Search

Simple Summary: Ovarian cancer (OVCA) is a fatal gynecological disease for which there is no early detection test. Glucose-regulated protein 78 (GRP78), a protein marker of stress, increases during chronic stress. Chronic stress has been suggested as a hallmark of cancer development. This study examined whether expression of GRP78 is associated with development of OVCA and whether GRP78 can predict OVCA at early stage. This study found GRP78 expression and its secretion in blood increased during OVCA development and progression. This study also developed a GRP78-targeted ultrasound scanning agent that detected ovarian tumors at early stages. Thus, a woman with high levels of GRP78 in her blood may be referred to have targeted-ultrasound scanning for confirming if she has ovarian tumors. These results will be a foundation for a clinical study to examine the feasibility of GRP78 as a potential marker of blood and ultrasound scanning for early detection of OVCA. Background: Understanding malignant transformation associated with ovarian cancer (OVCA) is important to establish early detection tests. This study examined whether expression of glucose-regulated protein 78 (GRP78, marker of cellular stress) increases during OVCA development, and whether GRP78 can be detected by targeted-transvaginal ultrasound (TVUS) imaging. Methods: Normal ovaries (n = 10), benign (n = 10) and malignant ovarian tumors at early (n = 8) and late stages (n = 16), hens with and without ovarian tumors at early and late stages (n = 10, each) were examined for GRP78 expression during OVCA development by immunohistochemistry, immunoblotting, gene expression and immunoassay. Feasibility of GRP78-targeted TVUS imaging in detecting early OVCA was examined. Results: Compared with normal ovaries and benign tumors, intensity of GRP78 expression was higher (p < 0.0001) in OVCA patients. Compared with normal (9007.76 ± 816.54 pg/mL), serum GRP78 levels were significantly higher (p < 0.05) in patients with early (12,730.59 ± 817.35 pg/mL) and late-stage OVCA (13,930.12 ± 202.35) (p < 0.01). Compared with normal (222.62 ± 181.69 pg/mL), serum GRP78 levels increased (p < 0.05) in hens with early (590.19 ± 198.18 pg/mL) and late-stage OVCA (1261.38 ± 372.85) (p < 0.01). Compared with non-targeted, GRP78-targeted imaging enhanced signal intensity of TVUS (p < 0.0001). Conclusions: Tissue and serum levels of GRP78 increase in association with OVCA. GRP78 offers a potential serum and imaging marker for early OVCA detection. [ABSTRACT FROM AUTHOR]

Long noncoding (lnc) RNAs regulate cancer progression. However, the importance of lncRNAs and how they are regulated in colorectal cancer (CRC) are unclear. We aim to evaluate the function of lncRNA ADAMTS9-AS2 in CRC and its fundamental mechanism. Levels of ADAMTS9-AS2, miR-27a-3p, and B-cell translocation gene 2 (BTG2) were measured by qPCR. Cell viability was analyzed by CCK-8 and colony formation. Migration and invasion were tested by transwell assay. The interactions among ADAMTS9-AS2, miR-27a-3p, BTG2, and YTHDF2 were analyzed by luciferase test, immunoblotting, RNA pull-down, or RNA immunoprecipitation (RIP). An animal model was adopted to assess ADAMTS9-AS2's function. Overexpressing ADAMTS9-AS2 inhibited cell migration, invasion, colony formation capacity, and proliferation in vitro. The direct targeting of miR-27a-3p by ADAMTS9-AS2 abrogated the latter's effect in CRC cells. BTG2 was identified a target of miR-27a-3p, and silencing BTG2 weakened miR-27a-3p's effect. Knocking down ADAMTS9-AS2 abolished sh-YTHDF2's inhibitory effect on cell proliferation and invasion. Finally, overexpressing ADAMTS9-AS2 restrained xenograft growth. M6A reader YTHDF2-mediated degradation of ADAMTS9-AS2 promotes colon carcinogenesis via miR-27a-3p/BTG2 axis. [ABSTRACT FROM AUTHOR]

Altered lipid metabolism is a hallmark of hepatocellular carcinoma (HCC), a common malignancy with a dismal prognosis against which there is a lack of effective therapeutic strategies. Bufalin, a classical Na + -K + -ATPase (NKA) inhibitor, shows a potent antitumor effect against HCC. However, the role of bufalin in regulating lipid metabolism-related pathways of HCC remains unclear. In this study, we examined the interaction between bufalin and its target molecule, ATP1A1/CA2, in vitro and in vivo and explored the intersected downstream pathways in silico. A multi-omics analysis of transcriptomics and metabolomics was employed to screen for potential action targets. The results were verified and correlated with the downstream lipid de novo synthesis pathway and the bufalin/ATP1A1/CA2 axis. We found that bufalin suppressed the ATP1A1/CA2 ratio in the treated HCC cells and showed a negative correlation with bufalin drug sensitivity. Functionally, ATP1A1 overexpression and CA2 down-regulation inhibited the bufalin-suppressed HCC proliferation and metastasis. Furthermore, down-regulation of CA2 induced epithelial-mesenchymal transition and bufalin resistance in HCC cells by up-regulating ATP1A1. Mechanistically, lipid metabolism-related signaling pathways were enriched in low ATP1A1 and high CA2 expression subgroups in GSEA. The multi-omics analysis also showed that bufalin was closely related to lipid metabolism. We demonstrated that bufalin inhibits lipogenesis and tumorigenesis by down-regulating SREBP-1/FASN/ACLY via modulating the ATP1A1/CA2 axis in HCC. [ABSTRACT FROM AUTHOR]

Objective To analyse pro-survival mechanisms elicited in RA synovial fibroblasts (RASFs) upon detachment from their extracellular matrix dependent on the disintegrin metalloproteinase ADAM15 and Yes-associated protein kinase 1 (YAP1). Methods Detachment-induced apoptosis was determined by caspase 3/7 assays. Immunofluorescent stainings, cell surface biotinylation and immunoblotting were applied to analyse phosphorylated kinases and subcellular localization of YAP1 and connective tissue growth factor (CTGF). Caspase and transwell transmigration assays served to study CTGF function. Results Silencing of ADAM15 or YAP1 in RASFs leads to significantly increased levels of detachment-induced caspase activity. In non-silenced RASFs detachment causes simultaneous ADAM15-enhanced phosphorylation of YAP1 at S127, known for promoting its cytoplasmic localization, and Src-dependent phosphorylation at tyrosine Y357. The majority of nuclear YAP1 leaves the nucleus shortly after cell detachment, but prolonged detachment causes a marked nuclear re-entry of YAP1, resulting in significantly increased synthesis of CTGF. The newly synthesized CTGF, however, is not detectable in the supernatant, but is bound to the outside of the plasma membrane. In vitro studies demonstrated autocrine binding of CTGF to the EGF receptor and β1 integrin, with concomitant triggering of survival kinases, AKT1, ERK1/2, Src and focal adhesion kinase. Functional studies revealed anti-apoptotic effects of CTGF on detached RASFs and an enhancement of their potential for endothelial transmigration using HUVEC-coated transwells. Conclusion The elucidation of a new molecular mechanism that protects RASFs in the highly pro-apoptotic environment of inflamed RA joints by promoting anoikis-resistance and transendothelial migration via ADAM15/YAP1-mediated CTGF upregulation uncovers potentially new targets for future therapeutic intervention. [ABSTRACT FROM AUTHOR]

Simple Summary: The compounds named PROTACs are formed by two fragments, which bring together a particular protein with a ubiquitinating enzyme. This allows ubiquitination and degradation of the targeted protein. Ubiquitination-mediated protein degradation is an important regulatory process to control the expression levels of proteins and maintain the homeostatic conditions in cells. Thus, by re-directing a mechanism that is normally used for cell regulation, PROTACs allow to remove specific proteins associated to particular diseases or pathological conditions. In this paper, we report a type of PROTAC that targets for degradation the protein named p38α, whose functions have been linked to cancer and other diseases. We show that these PROTACs effectively reduce p38α protein expression not only in several cancer cell lines but also in tumors generated in mice. These compounds may provide an attractive strategy to evaluate potential therapeutic applications for targeting p38α in the clinical context. We report a series of small molecule proteolysis-targeting chimeras (PROTACs) that target the protein kinase p38α for degradation. These PROTACs are based on a ligand of the VHL E3 ubiquitin ligase, which is linked to an ATP competitive inhibitor of p38α. We provide evidence that these compounds can induce the specific degradation of p38α, but not p38β and other related kinases, at nanomolar concentrations in several mammalian cell lines. We also show that the p38α-specific PROTACs are soluble in aqueous solutions and therefore suitable for their administration to mice. Systemic administration of the PROTACs induces p38α degradation only in the liver, probably due to the PROTAC becoming inactivated in that organ, but upon local administration the PROTACs induce p38α degradation in mammary tumors. Our compounds provide an alternative to traditional chemical inhibitors for targeting p38α signaling in cultured cells and in vivo. [ABSTRACT FROM AUTHOR]

