Neutrophil extracellular traps induced by chemotherapy inhibit tumor growth in murine models of colorectal cancer.

Neutrophil extracellular traps (NETs), a web-like structure of cytosolic and granule proteins assembled on decondensed chromatin, kill pathogens and cause tissue damage in diseases. Whether NETs can kill cancer cells is unexplored. Here, we report that a combination of glutaminase inhibitor CB-839 and 5-FU inhibited the growth of PIK3CA-mutant colorectal cancers (CRCs) in xenograft, syngeneic, and genetically engineered mouse models in part through NETs. Disruption of NETs by either DNase I treatment or depletion of neutrophils in CRCs attenuated the efficacy of the drug combination. Moreover, NETs were present in tumor biopsies from patients treated with the drug combination in a phase II clinical trial. Increased NET levels in tumors were associated with longer progression-free survival. Mechanistically, the drug combination induced the expression of IL-8 preferentially in PIK3CA-mutant CRCs to attract neutrophils into the tumors. Further, the drug combination increased the levels of ROS in neutrophils, thereby inducing NETs. Cathepsin G (CTSG), a serine protease localized in NETs, entered CRC cells through the RAGE cell surface protein. The internalized CTSG cleaved 14-3-3 proteins, released BAX, and triggered apoptosis in CRC cells. Thus, our studies illuminate a previously unrecognized mechanism by which chemotherapy-induced NETs kill cancer cells.

Monotropein inhibits colitis associated cancer through VDR/JAK1/STAT1 regulation of macrophage polarization.

Colorectal cancer (CRC) is a growing concern due to its high morbidity and mortality, and the search for effective and less toxic active substances against inflammatory bowel diseases has been a hot topic in the research and development of drugs against CRC. It is reported that monotropein isolated from the roots of Morinda officinalis, can improve Dextran Sodium Sulfate (DSS)-induced ulcerative colitis in mice, but its therapeutic effects and mechanisms for CRC treatment are still to be investigated. In the present study, we first used molecular docking, BLI, CESTA, and DARTS methods to detest whether monotropein targets VDR proteins. In addition, we used tumor cell conditioned co-culture and four models of macrophage polarisation to investigate the regulation of four macrophage polarisations by monotropein using RT-PCR, IF and western blot. Furthermore, we further validated the target of action of monotropein for the treatment of Azoxymethane (AOM)/DSS induced colitis associated cancer (CAC) using knockout animals. Meanwhile, we further explored the mechanism of action of monotropein in regulating polarisation by detecting JAK/STAT1-related genes and proteins. Molecular docking and biofilm interference techniques showed that monotropein bound to the VDR, and additional results from CESTA and DARTS suggested that VDR proteins are targets of monotropein. Furthermore, in tumor cell conditioned co-cultures or LPS + IFN-γ induced RAW264.7 cells, VDR translocation to the nucleus was reduced, JAK1/STAT1 signaling pathway proteins were up-regulated, and macrophages were polarised towards the M1-type after monotropein intervention. Animal models in which normal VDR or myeloid VDR was knocked out confirmed that JAK1 levels in intestinal tissues were increased after monotropein intervention, macrophages were polarised towards the M1 type, and CAC paracarcinomas were ameliorated. Taken together, the present study concluded that monotropein inhibited colitis-associated cancers through macrophage polarisation regulated by VDR/JAK1/STAT1.

Multiple Myeloma Therapy: Emerging Trends and Challenges.

Multiple myeloma (MM) is a complex hematologic malignancy characterized by the uncontrolled proliferation of clonal plasma cells in the bone marrow that secrete large amounts of immunoglobulins and other non-functional proteins. Despite decades of progress and several landmark therapeutic advancements, MM remains incurable in most cases. Standard of care frontline therapies have limited durable efficacy, with the majority of patients eventually relapsing, either early or later. Induced drug resistance via up-modulations of signaling cascades that circumvent the effect of drugs and the emergence of genetically heterogeneous sub-clones are the major causes of the relapsed-refractory state of MM. Cytopenias from cumulative treatment toxicity and disease refractoriness limit therapeutic options, hence creating an urgent need for innovative approaches effective against highly heterogeneous myeloma cell populations. Here, we present a comprehensive overview of the current and future treatment paradigm of MM, and highlight the gaps in therapeutic translations of recent advances in targeted therapy and immunotherapy. We also discuss the therapeutic potential of emerging preclinical research in multiple myeloma.

