Anti-thrombotic Effects Mediated by a Novel Dual-Target Peptide Inhibiting Both Platelet Aggregation and Thrombin Activity without Causing Bleeding.

BACKGROUND: Classical anticoagulants and antiplatelets are associated with high frequencies of bleeding complications or treatment failure when used as single agents. Thrombin plays an important role in the blood coagulation system. GP IIb/IIIa is the central receptor of platelets, which can recognize the Arg-Gly-Asp (RGD) sequence and activate platelets. MATERIAL AND METHODS: Molecular simulation and homology modeling were performed to design a novel dual-target anticoagulant short peptide (PTIP ). The activities of PTIP on coagulation and platelet in vitro were analyzed. The antithrombotic activity of PTIP was determined by pulmonary thromboembolism model, ferric chloride injury model and arteriovenous bypass thrombosis model. Bleeding effect and toxicity of PTIP were evaluated. RESULTS: We have constructed a novel dual-target peptide (PTIP) based on the direct thrombin inhibitor peptide (DTIP). PTIP was expressed at high levels in Pichia pastoris. PTIP interfered with thrombin-mediated coagulation and ADP-induced platelet aggregation in vitro. When injected intravenously or subcutaneously, PTIP showed potent and dose-dependent extension of aPTT and PT which were similar to DTIP; but only PTIP was capable of inhibiting platelet aggregation. PTIP (1.0 mg/kg) decelerated thrombosis formation in venous and arterial vessels induced by FeCl3 injury. PTIP (1.0 mg/kg) also prevented deep venous thrombosis and increased the survival rate associated with pulmonary thromboembolism. And PTIP effectively reduced thrombus length in arteriovenous bypass thrombosis model. Moreover, the antithrombotic dose of PTIP could not induce bleeding. CONCLUSION: These data establish that PTIP represents a novel antithrombotic agent whose effects involve both inhibition of platelet activation and reduction of fibrin generation. And PTIP not only can be used in venous thrombosis and arterial thrombosis, it can also replace the combined treatment of antiplatelet and anticoagulant drugs in thrombotic diseases.

MET exon 14 skipping mutation drives cancer progression and recurrence via activation of SMAD2 signalling.

BACKGROUND: c-Met encoded by the proto-oncogene MET, also known as hepatocyte growth factor (HGF) receptor, plays a crucial role in cellular processes. MET exon 14 skipping alteration (METΔ14EX) is a newly discovered MET mutation. SMAD2 is an important downstream transcription factor in TGF-ß pathway. Unfortunately, the mechanisms by which METΔ14EX leads to oncogenic transformation are scarcely understood. The relationship between METΔ14EX and SMAD2 has not been studied yet. METHODS: We generate METΔ14EX models by CRISPR-Cas9. In vitro transwell, wound-healing, soft-agar assay, in vivo metastasis and subcutaneous recurrence assay were used to study the role of METΔ14EX in tumour progression. RNA-seq, Western blotting, co-immunoprecipitation (CO-IP) and immunofluorescent were performed to explore the interaction between c-Met and SMAD2. RESULTS: Our results demonstrated that METΔ14EX, independent of HGF, can prolong the constitutive activation of c-Met downstream signalling pathways by impeding c-Met degradation and facilitating tumour metastasis and recurrence. Meanwhile, METΔ14EX strengthens the interaction between c-Met and SMAD2, promoting SMAD2 phosphorylation. Therapeutically, MET inhibitor crizotinib impedes METΔ14EX-mediated tumour metastasis by decreasing SMAD2 phosphorylation. CONCLUSIONS: These data elucidated the previously unrecognised role of METΔ14EX in cancer progression via activation of SMAD2 independent of TGF-ß, which helps to develop more effective therapies for such patients. METΔ14EX alteration significantly triggers tumour progression via activation of SMAD2 signalling that are involved in activating tumour invasion, metastasis and recurrence. On the left, in the MET wild-type (METWT), the juxtamembrane (JM) domain is involved in the regulation of tyrosine kinase activity, receptor degradation, and caspase cleavage. On the right, the METΔ14EX mutation leads to the loss of the juxtamembrane domain, resulting in an abnormal MET protein lacking a CBL-binding site. This causes the accumulation of truncated MET receptors followed by constitutive activation of the MET signalling pathway. Thus, the METΔ14EX-mutated protein has strong binding and phosphorylation to SMAD2, which results in the phosphorylation of a large number of SMAD2/3 proteins that combine with SMAD4 to form a complex in the nucleus, activating downstream signalling pathways, such as EMT and ECM remodelling, resulting in tumour progression and recurrence. TF transcription factor.

ASCL2 induces an immune excluded microenvironment by activating cancer-associated fibroblasts in microsatellite stable colorectal cancer.

