Discovery of Small and Bifunctional Molecules Targeting PD-L1/CD73 for Cancer Dual Immunotherapy.

In this work, a series of bifunctional PD-L1/CD73 (cluster of differentiation 73) small-molecule inhibitors were designed and synthesized. Among them, CC-5 showed the strongest PD-L1 inhibitory effects with an IC50 of 6 nM and potent anti-CD73 activity with an IC50 of 0.773 µM. The high PD-L1/CD73 inhibitory activity of CC-5 was further confirmed by SPR assays with KD of 182 nM for human PD-L1 and 101 nM for CD73, respectively. Importantly, CC-5 significantly suppressed tumor growth in a CT26 and B16-F10 tumor model with TGI of 64.3% and 39.6%, respectively. Immunohistochemical (IHC) and flow cytometry analysis of tumor-infiltrating lymphocytes (TILs) indicated that CC-5 exerted anticancer effects via activating the tumor immune microenvironment. Collectively, CC-5 represents the first dual PD-L1/CD73 inhibitor worthy of further research as a bifunctional immunotherapeutic agent.

Discovery of Novel Heterotricyclic Compounds as DNA-Dependent Protein Kinase (DNA-PK) Inhibitors with Enhanced Chemosensitivity, Oral Bioavailability, and the Ability to Potentiate Cancer Immunotherapy.

In this work, a novel series of heterotricyclic DNA-PK inhibitors were rationally designed, synthesized, and assessed for their biological activity. In the DNA-PK biochemical assay, most compounds displayed potent enzymatic activity, with IC50 values between 0.11 and 71.5 nM. Among them, SK10 exhibited the most potent DNA-PK-inhibitory activity (IC50 = 0.11 nM). Studies of the mechanism of action indicated that SK10 could lower γH2A.X expression levels and demonstrate optimal synergistic antiproliferative activity against Jurkat cells (IC50 = 25 nM) when combined with doxorubicin. Importantly, in CT26 and B16-F10 tumor-bearing mouse models, the combination therapies of SK10 with chemotherapeutic drug doxorubicin, a PD-L1 antibody, and SWS1 (a potent PD-L1 small-molecule inhibitor) demonstrated superior synergistic anticancer and potential immunomodulatory effects. Furthermore, SK10 possessed favorable in vivo pharmacokinetic properties [e.g., oral bioavailability (F) = 31.8%]. Taken together, SK10 represents a novel heterotricyclic DNA-PK inhibitor with antitumor immune effects and favorable pharmacokinetics.

Recent Progress in DNA Damage Response-Targeting PROTAC Degraders.

DNA damage response (DDR) defects in cells play a crucial role in tumor development by promoting DNA mutations. These mutations create vulnerabilities specific to cancer cells, which can be effectively targeted through synthetic lethality-based therapies. To date, numerous small molecule DDR inhibitors have been identified, and some of them have already been approved for clinical use. However, due to the complexity of the tumor microenvironment, mutations may occur in the amino acid residues of DDR targets. These mutations can affect the efficacy of small molecule inhibitors targeting DDR pathways. Therefore, researchers have turned their attention to next-generation DNA damage repair modulators, particularly those based on PROTAC technology. From this perspective, we overviewed the recent progress on DDR-targeting PROTAC degraders for cancer therapy. In addition, we also summarized the biological functions of different DDR targets. Finally, the challenges and future directions for DDR-target PROTAC degraders are also discussed in detail.

Breviscapine alleviates myocardial ischemia-reperfusion injury in diabetes rats.

