Mechanistic prediction and validation of Brevilin A Therapeutic effects in Lung Cancer.

BACKGROUND: Traditional Chinese medicine (TCM) has been found widespread application in neoplasm treatment, yielding promising therapeutic candidates. Previous studies have revealed the anti-cancer properties of Brevilin A, a naturally occurring sesquiterpene lactone derived from Centipeda minima (L.) A.Br. (C. minima), a TCM herb, specifically against lung cancer. However, the underlying mechanisms of its effects remain elusive. This study employs network pharmacology and experimental analyses to unravel the molecular mechanisms of Brevilin A in lung cancer. METHODS: The Batman-TCM, Swiss Target Prediction, Pharmmapper, SuperPred, and BindingDB databases were screened to identify Brevilin A targets. Lung cancer-related targets were sourced from GEO, Genecards, OMIM, TTD, and Drugbank databases. Utilizing Cytoscape software, a protein-protein interaction (PPI) network was established. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene set enrichment analysis (GSEA), and gene-pathway correlation analysis were conducted using R software. To validate network pharmacology results, molecular docking, molecular dynamics simulations, and in vitro experiments were performed. RESULTS: We identified 599 Brevilin A-associated targets and 3864 lung cancer-related targets, with 155 overlapping genes considered as candidate targets for Brevilin A against lung cancer. The PPI network highlighted STAT3, TNF, HIF1A, PTEN, ESR1, and MTOR as potential therapeutic targets. GO and KEGG analyses revealed 2893 enriched GO terms and 157 enriched KEGG pathways, including the PI3K-Akt signaling pathway, FoxO signaling pathway, and HIF-1 signaling pathway. GSEA demonstrated a close association between hub genes and lung cancer. Gene-pathway correlation analysis indicated significant associations between hub genes and the cellular response to hypoxia pathway. Molecular docking and dynamics simulations confirmed Brevilin A's interaction with PTEN and HIF1A, respectively. In vitro experiments demonstrated Brevilin A-induced dose- and time-dependent cell death in A549 cells. Notably, Brevilin A treatment significantly reduced HIF-1α mRNA expression while increasing PTEN mRNA levels. CONCLUSIONS: This study demonstrates that Brevilin A exerts anti-cancer effects in treating lung cancer through a multi-target and multi-pathway manner, with the HIF pathway potentially being involved. These results lay a theoretical foundation for the prospective clinical application of Brevilin A.

Pancancer analysis of the prognostic and immunological role of FANCD2: a potential target for carcinogenesis and survival.

Recent evidence has shed light on the significant role of FANCD2 in cancer initiation, development, and progression. However, a comprehensive pan-cancer analysis of FANCD2 has been lacking. In this study, we have conducted a thorough investigation into the expression profiles and prognostic significance of FANCD2, as well as its correlation with clinicopathological parameters and immune cell infiltration, using advanced bioinformatic techniques. The results demonstrate that FANCD2 is significantly upregulated in various common cancers and is associated with prognosis. Notably, higher expression levels of FANCD2 are linked to poor overall survival, as indicated by Cox regression and Kaplan-Meier analyses. Additionally, we have observed a decrease in the methylation of FANCD2 DNA in some cancers, and this decrease is inversely correlated with FANCD2 expression. Genetic alterations in FANCD2 predominantly manifest as mutations, which are associated with overall survival, disease-specific survival, disease-free survival, and progression-free survival in certain tumor types. Moreover, FANCD2 exhibits a strong correlation with infiltrating cell levels, immune checkpoint genes, tumor mutation burden (TMB), and microsatellite instability (MSI). Enrichment analysis further highlights the potential impact of FANCD2 on Fanconi anemia (FA) pathway and cell cycle regulation. Through this comprehensive pan-cancer analysis, we have gained a deeper understanding of the functions of FANCD2 in oncogenesis and metastasis across different types of cancer.

Identification of arnicolide C as a novel chemosensitizer to suppress mTOR/E2F1/FANCD2 axis in non-small cell lung cancer.

