Inhibitor SB203580 Research Articles

Abstract 5983: p38 kinase acts as a negative regulator of recurrence after surgery in a mammary carcinoma model, through apoptosis induction and proliferation inhibition

Abstract The p38 pathway can be activated by three different MAP kinase kinases: MKK3, MKK4 and MKK6, each one activated by different stimuli, like DNA damage, oxidative stress, proinflammatory cytokines or growth factors. In consequence, the responses mediated by this kinase are also significantly diverse. Its participation in such different processes makes it reasonable for the role of p38 in cancer to be a topic of current debate. Several authors reported a pro-tumorigenic role of p38, through the stimulation of tumor progression, epidermal to mesenchymal transition and migration and chemotherapy resistance, as a consequence of the transduction of pro-survival signals. Due to the above mentioned, the combination of p38 inhibitors and chemotherapy is frequently proposed as an interesting clinical approach for cancer treatment. In contrast, several reports underline the importance of p38 suppression of tumor initiation, metastasis inhibition through dormancy maintenance, and also a decisive role in the action mechanism of antitumor treatments under evaluation. Specifically, an important amount of evidence point p38 as a key apoptosis mediator in cancer cells, as a response to treatment with anticancer drugs included in different clinical and preclinical protocols. Previously we studied the effect of p38 inhibition in a mouse mammary carcinoma model named F3II. We described that in vitro inhibition of p38 provokes an increased in cell adhesion, cell spreading, proliferation in 3D cultures, ERK phosphorylation and Integrin α5 expression. In vivo inhibition also resulted in shortened latency times and increased tumor growth rates. In this work, we studied the effect of p38 inhibition in the development of recurrences after tumor surgery. When SB203580 inhibitor was administered after surgery, mice developed larger recurrences. Apoptotic cells in histological samples were detected by TUNEL analysis, showing that SB203580 treatment decreased the number of apoptotic cells in tumor recurrences. In addition, mitotic cell count in recurrence tissue showed that SB203580 treatment increased the mitotic index in the tumors. In conclusion, in this work we demonstrated that in F3II cell line, p38 inhibition promotes tumor recurrence development after surgery, mainly blocking the apoptotic response, and promoting cell duplication. Results presented in this work position p38 as a tumor suppressor kinase. The evidence presented here highlight the possible risk of the administration of a p38 inhibitor to oncological patients that may present newly disseminated cells, or a residual tumor mass that remains after treatment. From our perspective, candidates to be treated with new p38 inhibitors should be carefully selected according to the tumor type and the stage of the disease, since the response might be extremely variable between the different conditions. Citation Format: Carla S. Capobianco, Maria F. Gottardo, Johanna E. Sidabra, Julio A. Aguirre Ghiso, Daniel F. Alonso, Hernan G. Farina. p38 kinase acts as a negative regulator of recurrence after surgery in a mammary carcinoma model, through apoptosis induction and proliferation inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5983.

Abstract 5443: Modulating plasticity in aggressive variant prostate cancers

Abstract Aggressive Variant Prostate Cancers (AVPC) are a clinically and molecularly distinct subset of prostate cancers with a poor prognosis characterized by androgen-indifference, platinum-sensitivity and combined defects in Tp53, RB1 and PTEN, for which novel therapeutic approaches are urgently needed. Defects observed in these tumor suppressor pathways have been linked to lineage plasticity in genetically engineered mouse models. We hypothesized that this plasticity can be identified in human tumor samples, and can be modulated to serve as the foundation for effective combination therapies for the AVPC subset. We previously defined a gene expression signature derived from mouse embryonic day 6.5 that is representative of extensive cellular plasticity (PMID:26123483). We determined the plasticity scores in 12 patient-derived xenograft (PDX) models representative of the spectrum of prostate cancer biology (using available RNA sequencing profiles), and in 26 macrodissected tumor biopsies obtained from patients who received platinum-based chemotherapy for aggressive prostate cancer (using ALMAC Affymetrix gene expression microarray). As expected, the AVPC PDX models exhibited high plasticity scores as did 11 (42.3%) of the patient samples, associated with patient progression free- and overall survival. We next treated a well characterized AVPC PDX model (144-4) with the anti-EMT drug (p38MAPK inhibitor) SB203580 at 10mg/kg, and observed a decrease in stem-cell and EMT factors associated with plasticity. These data confirm the association between plasticity and AVPC in human tumor samples, and suggest that changes in plasticity can be measured in tumor samples to determine the value of its modulation in clinical trials. Acknowledgments: ALMAC Diagnostics Services, UK, and UTMDACC ATGC Core Lab (CCSG-CA016672-ATGC) for RNA sequencing services; UTMDACC Prostate Cancer SPORE (RS, SAM, AA), UTMDACC IRG (RS), and the UTMDACC Prostate Cancer Moonshot Program (AA) for financial support. Citation Format: Rama Soundararajan, Petra den Hollander, Peter Shepherd, Nora Navone, Daniele Lorenzini, Caddie Laberiano-Fernandez, Wei Lu, Jianling Zhou, Jingjing Liu, Jianhua Zhang, Luisa Maren Solis-Soto, Sendurai A. Mani, Ana Aparicio. Modulating plasticity in aggressive variant prostate cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5443.

