Butyrate enhances erastin-induced ferroptosis of osteosarcoma cells via regulating ATF3/SLC7A11 pathway.

Osteosarcoma (OS) is a highly fatal bone tumor characterized by high degree of malignancy and early lung metastasis. Traditional chemotherapy fails in improving the efficacy and survival rate of patients with OS. Butyrate (NaBu) has been reported as a new antitumor drug for inhibiting proliferation and inducing apoptosis in various cancer cells. However, the effect of NaBu on the ferroptosis of OS is still unknown. This study aimed to investigate whether NaBu promotes erastin-induced ferroptosis in OS cells and to uncover the underlying mechanism. Here, we found that NaBu significantly enhanced erastin-induced ferroptosis in vitro and in vivo. Compared with the group that erastin used alonely, pre-treating with NaBu exacerbated erastin-meditated GSH depletion, lipid peroxidation, and mitochondrial morphologic changes in OS cells. In a subcutaneous OS model, NaBu combined with erastin significantly reduced tumor growth and increased the levels of 4-HNE. Mechanistically, NaBu downregulated SLC7A11 transcription via regulating ATF3 expression. Overexpression of ATF3 facilitated erastin to induce ferroptosis, while ATF3 knockdown attenuated NaBu-induced ferroptosis sensitivity. In conclusion, our findings revealed a previously unidentified role of NaBu in erastin-induced ferroptosis by regulating SLC7A11, suggesting that NaBu may be a potential therapeutic agent for OS treatment.

Butyrate reverses ferroptosis resistance in colorectal cancer by inducing c-Fos-dependent xCT suppression.

Ferroptosis has emerged to be a promising approach in cancer therapies; however, colorectal cancer (CRC) is relatively insensitive to ferroptosis. Exactly how the gut microenvironment impacts the ferroptotic sensitivity of CRC remains unknown. Herein, by performing metabolomics, we discovered that butyrate concentrations were significantly decreased in CRC patients. Butyrate supplementation sensitized CRC mice to ferroptosis induction, showing great in vivo translatability. Particularly, butyrate treatment reduced ferroptotic resistance of cancer stem cells. Mechanistically, butyrate inhibited xCT expression and xCT-dependent glutathione synthesis. Moreover, we identified c-Fos as a novel xCT suppressor, and further elucidated that butyrate induced c-Fos expression via disrupting class I HDAC activity. In CRC patients, butyrate negatively correlated with tumor xCT expression and positively correlated with c-Fos expression. Finally, butyrate was found to boost the pro-ferroptotic function of oxaliplatin (OXA). Immunohistochemistry data showed that OXA non-responders exhibited higher xCT expression compared to OXA responders. Hence, butyrate supplementation is a promising approach to break the ferroptosis resistance in CRC.

Platelets reprogram monocyte functions by secreting MMP-9 to benefit postoperative outcomes following acute aortic dissection.

Platelets have a great ability to modulate immune responses. Monocyte-platelet aggregates (MPAs) are associated with the pathogenesis of cardiac disease. Notably, a low preoperative platelet count often indicates poor postoperative recovery following acute aortic dissection (AAD). The functions of platelets and MPAs in AAD, however, remain poorly understood. We found that, despite decreased platelet counts, platelets were also activated in AAD patients, with significant alterations in immune-modulating mediators. Of interest, monocytes in AAD patients had a suppressed immune status, which was correlated with poor outcomes following surgery. Interestingly, platelets preferentially aggregated with monocytes, and the levels of MPAs were related to recovery after surgical repair in AAD patients. Platelets restored suppressed monocyte functions in AAD patients by forming aggregates and partly by secreting matrix metalloproteinase-9 (MMP-9). Thus, the results point to a previously unknown mechanism for platelets involving monocyte reprogramming, which may improve postoperative outcomes following complex cardiovascular surgery.

Brain-Derived Neurotrophic Factor Precursor Contributes to a Proinflammatory Program in Monocytes/Macrophages After Acute Myocardial Infarction.

