Triple‑negative breast cancer (TNBC) is a lethal subtype of breast cancer with a poor prognosis and limited existing treatment options. The immune checkpoint inhibitor, anti‑programmed death ligand 1 (PD‑L1), has recently emerged as a promising alternative in treating TNBC. PD‑L1 is critical in tumor immune evasion and is therefore a key target for cancer immunotherapy. Although anti‑PD‑L1 therapy is effective in breast cancer based on clinical trials, the relationship between PD‑L1 expression levels and treatment response remains unclear. To investigate this, a 4T1 breast cancer cell line that stably overexpressed PD‑L1 was established and was used to create a tumor model in mice. Mice were treated with anti‑PD‑L1 antibodies, and tumor growth was compared between the control and treated groups. PD‑L1 overexpressing tumors did not exhibit an antitumor response to anti‑PD‑L1 therapy compared with the control tumors. Additionally, immune cell infiltration and activation were significantly altered, as shown by immunohistochemical staining and bulk RNA sequencing. In PD‑L1‑overexpressing tumors that did not respond to treatment, immune cell markers and antitumor immune pathways were downregulated. These results demonstrated that excessive PD‑L1 expression creates an immunosuppressive tumor microenvironment, which impairs the efficacy of anti‑PD‑L1 therapy. The present study suggests that excessive PD‑L1 expression reduces the effectiveness of antitumor immunotherapy, and that PD‑L 1 expression levels are essential in predicting the response to antitumor immunotherapy.

Andrographolide, fucoidan, or a combination of both compounds were evaluated to determine their effects on the antiviral response in the Atlantic salmon macrophage-like cell line (SHK-1) infected with infectious pancreatic necrosis virus (IPNV). We assessed the transcript expression levels of key molecules involved in the interferon (IFN)-dependent antiviral response, as well as the viral load in cells treated with these compounds. In non-infected cells, incubation with either fucoidan, andrographolide, or a mixture of both resulted in an increase in the transcript expression of IFNα1 and various interferon-stimulated genes (ISGs). In IPNV-infected cells, treatment with either fucoidan or andrographolide separately did not significantly enhance the antiviral response compared to that of infected cells that had not previously been treated with these compounds. In contrast, the combination of andrographolide and fucoidan led to a marked increase in the transcript expression of viperin and a significant reduction in viral load. Overall, combining andrographolide and fucoidan resulted in a greater reduction in IPNV viral load in infected cells than that noted when the compounds were administered individually. Our findings suggest that pre-incubation with this mixture promotes the establishment of a protective antiviral state against IPNV, likely mediated by an IFN-dependent response.

Breast cancer is a heterogeneous disease, and treatment resistance remains a critical challenge. Claudin-8 (CLDN8), a tight junction protein, has emerged as a potential indicator of therapeutic response and prognosis in breast cancer patients. In this study, we evaluated CLDN8 as a predictive biomarker and a potential therapeutic target. We analyzed CLDN8 gene expression in breast cancer patient cohorts to assess its association with clinical outcomes and response to therapy. We also established breast cancer cell models with altered CLDN8 expression to examine its effects on cell behavior and drug sensitivity. High CLDN8 expression was significantly associated with improved disease-free survival, particularly in estrogen receptor-negative patients (p = 0.007), suggesting a favorable prognostic role. Notably, tumors with elevated CLDN8 showed better outcomes in patients treated with surgery alone or endocrine therapy, whereas in those receiving chemotherapy (including neoadjuvant) or anti-HER2 therapy, high CLDN8 levels were paradoxically linked to poorer survival and therapy resistance. In vitro, CLDN8 knockdown reduced sensitivity to endocrine treatments, HER2-targeted agents, and chemotherapeutic drugs, mirroring clinical patterns. In conclusion, our findings identify CLDN8 as an important prognostic factor in breast cancer and as a novel predictor of treatment response. These results underscore the potential utility of CLDN8 status in guiding personalized therapy and highlight CLDN8 as a candidate target for overcoming treatment resistance in breast cancer.