Abstract 2218: High affinity and cleavage resistant CD16 enhances Trastuzumab mediated killing of gastric cancer targets by human placental CD34+-derived natural killer cells

Abstract Introduction: When targeting tumors with monoclonal antibodies, natural killer (NK) cells are key effectors of antibody dependent cellular cytotoxicity (ADCC) following recognition of antibody Fc by CD16. Celularity has developed a novel proprietary technology platform that enables production of an allogeneic, off-the-shelf, placental CD34+-derived NK cell therapy for the treatment of hematologic and solid tumor malignancies. Here we report the genetic modification of CD16 in NK cells for high binding affinity to IgG and resistance to protease cleavage, together with functional evaluation of anti-tumor ADCC activity against epidermal growth factor receptor 2 (HER2) positive gastric cancer cells in combination with Trastuzumab (anti-Her2). Methods: Human placental CD34+ cells were transduced with lentivirus expressing a high binding affinity (158Val) and protease resistant (197Pro) CD16 variant (CD16VP) and cultured for 35 days in the presence of cytokines, including thrombopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2, to generate CD16VP cells. The tumorigenic activity of CD16VP cells against HER2-positive gastric cancer cell lines NCI-N87 and OE19 was assessed in combination with Trastuzumab. ADCC activity was measured by real-time xCELLigence, and cytokine secretion by Luminex. The kinase inhibitors PD98059 (MEK/ERK), SB203580 (p38 MAPK), SP600125 (JNK), and Wortmannin (PI3K) were used to study the molecular mechanisms underlying cytotoxicity of CD16VP cells against gastric cancer. Results and conclusion: High transduction efficiency was achieved in multiple placental CD34+ donors with an average of 64.6 ± 10.3% (n=8) CD16 expression on NK cells compared to 12.1 ± 3.3% (n=8) on non-transduced cells. These cells expanded [7095 ± 2998-fold (n=8)] and differentiated into NK cells with >90% purity (CD56+CD3-). While treatment of PMA/ionomycin for 4 hours resulted in >89% CD16 cleavage on non-transduced cells, <11% cleavage was observed on CD16VP cells, demonstrating shedding resistance. CD16VP cells in the presence of Trastuzumab compared to IgG control, showed enhanced lysis of NCI-N87 and OE19 targets (97 ± 7% and 95 ± 2% vs. 55 ± 19% and 42 ± 14% respectively, p<0.01), secreted higher levels of GM-CSF, IFN-γ, and TNF-α, and demonstrated increased degranulation against OE19. The ADCC mediated cytotoxicity and degranulation were PI3K and JNK pathway-dependent as Wortmannin decreased ADCC by 82.2 ± 17.7% and degranulation by 43.9 ± 1.9%, and SP600125 decreased degranulation by 72.3 ± 1.2%. Our results demonstrate that CD16VP cells have enhanced Trastuzumab-mediated ADCC activity against gastric cancer tumor cell lines with resistance to CD16 cleavage. Development of CD16VP cells provides a combination therapeutic option by harnessing the anti-tumor activities of both targeted biologics and innate cytotoxicity of NK cells. Citation Format: Irene Raitman, Salvatore Rotondo, Tanel Mahlakõiv, Xuan Guo, Andrea DiFiglia, Hemlata Rana, Qian Ye, Srinivas Somanchi, William van der Touw, Lin Kang, Robert Hariri, Xiaokui Zhang. High affinity and cleavage resistant CD16 enhances Trastuzumab mediated killing of gastric cancer targets by human placental CD34+-derived natural killer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2218.

Genome-wide gene expression analysis of a murine model of prostate cancer progression: Deciphering the roles of IL-6 and p38 MAPK as potential therapeutic targets.