Factors associated with patient delay for older adults with benign prostatic hyperplasia: A descriptive qualitative study.

Based on the theory of planned behavior, the aim of this study was to describe the influencing factors of patient delay intentions and behaviors in benign prostatic hyperplasia (BPH) patients and to provide a reference for the development of a patient delay intention scale. This study was carried out over 4 months in 2021 in Daqing, Heilongjiang, China. The participants were 20 patients with BPH who were aged 60 to 82 years and experienced patient delay; participants were selected through a purposive sampling method. The data were collected via face-to-face semistructured interviews. Five main themes emerged from the interviews, including an insufficient understanding of symptoms, experiences of coping instead of seeking health care, negative attitudes toward care-seeking, the influence of others on decision-making for care-seeking, and obstacles to seeking health care. In conclusion, the patient delay intentions and behaviors of BPH patients are the result of a combination of many factors.

Circulating microbial content in myeloid malignancy patients is associated with disease subtypes and patient outcomes.

Although recent work has described the microbiome in solid tumors, microbial content in hematological malignancies is not well-characterized. Here we analyze existing deep DNA sequence data from the blood and bone marrow of 1870 patients with myeloid malignancies, along with healthy controls, for bacterial, fungal, and viral content. After strict quality filtering, we find evidence for dysbiosis in disease cases, and distinct microbial signatures among disease subtypes. We also find that microbial content is associated with host gene mutations and with myeloblast cell percentages. In patients with low-risk myelodysplastic syndrome, we provide evidence that Epstein-Barr virus status refines risk stratification into more precise categories than the current standard. Motivated by these observations, we construct machine-learning classifiers that can discriminate among disease subtypes based solely on bacterial content. Our study highlights the association between the circulating microbiome and patient outcome, and its relationship with disease subtype.

Eltrombopag inhibits TET dioxygenase to contribute to hematopoietic stem cell expansion in aplastic anemia.

Eltrombopag, an FDA-approved non-peptidyl thrombopoietin receptor agonist, is clinically used for the treatment of aplastic anemia, a disease characterized by hematopoietic stem cell failure and pancytopenia, to improve platelet counts and stem cell function. Eltrombopag treatment results in a durable trilineage hematopoietic expansion in patients. Some of the eltrombopag hematopoietic activity has been attributed to its off-target effects, including iron chelation properties. However, the mechanism of action for its full spectrum of clinical effects is still poorly understood. Here, we report that eltrombopag bound to the TET2 catalytic domain and inhibited its dioxygenase activity, which was independent of its role as an iron chelator. The DNA demethylating enzyme TET2, essential for hematopoietic stem cell differentiation and lineage commitment, is frequently mutated in myeloid malignancies. Eltrombopag treatment expanded TET2-proficient normal hematopoietic stem and progenitor cells, in part because of its ability to mimic loss of TET2 with simultaneous thrombopoietin receptor activation. On the contrary, TET inhibition in TET2 mutant malignant myeloid cells prevented neoplastic clonal evolution in vitro and in vivo. This mechanism of action may offer a restorative therapeutic index and provide a scientific rationale to treat selected patients with TET2 mutant-associated or TET deficiency-associated myeloid malignancies.

Silencing of HuR Inhibits Osteosarcoma Cell Epithelial-Mesenchymal Transition via AGO2 in Association With Long Non-Coding RNA XIST.

