Downstream Signaling Pathways Research Articles

Agonist Discovery for Membrane Proteins on Live Cells by Using DNA-encoded Libraries.

Identifying biologically active ligands for membrane proteins is an important task in chemical biology. We report an approach to directly identify small molecule agonists against membrane proteins by selecting DNA-encoded libraries (DELs) on live cells. This method connects extracellular ligand binding with intracellular biochemical transformation, thereby biasing the selection toward agonist identification. We have demonstrated the methodology with three membrane proteins: epidermal growth factor receptor (EGFR), thrombopoietin receptor (TPOR), and insulin receptor (INSR). A ∼30 million and a 1.033 billion-compound DEL were selected against these targets, and novel agonists with subnanomolar affinity and low micromolar cellular activities have been discovered. The INSR agonists activated the receptor by possibly binding to an allosteric site, exhibited clear synergistic effects with insulin, and activated the downstream signaling pathways. Notably, the agonists did not activate the insulin-like growth factor 1 receptor (IGF-1R), a highly homologous receptor whose activation may lead to tumor progression. Collectively, this work has developed an approach toward "functional" DEL selections on the cell surface and may provide a widely applicable method for agonist discovery for membrane proteins.

Soft Corals-derived Dihydrosinularin Attenuates Neuronal Apoptosis in 6-hydroxydopamine-induced Cell Model of Parkinson's Disease by Regulating the PI3K Pathway.

Parkinson's disease (PD) is an irreversible, progressive disorder that profoundly impacts both motor and non-motor functions, thereby significantly diminishing the individual's quality of life. Dihydrosinularin (DHS), a natural bioactive molecule derived from soft corals, exhibits low cytotoxicity and anti-inflammatory properties. However, the therapeutic effects of DHS on neurotoxins and PD are currently unknown. This study investigated whether DHS could mitigate 6-hydroxydopamine (6- OHDA)-induced neurotoxicity and explored the role of neuroprotective PI3K downstream signaling pathways, including that of AKT, ERK, JNK, BCL2, and NFκB, in DHS- mediated neuroprotection. We treated the human neuroblastoma cell line, SH-SY5Y, with the neurotoxin 6-OHDA to establish a cellular model of PD. Meanwhile, we assessed the anti-apoptotic and neuroprotective properties of DHS through cell viability, apoptosis, and immunostaining assays. Furthermore, we utilized the PI3K inhibitor LY294002 to validate the therapeutic target of DHS. Based on the physicochemical properties of DHS, it can be inferred that it has promising oral bioavailability and permeability across the blood-brain barrier (BBB). It was demonstrated that DHS upregulates phosphorylated AKT and ERK while downregulating phosphorylated JNK. Consequently, this enhances the expression of BCL2, which exerts a protective effect on neuronal cells by inhibiting caspase activity and preventing cell apoptosis. The inhibition of PI3K significantly reduced the relative protective activity of DHS in 6-OHDA-induced neurotoxicity, suggesting that the neuroprotective effects of DHS are mediated through the activation of PI3K signaling. By investigating the mechanisms involved in 6-OHDA-induced neurotoxicity, we provided evidence concerning the therapeutic potential of DHS in neuroprotection. Further research into DHS and its mechanisms of action holds promise for developing novel therapeutic strategies for PD.

Maternal exposure to Di-n-butyl phthalate (DBP) inhibit orexin receptor 1 (OX1R) expression to prevent Sertoli cells proliferation through the AKT signaling pathway.

