Sepsis is the leading cause of death in intensive care units worldwide. Current treatments of sepsis are largely supportive and clinical trials using specific pharmacotherapy for sepsis have failed ...to improve outcomes. Here, we used the lipopolysaccharide (LPS)-stimulated mouse RAW264.7 cell line and AlphaLisa assay for TNFa as a readout to perform a supervised drug repurposing screen for sepsis treatment with compounds targeting epigenetic enzymes, including kinases. We identified the SCH772984 compound, an extracellular signal-regulated kinase (ERK) 1/2 inhibitor, as an effective blocker of TNFa production in vitro. RNA-Seq of the SCH772984-treated RAW264.7 cells at 1, 4, and 24 h time points of LPS challenge followed by functional annotation of differentially expressed genes highlighted the suppression of cellular pathways related to the immune system. SCH772984 treatment improved survival in the LPS-induced lethal endotoxemia and cecal ligation and puncture (CLP) mouse models of sepsis, and reduced plasma levels of Ccl2/Mcp1. Functional analyses of RNA-seq datasets for kidney, lung, liver, and heart tissues from SCH772984-treated animals collected at 6 h and 12 h post-CLP revealed a significant downregulation of pathways related to the immune response and platelets activation but upregulation of the extracellular matrix organization and retinoic acid signaling pathways. Thus, this study defined transcriptome signatures of SCH772984 action in vitro and in vivo, an agent that has the potential to improve sepsis outcome.
Tyrosine phosphorylation is one of the basic mechanisms for signal transduction in the cell. Receptors exhibiting tyrosine kinase activity are widely involved in carcinogenesis and are negatively ...regulated by receptor protein tyrosine phosphatases (RPTP). Genes encoding different RPTPs are affected by aberrant epigenetic regulation in cancer. PTPRH (SAP-1) has been previously described to be overexpressed in colorectal cancer (CRC) and classified as an oncogenic factor. Previous microarray-based mRNA expression comparison of colorectal adenomas (AD), CRC and normal mucosa samples (NM) demonstrated that PTPRH tumor expression is the most reduced of all RPTP genes. qRT-PCR validation revealed gene downregulation for CRC (7.6-fold-change; P<0.0001) and AD (3.4-fold-change; P<0.0001) compared to NM. This was confirmed by immunohistochemical staining of tumor and NM sections as pronounced decrease of protein expression was observed in CRCs compared to the corresponding normal tissue. DNA methylation of two PTPRH promoter fragments was analyzed by pyrosequencing in a group of CRC, and AD patients as well as NM samples and CRC cell lines. The mean DNA methylation levels of these two regions were significantly higher in CRC than in NM. Both regions were highly methylated in SW480 and HCT116 cell lines contrary to unmethylated HT29 and COLO205. Cell lines with highly methylated promoters notably showed lower PTPRH expression levels, lower RNA II polymerase concentrations and higher levels of H3K27 trimethylation in the promoter and gene body, measured by chromatin immunoprecipitation. Cells were cultured with 5-aza-deoxycitidine and an increase in PTPRH expression was observed in SW480 and HCT116, whereas this was unchanged in the unmethylated cell lines. The results indicate that PTPRH is downregulated in colorectal tumors and its expression is epigenetically regulated via DNA methylation and chromatin modifications.
The family of PIM serine/threonine kinases includes three highly conserved oncogenes,
and
, which regulate multiple prosurvival pathways and cooperate with other oncogenes such as
. Recent genomic ...CRISPR-Cas9 screens further highlighted oncogenic functions of PIMs in diffuse large B-cell lymphoma (DLBCL) cells, justifying the development of small-molecule PIM inhibitors and therapeutic targeting of PIM kinases in lymphomas. However, detailed consequences of PIM inhibition in DLBCL remain undefined. Using chemical and genetic PIM blockade, we comprehensively characterized PIM kinase-associated prosurvival functions in DLBCL and the mechanisms of PIM inhibition-induced toxicity. Treatment of DLBCL cells with SEL24/MEN1703, a pan-PIM inhibitor in clinical development, decreased BAD phosphorylation and cap-dependent protein translation, reduced MCL1 expression, and induced apoptosis. PIM kinases were tightly coexpressed with MYC in diagnostic DLBCL biopsies, and PIM inhibition in cell lines and patient-derived primary lymphoma cells decreased MYC levels as well as expression of multiple MYC-dependent genes, including
. Chemical and genetic PIM inhibition upregulated surface CD20 levels in an MYC-dependent fashion. Consistently, MEN1703 and other clinically available pan-PIM inhibitors synergized with the anti-CD20 monoclonal antibody rituximab
, increasing complement-dependent cytotoxicity and antibody-mediated phagocytosis. Combined treatment with PIM inhibitor and rituximab suppressed tumor growth in lymphoma xenografts more efficiently than either drug alone. Taken together, these results show that targeting PIM in DLBCL exhibits pleiotropic effects that combine direct cytotoxicity with potentiated susceptibility to anti-CD20 antibodies, justifying further clinical development of such combinatorial strategies. SIGNIFICANCE: These findings demonstrate that inhibition of PIM induces DLBCL cell death via MYC-dependent and -independent mechanisms and enhances the therapeutic response to anti-CD20 antibodies by increasing CD20 expression.
