Genetic analyses had been performed utilizing the S.A.G.E. (Statistical Examination for Genetic Epidemiology) software package, variation five.3 (http://darwin.cwru.edu/sage/). Allele frequencies have been believed separately in the 4 ethnic groups employing the greatest probability technique implemented in the program FREQ. Mendelian inconsistencies had been recognized with the MARKERINFO software and inconsistent genotypes were coded as lacking. Glitches in connection specification ended up identified with the plan RELTEST. When required, a next romantic relationship testing program, RELPAIR model two..1, was enlisted to solve potential glitches involving sophisticated relationships. Multipoint identification by descent (IBD) allele sharing chances had been approximated by the technique of maximum likelihood, employing all available information in the pedigree as applied in the plan GENIBD. Multipoint IBD-sharing estimates are sturdy to misspecification of populace allele frequencies, as may possibly occur with admixed samples, due to the fact most of the parental details is inferred when the accessible info is substantial [27]. The Shannon info, as calculated by Merlin [28], accessible from the Illumina IV SNP panel was in no way considerably less than .7, and rarely much less than .8, other than at the telomeric locations (knowledge not demonstrated). Using the multipoint IBD sharing estimates, a genome-broad linkage scan for quantitative trait loci potentially influencing eGFR was carried out by the Haseman-Elston regression technique applied inside of the plan SIBPAL, employing the W4 weighting alternative to maximize electrical power. We converted the p values described by SIBPAL to LOD scores employing the one-sided chi-squared distribution with 1 diploma of freedom (i.e., a fifty:fifty mixture of distributions with and 1 levels of liberty), proper for a one particular-sided test. In principle, the sib pairs who are identical by descent (IBD) at a marker locus will be phenotypically equivalent for attributes affected by a close by joined gene. Evidence for linkage of eGFR was assessed with and with out incorporating covariate outcomes of diabetes length and physique mass index (BMI), entered in the regression product as the sibpair sum. Non-parametric multipoint linkage investigation was carried out independently in each and every ethnic group, and P values had been mixed across ethnicities in accordance to Fisher’s approach [29]. Table two. Scientific characteristics of the genotyped men and women.
Empirical P values have been attained for the significant linkage peaks utilizing the “simulation” selection in SIBPAL, which performs a permutation test. Association investigation was conducted as explained formerly [26] making use of the linear mixed design technique carried out in the S.A.G.E. program ASSOC. Final results were merged across ethnic groups using Fisher’s technique [26,29]. The SNPs employed in this investigation have been previously reported [26]. To assess the sensitivity of the affiliation examination to genetic admixture, the linear mixed model was equipped with and without adjustment for the initial two principal parts from a principal elements investigation employing five,547 SNPs from the Illumina IV panel with minimal allele frequencies of at the very least .05 in the blended sample. Principal factors ended up obtained through the smartpca system in EIGENSOFT [30].Many good quality handle measures ended up applied to figure out the closing established of markers for the linkage analysis. Briefly, SNPs ended up essential to have median GenCall scores (a measure of how close a genotype is to the centre of the cluster of other samples assigned to the very same genotypes) $.five, MAF (certain to ethnic group) $.05, and p worth for deviation from Hardy-Weinberg proportions ..001. Given that, LD between neighboring SNPs could create bias in estimates of IBD sharing amid kin, markers were screened these kinds of that pairwise | D9 | was considerably less than .three. After quality manage, a final marker set of SNPs qualifying for more genetic evaluation was determined as explained previously [24]. Table 1 lists the ethnicities of the three,960 subjects comprising three,547 sib pairs and 442 fifty percent-sib pairs from 4 ethnic groups in whom eGFR and genotypic information have been offered. Of these, forty.7%, 24.1%, 19.7%, and 15.5% ended up MA, AA, AI, and EA, respectively. Desk two shows the clinical attributes of genotyped individuals from each ethnic group.
Adjusting for the covariate outcomes of diabetes period and BMI, the genome-broad linkage scan in population-combined information recognized the strongest evidence for linkage of eGFR on chromosome 10p12.31 (P = 5.561024) at forty four cM near rs1339048 (Figures 1, 2a and Desk three). Evidence for linkage was mostly contributed by the AA and EA teams, with a smaller contribution from MA.
Focus Forming Unit (FFU) Reduction Assay
FFU reduction assays were performed as previously described [14]. Approximately 200 FFU of virus were incubated with peptide in serum-free DMEM for 1 hr at room temperature before infecting LLC-MK2 cell monolayers for 1 hr at 37uC, and overlaying with media containing 0.85% (w/v) Sea-Plaque Agarose (Cambrex Bio Science, Rockland, ME). Infected cells were incubated at 37uC with 5% CO2 for 2 days (yellow fever virus), 3 days (dengue virus 3 and 4, Russian spring summer encephalitis virus and Central European encephalitis virus) or 5 days (dengue virus 1and 2). Infected cultures were fixed with 10% (v/v) formalin, permeablized with 70% (v/v) ethanol, and foci were detected using mouse monoclonal antibodies against yellow fever virus (Chemicon, Temecula, CA), dengue (E60), or polyclonal anti-Kumlinge virus rED3 antisera, followed by horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (Pierce, Rockford, IL), and developed using AEC chromogen substrate (Dako, Carpinteria, CA) as previously described [15,29].
Virus Inhibition on C6/36 Cells
C6/36 monolayers were infected with approximately 7,600 FFU of dengue virus 2 at 37uC for 1 hr before being aspirated, complete culture media added, and incubated at 37uC and 5% CO2. After 72 hrs, RNA was isolated from cells using an RNeasy Mini Kit (Qiagen, Valencia, CA). qRT-PCR was performed as previously described [18].Materials and Methods Viruses and Cells
Dengue virus 1 (HI-1), dengue virus 2 (NGC-2), dengue virus 3 (H-78), dengue virus 4 (H-42), and yellow fever virus (17-D) were propagated in LLC-MK2 cells (American Type Culture Collection (ATCC), Manassas, VA, cat. no. CCL-7) [15]. Russian spring summer encephalitis virus (Sofjin), and Central European encephalitis virus (Hypr) were propagated in BHK-21 cells (ATCC, cat. no. CCL-10). C6/36 cells (ATCC, cat. no. CRL1660) were maintained in Dulbecco’s modified eagle medium (DMEM) with 10% fetal bovine serum (FBS), 100 mM Nonessential amino acids, 2 mM Glutamax, 100 U/ml penicillin G, 100 mg/ml streptomycin and 0.25 mg/ml amphotericin B, at 30uC with 5% CO2. For the cryo-electron microscopy studies, dengue virus 2 (16681) was grown in C6/36 cells and the tissue culture supernatant was collected on day 3-4, spun at 2,7046g for 10 minutes at 4uC. 8% PEG in NTE (120 mM NaCl, 12 mM Tris, pH 8.0, 1 mM EDTA) was added to the tissue culture supernatant and mixed. The solution was then allowed to sit overnight before the PEG precipitated virus was centrifuged at 14,6366g for 1 hr. The pellet was resuspended in 1 ml NTE buffer, loaded onto a 24% (w/v) sucrose cushion and centrifuged at 175,5876g for 90 min. Pellets were resuspended overnight in NTE before being loaded onto a 10-30% (w/v) potassium sodium tartrate step gradient and centrifuged at 175,5876g for 2 hrs. Purified virus was collected from the 20% potassium-tartrate fraction.