BackgroundProstate cancer (PCa) is the most commonly diagnosed cancer and the second leading cause of cancer-related deaths among adult males globally. The poor prognosis of PCa is largely due to late diagnosis of the disease when it has already progressed to an advanced stage marked by androgen-independence, thus necessitating new strategies for early detection and treatment. We construe that these direly needed advances are limited by our poor understanding of early events in the progression of PCa and that would thus represent ideal targets for early intervention. To begin to fill this void, we interrogated molecular “oncophenotypes” that embody the transition of PCa from an androgen-dependent (AD) to–independent (AI) state.MethodsTo accomplish this aim, we used our previously established AD and AI murine PCa cell lines, PLum-AD and PLum-AI, respectively, which recapitulate primary and progressive PCa morphologically and molecularly. We statistically surveyed global gene expressions in these cell lines by microarray analysis. Differential profiles were functionally interrogated by pathways, gene set enrichment and topological gene network analyses.ResultsGene expression analysis of PLum-AD and PLum-AI transcriptomes (n = 3 each), revealed 723 differentially expressed genes (392 upregulated and 331 downregulated) in PLum-AI compared to PLum-AD cells. Gene set analysis demonstrated enrichment of biological functions and pathways in PLum-AI cells that are central to tumor aggressiveness including cell migration and invasion facilitated by epithelial-to-mesenchymal transition (EMT). Further analysis demonstrated that the p38 mitogen-activated protein kinase (MAPK) was predicted to be significantly activated in the PLum-AI cells, whereas gene sets previously associated with favorable response to the p38 inhibitor SB203580 were attenuated (i.e., inversely enriched) in the PLum-AI cells, suggesting that these aggressive cells may be therapeutically vulnerable to p38 inhibition. Gene set and gene-network analysis also alluded to activation of other signaling networks particularly those associated with enhanced EMT, inflammation and immune function/response including, but not limited to Tnf, IL-6, Mmp 2, Ctgf, and Ptges. Accordingly, we chose SB203580 and IL-6 to validate their effect on PLum-AD and PLum-AI. Some of the common genes identified in the gene-network analysis were validated at the molecular and functional level. Additionally, the vulnerability to SB203580 and the effect of IL-6 were also validated on the stem/progenitor cell population using the sphere formation assay.ConclusionsIn summary, our study highlights pathways associated with an augmented malignant phenotype in AI cells and presents new high-potential targets to constrain the aggressive malignancy seen in the castration-resistant PCa.

Berberine inhibits proliferation and apoptosis of vascular smooth muscle cells induced by mechanical stretch via the PDI/ERS and MAPK pathways

AimsWe recently demonstrated that mechanical stretch increases the proliferation and apoptosis of vascular smooth muscle cells (VSMCs) by activating the protein disulfide isomerase (PDI) redox system, thus accelerating atherosclerotic lesion formation in the transplanted vein. At present, there are no efficient intervention measures to prevent this phenomenon. Berberine inhibits pathological vascular remodeling caused by hypertension, but the underlying mechanism is controversial. Herein, we investigate the role of berberine and the underlying mechanism of its effects on mechanical stretch-induced VSMC proliferation and apoptosis. Main methodsMouse VSMCs cultivated on flexible membranes were pretreated for 1 h with one of the following substances: berberine, PDI inhibitor bacitracin, MAPK inhibitors, or ERS inhibitor 4-PBA. VSMCs were then subjected to mechanical stretch. Immunofluorescence and western blot were used to detect proliferation and apoptosis, as well as to analyze signaling pathways in VSMCs. Key findingsOur results showed that berberine inhibits the PDI-endoplasmic reticulum stress system, thereby attenuating the simultaneous increase of VSMC proliferation and apoptosis in response to mechanical stretch. Interestingly, MAPK inhibitors PD98059, SP600125, and SB202190 significantly reduced the activation of ERS signaling cascades, and their combination with berberine had additive effects. The ERS inhibitor 4-PBA reduced PDI activation and ERS signaling, but not MAPK phosphorylation. Moreover, caspase-3 and caspase-12 were downregulated by berberine. SignificanceThese results illustrate a novel mechanism of action of berberine that has practical implications. Our data provide important insights for the prevention and treatment of vascular remodeling and diseases caused by mechanical stretching during hypertension.

Apoptotic and antihepatofibrotic effect of honokiol via activation of GSK3β and suppression of Wnt/β-catenin pathway in hepatic stellate cells.

Though honokiol, derived from the Magnolia tree, was known to suppress renal fibrosis, pulmonary fibrosis, non-alcoholic steatoheptitis, inflammation and cancers, the underlying antifibrotic mechanisms of honokiol are not fully understood in hepatic stellate cells until now. Thus, in the present study, inhibitory mechanism of honokiol on liver fibrosis was elucidated mainly in hepatic stellate cells (HSCs) by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell cycle analysis and western-blotting. Honokiol exerted cytotoxicity in LX-2, HSC-T6 and Hep-G2 cells. Honokiol increased sub G1 population and activated caspase 3 and cleaved poly (ADP-ribose) polymerase (PARP) in HSCs. Moreover, honokiol attenuated the expression of alpha smooth muscle actin (α-SMA), transforming growth factor beta 1 (TGF-β1), phospho-Smad3, phospho-AKT, cyclin D1, c-Myc, Wnt3a, β-catenin, and activated phosphorylation of glycogen synthase kinase 3 beta (GSK3β) in HSCs. Conversely, GSK3β inhibitor SB216763 reversed the effect of honokiol on PARP, α-SMA, phospho-GSK3β, β-catenin and sub G1 population in LX-2 cells. Overall, honokiol exerts apoptotic and antifibrotic effects via activation of GSK3β and inhibition of Wnt3a/β-catenin signalling pathway.