The retinal pigment epithelium (RPE) plays a crucial role in maintaining retinal homeostasis, and dysregulation of the transforming growth factor-beta (TGF-β) signaling pathways contributes to retinal fibrosis and inflammatory diseases, including proliferative vitreoretinopathy (PVR). Tacrolimus (FK506), an immunosuppressant, has shown potential antifibrotic properties, but its effects on TGF-β-related genes and microRNAs (miRNAs) in RPE cells remain unclear. Human RPE (H-RPE) cells were treated with lipopolysaccharide (LPS) to induce inflammation and subsequently exposed to tacrolimus. Gene and miRNA expression profiling related to TGF-β signaling pathways were conducted using microarrays, followed by Quantitative Reverse-Transcription Polymerase Chain Reaction (RT-qPCR) validation. Protein levels were assessed via enzyme-linked immunosorbent assay (ELISA), and interactions were analyzed using STRING database network analysis. Tacrolimus modulated key components of the TGF-β pathway, upregulating TGF-β2, TGF-β3, SMAD2, and SMAD4 while downregulating TGF-βR1 and SMAD7. JAK/STAT and MAPK pathways were also affected, indicating broad regulatory effects. miRNA profiling identified hsa-miR-200a-3p, hsa-miR-589-3p, hsa-miR-21, and hsa-miR-27a-5p as key regulators. STRING analysis confirmed strong functional interactions within the TGF-β network. In conclusion, tacrolimus modulates both canonical (upregulation of SMAD2/4 and downregulation of SMAD7) and non-canonical (JAK/STAT and MAPK) TGF-β signaling pathways in LPS-stimulated RPE cells. These changes collectively suggest a dual anti-inflammatory and anti-fibrotic effect. The increased TGF-β2 and decreased SMAD7 levels, alongside altered miRNA expression (e.g., downregulation of miR-200a-3p), indicate that tacrolimus may inhibit key profibrotic mechanisms underlying PVR. These findings support the potential therapeutic repurposing of tacrolimus in PVR and warrant further in vivo validation.

Arsenic, a potent metalloid contaminant of drinking water, is known for its ability to act as an initiator and modulator of disease in a variety of human tissues. Upon ingestion, arsenic is bio-transformed in the liver into a variety of metabolites, including arsenite. Arsenite permeates the blood-brain barrier (BBB), inducing oxidative stress that can be detrimental to brain neurons. As the primary glial cell at the BBB interface, astrocytes play a pivotal role in detoxifying xenobiotics such as arsenite via the production of the tripeptide antioxidant γ-glutamylcysteine, or glutathione (GSH). In this study, we assessed the mRNA levels of key components of the GSH synthetic pathway in astrocytes exposed to arsenite compared to vehicle controls. These components included xCT [substrate-specific light chain of the substrate importing transporter, system xc- (Sxc-)], glutamate-cysteine ligase [both catalytic (GCLC) and modifying (GCLM) subunits], and glutathione synthetase (GS). Additionally, we analyzed protein levels of some components by Western blotting and evaluated functional activity of Sxc- using a fluorescence-based cystine uptake assay. Finally, we utilized a luminescence-based glutathione assay to determine the intracellular and extracellular GSH content in arsenite-treated cells. Arsenite significantly increased xCT, GCLC, GCLM, and GS mRNA levels, an effect blocked by the transcriptional inhibitor actinomycin D (ActD). A corresponding increase in Sxc- activity was also observed in the arsenite treatment groups, along with significant increases in GCLC and GCLM protein expression. However, no increase in GS protein expression was detected. Finally, arsenite treatment significantly increased extracellular GSH levels, an effect which was also prevented by the inclusion of ActD. Overall, our study provides evidence that arsenite transcriptionally regulates several cellular processes necessary for GSH synthesis in primary cortical astrocyte cultures, thereby contributing to a better understanding of how this environmental toxicant influences antioxidant defenses in the brain. However, these results should be interpreted with caution regarding their applicability to vivo systems.

