STIL facilitates the development and malignant progression of triple-negative breast cancer through activation of Fanconi anemia pathway via interacting with KLF16.

BACKGROUND: STIL is an important cell cycle-regulating protein specifically recruited to the mitotic centrosome to promote the replication of centrioles in dividing cells. However, the potential role of STIL in the regulation of the biological functions of triple-negative breast cancer remains still unclear. METHODS: We screened for differentially expressed STIL in the Cancer Genome Atlas database. The expression of STIL protein in 10 pairs of breast cancer tissues and adjacent normal tissues was further assessed by western blotting. Functionally, the knockdown and overexpression of STIL have been used to explore the effects of STIL on breast cancer cell proliferation, migration, and invasion. Mechanistically, RNA-seq, dual-luciferase reporter assay, chromatin immunoprecipitation assay, mass spectrometry, immunoprecipitation assay, and DNA pull-down assay were performed. RESULTS: Breast cancer tissues and cells have higher STIL expression than normal tissues and cells. STIL knockdown impairs breast cancer cell growth, migration, and invasion, whereas STIL overexpression accelerates these processes. STIL promotes breast cancer progression by regulating FANCD2 expression, and exploration of its molecular mechanism demonstrated that STIL interacts with KLF16 to regulate the expression of FANCD2. CONCLUSIONS: Collectively, our findings identified STIL as a critical promoter of breast cancer progression that interacts with KLF16 to regulate Fanconi anemia pathway protein FANCD2. In summary, STIL is a potential novel biomarker and therapeutic target for breast cancer.

A genomic compendium of cultivated human gut fungi characterizes the gut mycobiome and its relevance to common diseases.

The gut fungal community represents an essential element of human health, yet its functional and metabolic potential remains insufficiently elucidated, largely due to the limited availability of reference genomes. To address this gap, we presented the cultivated gut fungi (CGF) catalog, encompassing 760 fungal genomes derived from the feces of healthy individuals. This catalog comprises 206 species spanning 48 families, including 69 species previously unidentified. We explored the functional and metabolic attributes of the CGF species and utilized this catalog to construct a phylogenetic representation of the gut mycobiome by analyzing over 11,000 fecal metagenomes from Chinese and non-Chinese populations. Moreover, we identified significant common disease-related variations in gut mycobiome composition and corroborated the associations between fungal signatures and inflammatory bowel disease (IBD) through animal experimentation. These resources and findings substantially enrich our understanding of the biological diversity and disease relevance of the human gut mycobiome.

Shikonin and chitosan-silver nanoparticles synergize against triple-negative breast cancer through RIPK3-triggered necroptotic immunogenic cell death.

Necroptotic immunogenic cell death (ICD) can activate the human immune system to treat the metastasis and recurrence of triple-negative breast cancer (TNBC). However, developing the necroptotic inducer and precisely delivering it to the tumor site is the key issue. Herein, we reported that the combination of shikonin (SHK) and chitosan silver nanoparticles (Chi-Ag NPs) effectively induced ICD by triggering necroptosis in 4T1 cells. Moreover, to address the lack of selectivity of drugs for in vivo application, we developed an MUC1 aptamer-targeted nanocomplex (MUC1@Chi-Ag@CPB@SHK, abbreviated as MUC1@ACS) for co-delivering SHK and Chi-Ag NPs. The accumulation of MUC1@ACS NPs at the tumor site showed a 6.02-fold increase compared to the free drug. Subsequently, upon reaching the tumor site, the acid-responsive release of SHK and Chi-Ag NPs from MUC1@ACS NPs cooperatively induced necroptosis in tumor cells by upregulating the expression of RIPK3, p-RIPK3, and tetrameric MLKL, thereby effectively triggering ICD. The sequential maturation of dendritic cells (DCs) subsequently enhanced the infiltration of CD8+ and CD4+ T cells in tumors, while inhibiting regulatory T cells (Treg cells), resulting in the effective treatment of primary and distal tumor growth and the inhibition of TNBC metastasis. This work highlights the importance of nanoparticles in mediating drug interactions during necroptotic ICD.

A high-affinity fluorescent probe for human uridine-disphosphate glucuronosyltransferase 1A9 function monitoring under environmental pollutant exposure.

