Estrogen plays some important roles in many physiological processes in animals. This hormone is used as a type of medication for humans and animals, including fish, but is associated with serious side effects and environmental persistence, which has led to a growing interest in phytoestrogens as an alternative. Phytoestrogens are compounds derived from plants that are structurally similar to estrogen and may exhibit similar behavior in the body. To date, no studies have investigated the activity of phytoestrogens in relation to the maturation of eels. In the present study, we investigated the effects of ten different plant extracts on vitellogenin (vtg) and estrogen receptor (esr1, esr2) gene expression in eel hepatocytes. As a result, Schisandra and Astragalus extracts induced higher levels of vtg mRNA expression compared to the other extracts. However, increased esr mRNA expression was observed only in the Schisandra and soybean extract-treated groups. The phytoestrogens known to be present in Schisandra and Astragalus were analyzed using HPLC. Schizandrin, gomisin A, and gomisin N were detected in Schisandra extract, and calycosin and formononetin were detected in Astragalus extract. We then examined whether these phytoestrogens could induce vtg mRNA expression in eel hepatocytes. As a result, gomisin N and formononetin significantly induced vtg mRNA expression. In conclusion, among the 10 plant extracts treated in this study, Schisandra and Astragalus extracts induced estrogenic activity in eel hepatocytes. These extracts were found to contain phytoestrogens, with gomisin N and formononetin identified as the primary active components responsible for the observed estrogenic activity in eel hepatocytes.

Purpose: Homocysteine (HCY) metabolism is regulated by the methionine cycle, which is essential for DNA methylation and is associated with the folate cycle. This study examines the alterations in DNA methylation signature including epigenetic age changes, measure cell-free DNA (cfDNA), and HCY concentrations, and identifies genetic markers that may influence homocysteine response following folic acid (FA) supplementation in individuals with hyperhomocysteinemia (HHC). Methods: Blood samples were obtained from 43 HHC patients undergoing FA supplementation. We quantified FA and HCY levels, separated plasma and white blood cell fractions, and evaluated global DNA methylation using LINE-1 bisulfite pyrosequencing. Biological age was determined using Illumina BeadArray technology, and whole-exome sequencing was performed to investigate the patients' genetic backgrounds. Results: Following FA supplementation, cfDNA levels significantly decreased and correlated positively with HCY (r = 0.2375). Elevated average LINE-1 methylation of cfDNA and PBMC-origin DNA was observed, with mean relative changes of 1.9% for both sample types. Regarding HCY levels, we categorized patients based on their response to FA supplementation. FA responders showed decreased HCY from 15.7 ± 5.5 to 11 ± 2.9 µmol/L, while in FA non-responders, an opposite trend was detected. The average biological age was reduced by 2.6 years, with a notable reduction observed in 80% of non-responders and 48% of responders. Sequencing identified mutations in several genes related to the one-carbon cycle, including MTRR, CHAT, and MTHFD1, with strong correlations to the non-responder phenotypes found in genes like PRMT3, TYMS, DNMT3A, and HIF3A. Conclusions: FA supplementation influences the HCY level, as well as affects the cfDNA amount and the DNA methylation pattern. However, genetic factors may play a crucial role in mediating individual responses to folate intake, emphasizing the need for personalized approaches in managing hyperhomocysteinemia.