Inhibition of oncogenic transcriptional programs is a promising therapeutic strategy. A substituted tricyclic benzimidazole, SEL120-34A, is a novel inhibitor of Cyclin-dependent kinase 8 (CDK8), ...which regulates transcription by associating with the Mediator complex. X-ray crystallography has shown SEL120-34A to be a type I inhibitor forming halogen bonds with the protein's hinge region and hydrophobic complementarities within its front pocket. SEL120-34A inhibits phosphorylation of STAT1 S727 and STAT5 S726 in cancer cells in vitro. Consistently, regulation of STATs- and NUP98-HOXA9- dependent transcription has been observed as a dominant mechanism of action in vivo. Treatment with the compound resulted in a differential efficacy on AML cells with elevated STAT5 S726 levels and stem cell characteristics. In contrast, resistant cells were negative for activated STAT5 and revealed lineage commitment. In vivo efficacy in xenotransplanted AML models correlated with significant repression of STAT5 S726. Favorable pharmacokinetics, confirmed safety and in vivo efficacy provide a rationale for the further clinical development of SEL120-34A as a personalized therapeutic approach in AML.
Expression of ZmCPK11, a member of the maize (Zea mays) calcium‐dependent protein kinases (CDPKs) family, is induced by mechanical wounding. A rapid increase of the activity of a 56‐kDa CDPK has been ...observed in damaged leaves. In the present work, it is shown that the 56‐kDa CDPK, identified as ZmCPK11, is also activated in non‐wounded leaves as an element of systemic wound response. Moreover, an increase of the enzyme's activity and induction of ZmCPK11 expression was observed after touching the leaves. To study the role of ZmCPK11 in wound and touch signaling, transgenic Arabidopsis thaliana plants in which c‐Myc‐ZmCPK11 was expressed under control of the CaMV 35S promoter were generated. Analysis of the transgenic plants showed that c‐Myc‐ZmCPK11 was activated upon wounding and touching. Furthermore, pre‐treatment with acetylsalicylic acid (acSA), an inhibitor of jasmonic acid (JA)‐dependent wound signaling, abolished the wound‐induced activation of ZmCPK11 in maize and the transgenic A. thaliana plants. Methyl jasmonate (MeJA) and linolenic acid (LA) stimulated the activity of ZmCPK11 as well as induced the expression of ZmCPK11 and other wound‐responsive genes, lipoxygenase 1 (ZmLOX1) and proteinase inhibitor 1 (ZmWIP1). These results indicate that ZmCPK11, regulated at the enzymatic and transcriptional level by LA and MeJA, is a component of touch‐ and wound‐induced pathway(s), participating in early stages of local and systemic responses.
Carcinogenesis involves altered cellular interaction and tissue morphology that partly arise from aberrant expression of cadherins. Mucin-like protocadherin is implicated in intercellular adhesion ...and its expression was found decreased in colorectal cancer (CRC). This study has compared MUPCDH (CDHR5) expression in three key types of colorectal tissue samples, for normal mucosa, adenoma, and carcinoma. A gradual decrease of mRNA levels and protein expression was observed in progressive stages of colorectal carcinogenesis which are consistent with reports of increasing MUPCDH 5′ promoter region DNA methylation. High MUPCDH methylation was also observed in HCT116 and SW480 CRC cell lines that revealed low gene expression levels compared to COLO205 and HT29 cell lines which lack DNA methylation at the MUPCDH locus. Furthermore, HCT116 and SW480 showed lower levels of RNA polymerase II and histone H3 lysine 4 trimethylation (H3K4me3) as well as higher levels of H3K27 trimethylation at the MUPCDH promoter. MUPCDH expression was however restored in HCT116 and SW480 cells in the presence of 5-Aza-2′-deoxycytidine (DNA methyltransferase inhibitor). Results indicate that μ-protocadherin downregulation occurs during early stages of tumourigenesis and progression into the adenoma-carcinoma sequence. Epigenetic mechanisms are involved in this silencing.