Cryo-electron Microscopy
1 mM DN59 in 10% (v/v) DMSO was mixed with 18 ml of mature dengue virus to give a final DN59 concentration of 100 mM with 1% (v/v) DMSO. The mixture was incubated at 37uC for 30 min, then 4uC for 2 hrs and frozen on holey carbon grids. Dengue virus without peptide and dengue virus incubated with DMSO only controls were also frozen. Images were collected with a Philips CM200 cryo-electron microscope using 200 KV, a magnification of 50,000, an electron dose of 25 e2/A2, and taken at about 4.3 to 7 mm out-of-focus. Thirty-eight DN59 treated dengue virus particles were selected for three-dimensional (3D) image reconstruction. Initial models for 3D reconstructions were generated using the program starticos in EMAN [30]. This program correlates each image with itself after rotating by 72u, 120u and the starting model is essentially a random model based on combining the three orientations related by icosahedral symmetry. Subsequently, thirty iterations were performed in which the orientation of each of the raw images was determined relative to the current model from the previous cycle using the program SPIDER [31]. The images were split into two groups for resolution estimation, by observing the point at which the Fourier shell coefficient fell below 0.5 [32].(Figure S3). Contours were chosen to only just avoid opening a hole in the capsid other than at the five-fold vertices.RNase Assay and qRT-PCRApproximately 1.4-2.96104 FFU of dengue virus 2 was incubated with DN59 for 1 hr at room temperature and then digested with micrococcal nuclease (New England BioLabs, Ipswich, MA) for 1 hr at 37uC.PBS, with 15 minutes of equilibration after each titration before measurements were made. Binding curves were obtained by taking the intensity at 335 nm for each spectra, minus the intensity of the appropriate peptide-free control sample.
Liposome Vesicle Leakage
The fluorescent dye 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and its obligate quencher p-xylene-bis-pyridinium bromide (DPX) were purchased from Invitrogen (Carlsbad, CA). Vesicles were prepared with ANTS/DPX entrapped inside where DPX quenches ANTS fluorescence [34]. Lipids were hydrated with buffer containing 50 mM ANTS and 12.5 mM DPX followed by extrusion and then gel filtration chromatography using Sephadex G-200 to exchange the external ANTS/DPX solution for buffer. In leakage experiments, 0.5 mM vesicles were mixed with peptide from 0.5 to 10 mM to give peptide to lipid ratios ranging from 1:50 to 1:1000. The increase in ANTS fluorescence after 1 hr incubation with peptide reports on vesicle leakage. A complete leakage control was achieved by the addition of 10 mM of the lytic bee venom peptide melittin.
Infectivity Inhibition Reversibility Assay
Similar to the FFU reduction assays, approximately 200 FFU of dengue virus 2 were incubated with 0 or 10 mM DN59 in a total volume of 100 ml serum-free DMEM for 1 hr at room temperature. Immediately before infecting LLC-MK2 cell monolayers, the virus/peptide mixtures were diluted with serum-free DMEM to 1 ml, reducing the concentration of DN59 to 1 mM.
Cell Toxicity Assays
Cytotoxicity of DN59 was measured by mitochondrial reductase activity using the TACSTM MTT cell proliferation assay (R&D Systems Inc., Minneapolis. MN). DN59 in serum-free DMEM was added to LLC-MK2, BHK, or C6/36 cells for 1 hr at 37uC, the solution was removed and the cells incubated at 37uC in complete medium with 5% CO2 for 24 hrs.
Results and Discussion Identification of WIKI as a Small Molecule Inhibitor of Wnt/?catenin Signaling
To make an assay for Wnt/?catenin signaling suitable for high throughput screening, we generated A375 melanoma cells stably infected with a ?catenin-activated luciferase reporter (BAR) [23,36] and selected populations in which luciferase activity is increased at least 4,000-fold by WNT3A. We tested the robustness of our assay by calculating the Z-factor (Z9) values [40] using probes that are known to enhance (U0126 [41], Riluzole [42], and GSK3B inhibitor IX [43]) or inhibit (XAV-939 [33]) Wnt/�catenin signaling (Figure S1A). For all control probes, we found the Z9 values to be greater than .45 (Figure S1A), a value considered robust in high throughput screening assays [40]. Following validation of our assay, we then screened A375 melanoma cells at two concentrations of a small molecule library in the presence of a twenty percent effective concentration (EC20) dose of WNT3A. We focused on small molecules that reduced expression of the luciferase reporter at a low dose (330 nM) and that did not kill cells at a high dose (10 mM) relative to controls treated with dimethyl sulfoxide (DMSO), with the expectation that these criteria would filter out compounds that inhibited BAR due to cellular toxicity. Five compounds met our criteria for further study by significantly decreasing Wnt/?catenin signaling without causing toxicity at either dose (Fig. 1A).