Uridine-disphosphate glucuronosyltransferase 1A9 (UGT1A9), an important detoxification and inactivation enzyme for toxicants, regulates the exposure level of environmental pollutants in the human body and induces various toxicological consequences. However, an effective tool for high-throughput monitoring of UGT1A9 function under exposure to environmental pollutants is still lacking. In this study, 1,3-dichloro-7-hydroxy-9,9-dimethylacridin-2(9H)-one (DDAO) was found to exhibit excellent specificity and high affinity towards human UGT1A9. Remarkable changes in absorption and fluorescence signals after reacting with UGT1A9 were observed, due to the intramolecular charge transfer (ICT) mechanism. Importantly, DDAO was successfully applied to monitor the biological functions of UGT1A9 in response to environmental pollutant exposure not only in microsome samples, but also in living cells by using a high-throughput screening method. Meanwhile, the identified pollutants that disturb UGT1A9 functions were found to significantly influence the exposure level and retention time of bisphenol S/bisphenol A in living cells. Furthermore, the molecular mechanism underlying the inhibition of UGT1A9 by these pollutant-derived disruptors was elucidated by molecular docking and molecular dynamics simulations. Collectively, a fluorescent probe to characterize the responses of UGT1A9 towards environmental pollutants was developed, which was beneficial for elucidating the health hazards of environmental pollutants from a new perspective.

Targeting Keap1 with Inulae Herba activated the Nrf2 receptor to alleviate LPS-mediated acute lung injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Inulae Herba (IH) is known as Jinfeicao recorded in Chinese Pharmacopoeia with effects of lowering qi and eliminating phlegm, and used for the treatment of pulmonary diseases. However, its protective mechanism on pulmonary diseases, especially acute lung injury (ALI), is still undefined. AIM OF THE STUDY: This study aimed to explore anti-inflammatory and anti-oxidation effects of IH and its underlying mechanism for treating ALI. MATERIALS AND METHODS: We constructed a lipopolysaccharide (LPS)-ALI mouse model to reveal the therapeutical effect of IH. Western blot, real-time quantitative PCR, flow cytometry, small RNA interference, immunohistochemical staining, and the dual-luciferase experiment were performed to study the mechanism of IH for treating ALI. RESULTS: IH attenuated LPS-mediated pathological changes (e.g. pneumonedema and pulmonary congestion) through inactivation of macrophages in an ALI mouse model. The result of flow cytometry demonstrated that IH regulated the homeostasis of M1 (CD80+CD206-) and M2 (CD80+CD206+) phenotype macrophages. Furthermore, IH suppressed mRNA expressions of M1 phenotype markers, such as iNOS and IL-6, whereas promoted mRNA expressions of M2 phenotype markers, such as ARG1 and RETNLA in LPS-mediated mice. Notably, IH targeted Keap1 to activate the Nrf2 receptor, exerting its anti-inflammatory and anti-oxidation effects proved by using immunohistochemical staining, dual-luciferase, and Keap1 knockdown technologies. CONCLUSION: These findings suggested that targeting Keap1 with IH alleviated LPS-mediated ALI, and it could serve as a herbal agent for developing anti-ALI drugs.

The p23 co-chaperone is a succinate-activated COX-2 transcription factor in lung adenocarcinoma tumorigenesis.

P23, historically known as a heat shock protein 90 (HSP90) co-chaperone, exerts some of its critical functions in an HSP90-independent manner, particularly when it translocates into the nucleus. The molecular nature underlying how this HSP90-independent p23 function is achieved remains as a biological mystery. Here, we found that p23 is a previously unidentified transcription factor of COX-2, and its nuclear localization predicts the poor clinical outcomes. Intratumor succinate promotes p23 succinylation at K7, K33, and K79, which drives its nuclear translocation for COX-2 transcription and consequently fascinates tumor growth. We then identified M16 as a potent p23 succinylation inhibitor from 1.6 million compounds through a combined virtual and biological screening. M16 inhibited p23 succinylation and nuclear translocation, attenuated COX-2 transcription in a p23-dependent manner, and markedly suppressed tumor growth. Therefore, our study defines p23 as a succinate-activated transcription factor in tumor progression and provides a rationale for inhibiting p23 succinylation as an anticancer chemotherapy.

BDE209-promoted Dio2 degradation in H4 glioma cells through the autophagy pathway, resulting in hypothyroidism and leading to neurotoxicity.