Liquid Platelet-Rich Fibrin and Heat-Coagulated Albumin Gel: Bioassays for TGF-β Activity.

Liquid platelet-rich fibrin (PRF) can be prepared by high centrifugation forces separating the blood into a platelet-poor plasma (PPP) layer and a cell-rich buffy coat layer, termed concentrated PRF (C-PRF). Heating the liquid PPP was recently introduced to prepare an albumin gel (Alb-gel) that is later mixed back with the concentrated liquid C-PRF to generate Alb-PRF. PRF is a rich source of TGF-β activity; however, the overall TGF-β activity in the PPP and the impact of heating the upper plasma layer remains unknown. Here, we investigated for the first time the in vitro TGF-β activity of all fractions of Alb-PRF. We report that exposure of oral fibroblasts with lysates of PPP and the buffy coat layer, but not with heated PPP, provoked a robust increase in the TGF-β target genes interleukin 11 and NADPH oxidase 4 by RT-PCR, and for IL11 by immunoassay. Consistent with the activation of TGF-β signaling, expression changes were blocked in the presence of the TGF-β receptor type I kinase inhibitor SB431542. Immunofluorescence and Western blot further confirmed that lysates of PPP and the buffy coat layer, but not heated PPP, induced the nuclear translocation of Smad2/3 and increased phosphorylation of Smad3. The immunoassay further revealed that PPP and particularly BC are rich in active TGF-β compared to heated PPP. These results strengthen the evidence that not only the cell-rich C-PRF but also PPP comprise a TGF-β activity that is, however, heat sensitive. It thus seems relevant to mix the heated PPP with the buffy coat C-PRF layer to regain TGF-β activity, as proposed during the preparation of Alb-PRF.

TRIM59 Promotes Retinoblastoma Progression by Activating the p38-MAPK Signaling Pathway.

PurposeRetinoblastoma is a malignant tumor of the developing retina that mostly occurs in children. Our study aimed to investigate the effect of tripartite motif-containing protein 59 (TRIM59) on retinoblastoma growth and the underlying mechanisms.MethodsWe performed bioinformatic analysis of three datasets (GSE24673, GSE97508, and GSE110811) from the Gene Expression Omnibus database. Quantitative reverse-transcription PCR and immunoblotting of three retinoblastoma cell lines were conducted to verify TRIM59 as a differentially expressed gene. Specific siRNAs were used to inhibit TRIM59 expression in the HXO-Rb44 cell line. A lentiviral vector was transfected into the Y79 cell line to overexpress TRIM59. The effects of TRIM59 on retinoblastoma cell proliferation, cell cycling, and apoptosis were explored in vitro using the abovementioned cell lines. The effect of TRIM59 expression on retinoblastoma cell proliferation was evaluated in a mouse xenograft tumor model.ResultsTRIM59 expression in three retinoblastoma cell lines was remarkably elevated compared with normal control. Knocking down TRIM59 expression remarkably suppressed cell proliferation and growth and promoted cell apoptosis in HXO-Rb44 cells, whereas TRIM59 overexpression promoted tumor progression in Y79 cells. Silencing TRIM59 also markedly inhibited in vivo tumor growth in the xenograft model. Mechanistic studies revealed that TRIM59 upregulated phosphorylated p38, p-JNK1/2, p-ERK1/2, and p-c-JUN expression in retinoblastoma cells. Notably, the p38 inhibitor SB203580 attenuated the effects of TRIM59 on cell proliferation, apoptosis, and the G1/S phase transition.ConclusionsTRIM59 plays an oncogenic role in retinoblastoma and exerts its tumor-promotive function by activating the p38–mitogen-activated protein kinase pathway.

Gadd45α affects retinal ganglion cell injury in chronic ocular hypertension rats by regulating p38MAPK pathway.