The promising antitumor effects of progesterone derivatives have been identified in many studies. However, the specific mechanism of action of this class of compounds has not been fully described. Therefore, in this study, we investigated the antiproliferative and (anti)estrogenic activities of novel pentacyclic derivatives and benzylidenes of the progesterone series. The antiproliferative effects of the compounds were evaluated on hormone-dependent MCF7 breast cancer cells using the MTT test. Estrogen receptor α (ERα) activity was assessed by a luciferase-based reporter assay. Immunoblotting was used to evaluate the expression of signaling proteins. All benzylidenes demonstrated inhibitory effects with IC50 values below 10 µM, whereas pentacyclic derivatives were less active. These patterns may be associated with the lability of the geometry of benzylidene molecules, which contributes to an increase in the affinity of interaction with the receptor. The selected compounds showed significant anti-estrogenic potency. Benzylidene 1d ((8 S,9 S,10R,13 S,14 S,17 S)-17-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-10,13-dimethyl-1,2,6,7,8,9,11,12,14,15-decahydrocyclopenta[a]phenanthren-3-one) was the most active in antiproliferative and anti-estrogenic assays. Apoptosis induced by compound 1d was accompanied by decreases in CDK4, ERα, and Cyclin D1 expression. Compounds 1d and 3d were characterized by high inhibitory potency against resistant breast cancer cells. Apoptosis induced by the leader compounds was confirmed by PARP cleavage and flow cytometry analysis. Compound 3d caused cell arrest in the G2/M phase. Further analysis of novel derivatives of the progesterone series is of great importance for medicinal chemistry, drug design, and oncology. [ABSTRACT FROM AUTHOR]

Simple Summary: Libertellenone T (B) is a natural product derived from the secondary metabolites of the endolichenic fungus, Pseudoplectania sp. In this study, we investigated the underlying molecular mechanisms that induce the apoptotic cell death of the colorectal cancer cell line, Caco2, in response to B. Our findings demonstrate that B induces Caco2 cell apoptosis via G2/M phase arrest and activation of ROS/JNK signaling. Moreover, B exhibited excellent synergistic effects when combined with the known and novel anticancer agents 5-FU and compound D, respectively. In light of this investigation, we propose that B is a promising potential chemotherapeutic agent against colorectal cancer. Colorectal cancer (CRC) is the third most deadly type of cancer in the world and continuous investigations are required to discover novel therapeutics for CRC. Induction of apoptosis is one of the promising strategies to inhibit cancers. Here, we have identified a novel compound, Libertellenone T (B), isolated from crude extracts of the endolichenic fungus from Pseudoplectania sp. (EL000327) and investigated the mechanism of action. CRC cells treated by B were subjected to apoptosis detection assays, immunofluorescence imaging, and molecular analyses such as immunoblotting and QRT-PCR. Our findings revealed that B induced CRC cell death via multiple mechanisms including G2/M phase arrest caused by microtubule stabilization and caspase-dependent apoptosis. Further studies revealed that B induced the generation of reactive oxygen species (ROS) attributed to activating the JNK signaling pathway by which apoptosis and autophagy was induced in Caco2 cells. Moreover, B exhibited good synergistic effects when combined with the well-known anticancer drug, 5-FU, and another cytotoxic novel compound D, which was isolated from the same crude extract of EL000327. Overall, Libertellenone T induces G2/M phase arrest, apoptosis, and autophagy via activating the ROS/JNK pathway in CRC. Thus, B may be a potential anticancer therapeutic against CRC that is suitable for clinical applications. [ABSTRACT FROM AUTHOR]

Simple Summary: Triple-negative breast cancer (TNBC) is an aggressive type of cancer that is challenging to treat due to the lack of hormonal receptors used to target cancer cells; due to this, TNBC patients have high mortality rates. Plant-derived compounds are being sought as potential adjuvants for common chemotherapy drugs, such as paclitaxel (Pac). To this end, this research aimed to study the prenylated stilbenoid arachidin-1 (A-1) as a potential adjuvant for Pac. Here, the cytotoxic and apoptosis induction effects of A-1 alone and combined with Pac were investigated in 2D TNBC cell cultures and a 3D TNBC spheroid model. The results illustrated that A-1, in low micromolar concentrations, combined with Pac, inhibited cell proliferation, induced apoptosis, and reduced spheroid growth. Furthermore, our findings suggest that A-1 combined with Pac has the potential to be developed as a novel plant-derived treatment for TNBC. Triple-negative breast cancer (TNBC) is one of the deadliest forms of breast cancer. Investigating alternative therapies to increase survival rates for this disease is essential. To this end, the cytotoxic effects of the prenylated stilbenoids arachidin-1 (A-1) and arachidin-3 (A-3), and non-prenylated resveratrol (RES) were evaluated in human TNBC cell lines as potential adjuvants for paclitaxel (Pac). A-1, alone or in combination with Pac, showed the highest cytotoxicity in TNBC cells. Apoptosis was further evaluated by measuring key apoptosis marker proteins, cell cycle arrest, and intracellular reactive oxygen species (ROS) generation. Furthermore, the cytotoxic effect of A-1 combined with Pac was also evaluated in a 3D spheroid TNBC model. The results showed that A-1 decreased the Pac IC50 approximately 2-fold in TNBC cells. The synergistic combination of A-1 and Pac arrested cells in G2/M phase and activated p53 expression. In addition, the combined treatment increased intracellular ROS generation and induced apoptosis. Importantly, the combination of A-1 with Pac inhibited TNBC spheroid growth. Our results demonstrated that A-1 in combination with Pac inhibited cell proliferation, induced apoptosis through mitochondrial oxidative stress, and reduced TNBC spheroid growth. These findings underscore the impactful effects of the prenylated stilbenoid A-1 as a novel adjuvant for Pac chemotherapy in TNBC treatment. [ABSTRACT FROM AUTHOR]

20(S)-protopanaxadiol (PPD), a metabolite of Panax ginseng, has multiple pharmacological properties. However, the effects of PPD against human gastric cancer have not been elucidated. Our purpose in this study was to investigate if PPD has anticancer effects against human gastric cancer in vitro. Cell viability, migration, clone formation, and invasion were assessed to explore the effects of PPD on cancer cells. PI and annexin V staining as well as immunoblotting were employed to determine if PPD-induced apoptosis and autophagy of MKN1 and MKN45 cells. The target of PPD was identified using immunoblotting, overexpression analysis, and flow cytometric analysis. PPD exhibited significantly suppressed cell viability, migration, colony formation, and invasion. Phosphorylation of Src and its down-stream effectors were inhibited by PPD. PPD-enhanced apoptosis and autophagy in a dose- and time-dependent manner by inhibiting Src. Collectively, our results demonstrate that PPD induces apoptosis and autophagy in gastric cancer cells in vitro by inhibiting Src. [ABSTRACT FROM AUTHOR]

Objective To explore and define the molecular cause(s) of a multi-generational kindred affected by Bechet's-like mucocutaneous ulcerations and immune dysregulation. Methods Whole genome sequencing and confirmatory Sanger sequencing were performed. Components of the NFκB pathway were quantified by immunoblotting, and function was assessed by cytokine production (IL-6, TNF-α, IL-1β) after lipopolysaccharide (LPS) stimulation. Detailed immunophenotyping of T-cell and B-cell subsets was performed in four patients from this cohort. Results A novel variant in the RELA gene, p. Tyr349LeufsTer13, was identified. This variant results in premature truncation of the protein before the serine (S) 536 residue, a key phosphorylation site, resulting in enhanced degradation of the p65 protein. Immunoblotting revealed significantly decreased phosphorylated [p]p65 and pIκBα. The decrease in [p]p65 may suggest reduced heterodimer formation between p50/p65 (NFκB1/RelA). Immunophenotyping revealed decreased naïve T cells, increased memory T cells, and expanded senescent T-cell populations in one patient (P1). P1 also had substantially higher IL-6 and TNF-α levels post-stimulation compared with the other three patients. Conclusion Family members with this novel RELA variant have a clinical phenotype similar to other reported RELA cases with predominant chronic mucocutaneous ulceration; however, the clinical phenotype broadens to include Behçet's syndrome and IBD. Here we describe the clinical, immunological and genetic evaluation of a large kindred to further expand identification of patients with autosomal dominant RELA deficiency, facilitating earlier diagnosis and intervention. The functional impairment of the canonical NFκB pathway suggests that this variant is causal for the clinical phenotype in these patients. [ABSTRACT FROM AUTHOR]

