Search

Objective and Background: This research aimed to examine the role of C‐X‐C motif chemokine ligand 5 (CXCL5) and C‐X‐C motif chemokine ligand 8 (CXCL8; also known as IL‐8) in neutrophilic inflammation triggered by peri‐implantitis and to shed light on the underlying mechanisms that link them to the development of this condition. Materials: This study included 40 patients who visited the Department of Periodontology at Kyungpook University Dental Hospital. They were divided into two groups based on their condition: healthy implant (HI) group (n = 20) and peri‐implantitis (PI) group (n = 20). Biopsy samples of PI tissue were collected from the patients under local anesthesia. HI tissue was obtained using the same method during the second implant surgery. To construct libraries for control and test RNAs, the QuantSeq 3′ mRNA‐Seq Library Prep Kit (Lexogen, Inc., Austria) was used according to the manufacturer's instructions. Samples were pooled based on representative cytokines obtained from RNA sequencing results and subjected to Reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR). Hematoxylin and eosin staining, and immunohistochemistry (IHC) analysis were performed to visually assess expression levels and analyze tissue histology. Student's t‐test was employed to conduct statistical analyses. Results: Initially, heatmaps were used to examine gene expression variations between the HI and PI groups based on the results of RNA sequencing. Notably, among various cytokines, CXCL5 and CXCL8 had the highest expression levels in the PI group compared with the HI group, and they are known to be associated with inflammatory responses. In the gingival tissues, the expression of genes encoding cytokines such as interleukin (IL)‐1β, tumor necrosis factor‐alpha (TNF)‐α, interleukin (IL)‐6, and CXCL5/CXCL8 was assessed via RT‐qPCR. The mRNA expression level of CXCL5/CXCL8 significantly increased in the PI group compared with the HI group (p <.045). Contrarily, the mRNA expression level of interleukin 36 receptor antagonist (IL36RN) significantly decreased (p <.008). IHC enabled examination of the distribution and intensity of CXCL5/CXCL8 protein expression within the tissue samples. Specifically, increased levels of CXCL5/CXCL8 promote inflammatory responses, cellular proliferation, migration, and invasion within the peri‐implant tissues. These effects are mediated through the activation of the PI3K/Akt/NF‐κB signaling pathway. Conclusions: This study found that the PI sites had higher gene expression level of CXCL8/CXCL5 in the soft tissue than HI sites, which could help achieve more accurate diagnosis and treatment planning. [ABSTRACT FROM AUTHOR]

Medulla Tetrapanacis (MT) is a commonly used herb to promote lactation and manage mastitis in lactating mothers. However, its anti-inflammatory and anti-bacterial effects are currently unknown. We hypothesized that MT water extract possesses anti-inflammatory and anti-bacterial effects by modulating macrophage polarization to reduce the release of inflammatory mediators and phagocytosis via inactivation of MAPKs pathways. The chemical composition of the MT water extract was analyzed by UPLC-Orbitrap-mass spectrometry. The anti-inflammatory and anti-bacterial properties of the MT water extract were examined using LPS-stimulated inflammation and Staphylococcus aureus infection model in RAW 264.7 cells, respectively. The underlying mechanism of action of the MT water extract was also investigated. We identified eight compounds by UPLC-Orbitrap-mass spectrometry that are abundant within the MT water extract. MT water extract significantly suppressed LPS-induced nitric oxide, TNF-α and IL-6 secretion in RAW 264.7 cells which was accompanied by the promotion of macrophage polarization from pro-inflammatory towards anti-inflammatory phenotypes. MT water extract significantly suppressed the LPS-induced MAPK activation. Finally, MT water extract decreased the phagocytic capacity of the RAW 264.7 cells against S. aureus infection. MT water extract could suppress LPS-induced inflammation by promoting macrophages towards an anti-inflammatory phenotype. In addition, MT also inhibited the growth of S. aureus. [ABSTRACT FROM AUTHOR]

Methylobacterium is a genus belonging to the pink-pigmented facultative methylotrophs, which can use C1 substrates as a sole carbon source. Methylorubrum extorquens (formerly Methylobacterium extorquens) is a potentially valuable bacteria in methanol-based bioindustry producing high-value-added chemicals. Thus, it is critical to understand the core metabolic pathways of M. extorquens strains for further advancement as a platform bacterium in the bioindustry. However, there are no reported systems biological approaches comparing different M. extorquens strains. This paper focuses on comparing the genomic properties of seven completely sequenced M. extorquens strains, including M. extorquens PA1, which was re-sequenced, and M. extorquens ATCC 55366, which was newly sequenced in this study. Pan-genome analysis indicated that a total of 10,431 ortholog clusters composed the pan-genome of these M. extorquens strains, including 3,507 core genome, 1,860 accessory genome, and 5,064 unique genome clusters. The functional annotation discovered that genes related to signal transduction were the most abundant in the pan-genome. Pan-genome analysis of two different habitat-specific groups revealed that the strains isolated from various soil environments had a higher percentage of genes involved in mobilome in their core genome. Subsequent exploration for secondary metabolite biosynthesis clusters denoted the unique ability of M. extorquens AM1 to produce various toblerols. M. extorquens PA1 could be a strong candidate for industrial strain development because of its minimal genome size with fully conserved central metabolism. This study provides the first inclusive insight into the differences in genomic characteristics of central metabolic networks of M. extorquens. [ABSTRACT FROM AUTHOR]

Simple Summary: Ubiquitin-specific Peptidase 13 (USP13) is highly amplified and promotes tumorigenesis and metastasis in ovarian cancer. However, post-translational modifications and their functions on USP13 are largely unknown. This study revealed that USP13 is phosphorylated at Thr122, which upregulates the stability of USP13 at a post-translation level. Notably, mutation of Thr122 diminished the effect of USP13 on the increased proliferation of ovarian cancer cells. Overall, we uncovered a new mechanism for the regulation of USP13 stability via a post-translational modification, suggesting novel therapeutics targeting USP13 in USP13-amplified cancers. Ubiquitin-specific Peptidase 13 (USP13) is a deubiquitinating enzyme that regulates the stability or function of its substrate. USP13 is highly amplified in human ovarian cancer, and elevated expression of USP13 promotes tumorigenesis and metastasis of ovarian cancer. However, there is little known about USP13 post-translational modifications and their role in ovarian cancer. Here, we found that USP13 is phosphorylated at Thr122 in ovarian cancer cells. Phosphorylated Thr122 (pT122) on endogenous USP13 was observed in most human ovarian cancer cells, and the abundance of this phosphorylation was correlated to the total level of USP13. We further demonstrated that Casein kinase 2 (CK2) directly interacts with and phosphorylates USP13 at Thr122, which promotes the stability of USP13 protein. Finally, we showed that Threonine 122 is important for cell proliferation of ovarian cancer cells. Our findings may reveal a novel regulatory mechanism for USP13, which may lead to novel therapeutic targeting of USP13 in ovarian cancer. [ABSTRACT FROM AUTHOR]

