatant using 0.25 ml of 100% ethanol by gently inverting the tube 5–8 times at room temperature for 1–3 min. The DNA was washed twice in 0.4 ml of 75% ethanol. The DNA was then solubilized in TE buffer (pH 8.0), and the final concentration was adjusted to 100 μg ml−1 (using a Gene Quant pro spectrophotometer). AP-site determinations were performed on the genomic DNA using an aldehyde reactive probe assay kit using the protocol provided by the manufacturer . Untreated cells were compared with cells exposed to either MMS alone, APE1 inhibitor alone or combination of MMS and APE1 inhibitor. DNA was extracted at 90 min and AP site quantified as described previously. All experiments were performed in triplicate.AP sites were quantified as described previously (μl of medium) were seeded into a 96-well plate. For HUVEC cells, 5 μl of 2% type 2 gelatine (Sigma) was added to the wells and the plates were preincubated for 20 min at 37°C before seeding of cells. For intrinsic cytotoxicity assessments, cells were incubated with varying concentrations of APE1 inhibitors and the MTS assay was performed on day 5. For potentiation experiments, cells were preincubated with a relatively nontoxic concentration of APE1 inhibitor for 24 h and then exposed to MMS, temozolomide or doxorubicin. Non-radioactive cell proliferation assay was conducted as described previously.To evaluate intrinsic cytotoxicity and to evaluate the potentiation of toxicity of cytotoxic agents by APE1 inhibitors, MTS assays were performed as per the manufacturer's recommendation . Briefly, 2000 cells per well  (ompounds). A totalompounds). The top50 for APE1 inhibition ranged between 30 nm to 50 μm. This report presents in silico, biochemical and cytotoxicity analyses of seven representative compounds. 5-Fluoro-1H-indole-2-carboxylic acid (compound 1) was originally identified using the ‘CRT0044876 (C)’ template. N-(3-benzooxazol-2-yl-4-hydroxy-phenyl)-2-(2-naphthyloxy)acetamide (compound 2), (3-(2-naphthyl)-5-phenyl-2,5-dihydropyrazol-1-yl]carbonylmethyl 5-nitrothiophene-2-carboxylate (compound 3), N-(4-fluorophenyl)-2-[4-phenylsulfonyl-2-(p-tolyl)oxazol-5-yl) sulfanyl-acetamide (compound 4) and N-dioxol-5-ylmethyl)-4-(2-oxo-4-(thiazol-2-ylcarbamoylmethylsulfanyl)-9-thia-3,5-diazabicyclo(4.3.0) nona-4,7,10-trien-3-yl)-butanamide (compound 5) was identified through the M3 template. 2-(1H-benzoimidazol-2-ylsulfanyl)-N-methyleneamino) acetamide 1,3-bisacetone (compound 6) was identified through the M2 template and 3-benzofuran-2-yl-2-benzothiazol-2-yl-3-oxo-propanenitrile (compound 7) was identified through M1 template. The chemical structures, consensus scores and biochemical profiles are summarised in 50=11 μM).Compounds were tested in the fluorescence APE1 cleavage assay . A totalE.coli orthologue of APE1 that performs AP site cleavage in a way similar to APE1 but has a structurally and mechanistically different active site  compared with the wild type (11 μM). The preferential inhibitory activity of compound 4 towards the D148E protein was also confirmed in radiolabelled oligonucleotide assays (data not shown). We were not able to demonstrate preferential inhibitory activity of other compounds either in fluorescence or radiolabelled assays.The D148E polymorphic variant of APE1 has been implicated in cancer predisposition including melanoma , consistM and kcat decreased at each inhibitor concentration (compared with no inhibitor) and the kcat/KM decreased at increasing inhibitor concentration. The data is consistent with uncompetitive inhibition.To evaluate potential mechanism of action of APE1 inhibitor, kinetic analysis was performed . As compM in the presence of the compound, though this is unlikely.However, we cannot exclude the possibility that compound 4 operates as a weak uncompetitive inhibitor (meaning it binds the protein–DNA substrate complex), as we observed a reproducibly lower Kin vivo. As AP sites are obligatory intermediates during the repair of MMS-induced base damage, accumulation of AP sites were also demonstrated in cells exposed to MMS alone. Moreover, AP-site accumulation in cells exposed to a combination of APE1 inhibitor and MMS was more than the cells exposed to either agent alone. Similar accumulation of AP sites was also demonstrated in melanoma cells.In order to test the biological activity of APE1 inhibitors under physiological conditions, analysis was then undertaken in melanoma cell lines  and glioma cell lines (U89MG and SNB-19). We