Cytochrome P450 F3 promotes colorectal cancer via inhibiting NRF2-mediated ferroptosis.

Cytochrome P450 F3 (CYP4F3) is recognized as a disease-associated immune response initiator that is involved in the synthesis of cholesterol, steroids, and lipids. This study identified the upregulation of CYP4F3 expression in colorectal cancer (CRC) and its association with poor patient prognosis through a comparative analysis between CRC tumor tissues with normal tissues from public databases. The overexpression of CYP4F3 in CT26.wt and SW620, promoted cell proliferation and migration, a reduction of cellular oxidative stress, an up-regulation of the oxidative stress-related pathway NRF2, and an inhibition of cellular ferroptosis. Additionally, inhibition of NRF2 activity stimulated cellular ferroptosis when CYP4F3 was overexpressed. Ferroptosis, characterized by iron-dependent lipid peroxidation, is a non-apoptotic way of cell death with a critical role in cancer development. When given a ferroptosis agonist to CYP4F3-overexpression CRC cells, NRF2 was activated, and cell proliferation and migration were reduced. Furthermore, the mice subcutaneously injected with CYP4F3-overexpression CT26.wt cells formed significantly larger tumors compared to the CYP4F3-vector CT26.wt cell group. This study systematically identified an important role of CYP4F3 in CRC development as a regulator of CRC cells to escape ferroptosis via NRF2, highlighting the significance of CYP4F3 as a potential therapeutic target for CRC.

Establishment of a neutrophil extracellular trap-related prognostic signature for colorectal cancer liver metastasis and expression validation of CYP4F3.

Liver metastasis stands as the primary contributor to mortality among patients diagnosed with colorectal cancer (CRC). Neutrophil extracellular traps (NETs) emerge as pivotal players in the progression and metastasis of cancer, showcasing promise as prognostic biomarkers. Our objective is to formulate a predictive model grounded in genes associated with neutrophil extracellular traps and identify novel therapeutic targets for combating CRLM. We sourced gene expression profiles from the Gene Expression Omnibus (GEO) database. Neutrophil extracellular trap-related gene set was obtained from relevant literature and cross-referenced with the GEO datasets. Differentially expressed genes (DEGs) were identified through screening via the least absolute shrinkage and selection operator regression and random forest modeling, leading to the establishment of a nomogram and subtype analysis. Subsequently, a thorough analysis of the characteristic gene CYP4F3 was undertaken, and our findings were corroborated through immunohistochemical staining. We identified seven DEGs (ATG7, CTSG, CYP4F3, F3, IL1B, PDE4B, and TNF) and established nomograms for the occurrence and prognosis of CRLM. CYP4F3 is highly expressed in CRC and colorectal liver metastasis (CRLM), exhibiting a negative correlation with CRLM prognosis. It may serve as a potential therapeutic target for CRLM. A novel prognostic signature related to NETs has been developed, with CYP4F3 identified as a risk factor and potential target for CRLM.

Single-cell profiling of peripheral blood and muscle cells reveals inflammatory features of juvenile dermatomyositis.

Introduction: Juvenile dermatomyositis (JDM) is a rare yet serious childhood systemic autoimmune condition that primarily causes skin rashes and inflammatory myopathy of the proximal muscles. Although the associated immune response involves the innate and adaptive arms, a detailed analysis of the pertinent immune cells remains to be performed. This study aims to investigate the dynamic changes of cell type, cell composition and transcriptional profiles in peripheral blood and muscle tissues, and in order to clarify the involvement of immune cells in the pathogenesis of JDM and provide a theoretical reference for JDM. Methods: Single-cell RNA sequencing combined with bioinformatic analyses were used to investigate the dynamic changes in cell composition and transcriptional profiles. Results: Analysis of 45,859 cells revealed nine and seven distinct cell subsets in the peripheral blood and muscle tissues respectively. IFITM2+ and CYP4F3+ monocytes were largely produced, and CD74+ smooth muscle cells (SMCs) and CCL19+ fibroblasts were identified as inflammatory-related cell subtypes in JDM patients, exhibiting patient-specific cell population heterogeneity.The dynamic gene expression patterns presented an enhanced type I interferon response in peripheral blood monocytes and T-cells, and SMCs and fibroblasts in muscle of untreated JDM patients. EGR1 and IRF7 may play central roles in the inflammation in both CD74+ SMCs and CCL19+ fibroblasts. Moreover, inflammatory-related monocytes could regulate T-cells, and the interaction between immune cells and SMCs or fibroblasts in muscle was enhanced under the inflammatory state. Conclusions: Immune dysregulation is one of the key pathogenic factors of JDM, and type I interferon responses are significantly enhanced in peripheral blood Monos and T cells as well as SMCs and fibroblasts. EGR1 and IRF7 may play central roles in the inflammation and are considered as potential therapeutic targets for JDM.

Preterm labor with and without chorioamnionitis is associated with activation of myometrial inflammatory networks: a comprehensive transcriptomic analysis.