Type 1 diabetes (T1D) is an autoimmune disease characterised by the destruction of the insulin-producing beta (β)-cells within the pancreatic islets. We have previously identified a novel parasite-derived molecule, termed Fasciola hepatica helminth defence molecule 1 (FhHDM-1), that prevents T1D development in non-obese diabetic (NOD) mice. In this study, proteomic analyses of pancreas tissue from NOD mice suggested that FhHDM-1 activated the PI3K/Akt signalling pathway, which is associated with β-cell metabolism, survival and proliferation. Consistent with this finding, FhHDM-1 preserved β-cell mass in NOD mice. Examination of the biodistribution of FhHDM-1 after intraperitoneal administration in NOD mice revealed that the parasite peptide localised to the pancreas, suggesting that it exerted a direct effect on the survival/function of β-cells. This was confirmed in vitro, as the interaction of FhHDM-1 with the NOD-derived β-cell line, NIT-1, resulted in increased levels of phosphorylated Akt, increased NADH and NADPH and reduced activity of the NAD-dependent DNA nick sensor, poly(ADP-ribose) polymerase (PARP-1). As a consequence, β-cell survival was enhanced and apoptosis was prevented in the presence of the pro-inflammatory cytokines that destroy β-cells during T1D pathogenesis. Similarly, FhHDM-1 protected primary human islets from cytokine-induced apoptosis. Importantly, while FhHDM-1 promoted β-cell survival, it did not induce proliferation. Collectively, these data indicate that FhHDM-1 has significant therapeutic applications to promote β-cell survival, which is required for T1D and T2D prevention and islet transplantation. Key messages: FhHDM-1 preserves β-cell mass in NOD mice and prevents the development of T1D. FhHDM-1 enhances phosphorylation of Akt in mouse β-cell lines. FhHDM-1 increases levels of NADH/NADPH in mouse β-cell lines in vitro. FhHDM-1 prevents cytokine-induced cell death of mouse β-cell lines and primary human β-cells in vitro via activation of the PI3K/Akt pathway. [ABSTRACT FROM AUTHOR]

Pancreatic cancer has a poor prognosis with a five-year survival rate of less than 10%. Moreover, chemotherapy is mostly rendered ineffective owing to chemotherapy resistance and cytotoxicity. Therefore, the development of effective therapeutic strategies and novel drugs against pancreatic cancer is an urgent need. Cucurbitacin D (CuD), a plant steroid derived from Trichosanthes kirilowii, is an anticancer agent effective against various cancer cell lines. However, the anticancer activity and molecular mechanism of CuD in pancreatic cancer remain unknown. Therefore, we aimed to investigate the anticancer activity and molecular mechanism of CuD in the human pancreatic cancer cell line, Capan-1. CuD induced cell cycle arrest at the G2/M phase, apoptosis, and reactive oxygen species generation in Capan-1 cell line. In addition, CuD induced the activation of the p38 MAPK signaling pathway that regulates apoptosis, which was also inhibited by N-acetyl-L-cysteine and the p38 inhibitor SB203580. These data suggest that CuD induces cell cycle arrest and apoptosis via the ROS/p38 pathway in Capan-1 pancreatic cancer cell line; hence, CuD is a promising candidate that should be explored further for its effectiveness as an anticancer agent against pancreatic cancer. [ABSTRACT FROM AUTHOR]

P4-ATPases are lipid flippases that catalyze the transport of phospholipids to create membrane phospholipid asymmetry and to initiate the biogenesis of transport vesicles. Here we show, for the first time, that lipid flippases are essential to dampen the inflammatory response and to mediate the endotoxin-induced endocytic retrieval of Toll-like receptor 4 (TLR4) in human macrophages. Depletion of CDC50A, the β-subunit that is crucial for the activity of multiple P4-ATPases, resulted in endotoxin-induced hypersecretion of proinflammatory cytokines, enhanced MAP kinase signaling and constitutive NF-κB activation. In addition, CDC50A-depleted THP-1 macrophages displayed reduced tolerance to endotoxin. Moreover, endotoxin-induced internalization of TLR4 was strongly reduced and coincided with impaired endosomal MyD88-independent signaling. The phenotype of CDC50A-depleted cells was also induced by separate knockdown of two P4-ATPases, namely ATP8B1 and ATP11A. We conclude that lipid flippases are novel elements of the innate immune response that are essential to attenuate the inflammatory response, possibly by mediating endotoxin-induced internalization of TLR4. [ABSTRACT FROM AUTHOR]

We describe the theoretical and computational framework for the Dynamic Signatures Generated by Regulatory Networks (DSGRN) database. The motivation stems from an urgent need to understand the global dynamics of biologically relevant signal transduction/gene regulatory networks that have at least 5 to 10 nodes, involve multiple interactions, and have decades of parameters. The input to the database computations is a regulatory network, i.e., a directed graph with edges indicating up or down regulation. A computational model based on switching networks is generated from the regulatory network. The phase space dimension of this model equals the number of nodes and the associated parameter space consists of one parameter for each node (a decay rate) and three parameters for each edge (low level of expression, high level of expression, and threshold at which expression levels change). Since the nonlinearities of switching systems are piecewise constant, there is a natural decomposition of phase space into cells from which the dynamics can be described combinatorially in terms of a state transition graph. This in turn leads to a compact representation of the global dynamics called an annotated Morse graph that identifies recurrent and nonrecurrent dynamics. The focus of this paper is on the construction of a natural computable finite decomposition of parameter space into domains where the annotated Morse graph description of dynamics is constant. We use this decomposition to construct an SQL database that can be effectively searched for dynamical signatures such as bistability, stable or unstable oscillations, and stable equilibria. We include two simple 3-node networks to provide small explicit examples of the type of information stored in the DSGRN database. To demonstrate the computational capabilities of this system we consider a simple network associated with p53 that involves 5 nodes and a 29-dimensional parameter space. [ABSTRACT FROM AUTHOR]

Non-alcoholic fatty liver disease (NAFLD), characterized by fat accumulation in liver, is closely associated with central obesity, over-nutrition and other features of metabolic syndrome, which elevate the risk of developing hepatocellular carcinoma (HCC). The Wnt/β-catenin signaling pathway plays a significant role in the physiology and pathology of liver. Up to half of HCC patients have activation of Wnt/β-catenin signaling. However, the mutation frequencies of CTNNB1 (encoding β-catenin protein) or other antagonists targeting Wnt/β-catenin signaling are low in HCC patients, suggesting that genetic mutations are not the major factor driving abnormal β-catenin activities in HCC. Emerging evidence has demonstrated that obesity-induced metabolic pathways can deregulate chromatin modifiers such as histone deacetylase 8 to trigger undesired global epigenetic changes, thereby modifying gene expression program which contributes to oncogenic signaling. This review focuses on the aberrant epigenetic activation of Wnt/ β-catenin in the development of NAFLD-associated HCC. A deeper understanding of the molecular mechanisms underlying such deregulation may shed light on the identification of novel druggable epigenetic targets for the prevention and/or treatment of HCC in obese and diabetic patients. [ABSTRACT FROM AUTHOR]