initially tested if these cell lines expressed APE1 protein. Robust APE1 expression was seen in these cell lines using western blot analyses  and 7A. 50>300 μM), the GI50 (cell growth inhibition) ranged between 1 and 50 μM for the other APE1 inhibitors: compound 2=50 μM, compound 3=1 μM, compound 4=17 μM (glioma cell lines) and 20 μM (melanoma cell lines), compound 5=14 μM, compound 6=50 μM and compound 7=40 μM. We investigated if at relatively non-toxic concentrations, APE1 inhibitors would potentiate the cytotoxicity of monofunctional alkylating agents MMS. APE1 inhibitors were then tested for their inherent toxicity in melanoma and glioma cell lines. Whereas compound 1 was non-toxic  and glioma cell line (U89MG) . SimilarIn order to investigate whether APE1 inhibitor was toxic to non-cancer cells, we conducted toxicity analysis in HUVEC endothelial cells. The overall prognosis of advanced melanoma and glioma remains poor and strategies to improve tumour response to chemotherapy remain a high priority. Blocking DNA repair may enhance cell kill in cancer and improve outcomes . APE1, ain silico  and a total of 1679 virtual hits with similarities to the templates were identified . Detailed biochemical screening showed that majority of the compounds conform to the M3 template, which bears an additional heteroaromatic sidechain that can access a subsidiary cleft in one branch of the ligand-binding groove  and decreased the kcat/KM implying uncompetitive inhibition. Future cocrytallization experiments in the presence of DNA are likely to provide further information regarding the exact mechanism of action of this compound. To assess potency and specificity of our compounds, we screened their ability to block AP-site cleavage activity using WCE. This is a good system to screen for compounds that may have non-specific binding to other cellular proteins. Compound 4 exhibited more than 90% inhibition in the WCE assays, implying strong potency and specificity. Although compound 3 blocked APE1-directed AP-site cleavage activity in purified APE1-based assay, it had no effect in the WCE assay. This implies that the compound has ‘off target’ non-specific protein-binding effect and suggests that it is unlikely to be a good development candidate.In this study, we also provide evidence for the first time that certain APE1 inhibitors may be more effective in blocking the endonuclease activity of the D148E polymorph (a common polymorph associated with cancer predisposition) compared with the wild type. The inability of six of the seven compounds examined to inhibit the activity of endonuclease IV provides presumptive evidence that the compounds indeed act by interaction with APE1 rather than by obscuring the abasic site on the DNA substrate. Moreover, the kinetics analysis has provided insight into the mechanism of action of the inhibitor. We have shown that compound 4 decreased Kin vivo in cells exposed to inhibitor, providing direct evidence of target inhibition in vivo. Intrinsic cytotoxicity for several of the inhibitors was demonstrated in glioma and melanoma cell lines, a finding consistent with the observation that APE1 downregulation in melanoma cell lines promotes apoptosis, although non-specific toxicity at higher doses of the compound cannot be excluded in our study (In order to provide further preclinical evidence that blocking the repair domain of APE1 is a potential treatment strategy, we conducted studies in glioma and melanoma cell lines. We confirmed APE1 expression in these cancer cell lines. We then confirmed accumulation of AP sites ur study , suggestFollowing our initial report, other investigators have identified various APE1 inhibitors for potential pharmaceutical application (


In conclusion, bortezomib induces autophagy in HCC through a CIP2A-PP2A-Akt-4EBP1 pathway.Previously, we reported that cancerous inhibitor of protein phosphatase 2A (CIP2A) mediates the apoptotic effect of bortezomib in hepatocellular carcinoma (HCC). Here, we report a proteasome-independent mechanism by which bortezomib induces autophagy in HCC. Our data indicate that bortezomib activated autophagy in a dose- and time- dependent manner in HCC cell lines including Huh-7, Sk-Hep1, and Hep3B. Bortezomib downregulated CIP2A, phospho-Akt (P-Akt) and phospho-4EBP1 (P-4EBP1) in a dose- and time-dependent manner in all tested HCC cells. Ectopic expression of CIP2A abolished the effect of bortezomib on autophagy. Co-treatment of bortezomib and calyculin