CircRAPGEF5 acts as a modulator of RAS/RAF/MEK/ERK signaling during colorectal carcinogenesis.

Mutations in oncogenes such as KRAS, NRAS and BRAF promote the growth and survival of tumors, while excessive RAS/RAF/MEK/ERK activation inhibits tumor growth. In this study we examined the precise regulatory machinery that maintains a moderate RAS/RAF/MEK/ERK pathway activation during CRC. Here, using bioinformatic analysis, transcriptomic profiling, gene silencing and cellular assays we discovered that a circular RNA, circRAPGEF5, is significantly upregulated in KRAS mutant colorectal cancer (CRC) cells. CircRAPGEF5 suppressed mutant and constitutively activated KRAS and the expression of the death receptor TNFRSF10A. Silencing of circRAPGEF5-induced RAS/RAF/MEK/ERK signaling hyperactivation and apoptosis in CRC cells suggesting that an upregulation of circRAPEF5 may suppress the expression of TNFRSF10A and aid CRC progression by preventing apoptosis, while the direct interactions between circRAPGEF5 and elements of the RAS/RAF/MEK/ERK pathway was not identified, which nevertheless can be the basis for future research. Moreover, EIF4A3, was observed to share a similar expression pattern with circRAPEF5 and demonstrated to be a major controller of circRAPGEF5 via the promotion of circRAPGEF5 circularization and its silencing reduced circRAPGEF5 levels. Taken together, our findings reveal a mechanism of accurate RAS/RAF/MEK/ERK signaling regulation during CRC progression maintained by upregulation of circRAPGEF5 which may be a plausible target for future clinical applications that seek to induce CRC cell apoptosis via the RAS/RAF/MEK/ERK signaling pathway.

Belamcanda chinensis extract inhibits non-small cell lung cancer proliferation and induces apoptosis via inhibiting the MAPK (Ras/Raf) and Akt pathways.

Non-small cell lung cancer (NSCLC) is associated with high mortality and morbidity rates. Despite major progress of treatment of NSCLC over the past few decades, the prognosis of advanced NSCLC is poor, with 5-year survival rates ranging from 2 % to 13 %. Belamcanda chinensis is a traditional Chinese medicine used to promote blood circulation, reduce swelling, heal ulcers, disperse lumps and tumors, and resolve blood stasis. In the present study, the anti-proliferative and pro-apoptotic effects and potential mechanisms of action of Belamcanda chinensis extract (BCE) in SPC-A1 and NCI-H460 NSCLC cells were investigated using MTS, flow cytometry, and western blotting. Also, xenograft model in vivo was established to investigate the anti-NSCLC effects of BCE. The compounds in BCE were quantified using gas chromatography-mass spectrometry (GC-MS). Twenty compounds were found in BCE, and BCE induced cell cycle arrest significantly inhibited the proliferation of NSCLC. Furthermore, BCE was found to induce Cyto C release and the activation of Caspase-3, -8, -9, PARP, ultimately inducing apoptosis in NSCLC cells through both exogenous and endogenous apoptotic pathways (the mitochondrial pathway). BCE also blocked the MAPK (Ras/Raf) and Akt signaling pathways, significantly downregulating the expression of Ras, Raf, Erk1/2, p-Erk1/2, Akt, and p-Akt proteins. Furthermore, BCE significantly inhibited the growth of NSCLC cells SPC-A1 in nude mice and downregulated Ras, Raf, Akt, and p-Akt expression in vivo. The antitumor effects of BCE suggest its potential clinical application in patients with NSCLC, especially in those bearing Ras or Raf mutations.

An Overview on Prevalence and Detection Approaches of BRAF V600E Mutation in Anaplastic Thyroid Carcinoma: A Systematic Review and Meta-Analysis.