We next asked whether any of the five compounds preferentially modulated Wnt/?catenin signaling by comparing the repression of BAR in A375 cells relative to luciferase reporters for the Nuclear Factor Kappa B (NF-kB), Transforming Growth Factor Beta (TGF?, and Retinoic Acid (RA) signaling pathways (Fig. 1B). Of the five candidate Wnt/?catenin inhibitors that we tested, WIKI4 (left panel, Fig. 1C) was the only inhibitor of BAR that did not also inhibit the reporters for NF-kB, TGF? and RA (Fig. 1B). Furthermore, WIKI4 has demonstrated activity in one of nine published assays (http://pubchem.ncbi.nlm.nih.gov/summary/ summary.cgi?cid = 2984337), supporting our contention that WIKI4 is not a general inhibitor of activity in high throughput screening assays. We then demonstrated that WIKI4 inhibits Wnt/?catenin signaling in several other cell lines, including DLD1 colorectal cancer cells (Fig. 1D), NALM6 B cells (Figure S1B), U2OS osteosarcoma cells (Figure S1B) and hESCs (Figure S1C). In all cell types tested, we observed that WIKI4 potently inhibited Wnt/?catenin signaling and that its half-maximal response dose was ,75 nM. We next investigated whether WIKI4 is sufficient to inhibit expression of Wnt/?catenin target genes in DLD1 colorectal carcinoma cells, which express a truncated form of the Wnt/atenin inhibitor APC [44]. We found that incubation of DLD1 cells overnight with either WIKI4 or the structurally distinct TNKS inhibitor, XAV-939 (right panel, Fig. 1C) [33], resulted in decreased steady-state abundance of AXIN2, and TNFRSF19 (Fig. 1E), which is consistent with WIKI4 acting as an inhibitor of Wnt/?catenin signaling. Furthermore, we observed that WIKI4 is sufficient to inhibit WNT3A-dependent increases in the expression of AXIN2 and TNFRSF19 in hESCs (Figure S1D, S1E). Thus we have identified WIKI4 as a new inhibitor of Wnt/ ?catenin signaling that regulates the pathway in several cell types. To determine which chemical groups in WIKI4 are required for its ability to inhibit Wnt/?catenin signaling, we next performed a structure activity relationship analysis (Figure S2). WIKI4 has a molecular weight of 522 and a calculated partition coefficient of 4.8, putting it near the limits of “druglikeness” by Lipinski’s Rule of Five [45]. WIKI4’s mass and complexity is greater than XAV939 (Fig. 1C), and identification of small active WIKI4 analogs could provide more opportunities for modification while maintaining its druglike properties. To identify less complex WIKI4 analogs and to determine which portions of WIKI4 are required for activity, we searched for commercially available analogs. We queried the ZINC [46] and eMolecule (www.emolecules.com) databases and identified 62 WIKI4 analogs for further testing (Table S3). We assayed the Wnt/?catenin inhibitory activity of a subset of these compounds (Figure S2). Our results indicate that the traizole’s 4-pyridyl and 4-methoxyphenyl groups tolerate some modification, but the latter group could not be removed (Figure S2A). Additionally, substitution of the 1,8-naphthalimide group with a phthalimade group eliminated activity as did replacement of the 1,8-naphthalimide group with a methyl or phenyl group (Figure S2B).WIKI4 Inhibits the Cellular Responses to Wnt/?catenin Signaling
We next asked whether cells treated with an effective dose of WIKI4 would show a reduction in Wnt/?catenin-mediated responses at the cellular level. As DLD1 colorectal cancer cells require ?catenin signaling for growth in limiting culture experiments [47], these cells provide an excellent functional model of the pathway in which to test small molecules. We found that WIKI4 inhibits growth of DLD1 cells relative to DMSO controls in media containing low serum (Fig. 2A).Figure 2. WIKI4 inhibits the functional outcomes of Wnt/?catenin signaling. (A) WIKI4 inhibits colony formation of DLD1 colorectal cancer cells. DLD1 cells were plated individually in 0.5% serum containing medium, and treated with the indicated concentrations of WIKI4 and XAV-939. This experiment is representative of three independent experiments and the error bars represent standard deviation of three technical replicates. (BF) WIKI4 prevents Wnt3A-dependent differentiation of H1 human embryonic stem cells (hESCs). (B) Culturing hESCs for six days with Wnt3A causes marked morphological changes that are rescued by treatment with WIKI4. Scalebar = 500 mm. expression of markers of undifferentiated hESCs following Wnt3A stimulus. hESCs were stimulated with the indicated treatments and expression of GCTM2 and CD9 was assessed by flow cytometry following six days of treatment. (D-F) The effect of WIKI4 treatment on the expression of genes that are altered during Wnt3A-dependent differentiation of hESCs was assessed by qPCR. hESCs were treated for the indicated conditions for six days, and then analyzed by qPCR for markers of undifferentiated stem cells (NANOG, POU5F1) (D), endoderm (SOX17, GATA6) (E), and mesoderm (T, KDR) (F). The data was normalized to 100,000 copies of GAPDH and plotted as a ratio to the untreated hESCs (cultured in KSR media). The data in the experiments presented in B-F are representative of three independent experiments and the error represents standard deviation of technical replicates. In B-F, LCM = control L cell CM, WNT3A = Wnt3a CM; both 50% (vol/vol) in KSR medium. that WIKI4 inhibits a known cellular response to Wnt/?catenin signaling. Given that cellular responses to Wnt/?catenin signaling are diverse and context-dependent, we next examined the effects of WIKI4 on hESCs. Activation of Wnt/?catenin signaling in hESCs alters their cell fate and causes them to differentiate into early mesoderm and endoderm lineage cells [16,17].
The Huh-seven AKT3 stages are not stunning thinking about the endogenous expression of miR-122 in these cells
Subsequent we searched bioinformatic prediction algorithms this sort of as miRanda, TargetMiner, DIANA-MicroT, UPennrna22, and miRDB for predicted targets of this miRNA. AKT3 was identified as a single of the candidate targets for hsa-miR-122-5p. Making use of a diverse bioinformatic algorithm, Tsai and colleagues also experienced earlier stated AKT3 as a prospective target of miR-122, while they did not check out this conversation [eighteen]. Considering that AKT is a crucial regulator in quite a few cancers, we decided to look into the sequence alignments amongst AKT3 3’UTR more, and found that in a few species, the human miR-122 in fact exhibits partial complementarity (Determine 2A). We then amplified the human AKT3 3’UTR by PCR and sub-cloned it into a luciferase reporter vector as illustrated in Figure 2B. This build was utilized for cotransfection with miR-122 build in SNU182 (cells missing endogenous miR-122 expression) and Huh7 (cells harboring some endogenous miR122 expression) mobile traces. A luciferase assay was then employed in analyzing no matter if miR-122 can bind to the 3’UTR of AKT3. Outcomes display that miR-122 expression remarkably decreased the firefly luciferase activity in SNU-182 cells indicating miR-122 binding to 3’UTR (Figure 2C).