Decabromodiphenyl ether (BDE209), the homologue with the highest number of brominates in polybrominated diphenyl ethers (PBDEs), is one of the most widespread environmental persistent organic pollutants (POPs) due to its mass production and extensive application in recent decades. BDE209 is neurotoxic, possibly related to its interference with the thyroid hormone (TH) system. However, the underlying molecular mechanisms of BDE209-induced TH interference and neurobehavioral disorders remains unknown. Here, we explored how BDE209 manipulated the major enzyme, human type II iodothyronine deiodinase (Dio2), that is most important in regulating local cerebral TH equilibrium by neuroglial cells, using an in vitro model of human glioma H4 cells. Clonogenic cell survival assay and LC/MS/MS analysis showed that BDE209 could induce chronic neurotoxicity by inducing TH interference. Co-IP assay, RT-qPCR and confocal assay identified that BDE209 destroyed the stability of Dio2 without affecting its expression, and promoted its binding to p62, thereby enhancing its autophagic degradation, thus causing TH metabolism disorder and neurotoxicity. Furthermore, molecular docking studies predicted that BDE209 could effectively suppress Dio2 activity by competing with tetraiodothyronine (T4). Collectively, our study demonstrates that BDE209-induced Dio2 degradation and loss of its enzymatic activity in neuroglial cells are the fundamental pathogenic basis for BDE209-mediated cerebral TH disequilibrium and neurotoxicity, providing a target of interest for further investigation using glial/neuronal cell co-culture system and in vivo models.

MiR-24-3p/Dio3 axis is essential for BDE47 to induce local thyroid hormone disorder and neurotoxicity.

BDE47 (2,2,4,4-tetrabromodiphenyl ether) is a member of the most important congeners of polybrominated diphenyl ethers (PBDEs) and has been identified as a developmental, reproductive and nervous system toxicant and endocrine system disruptor due to its frequent detection in human tissue and environmental samples. Our preliminary work suggested that high- and low-level of bromodiphenyl ethers have different effects on neuronal cells with differential targets of actions on neural tissues. In this study, we presented the underlying mechanism of BDE47 neurotoxicity from the perspective of thyroid hormone (TH) metabolism using in vitro model of human SK-N-AS neuronal cells. BDE47 could induce local TH metabolism disorder in neuronal cells by inhibiting the expression of the main enzyme, human type III iodothyronine deiodinase (Dio3). Further elucidation revealed that BDE47 effectively up-regulating miR-24-3p, which binds to the 3'-UTR of Dio3 and inhibits its expression. In addition, BDE47 could also inhibit the deiodinase activity of Dio3. Collectively, our study demonstrates the molecular mechanism of BDE47 regulating Dio3-induced TH metabolism disorder through inducing miR-24-3p, providing new clues for the role of miRNAs in neurodevelopmental toxicity mediated by environmental pollutants.

Macrophage Inactivation by Small Molecule Wedelolactone via Targeting sEH for the Treatment of LPS-Induced Acute Lung Injury.

Soluble epoxide hydrolase (sEH) plays a critical role in inflammation by modulating levels of epoxyeicosatrienoic acids (EETs) and other epoxy fatty acids (EpFAs). Here, we investigate the possible role of sEH in lipopolysaccharide (LPS)-mediated macrophage activation and acute lung injury (ALI). In this study, we found that a small molecule, wedelolactone (WED), targeted sEH and led to macrophage inactivation. Through the molecular interaction with amino acids Phe362 and Gln384, WED suppressed sEH activity to enhance levels of EETs, thus attenuating inflammation and oxidative stress by regulating glycogen synthase kinase 3beta (GSK3ß)-mediated nuclear factor-kappa B (NF-κB) and nuclear factor E2-related factor 2 (Nrf2) pathways in vitro. In an LPS-stimulated ALI animal model, pharmacological sEH inhibition by WED or sEH knockout (KO) alleviated pulmonary damage, such as the increase in the alveolar wall thickness and collapse. Additionally, WED or sEH genetic KO both suppressed macrophage activation and attenuated inflammation and oxidative stress in vivo. These findings provided the broader prospects for ALI treatment by targeting sEH to alleviate inflammation and oxidative stress and suggested WED as a natural lead candidate for the development of novel synthetic sEH inhibitors.

Rational design of a NIR fluorescent probe for carboxylesterase 1 detection during endoplasmic reticulum stress and drug-induced acute liver injury.

An endoplasmic reticulum targeting NIR fluorescent probe (ERBM) was developed for real-time monitoring of carboxylesterase 1 (CES1) and exhibited excellent ER location in living cell imaging. In addition, ERBM was applied to illustrate the regulation characteristics of CES1 under ER stress and acute liver injury models at the cell and animal level.

Biotransformation of 18ß-Glycyrrhetinic Acid by Human Intestinal Fungus Aspergillus niger RG13B1 and the Potential Anti-Inflammatory Mechanism of Its Metabolites.