Invasive Lobular Carcinoma (ILC), a distinct subtype of breast cancer, is hallmarked by E-Cadherin loss, slow proliferation, and strong hormone receptor positivity. ILC faces significant challenges in clinical management due to advanced stage at diagnosis, late recurrence, and development of resistance to endocrine therapy - a cornerstone of ILC treatment. To elucidate the mechanisms underlying endocrine resistance in ILC, ILC cell lines (MDA-MB-134-VI, SUM44PE) were generated to be resistant to tamoxifen, a selective estrogen receptor modulator. The tamoxifen-resistant (TAMR) cells exhibit a 2-fold increase in tamoxifen IC50 relative to parental cells. Metabolomics and RNA-sequencing revealed deregulation of alanine, aspartate, and glutamate metabolism, purine metabolism, and arginine and proline metabolism in TAMR cells. Among the fifteen commonly dysregulated genes in these pathways, low argininosuccinate synthase (ASS1) expression was identified in the TAMR cells and was significantly correlated with poor outcome in ILC patients, specifically in the context of endocrine therapy. Our study reveals methylation-mediated silencing of ASS1 in TAMR cells as a likely mechanism of downregulation. Demethylation restored ASS1 expression and correspondingly reduced tamoxifen IC50 toward parental levels. Nucleic acid biosynthesis is augmented in TAMR cells, evidenced by an increase in nucleotide intermediates. Both TAMR cell lines demonstrated increased expression of several nucleic acid biosynthesis enzymes, including PAICS, PRPS1, ADSS2, CAD, and DHODH. Furthermore, CAD, the key multifunctional protein of the de novo pyrimidine biosynthesis pathway, is differentially activated in TAMR cells. Treating TAMR cells with Decitabine, a demethylating agent, or Farudodstat, a pyrimidine biosynthesis inhibitor, markedly augmented the efficacy of tamoxifen. Collectively, our study unveils ASS1 downregulation as a novel mechanism underlying acquired tamoxifen resistance in ILC and establishes a metabolic link between ASS1 and nucleic acid biosynthesis. Restoring ASS1 expression or inhibiting pyrimidine biosynthesis reinstated tamoxifen sensitivity. ASS1 could be a potential biomarker and therapeutic target in tamoxifen-resistant ILC patients, warranting further investigation.

Tyrosine kinase inhibitors (TKIs) are the standard treatment for advanced hepatocellular carcinoma (HCC). However, their therapeutic efficacy is often limited by drug resistance, primarily driven by tumoral intrinsic mechanisms. In this study, we demonstrate that IFNγ in the tumor microenvironment can potentiate TKI response, and that ablation of IFNγ receptor on HCC cells leads to TKI resistance in vivo. Mechanistically, IFNγ synergizes with TKI to induce GSDME-mediated pyroptosis of HCC cells. The PERK-mediated unfolded protein response (UPR) protects HCC cells from TKI-induced pyroptosis. IFNγ attenuates PERK activation by inducing the expression of PDIA1, which alleviates the stress of protein unfolding. In vivo, PERK inhibition augments TKI therapy, and elevated PERK expression correlates with poor overall survival of patients with HCC. Moreover, IFNγ-producing CD8+ T cells can potentiate TKI efficacy. Combining PD-1 blockade to activate T-cell response with TKI therapy synergistically suppresses the growth of GSDME-expressing HCC tumors, which is further enhanced by the PERK inhibitor. Our findings reveal how IFNγ signaling modulates TKI response and demonstrate the potential of a sequential combination of ICB-mediated immunotherapy and TKI therapy for patients with GSDME+ HCC. T cell-derived IFNγ enhances TKI-induced pyroptosis in HCC. Mechanistic illustration of IFNγ secreted from CD8+ T cells enhancing TKI-induced GSDME-mediated pyroptosis in hepatocellular carcinoma via suppression of the PERK pathway. Created with BioRender.com.

We hypothesize that gold nanoclusters functionalized with adenosine triphosphate (AuNC@ATP) can inhibit schwannoma growth.

High-grade osteosarcoma (HGOS) is the most common primary malignant bone tumor in children and adolescents. Poor response to chemotherapy is linked to worse prognosis and increased risk of recurrence and metastasis. However, current assessment methods, such as tumor necrosis evaluation, are time-consuming and delay treatment decisions. Thus, identifying molecular pathways and predictive biomarkers is essential for guiding early therapeutic strategies. In this study, RNA-seq analysis of HGOS tissues revealed enrichment of cholesterol biosynthesis and mitotic pathways in poor responders. Additionally, high HMGCR expression, as analyzed from TCGA data, was associated with poor prognosis in sarcoma. Functional validation using SaOS-2 cells, which exhibited poor drug sensitivity and elevated HMGCR levels, demonstrated that simvastatin enhanced the efficacy of cisplatin and doxorubicin by inducing mitochondrial-mediated apoptosis and downregulating anti-apoptotic proteins. Simvastatin also reduced cell migration and invasion by suppressing epithelial-mesenchymal transition and extracellular matrix degradation. Mechanistically, simvastatin disrupted Ras prenylation and inhibited downstream oncogenic signaling pathways, including Akt/mTOR and Akt/GSK3, which regulate survival and metastasis-associated gene expression. These findings suggest that the cholesterol biosynthesis pathway particularly plays a critical role in chemoresistance and metastasis in HGOS and may serve as a promising predictive molecular target for guiding early therapeutic strategies.