Background: BRAF V600E mutation is proved critical in the progression and invasion of thyroid cancer, and as a prognostic biomarker. As anaplastic thyroid cancer (ATC) is a rare and aggressive form of thyroid cancer, this study was conducted to provide a view on prevalence of BRAF V600E as well as the best molecular diagnostic method in ATC patients. Methods: A comprehensive literature search was performed from their inception to Oct 2022 in PubMed, Scopus, Google Scholar, and Web of Science (WoS). The data of the prevalence of ATC were extracted. Moreover, the diagnostic feature of the available diagnostic tools was extracted to measure the sensitivity and specificity. To pool the prevalence data, we used meta-proportion analysis and diagnostic meta-analysis was conducted to determine the specificity and sensitivity of the immunohistochemistry method in detecting BRAF V600E mutation among patients with ATC. Results: Overall, 34 studies were included in this meta-analysis. The incidence of BRAF V600E was shown 33% in the 978 patients. The sensitivity and specificity of IHC in detecting BRAF V600E were detected 78.9% (95%CI: 60.1-97.2), and 69.7% (95%CI: 41.2-98.1), respectively. Conclusion: IHC had an acceptable prognostic profile for detecting BRAF V600E in ATC patients. The diagnosis of BRAF mutation is critical in clinical trials and may be helpful for choosing proper-targeted therapy strategies in ATC patients.

USP25 Promotes the Antimycobacterial Response of Macrophages Through Stabilizing B-Raf and C-Raf.

Ubiquitin-specific peptidase 25 (USP25) is one of the best-characterized deubiquitinating enzymes and plays a vital regulatory role in various biological processes, especially in cancer development and immune regulation. However, the exact role of USP25 and its underlying mechanisms in macrophage activation and immunogenicity during Mycobacterium tuberculosis infection remain unclear. In this study, we found that M tuberculosis infection induced USP25 expression in human and mouse macrophages. In particular, USP25 expression is elevated in multiple cell types, especially monocytes, in patients with tuberculosis. Additionally, USP25 deficiency in macrophages and mice resulted in compromised immunity against M tuberculosis infection, accompanied by reduced expressions of various proinflammatory cytokines and chemokines. Mechanistically, USP25 in macrophages promoted the activation of the ERK signaling pathway through deubiquitination and stabilization of B-Raf and C-Raf. These findings collectively suggest the critical roles of USP25 in M tuberculosis infection and its potential as a therapeutic target.

A Bioinformatic Analysis Predicts That Cannabidiol Could Function as a Potential Inhibitor of the MAPK Pathway in Colorectal Cancer.

Colorectal cancer (CRC), found in the intestinal tract, is initiated and progresses through various mechanisms, including the dysregulation of signaling pathways. Several signaling pathways, such as EGFR and MAPK, involved in cell proliferation, migration, and apoptosis, are often dysregulated in CRC. Although cannabidiol (CBD) has previously induced apoptosis and cell cycle arrest in vitro in CRC cell lines, its effects on signaling pathways have not yet been determined. An in silico analysis was used here to assess partner proteins that can bind to CBD, and docking simulations were used to predict precisely where CBD would bind to these selected proteins. A survey of the current literature was used to hypothesize the effect of CBD binding on such proteins. The results predict that CBD could interact with EGFR, RAS/RAF isoforms, MEK1/2, and ERK1/2. The predicted CBD-induced inhibition might be due to CBD binding to the ATP binding site of the target proteins. This prevents the required phosphoryl transfer to activate substrate proteins and/or CBD binding to the DFG motif from taking place, thus reducing catalytic activity.

Comparison of Relative Activity versus Relative Expression Factors (RAF versus REF) in Predicting Glucuronidation Mediated Drug Clearance Using Recombinant UGTs.

PURPOSE: Predicting the quantitative fraction of glucuronidation (fgluc) by individual UDP-glucuronosyltransferase enzymes (UGTs) is challenging due to the lack of selective inhibitors and inconsistent activity of recombinant UGT systems (rUGTs). Our study compares the relative expression versus activity factors (REF versus RAF) to predict fgluc based on rUGT data to human liver and intestinal microsomes (HLM and HIM). METHODS: REF scalars were derived from a previous in-house proteomics study for eleven UGT enzymes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B10, UGT2B15, and UGT2B17), whereas RAF was calculated by measuring activities in rUGTs to microsomes of selective UGT probe substrates. Protein-normalized activity factor (pnAF) values were generated after correcting activity of individual UGTs to their corresponding protein abundance. The utility of REF and RAF in predicting fgluc was assessed for three UGT substrates-diclofenac, vorinostat, and raltegravir. RESULTS: The REF values ranged from 0.02 to 1.75, RAF based on activity obtained in rUGTs to HLM/HIM were from 0.1 to 274. pnAF values were ~ 5 to 80-fold, except for UGT2B4 and UGT2B15, where pnAF was ~ 180 and > 1000, respectively. The results revealed confounding effect of differential specific activities (per pmol) of rUGTs in fgluc prediction. CONCLUSION: The data suggest that the activity of UGT enzymes was significantly lower when compared to their activity in microsomes at the same absolute protein amount (pmol). Collectively, results of this study demonstrate poor and variable specific activity of different rUGTs (per pmol protein), as determined by pnAF values, which should be considered in fgluc scaling.