BACKGROUND: Osteosarcoma (OS) is a highly malignant and aggressive bone tumor. This study was performed to explore the mechanisms of HuR (human antigen R) in the progression of OS. METHODS: HuR expression levels in OS tissues and cells were detected by immunohistochemistry and western blotting. HuR siRNA was transfected into SJSA-1 OS cells to downregulate HuR expression, and then cell proliferation, migration, and epithelial-mesenchymal transition (EMT) were evaluated. RNA immunoprecipitation was performed to determine the association of the long non-coding RNA (lncRNA) XIST and argonaute RISC catalytic component (AGO) 2 with HuR. Fluorescence in situ hybridization analysis was performed to detect the expression of lncRNA XIST. Western blotting and immunofluorescence assays were performed to observe AGO2 expression after HuR or/and lncRNA XIST knockdown. RESULTS: Knockdown of HuR repressed OS cell migration and EMT. AGO2 was identified as a target of HuR and silencing of HuR decreased AGO2 expression. The lncRNA XIST was associated with HuR-mediated AGO2 suppression. Moreover, knockdown of AGO2 significantly inhibited cell proliferation, migration, and EMT in OS. CONCLUSION: Our findings indicate that HuR knockdown suppresses OS cell EMT by regulating lncRNA XIST/AGO2 signaling.

Oxidative stress-induced mitophagy is suppressed by the miR-106b-93-25 cluster in a protective manner.

Increased reactive oxygen species levels in the mitochondrial matrix can induce Parkin-dependent mitophagy, which selectively degrades dysfunctional mitochondria via the autolysosome pathway. Phosphorylated mitofusin-2 (MFN2), a receptor of parkin RBR E3 ubiquitin-protein ligase (Parkin), interacts with Parkin to promote the ubiquitination of mitochondrial proteins; meanwhile, the mitophagy receptors Optineurin (OPTN) and nuclear dot protein 52 (NDP52) are recruited to damaged mitochondria to promote mitophagy. However, previous studies have not investigated changes in the levels of OPTN, MFN2, and NDP52 during Parkin-mediated mitophagy. Here, we show that mild and sustained hydrogen peroxide (H2O2) stimulation induces Parkin-dependent mitophagy accompanied by downregulation of the mitophagy-associated proteins OPTN, NDP52, and MFN2. We further demonstrate that H2O2 promotes the expression of the miR-106b-93-25 cluster and that miR-106b and miR-93 synergistically inhibit the translation of OPTN, NDP52, and MFN2 by targeting their 3' untranslated regions. We further reveal that compromised phosphorylation of MYC proto-oncogene protein (c-Myc) at threonine 58 (T58) (producing an unstable form of c-Myc) caused by reduced nuclear glycogen synthase kinase-3 beta (GSK3ß) levels contributes to the promotion of miR-106b-93-25 cluster expression upon H2O2 induction. Furthermore, miR-106b-mediated and miR-93-mediated inhibition of mitophagy-associated proteins (OPTN, MFN2, and NDP52) restrains cell death by controlling excessive mitophagy. Our data suggest that microRNAs (miRNAs) targeting mitophagy-associated proteins maintain cell survival, which is a novel mechanism of mitophagy control. Thus, our findings provide mechanistic insight into how miRNA-mediated regulation alters the biological process of mitophagy.

LAIR-1 overexpression inhibits epithelial-mesenchymal transition in osteosarcoma via GLUT1-related energy metabolism.