Simple Summary: Long non-coding RNAs (lncRNAs) have emerged as a significant player in various cancers, including pancreatic cancer. However, how lncRNAs are aberrantly expressed in cancers is largely unknown. We hypothesized that lncRNAs would be regulated by signaling pathways and contribute to malignant phenotypes of cancer. In this study, to understand the significance of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) for the expression of lncRNAs in pancreatic cancer, we performed comparative transcriptome analyses between pancreatic cancer cell lines with or without activation of MAPK. We identified 45 lncRNAs presumably associated with MAPK in pancreatic cancer cells; among these, LINC00941 was consistently upregulated by MAPK. The promoter of LINC00941 was determined and found to be preferentially associated with MAPK activity via ETS-1 binding site. TCGA data analysis indicated that high expression of LINC00941 was associated with poor prognosis of pancreatic cancer patients. Downstream targets of LINC00941 were involved in 44 biological processes, including the glycoprotein biosynthetic process, beta-catenin-TCF complex assembly, and histone modification. These results indicate that MAPK mediates the aberrant expression of lncRNAs, and LINC00941 is the most consistently promoted lncRNA by MAPK in pancreatic cancer. Therefore, MAPK-associated lncRNAs might represent both potentially valid therapeutic targets and diagnostic biomarkers. Long non-coding RNAs (lncRNAs) have emerged as a significant player in various cancers, including pancreatic cancer. However, how lncRNAs are aberrantly expressed in cancers is largely unknown. We hypothesized that lncRNAs would be regulated by signaling pathways and contribute to malignant phenotypes of cancer. In this study, to understand the significance of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), which is a major aberrant signaling pathway in pancreatic cancer, for the expression of lncRNAs, we performed comparative transcriptome analyses between pancreatic cancer cell lines with or without activation of MAPK. We identified 45 lncRNAs presumably associated with MAPK in pancreatic cancer cells; among these, LINC00941 was consistently upregulated by MAPK. The immediate genomic upstream region flanking LINC00941 was identified as a promoter region, the activity of which was found to be preferentially associated with MAPK activity via ETS-1 binding site. LINC00941 promoted cell proliferation in vitro. Moreover, TCGA data analysis indicated that high expression of LINC00941 was associated with poor prognosis of patients with pancreatic cancer. Transcriptomes comparing transcriptions between cells with and without LINC00941 knockdown revealed 3229 differentially expressed genes involved in 44 biological processes, including the glycoprotein biosynthetic process, beta-catenin-TCF complex assembly, and histone modification. These results indicate that MAPK mediates the aberrant expression of lncRNAs. LINC00941 is the lncRNA by MAPK most consistently promoted, and is implicated in the dismal prognosis of pancreatic cancer. MAPK-associated lncRNAs may play pivotal roles in malignant phenotypes of pancreatic cancer, and as such might represent both potentially valid therapeutic targets and diagnostic biomarkers. [ABSTRACT FROM AUTHOR]

Simple Summary: Solid tumors account for ~60% of pediatric, as well as adolescent and young adult (AYA), cancers, and outcomes for patients with these progressive diseases remain poor. This highlights the critical need to develop tumor models from patients with aggressive cancers so that oncogenic signatures can be identified for therapeutic testing. Thus, patient-derived xenografts (PDXs) were established from sarcoma and Wilms tumor patients at diagnosis or following treatment. Overall, the molecular landscape of serially passaged PDXs recapitulated the original tumor based on an integrated multi-OMICS pipeline that cross-validated cancer-associated pathways. Actionable mechanisms of tumor progression were identified. CDK4/6 and BETs were prioritized as biomarkers of therapeutic response for in vivo validation. In osteosarcoma PDXs harboring pertinent molecular signatures, inhibition of CDK4/6 or BETs decreased growth. This systematic approach that links patient disease history to data generated from its corresponding PDX provides a foundation to discover improved therapies for patients with high-risk cancers. Establishment of clinically annotated, molecularly characterized, patient-derived xenografts (PDXs) from treatment-naïve and pretreated patients provides a platform to test precision genomics-guided therapies. An integrated multi-OMICS pipeline was developed to identify cancer-associated pathways and evaluate stability of molecular signatures in a panel of pediatric and AYA PDXs following serial passaging in mice. Original solid tumor samples and their corresponding PDXs were evaluated by whole-genome sequencing, RNA-seq, immunoblotting, pathway enrichment analyses, and the drug–gene interaction database to identify as well as cross-validate actionable targets in patients with sarcomas or Wilms tumors. While some divergence between original tumor and the respective PDX was evident, majority of alterations were not functionally impactful, and oncogenic pathway activation was maintained following serial passaging. CDK4/6 and BETs were prioritized as biomarkers of therapeutic response in osteosarcoma PDXs with pertinent molecular signatures. Inhibition of CDK4/6 or BETs decreased osteosarcoma PDX growth (two-way ANOVA, p < 0.05) confirming mechanistic involvement in growth. Linking patient treatment history with molecular and efficacy data in PDX will provide a strong rationale for targeted therapy and improve our understanding of which therapy is most beneficial in patients at diagnosis and in those already exposed to therapy. [ABSTRACT FROM AUTHOR]

Simple Summary: p90RSK is a downstream effector protein of the MAPK pathway. In many human cancers, the MAPK pathway is constitutively activated due to oncogenic mutations of its components. Consequently, p90RSK is hyperactive and capable of hyper phosphorylating substrates involved in tumorigenesis. p90RSK belongs to the AGC kinase family, which phosphorylates substrates with an RXRXXS/T consensus motif. MDM2 protein presents this consensus in its sequence at serine 166. MDM2 is a ubiquitin ligase that negatively regulates the protein stability of p53. The aim of this study has been to verify whether p90RSK is capable of phosphorylating MDM2 in serine 166 and to investigate the role of p90RSK in regulating the p53 pathway, especially in thyroid tumors in which the MAPK pathway is constitutively active and in which the development of specific drugs aimed at inhibiting the kinase activity of p90RSK could represent a new way of inhibiting tumorigenesis. The expression level of the tumor suppressor p53 is controlled by the E3 ubiquitin ligase MDM2 with a regulatory feedback loop, which allows p53 to upregulate its inhibitor MDM2. In this manuscript we demonstrated that p90RSK binds and phosphorylates MDM2 on serine 166 both in vitro and in vivo by kinase assay, immunoblot, and co-immunoprecipitation assay; this phosphorylation increases the stability of MDM2 which in turn binds p53, ubiquitinating it and promoting its degradation by proteasome. A pharmacological inhibitor of p90RSK, BI-D1870, decreases MDM2 phosphorylation, and restores p53 function, which in turn transcriptionally increases the expression of cell cycle inhibitor p21 and of pro-apoptotic protein Bax and downregulates the anti-apoptotic protein Bcl-2, causing a block of cell proliferation, measured by a BrdU assay and growth curve, and promoting apoptosis, measured by a TUNEL assay. Finally, an immunohistochemistry evaluation of primary thyroid tumors, in which p90RSK is very active, confirms MDM2 stabilization mediated by p90RSK phosphorylation. [ABSTRACT FROM AUTHOR]

Simple Summary: Acute myeloid leukemia (AML) with mutations in the tumor suppressor gene TP53 is rapidly lethal for most patients. Here, we investigated the preclinical activity of TP-0903, a multikinase inhibitor that inhibits kinases with potential synthetical lethality in TP53 mutant AML. TP-0903 inhibited cell viability and induced apoptosis in multiple TP53 mutant AML cell lines at nanomolar concentrations in vitro. TP-0903, both alone and in combination with decitabine, the current standard of care, improved survival in two xenograft models of TP53 mutant AML. These results demonstrate that TP-0903 has activity in AML with TP53 dysfunction and support the clinical evaluation of TP-0903 in combination with decitabine in TP53 mutant AML. Acute myeloid leukemia (AML) with mutations in the tumor suppressor gene TP53 confers a dismal prognosis with 3-year overall survival of <5%. While inhibition of kinases involved in cell cycle regulation induces synthetic lethality in a variety of TP53 mutant cancers, this strategy has not been evaluated in mutant TP53 AML. Previously, we demonstrated that TP-0903 is a novel multikinase inhibitor with low nM activity against AURKA/B, Chk1/2, and other cell cycle regulators. Here, we evaluated the preclinical activity of TP-0903 in TP53 mutant AML cell lines, including a single-cell clone of MV4-11 containing a TP53 mutation (R248W), Kasumi-1 (R248Q), and HL-60 (TP 53 null). TP-0903 inhibited cell viability (IC50, 12–32 nM) and induced apoptosis at 50 nM. By immunoblot, 50 nM TP-0903 upregulated pChk1/2 and pH2AX, suggesting induction of DNA damage. The combination of TP-0903 and decitabine was additive in vitro, and in vivo significantly prolonged median survival compared to single-agent treatments in mice xenografted with HL-60 (vehicle, 46 days; decitabine, 55 days; TP-0903, 63 days; combination, 75 days) or MV4-11 (R248W) (51 days; 62 days; 81 days; 89 days) (p < 0.001). Together, these results provide scientific premise for the clinical evaluation of TP-0903 in combination with decitabine in TP53 mutant AML. [ABSTRACT FROM AUTHOR]

Though the first discovery of TRPV6 channel expression in various tissues took place in the early 2000s, reliable tools for its protein detection in various cells and tissues are still missing. Here we show the generation and validation of rabbit polyclonal anti-TRPV6 channel antibodies (rb79–82) against four epitopes of 15 amino acids. Among them, only one antibody, rb79, was capable of detecting the full-length glycosylated form of the TRPV6 channel at around 100 kDa. The generated antibody was shown to be suitable for all in vitro applications, such as immunoblotting, immunoprecipitation, immunocytochemistry, immunofluorescence, etc. One of the most important applications is immunohistochemistry using the paraffin-embedded sections from cancer resection specimens. Using prostate cancer resection specimens, we have confirmed the absence of the TRPV6 protein in both healthy and benign hyperplasia, as well as its expression and correlation to the prostate cancer grades. Thus, the generated rabbit polyclonal anti-TRPV6 channel antibody rb79 is suitable for all in vitro diagnostic applications and particularly for the diagnosis in clinics using paraffin-embedded sections from patients suffering from various diseases and disorders involving the TRPV6 channel. [ABSTRACT FROM AUTHOR]