Proficient mismatch repair or microsatellite stable (pMMR/MSS) colorectal cancers (CRCs) are vastly outnumbered by deficient mismatch repair or microsatellite instability-high (dMMR/MSI-H) tumors and lack a response to immune checkpoint inhibitors (ICIs). In this study, we reported two distinct expression patterns of ASCL2 in pMMR/MSS and dMMR/MSI-H CRCs. ASCL2 is overexpressed in pMMR/MSS CRCs and maintains a stemness phenotype, accompanied by a lower density of tumor-infiltrating lymphocytes (TILs) than those in dMMR/MSI CRCs. In addition, coadministration of anti-PD-L1 antibodies facilitated T cell infiltration and provoked strong antitumor immunity and tumor regression in the MC38/shASCL2 mouse CRC model. Furthermore, overexpression of ASCL2 was associated with increased TGFB levels, which stimulate local Cancer-associated fibroblasts (CAFs) activation, inducing an immune-excluded microenvironment. Consistently, mice with deletion of Ascl2 specifically in the intestine (Villin-Cre+, Ascl2 flox/flox, named Ascl2 CKO) revealed fewer activated CAFs and higher proportions of infiltrating CD8+ T cells; We further intercrossed Ascl2 CKO with ApcMin/+ model suggesting that Ascl2-deficient expression in intestinal represented an immune infiltrating environment associated with a good prognosis. Together, our findings indicated ASCL2 induces an immune excluded microenvironment by activating CAFs through transcriptionally activating TGFB, and targeting ASCL2 combined with ICIs could present a therapeutic opportunity for MSS CRCs.

Association of Interleukin-6 Polymorphisms with Schizophrenia and Depression: A Case-Control Study.

OBJECTIVE: Growing evidence suggests a crossover in genetic susceptibility to schizophrenia and depression. We aimed to investigate the association of the rs1800795 and rs1800796 polymorphisms of the IL-6 gene with schizophrenia and depression in the Han Chinese population, combined with IL-6 serum levels. METHODS: Gene sequencing and enzyme-linked immunosorbent assay were performed on 113 subjects with schizophrenia, 114 subjects with depression, and 110 healthy controls. RESULTS: Our findings showed that IL-6 concentrations in schizophrenia and depression groups were significantly higher than in the control group. The rs1800796 CC genotype and C allele were significantly associated with depression (P = .012 and P < .05, respectively). The rs1800796 CC and CG genotype was significantly associated with chronic schizophrenia (P = .020 and P = .009, respectively). Regarding the rs1800795 polymorphism, only one case of CG genotype was detected. The remainder were of the GG genotype. CONCLUSION: The IL-6 rs1800796 might serve as a protective factor for depression and schizophrenia in the Han Chinese population.

Autophagy Related Noncoding RNAs: Emerging Regulatory Factors of Gastric Cancer.

Gastric cancer (GC) is one of the most common malignant cancers that seriously affect human health. Autophagy is a highly conserved self-defense mechanism found to plays an important role in the occurrence, progression, drug resistance, and prognosis of GC. Noncoding RNAs (ncRNAs) play a critical role in the occurrence and development of a variety of diseases including GC. In recent years, increasing attention has been given to research on autophagy-related ncRNAs, such as miRNA, lncRNA, and circRNA in GC. Herein, we briefly summarize the roles, functions, and the research progress of autophagy and autophagy-related ncRNAs in GC with a focus on the potential application in GC tumorigenesis, development, prognosis, and drug resistance. We also discussed prospects of clinical application, future research direction, and challenges in future research of autophagy-related ncRNAs.

Design and fabrication of r-hirudin loaded dissolving microneedle patch for minimally invasive and long-term treatment of thromboembolic disease.

Cardiovascular disease is the leading cause of global mortality, with anticoagulant therapy being the main prevention and treatment strategy. Recombinant hirudin (r-hirudin) is a direct thrombin inhibitor that can potentially prevent thrombosis via subcutaneous (SC) and intravenous (IV) administration, but there is a risk of haemorrhage via SC and IV. Thus, microneedle (MN) provides painless and sanitary alternatives to syringes and oral administration. However, the current technological process for the micro mould is complicated and expensive. The micro mould obtained via three-dimensional (3D) printing is expected to save time and cost, as well as provide a diverse range of MNs. Therefore, we explored a method for MNs array model production based on 3D printing and translate it to micro mould that can be used for fabrication of dissolving MNs patch. The results show that r-hirudin-loaded and hyaluronic acid (HA)-based MNs can achieve transdermal drug delivery and exhibit significant potential in the prevention of thromboembolic disease without bleeding in animal models. These results indicate that based on 3D printing technology, MNs combined with r-hirudin are expected to achieve diverse customizable MNs and thus realize personalized transdermal anticoagulant delivery for minimally invasive and long-term treatment of thrombotic disease.

Development of bispecific anti-c-Met/PD-1 diabodies for the treatment of solid tumors and the effect of c-Met binding affinity on efficacy.