BACKGROUND: Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is a collagen receptor belonging to the immunoglobulin superfamily. Although previous studies have evaluated the biological role of LAIR in solid tumors, the precise mechanisms underlying the functions of LAIR-1 as a regulator of tumor biological functions remain unclear. METHODS: LAIR-1 expression was evaluated by immunohistochemical analysis using an osteosarcoma (OS) tissue microarray. Wound healing and transwell migration assays were performed to evaluate tumor cell migration. Quantitative real-time polymerase chain reaction (qPCR) and western blotting were conducted to detect the expression of epithelial-mesenchymal transition (EMT)-related molecules. RNA-sequencing (RNA-seq) was conducted to evaluate the mRNA expression profiles after overexpressing LAIR-1 in OS cells. Glucose transporter (Glut)1 expression in OS cells was evaluated by western blotting. RESULTS: LAIR-1 expression was significantly different between the T1 and T2 stages of OS tumors, and it inhibited OS cell migration. LAIR-1 expression was inversely correlated with the expression of Twist1, an EMT-associated transcription factor, via the Forkhead box O1 signal transduction pathway. Furthermore, RNA-seq and qPCR demonstrated that the expression of EMT energy metabolism-related molecules was significantly reduced after LAIR-1 overexpression. CONCLUSIONS: LAIR-1 overexpression decreased the expression of Glut1 and inhibited the expression of EMT-related molecules in OS cells. These findings provide new insights into the molecular mechanism underlying OS progression.

Deficiency of platelet adhesion molecule CD226 causes megakaryocyte development and platelet hyperactivity.

This study used constitutive CD226 gene knockout (KO) mice as a model to investigate the functions and mechanisms of CD226 in megakaryocyte (MK) maturation and platelet activation. Although CD226 deficiency did not cause MK polyploidization or platelet granule abnormalities, increased MK counts were detected in the femora bone marrow (BM) and spleen of CD226 KO mice. Particularly, CD226 KO mice have a more extensive membrane system in MKs and platelets than wild-type (WT) mice. We also demonstrated that CD226 KO mice displayed increased platelet counts, shortened bleeding time, and enhanced platelet aggregation. CD226 KO platelets had an increased mature platelet ratio compared to the control platelets. In addition, the observed reduction in bleeding time may be due to decreased nitric oxide (NO) production in the platelets. Platelet-specific CD226-deficient mice showed similar increased MK counts, shortened bleeding time, enhanced platelet aggregation, and decreased NO production in platelets. Furthermore, we performed middle cerebral artery occlusion-reperfusion surgery on WT and CD226 KO mice to explore the potential effect of CD226 on acute ischemia-reperfusion injury; the results revealed that CD226 deficiency led to significantly increased infarct area. Thus, CD226 is a promising candidate for the treatment of thrombotic disorders.

CD226 deficiency on regulatory T cells aggravates renal fibrosis via up-regulation of Th2 cytokines through miR-340.

In this study, we observed that deletion of CD226 on regulatory T cells (Tregs) precedes renal fibrosis in a mouse unilateral ureteral obstruction (UUO) model. First, we generated Treg-specific CD226 gene knockout mice (CD226fl/fl Foxp3YFP-Cre ). Next, CD226fl/fl Foxp3YFP-Cre mice and Foxp3YFP-Cre control mice were subjected to UUO surgery. Pathologic analysis and Sirius red and Masson's trichrome staining showed that the kidneys of CD226fl/fl Foxp3YFP-Cre mice following UUO showed much more severe interstitial fibrosis than Foxp3YFP-Cre control mice at days 10 and 20. Additionally, CD226fl/fl Foxp3YFP-Cre mice showed increased fibronectin expression, as demonstrated by immunohistochemistry (IHC) staining. Although Treg cell-restricted CD226 deficiency showed increased Foxp3+ expression, expression of the cell surface functional molecule CD103 was significantly reduced, indicating impaired homeostasis in the Tregs of CD226fl/fl Foxp3YFP-Cre mice. To better understand CD226 function, RNA sequencing (RNA-Seq) analysis was conducted in Tregs isolated from CD226fl/fl Foxp3YFP-Cre and Foxp3YFP-Cre mice. RNA-Seq data showed that the helper T cell (Th) 2-related cytokines IL-4 and IL-10 were significantly up-regulated in CD226 deficient Tregs. In addition, mRNA analysis of kidney samples from the mice following UUO by qPCR also showed increased IL-4 and IL-10 expression in CD226fl/fl Foxp3YFP-Cre mice, as well as elevated TGF-ß1 levels, indicating that CD226 deficiency in Tregs resulted in the acquisition of the ability to produce Th2 cytokines. Finally, we found that microRNA-340 (miR-340), which was down-regulated in Tregs isolated from CD226fl/fl Foxp3YFP-Cre mice, directly regulated IL-4 gene expression in vitro. These data suggest that the promotion of CD226 signaling on Tregs is a therapeutic target for renal disease.