To investigate the impact and the mechanism of Gadd45α mediating p38MAPK pathway on the retinal ganglion cells (RGCs) injury in chronic ocular hypertension (COH) rats. COH model in rats were established and intraocular pressure (IOP) was tested. Retrograde labeling was used for counting RGCs and TUNEL staining was performed for RGCs apoptosis. Western Blotting was conducted to examine the expression of Gadd45α and p38MAPK pathway. Besides, RGC-5 cells cultured in vitro were treated with H2O2. Cell viability was detected by CCK-8, ROS level tested by DCFH-DA assay, and cell apoptosis examined by flow cytometry. COH rats had increased expression of Gadd45α and p-p38/p38 protein 1-4weeks after surgery. Rats in COH group enhanced obviously in IOP, RGC apoptosis rate and the protein expression of Gadd45α, p-p38/p38, Bax/Bcl-2 and cleaved caspase-3, but declined appreciably in RGC counting. However, the above indicators of COH rats were effectively improved by Gadd45α shRNA treatment. Additionally, RGC-5 cells in H2O2 group reduced in cell viability and went up in ROS level and apoptosis rate. The H2O2-induced RGC-5 cells treated with Gadd45α shRNA were improved apparently in those indicators, and cells treated with pcDNA Gadd45α showed an opposite trend. Moreover, p38 MAPK inhibitor SB203580 can effectively reverse the damage of pcDNA Gadd45α from H2O2-induced RGC-5 cells. Silencing Gadd45α can reduce the RGC damage in COH rats by inhibiting p38MAPK pathway and such a protective role may be associated with the suppression of RGC apoptosis and the mitigation of oxidative stress.

The Effects of Lauric Acid on IPEC-J2 Cell Differentiation, Proliferation, and Death

Lauric acid (LA) has antimicrobial effects and the potential to replace antibiotics in feeds to prevent postweaning diarrhea and increase overall swine productivity. The effects of lauric acid on the intestinal epithelial cells remain unclear. This study investigates the effects of LA on pig intestinal epithelial cell line (IPEC-J2) differentiation, proliferation, and death and explores its underlying mechanisms. It was found that 0.25-0.1 mM LA promoted IPEC-J2 cell differentiation. At 1 mM or higher concentrations, it induced IPEC-J2 cell viability decreases, lipid accumulation, cell proliferation inhibition, and cell apoptosis. The cell death induced did not depend on caspase pathways. The data demonstrated that LA induced the IPEC-J2 cell autophagy and impaired autophagy flux and autophagy plays a role in protecting against LA induced-cell death. p38 MAPK inhibitor SB202190 attenuated LA-reduced IPEC-J2 cell viability. This associated with an increase in autophagy level and a decrease in lipid accumulations and FABPI levels. In summary, LA promoted the IPEC-J2 cell apoptosis depends on the p38 MAPK pathways and may involve autophagy and TG metabolism regulation.

Differential regulation of TGFβ type-I receptor expressions in TGFβ1-induced myofibroblast differentiation.

Fibroblast-to-myofibroblast (FibroMF) differentiation is crucial for embryogenesis and organ fibrosis. Although transforming growth factor-β (TGFβ) is the primary mediator of FibroMF differentiation, the type-I receptor (TGFβRI) responsible for this has not yet been confirmed. In the current study, we investigated the ALK1 and ALK5 expressions in TGFβ1-stimulated NIH 3T3 fibroblasts to compare with the data from the Gene Expression Omnibus (GEO) repository. In our results, whereas TGFβ1 treatment promoted FibroMF differentiation accompanied by increased ALK5 expression and reduced ALK1 expression, TGFβ1-induced FibroMF differentiation and increased α-smooth muscle actin (αSMA) and ALK5 expression were inhibited by co-treatment with ALK5 inhibitor SB431542. GEO database analysis indicated increased ALK5 expression and reduced ALK1 expression in fibrotic compared to normal mouse or human tissues correlating with organ fibrosis progression. Finally, the inhibitors of Akt, mTOR, and β-catenin suppressed TGFβ1-induced ALK5 expression, indicating that the Akt pathway promotes FibroMF differentiation via ALK5 expression and fibrosis.

The indirubin derivative 6-bromoindirubin-3′-glycerol-oxime ether (6BIGOE) potently modulates inflammatory cytokine and prostaglandin release from human monocytes through GSK-3 interference