BACKGROUND AND PURPOSE: The mammalian target of rapamycin (mTOR) pathway plays critical roles in intrinsic chemoresistance by regulating Fanconi anaemia complementation group D2 (FANCD2) expression. However, the mechanisms by which mTOR regulates FANCD2 expression and related inhibitors are not clearly elucidated. Extracts of Centipeda minima (C. minima) showed promising chemosensitizing effects by inhibiting FANCD2 activity. Here, we have aimed to identify the bioactive chemosensitizer in C. minima extracts and elucidate its underlying mechanism. EXPERIMENTAL APPROACH: The chemosensitizing effects of arnicolide C (ArC), a bioactive compound in C. minima, on non-small cell lung cancer (NSCLC) were investigated using immunoblotting, immunofluorescence, flow cytometry, the comet assay, small interfering RNA (siRNA) transfection and animal models. The online SynergyFinder software was used to determine the synergistic effects of ArC and chemotherapeutic drugs on NSCLC cells. KEY RESULTS: ArC had synergistic cytotoxic effects with DNA cross-linking drugs such as cisplatin and mitomycin C in NSCLC cells. ArC treatment markedly decreased FANCD2 expression in NSCLC cells, thus attenuating cisplatin-induced FANCD2 nuclear foci formation, leading to DNA damage and apoptosis. ArC inhibited the mTOR pathway and attenuated mTOR-mediated expression of E2F1, a critical transcription factor of FANCD2. Co-administration of ArC and cisplatin exerted synergistic anticancer effects in the A549 xenograft mouse model by suppressing mTOR/FANCD2 signalling in tumour tissues. CONCLUSION AND IMPLICATIONS: ArC suppressed DNA cross-linking drug-induced DNA damage response by inhibiting the mTOR/E2F1/FANCD2 signalling axis, serving as a chemosensitizing agent. This provides insight into the anticancer mechanisms of ArC and offers a potential combinatorial anticancer therapeutic strategy.

Centipeda minima active components and mechanisms in lung cancer.

BACKGROUND: Traditional Chinese medicine (TCM) has been extensively used for neoplasm treatment and has provided many promising therapeutic candidates. We previously found that Centipeda minima (C. minima), a Chinese medicinal herb, showed anti-cancer effects in lung cancer. However, the active components and underlying mechanisms remain unclear. In this study, we used network pharmacology to evaluate C. minima active compounds and molecular mechanisms in lung cancer. METHODS: We screened the TCMSP database for bioactive compounds and their corresponding potential targets. Lung cancer-associated targets were collected from Genecards, OMIM, and Drugbank databases. We then established a drug-ingredients-gene symbols-disease (D-I-G-D) network and a protein-protein interaction (PPI) network using Cytoscape software, and we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses using R software. To verify the network pharmacology results, we then performed survival analysis, molecular docking analysis, as well as in vitro and in vivo experiments. RESULTS: We identified a total of 21 C. minima bioactive compounds and 179 corresponding targets. We screened 804 targets related to lung cancer, 60 of which overlapped with C. minima. The top three candidate ingredients identified by D-I-G-D network analysis were quercetin, nobiletin, and beta-sitosterol. PPI network and core target analyses suggested that TP53, AKT1, and MYC are potential therapeutic targets. Moreover, molecular docking analysis confirmed that quercetin, nobiletin, and beta-sitosterol, combined well with TP53, AKT1, and MYC respectively. In vitro experiments verified that quercetin induced non-small cell lung cancer (NSCLC) cell death in a dose-dependent manner. GO and KEGG analyses found 1771 enriched GO terms and 144 enriched KEGG pathways, including a variety of cancer related pathways, the IL-17 signaling pathway, the platinum drug resistance pathway, and apoptosis pathways. Our in vivo experimental results confirmed that a C. minima ethanol extract (ECM) enhanced cisplatin (CDDP) induced cell apoptosis in NSCLC xenografts. CONCLUSIONS: This study revealed the key C. minima active ingredients and molecular mechanisms in the treatment of lung cancer, providing a molecular basis for further C. minima therapeutic investigation.