Sterigmatocystin declines mouse oocyte quality by inducing ferroptosis and asymmetric division defects.

BACKGROUND: Sterigmatocystin (STE) is a mycotoxin widely found in contaminated food and foodstuffs, and excessive long-term exposure to STE is associated with several health issues, including infertility. However, there is little information available regarding the effects of STE toxin on the female reproductive system, particularly concerning oocyte maturation. METHODS: In the present study, we investigated the toxic effects of STE on mouse oocyte maturation. We also used Western blot, immunofluorescence, and image quantification analyses to assess the impact of STE exposure on the oocyte maturation progression, mitochondrial distribution, oxidative stress, DNA damages, oocyte ferroptosis and asymmetric division defects. RESULTS: Our results revealed that STE exposure disrupted mouse oocyte maturation progression. When we examined the cellular changes following 100 µM STE treatment, we found that STE adversely affected polar body extrusion and induced asymmetric division defects in oocytes. RNA-sequencing data showed that STE exposure affects the expression of several pathway-correlated genes during oocyte meiosis in mice, suggesting its toxicity to oocytes. Based on the RNA-seq data, we showed that STE exposure induced oxidative stress and caused DNA damage in oocytes. Besides, ferroptosis and α-tubulin acetylation were also found in STE-exposed oocytes. Moreover, we determined that STE exposure resulted in reduced RAF1 protein expression in mouse oocytes, and inhibition of RAF1 activity also causes defects in asymmetric division of mouse oocytes. CONCLUSIONS: Collectively, our research provides novel insights into the molecular mechanisms whereby STE contributes to abnormal meiosis.

Promotion of Raf-1/ASK1 complex formation by corylin inhibits cell apoptosis in myocardial ischemia/reperfusion injury.

Effective treatment of myocardial ischemia-reperfusion (MIR) injury remains an unmet clinical need. Cardiomyocyte apoptosis is common at this stage and poses a significant risk. Corylin, a flavonoid compound extracted from Psoralea corylifolia L., has been shown to have anti-inflammatory, anticancer, and antiatherosclerotic properties. However, whether and how corylin affects MIR injury remain unclear. In this study, we explored the mechanism of corylin as a potent therapeutic agent for MI/R injury, using a left anterior descending (LAD) coronary artery ligation and oxygen-glucose deprivation and reperfusion (OGD/R) model in vivo and in vitro. TUNEL, Annexin-V/PI double staining,Ki67 immunohistochemistry, western blot analysis, and immunofluorescence were used to validate cell apoptosis level and Raf-1/ASK1 complex activity. The interaction between corylin and Raf-1/ASK1 complex was detected using molecular docking, corylin-Raf-1 binding assays, and coimmunoprecipitation (Co-IP). Moreover, TTC staining, echocardiography, HE staining, Masson trichrome staining and serological testing were performed to assess the cardioprotective effects of corylin in vivo. These findings showed that corylin reduces MIR injury-induced cardiomyocyte apoptosis and improves cardiac function. Mechanistically, corylin can interact with Raf-1 and promote the formation of the Raf-1/ASK1 complex, thus inhibiting cardiomyocyte apoptosis. In conclusion, our results demonstrate that corylin ameliorated cardiac dysfunction after MIR injury by reducing myocardial apoptosis.

Evaluation of Pan-RAF Inhibitor LY3009120 on Human Uveal Melanoma Cell Line 92-1.