TNF-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane protein capable of selectively inducing apoptosis in cancer cells by binding to its cognate receptors. Here, we examined the ...anticancer efficacy of a recently developed chimeric AD-O51.4 protein, a TRAIL fused to the VEGFA-originating peptide. We tested AD-O51.4 protein activity against human colorectal cancer (CRC) models and investigated the resistance mechanism in the non-responsive CRC models. The quantitative comparison of apoptotic activity between AD-O51.4 and the native TRAIL in nine human colorectal cancer cell lines revealed dose-dependent toxicity in seven of them; the immunofluorescence-captured receptor abundance correlated with the extent of apoptosis. AD-O51.4 reduced the growth of CRC patient-derived xenografts (PDXs) with good efficacy. Cell lines that acquired AD-O51.4 resistance showed a significant decrease in surface TRAIL receptor expression and apoptosis-related proteins, including Caspase-8, HSP60, and p53. These results demonstrate the effectiveness of AD-O51.4 protein in CRC preclinical models and identify the potential mechanism underlying acquired resistance. Progression of AD-O51.4 to clinical trials is expected.
The aim of this study was to evaluate the effect of the foliar fertilizer InCa on the chemical composition of fruits and leaves of sweet cherry cultivar ‘Burlat’. The study involved 14-year old sweet ...cherry trees of ‘Burlat’ cultivar, grafted onto ‘PHL-A’ rootstock and growing at 4×3 m spacing. The trees were sprayed with InCa calcium fertilizer based on CaT technology (manufacturer: Plant Impact). The fertilizer was applied three times, at a dose of 1.5 dm3∙ha. The first foliar fertilization was performed at the beginning of the flowering period, and the next two took place at fourteen-day intervals. The fruits were analysed for nitrate content, and both fruits and leaves were considered for dry weight and macro- and micronutrient analysis. The use of InCa fertilizer improved the dry matter and calcium content and reduced potassium, magnesium and cadmium level in the leaves and fruits of sweet cherry. The fertilizer facilitated narrowing K:Ca and K:(Ca + Mg) ratioes and extending of K:Mg, and especially Ca:Mg ratio in both leaves and fruits. Foliar supplementation with the investigated preparation resulted in an increase in nitrogen and manganese content, and a drop in phosphorus, copper, nickel and lead concentration in the leaves of the studied sweet cherry cultivar.
Three bioactive fractions isolated from Cerrena unicolor cultures-crude endopolysaccharide (c-EPS), laccase, and a subfraction of low-molecular weight secondary metabolites-were used to determine ...potential cytotoxic effects on the mouse melanoma B16-F10 cell line (American Type Culture Collection CRL-6475). The results obtained prove that all examined fractions exhibited activity against the investigated tumor cells. In addition, an evident immunomodulatory effect of the c-EPS fraction was observed. Our results show that the levels of 2 cytokines (tumor necrosis factor-a and chemokine ligand 2) in mouse inner medullary collecting duct mIMCD-3 cells (American Type Culture Collection CRL-2123) stimulated by c-EPS were significantly higher. A lipopolysaccharide model was used at the same concentration (10 μg/mL) as a positive control.
Prenyl ammonium iodides (Amino-Prenols, APs), semi-synthetic polyprenol derivatives were studied as prospective novel gene transfer agents.
AP-7, -8, -11 and -15 (aminoprenols composed of 7, 8, 11 or ...15 isoprene units, respectively) were examined for their capacity to form complexes with pDNA, for cytotoxicity and ability to transfect genes to cells.
All the carriers were able to complex DNA. The highest, comparable to commercial reagents, transfection efficiency was observed for AP-15. Simultaneously, AP-15 exhibited the lowest negative impact on cell viability and proliferation--considerably lower than that of commercial agents. AP-15/DOPE complexes were also efficient to introduce pDNA to cells, without much effect on cell viability. Transfection with AP-15/DOPE complexes influenced the expression of a very few among 44 tested genes involved in cellular lipid metabolism. Furthermore, complexes containing AP-15 and therapeutic plasmid, encoding the TIMP metallopeptidase inhibitor 2 (TIMP2), introduced the TIMP2 gene with high efficiency to B16-F10 melanoma cells but not to B16-F10 melanoma tumors in C57BL/6 mice, as confirmed by TIMP2 protein level determination.
Obtained results indicate that APs have a potential as non-viral vectors for cell transfection.
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