Background The imbalance of monocyte/macrophage polarization toward the preferential proinflammatory phenotype and a lack of normal inflammation resolution are present in acute myocardial infarction (AMI). Our previous study showed that upregulation of brain-derived neurotrophic factor precursor (proBDNF) in M2-like monocytes may contribute to the proinflammatory response in the Stanford type-A acute aortic dissection. The present study aimed to investigate the role of proBDNF signaling in monocytes/macrophages in the progress of AMI. Methods and Results We observed the upregulation of proBDNF in the proinflammatory monocytes of patients with AMI. The upregulation of proBDNF was also observed in the circulating proinflammatory Ly6Chigh monocytes and cardiac F4/80+CD86+ macrophages 3 days after AMI in a mice model. To neutralize proBDNF, the mice subjected to AMI were injected intraperitoneally with a monoclonal anti-proBDNF antibody. Echocardiography, 2,3,5-triphenyltetrazolium chloride staining, and positron emission tomography/computed tomography results demonstrate that monoclonal anti-proBDNF antibody treatment further impaired cardiac functions, increased infarct size, and exacerbated the proinflammatory state. Moreover, the level of proinflammatory Ly6Chigh in the blood and F4/80+CD86+ in the heart was further increased in monoclonal anti-proBDNF antibody mice. RNA sequencing revealed that matrix metalloprotease-9 protein level was dramatically increased, along with the activated proinflammatory-related cytokines. Matrix metalloprotease-9 inhibitor treatment attenuated the deteriorated effect of monoclonal anti-proBDNF antibody on cardiac function and infarct areas. Conclusions Our study shows that endogenous proBDNF in monocytes/macrophages may exert protective roles in cardiac remodeling after AMI by regulating matrix metalloprotease-9 activity.

Up-Regulation of ProBDNF/p75NTR Signaling in Spinal Cord Drives Inflammatory Pain in Male Rats.

Background: The spinal cord expresses brain-derived neurotrophic factor precursor (proBDNF) and its receptor pan neurotrophin receptor 75 (p75NTR). However, the role of spinal proBDNF signaling in the pathogenesis of inflammatory pain remains unknown. Methods: Rats were locally injected with complete Freund's adjuvant (CFA) to induce inflammatory pain. The proBDNF signal expression was detected by double-labeled immunofluorescence. ProBDNF protein, p75NTR extracellular domain (p75NTR-ECD), or monoclonal anti-proBDNF (McAb-proB) were administrated by intrathecal injection to investigate their effects on pain behavior. Paw withdrawal thermal latency (PWL) and paw withdrawal mechanical threshold (PWT) were performed to evaluate pain behavior. Immunoblotting, immunohistochemistry, and immunofluorescence were used to assess inflammation-induced biochemical changes. Results: CFA induced a rapid increase in proBDNF in the ipsilateral spinal cord, and immunofluorescence revealed that CFA-enhanced proBDNF was expressed in NeuN positive neurons and GFAP positive astrocytes. The administration of furin cleavage-resistant proBDNF via intrathecal injection (I.t.) significantly decreased the PWT and PWL, whereas McAb-proB by I.t. alleviated CFA-induced pain-like hypersensitivity in rats. Meanwhile, CFA administration triggered the activation of p75NTR and its downstream signaling extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor (NF)-kappaB p65 in the spinal cord. I.t. administration of p75NTR-ECD suppressed CFA-induced pain and neuroinflammation, including the expression of p-ERK1/2, p-p65, and the gene expression of tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6). Conclusion: Our study reveals that the activated proBDNF/p75NTRsignaling in the spinal cord contributes to the development of CFA-induced inflammatory pain. McAb-proB and p75NTR-ECD appear to be promising therapeutic agents for inflammatory pain.

Infiltrating circulating monocytes provide an important source of BMP4 at the early stage of spinal cord injury.

