BCKDK modification enhances the anticancer efficacy of CAR-T cells by reprogramming branched chain amino acid metabolism.

Altered branched chain amino acids (BCAAs), including leucine, isoleucine, and valine, are frequently observed in patients with advanced cancer. We evaluated the efficacy of chimeric antigen receptor (CAR) T cell-mediated cancer cell lysis potential in the immune microenvironment of BCAA supplementation and deletion. BCAA supplementation increased cancer cell killing percentage, while accelerating BCAA catabolism and decreasing BCAA transporter decreased cancer cell lysis efficacy. We thus designed BCKDK engineering CAR T cells for the reprogramming of BCAA metabolism in the tumor microenvironment based on the genotype and phenotype modification. BCKDK overexpression (OE) in CAR-T cells significantly improved cancer cell lysis, while BCKDK knockout (KO) resulted in inferior lysis potential. In an in vivo experiment, BCKDK-OE CAR-T cell treatment significantly prolonged the survival of mice bearing NALM6-GL cancer cells, with the differentiation of central memory cells and an increasing proportion of CAR-T cells in the peripheral circulation. BCKDK-KO CAR-T cell treatment resulted in shorter survival and a decreasing percentage of CAR-T cells in the peripheral circulation. In conclusion, BCKDK-engineered CAR-T cells exert a distinct phenotype for superior anticancer efficiency.

High expression of DEC2 distinguishes chondroblastic osteosarcoma and promotes tumour growth by activating the VEGFC/VEGFR2 signalling pathway.

Osteosarcoma (OS) is the most common primary malignant bone tumour in children and young adults. Account for 80% of all OS cases, conventional OS are characterized by the presence of osteoblastic, chondroblastic and fibroblastic cell types. Despite this heterogeneity, therapeutic treatment and prognosis of OS are essentially the same for all OS subtypes. Here, we report that DEC2, a transcriptional repressor, is expressed at higher levels in chondroblastic OS compared with osteoblastic OS. This difference suggests that DEC2 is disproportionately involved in the progression of chondroblastic OS, and thus, DEC2 may represent a possible molecular target for treating this type of OS. In the human chondroblastic-like OS cell line MNNG/HOS, we found that overexpression of DEC2 affects the proliferation of the cells by activating the VEGFC/VEGFR2 signalling pathway. Enhanced expression of DEC2 increased VEGFR2 expression, as well as increased the phosphorylation levels at sites Y951 and Y1175 of VEGFR2. On the one hand, activation of VEGFR2Y1175 enhanced cell proliferation through VEGFR2Y1175-PLCγ1-PKC-SPHK-MEK-ERK signalling. On the other hand, activation of VEGFR2Y951 decreased mitochondria-dependent apoptosis rate through VEGFR2Y951-VARP-PI3K-AKT signalling. Activation of these two signalling pathways resulted in enhanced progression of chondroblastic OS. In conclusion, DEC2 plays a pivotal role in cell proliferation and apoptosis-resistance in chondroblastic OS via the VEGFC/VEGFR2 signalling pathway. These findings lay the groundwork for developing focused treatments that target specific types of OS.

The effects of methylphenidate and atomoxetine on Drosophila brain at single-cell resolution and potential drug repurposing for ADHD treatment.