The adenomatous polyposis coli (APC) tumor suppressor is a multifunctional regulator of Wnt signaling and acts as a mobile scaffold at different cellular sites. APC was recently found to stimulate microtubule (MT) growth at the interphase centrosome; however, little is known about its dynamics and localization at this site. To address this, we analysed APC dynamics in fixed and live cells by fluorescence microscopy. In detergent-extracted cells, we discovered that APC was only weakly retained at the centrosome during interphase suggesting a rapid rate of exchange. This was confirmed in living cells by fluorescence recovery after photobleaching (FRAP), which identified two pools of green fluorescent protein (GFP)-APC: a major rapidly exchanging pool (~86%) and minor retained pool (~14%). The dynamic exchange rate of APC was unaffected by C-terminal truncations implicating a targeting role for the N-terminus. Indeed, we mapped centrosome localization to N-terminal armadillo repeat (ARM) domain amino acids 334-625. Interestingly, the rate of APC movement to the centrosome was stimulated by intact MTs, and APC dynamics slowed when MTs were disrupted by nocodazole treatment or knockdown of -tubulin. Thus, the rate of APC recycling at the centrosome is enhanced by MT growth, suggesting a positive feedback to stimulate its role in MT growth. [ABSTRACT FROM AUTHOR]

Background: Centipeda minima (Ebushicao) has been used for the treatment of various diseases, such as nasal allergies, rhinitis and sinusitis, nasopharyngeal carcinoma, cough, and headache. This study aims to investigate the anticancer activities of Centipeda minima ethanol extracts (CME) against nasopharyngeal carcinoma cell CNE-1 and their underlying mechanism. Methods: CNE-1 cells were treated with diferent concentrations (15-50 µg/mL) of CME for diferent time intervals (24, 48, and 72 h). Cytotoxicity of CME was determined by MTT assay. Cell morphological changes were observed by luorescence microscopy after HO 33258 staining. Cell cycle status was evaluated by low cytometry following pro- pidium iodide staining. Apoptosis was detected by low cytometry following annexin V-FITC/PI staining. The levels of apoptosis-associated and PI3K-Akt-mTOR signaling related proteins were measured by western blotting analysis. Results: CME (15-50 µg/mL) signiicantly inhibited the proliferation of CNE-1 in a dose- and time-dependent manner (P = 0.026 for 15 µg/mL, P < 0.001 for 25, 30, 40, and 50 µg/mL, respectively); the IC50 values (µg/mL) were 41.57 ± 0.17, 30.34 ± 0.06 and 24.98 ± 0.08 for 24, 48 and 72 h treatments, respectively. Signiicant morphological changes of CNE-1 cells displaying apoptosis were observed after CME treatment. CME showed low cytotoxicity toward normal LO2 cells. CNE-1 cells were arrested in the G2/M phase while treated with 15, 25, 40 µg/mL of CME, respectively (P = 0.032, P = 0.0053, P < 0.001). CME (15, 25, 40 µg/mL) down-regulated Bcl-2 expression (P = 0.032, P = 0.0074, P < 0.001), and up-regulated Bax (P = 0.026, P = 0.0056, P < 0.001) with activation of caspase-3, caspase-8, caspase-9, and PARP observed in CNE-1 cells (P = 0.015, P = 0.0067, P < 0.001 for caspase 3; P = 0.210, 0.028, < 0.001 for caspase 8; P = 0.152, 0.082, 0.0080 for caspase 9; P = 0.265, 0.0072, < 0.001 for PARP). CME suppressed the activation of the PI3K-AKT-mTOR pathway (P = 0.03, 0.0007, 0.004, 0.006, 0.022 for p-PI3K, p-Akt-Ser473, p-Akt-Thr308, p-mTOR-Ser2448, p-mTOR-Ser2481, respectively after 40 µg/mL of CME treated for 24 h). Conclusion: CME inhibited the proliferation of CNE-1 cells and activation of the PI3K-AKT-mTOR signaling pathway. [ABSTRACT FROM AUTHOR]

During limb development Pax3 positive myoblasts delaminate from the hypaxial dermomyotome of limb level somites and migrate into the limb bud where they form the dorsal and ventral muscle masses. Only then do they begin to differentiate and express markers of myogenic commitment and determination such as Myf5 and MyoD . However the signals regulating this process remain poorly characterised. We show that FGF18, which is expressed in the distal mesenchyme of the limb bud, induces premature expression of both Myf5 and MyoD and that blocking FGF signalling also inhibits endogenous MyoD expression. This expression is mediated by ERK MAP kinase but not PI3K signalling. We also show that retinoic acid (RA) can inhibit the myogenic activity of FGF18 and that blocking RA signalling allows premature induction of MyoD by FGF18 at HH19. We propose a model where interactions between FGF18 in the distal limb and retinoic acid in the proximal limb regulate the timing of myogenic gene expression during limb bud development. [ABSTRACT FROM AUTHOR]

The calcium-activated K+ (BKCa) channel is one of the potassium-selective ion channels that are present in the nervous and vascular systems. Ca2+ is the main regulator of BKCa channel activation. The BKCa channel contains two high affinity Ca2+ binding sites, namely, regulators of K+ conductance, RCK1 and the Ca2+ bowl. Lysophosphatidic acid (LPA, 1-radyl-2-hydroxy-sn-glycero-3-phosphate) is one of the neurolipids. LPA affects diverse cellular functions on many cell types through G proteincoupled LPA receptor subtypes. The activation of LPA receptors induces transient elevation of intracellular Ca2+ levels through diverse G proteins such as Gαq/11, Gαi, Gα12/13, and Gαs and the related signal transduction pathway. In the present study, we examined LPA effects on BKCa channel activity expressed in Xenopus oocytes, which are known to endogenously express the LPA receptor. Treatment with LPA induced a large outward current in a reversible and concentration-dependent manner. However, repeated treatment with LPA induced a rapid desensitization, and the LPA receptor antagonist Ki16425 blocked LPA action. LPA-mediated BKCa channel activation was also attenuated by the PLC inhibitor U-73122, IP3 inhibitor 2-APB, Ca2+ chelator BAPTA, or PKC inhibitor calphostin. In addition, mutations in RCK1 and RCK2 also attenuated LPA-mediated BKCa channel activation. The present study indicates that LPA-mediated activation of the BKCa channel is achieved through the PLC, IP3, Ca2+, and PKC pathway and that LPA-mediated activation of the BKCa channel could be one of the biological effects of LPA in the nervous and vascular systems [ABSTRACT FROM AUTHOR]

Combining immunotherapy with targeted therapy blocking oncogenic BRAFV600 may result in improved treatments for advanced melanoma. In this study, we developed a BRAFV600E-driven murine model of melanoma, SM1, which is syngeneic to fully immunocompetent mice. SM1 cells exposed to the BRAF inhibitor vemurafenib (PLX4032) showed partial in vitro and in vivo sensitivity resulting from the inhibition of MAPK pathway signaling. Combined treatment of vemurafenib plus adoptive cell transfer therapy with lymphocytes genetically modified with a T-cell receptor (TCR) recognizing chicken ovalbumin (OVA) expressed by SM1-OVA tumors or pmel-1 TCR transgenic lymphocytes recognizing gp100 endogenously expressed by SM1 resulted in superior antitumor responses compared with either therapy alone. T-cell analysis showed that vemurafenib did not significantly alter the expansion, distribution, or tumor accumulation of the adoptively transferred cells. However, vemurafenib paradoxically increased mitogen-activated protein kinase (MAPK) signaling, in vivo cytotoxic activity, and intratumoral cytokine secretion by adoptively transferred cells. Taken together, our findings, derived from 2 independent models combining BRAF-targeted therapy with immunotherapy, support the testing of this therapeutic combination in patients with BRAFV600 mutant metastatic melanoma. [ABSTRACT FROM AUTHOR]