Bone morphogenetic protein (BMP)4 plays a critical role in regulating neuronal and glial activity in the course of spinal cord injury (SCI). The underlying cause and cellular source of BMP4 accumulation at the injured spinal cord remain unclear. Here, we observed that plasma BMP4 levels are statistically higher in SCI patients than in healthy donors. When comparing rats in the sham group (T9 laminectomy without SCI) with rats in the SCI group, we found a persistent decline in BBB scores, together with necrosis and mononuclear cell accumulation at the contusion site. Moreover, during 2 weeks after SCI both plasma and cerebrospinal fluid levels of BMP4 displayed notable elevation, and a positive correlation. Importantly, percentages of circulating BMP4-positive (BMP4+) monocytes and infiltrating MDMs were higher in the SCI group than in the sham group. Finally, in the SCI+clodronate liposome group, depletion of monocytes effectively attenuated the accumulation of both BMP4+ MDMs and BMP4 in the injured spinal cord. Our results indicated that, following SCI, infiltrating MDMs provide an important source of BMP4 in the injured spinal cord and, therefore, might serve as a potential therapeutic target.

Systemic blockade of proBDNF inhibited the expansion and altered the transcriptomic expression in CD3+B220+ cells in MRL/lpr lupus mice.

OBJECTIVES: The overexpansion of CD3+B220+ cells is the hallmark and main pathological mechanism of clinical manifestations of spontaneously developed MRL/lpr mice, which are primarily used as a mouse model of SLE. Our recent report demonstrated that blocking brain-derived neurotrophic factor precursor (proBDNF) suppressed the antibody-secreting cell differentiation and proliferation and inhibited the progression of SLE; however, the effect of proBDNF blockade on these CD3+B220+ cells in MRL/lpr mice is unclear. METHODS: To explore the effect of proBDNF on CD3+B220+ cells, MRL/lpr mice at 12 weeks old were intraperitoneally injected with monoclonal anti-proBDNF antibody (McAb-proB) or control IgG continuously for 8 weeks. The manifestations in mice were observed, and peripheral blood and splenocytes were collected and analysed via flow cytometry at 20 weeks old. In addition, splenic CD3+B220+ cells were subjected to RNA sequencing (RNA-seq) analysis to identify transcriptomic alterations. RESULTS: CD3+B220+ cells in peripheral blood (p=0.0101) and spleen (p<0.0001) were expanded in MRL/lpr mice. Meanwhile, inhibition of proBDNF signalling reduced the percentage of CD3+B220+ cells in peripheral blood (p=0.0036) and spleen (p=0.0280), alleviated lymphadenopathy, reduced urine protein level (p<0.0001) and increased the body weight (p=0.0493). RNA-seq revealed 501 upregulated and 206 downregulated genes in splenic CD3+B220+ cells in McAb-proB-treated MRL/lpr mice compared with IgG-treated mice. The differentially expressed genes were found to be involved in apoptosis, tumour necrosis factor signalling, and T cell differentiation and proliferation. CONCLUSION: Systemic blockade of proBDNF inhibited the overexpansion of CD3+B220+ cells and altered their signals related to cell cycle, cell apoptosis and the immune response, which may contribute to the attenuation of disease symptoms in murine lupus.

Bioinformatics Profiling and Experimental Validation of 4 Differentially-Expressed LIM Genes in the Course of Colorectal-Adenoma-Carcinoma.

BACKGROUND LIM domain proteins play crucial roles in tumors by interacting with diverse proteins. However, their roles in the course of colorectal mucosa-adenoma-carcinoma remain unclear. This study aimed to depict their dynamic expression profiles and elucidate their potential functions in this transition course. MATERIAL AND METHODS Differentially-expressed LIM proteins (DELGs) in paired adenomas, carcinomas, and mucosae were identified using the GEO dataset (GSE 117606) and validated by immunohistochemistry using our tissue microarray. Kaplan-Meier survival analysis, WGCNA, module-trait analysis, and KEGG enrichment were conducted. The correlation of DELGs expression levels with immune infiltration was assessed using the ESTIMATE package and TISCH database. The role of DELGs of interest was validated using cell proliferation, migration, and invasion assays. RESULTS Four DELGs were identified - LMO3, FHL1, NEBL, and TGFB1I1 - all of which were of significance in prognosis. Module-trait correlation and KEGG enrichment revealed their involvement in cancer-related signaling. Immunohistochemistry showed gradual downregulation of LMO3 but upregulation of NEBL in the mucosa-adenoma-carcinoma sequence. The opposite expression patterns were observed for FHL1 and TGFB1I1 in tumor epithelium and mesenchyme. High expression levels of the DELGs were correlated with increased infiltration of NK, NKT, and macrophages, except for NEBL. Importantly, LMO3 inhibited proliferation, migration, and invasion of colon epithelial cells. CONCLUSIONS This study identified 4 differentially-expressed LIM genes - LMO3, FHL1, TGFB1I1, and NEBL - and revealed they were involved in the mucosa-adenoma-carcinoma sequence via regulating cancer-related pathways, influencing epigenetic field, or affecting immune infiltration. Our findings provide new insights into the roles of LIM proteins in the course of mucosa-adenoma-carcinoma.