Indirubin is a natural bis-indole alkaloid contained as active ingredient in the traditional Chinese remedy Danggui Longhui Wan. Indirubin and its 3′-oxime derivatives exhibit anti-cancer and anti-inflammatory properties and they inhibit glycogen synthase kinase (GSK)-3 in cell-free assays where 6-bromoindirubin-3′-oxime (6BIO) is among the most potent analogs. Here, we reveal 6-bromoindirubin-3′-glycerol-oxime ether (6BIGOE) as highly potent derivative able to inhibit pro-inflammatory cytokine, chemokine and prostaglandin (PG) release in human primary monocytes while increasing anti-inflammatory interleukin (IL)-10 levels. 6BIGOE suppressed lipopolysaccharide (LPS)-induced IL-1β and PGE2 release with IC50 of 0.008 and 0.02 µM, respectively, being ≥ 12-fold more potent than 6BIO. The effects of 6BIGOE are mediated via intracellular inhibition of GSK-3, where 6BIGOE again surpassed the effectiveness of 6BIO despite the higher potency of the latter in cell-free GSK-3 activity assays. Side-by-side comparison of 6BIGOE (0.1 µM) with the selective GSK-3 inhibitor SB216763 (5 µM) revealed congruent properties such as enrichment of β-catenin and suppression of cyclooxygenase (COX)-2 protein levels due to GSK–3 inhibition. Metabololipidomics using ultra-performance liquid chromatography-tandem mass spectrometry showed that 6BIGOE selectively decreases pro-inflammatory COX-derived product formation without marked modulation of other lipid mediators. In summary, 6BIGOE is a highly potent indirubin derivative in the cellular context that favorably modulates pro- and anti-inflammatory cytokines as well as COX-2-derived PG via interference with GSK-3.

TGF-βR inhibitor SB431542 restores immune suppression induced by regulatory B-T cell axis and decreases tumour burden in murine fibrosarcoma.

The contribution of immune cells in soft tissue sarcomas (STS) is not completely known and understanding their role is very essential for employing immunotherapy strategies. Here, we show that murine fibrosarcoma-conditioned medium promoted total spleen cell proliferation but inhibited T cell responses to mitogenic and allo-antigen-mediated stimulation. This increased proliferation was found to be in B cells resulting in generation of Breg further leading to Treg population. This was found to be the same in vitro and in vivo. The phenotype of these B cells was CD19+CD81+CD27+CD25+PD-L1hi and they secreted both IL-10 and TGF-β. These tumor evoked Bregs (tBreg), when co-cultured with B depleted T cells, suppressed their proliferation in response to anti-CD3/CD28 stimulation. tBreg-induced suppression of T cell responses wasnot abrogated by the inhibition or neutralization of IL-10 but by the small molecule inhibitor of TGFβ Receptor type I, SB431542. While SB531542 per se was not cytotoxic to tumor cells, administration of SB431542 in tumor-bearing mice (TBM) significantly reduced the tumor burden. In addition, the treatment significantly reduced Treg cells and rescued proliferation of T cells in response to mitogen and allo-antigen. Collectively, our results identify that tumor evoked Breg cells mediate T cell immune suppression through TGFβ-mediated pathway and that targeting the Breg-Treg axis can be potentially used as an immunotherapy agent.

Filamentous nestin and nonmuscle myosin IIB are associated with a migratory phenotype in neonatal rat cardiomyocytes.

Cardiomyocyte migration represents a requisite event of cardiogenesis and the regenerative response of the injured adult zebrafish and neonatal rodent heart. The present study tested the hypothesis that the appearance of the intermediate filament protein nestin in neonatal rat ventricular cardiomyocytes (NNVMs) was associated in part with the acquisition of a migratory phenotype. The cotreatment of NNVMs with phorbol 12,13-dibutyrate (PDBu) and the p38α/β mitogen-activated protein kinase inhibitor SB203580 led to the de novo synthesis of nestin. The intermediate filament protein was subsequently reorganized into a filamentous pattern and redistributed to the leading edge of elongated membrane protrusions translating to significant lengthening of NNVMs. PDBu/SB203580 treatment concomitantly promoted the reorganization of nonmuscle myosin IIB (NMIIB) located predominantly at the periphery of the plasma membrane of NNVMs to a filamentous phenotype extending to the leading edge of elongated membrane protrusions. Coimmunoprecipitation assay revealed a physical interaction between NMIIB and nestin after PDBu/SB203580 treatment of NNVMs. In wild-type and heterozygous NMIIB embryonic hearts at E11.5-E14.5 days, nestin immunoreactivity was identified in a subpopulation of cardiomyocytes elongating perpendicular to the compact myocardium, at the leading edge of nascent trabeculae and during thickening of the compact myocardium. In mutant embryonic hearts lacking NMIIB protein expression, trabeculae formation was reduced, the compact myocardium significantly thinner and nestin immunoreactivity undetectable in cardiomyocytes at E14.5 days. These data suggest that NMIIB and nestin may act in a coordinated fashion to facilitate the acquisition of a migratory phenotype in neonatal and embryonic cardiomyocytes.

MD1 deletion exaggerates cardiomyocyte autophagy induced by heart failure with preserved ejection fraction through ROS/MAPK signalling pathway.