BACKGROUND: The onset of preterm labor is associated with inflammation. Previous studies suggested that this is distinct from the inflammation observed during term labor. Our previous work on 44 genes differentially expressed in myometria in term labor demonstrated a different pattern of gene expression from that observed in preterm laboring and nonlaboring myometria. We found increased expression of inflammatory genes in preterm labor associated with chorioamnionitis, but in the absence of chorioamnionitis observed no difference in gene expression in preterm myometria regardless of laboring status, suggesting that preterm labor is associated with different myometrial genes or signals originating from outside the myometrium. Given that a small subset of genes were assessed, this study aimed to use RNA sequencing and bioinformatics to assess the myometrial transcriptome during preterm labor in the presence and absence of chorioamnionitis. OBJECTIVE: This study aimed to comprehensively determine protein-coding transcriptomic differences between preterm nonlaboring and preterm laboring myometria with and without chorioamnionitis. STUDY DESIGN: Myometria were collected at cesarean delivery from preterm patients not in labor (n=16) and preterm patients in labor with chorioamnionitis (n=8) or without chorioamnionitis (n=6). Extracted RNA from myometrial tissue was prepared and sequenced using Illumina NovaSeq. Gene expression was quantified by mapping the sequence reads to the human reference genome (hg38). Differential gene expression analysis, gene set enrichment analysis, and weighted gene coexpression network analysis were used to comprehensively interrogate transcriptomic differences and their associated biology. RESULTS: Differential gene expression analysis comparing preterm patients in labor with chorioamnionitis with preterm patients not in labor identified 931 differentially expressed genes, whereas comparing preterm patients in labor without chorioamnionitis with preterm patients not in labor identified no statistically significant gene expression changes. In contrast, gene set enrichment analysis and weighted gene coexpression network analysis demonstrated that preterm labor with and without chorioamnionitis was associated with enrichment of pathways involved in activation of the innate immune system and inflammation, and activation of G protein-coupled receptors. Key genes identified included chemotactic CYP4F3, CXCL8, DOCK2, and IRF1 in preterm labor with chorioamnionitis and CYP4F3, FCAR, CHUK, and IL13RA2 in preterm labor without chorioamnionitis. There was marked overlap in the pathways enriched in both preterm labor subtypes. CONCLUSION: Differential gene expression analysis demonstrated that myometria from preterm patients in labor without chorioamnionitis and preterm patients not in labor were transcriptionally similar, whereas the presence of chorioamnionitis was associated with marked gene changes. In contrast, comprehensive bioinformatic analysis indicated that preterm labor with or without chorioamnionitis was associated with innate immune activation. All causes of preterm labor were associated with activation of the innate immune system, but this was more marked in the presence of chorioamnionitis. These data suggest that anti-inflammatory therapy may be relevant in managing preterm labor of all etiologies.

A disease-associated missense mutation in CYP4F3 affects the metabolism of leukotriene B4 via disruption of electron transfer.

BACKGROUND: Cytochrome P450 4F3 (CYP4F3) is an ω-hydroxylase that oxidizes leukotriene B4 (LTB4), prostaglandins, and fatty acid epoxides. LTB4 is synthesized by leukocytes and acts as a chemoattractant for neutrophils, making it an essential component of the innate immune system. Recently, involvement of the LTB4 pathway was reported in various immunological disorders such as asthma, arthritis, and inflammatory bowel disease. We report a 26-year-old female with a complex immune phenotype, mainly marked by exhaustion, muscle weakness, and inflammation-related conditions. The molecular cause is unknown, and symptoms have been aggravating over the years. METHODS: Whole exome sequencing was performed and validated; flow cytometry and enzyme-linked immunosorbent assay were used to describe patient's phenotype. Function and impact of the mutation were investigated using molecular analysis: co-immunoprecipitation, western blot, and enzyme-linked immunosorbent assay. Capillary electrophoresis with ultraviolet detection was used to detect LTB4 and its metabolite and in silico modelling provided structural information. RESULTS: We present the first report of a patient with a heterozygous de novo missense mutation c.C1123 > G;p.L375V in CYP4F3 that severely impairs its activity by 50% (P < 0.0001), leading to reduced metabolization of the pro-inflammatory LTB4. Systemic LTB4 levels (1034.0 ± 75.9 pg/mL) are significantly increased compared with healthy subjects (305.6 ± 57.0 pg/mL, P < 0.001), and immune phenotyping shows increased total CD19+ CD27- naive B cells (25%) and decreased total CD19+ CD27+ IgD- switched memory B cells (19%). The mutant CYP4F3 protein is stable and binding with its electron donors POR and Cytb5 is unaffected (P > 0.9 for both co-immunoprecipitation with POR and Cytb5). In silico modelling of CYP4F3 in complex with POR and Cytb5 suggests that the loss of catalytic activity of the mutant CYP4F3 is explained by a disruption of an α-helix that is crucial for the electron shuffling between the electron carriers and CYP4F3. Interestingly, zileuton still inhibits ex vivo LTB4 production in patient's whole blood to 2% of control (P < 0.0001), while montelukast and fluticasone do not (99% and 114% of control, respectively). CONCLUSIONS: A point mutation in the catalytic domain of CYP4F3 is associated with high leukotriene B4 plasma levels and features of a more naive adaptive immune response. Our data provide evidence for the pathogenicity of the CYP4F3 variant as a cause for the observed clinical features in the patient. Inhibitors of the LTB4 pathway such as zileuton show promising effects in blocking LTB4 production and might be used as a future treatment strategy.