BACKGROUND/AIM: Uveal melanoma (UM) represents a prevailing primary intraocular malignancy, with a limited median overall survival among metastatic patients, and most tumors lack RAF/RAS mutations. The pan-RAF inhibitor LY3009120 has demonstrated valuable anti-tumor effects in a wide range of RAF/RASmut and wild-type (WT) tumor models. This study aimed to evaluate the antitumor effect of LY3009120 on 92-1 UM cell line. MATERIALS AND METHODS: The effect of the pan-RAF inhibitor LY3009120 on cell proliferation, metabolic activity, biomass, early and late apoptosis/necrosis, and morphology was characterized in vitro (0.1-5 µM for 48 h/72 h). Furthermore, targeted panel sequencing was used to characterize the mutational landscape of the human 92-1 UM cell line. RESULTS: LY3009120 showed a significant concentration-dependent anti-proliferative effect on 92-1 cells. Cell proliferation and viability were significantly reduced at the lowest effective concentration of 0.5 µM (at 48 and 72 h, p<0.001). Furthermore, LY3009120 caused significant early apoptosis and late apoptosis/necrosis in 92-1 cells at 5 µM. Except for TP53, NGS showed that all 49 additional analysed genes (Oncomine myeloid panel) of 92-1 were wild-type, including BRAF, NRAS, and KRAS. CONCLUSION: The pan-RAF inhibitor LY3009120 demonstrated a significant anti-tumor effect on human UM cell line 92-1 independent of the molecular BRAF and RAS mutational status.

Noninvasive prediction of BRAF V600E mutation status of pleomorphic xanthoastrocytomas with MRI morphologic features and diffusion-weighted imaging.

OBJECTIVES: Seeking a noninvasive predictor for BRAF V600E mutation status of pleomorphic xanthoastrocytomas (PXAs) is essential for their prognoses and therapeutic use of BRAF inhibitors. We aimed to noninvasively diagnose BRAF V600E-mutated PXAs using MRI morphologic, DWI and clinical parameters. METHODS: The clinical findings, anatomical MRI characteristics, and diffusion parameters of 36 pathologically confirmed PXAs were retrospectively analyzed, and BRAF V600E-mutated (n = 16) and wild-type (n = 20) groups were compared. A binary logistic-regression analysis was performed, and a ROC curve was calculated to determine the independent predictors of BRAF V600E mutation status, diagnostic accuracy, and optimal cut-off value. RESULTS: A comparison of findings between groups showed that BRAF V600E-mutated PXAs were more frequent in children and young adults (≤ 35 years; P = 0.042) who often had histories of seizures (P = 0.004). Furthermore, BRAF V600E-mutated PXAs generally presented as solitary masses (P = 0.024), superficial locations with meningeal attachment (P < 0.001), predominantly cystic with mural nodules (P = 0.005), and had greater minimal ADC ratio (ADCratio) values of the tumor and peritumoral edema (P < 0.001). Binary logistic regression showed that age ≤ 35 years, solitary mass, superficial locations with meningeal attachment, and a greater minimal ADCratio of the tumor were independent predictors of BRAF V600E-mutated PXAs. The combination of all four independent predictors resulted in the highest sensitivity (100%) and specificity (90%), with AUC = 0.984. CONCLUSION: The BRAF V600E mutation status of PXAs could be noninvasively predicted using clinical and MRI characteristics. CRITICAL RELEVANCE STATEMENT: The noninvasive diagnostic criteria for BRAF V600E-mutated PXAs could offer guidance for the administration of BRAF V600E mutation inhibitors in the future.

Retinol Binding Protein 4 Serves as a Potential Tumor Biomarker and Promotes Malignant Behavior in Gastric Cancer.

Background: Gastric cancer (GC) is a highly phenotypically heterogeneous disease and is caused by a combination of factors. Retinol binding protein 4 (RBP4) is a member of a family of lipid transport proteins that are involved in the transport of substances between cells and play a crucial role in a variety of cancers. However, the expression and role of RBP4 in GC remain unknown. Methods: In this study, we explored the expression, prognostic significance, immune microenvironment, drug responsiveness and function of associated signaling pathways of RBP4 in GC using web-based bioinformatics tools. Immunohistochemistry and real-time quantitative PCR were utilized to analyze the tissue and cell expression levels of RBP4. CCK-8, colony formation, EDU incorporation, wound healing and transwell assays were applied to demonstrate the effect of RBP4 on GC cell function. Flow cytometric detection of apoptosis after RBP4 knockdown. Nude mice xenograft model elucidates the role of RBP4 for GC in vivo. Related proteins of the RAS signaling pathway were analyzed by employing Western blot assays. Results: RBP4 is highly expressed in GC. RBP4 is closely associated with patient survival and sensitivity to a wide range of antitumor agents. Knockdown of RBP4 promoted apoptosis and inhibited cell proliferation, invasion and migration. RBP4 promotes GC tumorigenesis in vivo. Finally, RBP4 modulates the RAS/RAF/ERK axis. Conclusion: RBP4 may promote gastric carcinogenesis and development through the RAS/RAF/ERK axis and is expected to be a novel target for GC treatment.