Synaptic plasticity is the key mechanism underlying learning and memory. Neurexins are pre-synaptic molecules that play a pivotal role in synaptic plasticity, interacting with many different post-synaptic molecules in the formation of neural circuits. Neurexins are alternatively spliced at different splice sites, yielding thousands of isoforms with different properties of interaction with post-synaptic molecules for a quick adaptation to internal and external inputs. The endocannabinoid system also plays a central role in synaptic plasticity, regulating key retrograde signaling at both excitatory and inhibitory synapses. This study aims at elucidating the crosstalk between alternative splicing of neurexin and the endocannabinoid system in the hippocampus. By employing an ex vivo hippocampal system, we found that pharmacological activation of cannabinoid receptor 1 (CB1) with the specific agonist ACEA led to reduced neurotransmission, associated with increased expression of the Nrxn1-3 spliced isoforms excluding the exon at splice site 4 (SS4-). In contrast, treatment with the CB1 antagonist AM251 increased glutamatergic activity and promoted the expression of the Nrxn variants including the exon (SS4+) Knockout of the involved splicing factor SLM2 determined the suppression of the exon splicing at SS4 and the expression only of the SS4+ variants of Nrxns1-3 transcripts. Interestingly, in SLM2 ko hippocampus, modulation of neurotransmission by AM251 or ACEA was abolished. These findings suggest a direct crosstalk between CB1-dependent signaling, neurotransmission and expression of specific Nrxns splice variants in the hippocampus. We propose that the fine-tuned regulation of Nrxn1-3 genes alternative splicing may play an important role in the feedback control of neurotransmission by the endocannabinoid system.

We have previously demonstrated the ability of inhibitors of LSD1 and HDAC1 to block rod degeneration, preserve vision, maintain transcription of rod photoreceptor genes, and downregulate transcripts involved in cell death, gliosis, and inflammation in the mouse model of Retinitis Pigmentosa (RP), rd10. To extend our findings, we tested the hypothesis that this effect was due to altered chromatin structure by using a range of inhibitors of chromatin condensation to prevent photoreceptor degeneration in the rd10 mouse model. We used inhibitors for both G9A/GLP, which catalyzes methylation of H3K9, and EZH2, which catalyzes trimethylation of H3K27, and compared them to the actions of inhibitors of LSD1 and HDAC. All the inhibitors are likely to decondense chromatin and all preserve, to different extents, retinas from degeneration in rd10 mice, but they act through different metabolic pathways. One group of inhibitors, modifiers for LSD1 and EZH2, demonstrate a high level of maintenance of rod-specific transcripts, activation of Ca2+ and Wnt signaling pathways with the inhibition of antigen processing and presentation, immune response, and microglia phagocytosis. Another group of inhibitors, modifiers for HDAC and G9A/GLP, work through upregulation of NGF-stimulated transcription, while downregulating genes belong to immune response, extracellular matrix, cholesterol signaling, and programmed cell death. Our results provide robust support for our hypothesis that inhibition of chromatin condensation can be sufficient to prevent rod death in rd10 mice.