PURPOSE: To investigate the protective effect of breviscapine on myocardial ischemia-reperfusion injury (MIRI) in diabetes rats. METHODS: Forty rats were divided into control, diabetes, MIRI of diabetes, and treatment groups. The MIRI of diabetes model was established in the latter two groups. Then, the treatment group was treated with 100 mg/kg breviscapine by intraperitoneal injection for 14 consecutive days. RESULTS: After treatment, compared with MIRI of diabetes group, in treatment group the serum fasting blood glucose, fasting insulin, homeostasis model assessment of insulin resistance, and glycosylated hemoglobin levels decreased, the serum total cholesterol, triacylglycerol, and low-density lipoprotein cholesterol levels decreased, the serum high-density lipoprotein cholesterol level increased, the heart rate decreased, the mean arterial pressure, left ventricular ejection fraction, and fractional shortening increased, the serum cardiac troponin I, and creatine kinase-MB levels decreased, the myocardial tumor necrosis factor α and interleukin-6 levels decreased, the myocardial superoxide dismutase level increased, and the myocardial malondialdehyde level decreased (all P < 0.05). CONCLUSIONS: For treating MIRI of diabetes in rats, the breviscapine can reduce the blood glucose and lipid levels, improve the cardiac function, reduce the myocardial injury, and decrease the inflammatory response and oxidative stress, thus exerting the alleviating effect.

HDAC-targeting epigenetic modulators for cancer immunotherapy.

HDAC inhibitors, which can inhibit the activity of HDAC enzymes, have been extensively studied in tumor immunotherapy and have shown potential therapeutic effects in cancer immunotherapy. To date, numerous small molecule HDAC inhibitors have been identified, but many of them suffer from limited clinical efficacy and serious toxicity. Hence, HDAC inhibitor-based combination therapies, and other HDAC modulators (e.g. PROTAC degraders, dual-acting agents) have attracted great attention with significant advancements achieved in the past few years due to their superior efficacy compared to single-target HDAC inhibitors. In this review, we overviewed the recent progress on HDAC-based drug discovery with a focus on HDAC inhibitor-based drug combination therapy and other HDAC-targeting strategies (e.g. selective HDAC inhibitors, HDAC-based dual-target inhibitors, and PROTAC HDAC degraders) for cancer immunotherapy. In addition, we also summarized the reported co-crystal structures of HDAC inhibitors in complex with their target proteins and the binding interactions. Finally, the challenges and future directions for HDAC-based drug discovery in cancer immunotherapy are also discussed in detail.

Breviscapine alleviates myocardial ischemia-reperfusion injury in diabetes rats

Purpose: To investigate the protective effect of breviscapine on myocardial ischemia-reperfusion injury (MIRI) in diabetes rats. Methods: Forty rats were divided into control, diabetes, MIRI of diabetes, and treatment groups. The MIRI of diabetes model was established in the latter two groups. Then, the treatment group was treated with 100 mg/kg breviscapine by intraperitoneal injection for 14 consecutive days. Results: After treatment, compared with MIRI of diabetes group, in treatment group the serum fasting blood glucose, fasting insulin, homeostasis model assessment of insulin resistance, and glycosylated hemoglobin levels decreased, the serum total cholesterol, triacylglycerol, and low-density lipoprotein cholesterol levels decreased, the serum high-density lipoprotein cholesterol level increased, the heart rate decreased, the mean arterial pressure, left ventricular ejection fraction, and fractional shortening increased, the serum cardiac troponin I, and creatine kinase-MB levels decreased, the myocardial tumor necrosis factor α and interleukin-6 levels decreased, the myocardial superoxide dismutase level increased, and the myocardial malondialdehyde level decreased (all P < 0.05). Conclusions: For treating MIRI of diabetes in rats, the breviscapine can reduce the blood glucose and lipid levels, improve the cardiac function, reduce the myocardial injury, and decrease the inflammatory response and oxidative stress, thus exerting the alleviating effect.