Cancer cells are able to proliferate in an unregulated manner. There are several mechanisms involved that propel such neoplastic transformations. One of these processes involves bypassing cell death through changes in gene expression and, consequently, cell growth. This involves a complex epigenetic interaction within the cell, which drives it towards oncogenic transformations. These epigenetic events augment cellular growth by potentially altering chromatin structures and influencing key gene expressions. Therapeutic mechanisms have been developed to combat this by taking advantage of the underlying oncogenic mechanisms through chemical modulation. Camptothecin (CPT) is an example of this type of drug. It is a selective topoisomerase I inhibitor that is effective against many cancers, such as colorectal cancer. Previously, we successfully formulated a magnetic nanocarrier-conjugated CPT with β-cyclodextrin and iron NPs (Fe3O4) cross-linked using EDTA (CPT-CEF). Compared to CPT alone, it boasts higher efficacy due to its selective targeting and increased solubility. In this study, we treated HT29 colon cancer cells with CPT-CEF and attempted to investigate the cytotoxic effects of the formulation through an epigenetic perspective. By using RNA-Seq, several differentially expressed genes were obtained (p < 0.05). Enrichr was then used for the over-representation analysis, and the genes were compared to the epigenetic roadmap and histone modification database. The results showed that the DEGs had a high correlation with epigenetic modifications involving histone H3 acetylation. Furthermore, a subset of these genes was shown to be associated with the Wnt/β-catenin signaling pathway, which is highly upregulated in a large number of cancer cells. These genes could be investigated as downstream therapeutic targets against the uncontrolled proliferation of cancer cells. Further interaction analysis of the identified genes with the key genes of the Wnt/β-catenin signaling pathway in colorectal cancer identified the direct interactors and a few transcription regulators. Further analysis in cBioPortal confirmed their genetic alterations and their distribution across patient samples. Thus, the findings of this study reveal that colorectal cancer could be reversed by treatment with the CPT-CEF nanoparticle-conjugated nanocarrier through an epigenetic mechanism. [ABSTRACT FROM AUTHOR]

Due to the unavoidable nonbiological variations accompanying many experiments, it is imperative to consider a way of unravelling the functional interaction structure of a cellular network (e.g. signalling cascades or gene networks) by using the qualitative information of time-series experimental data instead of computation through the measured absolute values. In this spirit, we propose a very simple but effective method of identifying the functional interaction structure of a cellular network based on temporal ascending or descending slope information from given time-series measurements. From this method, we can gain insight into the acceptable measurement error ranges in order to estimate the correct functional interaction structure and we can also find guidance for a new experimental design to complement the insufficient information of a given experimental dataset. We developed experimental sign equations, making use of the temporal slope sign information from time-series experimental data, without a specific assumption on parameter perturbations for each network node. Based on these equations, we further describe the available specific information from each part of experimental data in detail and show the functional interaction structure obtained by integrating such information. In this procedure, we use only simple algebra on sign changes without complicated computations on the measured absolute values of the experimental data. The result is, however, verified through rigorous mathematical definitions and proofs. The present method provides us with information about the acceptable measurement error ranges for correct estimation of the functional interaction structure and it further leads to a new experimental design to complement the given experimental data by informing us about additional specific sampling points to be chosen for further required information. [ABSTRACT FROM AUTHOR]

Simple Summary: In our previous study, purinergic P2Y2 receptor (P2Y2R) activation by ATP was found to play an important role in tumor progression and metastasis by regulating various responses in cancer cells and modulating crosstalk between cancer cells and endothelial cells (ECs). Therefore, we expected that P2Y2R would play a critical role in radioresistance and enhanced tumor progression in radioresistant triple-negative breast cancer (RT-R-TNBC). However, interestingly, P2Y2R expression was slightly decreased in RT-R-TNBC cells, while the expression of A2AR was significantly increased both in RT-R-TNBC cells and in tumor tissues, especially triple negative breast cancer (TNBC) tissues of breast cancer (BC) patients. Thus, we aimed to investigate the role of adenosine A2A receptor (A2AR) and its signaling pathway in the progression of RT-R-TNBC. The results reveal for the first time the role of A2AR in the progression and metastasis of RT-R-BC cells and suggest that the adenosine (ADO)-activated intracellular A2AR signaling pathway is linked to the AKT-β-catenin pathway to regulate RT-R-BC cell invasiveness and metastasis. Recently, we found that the expressions of adenosine (ADO) receptors A2AR and A2BR and the ectonucleotidase CD73 which is needed for the conversion of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and the extracellular ADO level are increased in TNBC MDA-MB-231 cells and RT-R-MDA-MB-231 cells compared to normal cells or non-TNBC cells. The expression of A2AR, but not A2BR, is significantly upregulated in breast cancer tissues, especially TNBC tissues, compared to normal epithelial tissues. Therefore, we further investigated the role of ADO-activated A2AR and its signaling pathway in the progression of RT-R-TNBC. ADO treatment induced MDA-MB-231 cell proliferation, colony formation, and invasion, which were enhanced in RT-R-MDA-MB-231 cells in an A2AR-dependent manner. A2AR activation by ADO induced AKT phosphorylation and then β-catenin, Snail, and vimentin expression, and these effects were abolished by A2AR-siRNA transfection. In an in vivo animal study, compared to 4T1-injected mice, RT-R-4T1-injected mice exhibited significantly increased tumor growth and lung metastasis, which were decreased by A2AR-knockdown. The upregulation of phospho-AKT, β-catenin, Snail, and vimentin expression in mice injected with RT-R-4T1 cells was also attenuated in mice injected with RT-R-4T1-A2AR-shRNA cells. These results suggest that A2AR is significantly upregulated in BC tissues, especially TNBC tissues, and ADO-mediated A2AR activation is involved in RT-R-TNBC invasion and metastasis through the AKT-β-catenin pathway. [ABSTRACT FROM AUTHOR]