In our previous studies, we reported that myeloid differentiation protein 1 (MD1) serves as a negative regulator in several cardiovascular diseases. However, the role of MD1 in heart failure with preserved ejection fraction (HFpEF) and the underlying mechanisms of its action remain unclear. Eight‐week‐old MD1‐knockout (MD1‐KO) and wild‐type (WT) mice served as models of HFpEF induced by uninephrectomy, continuous saline or d‐aldosterone infusion and a 1.0% sodium chloride treatment in drinking water for 4 weeks to investigate the effect of MD1 on HFpEF in vivo. H9C2 cells were treated with aldosterone to evaluate the role of MD1 KO in vitro. MD1 expression was down‐regulated in the HFpEF mice; HFpEF significantly increased the levels of intracellular reactive oxygen species (ROS) and promoted autophagy; and in the MD1‐KO mice, the HFpEF‐induced intracellular ROS and autophagy effects were significantly exacerbated. Moreover, MD1 loss activated the p38‐MAPK pathway both in vivo and in vitro. Aldosterone‐mediated cardiomyocyte autophagy was significantly inhibited in cells pre‐treated with the ROS scavenger N‐acetylcysteine (NAC) or p38 inhibitor SB203580. Furthermore, inhibition with the autophagy inhibitor 3‐methyladenine (3‐MA) offset the aggravating effect of aldosterone‐induced autophagy in the MD1‐KO mice and cells both in vivo and in vitro. Our results validate a critical role of MD1 in the pathogenesis of HFpEF. MD1 deletion exaggerates cardiomyocyte autophagy in HFpEF via the activation of the ROS‐mediated MAPK signalling pathway.

Mesenchymal Stem Cells Attenuated Blood-Brain Barrier Disruption via Downregulation of Aquaporin-4 Expression in EAE Mice

Blood-brain barrier disruption is one of the hallmarks of multiple sclerosis. Mesenchymal stem cells showed great potential for the multiple sclerosis therapy. However, the effect of mesenchymal stem cells on blood-brain barrier in multiple sclerosis remains unclear. Here, we investigated whether mesenchymal stem cells transplantation protected blood-brain barrier integrity and further explored possible underlying mechanisms. Adult female C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein peptide33-55 (MOG33-55) to induce experimental autoimmune encephalomyelitis (EAE). Mesenchymal stem cells (5 × 105) were transplanted via tail vein at disease onset. In the cell culture, we examined lipopolysaccharide-induced AQP4 upregulation in astrocytes. Results indicated that mesenchymal stem cells therapy improved neurobehavioral outcomes in EAE mice, reduced inflammatory cell infiltration, IgG protein leakage, and demyelination in spinal cord. Mesenchymal stem cells therapy also increased tight junction protein expression. In addition, mesenchymal stem cells downregulated AQP4 and A2B adenosine receptor (A2BAR) expression in EAE mice in spinal cord. We found that MSCs-conditioned medium (MCM) reduced the expression of inflammatory cytokines, AQP4 and A2BAR in lipopolysaccharide-activated astrocytes. BAY-60-6583 (a selective A2BAR agonist) reversed the MCM-induced AQP4 downregulation and increased p38 MAPK phosphorylation. Furthermore, the upregulation effects of A2BAR agonist were eliminated when treated with p38 MAPK inhibitor SB203580. Thus, we concluded that mesenchymal stem cells alleviated blood-brain barrier disruption by downregulating AQP4 in multiple sclerosis, possibly through inhibiting the A2BAR/p38 MAPK signaling pathway. Our work suggests that mesenchymal stem cells exert beneficial effect through maintaining blood-brain barrier integrity in EAE mice.

MiR-34c downregulation leads to SOX4 overexpression and cisplatin resistance in nasopharyngeal carcinoma

BackgroundA major cause of disease-related death in nasopharyngeal carcinoma (NPC) is the development of distant metastasis (DM) despite combination chemoradiotherapy treatment. We previously identified and validated a four microRNA (miRNA) signature that is prognostic for DM. In this study, characterization of a key component of this signature, miR-34c, revealed its role in chemotherapy resistance.MethodsTwo hundred forty-six NPC patient biopsy samples were subject to comprehensive miRNA profiling and immunohistochemistry (IHC). Two human normal nasopharyngeal cell lines (immortalized; NP69 and NP460), as well as the NPC cell line C666–1, were used for miR-34c gain-of-function and loss-of-function experiments. Signaling pathways were assessed using quantitative real-time PCR (qRT-PCR) and Western blot. Cell viability was measured using the ATPlite assay.ResultsMiR-34c was downregulated in NPC patient samples, and confirmed in vitro to directly target SOX4, a master regulator of epithelial-to-mesenchymal transition (EMT). MiR-34c downregulation triggered EMT-representative changes in NP69 and NP460 whereby Snail, ZEB1, CDH2, and SOX2 were upregulated, while Claudin-1 and CDH1 were downregulated. Phenotypically, inhibition of miR-34c led to cisplatin resistance, whereas miR-34c over-expression sensitized NPC cells to cisplatin. TGFβ1 decreased miR-34c and increased SOX4 expression in vitro. The TGFβ receptor 1 inhibitor SB431542 reduced SOX4 expression and increased cisplatin sensitivity. Finally, IHC revealed that lower SOX4 expression was associated with improved overall survival in chemotherapy-treated NPC patients.ConclusionmiR-34c is downregulated in NPC. Repression of miR-34c was shown to increase SOX4 expression, which leads to cisplatin resistance, while TGFβ1 was found to repress miR-34c expression. Taken together, our study demonstrates that inhibition of the TGFβ1 pathway could be a strategy to restore cisplatin sensitivity in NPC.