Background: 1,2,3,4,6-Penta-O-galloyl-β-D-glucose (β-PGG) is a polyphenol ellagic compound with a variety of pharmacological effects and has an inhibitory effect on lots of cancers. Objective: To explore the antitumor effects and mechanism of 1,2,3,4,6-Penta-O-galloyl-β-D-glucose on human hepatocellular carcinoma HepG2 cells. Design: A network pharmacology method was first used to predict the possible inhibition of hepatocellular carcinoma growth by 1,2,3,4,6-Penta-O-galloyl-β-D-glucose (β-PGG) through the p53 signaling pathway. Next, the Cell Counting Kit (CCK-8) assay was performed to evaluate changes in the survival rate of human hepatocellular carcinoma HepG2 cells treated with different concentrations of the drug; flow cytometry was used to detect changes in cell cycle, apoptosis, mitochondrial membrane potential (MMP) and intracellular Ca2+ concentration; real-time fluorescence quantification and immunoblotting showed that the expression of P53 genes and proteins associated with the p53 signaling pathway was significantly increased by β-PGG treatment. Reasult: It was found that β-PGG significantly inhibited survival of HepG2 cells, promoted apoptosis, decreased MMP and intracellular Ca2+ concentration, upregulated P53 gene and protein expression, increased CASP3 expression, and induced apoptosis in HepG2 cells. Conclusion: This study has shown that network pharmacology can accurately predict the target of β-PGG's anti-hepatocellular carcinoma action. Moreover, it was evident that β-PGG can induce apoptosis in HepG2 cells by activating the p53 signaling pathway to achieve its anti-hepatocellular carcinoma effect in vitro. [ABSTRACT FROM AUTHOR]

Background: Baked carbohydrate-rich foods are the main source of acrylamide (AA) in the general population and are widely consumed by teenagers. Considering the crucial development of the reproductive system during puberty, the health risks posed by AA in adolescent males have raised public concern. Methods: In this study, we exposed 3-week-old male pubertal mice to AA for 4 weeks to evaluate its effect on spermatogenesis using computer-assisted sperm analysis (CASA) and historical analysis. Flow cytometric analysis and meiocyte spreading assay were conducted to assess meiosis in mice. The expression of meiosis-related proteins and double-strand break (DSB) proteins were evaluated by immunoblot analyses. Additionally, isolated spermatocytes were used to explore the role of resveratrol in AA-induced damages of meiosis. Results: Our results showed that AA decreased the testicular and epididymal indexes, reduced sperm count and motility, and induced morphological disruption of the testes in pubertal mice. Subsequent meiotic analysis revealed that AA increased the proportion of 4C spermatocytes and decreased the proportion of 1C spermatids. The expression levels of meiosis-related proteins (SYCP3, Cyclin A1 and CDK2) were downregulated, and signaling proteins (γH2AX, p-CHK2 and p-ATM) expression levels were upregulated in AA-treated mice testes. Similar expression patterns were observed in primary spermatocytes treated with AA and these effects were reversed significantly by resveratrol. Conclusions: Our results indicate that AA induces meiotic arrest via persistent activation of DSBs, which may contribute to AA-compromised spermatogenesis. Resveratrol could serve as a potential therapeutic agent against AA-induced meiotic toxicity. These data highlight the importance of natural product supplementation for treating AA-related reproductive toxicity. [ABSTRACT FROM AUTHOR]

Background: Long intergenic non-protein-coding RNA 173 (LINC00173) executes vital functions in various cancers. Nevertheless, its role and expression in nasopharyngeal carcinoma (NPC) have yet to be investigated. Here, we investigated its effects on the malignancy characteristics of NPC and elucidated the potential molecular mechanism of LINC00173 in NPC progression. Methods: Quantitative real-time reverse transcription-PCR (qRT-PCR) and immunoblotting were conducted to estimate the LINC00173, microRNA-765 (miR-765), and Gremlin 1 (GREM1) expressions in NPC cells and tissues. Cell counting kit-8 (CCK8), colony formation, and wound healing experiments were done to evaluate the proliferation, growth, and migration of NPC cells, respectively. The tumorous growth of NPC cells in vivo was assessed through the xenograft tumor experiment. Furthermore, the interactions among miR-765, LINC00173, and GREM1 were investigated through bioinformatics analyses, luciferase reporter and RNA immunoprecipitation chip assays. Results: An upregulated LINC00173 expression was found in NPC cell lines and tissues. The functional experiments uncovered that its downregulation repressed NPC cell proliferation, growth, and migration. In addition, LINC00173 knockdown hampered the NPC cells' tumorous growth in vivo. These effects could partially be reversed by downregulating miR-765. GREM1 is a downstream target of miR-765. GREM1 knockdown could repress the proliferation, growth, and migration of NPC cells. Nonetheless, these anti-tumor effects could be abolished by miR-765 downregulation. Mechanistically, LINC00173 increased the expression of GREM1 by binding with miR-765. Conclusions: LINC00173 functions as an oncogenic factor by binding with miR-765 to promote the progression of NPC via GREM1 upregulation. This study provides a novel insight into the molecular mechanisms involved in NPC progression. [ABSTRACT FROM AUTHOR]

The widespread use of acetaminophen (APAP) in children as an over-the-counter treatment can cause acute liver failure through accidental overdose or ingestion. Therefore, the current research sought to investigate the function of hemin in mitigating the acute hepatotoxic effect of APAP in rat offspring. Thirty-two rats were assigned into four groups: control, hemin, APAP, and hemin/APAP groups. Liver enzymes were measured in serum along with oxidative stress indicators, tumor necrosis factor-α (TNF-α), interleukin-1beta (IL-1β), total nitrites (NOx), and caspase 3 in liver. Immunoblotting of heme oxygenase-1 (HO-1), interleukin-6 (IL-6), Janus kinase 2 (Jak2), and signal transducer and activator of transcription 3 (STAT3) was carried out. The Bax/Bcl2 mRNA expression ratio was determined. A histological study and an immunohistochemical study of phosphorylated STAT3 were also done. Hemin reduced liver enzymes, MDA, TNF-α, NOx, caspase 3, IL-1β, p-STAT3 expression, p-Jak2 expression, IL-6 expression, and Bax/Bcl2 mRNA expression ratio. In contrast, hemin increased GSH, TAC, and the expression of HO-1, improving the histopathological picture of liver tissue. Thus, hemin could ameliorate APAP-induced hepatic toxicity in rat offspring through anti-oxidant, anti-apoptotic, and anti-inflammatory actions with a possible role for the IL-6/HO-1/Jak2/STAT3 pathway. Graphical Abstract [ABSTRACT FROM AUTHOR]

Copyright of Parasite (1252607X) is the property of EDP Sciences and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background: Despite the low prevalence of IgE sensitivity to fresh or boiled coconut milk and coconut oil, those may contain allergens of which the clinical significance remains undetermined. This study aimed to identify and compare allergens in fresh coconut milk (FCM), boiled coconut milk (BCM), unrefined wet-processed coconut oil (WPCO), and dry-processed coconut oil (DPCO) using sera from patients with allergy to coconut milk. Methods: The study included 18 patients with immediate hypersensitivity to coconut milk, including five who developed anaphylaxis. Sensitization was assessed by skin prick test and ImmunoCAPs using commercially available coconut extracts. Immunoblotting was performed to identify and compare allergen profiles. Results: Total sIgE levels and overall IgE reactivity of patients with anaphylaxis were higher compared to patients with allergy. Twelve allergens ranging from 5 to 128 kDa including six novel allergens with 5, 12, 47, 87, 110, and 128 kDa were visualized in immunoblots with FCM. Similarly, nine allergens of 5, 12, 17, 32, 35, 47, 87, 110, and 128 kDa were detected in BCM. One allergen (110 kDa) was discerned in all four extracts. Higher IgE prevalence was detected with three allergens of 55, 87, and 110 kDa. Conclusions: Allergens of BCM and unrefined coconut oil (WPCO and DPCO) were determined for the first time. Novel allergens of 87 and 110 kDa and the 55 kDa allergen have the highest potential to be used in Component Resolved Diagnostics. Further, these findings demonstrate that, patients who have an allergy to coconut milk could also react to boiled coconut milk and unrefined coconut oil. [ABSTRACT FROM AUTHOR]