The stimulant methylphenidate (MPH) and the non-stimulant atomoxetine (ATX) are frequently used for the treatment of attention-deficit/hyperactivity disorder (ADHD); however, the function of these drugs in different types of brain cells and their effects on related genes remain largely unknown. To address these questions, we built a pipeline for the simultaneous examination of the activity behavior and transcriptional responses of Drosophila melanogaster at single-cell resolution following drug treatment. We selected the Drosophila with significantly increased locomotor activities (hyperactivity-like behavior) following the administration of each drug in comparison with the control (same food as the drug-treated groups with 5% sucrose, yeast, and blue food dye solution) using EasyFlyTracker. Subsequently, single cell RNA sequencing (scRNASEQ) was used to capture the transcriptome of 82,917 cells, unsupervised clustering analysis of which yielded 28 primary cell clusters representing the major cell types in adult Drosophila brain. Indeed, both neuronal and glial cells responded to MPH and ATX. Further analysis of differentially expressed genes (DEGs) revealed distinct transcriptional changes associated with these two drugs, such as two well-studied dopamine receptor genes (Dop2R and DopEcR) were responsive to MPH but not to ATX at their optimal doses, in addition to genes involved in dopamine metabolism pathways such as Syt1, Sytalpha, Syt7, and Ih in different cell types. More importantly, MPH also suppressed the expression of genes encoding other neurotransmitter receptors and synaptic signaling molecules in many cell types, especially those for Glu and GABA, while the responsive effects of ATX were much weaker. In addition to monoaminergic neuronal transmitters, other neurotransmitters have also shown a similar pattern with respect to a stronger effect associated with MPH than with ATX. Moreover, we identified four distinct glial cell subtypes responsive to the two drugs and detected a greater number of differentially expressed genes associated with ensheathing and astrocyte-like glia. Furthermore, our study provides a rich resource of candidate target genes, supported by drug set enrichment analysis (P = 2.10E-4; hypergeometric test), for the further exploration of drug repurposing. The whole list of candidates can be found at ADHDrug ( http://adhdrug.cibr.ac.cn/ ). In conclusion, we propose a fast and cost-efficient pipeline to explore the underlying molecular mechanisms of ADHD drug treatment in Drosophila brain at single-cell resolution, which may further facilitate drug repurposing applications.

Identification of putative allosteric inhibitors of BCKDK via virtual screening and biological evaluation.

Abnormal accumulation of branched-chain amino acids (BCAAs) can lead to metabolic diseases and cancers. Branched-chain α-keto acid dehydrogenase kinase (BCKDK) is a key negative regulator of BCAA catabolism, and targeting BCKDK provides a promising therapeutic approach for diseases caused by BCAA accumulation. Here, we screened PPHN and POAB as novel putative allosteric inhibitors by integrating allosteric binding site prediction, large-scale ligand database virtual screening, and bioactivity evaluation assays. Both of them showed a high binding affinity to BCKDK, with Kd values of 3.9 µM and 1.86 µM, respectively. In vivo experiments, the inhibitors demonstrated superior kinase inhibitory activity and notable antiproliferative and proapoptotic effects on diverse cancer cells. Finally, bulk RNA-seq analysis revealed that PPHN and POAB suppressed cell growth through a range of signalling pathways. Taken together, our findings highlight two novel BCKDK inhibitors as potent therapeutic candidates for metabolic diseases and cancers associated with BCAA dysfunctional metabolism.

D2HGDH-mediated D2HG catabolism enhances the anti-tumor activities of CAR-T cells in an immunosuppressive microenvironment.

The effect of immunotherapy is limited by oncometabolite D-2-hydroxyglutarate (D2HG). D2HGDH is an inducible enzyme that converts D2HG into the endogenous metabolite 2-oxoglutarate. We aimed to evaluate the impairment of CD8 T lymphocyte function in the high-D2HG environment and to explore the phenotypic features and anti-tumor effect of D2HGDH-modified CAR-T cells. D2HG treatment inhibited the expansion of human CD8 T lymphocytes and CAR-T cells, increased their glucose uptake, suppressed effector cytokine production, and decreased the central memory cell proportion. D2HGDH-modified CAR-T cells displayed distinct phenotypes, as D2HGDH knock-out (KO) CAR-T cells exhibited a significant decrease in central memory cell differentiation and intracellular cytokine production, while D2HGDH over-expression (OE) CAR-T cells showed predominant killing efficacy against NALM6 cancer cells in high-D2HG medium. In vivo xenograft experiments confirmed that D2HGDH-OE CAR-T cells decreased serum D2HG and improved the overall survival of mice bearing NALM6 cancer cells with mutation IDH1. Our findings demonstrated that the immunosuppressive effect of D2HG and distinct phenotype of D2HGDH modified CAR-T cells. D2HGDH-OE CAR-T cells can take advantage of the catabolism of D2HG to foster T cell expansion, function, and anti-tumor effectiveness.

Cucurbitacin IIb attenuates cancer cachexia induced skeletal muscle atrophy by regulating the IL-6/STAT3/FoxO signaling pathway.