Urothelial carcinoma (UC) is a common genitourinary malignancy. Smoking, exposure to arsenic in drinking water, and age can increase the risk of developing UC. Neoadjuvant cisplatin-based chemotherapy prior to radical cystectomy is the standard treatment for the muscle invasive form of UC (MIUC). Tumors of the basal/squamous (Ba/Sq) subtype of MIUC are aggressive, express basal keratins (KRT5, 6, and 14), are associated with squamous differentiation (SD), and frequently develop chemotherapy resistance. The SOX2 transcription factor is a marker of UC stem cells, and its expression is associated with poor overall and disease-free survival. We hypothesized that the attenuation of SOX2 would reduce the expression of basal keratins and increase the chemotherapy response in human UC cells. For this study, we performed lentiviral knockdown (KD) of SOX2 expression in two separate arsenite (As3+)-transformed UROtsa (As_I, As_II), 5637, and RT4 cells. Cellular growth and colony-forming ability was inhibited in all UC cell lines after SOX2 KD. We demonstrate that SOX2 KD in the UC cells of the Ba/Sq subtype (As_I, As_II, 5637) decreased the expression of stem-associated proteins, oncoproteins, and basal keratins. Additionally, there was an induction of several luminal markers and enhanced cisplatin sensitivity following the repression of SOX2. Lastly, proteomics revealed reductions in lipid-, cholesterol-, and interferon-signaling pathways after SOX2 KD. This study provides a better understanding of the regulation of key genes responsible for defining the Ba/Sq subtype of UC and demonstrates that the inhibition of SOX2 improves chemotherapy response in UC.

Plant immunity against pathogens is primarily triggered by the perception of pathogen-associated molecular patterns (PAMPs). Ascaroside#18, a nematode-derived pheromone, is the first identified nematode-associated molecular pattern conferring broad-spectrum pathogen resistance. Recently, ascr#18 was shown to be recognised by the leucine-rich repeat receptor NILR1, linked to pattern-triggered immunity (PTI) against nematodes. However, the molecular mechanisms downstream of ascr#18 perception remain largely unknown. Here, we show that ascr#18 triggers an immune response that differs from the typical PTI features, with no reactive oxygen species burst or defence-related growth inhibition. Further analysis indicates that the ascr#18-associated resistance mechanism against cyst nematodes (CN) operates independently of the peroxisomal β-oxidation pathway. Transcriptome profiling of Arabidopsis roots treated with ascr#18 revealed strong effects on the regulation of auxin transport and signalling genes, while classical defence genes remained unchanged. These changes, particularly the downregulation of auxin-related genes, occur independently of NILR1. Analysis of CN feeding sites revealed that ascr#18 pretreatment reduced expression of the auxin influx carrier AUX1 and the auxin-responsive genes SAUR69 and IAA27. Promoter-reporter analysis confirmed reduced AUX1 expression in both nematode-infected and non-infected roots treated with ascr#18. Since nematode establishment and the associated feeding cell development are heavily dependent on the modulation of auxin signalling, our results suggest a novel defence mechanism based on its suppression. This mechanism reduces nematode susceptibility without activating classical PTI responses. Our results provide new insights into how plants fend off biotrophic pathogens and point to ways of developing novel strategies for controlling nematodes and other biotrophic pathogens.

Hormones such as abscisic acid and brassinosteroids ameliorate stress tolerance in plants. The present investigation demonstrates the importance of brassinolide, an active form of brassinosteroids, in sustaining photosynthesis under high light (HL). The addition of brassinolide to mesophyll protoplasts activates the mitochondrial electron transport chain (mETC), particularly through an alternative oxidase (AOX) pathway. Brassinolide promotes both respiration and photosynthesis, including PSII activity, under HL. Total respiration was enhanced, but brassinolide caused a differential modulation of the components of mETC. The capacity of the AOX pathway was significantly enhanced, while the capacity of the cytochrome oxidase (COX) pathway was decreased in response to brassinolide under HL. Further, the transcripts of alternative oxidase1A (AOX1A) were elevated more than cytochrome oxidase subunit 15 (COX15), and cellular reactive oxygen species (ROS) were raised marginally upon treatment with brassinolide under HL. Brassinolide enhanced the capacity of the AOX pathway in mETC to ensure an optimal cellular ROS, which in turn sustains photosynthesis. Thus, mETC plays an important role in optimizing photosynthesis under HL stress and highlights the potential of brassinolide in enhancing plant stress tolerance.