18ß-Glycyrrhetinic acid (GA) is a triterpenoid possessing an anti-inflammatory activity in vivo, while the low bioavailability limits its application due to its intestinal accumulation. In order to investigate the metabolism of GA in intestinal microbes, it was incubated with human intestinal fungus Aspergillus niger RG13B1, finally leading to the isolation and identification of three new metabolites (1-3) and three known metabolites (4-6) based on 1D and 2D NMR and high-resolution electrospray ionization mass spectroscopy spectra. Metabolite 6 could target myeloid differentiation protein 2 (MD2) to suppress the activation of nuclear factor-kappa B (NF-κB) signaling pathway via inhibiting the nuclear translocation of p65 to downregulate its target proteins and genes in lipopolysaccharide (LPS)-mediated RAW264.7 cells. Molecular dynamics suggested that metabolite 6 interacted with MD2 through the hydrogen bond of amino acid residue Arg90. These findings demonstrated that metabolite 6 could serve as a potential candidate to develop the new inhibitors of MD2.

MiR-146b-5p/TRAF6 axis is essential for Ginkgo biloba L. extract GBE to attenuate LPS-induced neuroinflammation.

Background: Neuroinflammation plays a crucial role in the pathogenesis and progression of various neurodegenerative diseases, including Alzheimer's disease. The Ginkgo biloba leaf extract (GBE) has been widely used to treat cerebral and peripheral blood circulation disorders. However, its potential targets and underlying mechanisms regarding neuroinflammation have not yet been characterized. Aims: The purpose of this study was to investigate and validate the anti-neuroinflammatory properties of GBE against lipopolysaccharide (LPS)-mediated inflammation and to determine the underlying molecular mechanisms. Methods: The effect of GBE on LPS-induced release of inflammatory cytokines was examined using ELISA and western blot assay. The effects of GBE on NF-κB binding activity and translocation were determined via luciferase, streptavidin-agarose pulldown, and immunofluorescence assays. The potential targets of GBE were screened from the GEO and microRNA databases and further identified via qPCR, luciferase, gene mutation, and western blot assays. Results: GBE significantly inhibited LPS-induced pro-inflammatory responses in BV-2 and U87 cells, with no obvious cytotoxicity. GBE significantly induced miR-146b-5p expression, which negatively regulated TRAF6 expression by targeting its 3'-UTR. Thus, due to TRAF6 suppression, GBE decreases the transcriptional activity of NF-κB and the expression of pro-inflammatory cytokines, such as interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, and cyclooxygenase (COX)-2, and finally reverses LPS-induced neuroinflammation. Conclusion: Our study revealed the anti-neuroinflammatory mechanism of GBE through the miR-146b-5p/TRAF6 axis and provided a theoretical basis for its rational clinical application.

Prdx6-induced inhibition of ferroptosis in epithelial cells contributes to liquiritin-exerted alleviation of colitis.

Inhibition of ferroptosis in intestinal epithelial cells ameliorates clinical symptoms and improves endoscopic presentations in inflammatory bowel disease (IBD). Licorice is used worldwide in food and medicine fields. Liquiritin, a flavonoid component in licorice, is an effective substance used as an anti-inflammatory, antioxidant food that has been shown to improve chemically induced colitis. Herein we evaluated the therapeutic effects of liquiritin on colitis and determined whether liquiritin could affect colitis by modulating ferroptosis in epithelial cells. A colitis model was induced in mice by oral administration with 2.5% DSS dissolved in drinking water. The results showed that liquiritin significantly alleviated symptoms, suppressed intestinal inflammation and restored the epithelial barrier function in the colitis mouse model. Liquiritin supplementation upregulated colonic ferritin expression, increased the storage of cellular iron, reduced the cellular iron level and further inhibited ferroptosis in epithelial cells from the colitis model. Pharmacological stimulation of ferroptosis largely blocked liquiritin-induced alleviation of colitis. Peroxiredoxin-6 (Prdx6) expression was significantly decreased in the DSS group, which was reversed by liquiritin treatment. Genetic or pharmacological silencing of Prdx6 largely reversed liquiritin-induced modulation of the ferritin/iron level and ferroptosis in epithelial cells. Molecular docking results showed that liquiritin could bind to Prdx6 through the hydrogen bond interaction with amino acid residues Thr208, Val206 and Pro203. In conclusion, liquiritin treatment largely alleviated DSS induced colitis by inhibiting ferroptosis in epithelial cells. Liquiritin negatively regulated ferroptosis in epithelial cells in colitis by activating Prdx6, increasing the expression of ferritin and subsequently reducing the cellular iron level.

Microbial transformation of capsaicin by several human intestinal fungi and their inhibitory effects against lysine-specific demethylase 1.