In vitro inhibitory effects of components from Salvia miltiorrhiza on catalytic activity of three human Arachidonic acid ω-hydroxylases.

CYP4 enzymes are involved in the metabolism of xenobiotics and endogenous molecules. 20-Hydroxyeicosatetraenoic acid (20-HETE), the arachidonic acid (AA) ω-hydroxylation metabolite catalyzed by CYP4A/4F enzymes, is implicated in various biological functions. The goal of this investigation is to examine the inhibitory effects of components from Salvia miltiorrhiza(Danshen) on AA ω-hydroxylation using recombinant CYP4A11, CYP4F2, CYP4F3B, and microsomal systems. Tanshinone IIA had noncompetitive inhibition on CYP4F3B (Ki = 4.98 µM). Cryptotanshinone (Ki = 6.87 µM) and tanshinone I (Ki = 0.42 µM) had mixed-type inhibition on CYP4A11. Dihydrotanshinone I had mixed-type inhibition on CYP4A11 (Ki = 0.09 µM), and noncompetitive inhibition on CYP4F2 (Ki = 4.25 µM) and CYP4F3B (Ki = 3.08 µM). Salvianolic acid A had competitive inhibition on CYP4A11 (Ki = 19.37 µM), and noncompetitive inhibition on CYP4F2 (Ki = 15.28 µM) and CYP4F3B (Ki = 6.45 µM). Salvianolic acid C had noncompetitive inhibition on CYP4F2 (Ki = 5.70 µM) and CYP4F3B (Ki = 18.64 µM). In human kidney, human liver or rat heart microsomes, 20-HETE formation was significantly inhibited (P < 0.05) by dihydrotanshinone I (5 and 20 µM) and salvianolic acid A (20 and 50 µM). Given that low plasma concentrations of Danshen components after oral administration, Danshen preparations may not play pharmacological roles by inhibiting AA ω-hydroxylases; however, as Danshen components may reach high concentration in human intestine, drugs that have an important pre-systemic metabolism by these CYP4A/4F enzymes should avoid being co-administered with Danshen preparations.

[Effects of ophiopogonin D on fatty acid metabolic enzymes in cardiomyocytes].

To explore the effect of ophiopogonin D on main fatty acid metabolic enzymes in human cardiomyocyte AC-16,so as to provide reference for cardiovascular protection mechanism and safe clinical application of Ophiopogon japonicus.CCK-8 (cell counting kit-8) was used to detect the effect of different concentrations of ophiopogonin D on the viability of cardiomyocytes.Meanwhile,the effect of different concentrations of ophiopogonin D on the morphology and quantity of cardiomyocytes was observed under microscope.The effect of ophiopogonin D on the mRNA expression of CYP2J2,CYP4F3,CYP4A11,CYP4A22 and CYP4F2 in cardiomyocytes was detected by RT-PCR.Western blot was used to detect the protein expression of CYP4F3 in different concentrations of ophiopogonin D.Compared with the control group,low-concentration ophiopogonin D had no effect on the viability of cardiomyocytes.However,ophiopogonin D with a concentration of higher than 20µmol·L~(-1)could promote the viability.Under the microscope,ophiopogonin D with a concentration of below 100µmol·L~(-1)had no significant effect on the morphology and number of cardiomyocytes.RT-PCR results showed that compared with the control group,5µmol·L~(-1)ophiopogonin D could slightly up-regulate mRNA expressions of CYP2J2 and CYP4F3,while high-concentration ophiopogonin D (10 and 20µmol·L~(-1)) could significantly induce mRNA expressions of CYP2J2and CYP4F3 in a dose-dependent manner (P<0.05).The same concentration of ophiopogonin D had a little effect on the mRNA expressions of CYP4A11,CYP4A22 and CYP4F2.Western blot results showed that 20µmol·L~(-1)ophiopogonin D could significantly induce the protein expression of CYP4F3 in a dose-dependent manner (P<0.05).Based on the above results,ophiopogonin D (less than100µmol·L~(-1)) has no effect on the viability of AC-16 cardiomyocytes.Ophiopogonin D (less than 100µmol·L~(-1)) can selectively induce the expressions of CYP2J2 and CYP4F3,regulate the metabolic pathway of fatty acid signaling molecules,and thus protecting the cardiovascular system.