In this review, the role of microRNA‑21 (miRNA‑21) as an oncogene in lung cancer was investigated. Studies have shown that miRNA‑21 can promote the progression of lung cancer by targeting downstream target genes, and its expression can be modulated by transcription factors, DNA methylation or competitive endogenous RNA as an upstream regulator. This review highlights that miRNA‑21 can promote the progression of lung cancer through multiple signaling pathways, with a focus on the PI3K/AKT, MEK/ERK, TGF‑β/SMAD, Hippo, NF‑κB and STAT3 signaling pathways. Mechanistically, miRNA‑21 plays an important role in the progression of lung cancer by regulating multiple biological processes, such as proliferation, invasion, metastasis, apoptosis and angiogenesis in lung cancer cells. Higher expression of miRNA‑21 is associated with chemotherapy, radiotherapy and immune resistance in lung cancer. Targeting these molecular pathways may be a novel therapeutic strategy for treating lung cancer. Additionally, miRNA‑21 can serve as a biomarker for lung cancer diagnosis, prognosis and treatment response. This review also summarized the following: i) Current methods employed to inhibit the expression of miRNA‑21 in lung cancer, including CRISPR/Cas9 technology; ii) the application of natural anticancer agents, oligonucleotides, small molecules and miRNA sponges; and iii) the nano‑delivery systems developed for miRNA‑21 inhibitors. Finally, the advancements in research on miRNA mimics and inhibitors in clinical trials, which may promote the application of miRNA‑21 in clinical trials in lung cancer, were discussed. Given that lung cancer is a considerable public health challenge, these studies provide new ways of treating patients with lung cancer.

Glioblastoma is the most aggressive type of brain tumor and is associated with a poor prognosis. First-line treatment is surgical resection followed by radiotherapy and temozolomide-based chemotherapy. However, the duration of treatment with temozolomide is limited due to both its toxicity and the development of drug resistance. The prognostic and predictive factor for response to temozolomide is the methylation status of the MGMT promoter. Indeed, loss of MGMT promoter methylation is a major cause of chemoresistance. However, the development of drug resistance is not only associated with changes in MGMT methylation. The entire epigenome changes and acquires specific properties necessary for tumor progression.

Biodentine is widely used as a pulp capping agent due to its biocompatibility and ability to stimulate reparative dentin formation. Considering the growing use of lasers in dental clinics, this study aimed to assess the effects of 450 and 980 nm diode laser irradiations combined with Biodentine on proliferation and the expression of pluripotent genes of dental pulp stem cells (DPSCs). DPSCs were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS. The experiment consisted of ten groups, each subjected to different conditions of diode laser irradiation at two wavelengths (450 nm and 980 nm) and energy densities (2 J/cm² and 4 J/cm²), with or without the addition of Biodentine extract. The groups included controls such as Biodentine extract alone and untreated DPSCs. Cell proliferation was assessed using MTT assays. The expression of pluripotent genes (OCT4, SOX2, NESTIN, and NANOG) was evaluated through real-time PCR. Data were analyzed by two-way ANOVA (P < 0.05). DPSCs exposed to 450 nm /4 J/cm² in combination of Biodentine showed significantly higher proliferation when compared to the Biodentine group at 24, 48, and 72 h (P < 0.005). Moreover, the 450 nm/4 J/cm² laser induced the highest expression of pluripotent genes in cells exposed to Biodentine, whereas the 980 nm/4 J/cm² laser group demonstrated the lowest gene expression. Application of Biodentine in combination with photobiomodulation (PBM) therapy can enhance DPSC proliferation and increase the expression of pluripotent genes. PBM may be optimized as an adjunctive treatment to Biodentine in regenerative endodontics, promoting more effective tissue regeneration and healing. However, this study was limited to in vitro conditions, without in vivo validation or assessment of mineralization potential, highlighting the need for further studies to confirm these findings in clinical settings.