Microrna-605-3p Inhibited the Growth and Chemoresistance of Osteosarcoma Cells via Negatively Modulating RAF1.

BACKGROUND: Osteosarcoma (OS) is the leading cancer-associated mortality in childhood and adolescence. Increasing evidence has demonstrated the key function of microRNAs (miRNAs) in OS development and chemoresistance. Among them, miRNA-605-3p acted as an important tumor suppressor and was frequently down-regulated in multiple cancers. However, the function of miR-650-3p in OS has not been reported. OBJECTIVE: The aim of this work is to explore the novel role of miR-605-3p in osteosarcoma and its possible involvement in OS chemotherapy resistance. METHOD: The expression levels of miR-605-3p in OS tissues and cells were assessed by reverse transcription quantitative PCR (RT-qPCR). The relevance of miR-605-3p with the prognosis of OS patients was determined by the Kaplan-Meier analysis. Additionally, the influence of miR-605-3p on OS cell growth was analyzed using the cell counting kit-8, colony formation assay, and flow cytometry. The mRNA and protein expression of RAF1 were detected by RT-qPCR and western blot. The binding of miR-605-3p with the 3'-UTR of RAF1 was confirmed by dual-luciferase reporter assay. RESULTS: Our results showed that miR-605-3p was markedly decreased in OS tissues and cells. A lower level of miR-605-3p was strongly correlated with lymph node metastasis and poor 5-year overall survival rate of OS patients. In vitro assay found that miR-605-3p suppressed OS cell proliferation and promoted cell apoptosis. Mechanistically, the proto-oncogene RAF1 was seen as a target of miR-605-3p and strongly suppressed by miR-605-3p in OS cells. Restoration of RAF1 markedly eliminated the inhibitory effect of miR-605-3p on OS progression, suggesting RAF1 as a key mediator of miR-605-3p. Consistent with the decreased level of RAF1, miR-605-3p suppressed the activation of both MEK and ERK in OS cells, which are the targets of RAF1. Moreover, lower levels of miR-605-3p were found in chemoresistant OS patients, and downregulated miR-605-3p increased the resistance of OS cells to therapeutic agents. CONCLUSION: Our data revealed that miR-605-3p serves as a tumor suppressor gene by regulating RAF1 and increasing the chemosensitivity of OS cells, which provided the novel working mechanism of miR-605-3p in OS. Engineering stable nanovesicles that could efficiently deliver miR-605-3p with therapeutic activity into tumors could be a promising therapeutic approach for the treatment of OS.

An overview of RAF kinases and their inhibitors (2019-2023).

Protein kinases (PKs) including RAF, perform a principal role in regulating countless cellular events such as cell growth, differentiation, and angiogenesis. Overexpression and mutation of RAF kinases are significant contributors to the development and spread of cancer. Therefore, RAF kinase inhibitors show promising outcomes as anti-cancer small molecules by suppressing the expression of RAF protein, blocking RAS/RAF interaction, or inhibiting RAF enzymes. Currently, there are insufficient reports about approving drugs with minimal degree of toxicity. Therefore, it is an urgent need to develop new RAF kinase inhibitors correlated with increased anticancer activity and lower cytotoxicity. This review outlines reported RAF kinase inhibitors for cancer treatment in patents and literature from 2019 to 2023. It highlights the available inhibitors by shedding light on their chemical structures, biochemical profiles, and current status. Additionally, we highlighted the hinge region-binding moiety of the reported compounds by showing the hydrogen bond patterns of representative inhibitors with the hinge region for each class. In recent years, RAF kinase inhibitors have gained considerable attention in cancer research and drug development due to their potential to be studied under clinical trials and their demonstration of various degrees of efficacy and safety profiles across different cancer types. However, addressing challenges related to drug resistance and safety represents a major avenue for the optimization and enhancement of RAF kinase inhibitors. Strategies to overcome such obstacles were discussed such as developing novel pan-RAF inhibitors, RAF dimer inhibitors, and combination treatments.