Studies have demonstrated that Sertoli cells are the direct target of Dibutyl phthalate (DBP). However, the role of neurotransmitter receptors is not elucidated. Based on our previous studies, maternal Sprague-Dawley (SD) rats in Gestation Day (GD) 14-18 and TM4 cells exposure to 750mg/kg/day and 100μM DBP were regarded as treated groups. Firstly, qRT-PCR array was used to determine the different expression of neurotransmitter receptors. We examined the OX1R expression on Rats in Control and DBP groups by immunohistochemistry. Real-time PCR and Western Blot were used to detect the protein and mRNA expression levels of OX1R in vivo and in vitro. The potential downstream signaling pathways were explored by analyzing the GSE99690 cohort. In addition, we extracted Primary Sertoli Cells (PSCs) from the testis of control group. The apoptosis-related proteins, AKT signaling pathway-related proteins and mRNA expressions were detected by Western Blot and Real-time PCR in PSCs. The validity of PSCs was measured by CCK-8 assay and flow cytometric analysis was used to demonstrate the apoptotic rates of PSCs after DBP exposure. The Orexin receptor 1 (OX1R) was screened out by qRT-PCR array. Our results showed that DBP could significantly suppress the OX1R expression of Sertoli cells in vivo and in vitro. Functional analysis showed the AKT signaling pathway was mediated by OX1R. The highly expressed apoptosis level and impaired cell activity were observed in PSCs, which can be reversed by Orexin A. Meanwhile, the p-AKT signaling pathway were hindered after DBP exposure while rescued in DBP+Orexin-A group. DBP can induce Sertoli cell apoptosis through its toxicological effect by suppressing OX1R and p-AKT expression, which provide a novel insight on the role of neurotransmitter receptors.

High extracellular matrix stiffness promotes the metastasis of nasopharyngeal carcinoma through STAT3/FGF1 positive feedback regulation activated by JAK2

The stiffness of the extracellular matrix (EM) is known to be associated with tumor metastasis. This study aims to clarify the function and mechanisms of EM stiffness in nasopharyngeal carcinoma (NPC) and to provide a potential therapeutic target for patients with NPC. The NPC cell lines were cultured under varying stiffnesses. Plate cloning, Transwell assays and scratch tests were conducted to compare the phenotypes. Transcriptome sequencing and enrichment analysis were performed to explore the key molecules and the downstream signaling pathways, which were verified via in vitro and in vivo experiments. Our study showed that the NPC cells cultured on a stiff substrate (50 ​kPa) had greater colony formation, invasion and migration abilities than those cultured on a soft substrate. Transcriptome sequencing showed that a stiff environment induced high expression of fibroblast growth factor 1 (FGF1), which suggested a poor prognosis in various malignancies. The phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway played an important role in NPC metastasis activated by the FGF1 autocrine pathway in a stiff environment. The increased FGF1 expression was regulated by the transcription factor STAT3, and a positive feedback regulation between them was observed. The use of the JAK inhibitor Ruxolitinib and JAK2 interference inhibited the activation of STAT3. In conclusion, JAK2 activates STAT3/FGF1 positive feedback regulation and promotes NPC metastasis via the PI3K/Akt signaling pathway in a high-stiffness environment. Hence, FGF1 can be used as a potential therapeutic target for patients with NPC.

Design, synthesis, molecular modeling and evaluation of 2,4-diaminopyrimidine analogues as promising colorectal cancer drugs

The potential of cyclin-dependent kinases (CDKs) as therapeutic targets in cancer treatment is well established. In this study, we present our investigation into a group of 2,4-diaminopyrimidine derivatives that potently inhibit CDK9 and are cytotoxic when tested in colorectal cancer cell lines. We designed and synthesized forty analogues by altering substitutions at C-2 and C-4 position of the pyrimidine system. Among them, compounds 16 h and 16j exhibited strong inhibitory potency against both CDK9 enzymes (IC50 = 11.4 ± 1.4 nM, IC50 = 10.2 ± 1.3 nM respectively) with a significant preference for one over the other, and cytotoxic potency (IC50 = 61 ± 2 nM, IC50 = 20 ± 1 nM respectively) against HCT-116 was discovered through substantial modifications to its structure. Further investigations revealed that compounds 16 h and 16j were directly bound to CDK9, resulting in the suppression of its downstream signaling pathway. This inhibition of cell proliferation occurred by impeding the progression of the cell cycle and inducing apoptosis in cells by suppressing the phosphoryl RNA pol II Ser2. Significantly, compound 16 h and 16j effectively suppressed tumor growth in a xenograft mouse model and exhibited no apparent toxicity. This indicates that CDK9 inhibitors hold great potential as a therapeutic approach for colorectal cancer treatment. Therefore, the aforementioned discoveries are vital for the development of CDK9 inhibitors for the treatment of cancer.