A, a PP2A inhibitor, reduced the effect of bortezomib on P-Akt, P-4EBP1, and autophagy. Increased phosphorylation of either Akt or 4EBP1 by ectopic overexpression protected cells from bortezomib-induced autophagy. Furthermore, we examined the effect of ΔBtz, a bortezomib derivative that closely resembles bortezomib structurally but has no proteasome activity, in HCC. Interestingly, ΔBtz demonstrated similar effects to bortezomib on autophagy, CIP2A, P-Akt and P-4EBP1, suggesting that the effect of bortezomib on autophagy is independent of proteasome inhibition. Moreover, our  Hepatocellular carcinoma (HCC) is the fifth most common solid tumor worldwide Bortezomib is the first in-class dipeptide boronate proteasome inhibitor specifically designated to target the 26S proteasome in vivo tumor formation It is well known that mammalian target of rapamycin (mTOR) is a key regulator of cell growth and autophagy In this study, we report a proteasome-independent mechanism by which bortezomib induces autophagy in HCC. Our present work suggests that bortezomib induces autophagy in HCC through a CIP2A-PP2A-Akt-4EBP1 pathway.in vitro studies, bortezomib at various concentrations was dissolved in DMSO and then added to cells in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum (FBS). The final DMSO concentration was 0.1% after addition to medium. Calyculin A and 3-Methyladenine (3-MA) were purchased from Cayman Chemical . Antibodies for immunoblotting such as anti-Akt1, were purchased from Santa Cruz Biotechnology . Other antibodies such as anti-LC3, -P-Akt (Ser473), -4EBP1, -P-4EBP1, -P-mTOR, -mTOR, -P-S6K, -S6K, anti-S6, -P-S6, -NF-κB, -IκB-α, -ATG3, -ATG5, -ATG7, and -Beclin 1 were from Cell Signaling .Bortezomib (Velcade®) was kindly provided by Millennium Pharmaceuticals. For 2 in air. western blot analysis was performed as previously reported.The Sk-Hep1 and Hep3B cell lines were obtained from American Type Culture Collection . The Huh-7 HCC cell line was obtained from the Health Science Research Resources Bank . Cells were maintained in DMEM supplemented with 10% FBS, 100 units/mL, penicillin G, 100 µg/mL streptomycin sulfate, and 25 µg/mL amphotericin B in a 37°C humidified incubator and an atmosphere of 5% CO3-II. Briefly, Huh7 and Hep3B were seeded in a 6-cm dish. After being washed with PBS, cells were treated with 800 nM bortezomib for 16 and 24 hours, fixed with ice-cold 4% paraformaldehyde, and then treated with ice-cold 100% methanol. The fixed cells were subsequently incubated with the primary antibody rabbit anti-LC3II  in blocking solution  for 1 hour at room temperature, and then stained with anti-rabbit IgG (H+L), F(ab′)2 Fragment .Drug-induced autophagy was assessed by several assays including: (1) Western blot analysis of microtubule-associated protein 1 light chain 3 (LC3-II) as described previously; (2) Immunofluorescence of LC3-II. Briefly, Huh7 cells were seeded in a 6-cm dish. After being washed with PBS, cells were treated with 800 nM bortezomib for 16 hours, and fixed with ice-cold 4% paraformaldehyde. The fixed cells were subsequently incubated with the primary antibody rabbit anti-LC3II , in blocking solution  for 1 hour at room temperature and then stained with anti-rabbit IgG (H+L), F(ab′)2 Fragment  and DAPI. Cells were examined under a LEICA DM2500 fluorescence microscope; (3) Flow cytometry analysis of LCHuh7 cells, grown on coverslips, were transfected with EGFP-LC3 plasmid, followed by 400 nM bortezomib treatment for 24 hours. The cells were then rapidly washed with PBS and fixed at room temperature for 15 minutes with 4% paraformaldehyde. After being washed with PBS twice, the cells were blocked with 1% bovine serum albumin in TTBS and then mounted. The subcellular distribution of EGFP-LC3 was observed under a fluorescence microscope (LEICA DM2500). For acridine orange staining, SK Hep1 cells were seeded in a 6-cm dish. After being washed with PBS, cells were treated with 800 nM bortezomib for 16 hours, fixed with ice-cold 4% paraformaldehyde for 30 minutes at room temperature then stained with acridine orange (5 µg/ml) for 5 minutes at room temperature. Cells were examined under a LEICA DM2500 fluorescence microscope.CIP2A cDNA (KIAA1524) was purchased from Origene . Briefly, following transfection, cells were incubated in the presence of G418 (0.78 mg/mL). After 8 weeks of selection, surviving colonies, i.e., those arising from stably transfected cells, were selected and individually