CD226 is involved in megakaryocyte activation and early-stage differentiation.

This study was conducted to investigate the effect of CD226 on the differentiation, activation, and polyploidization of megakaryocytes (MKs) and explore the potential mechanism. Dami (megakaryocyte line) cell maturation was induced by phorbol 12-myristate 13-acetate. CD226 was silenced by infection with a CD226-specific shRNA lentiviral vector. The mRNA level of CD226 was detected by qRT-PCR. The expressions of Dami cells surface CD226, MK specific markers CD41 and CD62P, and DNA ploidy in Dami cells and CD226 knockdown (KD) cells were evaluated by flow cytometry. The effect of CD226 on the expression of megakaryocyte-associated transcription factors was measured by western blot and confocal analysis. Transfection with CD226 shRNA lentivirus dramatically decreased the level of CD226 and expression of CD62 P in Dami cells. Silencing of CD226 caused morphological changes and differentiation retardation in low-ploidy MK. Furthermore, CD226 knockout (KO) mice exhibited increased 2N-4N low-ploidy MK and decreased ≥8N polyploidy. Interestingly, silencing of CD226 in megakaryocytic cells down-regulated the expression of early stage transcription factors includes GATA-binding factor 1 (GATA-1) and friend leukemia integration 1 (FLI-1), but not late-stage nuclear factor, erythroid 2 (NF-E2). CD226 is involved in MKs activation and polyploidy cell cycle control.

[New processing procedure for Croton tiglium with study on comparison of Croton tiglium and processed product].

OBJECTIVE: To build a new processing procedure for Croton tiglium, providing a more simple, efficient and safe way of processing. METHODS: Used the contents of isoguanosine and toxic protein in Croton tiglium as the indexes to investigate the effect of different temperature, thickness and baked time on processing for Croton tiglium. After established all factors and levels, processed a batch of Croton tiglium under optimum processing conditions and compared it with raw Croton tiglium in the test of acute toxicity and gastrointestinal propulsive motility. RESULTS: The parameters of optimum processing were as follows:the temperature was set at 180 degrees C, the thickness of placement was 3 cm and baked time was 90 min. The LD50 value of raw Croton tiglium and the processed Croton tiglium was 888 mg/kg and 2139 mg/kg respectively. CONCLUSION: The processing procedure is simple, affordable, safe and efficient, deserved to promote for application.

[Screening of anti-tumor parts from Tripterygium hypoglaucum].

OBJECTIVE: To screen the anti-tumor active parts from Tripterygium hypoglaucum by anti-tumor experimental model in vivo and in vitro. METHOD: Ethanol extraction was separated and purified by column chromatography of ion polymeric adsorbent and macroporous adsorptive resins. MTT assay and the inhibition effect to S180 solid tumor were used to detect anti-tumor activity of each separation. RESULT: There are anti-tumor activities in the ethanol extraction, total alkaloids, and macroporous resin absorption in vivo and vitro. Minimum IC50 of Total alkaloids was 29.90 mg L(-1), and S180 solid tumor inhibition ratio of different dose of 25, 50, 100 mg kg(-1) were 38.10%, 50.60%, 60.71% respectively. Minimum IC50 of macroporous resin absorption was 98.56 mg L(-1), and S180 solid tumor inhibition ratio of different dose of 100, 200, 400 mg kg(-1) were 42.96%, 53.57%, 63.79% respectively. Water solubility position had no effect in vivo and vitro. CONCLUSION: T. hypoglaucum has fine anti-tumor activity in vivo and in vitro, and total alkaloids are the main part of anti-tumor active part.