miR-122 immediately binds to the 3’UTR of hsa-AKT3. (A) Sequence alignments of miR-122 with 3’UTR of AKT3 from 3 mammalian species demonstrates partial complementarity. (B) Schematic representation describing the 3’UTR luciferase reporter assay. The assay was carried out simultaneously in SNU-182 and Huh-7 cells, above-expressing miR-122 GFP or the GFP vector on your own, as well as parental cells co-transfected with the pGL3-3’UTR build made up of AKT3 3’UTR. Luciferase assays were carried out 48 hours soon after transfection working with the Twin-Luciferase Reporter Assay Process (Promega). Firefly luciferase activity was normalized to Renilla luciferase exercise to account for versions in transfection performance. Firefly luciferase activity will be lowered if there is a immediate binding involving miR-122 and the 3’UTR of AKT3 sequence inserted in the vector. (C) Luciferase exercise was calculated in SNU-182 and Huh-seven parental, miR-122-GFP and GFP more than-expressing cells transfected with the luciferase reporter 3’UTR build or vector on your own.in Figure one and Determine 3A, miR-122 expression is significantly diminished in the HCC mobile lines in contrast to that in standard liver. Concurrently, AKT3 expression level is up-controlled in all 3 HCC cell traces (Hep3B2, SNU-182 and SNU-475) with tiny or no expression of miR-122 (Determine 3A). Curiously, in the hepatoblastoma HepG2 cells and the HCV-reworked Huh-seven strains, AKT3 is not more than-expressed in comparison to standard liver tissue (Determine 3A). The Huh-seven AKT3 ranges are not surprising thinking about the endogenous expression of miR-122 in these cells. As expected because of to a absence of miR-122 binding website, though remarkably homologous, AKT1 and AKT2 mRNA levels only confirmed slight raises in the HCC cell strains in comparison to usual liver (Figure 3B). Equivalent to the observations made for AKT3 transcript amounts, AKT3 protein amounts ended up also substantially higher in SNU182 and SNU-475 HCC cell lines (Figure 3C). These outcomes point out that miR-122 amount is inversely correlated to the AKT3 mRNA and protein levels in the HCC mobile strains.
We upcoming examined the effects of miR-122 more than expression in human HCC cell lines, SNU-182, SNU-475, Hep3B2, and Huh-seven. miR-122 was sub-cloned in a lentiviral expression vector and was properly in excess of expressed in these cell lines (Figure 4A). As predicted, over-expression of miR-122 reduced the two the mRNA and protein amounts of AKT3 in SNU-182 cells as shown in Figure 4A. Very similar facts was collected from the SNU-475, and Hep3B2 (info not demonstrated). In Huh-7 cells, which convey some endogenous miR-122, more than-expression of miR122 also diminished AKT3 protein levels but this modify was only visible on the immunoblot with lengthy exposure time owing to the reduced endogenous AKT3 stages in this cell line (Determine 4A). To affirm specificity, we also examined alterations in the other two AKT relatives members in these miR122 transduced cells. Above-expression of miR-122 in SNU182 and Huh-seven did not substantially change the AKT1 or AKT2 expression, as shown in Figure 4B, yet again suggesting that miR-122 exclusively targets AKT3. Consequently, these final results guidance the hypothesis that miR-122 negatively regulates AKT3 translation in HCC mobile traces.
Experiments 4? were conducted to determine effects of MG132 on oocyte nuclear maturation (Experiments 4 and 5) and fertilization rate (Experiment 6). For Experiment 4, COCs were treated with vehicle or 10 mM MG132 for the first 6 h of maturation and nuclear maturation was examined at 16 h after initiation of maturation. The experiment was replicated three times with 20?5 COCs per treatment for each replicate. For Experiments 5 and 6, COCs were untreated or treated with 10 mM MG132 at two times (0? h of maturation, 16?2 h of maturation, or at both times) using a 262 factorial arrangement of treatments and procedures as described for Experiment 3. The endpoints were nuclear maturation at 22 h of maturation (Experiment 5) or sperm penetration at 18 h after exposure to sperm (Experiment 6). Experiment 5 was replicated three times with 20?5 COCs per treatment for each replicate. Experiment 6 was replicated four times with 20?0 COCs per treatment for each replicate. Data were analyzed statistically as follows. For each replicate, percentage data (for example, percentage of oocytes that cleaved and percentage of cleaved embryos that became blastocysts) were calculated for all oocytes or embryos within the same treatment. Thus, the group of oocytes treated alike within each replicate was the experimental unit. Statistical analyses were performed using the Statistical Analysis System (version 9.2; SAS Institute Inc., Cary, NC, USA). Data were analyzed using the General Linear Models procedure. For main effects with more than 1 degree of freedom, the pdiff mean separation procedure was used when main effects or interactions differed at P,0.10. Percentage data were arcsine-transformed prior to analysis to maintain homogeneity of variance. Results are expressed as least-squares means 6 standard error of the mean (SEM) of the untransformed data.
Effect of MG132 on the Oocyte Proteome (Experiment 7)
Oocytes were matured as described above. After 16 h of maturation, COCs were placed in fresh medium containing 10 mM MG132 or vehicle. The COCs were denuded after 22 h of maturation by vortexing after treatment with hyaluronidase. Those oocytes in which a polar body was evident by light microscopy were retained and processed for protein extraction.
The zona pellucida was removed by treatment for 5 min with 0.1% (w/v) protease from Streptomyces griseus followed by mechanical shearing. Three biological replicates were included for both vehicle and MG132 groups. A biological replicate represented a pool of polarbody-extruded oocytes collected from several oocyte maturation procedures. The number of oocytes per pool was 225 for replicate 1, 225 for replicate 2 and 1000 for replicate 3. Oocytes were suspended in 10 mM KPO4, pH 7.4 containing 1 mg/ml polyvinyl alcohol and 1% (w/v) protease inhibitor cocktail (Sigma) and stored at 270uC until processing. Total protein was isolated from pooled oocytes and purified as described elsewhere [10]. The protein concentration was determined using the BCAH Protein Assay (Thermo, Rockford, IL, USA). For each sample (regardless of the number of starting oocytes), 100 mg protein was dissolved in protein buffer [0.2% (w/v) sodium dodecyl sulfate, 8 M urea, and 10 mM Triton X-100). The samples were reduced, alkylated, trypsin-digested, and labeled following the manufacturer’s instructions for the iTRAQ Reagents 4-plex kit (AB Sciex Inc., Foster City, CA, USA). To verify the tag efficiency of the iTRAQ method, iTRAQ tags 114 and 115 were used to label control samples and tags 116 and 117 were used to label MG132 groups. Two iTRAQ procedures were conducted. In Set 1, one control and one MG132 sample were analyzed twice to determine technical replication. In Set 2, two biological replicates of each treatment were analyzed. Proteins were identified using an off-line 2D liquid chromatography-MS/MS method with strong cation exchange (SCX) chromatography as a first step to fractionate the oocyte proteome (Figure S1). The tryptic peptide mixtures were lyophilized, dissolved in SCX solvent A [25% (v/v) acetonitrile, 10 mM ammonium formate, and 0.1% (v/v) formic acid, pH 2.8], and fractionated using an Agilent HPLC system 1260 with a polysulfoethyl A column (2.1 6 100 mm, 5 mm, 300 ?A; PolyLC, Columbia, MD, USA). Tryptic peptides were separated with a LC Packing C18 Pep Map HPLC column (Dionex, San Francisco, CA, USA), and a hybrid quadrupoleTOF QSTAR Elite MS/MS system (AB Sciex Inc., Framingham, MA, USA) was used for data acquisition.