Centipeda minima extract sensitizes lung cancer cells to DNA-crosslinking agents via targeting Fanconi anemia pathway.

BACKGROUND: Intrinsic and acquired chemoresistance remains a critical challenge in lung cancer chemotherapy. Fanconi anemia (FA) pathway plays an important role in antagonizing the cytotoxic effects of chemotherapeutics by repairing DNA damage. We recently demonstrated that the traditional Chinese medicinal herb, Centipeda minima (C. minima), possessed anti-inflammatory and antioxidant properties. However, the potential anticancer application of C. minima and the underlying mechanisms remain unclear. PURPOSE: We aimed to investigate the combined anticancer effects of the ethanol extract of C. minima (ECM) and DNA-crosslinking agents on non-small cell lung cancer (NSCLC) and elucidate the underlying mechanisms. METHODS: Cell viability and flow cytometry assay were performed to determine the synergistic cytotoxicity of ECM and DNA-crosslinking agents, cisplatin (CDDP) or mitomycin C (MMC), in NSCLC cells. Western blotting and immunofluorescence were conducted to examine the effects of ECM on protein expression in DNA damage repair pathway. Comet assay was applied to evaluate DNA damage levels. Subcutaneous xenografts of NSCLC were established to evaluate the combined anticancer effects of ECM and CDDP. RESULTS: Combined treatments with ECM and DNA-crosslinking agents exhibited synergistic cytotoxic effects against A549 and H1299 cells. FANCD2 was highly expressed in NSCLC that correlates with poor prognosis of NSCLC patients, based on the online database analysis. ECM significantly inhibited DNA damage-induced monoubiquitination and nuclear foci formation of FANCD2, thereby sensitizing NSCLC to CDDP- or MMC-induced DNA damage and apoptosis, as evidenced by increased expression of γ-H2AX, increased cleavage of caspases-3 and PARP, and enhanced Annexin V-FITC/PI staining. Further, ECM can also decrease the protein level of FANCD2 that contributes to the chemosensitizing effects. Moreover, ECM significantly attenuated CDDP-mediated S-phase arrest by antagonizing the activation of ATR/Chk1 pathway in NSCLC cells. Animal experiments further demonstrated that ECM and CDDP combination treatment synergistically inhibited tumor growth by decreasing FANCD2 protein level in tumor tissues. CONCLUSION: Our results demonstrated that ECM can inhibit DNA-crosslinking agents-induced activation of FA pathway by attenuating both the expression and monoubiquitination of FANCD2. ECM and CDDP combination therapy exhibited synergistic anticancer effects both in vitro and in vivo, indicating that ECM and its active components might serve as novel anticancer drugs in the combination chemotherapy.

Phytochemical library screening reveals betulinic acid as a novel Skp2-SCF E3 ligase inhibitor in non-small cell lung cancer.

Skp2 is overexpressed in multiple cancers and plays a critical role in tumor development through ubiquitin/proteasome-dependent degradation of its substrate proteins. Drugs targeting Skp2 have exhibited promising anticancer activity. Here, we identified a plant-derived Skp2 inhibitor, betulinic acid (BA), via high-throughput structure-based virtual screening of a phytochemical library. BA significantly inhibited the proliferation and migration of non-small cell lung cancer (NSCLC) through targeting Skp2-SCF E3 ligase both in vitro and in vivo. Mechanistically, BA binding to Skp2, especially forming H-bonds with residue Lys145, decreases its stability by disrupting Skp1-Skp2 interactions, thereby inhibiting the Skp2-SCF E3 ligase and promoting the accumulation of its substrates; that is, E-cadherin and p27. In both subcutaneous and orthotopic xenografts, BA significantly inhibited the proliferation and metastasis of NSCLC through targeting Skp2-SCF E3 ligase and upregulating p27 and E-cadherin protein levels. Taken together, BA can be considered a valuable therapeutic candidate to inhibit metastasis of NSCLC.