Aflatoxins, particularly Aflatoxin B1 (AFB1), produced by Aspergillus flavus and other species, pose significant health risks due to their carcinogenic properties. This study investigates the inhibitory effects of Protocatechuic Acid (PCA) on mycotoxigenic fungi and AFB1 production. PCA demonstrated significant dose-dependent antifungal activity against various Aspergillus species, with A. flavus showing inhibition zones ranging from 5.3 mm to 16.7 mm at concentrations of 50 µg/ml to 250 µg/ml, while A. ochraceus exhibited the highest sensitivity, with zones up to 23.6 mm. Additionally, PCA effectively reduced AFB1 production in liquid media, achieving up to 80.21% inhibition at 250 µg/ml, and decreased the mycelial weight of A. flavus by 60.8%. Gene expression analysis revealed that PCA significantly downregulated the expression of the AFB1 biosynthetic genes nor-1 (95% reduction) and omt-A (74% reduction), suggesting that PCA disrupts multiple stages of aflatoxin synthesis. Furthermore, PCA demonstrated efficacy in controlling AFB1 contamination in postharvest corn grains, with inhibition percentages of 44.8%, 55.7%, and 64.6% at 150, 200, and 250 µg/ml, respectively. These findings indicate PCA's potential as a natural antifungal agent, offering promising applications in food safety and postharvest management.

Probiotics are beneficial microorganisms that modulate various signaling pathways to improve human health status. In this study we aimed to evaluate the precise molecular effects of Lactobacillus spp., Bifidobacterium spp., and a mixture of our native potential probiotics on the autophagy signaling pathway during the presence of inflammation. The evaluation of autophagy gene expression was performed after exposing the HT -29 cell line with the sonicated pathogens and probiotics, before and simultaneously with inflammation induction by quantitative real-time polymerase chain reaction (qPCR) and cytokine assays. The results of the current study showed that our native potential probiotic cocktails could upregulate the expression level of the autophagy genes including pik3c3, atg14, beclin, pik3r4, atg5, atg16, atg7, and atg3 compared with sonicated pathogen treatments, and also these native potential probiotic strains could exert anti-inflammatory effects, especially before inflammation induction. In conclusion, our native potential probiotic cocktail indicated the preventive and therapeutic effect on inflammation, but our selected probiotics could affect autophagy genes stronger before inflammation compared to expose simultaneously with inflammation. Therefore, the administration of probiotics as a prophylactic agent with the least side effects could be considered a suitable treatment for patients with inflammatory-related disease, even before or at the beginning of inflammation.

Previous studies have shown that Ras-Related Nuclear Protein (RAN), a member of the RAS superfamily, is a small GTPase and an important oncogene in several cancers. However, its role in multiple myeloma (MM) and its potential contribution to drug resistance remain undetermined. Bioinformatics was employed to analyze differentially expressed genes in MM samples. RT-qPCR and western blotting were utilized for protein transcription and expression analysis. The CCK-8 assay was adopted to evaluate cell proliferation, and in vivo animal experiments were conducted to validate the results. The findings reveal that RAN represents one of the most significantly aberrant genes in MM, with its expression significantly elevated in both MM tissues and cells. Genetic manipulation experiments demonstrated that RAN promotes MM cell proliferation by activating the Wnt/PCP pathway. Concurrently, RAN governs the response of MM cells to the anti-cancer drug bortezomib (BTZ). Knockdown of RAN leads to increased sensitivity to BTZ. Mechanistic studies indicate that RAN influences drug response by regulating the activation of the JNK/c-Jun axis, thereby affecting the therapeutic response of MM cells. In summary, the upregulated expression of RAN in MM leads to BTZ resistance via activation of the Wnt/PCP pathway, potentially serving as a novel therapeutic target for MM.