The main features of cancer cachexia include skeletal muscle atrophy, which can significantly reduce the quality of life of patients. Clinical treatment of cancer cachexia is mainly based on nutritional therapy and physical exercise; medication only improves appetite but does not reverse the symptoms of skeletal muscle wasting. In this work, we systematically studied the underlying molecular mechanisms by which cucurbitacin IIb (CuIIb) ameliorates muscle wasting in cancer cachexia both in vitro and in vivo. CuIIb significantly ameliorated the chief features of cancer cachexia in vivo, alleviating weight loss, food intake, muscle wasting, adipose tissue depletion, and organ weight reductions. In vitro, CuIIb (10 and 20 µM) dose-dependently attenuated conditioned medium (CM)-induced C2C12 myotube atrophy. Collectively, our findings demonstrated that CuIIb prevented the upregulation of the E3 ubiquitin ligase muscle atrophy Fbox protein (MAFbx), myosin heavy chain (MyHC), and myogenin (MyoG) and impacted protein synthesis and degradation. In addition, CuIIb decreased the phosphorylation of Tyr705 in STAT3 by regulating the IL-6/STAT3/FoxO pathway to reduce skeletal muscle atrophy in cancer cachexia.

Scutellarin Attenuates Doxorubicin-Induced Cardiotoxicity by Inhibiting Myocardial Fibrosis, Apoptosis and Autophagy in Rats.

The anthracycline antibiotic doxorubicin (DOX) is an effective anticancer agent, but its clinical use is limited by dose-dependent cardiotoxicity. Scutellarin (SCU), a natural polyphenolic flavonoid, is used as a cardioprotective agent for infarction and ischemia-reperfusion injury. This study investigated the beneficial effect of SCU on DOX-induced chronic cardiotoxicity. Rats were injected intraperitoneally (i. p.) with DOX (2.5 mg/kg) twice a week for four weeks and then allowed to rest for two weeks to establish the chronic cardiotoxicity animal model. A dose of 10 mg/kg/day SCU was injected i. p. daily for six weeks to attenuate cardiotoxicity. SCU attenuated DOX-induced elevated oxidative stress levels and cardiac troponin T (cTnT), decreased left ventricular ejection fraction (LVEF) and fractional shortening (LVFS), elevated isovolumic relaxation time (IVRT), electrophysiology and histopathological alterations. In addition, SCU significantly attenuated DOX-induced cardiac fibrosis and reduced extracellular matrix (ECM) accumulation by inhibiting the TGF-ß1/Smad2 signaling pathway. Furthermore, SCU also prevented against DOX-induced apoptosis and autophagy as evidenced by upregulation of Bcl-2, downregulation of Bax and cleaved caspase-3, inhibited the AMPK/mTOR pathway. These results revealed that the cardioprotective effect of SCU on DOX-induced chronic cardiotoxicity may be attributed to reducing oxidative stress, myocardial fibrosis, apoptosis and autophagy.

Daidzein alleviates cisplatin-induced muscle atrophy by regulating Glut4/AMPK/FoxO pathway.

Cisplatin (DDP) is widely used in cancer treatment, but DDP can cause skeletal muscle atrophy and cachexia. This study explored the effect and mechanism of daidzein (DAI) in reducing DDP-induced skeletal muscle atrophy and cachexia in vivo and in vitro. DAI alleviated the weight, food intake, muscle, adipose tissue, kidney weight and forelimb grip of LLC tumour-bearing mice after DDP treatment, and did not affect the antitumour effect of DDP. DAI can reduce the decrease of the cross-sectional area of skeletal muscle fibre-induced by DDP and prevent the change of fibre type proportion. In skeletal muscle, it can inhibit Glut4/AMPK/FoxO pathway, down-regulate the expression of atrogin1 and MuRF1, and inhibit skeletal muscle protein degradation. In DDP treated C2C12 myotubes, DAI could inhibit Glut4/AMPK/FoxO pathway to reduce myotubes atrophy, while AMPK agonist MK-3903 could reverse the protective effect of DAI. These results suggest that DAI can alleviate DDP-induced skeletal muscle atrophy by downregulating the expression of Atrogin1 and MuRF1 through the regulation of Glut4/AMPK/FoxO pathway.

Imperatorin alleviates cancer cachexia and prevents muscle wasting via directly inhibiting STAT3.