Elevated expansion of follicular helper T cells in peripheral blood from children with acute measles infection.

BACKGROUND: Measles outbreaks have threatened the global elimination and eradication of measles in recent years. Measles virus (MeV)-specific antibodies are successful in clearing MeV infection. Follicular helper T (Tfh) cells play a crucial role in promoting antibody production. This study investigated the potential role of Tfh cells in peripheral blood mononuclear cells (PBMCs) from children with acute MeV infection. RESULTS: The frequencies of CXCR5+CD4+ Tfh, ICOShigh Tfh, and PD-1high Tfh cells in PBMCs and levels of IL-6 and IL-21 in plasma were significantly elevated in patients with acute MeV infection. Moreover, a positive correlation was discovered among the frequency of ICOShigh Tfh cells, plasma levels of IL-21 and optical density (OD) values of MeV-specific IgM antibodies in the patients with acute MeV infection. However, elevated plasma MeV-specific NAb titres were not associated with the frequency of Tfh, ICOShigh Tfh, or PD-1high Tfh cells in the patients with acute MeV infection. CONCLUSION: These results suggest that an elevated Tfh cell frequency and associated molecules possibly play a key role in children with acute MeV infection, which contributes to the prevention and treatment of MeV infection in children.

Overexpression of microRNA-9 enhances cisplatin sensitivity in hepatocellular carcinoma by regulating EIF5A2-mediated epithelial-mesenchymal transition.

We investigated the role of microRNA (miR)-9 in modulating chemoresistance in hepatocellular carcinoma (HCC) cells. MiR-9 was overexpressed or knocked down in HCC cell lines. Cell viability, cell proliferation, the expression of EIF5A2 and the epithelial-mesenchymal transition (EMT)-related proteins were examined. HCC cells overexpressing miR-9 were more sensitive to cisplatin; miR-9 knockdown yielded the opposite result. The in vivo nude mouse HCC xenograft tumors yielded the same results. EIF5A2 was identified as a potential target of miR-9, where miR-9 regulated EIF5A2 expression at mRNA and protein level. EIF5A2 knockdown reversed miR-9 inhibition-mediated cisplatin resistance. Altering miR-9 and EIF5A2 expression changed E-cadherin and vimentin expression. Furthermore, EIF5A2 mediated miR-9 EMT pathway regulation, indicating that miR-9 can enhance cisplatin sensitivity by targeting EIF5A2 and inhibiting the EMT pathway. Targeting miR-9 may be useful for overcoming drug resistance in HCC.

The regulatory role of ProBDNF in monocyte function: Implications in Stanford type-A aortic dissection disease.

Brain-derived neurotrophic factor precursor (proBDNF) has been reported to strengthen the dysfunction of monocytes/macrophages in animal studies. However, it is still unknown the roles of proBDNF in the dysfunction of monocytes in the inflammatory diseases in humans. In the present study, we showed that proBDNF and pan neurotrophic receptor p75 were significantly upregulated in monocytes from healthy donors (HD) after lipopolysaccharide treatment. Exogenous proBDNF treatment upregulated CD40 and proinflammatory cytokines expression in monocytes including interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α. In Stanford type-A acute aortic dissection (AAD) patients, proBDNF was upregulated in CD14+ CD163+ CX3CR1+ M2- but not CD14+ CD68+ CCR2+ M1-like monocytes. In addition, sera from AAD patients activated gene expression of proinflammatory cytokines in cultured PBMCs from HD, which was attenuated by proBDNF monoclonal antibody (Ab-proB) treatment. These findings suggested that upregulation of proBDNF in M2-like monocytes may contribute to the proinflammatory response in the AAD.