The NOS1AP gene encodes the nitric oxide synthase 1 adaptor protein (NOS1AP), which binds to neuronal nitric oxide synthase (nNOS) and regulates nitric oxide (NO) production by dissociating nNOS from NMDA receptors (NMDARs). Notably, NOS1AP expression is upregulated upon NMDAR activation; however, there is no available data regarding its production under the receptor inhibition. The NOS1AP gene is also 1 among more than 1000 genes that are presumed to be associated with the development of schizophrenia. Various animal models of this disorder have been developed, some of which are based on the use of NMDAR antagonists such as dizocilpine (MK-801). In this study, we investigated the expression and production of NOS1AP in rats injected with a low dose of dizocilpine (0.1 mg/kg), as well as in SH-SY5Y and HEK293T cell lines treated with varying concentrations of the same compound (10-200 µM). According to our results, neither the expression of the NOS1AP gene nor the production of NOS1AP protein was affected by dizocilpine treatment.

Biochanin A, a naturally occurring isoflavone derived from legumes, possesses anti-inflammatory, estrogenic, and anticancer activities. In this study, we investigated the cytotoxic effects and underlying molecular mechanisms of Biochanin A in acute myeloid leukemia (AML) cell lines, U937 and THP-1, using in vitro cytotoxicity assays, RNA sequencing, and bioinformatic analyses. Biochanin A induced dose-dependent apoptosis, as evidenced by caspase-7 activation and PARP1 cleavage. Over-representation analysis (ORA) revealed that differentially expressed genes (DEGs) were significantly enriched in pathways related to inflammatory responses, DNA replication, and cell cycle regulation. Gene set enrichment analysis (GSEA) further confirmed the upregulation of apoptosis- and inflammation-related pathways and the downregulation of MYC targets, cholesterol biosynthesis, and G2/M checkpoint gene sets. RT-qPCR analysis demonstrated that Biochanin A downregulated oncogenes such as RUNX1, BCL2, and MYC while upregulating CHOP (GADD153), CDKN1A (p21), and SQSTM1 (p62), contributing to apoptosis and cell cycle arrest across both cell lines. Notably, Biochanin A downregulated PLK1 and UHRF1 in THP-1 cells, indicating a disruption of mitotic progression and epigenetic regulation. In contrast, in U937 cells, Biochanin A upregulated TXNIP and downregulated CCND2, highlighting the involvement of oxidative stress and G1/S cell cycle arrest. These findings support the potential of Biochanin A as a promising therapeutic candidate for AML through both shared and distinct regulatory pathways.

The prognostic and predictive role of the human equilibrative nucleoside transporter 1 (hENT-1) has emerged in different cancer types, including intrahepatic cholangiocarcinoma (iCCA), but the mechanisms regulating its expression are poorly understood. Here, we investigated the link between p53 status and hENT-1 regulation in 38 iCCA patients and cell line models; the predictive role of p53 status in response to adjuvant gemcitabine was also investigated. A positive association between mutant p53 cells and hENT-1 expression was observed in iCCA tissue samples; furthermore, patients receiving adjuvant gemcitabine and expressing mutant p53 cells > 4% in tumor tissue had a longer disease-free survival (DFS) than patients expressing mutant p53 cells ≤ 4% (median 18.5 vs. 6 months, p = 0.0229). In iCCA cell line models, transient knockdown of mutant p53 resulted in a decrease in hENT-1 mRNA and protein expression; similarly, restoration of wild-type p53 function induced a significant reduction in hENT-1 mRNA and protein expression. Overall, these findings support a role of p53 status in the regulation of hENT-1 expression, suggesting an opposite effect (activating versus repressive) of mutant and wild-type p53 protein. Furthermore, although the present study should be considered as preliminary, our findings suggest a predictive role of p53 status in iCCA patients treated with gemcitabine, thus deserving future investigations in additional cohorts of cancer patients.