Skeletal muscle wasting is the most remarkable phenotypic feature of cancer cachexia that increases the risk of morbidity and mortality. Imperatorin (IMP), a main bioactive component of Angelica dahurica Radix, has been reported to possess several pharmacological effects including potential anti-colitis, anti-arthritis and anti-tumor activities. In this work, we demonstrated that IMP is a promising agent for the treatment of muscle wasting in cancer cachexia. IMP (5-20 µM) dose-dependently attenuated TCM-induced C2C12 myotube atrophy and prevented the induction of E3 ubiquitin ligases muscle RING-finger containing protein-1 (MuRF1) and muscle atrophy Fbox protein (Atrogin-1/MAFbx). Moreove, IMP administration significantly improved chief features of cancer cachexia in vivo, with significant prevention of the loss of body weight and deleterious wasting of multiple tissues, including skeletal muscle, fat and kidney and decreased expression of MuRF1 and Atrogin-1 in cachectic muscles. Cellular signaling pathway analysis showed that IMP selectively inhibited the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in vitro and in vivo, and surface plasmon resonance (SPR) affinity experiments further demonstrated IMP bound to STAT3 in a concentration-dependent resonance manner. Molecular docking results revealed that IMP binds to the SH2 domain of STAT3, forming a hydrogen bond interaction with Arg-609, and a Sigma-Pi interaction with Lys-591. Mechanism analysis demonstrated that STAT3 overexpression markedly weakens the improvements of IMP on myotube atrophy and muscle wasting of cancer cachexia, indicating that STAT3 mediated the therapeutic effect of IMP. All these favorable results indicated that IMP is a new potential therapeutic candidate for cancer cachexia.

Germanicol induces selective growth inhibitory effects in human colon HCT-116 and HT29 cancer cells through induction of apoptosis, cell cycle arrest and inhibition of cell migration.

PURPOSE: The main aim of this research was to evaluate the anticancer and apoptotic effects of germanicol - a natural triterpene - in HCT-116 and HT29 human colon cancer cells and deciphering its mode of action by studying its effect on the cell cycle and cell migration. METHODS: Cell cytotoxicity was evaluated by MTT assay, while cell death was assessed by LDH assay. Fluorescence microscopy, using DAPI and acridine orange/ethidium bromide (AO-ETBR), was carried out to evaluate the effect of germanicol on cellular morphology and apoptosis induction. Apoptosis quantification was performed by Annexin V-FITC assay, while cell cycle analysis was performed by flow cytometry using propidium iodide (PI). RESULTS: The results revealed that germanicol showed selective, potent and dose-dependent cytotoxicity in HCT-116 and HT29 human colon cancer cells, while it showed lower cytotoxicity in normal colon cells (human colon fibroblast, CCD-18Co). LDH assay also showed that germanicol induced dose-dependent cell death in HCT-116 and HT29 cells. Fluorescence microscopy revealed that germanicol induced apoptosis via chromatin condensation and DNA damage in HCT-116 colon cancer cells. It also revealed that the percentage of cells with orange and red fluorescence increased when adding a germanicol dose, indicating apoptosis. Germanicol also inhibited cancer cell migration. CONCLUSION: The current findings reveal that germanicol exhibits selective antiproliferative activity against two human colon cancer cells. The normal cell line was less affected by the drug, as compared to the two cancer cell lines, indicating that germanicol will not target normal living cells. The antiproliferative effect was shown to be mediated through the induction of apoptosis and suppression of cell migration.

A sensitive and high-throughput LC-MS/MS method for inhibition assay of seven major cytochrome P450s in human liver microsomes using an in vitro cocktail of probe substrates.