A miR-9-5p/FOXO1/CPEB3 Feed-Forward Loop Drives the Progression of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide, but its regulatory mechanism remains unclear and potential clinical biomarkers are still lacking. Co-regulation of TFs and miRNAs in HCC and FFL module studies may help to identify more precise and critical driver modules in HCC development. Here, we performed a comprehensive gene expression and regulation analysis for HCC in vitro and in vivo. Transcription factor and miRNA co-regulatory networks for differentially expressed genes between tumors and adjacent tissues revealed the critical feed-forward loop (FFL) regulatory module miR-9-5p/FOXO1/CPEB3 in HCC. Gain- and loss-of-function studies demonstrated that miR-9-5p promotes HCC tumor proliferation, while FOXO1 and CPEB3 inhibit hepatocarcinoma growth. Furthermore, by luciferase reporter assay and ChIP-Seq data, CPEB3 was for the first time identified as a direct downstream target of FOXO1, negatively regulated by miR-9-5p. The miR-9-5p/FOXO1/CPEB3 FFL was associated with poor prognosis, and promoted cell growth and tumor progression of HCC in vitro and in vivo. Our study identified for the first time the existence of miR-9-5p/FOXO1/CPEB3 FFL and revealed its regulatory role in HCC progression, which may represent a new potential target for cancer therapy.

From design to clinic: Engineered peptide nanomaterials for cancer immunotherapy.

Immunotherapy has revolutionized the field of cancer therapy. Nanomaterials can further improve the efficacy and safety of immunotherapy because of their tunability and multifunctionality. Owing to their natural biocompatibility, diverse designs, and dynamic self-assembly, peptide-based nanomaterials hold great potential as immunotherapeutic agents for many malignant cancers, with good immune response and safety. Over the past several decades, peptides have been developed as tumor antigens, effective antigen delivery carriers, and self-assembling adjuvants for cancer immunotherapy. In this review, we give a brief introduction to the use of peptide-based nanomaterials for cancer immunotherapy as antigens, carriers, and adjuvants, and to their current clinical applications. Overall, this review can facilitate further understanding of peptide-based nanomaterials for cancer immunotherapy and may pave the way for designing safe and efficient methods for future vaccines or immunotherapies.

Coumarin 1,4-enedione for selective detection of hydrazine in aqueous solution and fluorescence imaging in living cells.

Hydrazine is a widely used but highly toxic chemical reagent, and the development of a fluorescent probe for hydrazine detection is very meaningful. In this study, a novel coumarin-derived fluorescent probe containing a 1,4-enedione moiety for hydrazine detection was developed. The recognition of hydrazine with the probe brings about obvious fluorescence enhancement over other environmentally relevant ions and amine-containing species. The limit of detection for hydrazine is 2.7×10-8 M in aqueous solution. The fluorescence enhancement was ascribed to the cyclization reaction of the 1,4-enedione moiety of the probe and hydrazine which form a six-membered pyridazine ring and intramolecular charge transfer (ICT) mechanism. The mass spectrometry (MS), nuclear magnetic resonance (NMR) analysis and theoretical calculations confirmed the recognition produced. The probe can be used to determine trace hydrazine in real water samples. More importantly, the probe also showed good potential in detecting hydrazine by imaging of living HeLa cells.

Tissue-derived extracellular vesicles: Research progress from isolation to application.

Extracellular vesicles (EVs) are the structures that all cells release into the environment. They are separated by a lipid bilayer and contain the cellular components that release them. To date, most studies have been performed on EVs derived from cell supernatants or different body fluids, while the number of studies on EV isolation directly from tissues is still limited. Studies of EV isolation directly from tissues may provide us with better information. This review summarizes the role of EV in the extracellular matrix, the protocol for isolation of EV in the tissue interstitium, and the application of the protocol in different tissues.

miR-198 inhibits the progression of renal cell carcinoma by targeting BIRC5.