The Role of Tissue Factor In Signaling Pathways of Pathological Conditions and Angiogenesis.

Tissue factor (TF) is an integral transmembrane protein associated with the extrinsic coagulation pathway. TF gene expression is regulated in response to inflammatory cytokines, bacterial lipopolysaccharides, and mechanical injuries. TF activity may be affected by phosphorylation of its cytoplasmic domain and alternative splicing. TF acts as the primary initiator of physiological hemostasis, which prevents local bleeding at the injury site. However, aberrant expression of TF, accompanied by the severity of diseases and infections under various pathological conditions, triggers multiple signaling pathways that support thrombosis, angiogenesis, inflammation, and metastasis. Protease-activated receptors (PARs) are central in the downstream signaling pathways of TF. In this study, we have reviewed the TF signaling pathways in different pathological conditions, such as wound injury, asthma, cardiovascular diseases (CVDs), viral infections, cancer and pathological angiogenesis. Angiogenic activities of TF are critical in the repair of wound injuries and aggressive behavior of tumors, which are mainly performed by the actions of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1 (HIF1-α). Pro-inflammatory effects of TF have been reported in asthma, CVDs and viral infections, including COVID-19, which result in tissue hypertrophy, inflammation, and thrombosis. TF-FVII induces angiogenesis via clotting-dependent and -independent mechanisms. Clottingdependent angiogenesis is induced via the generation of thrombin and cross-linked fibrin network, which facilitate vessel infiltration and also act as a reservoir for endothelial cells (ECs) growth factors. Expression of TF in tumor cells and ECs triggers clotting-independent angiogenesis through induction of VEGF, urokinase-type plasminogen activator (uPAR), early growth response 1 (EGR1), IL8, and cysteine-rich angiogenic inducer 61 (Cyr61).

NAT10 Overexpression Promotes Tumorigenesis and Epithelial-Mesenchymal Transition Through AKT Pathway in Gastric Cancer.

N-acetyltransferase 10 (NAT10), the only RNA cytosine acetyltransferase known in humans, contributes to cancer tumorigenesis and progression. This study aims to investigate the effect of NAT10 on the malignant biological properties of gastric cancer (GC) and its underlying mechanism. The expression and prognostic significance of NAT10 in GC were analyzed using The Cancer Genome Atlas (TCGA) and Sun Yat-sen University (SYSU) cohorts. The influence of NAT10 on the malignant biological behaviors of GC was detected by Cell Counting Kit-8 (CCK-8) assay, plate colony formation assay, 5-ethynyl-2'-deoxyuridine (EdU), Transwell migration and invasion assays, scratch wound assay, flow cytometric analysis, and animal studies. The overall level of N4 acetylcytidine (ac4C) in GC was detected by liquid chromatography with tandem mass spectrometry (LC-MS/MS). The downstream signal pathways of NAT10 were analyzed by Gene Set Enrichment Analysis (GSEA) and verified by Western blot (WB) and immunofluorescence (IF). The significant upregulation of NAT10 expression in GC was associated with a poor prognosis. The knockdown of NAT10 markedly suppressed GC cell proliferation, migration, invasion, and cell cycle progression. Downregulating NAT10 reduced ac4C levels and inhibited AKT phosphorylation and epithelial-mesenchymal transition (EMT) in GC. NAT10 functions as an oncogene and may provide a new therapeutic target in GC.

Huangqi-Danshen Decoction Against Renal Fibrosis in UUO Mice via TGF-β1 Induced Downstream Signaling Pathway.