693 p38 signaling regulates human cutaneous metastatic melanoma (MM) invasion and MM-dependent disruption of keratinocyte differentiation

Advancing our understanding of MM invasion mechanisms is vital for developing new mechanism-based therapies and improving MM outcomes. Here we describe an optimized organotypic human skin equivalent co-culture system of primary epidermal keratinocytes, MM cells, and stromal fibroblasts recently developed in our laboratory in order to reliably model early invasive behavior of MM as well as melanoma-keratinocyte crosstalk in the tissue microenvironment that more accurately reflects human disease pathology. p38 kinases p38α/δ are the predominant p38 isoforms in keratinocytes, while p38α/β are the most abundant p38s in MM cells. However, the roles of p38 kinases in regulation of human cutaneous MM invasion or in control of melanoma-keratinocyte communication remain to be elucidated. Our data showed that in human skin equivalents harboring human A375 MM cells, pharmacologic inhibition of p38α/β isoforms with specific inhibitor SB203580 led to increased invasion of A375 cells into the dermis, as manifest by significantly increased size of the dermal nests of A375 cells relative to size of those in control vehicle-treated cultures. The hyper-invasive MM phenotype observed in skin equivalents treated with SB203580 was reversed by treatment with potent pan-p38 inhibitor Compound 62 back to the levels displayed by the control cultures. These data suggest that p38α/β function to restrict MM invasion, and support a role for keratinocyte p38δ in promoting MM invasion in this model system. Furthermore, reflecting effect of melanoma on keratinocyte differentiation as observed in human disease, skin equivalents harboring A375 MM cells displayed a marked disruption of keratinocyte differentiation program as evidenced by reduced cornification, absence of granular layer, and severely diminished expression of differentiation markers. Pan-p38 inhibition partially restored keratinocyte differentiation, supporting a role for p38 signaling in MM-dependent loss of the latter in this system.

Combination of Dichloroacetate and Atorvastatin Regulates Excessive Proliferation and Oxidative Stress in Pulmonary Arterial Hypertension Development via p38 Signaling.

Pulmonary arterial hypertension (PAH) is a lethal disease generally characterized by pulmonary artery remodeling. Mitochondrial metabolic disorders have been implicated as a critical regulator of excessively proliferative- and apoptosis-resistant phenotypes in pulmonary artery smooth muscle cells (PASMCs). Dichloroacetate (DCA) is an emerging drug that targets aerobic glycolysis in tumor cells. Atorvastatin (ATO) is widely used for hyperlipemia in various cardiovascular diseases. Considering that DCA and ATO regulate glucose and lipid metabolism, respectively, we hypothesized that the combination of DCA and ATO could be a potential treatment for PAH. A notable decrease in the right ventricular systolic pressure accompanied by reduced right heart hypertrophy was observed in the DCA/ATO combination treatment group compared with the monocrotaline treatment group. The DCA/ATO combination treatment alleviated vascular remodeling, thereby suppressing excessive PASMC proliferation and macrophage infiltration. In vitro, both DCA and ATO alone reduced PASMC viability by upregulating oxidative stress and lowering mitochondrial membrane potential. Surprisingly, when combined, DCA/ATO was able to decrease the levels of reactive oxygen species and cell apoptosis without compromising PASMC proliferation. Furthermore, suppression of the p38 pathway through the specific inhibitor SB203580 attenuated cell death and oxidative stress at a level consistent with that of DCA/ATO combination treatment. These observations suggested a complementary effect of DCA and ATO on rescuing PASMCs from a PAH phenotype through p38 activation via the regulation of mitochondrial-related cell death and oxidative stress. DCA in combination with ATO may represent a novel therapeutic strategy for PAH treatment.

Correction: The Natural Pesticide Dihydrorotenone Induces Human Plasma Cell Apoptosis by Triggering Endoplasmic Reticulum Stress and Activating p38 Signaling Pathway.

[This corrects the article DOI: 10.1371/journal.pone.0069911.].