Simple Summary: Epidemiological and experimental studies have demonstrated that aspirin (acetylsalicylic acid) may prevent the incidence of some types of human cancer, including colorectal cancer and hepatocellular carcinoma (HCC). In addition, preclinical studies indicate that aspirin in combination with other treatments may achieve a more significant anti-cancer effect for established tumors. This study aims to improve the anti-cancer effect of aspirin by targeting signaling pathways related to aspirin and its targets. We find that aspirin may induce cAMP–PKA–CREB/ATF1 signaling in HCC via AMPK and its downstream target carbamoyl-phosphate synthase 1 (CPS1). Blockade of PKA–CREB/ATF1 signaling by the natural agent berbamine could sensitize HCC to aspirin. This research indicates that the combination of two inexpensive drugs, aspirin and berbamine, holds promise in preventing and treating HCC. Aspirin can prevent or inhibit inflammation-related cancers, such as colorectal cancer and hepatocellular carcinoma (HCC). However, the effectiveness of chemotherapy may be compromised by activating oncogenic pathways in cancer cells. Elucidation of such chemoresistance mechanisms is crucial to developing novel strategies to maximize the anti-cancer effects of aspirin. Here, we report that aspirin markedly induces CREB/ATF1 phosphorylation in HCC cells, which compromises aspirin's anti-HCC effect. Inhibition of AMP-activated protein kinase (AMPK) abrogates the induction of CREB/ATF1 phosphorylation by aspirin. Mechanistically, activation of AMPK by aspirin results in decreased expression of the urea cycle enzyme carbamoyl-phosphate synthase 1 (CPS1) in HCC cells and xenografts. Treatment with aspirin or CPS1 knockdown stimulates soluble adenylyl cyclase expression, thereby increasing cyclic AMP (cAMP) synthesis and stimulating PKA–CREB/ATF1 signaling. Importantly, abrogation of aspirin-induced CREB/ATF1 phosphorylation could sensitize HCC to aspirin. The bis-benzylisoquinoline alkaloid berbamine suppresses the expression of cancerous inhibitor of protein phosphatase 2A (CIP2A), leading to protein phosphatase 2A-mediated downregulation of CREB/ATF1 phosphorylation. The combination of berbamine and aspirin significantly inhibits HCC in vitro and in vivo. These data demonstrate that the regulation of cAMP-PKA-CREB/ATF1 signaling represents a noncanonical function of CPS1. Targeting the PKA–CREB/ATF1 axis may be a strategy to improve the therapeutic effects of aspirin on HCC. [ABSTRACT FROM AUTHOR]

Simple Summary: Cancer cachexia is a systemic inflammatory disease characterized by the loss of muscle and fat and occurs in 50–80% of cancer patients. In cancer cachexia, the tumor tissues interact with other tissues and organs using secretory factors. Differentiated immune cells from hematopoietic stem cells (HSCs) of the bone marrow contribute to the systemic inflammation and may be affected by these intertissue interactions. However, the significant changes that occur in the bone marrow and the underlying mechanisms are still unclear. Here, we investigated the effects of cancer cachexia on bone and stem cells that reside in the bone marrow using a lung cancer cachexia animal model. Cancer cachexia induces bone loss and impairs the properties of the bone marrow mesenchymal stem cells via JAK/STAT and glucocorticoid signaling. Our findings provide new insights for developing a novel therapeutic strategy for cancer cachexia. Cancer cachexia is a multifactorial systemic inflammation disease caused by complex interactions between the tumor and host tissues via soluble factors. However, whether cancer cachexia affects the bone marrow, in particular the hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs), remains unclear. Here, we investigated the bone marrow and bone in a cancer cachexia animal model generated by transplanting Lewis lung carcinoma cells. The number of bone marrow mononuclear cells (BM-MNCs) started to significantly decrease in the cancer cachectic animal model prior to the discernable loss of muscle and fat. This decrease in BM-MNCs was associated with myeloid skewing in the circulation and the expansion of hematopoietic progenitors in the bone marrow. Bone loss occurred in the cancer cachexia animal model and accompanied the decrease in the bone marrow MSCs that play important roles in both supporting HSCs and maintaining bone homeostasis. Glucocorticoid signaling mediated the decrease in bone marrow MSCs in the cancer cachectic environment. The cancer cachexia environment also skewed the differentiation of the bone marrow MSCs toward adipogenic fate via JAK/STAT as well as glucocorticoid signaling. Our results suggest that the bone loss induced in cancer cachexia is associated with the depletion and the impaired differentiation capacity of the bone marrow MSCs. [ABSTRACT FROM AUTHOR]

Simple Summary: The prognosis of esophageal squamous cell carcinoma (ESCC) patients is poor, with a five-year survival of 15–34%. We examined the expression of STAT3α and STAT3β in pretreatment tumor biopsies of 105 ESCC patients who received concurrent chemoradiotherapy (CCRT) by immunohistochemistry. The data showed that ESCC patients who demonstrate both high STAT3α expression and high STAT3β expression in the cytoplasm have a significantly better survival rate. Moreover, the ESCC patients with high STAT3β expression have a complete response to concurrent chemoradiotherapy. STAT3β-overexpressed ESCC cell lines exhibit CCRT (platinum plus radiation therapy) sensitivity, resulting in cell death. RNA sequencing found that ESCC cells highly expressing STAT3β undergo necrosis after CCRT. In summary, STAT3β could be potentially used to predict the response to CCRT, which may provide an important insight into the treatment of ESCC. Concurrent chemoradiotherapy (CCRT), especially platinum plus radiotherapy, is considered to be one of the most promising treatment modalities for patients with advanced esophageal cancer. STAT3β regulates specific target genes and inhibits the process of tumorigenesis and development. It is also a good prognostic marker and a potential marker for response to adjuvant chemoradiotherapy (ACRT). We aimed to investigate the relationship between STAT3β and CCRT. We examined the expression of STAT3α and STAT3β in pretreatment tumor biopsies of 105 ESCC patients who received CCRT by immunohistochemistry. The data showed that ESCC patients who demonstrate both high STAT3α expression and high STAT3β expression in the cytoplasm have a significantly better survival rate, and STAT3β expression is an independent protective factor (HR = 0.424, p = 0.003). Meanwhile, ESCC patients with high STAT3β expression demonstrated a complete response to CCRT in 65 patients who received platinum plus radiation therapy (p = 0.014). In ESCC cells, high STAT3β expression significantly inhibits the ability of colony formation and cell proliferation, suggesting that STAT3β enhances sensitivity to CCRT (platinum plus radiation therapy). Mechanistically, through RNA-seq analysis, we found that the TNF signaling pathway and necrotic cell death pathway were significantly upregulated in highly expressed STAT3β cells after CCRT treatment. Overall, our study highlights that STAT3β could potentially be used to predict the response to platinum plus radiation therapy, which may provide an important insight into the treatment of ESCC. [ABSTRACT FROM AUTHOR]

Abstract: The catalytic subunit of acetylcholinesterase (AChET) interacts with proline-rich membrane anchor (PRiMA) to form PRiMA-linked G4 AChE on membrane surface for its cholinergic function. Cultured PC12 cells expressed the transcripts encoding AChET and PRiMA I, but the expression of PRiMA II transcript was below detection. Upon the treatment of dibutyryl-cAMP (Bt2-cAMP) and forskolin in cultured cells to stimulate the cAMP-dependent signaling pathway, the mRNA expressions of both AChET and PRiMA I, as well as the enzymatic activity were up-regulated. More importantly, sucrose density gradient analysis revealed that both G1 and G4 AChE isoforms were increased in the Bt2-cAMP-treated cultures. These results suggest that the regulation of PRiMA-linked G4 AChE in terms of gene transcription and molecular assembly in the cultured PC12 cells could be mediated by a cAMP-dependent signaling mechanism. [Copyright &y& Elsevier]