Huangqi-Danshen decoction (HDD) is a Chinese medicinal herb pair with good efficacy in treating chronic kidney disease, but its mechanism needs to be clarified. To uncover the underlying mechanism of HDD antagonizing renal fibrosis through network pharmacology (NP) analysis and experimental validation. The chemical components of water extract of HDD were analyzed by combining the ultra-high performance liquid chromatography coupled with Q-Exactive mass spectrum analysis (UHPLC-QE-MS) and HERB database. NP was used to identify core common targets of HDD components and renal fibrosis. Subsequently, male C57BL/6 mice were divided into Sham, unilateral ureteral obstruction (UUO) and UUO+HDD groups. Renal function, histopathology, Western blotting, and immunohistochemistry analyses were used to evaluate the protective effect of HDD on UUO mice. The effects of HDD on signaling pathways were validated in both UUO mice and transforming growth factor-β1 (TGF-β1)-induced HK-2 cells. By combining UHPLC-QE-MS analysis and HERB database, 25 components were screened in HDD extract. There were 270 intersection targets of the 25 components and renal fibrosis. Based on their scores in protein-protein interaction analysis and degree values in component-pathway-target triadic network, 6 core common targets of the 25 components and renal fibrosis were identified, namely phosphoinositide 3-kinase (PI3K), signal transducer and activator of transcription 3(Stat3), non-receptor tyrosine kinase Src (Src), epidermal growth factor receptor (EGFR), matrix metalloproteinase 9 (MMP9), and MMP2. HDD ameliorated renal tubular damage and collagen deposition and downregulated fibrosis-related proteins expression in UUO mice. Furthermore, HDD was demonstrated to reduce PI3K, Stat3, Src, EGFR, and MMP2 expressions, and enhance MMP9 expression in the kidney of UUO mice and in TGF-β1-induced HK-2 cells. HDD can alleviate renal fibrosis which may be related to regulating the expression of essential proteins in the epithelial-mesenchymal transition and extracellular matrix production/degradation signaling pathways.

Reserpine, a novel N6-methyladenosine regulator, reverses Lenvatinib resistance in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is an aggressive malignancy and a growing global health problem. Reserpine (Res), a plant-derived hypertension drug, has been reported to possess anti-tumor efficacy. However, the role and function of Res in N6-methyladenosine (m6A) regulation and Lenvatinib (Len) resistance in HCC have not been clarified. PurposeTo verify whether Res can be used as a natural small-molecule regulator of m6A to reverse Len resistance in HCC. MethodsDot blotting, Western blotting and m6A quantification were used to compare and analyze the differential expression of m6A and its methyltransferase METTL3. Western blotting, Real-Time PCR (RT-PCR), cellular thermal shift assay (CETSA) and molecular docking were used to explore the mechanism of interaction between Res and m6A. The effects of Res on the biological characteristics of Lenvatinib-resistant HCC cells were investigated through CCK-8, clone formation, and Transwell assays. Cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models were used to assess the ability of Res to reverse Len resistance in vivo. MeRIP m6A sequencing, PATHWAY analysis and Western blotting were used to analyze the downstream signaling pathways and genes involved in Res-mediated reversal of Len resistance. ResultsLen resistance in HCC is related to the increased m6A level and the high expression of METTL3. Res affects the activity of METTL3 protein by binding to it, thereby downregulating the level of m6A. In vitro study showed that Res can sensitize HCC cells to the anti-tumor effects of Len treatment, including blocking proliferation, inhibiting migration, and inducing apoptosis. Len-resistant CDX and PDX models revealed that Res can reverse the resistant phenotype, with the tumor inhibition rates of 77.46 % and 62.1 %, respectively, when combined with Len treatment. Analysis of xenograft tissues showed that the combination of Res and Len down-regulates the m6A level, reduces proliferation biomarkers, and induces apoptosis, which is consistent with the in vitro data. Mechanistically, our preliminary results indicate that Res can up-regulate the SMAD3 level by down-regulating m6A in Len-resistant cells. ConclusionsReserpine, a small-molecule regulator of m6A, reverses Lenvatinib-resistant phenotypes, including proliferation, migration and anti-apoptosis, in vitro and in vivo by targeting SMAD3 and down-regulating the m6A level in HCC.