Diversity and bioactivities of fungal endophytes from Distylium chinense, a rare waterlogging tolerant plant endemic to the Three Gorges Reservoir.

BACKGROUND: The present study involves diversity and biological activities of the endophytic fungal community from Distylium chinense, a rare waterlogging tolerant plant endemic to the Three Gorges Reservoir. This study has been conducted hypothesizing that the microbial communities in the TGR area would contribute to the host plant tolerating a range of abiotic stress such as summer flooding, infertility, drought, salinity and soil erosion etc., and they may produce new metabolites, which may possess plentiful bioactive property, especially antioxidant activity. Therefore in the current study, the antioxidant, antimicrobial and anticancer activities of 154 endophytes recovered from D. chinense have been investigated. Furthermore, the active metabolites of the most broad-spectrum bioactive strain have also been studied. RESULTS: A total of 154 fungal endophytes were isolated from roots and stems. They were categorized into 30 morphotypes based on cultural characteristics and were affiliated with 27 different taxa. Among these, the most abundant fungal orders included Diaporthales (34.4%) and Botryosphaeriales (30.5%), which were predominantly represented by the species Phomopsis sp. (24.7%) and Neofusicoccum parvum (23.4%). Fermentation extracts were evaluated, screening for antioxidant, antimicrobial and anticancer activities. Among the 154 isolates tested, 99 (64.3%) displayed significant antioxidant activity, 153 (99.4%) exhibited inclusive antimicrobial activity against at least one tested microorganism and 27 (17.5%) showed exclusive anticancer activity against one or more cancer cell lines. Specifically, the crude extract of Irpex lacteus DR10-1 exhibited note-worthy bioactivities. Further chemical investigation on DR10-1 strain resulted in the isolation and identification of two known bioactive metabolites, indole-3-carboxylic acid (1) and indole-3-carboxaldehyde (2), indicating their potential roles in plant growth promotion and human medicinal value. CONCLUSION: These results indicated that diverse endophytic fungal population inhabits D. chinense. One of the fungal isolate DR10-1 (Irpex lacteus) exhibited significant antioxidant, antimicrobial and anticancer potential. Further, its active secondary metabolites 1 and 2 also showed antioxidant, antimicrobial and anticancer potential.

Enhanced production of unusual triterpenoids from Kadsura angustifolia fermented by a symbiont endophytic fungus, Penicillium sp. SWUKD4.1850.

Highly oxygenated schitriterpenoids are interesting for study of their structures, bioactivities and synthesis. From Kadsura angustifolia fermented by an associated symbiotic endophytic fungus, Penicillium sp. SWUKD4.1850, nine undescribed triterpenoids, kadhenrischinins A-H, and 7ß-schinalactone C together with four known triterpenoids, henrischinins A and B, schinalactone C and nigranoic acid were isolated and established by the extensive 1D-, 2D-NMR, HR-ESI-MS and ECD data analysis. Except nigranoic acid, all these metabolites have been first detected in non-fermented K. angustifolia. Structurally, kadhenrischinins A-D belong to the relatively rare class of highly oxygenated schitriterpenoids that contain a unique 3-one-2-oxabicyclo [3,2,1]-octane motif, while kadhenrischinins E-H feature a cyclopentane ring in a side chain rarely found in the family Schisandraceae. These results indicated that fermentation of K. angustifolia with SWUKD4.1850 induced the production of highly oxygenated schitriterpenoids from nigranoic acid, which provided a guidance to obtain desired compounds from those plants initially thought not to produce. This is the first report on the fermentation of K. angustifolia medical plant and the first discovery of highly oxygenated schitriterpenoids by microbial technology.