Pulmonary lymphoepithelioma-like carcinoma (LELC) is a subtype of non-small cell lung cancer (NSCLC) characterized by marked lymphocytic infiltration and association with Epstein–Barr virus (EBV). The molecular basis underlying the disease remains unclear. We sought to study the molecular landscape by multiple approaches including whole genomic sequencing, capture-based targeted sequencing, fluorescent in situ hybridization and immunohistochemistry. Tumor cells from 57 EBV-positive pulmonary LELCs were isolated by careful microdissection prior to genomic sequencing. Integrated analysis revealed a distinct genomic landscape of low TP53 mutation rate (11%), low incidence of known drivers in the RTK/RAS/RAF (11%) and PI3K/AKT/mTOR pathways (7%), but enriched for loss-of-function mutations in multiple negative regulators of the NF-κB pathway. High level programmed cell death ligand-1 (PD-L1) expression was shown with 47% and 79% of the cases showing positive PD-L1 immunoreactivity at ≥50% and ≥1% tumor proportion score, respectively. Subsets of the patients with actionable fibroblast growth factor receptor 3 (FGFR3) aberrations (4%) and mismatch repair deficiency (4%) were potentially eligible for precision medicine. Pulmonary LELC showed a distinct genomic landscape, different from major NSCLC subtypes but resembled that of EBV-associated nasopharyngeal carcinoma. Our work facilitated the understanding of molecular basis underlying pulmonary LELC to explore potential therapeutic options. [ABSTRACT FROM AUTHOR]

Cholesterol plays an important role in cellular homeostasis by maintaining the rigidity of cell membranes, providing a medium for signaling transduction, and being converted into other vital macromolecules, such as sterol hormones and bile acids. Epidemiological studies have shown the correlation between cholesterol content and cancer incidence worldwide. Accumulating evidence has shown the emerging roles of the dysregulation of cholesterol metabolism in cancer development. More specifically, recent reports have shown the distinct role of cholesterol in the suppression of immune cells, regulation of cell survival, and modulation of cancer stem cells in cancer. Here, we provide a comprehensive review of the epidemiological analysis, functional roles, and mechanistic action of cholesterol homeostasis in regard to its contribution to cancer development. Based on the existing data, cholesterol homeostasis is identified to be a new key player in cancer pathogenesis. Lastly, we also discuss the therapeutic implications of natural compounds and cholesterol-lowering drugs in cancer prevention and treatment. In conclusion, intervention in cholesterol metabolism may offer a new therapeutic avenue for cancer treatment. [ABSTRACT FROM AUTHOR]

Non-small-cell lung cancer (NSCLC) is the most common lung cancer subtype and accounts for more than 80% of all lung cancer cases. Epidermal growth factor receptor (EGFR) phosphorylation by binding growth factors such as EGF activates downstream prooncogenic signaling pathways including KRAS-ERK, JAK-STAT, and PI3K-AKT. These pathways promote the tumor progression of NSCLC by inducing uncontrolled cell cycle, proliferation, migration, and programmed death-ligand 1 (PD-L1) expression. New cytotoxic drugs have facilitated considerable progress in NSCLC treatment, but side effects are still a significant cause of mortality. Gallic acid (3,4,5-trihydroxybenzoic acid; GA) is a phenolic natural compound, isolated from plant derivatives, that has been reported to show anticancer effects. We demonstrated the tumor-suppressive effect of GA, which induced the decrease of PD-L1 expression through binding to EGFR in NSCLC. This binding inhibited the phosphorylation of EGFR, subsequently inducing the inhibition of PI3K and AKT phosphorylation, which triggered the activation of p53. The p53-dependent upregulation of miR-34a induced PD-L1 downregulation. Further, we revealed the combination effect of GA and anti-PD-1 monoclonal antibody in an NSCLC-cell and peripheral blood mononuclear–cell coculture system. We propose a novel therapeutic application of GA for immunotherapy and chemotherapy in NSCLC. [ABSTRACT FROM AUTHOR]

Spontaneous tumors in pet dogs represent a valuable but undercharacterized cancer model. To better use this resource, we performed an initial global comparison between proliferative and invasive colorectal tumors from 20 canine cases, and evaluated their molecular homology to human colorectal cancer (CRC). First, proliferative canine tumors harbor overactivated WNT/β-catenin pathways and recurrent CTNNB1 (β-catenin) mutations S45F/P, D32Y and G34E. Invasive canine tumors harbor prominent fibroblast proliferation and overactivated stroma. Both groups have recurrent TP53 mutations. We observed three invasion patterns in canine tumors: collective, crypt-like and epithelial–mesenchymal transition (EMT). We detected enriched Helicobacter bilis and Alistipes finegoldii in proliferative and crypt-like tumors, but depleted mucosa-microbes in the EMT tumor. Second, guided by our canine findings, we classified 79% of 478 human colon cancers from The Cancer Genome Atlas into four subtypes: primarily proliferative, or with collective, crypt-like or EMT invasion features. Their molecular characteristics match those of canine tumors. We showed that consensus molecular subtype 4 (mesenchymal) of human CRC should be further divided into EMT and crypt-like subtypes, which differ in TGF-β activation and mucosa-microbe content. Our canine tumors share the same pathogenic pathway as human CRCs. Dog-human integration identifies three CRC invasion patterns and improves CRC subtyping. [ABSTRACT FROM AUTHOR]

Most patients with non-small-cell lung cancer present with advanced stage disease, for which standard therapy would be a platinum-based doublet combination. Novel targeted therapies that interfere with specific molecular signaling pathways have emerged as a new standard option for selected patients with epidermal growth factor receptor (EGFR) mutation. Clinical trials have demonstrated significant ethnic variation in tumor response to EGFR tyrosine kinase inhibitors (TKIs) and incidence of EGFR mutations, with a significantly higher incidence of activated EGFR mutation in Asia. This review discusses the variations and the implications for treatment. [ABSTRACT FROM AUTHOR]

The Ras-Raf-MEK-ERK pathway (or ERK pathway) is an important signal transduction system involved in the control of cell proliferation, survival and differentiation. However, the dynamic regulation of the pathway by positive- and negative-feedback mechanisms, in particular the functional role of Raf kinase inhibitor protein (RKIP) are still incompletely understood. RKIP is a physiological endogenous inhibitor of MEK phosphorylation by Raf kinases, but also participates in a positive-feedback loop in which ERK can inactivate RKIP. The aim of this study was to elucidate the hidden dynamics of these feedback mechanisms and to identify the functional role of RKIP through combined efforts of biochemical experiments and in silico simulations based on an experimentally validated mathematical model. We show that the negative-feedback loop from ERK to SOS plays a crucial role in generating an oscillatory behavior of ERK activity. The positive-feedback loop in which ERK functionally inactivates RKIP also enhances the oscillatory activation pattern of ERK. However, RKIP itself has an important role in inducing a switch-like behavior of MEK activity. When overexpressed, RKIP also causes delayed and reduced responses of ERK. Thus, positive- and negative-feedback loops and RKIP work together to shape the response pattern and dynamical characteristics of the ERK pathway. [ABSTRACT FROM AUTHOR]