A sensitive and high-throughput LC-MS/MS method was established and validated for the simultaneous quantification of seven probe substrate-derived metabolites (cocktail assay) for assessing the in vitro inhibition of cytochrome P450 (CYP) enzymes in pooled human liver microsomes. The metabolites acetaminophen (CYP1A2), hydroxy-bupropion (CYP2B6), n-desethyl-amodiaquine (CYP2C8), 4'-hydroxy-diclofenac (CYP2C9), 4'-hydroxy-mephenytoin (CYP2C19), dextrorphan (CYP2D6) and 1'-hydroxy-midazolam (CYP3A4/5), together with the internal standard verapamil, were eluted on an Agilent 1200 series liquid chromatograph in <7 min. All metabolites were detected by an Agilent 6410B tandem mass spectrometer. The concentration of each probe substrate was selected by substrate inhibition assay that reduced potential substrate interactions. CYP inhibition of seven well-known inhibitors was confirmed by comparing a single probe substrate assay with cocktail assay. The IC50 values of these inhibitors determined on this cocktail assay were highly correlated (R(2) > 0.99 for each individual probe substrate) with those on single assay. The method was selective and showed good accuracy (85.89-113.35%) and between-day (RSD <13.95%) and within-day (RSD <9.90%) precision. The sample incubation extracts were stable at 25 °C for 48 h and after three freeze-thaw cycles. This seven-CYP inhibition cocktail assay significantly increased the efficiency of accurately assessing compounds' potential inhibition of the seven major CYPs in drug development settings.

Baicalin, a component of Scutellaria baicalensis, alleviates anorexia and inhibits skeletal muscle atrophy in experimental cancer cachexia.

Inflammatory responses are key contributors to cancer cachexia and foster a complex cascade of biological outcomes. Baicalin is a natural compound derived from Scutellaria baicalensis that possesses anti-inflammatory properties in many diseases; therefore, the aim of this study was to verify whether baicalin could ameliorate cachexia in a CT26 adenocarcinoma-induced model. Tumour-bearing and control mice were injected with CT26 adenocarcinoma cells and phosphate-buffered saline (PBS), respectively, and baicalin was administered intraperitoneally for 15 days. During the study, food intake, body weight, major organ weight, gastrocnemius muscle weight, tibialis muscle weight, epididymal fat weight and serum cytokine levels were measured and evaluated. Additionally, the expression of two E3 ubiquitin ligases and NF-κB pathway proteins were detected by Western blot. The total food intake in tumour-bearing mice receiving baicalin from days 1-16, as well as the average food intake on days 10-16, were less than normal but were significantly higher than in vehicle-treated tumour-bearing mice. Loss of tumour-free body mass in vehicle-treated tumour-bearing mice was significantly increased compared with control mice and tumour-bearing mice receiving baicalin. Serum cytokines, including tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6), were lowered in tumour-bearing mice treated with baicalin. Gastrocnemius muscle, epididymal fat, heart and kidney weight were significantly greater in the baicalin treatment groups compared with the vehicle-treated tumour-bearing mice. In addition, the expression of two E3 ubiquitin ligases, as well as phospho-p65, was significantly downregulated, whereas the expression of IκBα was up-regulated in tumour-bearing mice treated with baicalin, as determined by Western blotting. The present study demonstrates that baicalin effectively ameliorates anorexia by inhibiting cytokine expression and prevents skeletal muscle atrophy most likely by inhibiting activation of NF-κB in an experimental cancer cachexia model, suggesting that baicalin represents a promising natural medicine for treating cancer-induced cachexia.

In vitro inhibitory effects of scutellarin on six human/rat cytochrome P450 enzymes and P-glycoprotein.

Inhibition of cytochrome P450 (CYP) and P-glycoprotein (P-gp) are regarded as the most frequent and clinically important pharmacokinetic causes among the various possible factors for drug-drug interactions. Scutellarin is a flavonoid which is widely used for the treatment of cardiovascular diseases. In this study, the in vitro inhibitory effects of scutellarin on six major human CYPs (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and six rat CYPs (CYP1A2, CYP2C7, CYP2C11, CYP2C79, CYP2D4, and CYP3A2) activities were examined by using liquid chromatography-tandem mass spectrometry. Meanwhile, the inhibitory effects of scutellarin on P-gp activity were examined on a human metastatic malignant melanoma cell line WM-266-4 by calcein-AM fluorometry screening assay. Results demonstrated that scutellarin showed negligible inhibitory effects on the six major CYP isoenzymes in human/rat liver microsomes with almost all of the IC50 values exceeding 100 µM, whereas it showed values of 63.8 µM for CYP2C19 in human liver microsomes, and 63.1 and 85.6 µM for CYP2C7 and CYP2C79 in rat liver microsomes, respectively. Scutellarin also showed weak inhibitory effect on P-gp. In conclusion, this study demonstrates that scutellarin is unlikely to cause any clinically significant herb-drug interactions in humans when co-administered with substrates of the six CYPs (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and P-gp.