Astragaloside IV relieves passive heymann nephritis and podocyte injury by suppressing the TRAF6/NF-κb axis.

The pathogenesis of membranous nephropathy (MN) involves podocyte injury that is attributed to inflammatory responses induced by local immune deposits. Astragaloside IV (AS-IV) is known for its robust anti-inflammatory properties. Here, we investigated the effects of AS-IV on passive Heymann nephritis (PHN) rats and TNF-α-induced podocytes to determine the underlying molecular mechanisms of MN. Serum biochemical parameters, 24-h urine protein excretion and renal histopathology were evaluated in PHN and control rats. The expression of tumor necrosis factor receptor associated factor 6 (TRAF6), the phosphorylation of nuclear factor kappa B (p-NF-κB), the expression of associated proinflammatory cytokines (TNF-α, IL-6 and IL-1ß) and the ubiquitination of TRAF6 were measured in PHN rats and TNF-α-induced podocytes. We detected a marked increase in mRNA expression of TNF-α, IL-6 and IL-1ß and in the protein abundance of p-NF-κB and TRAF6 within the renal tissues of PHN rats and TNF-α-induced podocytes. Conversely, there was a reduction in the K48-linked ubiquitination of TRAF6. Additionally, AS-IV was effective in ameliorating serum creatinine, proteinuria, and renal histopathology in PHN rats. This effect was concomitant with the suppression of NF-κB pathway activation and decreased expression of TNF-α, IL-6, IL-1ß and TRAF6. AS-IV decreased TRAF6 levels by promoting K48-linked ubiquitin conjugation to TRAF6, which triggered ubiquitin-mediated degradation. In summary, AS-IV averted renal impairment in PHN rats and TNF-α-induced podocytes, likely by modulating the inflammatory response through the TRAF6/NF-κB axis. Targeting TRAF6 holds therapeutic promise for managing MN.

Investigation into the anti-inflammatory mechanism of Pothos chinensis (Raf.) Merr. By regulating TLR4/MyD88/NF-κB pathway: Integrated network pharmacology, serum pharmacochemistry, and metabolomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Inflammation is directly related to disease progression and contributes significantly to the global burden of disease. Pothos chinensis (Raf.) Merr. (PCM) is commonly used in Yao medicine in China to treat tumors, and orthopedic illnesses such as knee osteoarthritis, and rheumatic bone discomfort. PCM was found to have significant anti-inflammatory properties in previous studies. AIM OF THE STUDY: To explore the active compounds of PCM and their anti-inflammatory pharmacological mechanisms through an integrated strategy of serum pharmacochemistry, network pharmacology, and serum metabolomics. MATERIALS AND METHODS: The qualitative and quantitative analyses of the chemical components of PCM were performed using UPLC-QTOF-MS/MS and UPLC, respectively, and the prototype components of PCM absorbed into the blood were analyzed. Based on the characterized absorbed into blood components, potential targets and signaling pathways of PCM anti-inflammatory were found using network pharmacology. Furthermore, metabolomics studies using UPLC-QTOF-MS/MS identified biomarkers and metabolic pathways related to the anti-inflammatory effects of PCM. Finally, the hypothesized mechanisms were verified by in vivo and in vitro experiments. RESULTS: Forty chemical components from PCM were identified for the first time, and seven of them were quantitatively analyzed, while five serum migratory prototype components were found. Network pharmacology KEGG enrichment analysis revealed that arachidonic acid metabolism, Tyrosine metabolism, TNF signaling pathway, NF-κB signaling pathway, and phenylalanine metabolism were the main signaling pathways of PCM anti-inflammatory. Pharmacodynamic results showed that PCM ameliorated liver injury and inflammatory cell infiltration and downregulated protein expression of IL-1ß, NF-κB p65, and MyD88 in the liver. Metabolomics studies identified 53 different serum metabolites, mainly related to purine and pyrimidine metabolism, phenylalanine metabolism, primary bile acid biosynthesis, and glycerophospholipid metabolism. The comprehensive results demonstrated that the anti-inflammatory modulatory network of PCM was related to 5 metabolites, 3 metabolic pathways, 7 targets, and 4 active components of PCM. In addition, molecular docking identified the binding ability between the active ingredients and the core targets, and the anti-inflammatory efficacy of the active ingredients was verified by in vitro experiments. CONCLUSION: Our study demonstrated the anti-inflammatory effect of PCM, and these findings provide new insights into the active ingredients and metabolic mechanisms of PCM in anti-inflammation.