Klhl31 is a member of the Kelch-like family in vertebrates, which are characterized by an amino-terminal b road complex t ram-track, b ric-a-brac/ po xvirus and z inc finger (BTB/POZ) domain, carboxy-terminal Kelch repeats and a central linker region (Back domain). In developing somites Klhl31 is highly expressed in the myotome downstream of myogenic regulators (MRF), and it remains expressed in differentiated skeletal muscle. In vivo gain- and loss-of-function approaches in chick embryos reveal a role of Klhl31 in skeletal myogenesis. Targeted mis-expression of Klhl31 led to a reduced size of dermomyotome and myotome as indicated by detection of relevant myogenic markers, Pax3, Myf5, myogenin and myosin heavy chain (MF20). The knock-down of Klhl31 in developing somites, using antisense morpholinos (MO), led to an expansion of Pax3, Myf5, MyoD and myogenin expression domains and an increase in the number of mitotic cells in the dermomyotome and myotome. The mechanism underlying this phenotype was examined using complementary approaches, which show that Klhl31 interferes with β-catenin dependent Wnt signaling. Klhl31 reduced the Wnt-mediated activation of a luciferase reporter in cultured cells. Furthermore, Klhl31 attenuated secondary axis formation in Xenopus embryos in response to Wnt1 or β-catenin. Klhl31 mis-expression in the developing neural tube affected its dorso-ventral patterning and led to reduced dermomyotome and myotome size. Co-transfection of a Wnt3a expression vector with Klhl31 in somites or in the neural tube rescued the phenotype and restored the size of dermomyotome and myotome. Thus, Klhl31 is a novel modulator of canonical Wnt signaling, important for vertebrate myogenesis. We propose that Klhl31 acts in the myotome to support cell cycle withdrawal and differentiation. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance Lonicera japonica Thunberg is a traditional herbal medicine widely used in East Asia as an anti-bacterial, anti-inflammatory, and antiviral agent. This study was designed to investigate the effects of HS-23, ethanol extract of the dried flower buds of Lonicera japonica, in experimental models of sepsis and elucidate the mechanisms of action of HS-23. Materials and methods Male ICR mice were intravenously administered HS-23 (20 and 40 mg/kg) for 0 (immediately) and 24 h after cecal ligation and puncture (CLP) for survival tests, and HS-23 (40 mg/kg) immediately after CLP for biochemical assays. Results HS-23 improved sepsis-induced mortality, enhanced bacterial clearance, and attenuated multiple organ failure. The mechanisms of action of HS-23 included attenuation of increased toll-like receptor (TLR)4 protein and mRNA expression. HS-23 suppressed sepsis-induced increases in protein expression of myeloid differentiation primary response protein 88, p38 and c-Jun N-terminal kinase in both liver and lung, as well as TIR-domain-containing adapter-inducing interferon-β and interferon regulatory transcription factor 3 protein expression in liver. Conclusion The results of this study revealed that HS-23 attenuated sepsis through suppression of TLR signaling pathways. Therefore, our findings suggest that HS-23 might be useful as a potential therapeutic agent for treatment of sepsis. [ABSTRACT FROM AUTHOR]

The invasion of luminal antigens and an aberrant immune response resulting from a disrupted physical epithelial barrier are the key characteristics of ulcerative colitis (UC). The restoration of damaged epithelial function is crucial for maintaining mucosal homeostasis and disease quiescence. Current therapies for UC primarily focus on suppressing inflammation. However, most patients fail to respond to therapy or develop secondary resistance over time, emphasizing the need to develop novel therapeutic targets for UC. Our study aimed to identify the potential targets of a novel modified herbal formula from the Zhen Wu Decoction, namely CDD-2103, which has demonstrated promising efficacy in treating chronic colitis. The effect of CDD-2103 on epithelial barrier function was examined using in vitro and ex vivo models of tissue injury, as well as a chronic colitis C57BL/6 mouse model. Transcriptomic analysis was employed to profile gene expression changes in colonic tissues following treatment with CDD-2103. Our in vivo experiments demonstrated that CDD-2103 dose-dependently reduced disease severity in mice with chronic colitis. The efficacy of CDD-2103 was mediated by a reduction in goblet cell loss and the enhancement of tight junction protein integrity. Mechanistically, CDD-2103 suppressed epithelial cell apoptosis and tight junction protein breakdown by activating the soluble guanynyl cyclase (sGC)-mediated cyclic guanosine monophosphate (cGMP)/PKG signaling cascade. Molecular docking analysis revealed strong sGC ligand recognition by the CDD-2103-derived molecules, warranting further investigation. Our study revealed a novel formulation CDD-2103 that restores intestinal barrier function through the activation of sGC-regulated cGMP/PKG signaling. Furthermore, our findings suggest that targeting sGC can be an effective approach for promoting mucosal healing in the management of UC. Among those with Ulcerative colitis (UC), intestinal barrier disruption is a key pathophysiological event. CDD-2103 inhibited the death of epithelial cells and the degradation of tight junction proteins by initiating the soluble guanynyl cyclase (sGC)-mediated cyclic guanosine monophosphate (cGMP)/PKG signal trail. [Display omitted] • CDD-2103 improved intestinal barrier function of colitis by activating cGMP-PKG signaling. • CDD-2103 reversed goblet cell loss and enhanced tight junction protein integrity. • CDD-2103 activated guanylyl cyclase-α1 regulated cGMP-PKG signaling cascade. • Binding of CDD-2103 compounds to soluble guanynyl cyclase support the activity of CDD-2103. [ABSTRACT FROM AUTHOR]

Diquafosol is known as a purinergic P2Y2 receptor (P2Y2R) agonist that stimulates water and mucin secretion from conjunctival epithelial cells and goblet cells, leading to tear film stability in dry eye. However, its effect on corneal epithelial healing has not yet been elucidated. The aim of the present study was to evaluate the effect of diquafosol on corneal epithelial healing in vivo and on P2Y2R-related downstream signaling pathways in vitro . We administered 3% diquafosol ophthalmic solution on 3 mm-diameter epithelial defects made in rat corneas and assessed the wound closure over time. Corneal epithelial healing was significantly accelerated in diquafosol-treated eyes compared to control eyes at 12 and 24 h. During wound healing, P2Y2R staining appeared stronger in the re-epithelized margin near the wound defect. To evaluate whether diquafosol stimulates epidermal growth factor receptor/extracellular-signal-regulated kinase (EGFR/ERK)-related cell proliferation and migration, simian virus 40-transfected human corneal epithelial (THCE) cells were used for in vitro experiments. Cell proliferation was accelerated by diquafosol at concentrations from 20 to 200 μM during 48 h, but inhibited at concentrations over 2000 μM. The intracellular calcium ([Ca 2+ ] i ) elevation was measured in diquafosol (100 μM)-stimulated cells using Fluo-4/AM ([Ca 2+ ] i indicator). [Ca 2+ ] i elevation was observed in diquafosol-stimulated cells regardless of the presence of calcium in media, and suramin pretreatment inhibited the calcium response. The effect of diquafosol on phosphorylation of EGFR, ERK and Akt, and cell migration was determined by western blotting and in vitro cell migration assay. Diquafosol induced phosphorylation of EGFR at 2 min post-stimulation, and phosphorylation of ERK at 5 min post-stimulation. Phosphorylation of ERK was attenuated in cells pretreated with suramin or BAPTA/AM ([Ca 2+ ] i chelator), and partially with AG1478 (EGFR inhibitor). Likewise, diquafosol-treated cells showed acceleration of gap closure in cell migration assay, which was inhibited by suramin, BAPTA/AM, AG1478, and U0126 (MEK inhibitor). These studies demonstrate that diquafosol is effective in promoting corneal epithelial wound healing and that this effect may result from ERK-stimulated cell proliferation and migration via P2Y2R-mediated [Ca 2+ ] i elevation. [ABSTRACT FROM AUTHOR]