BACKGROUND: miR-198 is involved in the formation, migration, invasion, and metastasis of various malignant cancers. However, the function and mechanism of action of miR-198 in the tumorigenesis of renal cell carcinoma (RCC) remain elusive. Here, we aimed to explore the role of miR198 in RCC. METHODS: Immunohistochemistry was performed to estimate the level of survivin in RCC sections. Quantitative real-time polymerase chain reaction was performed to determine the expression level of miR-198 in fresh RCC tissues. Furthermore, the target relationship between miR-198 and BIRC5 was predicted using the TargetScanHuman 7.2 database and verified via dual-luciferase reporter assay and western blotting. The effects of miR-198 on the viability, apoptosis, invasion, and migration of A498 and ACHN cells were studied using Cell Counting Kit-8, flow cytometry, transwell migration assay, and wound healing assay, respectively. Additionally, a xenograft nude mouse model was established to evaluate the effect of miR-198 on RCC tumorigenesis. RESULTS: The expression levels of BIRC5 and miR-198 were respectively higher and lower in RCC tissues than those in normal adjacent tissues. Furthermore, miR-198 could inhibit luciferase activity and reduce the protein level of survivin without affecting the BIRC5 mRNA levels. miR-198 inhibited cell viability, migration, and invasion and promoted cell apoptosis; co-transfection with BIRC5 could rescue these effects. Moreover, miR-198 could repress tumor growth in the xenograft nude mouse model of RCC. CONCLUSIONS: Our study demonstrates that miR-198 suppresses RCC progression by targeting BIRC5.

[Network pharmacology-based study of the therapeutic mechanism of resveratrol for Alzheimer's disease].

OBJECTIVE: To investigate the therapeutic mechanism of resveratrol (RES) for Alzheimer's disease (AD) in light of network pharmacology. METHODS: We searched PubChem, BATMAN-TCM, Genecards, AD, TTD, String 11.0, AlzData, SwissTargetPrediction, Metascape and other databases for the therapeutic targets of RES and human AD-related targets. The intersection was determined using Venny 2.1 to obtain the therapeutic targets of RES for AD. The protein-protein interaction (PPI) network was constructed, the gene ontology (GO) was enriched and the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG pathway) were analyzed. Cytoscape 3.7.1 software was used to construct a target-signaling pathway network of RES in the treatment of AD. Molecular docking verification was carried out on SwissDock (http://www.swissdock.ch/docking). We examined a 293Tau cell model of AD for changes in protein levels of pS396, pS199, Tau5, CDK5, glycogen synthase kinase 3ß (GSK3ß) and p-GSK3ß in response to RES treatment using Western blotting. RESULTS: We obtained 182 targets of RES, 525 targets related to AD, and 36 targets of RES for AD treatment, among which 34.6% of the targets were protein-modifying enzymes, 27.7% were metabolite invertase, 13.8% were gene-specific transcriptional regulators, and 10.3% were transporters. The core key targets of RES in the treatment of AD included INS, APP, ESR1, MMP9, IGF1R, CACNA1C, MAPT (microtubule- associated protein Tau), MMP2, TGFB1 and GSK3B. Enrichment analysis of GO biological process suggested that the biological function of RES in AD treatment mainly involved the response to ß-amyloid protein, positive regulation of transferase activity, the transmembrane receptor protein tyrosine kinase signaling pathway, regulation of behavior, learning or memory, aging, and transmembrane transport. KEGG pathway enrichment analysis showed that the most significantly enriched signaling pathways were AD pathway, PI3K-AKT signaling pathway, cGMP-PKG signaling pathway, and MAPK signaling pathway. Molecular docking results showed that RES had strong binding with ESR1, GSK3B, MMP9, IGF1R, APP and INS. In the cell model of AD, treatment with 50 µmol/L RES for 12 h significantly reduced the levels of pS396 and pS199 by regulating CDK5 and GSK3ß activity (P < 0.001). CONCLUSIONS: RES produces therapeutic effects on AD by acting on multiple targets and affecting multiple signaling pathways and improves AD-associated pathologies via a direct action on Aß and Tau pathological processes.

Decreased expression of apoptosis-inducing factor in renal cell carcinoma is associated with poor prognosis and reduced postoperative survival.