The MS/MS data were processed by a thorough search considering biological modification and amino acid substitution against the National Center for Biotechnology Information nonredundant Bos taurus fasta database (83,655 entries) and uniprot B. taurus database (33,808 entries) under the ParagonTM algorithm [11] using ProteinPilot v.4.2 software (Applied Biosystems). After searching MS/MS spectra against these databases, results were combined into each group. Animal species, fixed modification of methylmethane thiosulfate-labeled cysteine, fixed iTRAQ modification of amine groups in the N-terminus and lysine, and variable iTRAQ modifications of tyrosine were considered. The ProteinPilot cutoff score was set to 1.3 (a confidence level of 95%), and the false discovery rate (FDR) was estimated by performing the search against concatenated databases containing both forward and reverse sequences (Table S1). For protein quantification, we only considered MS/MS spectra that were unique to a particular protein and where the sum of the signal-to-noise ratio for all of the peak pairs was .9 (software default settings, AB Sciex Inc.). The accuracy of each protein ratio was calculated by the ProGroup analysis in the software to determine whether the protein is significantly differentially expressed [12]. To be identified as being significantly differentially expressed, a protein must have been quantified with at least three spectra, the fold change was .1.2 or ,0.8, and the P value for vehicle vs MG132 was ,0.05 as determined with Fisher’s combined probability test [13] (Fisher, 1948).
First, riluzole does not induce C-terminal Smad phosphorylation, suggesting that the TGFb/receptor complexes are not engagedTable 1. Genes whose expression was regulated by riluzole in the WM793 cell line using the Human TGFb/BMP Signaling Pathway RT2 ProfilerTM PCR Array.The fold increase or decrease (comparing to control) was above 2 for these four genes. BMP3: bone morphogenetic protein 3; GDF5: growth differentiation factor 5; INHBB: inhibin, beta B; PLAU: plasminogen activator, urokinase.upon riluzole treatment. In contrast, the initial step after TGFb activation is the C-terminal phosphorylation of Smad2 and Smad3, and this step is required for the TGFb-induced Smad3 phosphorylation [20]. Second, riluzole does not affect the expression of TGFb1, TGFb2 or TGFb3 as shown by real time RT-PCR (data not shown). Therefore, this does not support the possible hypothesis that riluzole increases Smad linker phosphorylation by inducing TGFb production. Finally, in contrast to TGFb-induced Smad linker phosphorylation, the TbRI inhibitor, SB431542, did not inhibit the riluzole-induced Smad linker phosphorylation. The effect of riluzole on the linker phosphorylation of Smad2 and Smad3, downstream effectors of TGFb, will likely modulate TGFb signaling and the expression of TGFb target genes. Our previous report suggested that Smad3 linker phosphorylation might contribute to the resistance to TGFb-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 TGFb 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 TGFb prooncogenic effects [10]. This model is in accordance with the well-documented fact that not all Smad transcriptional activities have been disrupted in melanoma cells [1]. It is now clear that Smad transcriptional activities are modulated by phosphorylation at their linker domain, but the nature of this modulation will likely depend on the promoter of each TGFb target gene, of the other transcription factors (repressors, activators), binding this promoter and the consequences of these phosphorylation events on the interaction between linker phosphorylated Smad and these other transcription factors. In addition, the identity of the sites phosphorylated in Smad2 and Smad3 (Threonine 220, Serines 245,250 and 255 for Smad2; Thr179 and Serines 204, 208 and 213 for Smad3) will play a role in the modulation of the TGFb target genes. We have shown that riluzole induces the phosphorylation of the cluster of serines (245/250/255) in Smad2 and serine 204 in Smad3 via GSK3, in the majority of the melanoma cell lines analyzed. The exact consequences of these phosphorylation events on Smad2 and Smad3 transcriptional activities will be promoter dependent, as mentioned previously. We initiated the characterization of genes associated with the TGFb signaling pathway and whose expression was modulated by riluzole. Our goal was to define possible mediators of riluzole action downstream of the TGFb signaling pathway. Since TGFb 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 TGFb 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 TGFb 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 [26,27]. We characterized two genes whose expression was upregulated by riluzole. The first one codes for inhibin beta B. Two inhibin beta B subunits form a homodimer called activin B, which is a member of the TGFb superfamily [47]. The fact that riluzole upregulates the expression of activin B could potentially have a negative impact on riluzole response since activin is suggested to play an active role in several carcinomas and glioma migration, invasion and progression [53,54,55,56,57]. In addition, one study suggested that melanoma cells might be resistant to the growth inhibitory and pro-apoptotic effects of activin [58].
Resembling the dual function of TGFb in melanoma, melanoma cells would be resistant to activin-mediated tumor suppression, but would utilize activin to promote their migration and metastasis. The second gene whose expression was upregulated by riluzole was PLAU coding for the urokinase plasminogen activator, described as a TGFb target gene [48,49]. It was shown that tumor growth was retarded in uPA-deficient mice [59,60]. uPA belongs to the uPAS system, which plays multiple roles in the neoplastic evolution, including angiogenesis, tumor cell proliferation, adhesion, migration, intravasation and growth at the metastatic site [61]. As suggested earlier regarding the upregulation of activin expression by riluzole, riluzole positive regulation of PLAU might contribute to the mixed responses to riluzole and the failure of some patients to respond to riluzole in clinical trials [26,27]. At this point of the study, we cannot directly link the riluzoleinduced linker phosphorylation of Smad2 and Smad3 to the upregulation of INHBB and PLAU expression. This would require an extensive analysis using stable transfectants expressing wildtype 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 TGFb signaling pathway. We then focused on the TGFb 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 riluzolebased therapy.