Capsaicin widely exists in the Capsicum genus (e.g., hot peppers) and is commonly used as a food additive or medicinal material. In this work, microbial transformation of capsaicin was performed based on the three cultivated human intestinal fungi. Fourteen metabolites were obtained, and their chemical structures were elucidated by spectroscopic data analysis, including 13 compounds with undescribed structures. Hydroxylation, lactylation, succinylation, citric acylation, and acetylation were observed for these microbial metabolites derived from capsaicin, which indicated diverse catalytic characteristics of human intestinal fungi. In an in vitro bioassay, four metabolites and capsaicin inhibited the activity of lysine-specific demethylase 1 (LSD1) with a more than 70% inhibitory rate at 10 µM. In particular, 9,5'-dihydroxycapsaicin displayed the strongest inhibitory effect with an IC50 of 1.52 µM. Therefore, capsaicin analogs displayed potential application as LSD1 inhibitors against the invasion and migration of cancer cells.

A practical strategy to develop isoform-selective near-infrared fluorescent probes for human cytochrome P450 enzymes.

Currently, the development of selective fluorescent probes toward targeted enzymes is still a great challenge, due to the existence of numerous isoenzymes that share similar catalytic capacity. Herein, a double-filtering strategy was established to effectively develop isoenzyme-specific fluorescent probe(s) for cytochrome P450 (CYP) which are key enzymes involving in metabolism of endogenous substances and drugs. In the first-stage of our filtering approach, near-infrared (NIR) fluorophores with alkoxyl group were prepared for the screening of CYP-activated fluorescent substrates using a CYPs-dependent incubation system. In the second stage of our filtering approach, these candidates were further screened using reverse protein-ligand docking to effectively determine CYP isoenzyme-specific probe(s). Using our double-filtering approach, probes S9 and S10 were successfully developed for the real-time and selective detection of CYP2C9 and CYP2J2, respectively, to facilitate high-throughput screening and assessment of CYP2C9-mediated clinical drug interaction risks and CYP2J2-associated disease diagnosis. These observations suggest that our strategy could be used to develop the isoform-specific probes for CYPs.

Visual Sensing of ß-Glucosidase From Intestinal Fungus in the Generation of Cytotoxic Icarisid II.

ß-Glucosidase (ß-Glc) is an enzyme capable of the selective hydrolysis of the ß-glycosidic bond of glycosides and glycans containing glucose. ß-Glc expressed by intestinal microbiota has attracted increasing levels of interest, due to their important roles for the metabolism of exogenous substances in the gut. Using the 2-((6-hydroxy-2,3-dihydro-1H-xanthen-4-yl)methylene)malononitrile fluorophore (DXM-OH, λem 636 nm) and the recognition group ß-Glucose, an enzymatic activatable turn-on fluorescent probe (DXM-Glc) was developed for the selective and sensitive sensing of ß-Glc. In addition, DXM-Glc could be used to sense endogenous ß-Glc in living fungal cells. Using DXM-Glc, Pichia terricola M2 was identified as a functional intestinal fungus with ß-Glc expression. P. terricola M2 could transform the flavone glycoside Icariin to Icariside Ⅱ efficiently, which confirmed the metabolism of glycosides in the gut mediated by fungi. Furthermore, Icariside Ⅱ could inhibit the proliferation of human endometrial cancer cells (RL 95-2 and ishikawa) significantly, suggesting the metabolic activation of Icariin by intestinal fungi in vivo. Therefore, DXM-Glc as a probe for ß-Glc provided a novel technique for the investigation of the metabolism of bioactive substances by intestinal microbiota.

A strategy for the rapid discovery and validation of active diterpenoids as quality markers in different habitats of Langdu using ultrahigh-performance liquid chromatography-tandem mass spectrometry with multivariate statistical analysis.

Langdu, known as a traditional Chinese medicine, was identified as the roots of species of Euphorbia ebracteolata Hayata and Euphorbia fischeriana Steud, displaying anti-tuberculosis activity. To clarify the potent quality markers of Langdu, this research first developed a fast and sensitive ultrahigh-performance liquid chromatography-tandem mass spectrometry method for the quantification of 13 diterpenoids in Langdu. The developed method was further applied in the analyses of 12 authentic E. ebracteolata and E. fischeriana samples collected in northern and southeastern China. Then, the anti-tuberculosis evaluation of 12 batches of Langdu samples was performed in vitro. Finally, partial least squares discrimination analysis was used in the discrimination of E. ebracteolata and E. fischeriana from different origins and processing methods. Jolkinolide A (1), jolkinolide E (3), yuexiandajisu D (6), and ebractenone A (11) were identified as key, potent diterpenoids for the quality control of E. ebracteolata Hayata and E. fischeriana Steud. The present study established a qualitative chemical analysis method for Langdu (E. ebracteolata and E. fischeriana) and suggested the key bioactive components that will improve qualitative control methodology for this important medicine.