Stem cell (SC) therapy has become a potential treatment modality for pulmonary artery hypertension (PAH), but the efficacy of human SC and priming effects have not yet been established. The mobilization and homing of hematopoietic stem cells (HSCs) are modulated by priming factors that include a bioactive lipid, sphingosine-1-phosphate (S1P), which stimulates CXCR4 receptor kinase signaling. Here, we show that priming human mesenchymal stem cells (MSCs) with S1P enhances their therapeutic efficacy in PAH. Human MSCs, similar to HSCs, showed stronger chemoattraction to S1P in transwell assays. Concomitantly, MSCs treated with 0.2 μM S1P showed increased phosphorylation of both MAPKp42/44 and AKT protein compared with nonprimed MSCs. Furthermore, S1P-primed MSCs potentiated colony forming unit-fibroblast, anti-inflammatory, and angiogenic activities of MSCs in culture. In a PAH animal model induced by subcutaneously injected monocrotaline, administration of human cord blood-derived MSCs (hCB-MSCs) or S1P-primed cells significantly attenuated the elevated right ventricular systolic pressure. Notably, S1P-primed CB-MSCs, but not unprimed hCB-MSCs, also elicited a significant reduction in the right ventricular weight ratio and pulmonary vascular wall thickness. S1P-primed MSCs enhanced the expression of several genes responsible for stem cell trafficking and angiogenesis, increasing the density of blood vessels in the damaged lungs. Thus, this study demonstrates that human MSCs have potential utility for the treatment of PAH, and that S1P priming increases the effects of SC therapy by enhancing cardiac and vascular remodeling. By optimizing this protocol in future studies, SC therapy might form a basis for clinical trials to treat human PAH. [ABSTRACT FROM AUTHOR]

The BRAF mutant, BRAFV600E, is expressed in nearly half of melanomas, and oral BRAF inhibitors induce substantial tumor regression in patients with BRAFV600E metastatic melanoma. The inhibitors are believed to work primarily by inhibiting BRAFV600E-induced oncogenic MAPK signaling; however, some patients treated with BRAF inhibitors exhibit increased tumor immune infiltration, suggesting that a combination of BRAF inhibitors and immunotherapy maybe beneficial. We used two relatively resistant variants of BrafV600E-driven mouse melanoma (SMI and SM1WT1) and melanoma-prone mice to determine the role of host immunity in type I BRAF inhibitor PLX4720 antitumor activity. We found that PLX4720 treatment downregulated tumor Ccl2 gene expression and decreased tumor CCL2 expression in both BrafV600E mouse melanoma transplants and in de novo melanomas in a manner that was coincident with reduced tumor growth. While PLX4720 did not directly increase tumor immunogenicity, analysis of SMI tumor-infiltrating leukocytes in PLX4720-treated mice demonstrated a robust increase in CD8+T/FoxP3+CD4+ T cell ratio and NK cells. Combination therapy with PLX4720 and anti-CCL2 or agonistic anti-CD137 antibodies demonstrated significant antitumor activity in mouse transplant and de novo tumorigenesis models. These data elucidate a role for host CCR2 in the mechanism of action of type I BRAF inhibitors and support the therapeutic potential of combining BRAF inhibitors with immunotherapy. [ABSTRACT FROM AUTHOR]

Summary: MET amplification activates ERBB3/PI3K/AKT signaling in EGFR mutant lung cancers and causes resistance to EGFR kinase inhibitors. We demonstrate that MET activation by its ligand, HGF, also induces drug resistance, but through GAB1 signaling. Using high-throughput FISH analyses in both cell lines and in patients with lung cancer, we identify subpopulations of cells with MET amplification prior to drug exposure. Surprisingly, HGF accelerates the development of MET amplification both in vitro and in vivo. EGFR kinase inhibitor resistance, due to either MET amplification or autocrine HGF production, was cured in vivo by combined EGFR and MET inhibition. These findings highlight the potential to prospectively identify treatment naive, patients with EGFR-mutant lung cancer who will benefit from initial combination therapy. [Copyright &y& Elsevier]

Abstract: β-catenin is a key mediator of the Wnt signaling process and accumulates in the nucleus and at the membrane in response to Wnt-mediated inhibition of GSK-3β. In this study we used live cell photobleaching experiments to determine the dynamics and rate of recruitment of β-catenin at membrane adherens junctions (cell adhesion) and membrane ruffles (cell migration). First, we confirmed the nuclear-cytoplasmic shuttling of GFP-tagged β-catenin, and found that a small mobile pool of β-catenin can move from the nucleus to membrane ruffles in NIH 3T3 fibroblasts with a t 0.5 of ~30 s. Thus, β-catenin can shuttle between the nucleus and plasma membrane. The localized recruitment of β-catenin-GFP to membrane ruffles was more rapid, and the strong recovery observed after bleaching (mobile fraction 53%, t 0. 5 ~5 s) is indicative of high turnover and transient association. In contrast, β-catenin-GFP displayed poor recovery at adherens junctions in MDCK epithelial cells (mobile fraction 10%, t 0. 5 ~8 s), indicating stable retention at these membrane structures. We previously identified IQGAP1 as an upstream regulator of β-catenin at the membrane, and this is supported by photobleaching assays which now reveal IQGAP1 to be more stably anchored at membrane ruffles than β-catenin. Further analysis showed that LiCl-mediated inactivation of the kinase GSK-3β increased β-catenin membrane ruffle staining; this correlated with a faster rate of recruitment and not increased membrane retention of β-catenin. In summary, β-catenin displays a high turnover rate at membrane ruffles consistent with its dynamic internalization and recycling at these sites by macropinocytosis. [Copyright &y& Elsevier]

Abstract: Acetylcholinesterase (AChE) is a highly polymorphic enzyme. Alternative splicing in the 3′ region of the primary transcript generates different subunits that contain the same catalytic domain but with distinct carboxyl termini. In mammals, the AChER variant produces a soluble monomer that is up-regulated in the brain during stress. The AChEH variant produces a GPI-anchored dimer that is mainly expressed in blood cells, while AChET variant is largely predominant in the brain and muscle. AChET subunits associate with a collagen tail subunit (ColQ) forming asymmetric AChE species (A4, A8, and A12 AChE) in muscle, and also form amphiphilic tetramers associated with a proline-rich membrane anchor (PRiMA) as globular AChE (G4 AChE) in brain and muscle. The formation of these AChE forms depends on the physiological status of the muscles, and on the innervating nerves. The motor nerves achieve this regulation by two distinct mechanisms: release of the trophic factor calcitonin gene-related peptide (CGRP) and nerve-evoked electrical activity, which differentially regulate the expression levels of AChET, PRiMA and ColQ via different downstream signaling cascades. The regulatory mechanisms provided by the nerve are important to account for the different expression patterns of AChE and associated proteins in fast- and slow-twitch muscles. [Copyright &y& Elsevier]