PGC-1a is a nicely regarded master regulator of mitochondrial oxidative metabolic rate [33]
PD is a renal replacement therapy that, instead of hemodialysis, is related with much better preservation of residual renal purpose, preliminary survival edge, diminished erythropoiesis stimulatory agent demands and preservation of vascular obtain websites [26?8]. Even so, the use of “unphysiologic” typical PD fluids (characterised by acidic pH, higher lactate concentrations, significant osmolality, large glucose concentrations, and contamination by glucose degradation solutions) could lead to the onset/ progress of several adverse results [29?two] and to the activation of oxidative anxiety [7]. Though effectively described, the molecular mechanisms associated to latter situation are however not totally known. To much better deal with this level, we targeted on the Peroxisome proliferator-activated receptor gamma coactivator one alpha (PGC1a)-associated intracellular equipment. PGC-1a is a properly identified grasp regulator of mitochondrial oxidative fat burning capacity [33]. Its expression looks finely tuned to mirror mobile electricity wants, with situations of elevated electricity calls for inducing its expression [34,35]. PGC-1a performs this undertaking by coactivating a large amount of transcription factors, which includes, among other people, nuclear respiratory transcription issue one (NRF-1) and in this way, it regulates the action of quite a few nuclear genes encoding mitochondrial proteins [36]. Curiously, our RT-PCR experiments demonstrated that the expression amounts of PGC-1a, NRF-one and the other analyzed downstream target genes were considerably down-regulated in PD sufferers compared to HS. Therefore PBMCs of CKD individuals showed a particular down-regulation of various nuclear-encoded genes involved in the mitochondrial biogenesis and features (TFAM, COX6C, COX7C, UQCRH and MCAD). TFAM has a critical biological role mainly because, right after migration into mitochondria, it regulates mitochondrial DNA transcription and replication [37]. COX6C and COX7C encode for two subunits of the cytochrome c oxidase (COX or Intricate IV), UQCRH is a ingredient of the ubiquinol-cytochrome c reductase intricate (advanced III), which catalyzes electron transfer from succinate and nicotinamide adenine dinucleotide-connected dehydrogenases to cytochrome c [38]. MCAD, then, is an oxidoreductase enzyme regulated by PGC1-a, that catalyzes the very first step of mitochondrial fatty acid beta-oxidation [39]. Primarily based on prior literature evidences reporting that reactive oxygen species may possibly induce PGC-1a down-regulation [forty], we assumed that this organic/biochemical intricate, through a mitigation of the mitochondrial OXPHOS action, could signify a protecting adaptive reaction from chronic mobile perturbation linked to the kidney disorder-related oxidative injuries. Our CKD people, in truth, showed better plasma concentration of Malondialdehyde (MDA), a thiobarbituric acid reactive substance (TBARS) frequently identified as a marker of oxidative pressure [41], in comparison to healthy controls. Furthermore, our benefits verified previous literature evidences reporting that state-of-the-art CKD “per se” mostly by the accumulation of a number of circulating uremic toxins (e.g., indoxyl sulfate, p-cresyl sulfate) [42,43] and the conversation of PBMCs with bio-incompatible dialysis equipment can trigger their activation with imbalance between pro- and anti-oxidant pursuits ensuing in high oxidative tension [eight,44]. This hypothesis was also in element confirmed by our acquiring of an additional activated cellular anti-oxidant machinery in PBMCs of PD patients. Especially, Nuclear issue erythroid-derived two-like 2 (NRF2 or NFE2L2), a transcription issue regulating the expression of quite a few antioxidant/detoxifying enzymes, and just one of its down-stream target genes superoxide dismutase-2 mitochondrial (SOD2) [forty five] resulted substantially up-controlled in our CKD-PD populace. SOD2 binds to the superoxide byproducts of oxidative phosphorylation and converts them to hydrogen peroxide and diatomic oxygen [46]. Thus, all alongside one another, our outcomes, while generated on a little but nicely picked patients’ population, unveiled, for the 1st time, a fine regulated intracellular biochemical program associated to oxidative anxiety response in CKD sufferers. It is plausible that this redox-dependent system could have a pivotal function in antioxidant protection cellular tactic happening in cells of these patients (Figure 9). Nevertheless, more research are needed to superior delineate all the biological/biochemical mechanisms concerned. The primary limitation of our examine is the lack of the assessment of all prospective medical variables able to influence the “mitochondrial” transcriptomic profile largely thanks to the time and value consuming of a international analytic/research strategy. Moreover, our in vitro model unsuccessful to corroborate our in vivo findings (See File S1 and Figure S1, 2, 3 and four). In reality, PBMCs stimulated with high glucose PD dialysis solutions showed the upregulation of all the earlier analyzed genes. These contradictory results plainly shown the unquestionable complexity of this machinery in which all together uremia, microinflammation, subclinical/medical peritoneal infections, acidosis, electrolytic unbalance contribute to the onset and improvement of this organic/clinical condition. Thus, we solid motivate a collaborative global exploration system to handle these factors. Eventually, we can not exclude that, in long term, the modulation of this machinery could flip on as a valuable point of therapeutic intervention to decrease oxidative tension-relevant medical difficulties in CKD clients in equally conservative and dialysis cure.
Oxygen availability regulates embryonic progress by way of oxygen sensing pathways and intracellular redox state modifications
Mitochondria function as `cellular electricity plants’, building adenosine triphosphate (ATP) that is expected for mobile survival and function. Nevertheless, mitochondria have a lot of other functions in addition to the manufacturing of ATP, which includes roles in sign transduction and the regulation of cellular calcium homeostasis [1]. Mitochondrial dysfunction is involved in pathogenesis of many neurological abnormalities such as neurodegenerative conditions [two], hypoxic brain personal injury [3] and psychiatric disorders [4]. For the duration of the process of differentiation of neurons from neural progenitor cells (NPCs) mitochondrial biogenesis happens to supply mitochondria for the developing dendrites and axon [five,six]. Simply because neurons are excitable cells and practical experience recurring bouts of membrane depolarization and Na+ and Ca2+ inflow, they have a substantially higher aerobic metabolic amount than NPCs [seven]. Mainly because mitochondrial respiration generates superoxide anion radical, cells with higher amounts of lively mitochondria, these kinds of as neurons and myocytes, create far more superoxide than non-excitable cells [one,8]. When extreme and uncontrolled generation of superoxide and other reactive oxygen species (ROS) can consequence in mobile harm and death [1], reduced nontoxic degrees perform essential roles in regulating several physiological mobile procedures [nine]. Oxygen availability regulates embryonic progress by way of oxygen sensing pathways and intracellular redox state improvements. Comparatively minimal oxygen rigidity and minimal endogenous ROS amounts retain the viability and self-renewal potential of stem cells [10]. Mitochondrial superoxide, created during electron transport chain (And many others) exercise, is the major supply of intracellular ROS. We previously described that excitable cells (cardiac myocytes and neurons) exhibit intermittent bursts of superoxide creation (superoxide flashes), the frequency of which is modulated by oxygen rigidity [8]. In distinction, NPCs show very low amounts of superoxide flashes, and when the superoxide flash frequency is increased the proliferation of the NPCs is diminished [eleven]. The mechanism by which mitochondrial superoxide flashes negatively control NPC proliferation involves inhibition of extracellular signal controlled kinases (ERKs) [eleven]. Collectively, the readily available data propose that improved aerobic activity is linked with greater bursts of mitochondrial superoxide generation which, in flip, inhibits NPC proliferation. Because cessation of mobile division is a prerequisite for neuronal differentiation, we decided no matter whether superoxide flashes management the approach of differentiation of NPCs into neurons. In contrast to basal amounts of superoxide generation, mitochondrial superoxide flashes are triggered by opening of mitochondrial membrane permeability changeover pores (mPTP) [eight]. The molecular composition of the proteins comprising the mPTP has not been founded, but rising proof factors to the F()-F(1) ATP synthase (the previous complex in the Etc) as currently being a main ingredient of the mPTP [twelve]. A protein that is linked with mPTP is cyclophilin D, which is a molecular concentrate on of cycosporin A, a drug that inhibits mPTP opening [13]. Sustained mPTP opening mediates apoptosis, a sort of programmed mobile loss of life in which cytochrome c is introduced from mitochondria and cell death effector proteins referred to as caspases are activated [fourteen]. In contrast, a transient “flickering” manner of mPTP opening can take place, and may possibly participate in roles in ROS-mediated signaling and adaptive mobile pressure responses [fifteen,sixteen]. The prevalence of superoxide flashes demands transient mPTP opening and Etc activity, suggesting a practical coupling of mPTP and the And so on [8]. In the existing analyze we offer evidence that mPTP-mediated superoxide flashes advertise the differentiation of neurons from NPCs.the brokers have been prepared as 1000x stocks in DMSO. Treatments were administered by immediate dilution into the lifestyle medium and an equivalent volume of car or truck was added to regulate cultures.