Pentachlorophenol impairs the antimicrobic capability of blood clam via undermining humoral immunity and disrupting humoral-cellular crosstalk

Due to past massive usage and persistent nature, pentachlorophenol (PCP) residues are prevalent in environments, posing a potential threat to various organisms such as sessile filter-feeding bivalves. Although humoral immunity and its crosstalk with cellular one are crucial for the maintaining of robust antimicrobic capability, little is known about the impacts of PCP on these critical processes in bivalve mollusks. In this study, pathogenic bacterial challenge and plasma antimicrobic capability assays were carried out to assess the toxic effects of PCP on the immunity of a common bivalve species, blood clam (Tegillarca granosa). Moreover, the impacts of PCP-exposure on the capabilities of pathogen recognition, hemocyte recruitment, and pathogen degradation were analyzed as well. Furthermore, the activation status of downstream immune-related signalling pathways upon PCP exposure was also assessed. Data obtained illustrated that 28-day treatment with environmentally realistic levels of PCP resulted in evident declines in the survival rates of blood clam upon Vibrio challenge along with markedly weakened plasma antimicrobic capability. Additionally, the levels of lectin and peptidoglycan-recognition proteins (PGRPs) in plasma as well as the expression of pattern recognition receptors (PRRs) in hemocytes were found to be significantly inhibited by PCP-exposure. Moreover, along with the downregulation of immune-related signalling pathway, markedly fewer chemokines (interleukin 8 (IL-8), IL-17, and tumor necrosis factor α (TNF-α)) in plasma and significantly suppressed chemotactic activity of hemocytes were also observed in PCP-exposed blood clams. Furthermore, compared to that of the control, blood clams treated with PCP had markedly lower levels of antimicrobic active substances, lysozyme (LZM) and antimicrobial peptides (AMP), in their plasma. In general, the results of this study suggest that PCP exposure could significantly impair the antimicrobic capability of blood clam via undermining humoral immunity and disrupting humoral-cellular crosstalk.

DUS4L suppresses invasion and metastasis in LUAD via modulation of PI3K/AKT and ERK/MAPK signaling through GRB2

ObjectiveLimited research has focused on the role of dihydrouridine synthases (DUS) family members in human tumors. Our previous findings indicated an impact of dihydrouridine synthase 4 like (DUS4L) on cell proliferation and apoptosis in lung adenocarcinoma (LUAD) A549 cell, yet its broader functions and regulatory mechanisms in LUAD remain elusive. MethodsUsing a LUAD tissue microarray and immunohistochemical (IHC) staining, we validated variations in DUS4L protein expression levels among LUAD patients and assessed its clinical significance. Additional experiments using short hairpin RNA (shRNA) against DUS4L (sh-DUS4L-2), LUAD cell lines, cell function assays (including wound healing, transwell migration and invasion, colony formation, and apoptosis assays), and mouse tumor xenografts were performed to examine the biological roles of DUS4L in LUAD progression. RNA sequencing, proteomic analyses, mass spectrometry, and co-immunoprecipitation experiments were conducted to identify and validate DUS4L-regulated downstream target genes and signaling pathways. ResultsWe identified a consistent upregulation of DUS4L in LUAD tissues. In vitro and in vivo experiments underscored the inhibitory effect of DUS4L downregulation on LUAD progression, including migration, invasion, and proliferation. Mechanistically, DUS4L was found to interact with the signaling molecule GRB2, promoting LUAD progression and metastasis by inducing epithelial-mesenchymal transition (EMT) via the PI3K/AKT and ERK/MAPK pathways. ConclusionOur results establish the functional role of DUS4L in driving the progression and metastasis of LUAD, implicating its potential as a candidate therapeutic target for LUAD.

HIV1 gp120 activates microglia via TLR2-NF-κB signaling to up-regulate inflammatory cytokine expression and induce neuropathic pain