Apoptosis-inducing factor (AIF) serves a crucial role in cell death and is involved in several types of cancer, including kidney cancer. The present study aimed to explore the association between AIF expression and patient survival based on tumor grades. AIF expression in 96 patients with renal cell carcinoma (RCC) was investigated using immunohistochemistry. Negative AIF expression was determined in 80 patients (83.3%). mRNA expression of AIF was analyzed in RCC and adjacent tissue samples from 15 patients. AIF mRNA expression in RCC tissues were significantly lower compared with that in adjacent tissues. Analysis of histopathological grades revealed that AIF expression was negatively associated with RCC grade, with AIF expression in Grade II tumors being lower than Grade I types, but higher than Grade III. Finally, 68 patients were followed up for 6-118 months, and it was revealed that the overall postoperative survival rate of patients with negative AIF expression was significantly lower compared with those those with positive AIF expression. These results suggest that decreased AIF expression could be associated with worsening RCC grade. Therefore, reduced AIF expression may potentially help diagnose RCC and distinguish tumor grades.

Application of Somatic Embryogenesis in Woody Plants.

Somatic embryogenesis is a developmental process where a plant somatic cell can dedifferentiate to a totipotent embryonic stem cell that has the ability to give rise to an embryo under appropriate conditions. This new embryo can further develop into a whole plant. In woody plants, somatic embryogenesis plays a critical role in clonal propagation and is a powerful tool for synthetic seed production, germplasm conservation, and cryopreservation. A key step in somatic embryogenesis is the transition of cell fate from a somatic cell to embryo cell. Although somatic embryogenesis has already been widely used in a number of woody species, propagating adult woody plants remains difficult. In this review, we focus on molecular mechanisms of somatic embryogenesis and its practical applications in economic woody plants. Furthermore, we propose a strategy to improve the process of somatic embryogenesis using molecular means.

MiR-144 regulates hematopoiesis and vascular development by targeting meis1 during zebrafish development.

Hematopoiesis is a dynamic process by which peripheral blood lineages are developed. It is a process tightly regulated by many intrinsic and extrinsic factors, including transcriptional factors and signaling molecules. However, the epigenetic regulation of hematopoiesis, for example, regulation via microRNAs (miRNAs), remains incompletely understood. Here we show that miR-144 regulates hematopoiesis and vascular development in zebrafish. Overexpression of miR-144 inhibited primitive hematopoiesis as demonstrated by a reduced number of circulating blood cells, reduced o-dianisidine staining of hemoglobin, and reduced expression of hbαe1, hbße1, gata1 and pu.1. Overexpression of miR-144 also inhibited definitive hematopoiesis as shown by reduced expression of runx1 and c-myb. Mechanistically, miR-144 regulates hematopoiesis by repressing expression of meis1 involved in hematopoiesis. Both real-time RT-PCR and Western blot analyses showed that overexpression of miR-144 repressed expression of meis1. Bioinformatic analysis predicts a target binding sequence for miR-144 at the 3'-UTR of meis1. Deletion of the miR-144 target sequence eliminated the repression of meis1 expression mediated by miR-144. The miR-144-mediated abnormal phenotypes were partially rescued by co-injection of meis1 mRNA and could be almost completely rescued by injection of both meis1 and gata1 mRNA. Finally, because meis1 is involved in vascular development, we tested the effect of miR-144 on vascular development. Overexpression of miR-144 resulted in abnormal vascular development of intersegmental vessels in transgenic zebrafish with Flk1p-EGFP, and the defect was rescued by co-injection of meis1 mRNA. These findings establish miR-144 as a novel miRNA that regulates hematopoiesis and vascular development by repressing expression of meis1.

Role of microRNA-27a in down-regulation of angiogenic factor AGGF1 under hypoxia associated with high-grade bladder urothelial carcinoma.