Monolayer NPCs were fastened in a resolution of four% paraformaldehyde in PBS for 20 minutes. Cells were being permeabilized and preincubated with blocking resolution (.three% Triton X-a hundred and 10% usual goat serum in PBS) for 30 min, and then incubated right away at four with antibodies against III tubulin (Tuj1 mouse, 1:250 Sigma, St. Louis, MO), Sox2 (one:200, Chemicon, Temecula, CA) and glial fibrillary acidic protein (GFAP mouse, 1:250 Sigma, St. Louis, MO) antibodies. Then the cells were being incubated with anti-mouse secondary antibodies (FITC-conjugated donkey IgG, 1:500, InVitrogen, Carlsbad, CA) in blocking solution for 2 h at home temperature. The cells have been counterstained with .02% propidium iodide (PI) and 1% RNAse in PBS for ten min they ended up then washed with PBS and mounted on microscope slides in an anti-fade medium (Vector Laboratories, Burlingame, CA). To measure the proportion of just about every mobile form, 300-500 cells in 4 random fields were being counted, and the percentage of cells that had been immunoreactive with every single antibody was calculated. Images have been obtained making use of a Zeiss LSM 510 confocal microscope with a 40X aim.
However, more experiments are necessary to determine if the BFAinsensitive pathway described here is indeed independent of the Golgi. Our results are consistent with a previous report where two distinct targeting pathways were proposed for TIP3;1 and BP-80 using a transient assay in tobacco protoplasts [17], and for a BFAinsensitive pathway for TIP3;1 trafficking in the same system [21]. Multiple pathways for membrane proteins to the vacuole were also identified in transient assays using tobacco leaf epidermis. In that system, the calcineurin binding protein CBL6 was trafficked in a COPII-independent manner, while aTIP/TIP3;1 and Vam3/ SYP22 were not [23]. It is unclear if the targeting of TIP3;1 via the BFA-insensitive pathway is also COPII-dependent in Arabidopsis. While BFA treatments may be used to differentiate these two pathways as well, BFA affects all Golgi-dependent pathways and it also has major effects on endomembrane morphology and endocytosis [53,61]. The lack of effects of C834 on the localization of at least twelve endomembrane markers suggests that C834 is a unique new tool that may be used for targeted inhibition of the BFA-insensitive pathway for tonoplast proteins in Arabidopsis roots. C834 may inhibit an important component of the endomembrane system involved in the BFA-insensitive pathway for membrane proteins. Identification of the C834 target(s) may shed light into the mechanisms of this pathway and its interactions with other trafficking pathways. However, the possibility exists that, in contrast to that of hypocotyls and protoplasts, the trafficking of the TIP3;1-YFP and GFP-TIP2;1 is Golgi-dependent in Arabidopsis roots, and in this case C834 would target a highly specific but unknown mechanism for TIP3;1 and TIP2;1 traffic to the vacuole. If this were the case, this would add one more layer of complexity to the regulation of the endomembrane system. We cannot fully exclude the possibility that C834 acts by enhancing a retrograde traffic of tonoplast proteins from the vacuole back to the ER instead of acting as an inhibitor of anterograde traffic. However, the loss of TIP3;1-YFP and GFPTIP2;1 from the vacuolar membrane during C834 treatments are not consistent with this possibility, as proteins that reach the ER by these means would be expected to eventually enter the anterograde pathway and reach the vacuole to some extent. More mechanistic and detailed analysis of the C834 inhibition could be used to differentiate between these possibilities, and our lab is moving in this direction.
Abstract
The experimental compound SU5416 went as far as Phase III clinical trials as an anticancer agent, putatively because of its activity as a VEGFR-2 inhibitor, but showed poor results. Here, we show that SU5416 is also an aryl hydrocarbon receptor (AHR) agonist with unique properties. Like TCDD, SU5416 favors induction of indoleamine 2,3 dioxygenase (IDO) in immunologically relevant populations such as dendritic cells in an AHR-dependent manner, leading to generation of regulatory T-cells in vitro. These characteristics lead us to suggest that SU5416 may be an ideal clinical agent for treatment of autoimmune diseases and prevention of transplant rejection, two areas where regulatory ligands of the AHR have shown promise. At the same time, AHR agonism might represent a poor characteristic for an anticancer drug, as regulatory T-cells can inhibit clearance of cancer cells, and activation of the AHR can lead to upregulation of xenobiotic metabolizing enzymes that might influence the half-lives of co-administered chemotherapeutic agents. Not only does SU5416 activate the human AHR with a potency approaching 2,3,7,8-tetrachlorodibenzo-p-dioxin, but it also activates polymorphic murine receptor isoforms (encoded by the Ahrd and Ahrb1 alleles) with similar potency, a finding that has rarely been described and may have implications in identifying true endogenous ligands of this receptor.
Citation: Mezrich JD, Nguyen LP, Kennedy G, Nukaya M, Fechner JH, et al. (2012) SU5416, a VEGF Receptor Inhibitor and Ligand of the AHR, Represents a New Alternative for Immunomodulation. PLoS ONE 7(9): e44547. doi:10.1371/journal.pone.0044547 ~ Editor: Jose Carlos Alves-Filho, University of Sao Paulo, Brazil Received March 6, 2012; Accepted August 8, 2012; Published September 6, 2012 Copyright: ?2012 Mezrich et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by Grant 1UL1RR025011 from the Clinical and Translational Science Award program of the National Center for Research Resources, National Institutes of Health (JDM), National Institutes of Environmental Health Services Grant R37ES005703 (CAB), National Cancer Institute Grant P30CA014520 (CAB). JDM is a John Merrill Grant Scholar of the American Society of Nephrology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: CAB has served as a scientific consultant to Dow Chemical Co. on issues related to dioxin toxicity. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. There are no patents, products in development or marketed products.