The Pan-Cancer Analysis Uncovers the Prognostic and Immunotherapeutic Significance of CD19 as an Immune Marker in Tumor.

Background: The specific cytotoxic effects of anti-CD19 chimeric antigen receptor (CAR) T-cell therapy have led to impressive outcomes in individuals previously treated for B-cell malignancies. However, the specific biological role of CD19(+) target cells, which exert antitumor immunity against some solid tumors, remains to be elucidated. Methods: We collected information regarding the level of CD19 mRNA and protein expression from various databases including The Cancer Genome Atlas (TCGA), Tumor Immune Estimation Resource (TIMER), Genotype-Tissue Expression (GTEx), and Human Protein Atlas (HPA) for both tumor and normal samples. To evaluate the patient's prognosis according to CD19 expression, a Kaplan-Meier (KM) analysis and univariate Cox regression were performed. Furthermore, using the Estimation of Stromal and Immune Cells in Malignant Tumor Tissues Using the Expression Data (ESTIMATE) algorithm, we estimated the ratio of immune cells infiltrating malignant tumor tissues. Afterward, the GSCALite repository was employed to evaluate the vulnerability of tumors expressing CD19 to drugs used in chemotherapy. To validate the results in clinical samples of certain cancer types, immunohistochemistry was then performed. Results: Most tumor types exhibited CD19 expression differently, apart from colon adenocarcinoma (COAD). The early diagnostic value of CD19 has been demonstrated in 9 different tumor types, and the overexpression of CD19 has the potential to extend the survival duration of patients. Multiple tumors showed a positive correlation between CD19 expression and tumor mutation burden (TMB), microsatellite instability (MSI), and ESTIMATE score. Furthermore, a direct association was discovered between the expression of CD19 and the infiltration of immune cells, particularly in cases of breast invasive carcinoma (BRCA). Moreover, CD19 is highly sensitive to a variety of chemotherapy drugs. Conclusion: The study reveals the potential of CD19 as both a predictive biomarker and a target for different cancer immunotherapies.

Network pharmacology and experimental validation to study the potential mechanism of Tongguanteng injection in regulating apoptosis in osteosarcoma.

OBJECTIVE: The main objectives of this study were to identify the active components of Tongguanteng injection (TGT) and investigate the preclinical efficacy and mechanism of TGT on osteosarcoma using a combination of network pharmacology and experimental validation. METHODS: To identify the active constituents and targets of TGT against osteosarcoma using network pharmacology, we constructed a network consisting of an 'active ingredient-disease-target-pathway' and a protein-protein interaction (PPI) network. The target organ network was utilized to investigate the distribution of core targets in tissues. Afterwards, the core targets underwent Gene ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The binding energy between receptors and ligands was compared using molecular docking. In addition, SwissADME was employed to forecast the pharmacokinetic characteristics of the substances. Finally, real-time polymerase chain reaction (RT-PCR), cell proliferation assay, morphological analysis, apoptosis assay, mitochondrial membrane potential (MMP) detection, and Western blotting were utilized to confirm the potential mechanisms of TGT treatment in osteosarcoma cell lines 143B and SAOS2. RESULTS: A total of 54 chemical constituents of TGT and 71 targets associated with osteosarcoma were acquired. Through the molecular docking technology, Tenacigenin B, Marsdekoiside, Taraxasterol, Tenacissoside G, Tenacissoside L, and Tenacissoside J were identified as the primary active components of TGT among the various compounds. Analysis of target organs suggests that TGT may play an anti-osteosarcoma role through immune regulation. The GO and KEGG enrichment analysis revealed that TGT could trigger osteosarcoma cell apoptosis by inhibiting the HIF-1 signalling pathway and modulating PD-1 expression and the PD-1 checkpoint pathway in cancer. SwissADME database predicted that Tenacigenin B and Taraxasterol had the best drug-likeness. In vitro studies also demonstrated that TGT suppressed the activity and induced alterations in the morphology of osteosarcoma cells. It decreased MMP levels, triggered apoptosis by increasing Bax expression and Caspase-3 activity, and decreased Bcl-2 expression, thereby exerting an anti-osteosarcoma effect. In the meantime, RT-PCR tests demonstrated that TGT could control immune response against tumors and hinder the proliferation and spread of cancerous cells by impacting the levels of critical factors, including JUN, HSP90AA1, HDAC1, and CDK1. CONCLUSION: The study accurately anticipated the active components, targets, and pathways of TGT in the management of osteosarcoma. The molecular mechanism of TGT-induced apoptosis in osteosarcoma cells was demonstrated by in vitro experiments. These results provide theoretical and technical support for TGT as a clinical adjuvant drug for osteosarcoma.