Delta-9-tetrahydrocannabinol (Δ-9-THC or THC), the primary psychoactive constituent of cannabis, can lead to adverse health conditions, including mental health issues, brain impairment, and cardiac and respiratory problems. The amount of THC in cannabis has steadily climbed over the past few decades, with today's cannabis having three times the concentration of THC compared to 25 years ago. Inhalation is a major route of exposure, allowing substances to enter the body via the respiratory tract. THC exposure causes cell death in the airway epithelium; however, the molecular underpinning of THC exposure-induced bronchial epithelial cell death is not clearly understood. To address the mechanisms involved in this process, the present study examined the cell viability, oxidative stress, lipid peroxidation, and transcriptional alterations caused by various concentrations of Δ-9-THC (0, 800, 1000, 1200, and 1500 ng/mL) in a human bronchial epithelial cell line (BEAS-2B) in vitro. Δ-9-THC exposure caused a significant dose-dependent decrease in cell viability after 24 h exposure. Transcriptome analysis showed a distinct dose-dependent response. HIF-1 signaling, ferroptosis, AMPK signaling, and immunogenic pathways were activated by Δ-9-THC-upregulated genes. Glutathione and fatty acid metabolic pathways were significantly altered by Δ-9-THC-dependent downregulated genes. Ingenuity Pathway Analysis (IPA) revealed several top canonical pathways altered by Δ-9-THC exposure, including ferroptosis, NRF-2-mediated oxidative stress response, caveolar-mediated endocytosis (loss of cell adhesion to the substrate), tumor microenvironment, HIF1alpha signaling, and the unfolded protein response pathway. Δ-9-THC-induced cell death was ameliorated by inhibiting the ferroptosis pathway, whereas treatments with ferroptosis agonist exacerbated the cell death process, suggesting that Δ-9-THC-induced bronchial epithelial cell death potentially involves the ferroptosis pathway.

Foxtail millet (Setaria italica L.), a representative C4 species, is recognized for its efficient nutrient utilization and robust abiotic stress responses. However, the molecular mechanisms mediating its tolerance to biotic stresses are poorly understood. In this study, we investigated the root transcriptomic response of foxtail millet to the damage-associated molecular pattern (DAMP), the plant elicitor peptide 1 (Pep1). Transcriptome analysis of Pep1-treated roots identified 401 differentially expressed genes (DEGs), comprising 144 up-regulated and 257 down-regulated genes. Gene Ontology (GO) enrichment analysis revealed a significant enrichment of 'peroxidase activity'. This finding was corroborated by DAB staining, which confirmed H2O2 accumulation, along with elevated malondialdehyde (MDA) levels, collectively indicating oxidative stress. Notably, Pep1 treatment also resulted in a marked up-regulation of the pathogenesis-related protein 1 (PR1) gene in leaves, suggesting the activation of systemic acquired resistance. Together, these results demonstrate that Pep1 triggers substantial transcriptional reprogramming in roots, induces oxidative stress, and activates systemic defense signaling in foxtail millet.

In fibrotic skeletal muscles, excessive extracellular matrix (ECM) deposition is a result of increased activation and decreased apoptosis of myofibroblasts. The aim of this study is to investigate whether treatment with quercetin, kaempferol or capsaicin can reduce the transforming growth factor-beta 1 (TGF-β1)-induced myofibroblast differentiation and fibrotic ECM expression in differentiated C2C12 cells. Two-day-differentiated C2C12 cells were treated with TGF-β1 for 48 h to induce myofibroblast differentiation. Twenty-four hours before (pre-treatment) and for forty-eight hours with (co-treatment) TGF-β1 treatment, cells were exposed to quercetin (25, 50 µM), kaempferol (10, 25, 50 µM) or capsaicin (25, 50 µM). The immunofluorescence intensity of alpha smooth muscle actin (αSMA) and collagen type I/III gene expression were assessed as myofibroblast markers. MyoD immunofluorescence intensity was measured as a myogenic marker. Co-treatment of TGF-β1 with the phytochemicals was most effective, resulting in a decreased number of αSMA-positive cells (all three compounds), decreased collagen type I (kaempferol, capsaicin) and type III (kaempferol) gene expression, and increased MyoD (kaempferol, capsaicin) protein expression compared to TGF-β1 treatment. This study demonstrates that treatment with quercetin, kaempferol or capsaicin can reduce myofibroblast markers. This suggests a possible anti-fibrotic effect of the phytochemicals in skeletal muscle.