Deciphering the pathogenic role of rare RAF1 heterozygous missense mutation in the late-presenting DDH.

Background: Developmental Dysplasia of the Hip (DDH) is a skeletal disorder where late-presenting forms often escape early diagnosis, leading to limb and pain in adults. The genetic basis of DDH is not fully understood despite known genetic predispositions. Methods: We employed Whole Genome Sequencing (WGS) to explore the genetic factors in late-presenting DDH in two unrelated families, supported by phenotypic analyses and in vitro validation. Results: In both cases, a novel de novo heterozygous missense mutation in RAF1 (c.193A>G [p.Lys65Glu]) was identified. This mutation impacted RAF1 protein structure and function, altering downstream signaling in the Ras/ERK pathway, as demonstrated by bioinformatics, molecular dynamics simulations, and in vitro validations. Conclusion: This study contributes to our understanding of the genetic factors involved in DDH by identifying a novel mutation in RAF1. The identification of the RAF1 mutation suggests a possible involvement of the Ras/ERK pathway in the pathogenesis of late-presenting DDH, indicating its potential role in skeletal development.

Spontaneous multifocal pyogenic granulomas.

Cutaneous pyogenic granulomas (PGs) are common, benign vascular tumors of uncertain pathogenesis; however, a growing body of literature suggests that the formation of PGs may be secondary to genetic alterations in both the Ras/Raf/MAPK and PI3K/Akt/mTOR pathways. We present three cases of spontaneous multifocal PGs that first presented in infancy, were not associated with other vascular anomalies or discernable etiology, harbored somatic genetic variants in the Ras/Raf/MAPK pathway (NRAS n = 2, FGFR1 n = 1), were refractory to treatment with beta-blockers and mTOR inhibitors, and responded best to pulsed dye laser. We propose the term "spontaneous multifocal PGs" to describe this entity.

Trametinib boosts palbociclib's efficacy in breast cancer via autophagy inhibition.

Breast cancer, a predominant global health issue, requires ongoing exploration of new therapeutic strategies. Palbociclib (PAL), a well-known cyclin-dependent kinase (CDK) inhibitor, plays a critical role in breast cancer treatment. While its efficacy is recognized, the interplay between PAL and cellular autophagy, particularly in the context of the RAF/MEK/ERK signaling pathway, remains insufficiently explored. This study investigates PAL's inhibitory effects on breast cancer using both in vitro (MCF7 and MDA-MB-468 cells) and in vivo (tumor-bearing nude mice) models. Aimed at elucidating the impact of PAL on autophagic processes and exploring the potential of combining it with trametinib (TRA), an MEK inhibitor, our research seeks to address the challenge of PAL-induced drug resistance. Our findings reveal that PAL significantly decreases the viability of MCF7 and MDA-MB-468 cells and reduces tumor size in mice while showing minimal cytotoxicity in MCF10A cells. However, PAL also induces protective autophagy, potentially leading to drug resistance via the RAF/MEK/ERK pathway activation. Introducing TRA effectively neutralized this autophagy, enhancing PAL's anti-tumor efficacy. A combination of PAL and TRA synergistically reduced cell viability and proliferation, and in vivo studies showed notable tumor size reduction. In conclusion, the PAL and TRA combination emerges as a promising strategy for overcoming PAL-induced resistance, offering a new horizon in breast cancer treatment.

Therapeutic strategies targeting the epidermal growth factor receptor signaling pathway in metastatic colorectal cancer.

More than 1.9 million new colorectal cancer (CRC) cases and 935000 deaths were estimated to occur worldwide in 2020, representing about one in ten cancer cases and deaths. Overall, colorectal ranks third in incidence, but second in mortality. More than half of the patients are in advanced stages at diagnosis. Treatment options are complex because of the heterogeneity of the patient population, including different molecular subtypes. Treatments have included conventional fluorouracil-based chemotherapy, targeted therapy, immunotherapy, etc. In recent years, with the development of genetic testing technology, more and more targeted drugs have been applied to the treatment of CRC, which has further prolonged the survival of metastatic CRC patients.