Hypoxia stimulates angiogenesis under a variety of pathological conditions, including malignant tumors by inducing expression of angiogenic factors such as VEGFA. Surprisingly, here we report significant association between down-regulation of a new angiogenic factor AGGF1 and high-grade urothelial carcinoma. The proportion of strong AGGF1 expression cases was significantly lower in the high-grade urothelial carcinoma group than that in the low-grade urothelial carcinoma group (P=1.40×10(-5)) or than that in the normal urothelium tissue group (P=2.11×10(-4)). We hypothesized that tumor hypoxia was responsible for differential expression of the AGGF1 protein in low- and high-grade urothelial carcinomas, and therefore investigated the molecular regulatory mechanism for AGGF1 expression under hypoxia. Under hypoxic conditions, AGGF1 protein levels declined without any change in mRNA levels and protein stability. Hypoxia-induced down-regulation of AGGF1 was mediated by miR-27a. Overexpression of miR-27a suppressed AGGF1 expression through translational inhibition, but not by RNA degradation. Moreover, the hypoxia-induced decrease of AGGF1 expression disappeared after miR-27a expression was inhibited. Furthermore, down-regulation of AGGF1 reduced hypoxia-induced apoptosis in cancer cells. Taken together, the results of this study indicate that (1) hypoxia down-regulates expression of the AGGF1 protein, but not AGGF1 mRNA, by inducing expression of miR-27a; (2) Down-regulation of AGGF1 had an apparent protective role for cancer cells under hypoxia; (3) Down-regulation of the AGGF1 protein confers a significant risk of high-grade human urothelial bladder carcinoma.

Meta-analysis identifies robust association between SNP rs17465637 in MIA3 on chromosome 1q41 and coronary artery disease.

Several large-scale meta-GWAS identified significant association between SNP rs17465637 in the MIA3 gene and coronary artery disease (CAD) in the European ancestry populations. However, three follow-up replication studies in the Chinese populations yielded inconsistent results. In order to unequivocally determine whether SNP rs17465637 is associated with CAD, we performed an independent case control association study in the Chinese Han population and a follow-up large scale meta-analysis for SNP rs17465637. Our study included 2503 CAD patients and 2920 non-CAD controls of the Chinese Han origin. A significant association was found between SNP rs17465637 and CAD (P = 0.01, OR = 1.11). Meta-analysis included 7263 CAD patients and 8347 controls combined from five Asian populations. The association between SNP rs17465637 and CAD became highly significant (P = 4.97 × 10(-5), OR = 1.11). Similar analysis also identified significant association between SNP rs17465637 and MI (2424 cases vs. 6,536controls; P = 5.00 × 10(-3), OR = 1.10). We conclude that SNP rs17465637 in MIA3 is indeed a genetic risk factor for CAD across different ethnic populations.

Transcriptional activation of the Prox1 gene by HIF-1α and HIF-2α in response to hypoxia.

Prox1 encodes a homeobox transcription factor critical to organ development, but its regulation is poorly understood. Here, we show that Prox1 expression is induced by hypoxia, and controlled by a hypoxia-response element (HRE) at the Prox1 promoter/regulatory region and HIF-1α/HIF-2α. EMSA and ChIP assays demonstrated the direct interaction of the HRE with HIF-1α or HIF-2α. Overexpression of HIF-1α or HIF-2α increased activation of the Prox1 promoter, whereas knockdown of HIF-1α or HIF-2α inhibited the activation. These data reveal a novel molecular mechanism for regulation of Prox1 expression in response to hypoxia and provide new insights into Prox1-controlled processes such as lymphangiogenesis.

Phytoremediation of trichlorophenol by Phase II metabolism in transgenic Arabidopsis overexpressing a Populus glucosyltransferase.

Trichlorophenol (TCP) and its derivatives are introduced into the environment through numerous sources, including wood preservatives and biocides. Environmental contamination by TCPs is associated with human health risks, necessitating the development of cost-effective remediation techniques. Efficient phytoremediation of TCP is potentially feasible because it contains a hydroxyl group and is suitable for direct phase II metabolism. In this study, we present a system for TCP phytoremediation based on sugar conjugation by overexpressing a Populus putative UDP-glc-dependent glycosyltransferase (UGT). The enzyme PtUGT72B1 displayed the highest TCP-conjugating activity among all reported UGTs. Transgenic Arabidopsis demonstrated significantly enhanced tolerances to 2,4,5-TCP and 2,4,6-TCP. Transgenic plants also exhibited a strikingly higher capacity to remove TCP from their media. This work indicates that Populus UGT overexpression in Arabidopsis may be an efficient method for phytoremoval and degradation of TCP. Our findings have the potential to provide a suitable remediation strategy for sites contaminated by TCP.