To explore the mechanism by which γ-aminobutyric acid (GABA) regulates the response of different salt-sensitive tomato seedlings under salt stress conditions, we used the previously selected salt-sensitive tomato 'M82' and the salt-tolerant introgression line 'IL-7-5-5'. The following three treatments were set up: (1) a normal nutrient solution concentration as the control, (2) a nutrient solution with 200 mmol·L-1 NaCl, and (3) a nutrient solution with 200 mmol·L-1 NaCl and 35 mmol·L-1 GABA. The concentration of the reactive oxygen species metabolism-related compounds and antioxidant enzyme activity in the leaves of tomato seedlings subjected to the different treatments were measured, and transcriptome and metabolome analyses were conducted. After adding GABA, the SOD, POD, and APX activity in the leaves of the 'M82' seedlings significantly increased, while the GR activity significantly decreased. In the 'IL-7-5-5' seedlings, the CAT, APX, and GR activity significantly increased. The combined results from the transcriptome and metabolome analysis in leaves indicated that in 'M82' seedlings, 52 metabolic pathways were enriched, which included plant signal transduction pathways, phenylpropanoid biosynthesis pathways, and amino sugar and nucleotide sugar metabolism pathways. In the salt-tolerant 'IL-7-5-5' seedling leaves, 59 metabolic pathways were enriched, which included plant signal transduction pathways, amino acid biosynthesis pathways, and carbon metabolism pathways. A further analysis revealed that both varieties had a higher number of differentially enriched genes and differential metabolites belonging to the plant hormone signal transduction and amino acid biosynthesis pathways, indicating that GABA enhances the salt tolerance of tomato seedlings by regulating these two mechanisms.

Azvudine (FNC) is a novel cytidine analogue that is widely used in the treatment of infectious diseases such as AIDS and COVID-19. Previous studies have demonstrated its anticancer activity in various cancer cell lines, including non-Hodgkin's lymphomas and lung adenocarcinoma cell lines. However, its effects on hepatocellular carcinoma (HCC) and the underlying mechanisms remain unclear. This study aimed to investigate the anti-epithelial-mesenchymal transition (anti-EMT) activity of FNC and evaluate its potential application in HCC treatment. We found that FNC significantly inhibits the migration of the liver cancer cell line Huh7 by downregulating key EMT markers, such as matrix metalloproteinases (MMPs) and E-cadherin, at both the transcriptional and protein expression levels. Notably, we found that FNC inhibits HEY proteins, particularly HEY1, a transcriptional regulator of the Notch signalling pathway that is overexpressed in approximately 50% of HCC patients. To identify the primary target of FNC, microscale thermophoresis (MST) and molecular dynamics (MD) simulations were performed, revealing that FNC directly binds to Jagged1. This study provides valuable insights into the therapeutic potential of FNC in HCC treatment and elucidates its underlying mechanisms.

Emergent cancer drug resistance and further metastasis can mainly be attributed to altered expression levels and functional activities of multiple genes of cancer cells under chemotherapy. In response to challenge with anticancer drugs, enhanced ceramide glycosylation catalyzed by glucosylceramide synthase (GCS) confers drug resistance and enrichment with cancer stem cells. p53 mutations, which gain function in tumor progression, are prevalently extant in ovarian cancers. Via integrated gene expression assessments, we characterized GCS-responsive genes in ovarian cancer cells treated with dactinomycin. NCI/ADR-RES cells dominantly expressed a p53 mutant (7 aa deleted in exon-5) and displayed anti-apoptosis; however, silencing GCS expression rendered these cells sensitive to dactinomycin-induced apoptosis. Microarray analyses of NCI/ADR-RES and its GCS transfected sublines found that elevated GCS expression or ceramide glycosylation was associated with altered expression of 41 genes, notably coding for ABCB1, FGF2, ALDH1A3, apolipoprotein E, laminin 2, chemokine ligands, and IL6, with cellular resistance to induced apoptosis and enrichment with cancer stem cells, promoting cancer progression. These findings were further corroborated through integrated genomic analyses of ovarian cancer from The Cancer Genome Atlas (TCGA) and cancer resistance to platinum-based chemotherapy. Altogether, our present study indicates that altered ceramide glycosylation can modulate expression of these GCS-responsive genes and alter cancer cell attributes under chemotherapy.