amplified. HCC cells with stable expression of CIP2A-myc were then treated with drugs, harvested, and processed for western blot analysis.ad libitum. All experimental procedures using these mice were performed in accordance with protocols approved by the Institutional Laboratory Animal Care and Use Committee of National Taiwan University (Permit Number: 20080205). Each mouse was inoculated s.c. in the dorsal flank with 1×106 Huh-7 cells suspended in 0.1 ml of serum-free medium containing 50% Matrigel . When tumors reached 200–300 mm3, mice received an intraperitoneal injection of bortezomib (1 mg/kg body weight) or ΔBtz (1 mg/kg body weight) twice weekly for the duration of treatment. Controls received vehicle.Male NCr athymic nude mice (5–7 weeks of age) were obtained from the National Laboratory Animal Center . The mice were housed in groups and maintained under standard laboratory conditions on a 12-h light-dark cycle. They were given access to sterilized food and water t test in SPSS for Windows 11.5 software .Tumor growth data points are reported as mean tumor volume ± SE. Comparisons of mean values were performed using the independent samples left, bortezomib increased the levels of LC3-II significantly after bortezomib treatment of 24 hours. Moreover, bortezomib-induced autophagy was also confirmed by LC3-II immunofluoresence staining (right). The stimulatory effect of bortezomib on autophagy was confirmed by an increase in the GFP-LC3 dots in the cells treated with the drug, as examined by GFP-LC3 assay (top), and also by an increase in acridine orange staining for acidic vesicular organelles (bottom). These data indicate that bortezomib significantly activates autophagy in HCC.To investigate the effect of bortezomib on autophagy in HCC, we first examined the expression of microtubule-associated protein 1 light chain 3 (LC3-II), a LC3-phosphatidyl-ethanolamine conjugate, that is a hallmark of autophagy. As shown in staining right. TC3 assay top, andganelles bottom. Our previous work identified that inhibition of CIP2A is the major determinant of bortezomib-induced apoptosis in HCC cells. To further explore the mechanism by which bortezomib induced autophagy in HCC, we next examined whether CIP2A plays a role in bortezomib-induced autophagy in HCC. As shown in left). overexpression of Akt1 abolished bortezomib-induced autophagy significantly (middle). Ectopic expression of 4EBP1 also reduced the effect of bortezomib on autophagy in Huh-7. These data indicate that the CIP2A-PP2A-Akt-4EBP1 signaling pathway plays a key role in mediating bortezomib's effect on autophagy in HCC. Furthermore, we examined the effect of a combination of bortezomib and rapamycin, a mTOR inhibitor, on autophagy. Our data showed co-treatment with rapamycin and bortezomib increased autophagy significantly (left). Furthermore, 3-MA, an autophagy inhibitor, reversed the autophagic effect of bortezomib in Huh-7 cells (right). Notably, we have performed all the experiments above . Compared with bortezomib, ΔBtz had little effect on proteasome inhibition (middle) or on NF-κB inhibition (right). NF-κB is a known proteasome inhibitor target. Interestingly, ΔBtz showed significant effects on CIP2A, P-Akt, and P-4EBP1 in a concentration- and time-dependent manner in HCC similar to bortezomib (left & middle). Co-treatment with the autophagy inhibitor 3-MA reduced the autophagic effect of ΔBtz (right). ΔBtz increased the expression of LC3-II (left), and also increased acridine orange staining for acidic vesicular organelles (middle). Using an electron microscopy technique, the numbers of autophagic vacuoles were shown to be markedly increased in Sk-Hep1 cells treated with 800 nM bortezomib or ΔBtz, but not in the control (right). Moreover, we examined the effects of other proteasome inhibitors MG132 and lactacystin, on autophagy and CIP2A. Our data showed that neither MG132 nor lactacystin had significant effects on autophagy or CIP2A (left). However, as expected, these two proteasome inhibitors exhibited significant proteasome inhibition activity (right). These data indicate that the effect of bortezomib on autophagy is independent of the proteasome.To examine whether the effect of bortezomib on autophagy and CIP2A is related to its proteasome inhibition activity, we synthesized a bortezomib derivative (ΔBtz), which is similar to bortezomib structurally except that it lacks a boronic acid functional group, a critical contributor to proteasome activity left. Cohibition middle ohibition right. N of ΔBtz right. Δf LC3-II left, anganelles middle.  