HIV associated neuropathic pain (HANP) is a common complication of AIDS. Intrathecal injection of recombinant HIV-1 gp120 in mice is a well-known model. Previous RNA sequencing revealed spinal TLR2 acts as a differentially expressed gene in HANP mice. The spinal TLR2 is involved in HANP, but its role and underlying mechanism remains unclear. In this study the transcription, expression and distribution characteristics of TLR2 in the spinal cord of HANP male mice have been analyzed by qRT-PCR, Western blotting, and immunofluorescent staining. We found that TLR2 expression was upregulated in the spinal dorsal horn and mainly distributed in microglial cells, and blocking TLR2 relieved pain of HANP mice. Following stimulation by gp120 microglial cells upregulate TLR2 expression and become activated. The activation stimulates their differentiation into the M1 type, increasing IL-1β and TNF-α expression while inhibiting IL-10 expression. Silencing the Tlr2 gene slows down the activation, polarization, and secretion of pro-inflammatory factors in microglial cells induced by gp120, and enhances the expression of anti-inflammatory factors. Further analysis of the impact of gp120 on downstream signaling pathways of TLR2 in microglial cells, including NF-κB, MAPK (p38MAPK, ERK, and JNK) and PI3K/AKT, revealed that TLR2-NF-κB signaling plays a crucial role in the activation and polarization of microglial cells by gp120. Activation of NF-κB signaling aggravates pain in HANP mice, while blocking it lightens pain. This data indicates that gp120, through the TLR2-NF-κB signaling, activates spinal microglial cells, promotes the secretion of inflammatory cytokines, leading to HANP. This provides new targets to develop drugs for HANP.

The Role of Redox Environment in Tumors.

Reactive Oxygen Species (ROS) and the redoxin system regulate the redox environment. Thus, they mediate various physiological and pathological processes involved in tumor occurrence and development by activating redox-sensitive genes and regulating redox signaling pathways, including tumor cell proliferation, migration, invasion, and various cell death types. Therefore, the mechanism underlying redox environment regulation must be clarified to accurately target this mechanism and improve the effect of tumor treatment. This review introduces redox-sensitive transcription factors and their activated downstream signaling pathways, and the application of inhibitors targeting related transcription factors in tumor therapy.

EEF1B2 regulates the proliferation and apoptosis of human spermatogonial stem cell lines through TAF4B

BackgroundSpermatogonial stem cells (SSCs) are essential for male fertility, maintaining sperm production throughout life. While mouse SSCs have been studied extensively, the mechanisms regulating human SSCs are less understood. ObjectivesTo investigate the role of EEF1B2 in regulating human SSC proliferation and apoptosis. Material and methodsSingle cell RNA sequencing (scRNA-seq) analysis was utilized to investigate the differentially expressed genes of SSC. The distribution of EEF1B2 in the human testis was examined using immunofluorescence and immunohistochemistry techniques. Cell proliferation, DNA replication, and self-renewal were analyzed using CCK8, EdU, Western blot, and flow cytometry. RNA sequencing was employed to analyze the downstream target molecules and signaling pathways of EEF1B2. ResultsIn this study, we analyzed single-cell sequencing data from human testicular samples and identified EEF1B2 as a protein highly expressed in SSCs, with expression decreasing during development. Immunohistochemistry and immunofluorescence confirmed this pattern and showed co-localization with the proliferation marker KI67. Knockdown of EEF1B2 in human SSC lines impaired proliferation and viability, reducing self-renewal proteins like PLZF and CCNE1. RNA sequencing revealed decreased TAF4B following EEF1B2 knockdown, which could be rescued by replenishing TAF4B. Testicular SSCs from non-obstructive azoospermia (NOA) patients also showed reduced EEF1B2. Discussion and conclusionOur findings reveal a novel regulatory mechanism involving EEF1B2 and TAF4B in human SSCs, suggesting EEF1B2 deficiency may contribute to male infertility.

Rosa roxburghii Tratt (Cili) has a more effective capacity in alleviating DSS-induced colitis compared to Vitamin C through B cell receptor pathway

Rosa roxburghii Tratt (RRT), a traditional Chinese plant known as the ’King of Vitamin C (VitC; ascorbic acid, AsA)’, contains a wealth of nutrients and functional components, including polysaccharides, organic acids, flavonoids, triterpenes, and high superoxide dismutase (SOD) activity. The various functional components of RRT suggest that it may theoretically have a stronger potential for alleviating colitis compared to VitC. This study aims to verify whether RRT has a stronger ability to alleviate colitis than equimolar doses of VitC and to explore the mechanisms underlying this improvement. Results showed that RRT significantly mitigated body weight loss, intestinal damage, elevated inflammation levels, and compromised barriers in mice induced by Dextran sulfate sodium (DSS). Additionally, RRT enhanced the diversity and composition of intestinal microbiota in these DSS-induced mice. Colon RNA sequencing analysis revealed that compared to VitC, RRT further downregulated multiple immune-related signaling pathways, particularly the B cell receptor (BCR) pathway, which is centered around genes like Btk and its downstream PI3K-AKT, NF-κB, and MAPK signaling pathways. Correlation analysis between microbiota and genes demonstrated a significant relationship between the taxa improved by RRT and the key genes in the BCR and its downstream signaling pathways. Overall, RRT exhibited superior capabilities in alleviating DSS-induced colitis compared to VitC by decreasing intestinal inflammation and modulating BCR and its downstream signaling pathways, potentially regulated by the improved intestinal microbiota.