Opioid use induces neurobiological adaptations throughout mesolimbic brain regions, such as the orbitofrontal cortex (OFC), which mediates decision-making and emotional-cognitive regulation. Previously, we showed that a circular RNA (circRNA) species, rno_circGrin2b_011731 (circGrin2b), is upregulated in the OFC of rats following chronic self-administration (SA) of the opioid heroin. circGrin2b is derived from Grin2b, which encodes the regulatory subunit of the glutamate ionotropic NMDA receptor, GluN2B. However, the upstream regulatory mechanisms of circGrin2b biogenesis and the downstream consequences of circGrin2b dysregulation remain unknown. We hypothesized that opioid-induced elevation of circGrin2b is accompanied by regulation of circRNA biogenesis enzymes, and that circGrin2b may sponge microRNAs (miRNAs), as miRNA sponging is a well-described characteristic of circRNAs. To test these hypotheses, we established an in vitro primary cortical cell culture model to examine alterations in circGrin2b expression following exposure to the opioid morphine. We measured mRNA expression of known circRNA splicing factors and observed significant downregulation of Fused in Sarcoma (Fus), a negative regulator of circRNA biogenesis, following 90 min or 24 h of morphine exposure. Downregulation of Fus at 24 h post-morphine was accompanied by upregulation of circGrin2b and downregulation of miR-26b-3p, a predicted miRNA target of circGrin2b. Luciferase reporter assays confirmed interaction of miR-26b-3p with circGrin2b. Finally, we report a significant negative relationship between circGrin2b and miR-26b-3p expression in the OFC of rats following heroin SA. We conclude that regulation of circGrin2b is an opioid-induced neuroadaptation that may impact downstream signaling of miRNA pathways in the frontal cortex.

Aluminum (Al) stress is typical for acidic soils and may affect cereals' yield. Al tolerance in triticale is mostly affected by the aluminum-activated malate transporter (ALMT) gene (7R) and some other QTLs (3R, 5R, and 6R). The trait is heritable in about 36% of cases, indicating that epigenetic factors may impact the phenomenon. This study demonstrates that utilizing different methods to quantify DNA methylation changes induced by Al stress results in detail differences, and the results evaluated should be compared critically. The Common and the basic General approaches are sufficient if general information is needed. The General (extended variant) approach may deliver data on methylation changes affecting symmetric sequence contexts. The markers assigned to DN-CG, DM-CG, and DN-CHG were suggested as the most important in explaining Al tolerance in triticale. Analysis of the maps constructed based on root tips and leaf tissues showed different densities of the epigenetic markers but reflected the comparable patterns of their distribution, supporting the hypothesis that Al stress could be transmitted to other plant tissues due to somatic memory. Methylation changes occur throughout the genome and are not associated with specific genes related to aluminum stress.

Molecular mechanisms responsible for the poor prognosis in patients with intrahepatic cholangiocarcinoma (CCA) are still unknown, but stem cell marker Cluster Differentiation 90 (CD90) has been reported to be associated with a more aggressive cancer phenotype. In this scenario, the TGF-β1 signaling pathway likely has a role as master gene regulator. Aim of the study is to investigate the role of CD90 in iCCA aggressiveness. The molecular profile of HuCCT1/CD90+ and HuCCT1/CD90- cells was obtained through transcriptomic analysis (NGS). Bioinformatic data were confirmed in both cell lines by qRT-PCR and Western blot. Cells were treated with Gemcitabine in monotherapy or in combination with Galunisertib, a selective inhibitor of TGF-βRI, in 2D and 3D models. HuCCT1/CD90+ cells are more proliferative, less migratory, and resistant to Gemcitabine treatment. HuCCT1/CD90+ cells also express lower levels of TGF-β1 compared to /CD90- cell lines. Finally, HuCCT1/CD90+ cells are resistant to Gemcitabine, while the combination of Gemcitabine and Galunisertib displays a synergistic effect on HuCCT1/CD90+ cell proliferation. These results underline that CD90-induced Gemcitabine resistance can be overcome by adding a TGFβ1 inhibitor such as Galunisertib, thereby moving further toward a precision medicine approach in patients with iCCA.