Identification of pseudolaric acid B as a novel Hedgehog pathway inhibitor in medulloblastoma.

Aberrant activation of the Hedgehog (Hh) pathway is implicated in the pathogenesis and development of multiple cancers, especially Hh-driven medulloblastoma (MB). Smoothened (SMO) is a promising therapeutic target of the Hh pathway in clinical cancer treatment. However, SMO mutations frequently occur, which leads to drug resistance and tumor relapse. Novel inhibitors that target both the wild-type and mutant SMO are in high demand. In this study, we identified a novel Hh pathway inhibitor, pseudolaric acid B (PAB), which significantly inhibited the expression of Gli1 and its transcriptional target genes, such as cyclin D1 and N-myc, thus inhibiting the proliferation of DAOY and Ptch1+/- primary MB cells. Mechanistically, PAB can potentially bind to the extracellular entrance of the heptahelical transmembrane domain (TMD) of SMO, based on molecular docking and the BODIPY-cyclopamine binding assay. Further, PAB also efficiently blocked ciliogenesis, demonstrating the inhibitory effects of PAB on the Hh pathway at multiple levels. Thus, PAB may overcome drug-resistance induced by SMO mutations, which frequently occurs in clinical setting. PAB markedly suppressed tumor growth in the subcutaneous allografts of Ptch1+/- MB cells. Together, our results identified PAB as a potent Hh pathway inhibitor to treat Hh-dependent MB, especially cases resistant to SMO antagonists.

Systematic Elucidation of the Mechanism of Quercetin against Gastric Cancer via Network Pharmacology Approach.

This study was aimed at elucidating the potential mechanisms of quercetin in the treatment of gastric cancer (GC). A network pharmacology approach was used to analyze the targets and pathways of quercetin in treating GC. The predicted targets of quercetin against GC were obtained through database mining, and the correlation of these targets with GC was analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Next, the protein-protein interaction (PPI) network was constructed, and overall survival (OS) analysis of hub targets was performed using the Kaplan-Meier Plotter online tool. Finally, the mechanism was further analyzed via molecular docking of quercetin with the hub targets. Thirty-six quercetin-related genes were identified, 15 of which overlapped with GC-related targets. These targets were further mapped to 319 GO biological process terms and 10 remarkable pathways. In the PPI network analysis, six hub targets were identified, including AKT1, EGFR, SRC, IGF1R, PTK2, and KDR. The high expression of these targets was related to poor OS in GC patients. Molecular docking analysis confirmed that quercetin can bind to these hub targets. In conclusion, this study provided a novel approach to reveal the therapeutic mechanisms of quercetin on GC, which will ease the future clinical application of quercetin in the treatment of GC.

A Systematic Review of the Mechanisms Underlying Treatment of Gastric Precancerous Lesions by Traditional Chinese Medicine.

Gastric precancerous lesions (GPLs) are an essential precursor in the occurrence and development of gastric cancer, known to be one of the most common and lethal cancers worldwide. Traditional Chinese medicine (TCM) has a positive prospect for the prevention and therapy of GPL owing to several advantages including a definite curative effect, fewer side effects compared to other treatments, multiple components, and holistic regulation. Despite these characteristic advantages, the mechanisms of TCM in treating GPL have not been fully elucidated. In this review, we summarize the current knowledge with respect to herbal formulations and the therapeutic mechanisms of TCM active ingredients for GPL. This paper elaborates on the mechanisms of TCM underlying the prevention and treatment of GPL, specifically those that are linked to anti-H. pylori, anti-inflammation, antiproliferation, proapoptotic, antioxidation, antiglycolytic, and antiangiogenesis effects.

Centipeda minima extract exerts antineuroinflammatory effects via the inhibition of NF-κB signaling pathway.