Although the blockade of the programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) pathway has become a promising treatment strategy for several types of cancers, the constitutive activation of c-Met in tumors may cause a low overall response rate to PD-1 inhibitors. Increasing evidence indicates that the dual inhibition of c-Met and PD-1 could improve the efficacy of anti-PD-1/PD-L1 monoclonal antibodies for tumor immunotherapy. In this study, we developed two bispecific single-chain diabodies targeting c-Met and PD-1 for the treatment of solid tumors based on protein homology modeling, and we identified that the binding affinity of diabody-mp to c-Met was 50-folds higher than that of diabody-pm. The results of in vitro studies revealed that both diabodies suppressed HGF-induced proliferation, migration, and invasion of tumor cells, inhibiting the activation of c-Met signaling by antagonizing HGF binding to c-Met. Moreover, they promoted T cell activation by blocking the PD-1 pathway, mediating tumor cellular cytotoxicity through T cell engagement. In vivo studies with mice models demonstrated that diabody-mp exhibited higher therapeutic efficacy than other structural antibodies, greatly enhancing the survival of c-Met-positive tumor-bearing mice compared to single or combined c-Met and PD-1 blockade therapy. Furthermore, diabody-mp, which had a higher c-Met binding affinity, showed better anti-tumoral activity than diabody-pm, which had a lower c-Met binding affinity. In conclusion, bispecific anti-PD-1/c-Met diabody-mp, with high c-Met-associated affinity, inhibited tumor growth by activating T cells, suggesting its therapeutic potential for c-Met-positive solid tumors.

Dual-function chimeric antigen receptor T cells targeting c-Met and PD-1 exhibit potent anti-tumor efficacy in solid tumors.

Purpose Programmed cell death 1 (PD-1), which is upregulated under the continuous induction of the tumor microenvironment, causes chimeric antigen receptor (CAR)-T cell hypofunction via interaction with programmed death ligand 1 (PD-L1). This study aimed to construct CAR-T cells that are resistant to PD-1 inhibition to improve the effect of CAR-T cells in solid tumors. Methods We constructed a type of dual-function CAR-T cell that targets tumor-associated antigen c-Met and blocks the binding of PD-1 with PD-L1. The expression of c-Met, PD-L1, and inhibitory receptors was measured using flow cytometry. The cytotoxicity, cytokine release, and differentiation level of CAR-T cells were determined using lactate dehydrogenase release assay, enzyme-linked immunosorbent assay, and flow cytometry, respectively. The levels of p-Akt, p-MAPK, caspase-3, and Bcl2 were detected by western blot. The in vivo anti-tumor effect was evaluated using tumor xenograft models. Results Dual-function CAR-T cells could mediate enhanced active signals upon encountering target antigens and had targeted cytotoxicity to target cells. However, the cytotoxicity of c-Met-CAR-PD-1+ T cells was impaired due to the interaction of PD-1 with PD-L1. By blocking the binding of PD-1 and PD-L1, the novel dual-function CAR-PD-1+ T cells could maintain cytotoxicity to PD-L1+ tumor cells. In tumor tissue, the dual-function CAR-T cells showed lower inhibitory receptor expression and lower differentiation characteristics, which resulted in potent anti-tumor effects and prolonged survival in PD-L1+ tumor xenograft models compared to single-target CAR-T cells. Conclusion These results confirm that the novel dual-function CAR-T cells exhibit stronger anti-tumor activity against solid tumors than traditional single-target CAR-T cells and present a new approach that enhance the activity of CAR-T cells in solid tumors.

Tobacco Smoke Plays an Important Role in Initiation and Development of Lung Cancer by Promoting the Characteristics of Cancer Stem Cells.

Lung cancer is one of the most common causes of cancer-related deaths worldwide. Tobacco smoke is the single greatest risk factor of lung cancer. Although enormous progress in understanding the molecular mechanisms by which tobacco smoke leading to lung cancer has been made, the molecular pathogenesis remains largely unclear. Cancer stem cells have been implicated in cancer initiation, development, and drug resistance. In this review, we reviewed the relationship between tobacco smoke and lung cancer, the key role of cancer stem cells in lung cancer and other tumors. More importantly, we elucidate the mechanism of tobacco smoke promoting lung cancer from the perspective of the characteristics of cancer stem cells induced by tobacco smoke.

VCAM1 Promotes Tumor Cell Invasion and Metastasis by Inducing EMT and Transendothelial Migration in Colorectal Cancer.

Vascular cell adhesion molecular 1 (VCAM1), an important member of the immunoglobulin superfamily, is related to the development of malignant tumors, such as breast cancer, melanoma, and renal clear cell carcinoma. However, the molecular role and mechanism of VCAM1 in the regulation of the progression of colorectal cancer (CRC) has rarely been studied. The results of IHC and RT-PCR analyses proved that VCAM1 was upregulated in human CRC tissues compared with matched adjacent normal intestinal epithelial tissues. Moreover, analysis of data from the TCGA and Gene Expression Omnibus (GEO) databases revealed that a higher level of VCAM1 was strongly correlated with poor differentiation, metastasis, and short survival in CRC patients. Furthermore, VCAM1 significantly influenced the invasion and metastasis of CRC cells in vitro and in vivo and activated the EMT program, by which cancer cells adhere to the endothelium and cross the vessel wall by forming pseudopodia and invadopodia. The current findings demonstrate that VCAM1 promotes tumor progression in CRC.