Introduction: Hyperphosphorylated Tau formed neurofibrillary tangles was one of the major neuropathological hallmarks of Alzheimer’s disease (AD). Dysfunctional insulin signaling in brain is involved in AD. However, the effect of Tau pathology on brain insulin resistance remains unclear. This study explored the effects of overexpressing wild-type Tau (WTau) or Tau with pseudophosphorylation at AT8 residues (PTau) on the insulin signaling pathway (ISP). Methods: 293T cells or SY5Y cells overexpressing WTau or PTau were treated with or without insulin. The elements in ISP or the regulators of IPS were analyzed by immunoblotting, immunofluorescent staining and coimmunoprecipitation. Akt inhibitor MK2206 was used for evaluating the insulin signaling to downstream of mTOR in Tau overexpressing cells. The effects of anti-aging drug lonafarnib on ISP in WTau or PTau cells were also analyzed with immunoblotting. Considering lonafarnib is an inhibitor of FTase, the states of Rhes, one of FTase substrate in WTau or PTau cells were analyzed by drug affinity responsive target stability (DARTS) assay and the cellular thermal shift assay (CETSA). Results: WTau or PTau overexpression in cells upregulated basal activity of elements in ISP in general. However, overexpression of WTau or PTau suppressed the ISP signaling transmission responses induced by insulin simulation, appearing relative higher response of IRS-1 phosphorylation at tyrosine 612 (IRS-1 p612) in upstream IPS, but a lower phosphorylation response of downstream IPS including mTOR, and its targets 4EPB1 and S6. This dysregulation of insulin evoked signaling transmission was more obvious in PTau cells. Suppressing Akt with MK2206 could compromise the levels of p-S6 and p-mTOR in WTau or PTau cells. Moreover, the changes of phosphatases detected in WTau and PTau cells may be related to ISP dysfunction. In addition, the effects of lonafarnib on the ISP in SY5Y cells with WTau and PTau overexpression were tested, which showed that lonafarnib treatment resulted in reducing the active levels of ISP elements in PTau cells but not in WTau cells. The differential effects are probably due to Tau phosphorylation modulating lonafarnib-induced alterations in Rhes, as revealed by DARTS assay. Conclusion and discussion: Overexpression of Tau or Tau with pseudophosphorylation at AT8 residues could cause an upregulation of the basal/ tonic ISP, but a suppression of insulin induced the phasic activation of ISP This dysfunction of ISP was more obvious in cells overexpressing pseudophosphorylated Tau. These results implied that the dysfunction of ISP caused by Tau overexpression might impair the physiological fluctuation of neuronal functions in AD. The different effects of lonafarnib on ISP between WTau and PTau cells, indicating that Tau phosphorylation mediates an additional effect on ISP. This study provided a potential linkage of abnormal expression and phosphorylation of Tau to the ISP dysfunction in AD. [ABSTRACT FROM AUTHOR]

Simple Summary: Reciprocal interactions between tumor and host cells in the tumor microenvironment critically influence the clinical outcome in ovarian carcinoma patients. Therefore, the identification of factors triggering central communication pathways controlling tumor growth and metastasis is highly relevant. This study was conducted to uncover the contribution of lipid mediators to this signaling network by different cell types in the tumor microenvironment and subsequent functional evaluation of clinically relevant candidates. We found that prostacyclin is mainly secreted by cancer-associated fibroblast and selectively acts on prostacyclin receptor-expressing macrophages to induce pro-tumorigenic and immunosuppressive features. Our findings improve the understanding of the tumor-promoting role of prostacyclin in ovarian carcinoma and identify prostacyclin synthesis in cancer-associated fibroblast as a potential target for improved treatment approaches. Metastasis of high-grade ovarian carcinoma (HGSC) is orchestrated by soluble mediators of the tumor microenvironment. Here, we have used transcriptomic profiling to identify lipid-mediated signaling pathways encompassing 41 ligand-synthesizing enzymes and 23 cognate receptors in tumor, immune and stroma cells from HGSC metastases and ascites. Due to its strong association with a poor clinical outcome, prostacyclin (PGI2) synthase (PTGIS) is of particular interest in this signaling network. PTGIS is highly expressed by cancer-associated fibroblasts (CAF), concomitant with elevated PGI2 synthesis, whereas tumor-associated macrophages (TAM) exhibit the highest expression of its surface receptor (PTGIR). PTGIR activation by PGI2 agonists triggered cAMP accumulation and induced a mixed-polarization macrophage phenotype with altered inflammatory gene expression, including CXCL10 and IL12A repression, as well as reduced phagocytic capability. Co-culture experiments provided further evidence for the interaction of CAF with macrophages via PGI2, as the effect of PGI2 agonists on phagocytosis was mitigated by cyclooxygenase inhibitors. Furthermore, conditioned medium from PGI2-agonist-treated TAM promoted tumor adhesion to mesothelial cells and migration in a PTGIR-dependent manner, and PTGIR activation induced the expression of metastasis-associated and pro-angiogenic genes. Taken together, our study identifies a PGI2/PTGIR-driven crosstalk between CAF, TAM and tumor cells, promoting immune suppression and a pro-metastatic environment. [ABSTRACT FROM AUTHOR]

Simple Summary: Two vascular endothelial growth factor (VEGF) receptors, FLT-1 and KDR, are expressed preferentially in proliferating endothelium. There is increasing evidence that soluble VEGF receptor domains that compete for VEGF binding may inhibit tumor angiogenesis, growth and metastatic spread. We tested whether soluble FLT-1 (sFLT-1) could inhibit breast cancer tumor growth in mice by blocking angiogenesis. A viral vector carrying the sflt-1 cDNA (Adsflt) was used to overexpress the receptor in MCF-7 cells, which produce VEGF. After six weeks, tumors were treated either with Adsflt or a negative control virus. After six months, average tumor volume in the Adsflt-treated group was decreased by 91% and vascular density was reduced 50%, relative to negative controls (p < 0.05), yet mice treated with Adsflt showed no delay in healing cutaneous wounds. These results provide evidence that viral sFLT-1 overexpression may be an effective anti-angiogenic therapy for cancer without the risk of systemic anti-angiogenic effects. Two vascular endothelial growth factor (VEGF) receptors, FLT-1 and KDR, are expressed preferentially in proliferating endothelium. There is increasing evidence that recombinant, soluble VEGF receptor domains interfering with VEGF signaling may inhibit in vivo neoangiogenesis, tumor growth and metastatic spread. We hypothesized that a soluble form of FLT-1 receptor (sFLT-1) could inhibit the growth of pre-established tumors via an anti-angiogenic mechanism. A replication-deficient adenovirus (Ad) vector carrying the sflt-1 cDNA (Adsflt) was used to overexpress the sFLT-1 receptor in a breast cancer animal model. MCF-7 cells, which produce VEGF, were used to establish solid tumors in the mammary fat pads of female nude mice. After six weeks, tumors were injected either with Adsflt or a negative control virus (AdCMV.βgal). After six months, average tumor volume in the Adsflt-infected group (33 ± 22 mm3) decreased by 91% relative to that of the negative control group (388 ± 94 mm3; p < 0.05). Moreover, 10 of 15 Adsflt-infected tumors exhibited complete regression. The vascular density of Adsflt-infected tumors was reduced by 50% relative to that of negative controls (p < 0.05), which is consistent with sFLT-1-mediated tumor regression through an anti-angiogenic mechanism. Moreover, cell necrosis and fibrosis associated with long-term regression of Adsflt–infected tumors were preceded by apoptosis of tumor vascular endothelial cells. Mice treated with Adsflt intratumorally showed no delay in the healing of cutaneous wounds, providing preliminary evidence that Ad-mediated sFLT-1 overexpression may be an effective anti-angiogenic therapy for cancer without the risk of systemic anti-angiogenic effects. [ABSTRACT FROM AUTHOR]

Background: To investigate specific brain regions and neural circuits that are responsible for migraine chronification. Methods: We established a mouse model of chronic migraine with intermittent injections of clinically-relevant dose of nitroglycerin (0.1 mg/kg for 9 days) and validated the model with cephalic and extracephalic mechanical sensitivity, calcitonin gene-related peptide (CGRP) expression in trigeminal ganglion, and responsiveness to sumatriptan or central CGRP blockade. We explored the neurons that were sensitized along with migraine chronification and investigated their roles on migraine phenotypes with chemogenetics. Results: After repetitive nitroglycerin injections, mice displayed sustained supraorbital and hind paw mechanical hyperalgesia, which lasted beyond discontinuation of nitroglycerin infusion and could be transiently reversed by sumatriptan. The CGRP expression in trigeminal ganglion was also upregulated. We found the pERK positive cells were significantly increased in the central nucleus of the amygdala (CeA), and these sensitized cells in the CeA were predominantly protein kinase C-delta (PKC-δ) positive neurons co-expressing CGRP receptors. Remarkably, blockade of the parabrachial nucleus (PBN)-CeA CGRP neurotransmission by CGRP8–37 microinjection to the CeA attenuated the sustained cephalic and extracephalic mechanical hyperalgesia. Furthermore, chemogenetic silencing of the sensitized CeA PKC-δ positive neurons reversed the mechanical hyperalgesia and CGRP expression in the trigeminal ganglion. In contrast, repetitive chemogenetic activation of the CeA PKC-δ positive neurons recapitulated chronic migraine-like phenotypes in naïve mice. Conclusions: Our data suggest that CeA PKC-δ positive neurons innervated by PBN CGRP positive neurons might contribute to the chronification of migraine, which may serve as future therapeutic targets for chronic migraine. [ABSTRACT FROM AUTHOR]