control right. Mor CIP2A left. Hoactivity right. Tin vivo effect of bortezomib on HCC xenograft tumors. Tumor-bearing mice were treated with vehicle or bortezomib or ΔBtz. Both drugs were injected intraperitoneally at 1.0 mg/kg twice a week for 2 weeks. All animals tolerated the treatments well without observable signs of toxicity and had stable body weights throughout the course of the study. No gross pathologic abnormalities were noted at necropsy. Our data indicated that bortezomib and ΔBtz showed similar effects on Huh-7 tumor growth. The size of tumor was reduced significantly after two weeks of treatment. To correlate biological response with the mechanism of action identified in vitro, the effect of bortezomib on autophagy in HCC tumors was examined. As shown in in vivo and, importantly, suggest that bortezomib induces autophagy in HCC via a proteasome-independent mechanism.To confirm whether the effect of bortezomib on autophagy has potentially relevant clinical implications, we assessed the left) despite showing significant proteasome inhibition activity (right). These data clearly indicate that the effect of bortezomib on autophagy can be independent of proteasome inhibition. Notably, the response of each cell line to the induction of LC3-I/LC3II by bortezomib was different , and increases understanding of oncogenic proteins such as Ras, Akt and ERK that regulate autophagy activity right. Tifferent  and was ifferent . One posThe proteasome and the autophagy-lysosome pathways are considered the two main routes for eukaryotic intracellular protein clearance and their linkage and interactions have been recent subjects of interest CIP2A gene by DNA methylation or micro-RNA machinery, or affecting other as yet unknown molecules that may regulate CIP2A expression.In addition to the finding that oncogenic CIP2A mediates bortezomib induced autophagy, this study identified p-4EBP-1 as a participant in bortezomib induced autophagy downstream of p-Akt. The role of p-4EBP1 was validated by ectopic expression of 4EBP1, which resulted in an increase in p-4EBP1, and subsequently reduced the effect of bortezomib on autophagy . 4EBP-1 In conclusion, bortezomib induces autophagy in HCC cells through a novel proteasome-independent mechanism: CIP2A-dependent p-4EBP-1 downregulation. This study identifies the novel oncoprotein CIP2A as a major mediator of HCC cells to bortezomib-induced autophagy and suggests that CIP2A may be a potential new drug target in HCC. Furthermore, discovery of compounds/drugs acting as CIP2A inhibitors may have therapeutic potential in HCC therapy.


In prior studies, we explored the role of the cGMP-dependent protein kinase I alpha (PKGIα) in regulating cardiac remodeling by studying mice with mutations in the PKGIα leucine zipper (LZ) domain , in whicJNK activation (phosphorylation) increased from 1.0 ± 0.3 ADUs in WT sham hearts to 3.5 ± 0.6 ADUs in WT 48 hour TAC hearts (p<0.001). However, JNK phosphorylation did not increase significantly in LZM hearts post TAC . Similar trends in JNK phosphorylation were observed at 24 hours post-TAC and 7 days post-TAC. Loss of TAC-induced JNK phosphorylation in LZM mice was accompanied by decreased TAC-induced activation of MAP Kinase Kinase 4 (MKK4) compared with WT controls at 48 hours post-TAC.We next explored upstream JNK regulators as potential novel PKGIα interacting molecules. In GST pulldown experiments with cardiac lysates from WT or LZM mice, the MAPKKK Mixed Lineage Kinase 3 (MLK3) bound to the WT PKGIα LZ domain, but not the mutant PKGIα LZ domain, supporting complex formation between PKGIα LZ and MLK3. Immunoprecipitation studies with WT cardiac lysates confirmed an interaction between these two signaling molecules. We next tested whether MLK3 is required for cGMP-PKGI-dependent JNK activation in cultured adult mouse cardiac myocytes (CM) following siRNA knockdown of MLK3. MLK3 knockdown blocked 8-Bromo-cGMP-stimulated JNK activation, while control siRNA had no effect on 8-Bromo-cGMP-stimulated JNK activation.These findings support that the cGMP pathway recruits the JNK signaling pathway by complex formation between PKGIα and MLK3, leading to activation of MKK4 and JNK in the heart. These findings define a novel mechanism by which cGMP and PKGIα inhibit pathological remodeling in vivo, and identify MLK3 as a potentially important therapeutic target in heart failure.