Ovarian cancer is a deadly gynecological malignancy often diagnosed at an advanced stage, with few treatment options available. Polyphyllin I (PPI), the primary active ingredient of Paris polyphylla, has shown excellent anti-cancer activity against various cancers. However, studies on PPI use and ovarian cancer are limited, and the potential mechanisms remain unclear. This study explored the effects of anti-ovarian cancer and the potential mechanisms of action of PPI. Methods such as MTT, colony formation, three-dimensional spheroid formation, flow cytometry, and western blotting were employed to assess the anti-proliferative effects of PPI on different ovarian cancer cell lines. RNA sequencing was used to discover potential mechanisms by evaluating the effects of PPI on transcriptional signatures and identifying differences in gene expression patterns. The results indicate that PPI demonstrated dose- and time-dependent anti-proliferative effects on ovarian cancer cell lines. PPI also up-regulated 1518 genes and down-regulated 800 genes. The genes showed enrichment in the "apoptosis" gene sets, while exhibiting negative enrichment in the "cell cycle" and "Myc signaling pathway" gene sets. PPI caused cell cycle arrest at the G0/G1 phase by increasing the expression of p21 and p27 and inhibiting the activity of the CDK2/cyclin E complex. PPI triggered cell apoptosis, which was associated with elevated cellular reactive oxygen species and mitochondrial membrane potential depolarization. Finally, PPI reduced c-Myc protein levels and affected mRNA levels of c-Myc pathway-related genes. PPI effectively suppressed ovarian cancer cell proliferation, indicating its potential as a treatment option for ovarian cancer.

Cocaine use disorder is characterized by persistent drug-seeking behavior and a high risk of relapse, driven in part by lasting molecular and circuit adaptations in the nucleus accumbens. To explore the transcriptomic changes underlying these alterations, we employed fluorescence-activated nucleus sorting coupled with single-nucleus RNA sequencing to analyze D1 and D2 medium spiny neurons in this brain region of male mice subjected to acute cocaine exposure or to prolonged withdrawal from repeated cocaine exposure without or with an acute cocaine rechallenge. This approach allowed us to precisely delineate and contrast transcriptionally distinct neuronal subpopulations─or ensembles─across various treatment conditions. We identified significant heterogeneity within both D1 and D2 MSNs, revealing distinct clusters with unique transcriptional profiles. Notably, we identified a discrete D1 MSN population characterized by the upregulation of immediate early genes, as well as another group of D1 MSNs linked to prolonged withdrawal, uncovering novel regulators of withdrawal-related transcriptome dynamics. Our findings provide a high-resolution transcriptomic map of D1 and D2 MSNs, illustrating the dynamic changes induced by cocaine exposure and withdrawal. These insights into the molecular mechanisms underlying cocaine use disorder highlight potential targets for therapeutic intervention aimed at preventing relapse.

Cannabis is among the most used illicit substances in the world, and approximately 10% of regular cannabis users are estimated to be susceptible to developing cannabis use disorder (CUD). We examined the effect of different concentrations of delta-9-tetrahydrocannabinol (THC) on the epigenetic DNA modifiers DNA methyltransferases (DNMTs) and ten-eleven translocation enzymes (TETs); cannabinoid CB1 and CB2 receptors; and the cytokines IL-1β, IL-6, IL-10, and TNF-α. We used two in vitro study designs on human peripheral blood mononuclear cells (PBMCs) collected from healthy donors: (a) repeated THC incubations and (b) repeated THC incubations followed by an "abstinence" period and a THC challenge incubation. We observed no significant effects on DNMTs and TETs mRNA levels, enzymatic activity, or CB1 and CB2 mRNA levels at an average THC concentration (50 ng/ml, n = 8 donors). However, repeated incubations at a high THC concentration (200 ng/ml, n = 16 donors) significantly downregulated DNMTs and upregulated TETs, CB1, and CB2 mRNA levels. Both THC concentrations upregulated the gene expression of IL-1β, IL-6, and IL-10, but had no effect on TNF-α gene expression. At the genome-wide level, repeated THC incubations resulted in a significant number of differentially hydroxymethylated genes being hyperhydroxymethylated. An additional THC challenge shifted the hyperhydroxymethylated state to hypohydroxymethylation. The genes with the strongest associations with THC exposure were found to be functionally significant for various signaling pathways. These findings suggest that repeated incubations with high concentrations of THC may affect the expression of genes critical for the development of CUD through aberrant demethylation.