Purpose: By-products from farmed fish contain large amounts of proteins and may be used for human consumption. The purpose of this study was to investigate cardiometabolic effects and metabolic tolerance in mice consuming fishmeal from salmon by-products, salmon filet or beef. Methods: Female C57BL/6J mice were fed chow, as a healthy reference group, or a high-fat diet for 10 weeks to induce obesity and glucose intolerance. Obese mice were subsequently given isocaloric diets containing 50% of the dietary protein from salmon fishmeal, salmon filet or beef for 10 weeks. Mice were subjected to metabolic phenotyping, which included measurements of body composition, energy metabolism in metabolic cages and glucose tolerance. Lipid content and markers of hepatic toxicity were determined in plasma and liver. Hepatic gene and protein expression was determined with RNA sequencing and immunoblotting. Results: Mice fed fishmeal, salmon filet or beef had similar food intake, energy consumption, body weight gain, adiposity, glucose tolerance and circulating levels of lipids and hepatic toxicity markers, such as p-ALT and p-AST. Fishmeal increased hepatic cholesterol levels by 35–36% as compared to salmon filet (p = 0.0001) and beef (p = 0.005). This was accompanied by repressed expression of genes involved in steroid and cholesterol metabolism and reduced levels of circulating Pcsk9. Conclusion: Salmon fishmeal was well tolerated, but increased hepatic cholesterol content. The high cholesterol content in fishmeal may be responsible for the effects on hepatic cholesterol metabolism. Before introducing fishmeal from salmon by-products as a dietary component, it may be advantageous to reduce the cholesterol content in fishmeal. [ABSTRACT FROM AUTHOR]

Summary: Light regulates the subcellular localization of plant photoreceptors, a key step in light signaling. Ultraviolet‐B radiation (UV‐B) induces the plant photoreceptor UV RESISTANCE LOCUS 8 (UVR8) nuclear accumulation, where it regulates photomorphogenesis. However, the molecular mechanism for the UV‐B‐regulated UVR8 nuclear localization dynamics is unknown.With fluorescence recovery after photobleaching (FRAP), cell fractionation followed by immunoblotting and co‐immunoprecipitation (Co‐IP) assays we tested the function of UVR8‐interacting proteins including CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), REPRESSOR OF UV‐B PHOTOMORPHOGENESIS 1 (RUP1) and RUP2 in the regulation of UVR8 nuclear dynamics in Arabidopsis thaliana.We showed that UV‐B‐induced rapid UVR8 nuclear translocation is independent of COP1, which previously was shown to be required for UV‐B‐induced UVR8 nuclear accumulation. Instead, we provide evidence that the UV‐B‐induced UVR8 homodimer‐to‐monomer photo‐switch and the concurrent size reduction of UVR8 enables its monomer nuclear translocation, most likely via free diffusion. Nuclear COP1 interacts with UV‐B‐activated UVR8 monomer, thereby promoting UVR8 nuclear retention. Conversely, RUP1and RUP2, whose expressions are induced by UV‐B, inhibit UVR8 nuclear retention via attenuating the UVR8–COP1 interaction, allowing UVR8 to exit the nucleus.Collectively, our data suggest that UV‐B‐induced monomerization of UVR8 promotes its nuclear translocation via free diffusion. In the nucleus, COP1 binding promotes UVR8 monomer nuclear retention, which is counterbalanced by the major negative regulators RUP1 and RUP2. [ABSTRACT FROM AUTHOR]

Abstract: Complex regional pain syndrome (CRPS) is an inadequate local response after a limb trauma, which leads to severe pain and autonomic and trophic changes of the affected limb. Autoantibodies directed against human β2 adrenergic and muscarinic M2 receptors (hβ2AR and hM2R) have been described in CRPS patients previously. We analyzed sera from CRPS patients for autoantibodies against hβ2AR, hM2R, and endothelial cells and investigated the functional effects of purified IgG, derived from 13 patients with CRPS, on endothelial cells. Eleven healthy controls, 7 radial fracture patients without CRPS, and 10 patients with peripheral arterial vascular disease served as control subjects. The CRPS-IgG, but not control IgG, bound to the surface of endothelial cells ( P < 0.001) and to hβ2AR and hM2R ( P < 0.05), the latter being reversed by adding β2AR and M2R antagonists. The CRPS-IgG led to an increased cytotoxicity and a reduced proliferation rate of endothelial cells, and by adding specific antagonists, the effect was neutralized. Regarding second messenger pathways, CRPS-IgG induced ERK1/2, p38, and STAT1 phosphorylation, whereas AKT phosphorylation was decreased at the protein level. In addition, increased expression of adhesion molecules (ICAM-1 and VCAM-1) on the mRNA level was induced by CRPS-IgG, thus inducing a pro-inflammatory condition of the endothelial cells. Our results show that patients with CRPS not only develop autoantibodies against hβ2AR and hM2R, but these antibodies also interfere with endothelial cells, inducing functional effects on these in vitro, and thus might contribute to the pathophysiology of CRPS. [ABSTRACT FROM AUTHOR]

Objectives Anti‐TIF1-gamma autoantibodies can be detected with immunoprecipitation (IP), line blot (LB) and ELISA. We compared assay performance in patients with DM and the potential of these assays to detect anti-TIF1-gamma positive cancer-associated DM (CADM). Methods We included sera from 131 patients with DM followed at Karolinska University Hospital, Stockholm, Sweden and 82 healthy controls. Serum samples taken at DM diagnosis were tested for anti-TIF1-gamma autoantibodies with IP, two ELISAs (in-house and commercial) and LB. Cancer diagnosis and dates were obtained from the Swedish national cancer register. CADM was defined as a malignancy that developed within 3 years of DM diagnosis. Results Anti-TIF1-gamma autoantibodies were detected in 19/101 (18.8%), 15/113 (13.2%), 34/131 (26%) and 45/131 (34.4%) of the patients with IP, LB, in-house and commercial ELISA, respectively. The anti-TIF1-gamma results from the in-house ELISA were confirmed with IP in 93 of 101 (92%) cases, κ = 0.76, with a commercial ELISA in 110 of 131 (84%) cases, κ = 0.63, and with LB in 101 of 113 (89.3%) cases, κ = 0.67. Anti-TIF1-gamma results with IP were confirmed with LB in 85 of 92 (92.4%) cases, κ = 0.73. For detecting CADM, the anti-TIF1-gamma in-house ELISA had a sensitivity of 58% and specificity of 86%, the commercial ELISA had a sensitivity of 63% and specificity of 82%, IP had a sensitivity of 52% and specificity of 92%, LB had a sensitivity of 40% and specificity of 96%. Conclusion The two anti-TIF1-gamma ELISA assays had advantages both for autoantibody detection and to identify anti-TIF1-gamma-positive CADM. [ABSTRACT FROM AUTHOR]

(1) Background: This investigation aimed at developing a series of c-Met-targeting cabozantinib-based PROTACs. (2) Methods: Purification of intermediate and target compounds was performed using column chromatography, in vitro antiproliferation activity was measured using a standard MTT assay and a c-Met degradation assay was performed via the immunoblotting technique. (3) Results: Several compounds exhibited antiproliferative activity towards different cell lines of breast cancer (T47D, MDA-MB-231, SKBR3, HCC1954 and MCF7) at the same level as parent cabozantinib and 7-demethyl cabozantinib. Two target conjugates, bearing a VHL-ligand as an E3-ligase binding moiety and glycol-based linkers, exhibited the effective inhibition of c-Met phosphorylation and an ability to decrease the level of c-Met in HCC1954 cells at micromolar concentrations. (4) Conclusions: Two compounds exhibit c-Met inhibition activity in the nanomolar range and can be considered as PROTAC molecules due to their ability to decrease the total level of c-Met in HCC1954 cells. The structures of the offered compounds can be used as starting points for further evaluation of cabozantinib-based PROTACs. [ABSTRACT FROM AUTHOR]

Simple Summary: In vivo studies have shown that 3D neurosphere formation is a significant predictor of clinical outcome in glioma patients, and is a robust, independent predictor of glioma tumor progression. However, neurosphere assays, in common with other in vitro assays, are associated with some limitations. Little is known about the upstream signaling events triggered and that lead to a cancer stem cell molecular signature, even less is known on how anticancer diet-derived molecules can target such events. Here, we provide assessment of several signal transducing events involved in the acquisition of an in vitro stemness phenotype, in comparison to 2D monolayer cultures of brain cancer cells. We further identify a new signaling axis that can become a target for future therapies. Background: Three-dimensional in vitro neurospheres cultures recapitulate stemness features associated with poor clinical outcome in glioblastoma patients. They are commonly used to address brain cancer stem cell (CSC) signal transducing biology that regulates spheroids formation and stemness phenotype, and to assess the in vitro pharmacological impact of chemotherapeutic drugs. Objective: Here, we addressed the role of a new signaling axis involved in the regulation of in vitro spheroids formation and assessed the chemopreventive ability of diet-derived epigallocatechin gallate (EGCG) to impact the processes that govern the acquisition of spheroids CSC stemness traits. Methods: Neurospheres were generated from adherent human U87 glioblastoma cancer cell cultures under conditions that recapitulate stemness features. Total RNA and protein lysates were isolated for gene expression by RT-qPCR and protein expression by immunoblot. Transcriptomic analysis was performed through RNA-Seq. Results: Compared to their parental adherent cells, tumorspheres expressed increased levels of the CSC markers NANOG, SOX2, PROM1 (CD133), as well as of the epithelial-to-mesenchymal transition (EMT) markers Fibronectin, SNAI1, and 37/67 kDa laminin-1 receptor ribosomal protein SA (RPSA). Increased PROM1, SOX2, Fibronectin, and RPSA transcripts level were also observed in clinical grade IV glioblastoma tissues compared to normal tissue. EGCG treatment reduced dose-dependently tumorspheres size and inhibited the transcriptional regulation of those genes. An apoptotic signature was also found in spheroids with increased signal transducing events involving GSK3α/β, RSK, and CREB. These were repressed upon RPSA gene silencing and partially by SNAI1 silencing. Conclusion: This work highlights a signaling axis linking RPSA upstream of SNAIL in neurospheres genesis and supports the chemopreventive impact that diet-derived EGCG may exert on the acquisition of CSC traits. [ABSTRACT FROM AUTHOR]