Establishment and validation of a 7-microRNA prognostic signature for non-small cell lung cancer.

PURPOSE: A series of microRNAs (miRNAs) have been identified as associated with the survival of patients with non-small-cell lung cancer (NSCLC). The aim of the present study was to explore whether combination of these experimentally validated individual miRNA biomarkers could be used to further increase their prognostic power in NSCLC. PATIENTS AND METHODS: Based on previously validated NSCLC prognostic miRNAs, gene signatures that could discriminate high-risk subgroups with poor clinical outcome in four NSCLC miRNA expression datasets (GSE13937, GSE16025, The Cancer Genome Atlas Lung Adenocarcinoma, and TCGA lung squamous cell carcinoma) were developed using the SurvMicro tool. The potential of the miRNA signature established was validated by quantitative real-time PCR analysis of clinical NSCLC samples, and its prognostic power evaluated using the survivalROC method. RESULTS: We developed two miRNA signatures with prognostic significance for NSCLC, comprising 12- and 7-miRNAs. The 7-miRNA signature (miR-148b, miR-365, miR-32, miR-375, miR-21, miR-125b, and miR-155) was a subset of a 12-miRNA set that retained prognostic power across NSCLC cohorts. Compared with previously established miRNA signatures, our 7-miRNA signature has similar potential, while comprising fewer miRNA components. The prognostic ability of the 7-miRNA signature was validated experimentally in an independent NSCLC cohort using real-time PCR (HR=3.4847, 95% CI=1.3693-8.8680, P=0.0092), and this signature, combined with tumor pathological stage, had superior prognostic ability compared with tumor stage alone. CONCLUSION: Our data indicate that the established 7-miRNA signature is simple, robust, and may have greater clinical prognostic utility for patients with NSCLC.

Overexpression of Wilms' tumor 1 in skin lesions of psoriasis is associated with abnormal proliferation and apoptosis of keratinocytes.

Psoriasis vulgaris (PV) is a chronic inflammatory skin disease, which is characterized by the abnormal proliferation and apoptosis of keratinocytes. Previous studies have demonstrated that transcription factor Wilms' tumor 1 (WT1) is involved in a number of pathophysiological processes, including organ development, tumorigenesis and cell proliferation. However, the role of WT1 in PV remains unclear. In the present study, WT1 expression was analyzed by reverse transcription­quantitative polymerase chain reaction and western blot analyses. WT1 was stably overexpressed or inhibited in HaCaT cells using Lipofectamine® 2000, and cell proliferation and apoptosis were determined using a Cell Counting Kit­8 or Fluorescein Isothiocyanate Annexin V Apoptosis Detection kit II, respectively. We demonstrated that compared with normal controls, the mRNA and protein expression levels of WT1 were significantly increased in non­lesional skins (human, P<0.0001 and P=0.0291, respectively; mouse, P=0.0020 and P=0.0150, respectively) and lesional skins (human, P<0.0001 and P=0.0060, respectively; mouse, P=0.0010 and P=0.0172, respectively) of patients with PV, in addition to the imiquimod (IMQ)­induced psoriasis­like mouse model. WT1 mRNA and protein expression levels in lesional skins were slightly increased compared with those in non­lesional skins from patients with psoriasis (P=0.2510 and P=0.1690, respectively) and IMQ­treated mice (P=0.9590 and P=0.2552, respectively), although there were no statistical differences. Knockdown of WT1 inhibited the proliferation of HaCaT cells [day (D)4, P=0.0454; D5, P=0.0021] and promoted their apoptosis (P=0.0007), while overexpressing WT1 exhibited the opposite effects (proliferation D3, P=0.0216; D4, P=0.0356; D5, P=0.0188; apoptosis, P=0.0003). Furthermore, it was identified that the inflammatory cytokines interleukin­17A (IL­17A), interferon­Î³ and IL­22 induced the overexpression of WT1 in HaCaT cells. The results of the present study suggested that inflammatory cytokine­induced WT1 overexpression may promote the formation of psoriatic skin lesions via regulation of the proliferation and apoptosis of keratinocytes.