Introduction
Agonists of the aryl hydrocarbon receptor (AHR) have been of interest to the pharmaceutical industry for many years. This interest originally stemmed from the observation that the AHR is a ligand-activated transcription factor that regulates the adaptive metabolism of xenobiotics [1] and because receptor binding is a known step in the carcinogenic and toxic action of environmental pollutants like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) [2]. Thus, agonism of the AHR has commonly been considered a signature for drugs that upregulate phase-I and phase-II metabolic systems and also for chemicals with pharmacological similarity to a known human carcinogen. As a result, AHR agonism has largely been considered a hazard signature for environmental chemicals and drugs in the pharmaceutical pipeline. Recent insights related to the normal physiological role of the AHR are changing our view of receptor agonism to one where agonism might be considered to hold therapeutic value. A number of recent reports are identifying new biological processes that might be influenced by endogenous receptor ligands. For example,
descriptions of mice harboring a null allele at the Ahr locus indicate that receptor signaling plays an important role in normal cardiovascular development and function [3,4].
Abstract
Glioblastoma multiforme (GBM) is the most common intracranial cancer but despite recent advances in therapy the overall survival remains about 20 months. Whole genome exon sequencing studies implicate mutations in the receptor tyrosine kinase pathways (RTK) for driving tumor growth in over 80% of GBMs. In spite of various RTKs being mutated or altered in the majority of GBMs, clinical studies have not been able to demonstrate efficacy of molecular targeted therapies using tyrosine kinase inhibitors in GBMs. Activation of multiple downstream signaling pathways has been implicated as a possible means by which inhibition of a single RTK has been ineffective in GBM. In this study, we sought a combination of approved drugs that would inhibit in vitro and in vivo growth of GBM oncospheres. A combination consisting of gefitinib and sunitinib acted synergistically in inhibiting growth of GBM oncospheres in vitro. Sunitinib was the only RTK inhibitor that could induce apoptosis in GBM cells. However, the in vivo efficacy testing of the gefitinib and sunitinib combination in an EGFR amplified/ PTEN wild type GBM xenograft model revealed that gefitinib alone could significantly improve survival in animals whereas sunitinib did not show any survival benefit. Subsequent testing of the same drug combination in a different syngeneic glioma model that lacked EGFR amplification but was more susceptible to sunitinib in vitro demonstrated no survival benefit when treated with gefitinib or sunitinib or the gefitinib and sunitinib combination. Although a modest survival benefit was obtained in one of two animal models with EGFR amplification due to gefitinib alone, the addition of sunitinib, to test our best in vitro combination therapy, did not translate to any additional in vivo benefit. Improved targeted therapies, with drug properties favorable to intracranial tumors, are likely required to form effective drug combinations for GBM.
Citation: Joshi AD, Loilome W, Siu I-M, Tyler B, Gallia GL, et al. (2012) Evaluation of Tyrosine Kinase Inhibitor Combinations for Glioblastoma Therapy. PLoS ONE 7(10): e44372. doi:10.1371/journal.pone.0044372 Editor: Maciej S. Lesniak, The University of Chicago, United States of America Received December 9, 2011; Accepted August 6, 2012; Published October 2, 2012 Copyright: ?2012 Joshi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This project was supported by National Institutes of Health (NIH) Grant R01 NS052507, the Virginia and D.K. Ludwig Fund for Cancer Research, and the Children’s Cancer Foundation. GJR is supported by the Irving J. Sherman M.D. Neurosurgery Research Professorship. AJ was supported by Matt Trainham ABTA basic research fellowship (http://www.abta.org/). WL was supported by the Faculty of Medicine at Khon Kaen University, Thailand. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
Introduction
Improving therapy for patients with Glioblastoma multiforme (GBM) is one of the biggest challenges in oncology. Although molecular targeting has shown success in many cancers, targeted therapy for GBM has yet to demonstrate an appreciable clinical survival benefit [1,2]. For example, targeting of Epidermal Growth Factor Receptor (EGFR) with small molecules or monoclonal antibodies has been reported to offer no survival benefit [1], despite the fact that EGFR is the most common genomically altered oncogene in GBM, and targeting EGFR has shown benefit in other cancers. So an important question is: can targeted therapy provide a benefit to GBM patients? The oncogenic receptor tyrosine kinases (RTKs) that are mutated in GBM are obvious molecular targets and many small molecule inhibitors of the RTKs are available. A mutation analysis of over 20,000 gene coding regions in GBM genomes confirmed that the RTK/PI3K/AKT pathway is one of the most frequently altered groups of genes in GBM [3]. The commonly altered genes include EGFR (40% approximate frequency), PTEN (37%), PIK3CA(13%), PIK3R1 (8%) and PDGFRA (8%) [3,4]. Over 80% of glioblastomas have an acquired alteration in the RTK/PI3K/AKT pathway with about 40% of tumors having some alteration in EGFR [3,5] suggesting that scarcity of a prevalent alteration is not the problem with targeted therapy in most GBMs. However, in spite of recent advances in development of targeted therapies, RTK inhibitors have shown negligible success against GBMs. Lack of successful therapies against GBMs using RTK inhibitors raises several questions. Are the molecular targeting agents reaching and inhibiting the presumed target effectively in GBM? What are the resistance mechanisms involved if the inhibitors are reaching the tumor in effective concentrations? Growth signaling through alternate pathways, as well as tumor heterogeneity could be two of many factors involved in tumor resistance mechanisms. In the following study, we tried to evaluate a series of RTK inhibitors in GBM systems in vitro and in vivo to determine if we could find a combination of RTK inhibitors that would be more successful than a single agent. The premise of the work was to evaluate approved inhibitors designed to target the most frequently activated tyrosine kinases in GBMs. The best in vitropair of drugs inhibited GBM oncospheres synergistically was gefitinib and sunitinib. However, the improved activity of RTK combination did not perform as predicted in vivo. Gefitinib alone had a significant but modest survival benefit in a GBM xenograft mouse model mouse model. Moreover, in vivo evaluation of the same drugs in a syngeneic rat model of GBM failed to provide any survival benefit. Although the single agent therapy might show activity in certain genetic backgrounds, combinations that effectively target multiple RTK pathways in an intracranial target are needed.