Simple Summary: Currently; there are no reports on the role of STIL in bladder cancer. Using public databases; we observed that STIL is highly expressed in bladder cancer and is closely related to the cell cycle. The results of tumor formation experiments on UMUC3 and T24 bladder cancer cell lines indicate that STIL promotes the growth of bladder cancer cells in vivo and in vitro. Mechanistically, the cell cycle after STIL knockout was arrested in the G0/G1 phase; and cell cycle-related proteins (cyclin D1 and CDK2/4/6) were also reduced. RNA-seq results and immunoblotting experiments confirmed that STIL enhanced the PI3K/AKT/mTOR pathway, resulting in increased c-myc expression which ultimately promoted the occurrence and progression of bladder cancer. Our results suggest that STIL may be a promising potential therapeutic target for bladder cancer. SCL/TAL1 interrupting locus (STIL) regulates centriole replication and causes chromosome instability, which is closely related to malignant tumors. The purpose of our study was to investigate the role of STIL in bladder cancer (BC) tumorigenesis for the first time. The public database indicated that STIL is highly expressed and correlated with the cell cycle in BC. Immunohistochemistry staining showed that STIL expression is significantly elevated in BC tissues compared with paracancer tissues. CRISPR-Cas9 gene editing technology was used to induce BC cells to express STIL-specific sgRNA, revealing a significantly delayed growth rate in STIL knockout BC cells. Moreover, cell cycle arrest in the G0/G1 phase was triggered by decreasing STIL, which led to delayed BC cell growth in vitro and in vivo. Mechanically, STIL knockout inhibited the PI3K/AKT/mTOR pathway and down-regulated the expression of c-myc. Furthermore, SC79 (AKT activating agent) partially reversed the inhibitory effects of STIL knockout on the proliferation and migration of BC cells. In conclusion, STIL enhanced the PI3K/AKT/mTOR pathway, resulting in increased expression of c-myc, ultimately promoting BC occurrence and progression. These results indicate that STIL might be a potential target for BC patients. [ABSTRACT FROM AUTHOR]

Traditional sanitation practices remain the main strategy for controlling Bombyx mori infections caused by microsporidia Nosema bombycis. This actualizes the development of new approaches to increase the silkworm resistance to this parasite. Here, we constructed a mouse scFv library against the outer loops of N. bombycis ATP/ADP carriers and selected nine scFv fragments to the transporter, highly expressed in the early stages of the parasite intracellular growth. Expression of selected scFv genes in Sf9 cells, their infection with different ratios of microsporidia spores per insect cell, qPCR analysis of N. bombycis PTP2 and Spodoptera frugiperda COXI transcripts in 100 infected cultures made it possible to select the scFv fragment most effectively inhibiting the parasite growth. Western blot analysis of 42 infected cultures with Abs against the parasite β-tubulin confirmed its inhibitory efficiency. Since the VL part of this scFv fragment was identified as a human IgG domain retained from the pSEX81 phagemid during library construction, its VH sequence should be a key antigen-recognizing determinant. Along with the further selection of new recombinant Abs, this suggests the searching for its natural mouse VL domain or "camelization" of the VH fragment by introducing cysteine and hydrophilic residues, as well as the randomization of its CDRs. [ABSTRACT FROM AUTHOR]

The green-lipped mussel (Perna canaliculus) originates from New Zealand. To preserve the health benefits of green-lipped mussel meat, it is freeze-dried to make a long-lasting powder. The powder is used to treat arthritis because of its potential anti-inflammatory properties. The report describes a 54-year-old woman who developed immediate rhinoconjunctival and respiratory symptoms after inhaling green-lipped mussel powder she gave to her dog for arthritis. A skin prick test with green-lipped mussel powder was performed. Protein extracts from P canaliculus were separated by sodium dodecyl-sulfate polyacrylamide (SDS) gel electrophoresis and probed with serum from patients and serum preincubated with green-lipped mussel extract. Bound immunoglobulin E (IgE) was detected by specific anti-human-IgE antibodies, and IgE-binding proteins were subsequently identified by liquid chromatography and mass spectrometry. The skin prick test was positive for green-lipped mussel. Specific IgE against green-lipped mussel extract was detected using Western immunoblotting. These potential allergenic proteins were identified by mass spectrometry as actin, tropomyosin, and paramyosin. All three allergens are reported for the first time for P canaliculus. Actin is a major allergen in Paphia textile, paramyosin in Sarcoptes scarbiei, and tropomyosin in Haliotis discus. For all IgE-binding proteins, the software AllCatPro predicted high allergenicity, supporting our conclusion that these proteins from P canaliculus may also be allergenic. The identification of allergens from P canaliculus provides the opportunity for specific tests to assess the frequency of allergic reactions to P canaliculus. [ABSTRACT FROM AUTHOR]

Introduction: Fibrosis of renal tubules is the final common pathway in diabetic nephropathy and develops in the face of tubular injury and fibroblast activation. Aberrant connexin 43 (Cx43) hemichannel activity has been linked to this damage under euglycaemic conditions, however, its role in glycaemic injury is unknown. This study investigated the effect of a Cx43 blocker (Tonabersat) on hemichannel activity and cell–cell interactions within and between tubular epithelial cells and fibroblasts in an in vitro model of diabetic nephropathy. Methods: Human kidney (HK2) proximal tubule epithelial cells and medullary fibroblasts (TK173) were treated in low (5 mM) or high (25 mM) glucose ± transforming growth factor beta‐1 (TGFβ1) ± Tonabersat in high glucose. Carboxyfluorescein dye uptake and ATPlite luminescence assessed changes in hemichannel‐mediated ATP release, while immunoblotting determined protein expression. Co‐incubation with the ATP‐diphosphohydrolase apyrase or a P2X7R inhibitor (A438079) assessed ATP‐P2X7R signalling. Indirect co‐culture with conditioned media from the alternate cell type evaluated paracrine‐mediated heterotypic interactions. Results: Tonabersat partially negated glucose/TGFβ1‐induced increases in Cx43 hemichannel‐mediated ATP release and downstream changes in adherens junction and extracellular matrix (ECM) protein expression in HK2 and TK173 cells. Apyrase and A438079 highlighted the role for ATP‐P2X7R in driving changes in protein expression in TK173 fibroblasts. Indirect co‐culture studies suggest that epithelial cell secretome increases Tonabersat‐sensitive hemichannel‐mediated dye uptake in fibroblasts and downstream protein expression. Conclusion: Tonabersat‐sensitive hemichannel‐mediated ATP release enhances TGFβ1‐driven heterotypic cell–cell interaction and favours myofibroblast activation. The data supports the potential benefit of Cx43 inhibition in reducing tubulointerstitial fibrosis in late‐stage diabetic nephropathy. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER) is a critical organelle that preserves the protein homeostasis of cells. Under various stress conditions, cells evolve a degree of capacity to maintain ER proteostasis, which is usually augmented in tumor cells, including colorectal cancer (CRC) cells, to bolster their survival and resistance to apoptosis. Bortezomib (BTZ) is a promising drug used in CRC treatment; however, its main limitation result from drug resistance. Here, we identified the role of tripartite motif-containing protein 59 (TRIM59)–a protein localized on the ER membrane– in the prevention of BTZ-mediated CRC killing. Depletion of TRIM59 is associated with the enhancement of ER stress and a remarkable increase in unfolded protein response (UPR) signaling. Besides, TRIM59 strengthens ER-associated degradation (ERAD) and alleviates the generation of ROS. Of note, TRIM59 knockdown synergizes with the anti-cancer effect of BTZ both in vitro and in vivo. Our findings revealed a role for TRIM59 in the ER by guarding ER proteostasis and represents a novel therapeutic target of CRC. [ABSTRACT FROM AUTHOR]

Connexins are essential gap junction proteins that play pivotal roles in intercellular communication in various organs of mammals. Connexin-43 (Cx43) is expressed in various components of the immune system, and there is extensive evidence of its participation in inflammation responses. The involvement of Cx43 in macrophage functionality involves the purinergic signaling pathway. Macrophages contribute to defenses against inflammatory reactions such as bacterial sepsis and peritonitis. Several assays can identify the presence and activity of Cx43 in macrophages. Real-time polymerase chain reaction (PCR) can measure the relative mRNA expression of Cx43, whereas western blotting can detect protein expression levels. Using immunofluorescence assays, it is possible to analyze the expression and observe the localization of Cx43 in cells or tissues. Moreover, connexin-mediated gap junction intercellular communication can be evaluated using functional assays such as microinjection of fluorescent dyes or scrape loading-dye transfer. The use of selective inhibitors contributes to this understanding and reinforces the role of connexins in various processes. Here, we discuss these methods to evaluate Cx43 and macrophage gap junctions. [ABSTRACT FROM AUTHOR]