Antagonist anti-LIF antibody derived from naive human scFv phage library inhibited tumor growth in mice

BackgroundLeukemia inhibitory factor (LIF) is a multifunctional member of the IL-6 cytokine family that activates downstream signaling pathways by binding to the heterodimer consisting of LIFR and gp130 on the cell surface. Previous research has shown that LIF is highly expressed in various tumor tissues (e.g. pancreatic cancer, breast cancer, prostate cancer, and colorectal cancer) and promotes cancer cell proliferation, migration, invasion, and differentiation. Moreover, the overexpression of LIF correlates with poor clinicopathological characteristics. Therefore, we hypothesized that LIF could be a promising target for the treatment of cancer. In this work, we developed the antagonist antibody 1G11 against LIF and investigated its anti-tumor mechanism and its therapeutic efficacy in mouse models.ResultsA series of single-chain variable fragments (scFvs) targeting LIF were screened from a naive human scFv phage library. These scFvs were reconstructed in complete IgG form and produced by the mammalian transient expression system. Among the antibodies, 1G11 exhibited the excellent binding activity to human, cynomolgus monkey and mouse LIF. Functional analysis demonstrated 1G11 could block LIF binding to LIFR and inhibit the intracellular STAT3 phosphorylation signal. Interestingly, 1G11 did not block LIF binding to gp130, another LIF receptor that is involved in forming the receptor complex together with LIFR. In vivo, intraperitoneal administration of 1G11 inhibited tumor growth in CT26 and MC38 models of colorectal cancer. IHC analysis demonstrated that p-STAT3 and Ki67 were decreased in tumor tissue, while c-caspase 3 was increased. Furthermore, 1G11 treatment improves CD3+, CD4 + and CD8 + T cell infiltration in tumor tissue.ConclusionsWe developed antagonist antibodies targeting LIF/LIFR signaling pathway from a naive human scFv phage library. Antagonist anti-LIF antibody exerts antitumor effects by specifically reducing p-STAT3. Further studies revealed that anti-LIF antibody 1G11 increased immune cell infiltration in tumor tissues.

Engineering aptamers to enhance their interaction with protein target for selective inhibition of cell surface receptors

Cell surface receptors play a key role in intracellular signaling, and their overexpression and activation are among the drivers of multiple diseases. Selective inhibition of cell surface receptors is important for regulating intracellular signaling pathways and cell behavior. Here, we design engineered aptamers to selectively inhibit receptor function. In this strategy, the aptamer specifically recognizing the extracellular structural domain of the EGFR, was conjugated to an adamantane moiety through linking arms of various lengths in order to obtain better performances toward EGFR. These interactions inhibit EGFR dimerization, thereby impeding the activation of downstream signaling pathways. It is shown that the adamantane-modified aptamers exhibit superior inhibition of downstream effector proteins relative to the unmodified aptamers. The optimal inhibitory effect was observed with a linker arm of 40 T-base in length. Notably, the best-performing adamantane-modified aptamer specifically binds to A549 cells with a dissociation constant (22.6 ± 4.5 nM) that is approximately 4-fold lower than that of the parent EGFR aptamer (94.4 ± 21.9 nM). We further combine the use of the adamantane-modified aptamer with that of genistein, a natural isoflavone compound with EGFR tyrosine kinase inhibition activity, to enhance the inhibitory effect on EGFR and its downstream signaling employing a synergistic action. This study is expected to provide a versatile approach for the improvement of existing aptamers obtaining increased selective inhibition of cell surface receptors.