Triple-negative breast cancer (TNBC) is the most malignant subtype of breast cancer (BC), characterized by limited treatment options and poor clinical outcomes. Aberrant FGFR signaling has been implicated in TNBC; however, the therapeutic potential of targeting FGFRs for TNBC treatment remains unclear. This study investigated the anti-cancer activity of the selective pan-FGFR inhibitor Erdafitinib and its underlying mechanisms using both in vitro and in vivo models. The results demonstrated that Erdafitinib suppressed TNBC tumorigenicity by promoting FGFR1/4 degradation, generating reactive oxygen species (ROS), inducing DNA damage, and ultimately triggering cell death. Mechanistic analyses revealed that Erdafitinib facilitated FGFR1/4 degradation through ubiquitination, enhanced interaction between TRIM25 and FGFR1/4, and subsequent lysosomal degradation. Furthermore, RNA-seq data from the TCGA and GEO databases, along with paired tumor tissues from TNBC patients, indicated that FGFR4 was significantly upregulated in TNBC. Notably, co-knockdown of FGFR1 and FGFR4 induced cytotoxicity in MDA-MB-231 cells, highlighting the therapeutic relevance of FGFR1/4 degradation by Erdafitinib in TNBC. These findings provide novel insights into the mechanisms underlying the anti-cancer efficacy of Erdafitinib, supporting its potential as a promising therapeutic agent for TNBC.

Prostate cancer (PCa), a common urinary malignancy, is the leading cause of mortality and morbidity among men worldwide. Curcuma amada extract has demonstrated antitumor properties in preclinical models of various cancers, however, its mechanisms against prostate cancer remain unclear. The current study aims to investigate the underlying mechanism of C. amada rhizome extract (CARE) in treating PCa through network pharmacology, bioinformatics analysis and in-vitro experiments. UHPLC-QTOF-HRMS/MS detected 16 phytoconstituents in C. amada, with 15 constituents passing drug-likeness criteria. Public databases identified 1,311 CARE and 473 PCa related targets, with 59 overlapping targets. PPI analysis revealed P53, CTNNB1, EGFR, AKT1, ESR1, HIF1A, CCND1, PIK3CA, and BCL2 as hub targets. Further,4-hydroxycinnamic acid, 13-hydroxylabda-8(17),14-dien-18-oic acid, labda-8(17),12-diene-15,16-dial, zederone, zedoarondiol, zerumin A and caffeic acid were identified as core compounds with high degree values. GO and KEGG analysis identified targets primarily associated with apoptosis and PI3K-AKT signalling pathway. Molecular docking confirmed good binding potential of core compounds with key hub targets, while molecular dynamics (MD) simulation validated the stability of these interactions with minimal fluctuations throughout the simulation. Additionally, mRNA expression levels, immune infiltration and genetic alteration of the hub targets were analyzed. CARE significantly inhibited the proliferation of PC-3 cells, induced apoptosis, and caused G2/M phase arrest. In addition, qRT-PCR analysis revealed that CARE was able to suppress mRNA expression of genes involved in the PI3K-AKT signalling pathway. Thus, the study highlights the underlying mechanism of CARE as a promising treatment option for prostate cancer.

Valproate (VPA), a drug used to treat neurological disorders such as epilepsy, bipolar disorder, and migraines, induces inositol depletion in the brain by preventing its synthesis, which is a hypothesized therapeutic mechanism for mood stabilizing drugs. However, the mechanism by which this occurs is not known. VPA treatment reduces activity of the enzyme that catalyzes the rate-limiting step of inositol synthesis, myo-inositol-3-phosphate synthase (MIPS). Utilizing the yeast model, we report that VPA induces repression of the MIPS-encoding gene INO1 by increasing nuclear translocation of the transcriptional repressor Opi1. In addition, VPA rapidly inhibits TORC1, an activator of INO1 expression, and increased TORC1 activity partially rescues expression of INO1. VPA also rapidly activates Snf1, a known regulator of INO1. However, neither TORC1 inhibition nor Snf1 activation is required for repression of INO1 by VPA. We postulate that it is likely that the pleiotropic effects of VPA not only bring about the initial decrease in MIPS protein levels by repressing INO1 transcription but also by preventing recovery of inositol synthesis by downregulating MIPS translation. These independent effects of VPA underlie the complex mechanisms that collectively induce inositol depletion.