BACKGROUND: Centipeda minima (L.) A.Br. (C. minima) has been used in traditional Chinese herbal medicine to treat nasal allergy, diarrhea, asthma and malaria for centuries. Recent pharmacological studies have demonstrated that the ethanol extract of C. minima (ECM) and several active components possess anti-bacterial, anti-arthritis and anti-inflammatory properties. However, the effects of ECM on neuroinflammation and the underlying mechanisms have never been reported. PURPOSE: The study aimed to examine the potential inhibitory effects of ECM on neuroinflammation and illustrate the underlying mechanisms. METHODS: High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was performed to qualify the major components of ECM; BV2 and primary microglial cells were used to examine the anti-inflammatory activity of ECM in vitro. To evaluate the anti-inflammatory effects of ECM in vivo, the mice were orally administrated with ECM (100, 200 mg•kg-1•d-1) for 2 days before cotreatment with LPS (2 mg•kg-1•d-1, ip) for an additional 3 days. The mice were sacrificed the day after the last treatment and the hippocampus was dissected for further experiments. The expression of inflammatory proteins and the activation of microglia were respectively detected by real-time PCR, ELISA, Western blotting and immunofluorescence. RESULTS: HPLC-MS/MS analysis confirmed and quantified seven chemicals in ECM. In BV2 and primary microglial cells, ECM inhibited the LPS-induced production of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), thus protecting HT22 neuronal cells from inflammatory damage. Furthermore, ECM inhibited the LPS-induced activation of NF-κB signaling pathway and subsequently attenuated the induction of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2), NADPH oxidase 2 (NOX2) and NADPH oxidase 4 (NOX4), leading to the decreased production of nitrite oxide, prostaglandin E2 (PGE2) and reactive oxygen species (ROS). In an LPS-induced neuroinflammatory mouse model, ECM was found to exert anti-inflammatory activity by decreasing the production of proinflammatory mediators, inhibiting the phosphorylation of NF-κB, and reducing the expression of COX2, iNOS, NOX2 and NOX4 in the hippocampal tissue. Moreover, LPS-induced microglial activation was markedly attenuated in the hippocampus, while ECM at a high dose possesses a stronger anti-inflammatory activity than the positive drug dexamethansone (DEX). CONCLUSION: These findings demonstrate that ECM exerts antineuroinflammatory effects via attenuating the activation of NF-κB signaling pathway and inhibiting the production of proinflammatory mediators both in vitro and in vivo. C. minima might become a novel phytomedicine to treat neuroinflammatory diseases.

A Systems Pharmacology Approach for Identifying the Multiple Mechanisms of Action of the Wei Pi Xiao Decoction for the Treatment of Gastric Precancerous Lesions.

The Wei Pi Xiao (WPX) decoction, based on the theory of traditional Chinese medicine, has been widely used for the treatment of gastric precancerous lesions (GPL). Although WPX is known to be effective for the treatment of GPL, its active ingredients, cellular targets, and the precise molecular mechanism of action are not known. This study aimed to identify the multiple mechanisms of action of the WPX decoction in the treatment of GPL. The active compounds, drug targets, and the key pathways involved in the therapeutic effect of WPX in the treatment of GPL were analyzed by an integrative analysis pipeline. The information pertaining to the compounds present in WPX and their disease targets was obtained from TCMSP and GeneCards, respectively. The mechanisms underlying the therapeutic effect of WPX were investigated with gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. A total of 82 bioactive compounds and 146 related targets were identified in this study. Following target analyses, the targets were further mapped to 26 key biological processes and 21 related pathways to construct a target-pathway network and an integrated GPL pathway. The study demonstrated that the WPX formula primarily treats the dysfunctions of GPL arising from cell proliferation, apoptosis, and mucosal inflammation, which offered a novel insight into the pathogenesis of GPL and revealed the molecular mechanism underlying the therapeutic effects of the WPX formula in GPL. This study offers a novel approach for the systematic investigation of the mechanisms of action of herbal medicines, which will provide an impetus to the GPL drug development pipeline.