Interleukin-33 promotes intrauterine adhesion formation in mice through the mitogen-activated protein kinase signaling pathway

IL-33 belongs to the inflammatory factor family and is closely associated with the inflammatory response. However, its role in the development of intrauterine adhesions (IUAs) remains unclear. In this study, the role of IL-33 in the formation of IUAs after endometrial injury was identified via RNA sequencing after mouse endometrial organoids were transplanted into an IUA mouse model. Major pathological changes in the mouse uterus, consistent with the expression of fibrotic markers, such as TGF-β, were observed in response to treatment with IL-33. This finding may be attributed to activation of the phosphorylation of downstream MAPK signaling pathway components, which are activated by the release of IL-33 in macrophages. Our study provides a novel mechanism for elucidating IUA formation, suggesting a new therapeutic strategy for the prevention and clinical treatment of IUAs.

The Pathophysiological Mechanism and Clinical Treatment of Polycystic Ovary Syndrome: A Molecular and Cellular Review of the Literature.

Polycystic ovary syndrome (PCOS) is a prevalent metabolic disorder among women of reproductive age, characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovaries. The pathogenesis of PCOS involves a complex interplay of genetic and environmental factors, including insulin resistance (IR) and resultant hyperinsulinemia. Insulin receptors, primarily in skeletal muscle, liver, and adipose tissue, activate downstream signaling pathways like PI3K-AKT and MAPK-ERK upon binding. These pathways regulate glucose uptake, storage, and lipid metabolism. Genome-wide association studies (GWASs) have identified several candidate genes related to steroidogenesis and insulin signaling. Environmental factors such as endocrine-disrupting chemicals and lifestyle choices also exacerbate PCOS traits. Other than lifestyle modification and surgical intervention, management strategies for PCOS can be achieved by using pharmacological treatments like antiandrogens, metformin, thiazolidinediones, aromatase inhibitor, and ovulation drugs to improve insulin sensitivity and ovulatory function, as well as combined oral contraceptives with or without cyproterone to resume menstrual regularity. Despite the complex pathophysiology and significant economic burden of PCOS, a comprehensive understanding of its molecular and cellular mechanisms is crucial for developing effective public health policies and treatment strategies. Nevertheless, many unknown aspects of PCOS, including detailed mechanisms of actions, along with the safety and effectiveness for the treatment, warrant further investigation.

Hydrogel biomaterials that stiffen and soften on demand reveal that skeletal muscle stem cells harbor a mechanical memory

Muscle stem cells (MuSCs) are specialized cells that reside in adult skeletal muscle poised to repair muscle tissue. The ability of MuSCs to regenerate damaged tissues declines markedly with aging and in diseases such as Duchenne muscular dystrophy, but the underlying causes of MuSC dysfunction remain poorly understood. Both aging and disease result in dramatic increases in the stiffness of the muscle tissue microenvironment from fibrosis. MuSCs are known to lose their regenerative potential if cultured on stiff plastic substrates. We sought to determine whether MuSCs harbor a memory of their past microenvironment and if it can be overcome. We tested MuSCs in situ using dynamic hydrogel biomaterials that soften or stiffen on demand in response to light and found that freshly isolated MuSCs develop a persistent memory of substrate stiffness characterized by loss of proliferative progenitors within the first three days of culture on stiff substrates. MuSCs cultured on soft hydrogels had altered cytoskeletal organization and activity of Rho and Rac guanosine triphosphate hydrolase (GTPase) and Yes-associated protein mechanotransduction pathways compared to those on stiff hydrogels. Pharmacologic inhibition identified RhoA activation as responsible for the mechanical memory phenotype, and single-cell RNA sequencing revealed a molecular signature of the mechanical memory. These studies highlight that microenvironmental stiffness regulates MuSC fate and leads to MuSC dysfunction that is not readily reversed by changing stiffness. Our results suggest that stiffness can be circumvented by targeting downstream signaling pathways to overcome stem cell dysfunction in aged and disease states with aberrant fibrotic tissue mechanics.