The knowledge of the mechanisms affecting the process of berry fruit ripening is important, not only to correctly determine the appropriate date of harvest, at which the fruit is most palatable and characterized by adequate shelf-life stability, but also, to develop new strategies of regulating the ripening process before harvesting. It is recognized that berry fruit quality and its post-harvest shelf-life depend on the cellular redox homeostasis. We, therefore, decided to conduct a comprehensive analysis of the level of oxidative stress status in the raspberry fruit proper at different stages of fruit ripening, from green to overripe fruit. We assessed both the level of typical oxidative stress markers, i.e. ROS production, expression of antioxidant enzymes, degree of cell damage, as well as the expression of selected proteins involved in the energy, glutathione, and polyphenols metabolism. We found two stage-related peaks of ROS production. The first - in green fruit, which corresponded to the maximum expression of antioxidant enzymes (MnSOD, CAT), PARP-1, as well as proteins related to glutathione biosynthesis (GS, γ-GC), autophagy (ATG-8) and ubiquitination (UBQ-11). The second one, in the overripe fruit, was responsible for the intensification of oxidative modifications of cell components, i.e. lipids, proteins, and DNA, as well as the loss of low molecular-weight antioxidants. The fruit ripening process, in turn, was manifested by a strong increase in the expression of proteins involved in the biosynthesis of polyphenols (PAL, CHS), cellular respiration (ACO-1, Cyt-C-ox, ATPase), ethylene biosynthesis and signaling (SAMs, EIN-2) and increasing amounts of ABA. [Display omitted] • Raspberry ripening is regulated by ROS, ABA and ethylene. • Autophagy and ubiquitination occur most intensively in green fruit. • Fruit ripening is associated with an increase in PAL and CHS expression. • The greatest degree of damage of cell components is in overripe fruit. [ABSTRACT FROM AUTHOR]

This study aims to elucidate the potential targets and molecular mechanisms underlying the anticancer effects of Red fermented rice extract using molecular simulation techniques. The inhibitory effects of different elution fractions of Red fermented rice extract on A549 and MCF-7 cell proliferation were evaluated through CCK-8 assays. Liquid chromatography-mass spectrometry (LC-MS) was employed to elucidate the structural information of active components, while molecular simulation techniques aided in identifying target proteins based on small molecule structures. Protein immunoblotting was utilized to investigate the mechanisms of action of relevant targets. The study found that the petroleum ether-ethyl acetate and ethyl acetate elution fractions of Red fermented rice extract significantly inhibited A549 and MCF-7 cell proliferation, with stronger effects observed on A549 cells. LC-MS structural analysis identified 25 small molecule structures. Molecular simulations successfully revealed interaction between active elution fractions of Red fermented rice extract and the cancer-related protein FGFR1. Further investigation into the phosphorylation of FGFR1 and its downstream pathway targets PI3K/AKT demonstrated that the active elution fractions exerted their anticancer activity by inhibiting the phosphorylation of FGFR1, PI3K, and AKT proteins. This comprehensive study, integrating CCK-8 assays, LC-MS, molecular simulation techniques, and protein immunoblotting, provides a deep understanding of the anticancer mechanisms of Red fermented rice extract, guiding its further development and clinical application. [Display omitted] • Hongqu extract demonstrates significant anticancer activity against A549 and MCF-7 cell lines. • Petroleum ether-ethyl acetate and ethyl acetate elution fractions exhibit potent inhibitory effects. • Molecular simulation identifies interaction between active fractions and cancer-related protein FGFR1. • Investigation reveals inhibition of FGFR1 phosphorylation and downstream PI3K/AKT pathway. • Protein immunoblotting validates the mechanism, indicating suppression of tumor cell proliferation. • Comprehensive use of assays and techniques provides a deep understanding of Hongqu extract's anticancer mechanisms. • Findings offer valuable insights for the further development and clinical exploration of Hongqu extract. [ABSTRACT FROM AUTHOR]

Neurocysticercosis is the leading cause for acquired epilepsy worldwide, and it is caused by the larval stage of the parasite Taenia solium. Several proteins of this stage have been characterized and studied to understand the parasite-host interaction, however, the proteins from the early cysticercus stages (the postoncospheral form) have not yet been characterized. The study of the postoncospheral form proteins is important to understand the host-parasite relationship in the early stages of infection. The aim of this work was to identify postoncospheral form antigenic proteins using sera from neurocysticercosis patients. T. solium activated oncospheres were cultured in HCT-8 cells to obtain the postoncospheral form. Soluble total and excretory/secretory proteins were obtained from the postoncospheral form and were incubated with both pool sera and individual serum of neurocysticercosis positive human patients. Immunoblotting showed target antigenic proteins with apparent molecular weights of 23 kDa and 46–48 kDa. The 46–48 kDa antigen bands present in soluble total and excretory/secretory postoncospheral form proteins were analyzed by LC-MS/MS; proteins identified were: nuclear elongation factor 1 alpha, enolase, unnamed protein product/antigen diagnostic GP50, calcium binding protein calreticulin precursor and annexin. The postoncospheral form expresses proteins related to interaction with the host, some of these proteins are predicted to be exosomal proteins. In conclusion, postoncospheral proteins are consistent targets of the humoral immune response in human and may serve as targets for diagnosis and vaccines. • Neurocysticercosis patients develop antibodies against Taenia solium postoncospheral proteins. • Major proteins detected by these antibodies are: nuclear elongation factor 1 alpha, enolase, antigen GP50, calreticulin precursor and annexin. • The annexin is a newly identified postoncospheral form protein, not previously reported in T. solium. [ABSTRACT FROM AUTHOR]

Interleukin (IL)-23 inhibitor monoclonal antibodies shown significant efficacy in treating autoimmune diseases. DNA or RNA aptamers exhibit comparable specificity to antibodies, are cost-effective, non-immunogenic, and do not have batch to batch variation. This study aimed to characterize a single-stranded DNA (ssDNA) aptamer targeting human IL-23. The alpha subunit of IL-23 (P19) and intact IL-23 were cloned, expressed, and the proteins finally were purified through Ni2+-iminodiacetic acid affinity chromatography. The selection and characterization of ssDNA aptamer against P19 were conducted using the protein-systematic evolution of ligands by exponential enrichment (SELEX). Dot blot assay was carried out to monitor binding of the aptamer output of SELEX rounds, to P19 protein. The dissociation constant (Kd) of aptamers with positive results in dot blot assay, determined based on their binding to IL-23 using an ELISA method. Recombinant P19 and IL-23 proteins were 26 and 72 kDa, respectively, observed on SDS-PAGE.12 %. The aptamers output from 7, 8, 9, 10, 11, and 12 rounds of the SELEX was monitored by dot blot assay, revealing that the aptamer from the round 8 has stronger luminescent signal and was selected for TA-cloning. After analyzing the biotinylated aptamers from clones, positive clones in dot blot assay and ELISA were sequenced. Finally, the Kd calculation revealed three aptamers with high affinity, named A23P3, A23P6, and A23P15 with Kd values of 1.37, 2.139, and 2.88 nM, respectively. Results of this study introduced three specific anti-IL-23 ssDNA aptamers with high affinity, which could be utilized for therapeutic and diagnostic purposes. • Interleukin (IL)-23 plays a crucial role in development of autoimmune conditions like psoriasis. • A23P3, A23P6, and A23P15 DNA aptamers binding to IL-23 were identified using protein-SELEX method. • Dissociation constants for the discovered aptamers were in the low nanomolar range (1.37–2.88 nM). [ABSTRACT FROM AUTHOR]

Scant information is available on the immunological aspect of Linguatula serrata causing linguatulosis in humans and animals. The present study aimed to analyze the content of crude somatic extracts and excretory-secretory products of L. serrata nymphs to detect the immune response of sheep and immunogenic proteins of the parasite. After collecting the nymphs, somatic extracts were prepared by sonication. Excretory secretory products were prepared by the incubation of nymphs in RPMI medium at 37°C with 5% CO2. Somatic and excretory-secretory proteins were isolated using SDS-PAGE. The immunogenic properties of the resulting proteins were determined using immunoblotting and positive sera from sheep infected with visceral linguatulosis. The total content of somatic extracts and excretory-secretory products of L. serrata nymphs analyzed by SDS-PAGE (12% gel) revealed two protein patterns with more than 18 and 9 strong bands, respectively. Immunoblots using sera samples of sheep infected with the parasite, somatic extracts and excretory-secretory products demonstrated 12 and 3 antigenic proteins with molecular weights mostly in the range of 24-100 kDa and an antigen more than 180 kDa. Three common immunodominant antigenic proteins with molecular weights of 38 and 57, as well as an antigen of more than 180 kDa, were detected in the somatic extracts and excretory-secretory products of L. serrata nymphs in sheep with visceral linguatulosis. These antigens can be considetered prime candidates for future serodiagnosis and immunoprotective studies of the parasite.