Cellular and Molecular Insights into the Divergence of Neural Stem Cells on Matrigel and Poly-l-lysine Interfaces.

Poly-l-lysine (PLL) and Matrigel, both classical coating materials for culture substrates in neural stem cell (NSC) research, present distinct interfaces whose effect on NSC behavior at cellular and molecular levels remains ambiguous. Our investigation reveals intriguing disparities: although both PLL and Matrigel interfaces are hydrophilic and feature amine functional groups, Matrigel stands out with lower stiffness and higher roughness. Based on this diversity, Matrigel surpasses PLL, driving NSC adhesion, migration, and proliferation. Intriguingly, PLL promotes NSC differentiation into astrocytes, whereas Matrigel favors neural differentiation and the physiological maturation of neurons. At the molecular level, Matrigel showcases a wider upregulation of genes linked to NSC behavior. Specifically, it enhances ECM-receptor interaction, activates the YAP transcription factor, and heightens glycerophospholipid metabolism, steering NSC proliferation and neural differentiation. Conversely, PLL upregulates genes associated with glial cell differentiation and amino acid metabolism and elevates various amino acid levels, potentially linked to its support for astrocyte differentiation. These distinct transcriptional and metabolic activities jointly shape the divergent NSC behavior on these substrates. This study significantly advances our understanding of substrate regulation on NSC behavior, offering novel insights into optimizing and targeting the application of these surface coating materials in NSC research.

Dl-3-n-butylphthalide promotes microglial phagocytosis and inhibits microglial inflammation via regulating AGE-RAGE pathway in APP/PS1 mice.

Alzheimer's disease (AD) stands as the most prevalent neurodegenerative condition worldwide, and its correlation with microglial function is notably significant. Dl-3-n-butylphthalide (NBP), derived from the seeds of Apium graveolens L. (Chinese celery), has demonstrated the capacity to diminish Aß levels in the brain tissue of Alzheimer's transgenic mice. Despite this, its connection to neuroinflammation and microglial phagocytosis, along with the specific molecular mechanism involved, remains undefined. In this study, NBP treatment exhibited a substantial improvement in learning deficits observed in AD transgenic mice (APP/PS1 transgenic mice). Furthermore, NBP treatment significantly mitigated the total cerebral Aß plaque deposition. This effect was attributed to the heightened presence of activated microglia surrounding Aß plaques and an increase in microglial phagocytosis of Aß plaques. Transcriptome sequencing analysis unveiled the potential involvement of the AGE (advanced glycation end products) -RAGE (receptor for AGE) signaling pathway in NBP's impact on APP/PS1 mice. Subsequent investigation disclosed a reduction in the secretion of AGEs, RAGE, and proinflammatory factors within the hippocampus and cortex of NBP-treated APP/PS1 mice. In summary, NBP alleviates cognitive impairment by augmenting the number of activated microglia around Aß plaques and ameliorating AGE-RAGE-mediated neuroinflammation. These findings underscore the related mechanism of the crucial neuroprotective roles of microglial phagocytosis and anti-inflammation in NBP treatment for AD, offering a potential therapeutic target for the disease.

Evaluation of the pharmacokinetics and cardiotoxicity of doxorubicin in rat receiving nilotinib.

Doxorubicin (DOX) is a potent chemotherapy drug with a narrow therapeutic window. Nilotinib, a small-molecule Bcr-Abl tyrosine kinase inhibitor, was reported to reverse multidrug resistance (MDR) mediated by P-glycoprotein (P-gp) transmembrane transporters. The present study aimed to investigate nilotinib's affection on the steady-state pharmacokinetics, disposition and cardiotoxicity of DOX. A total of 24 male Sprague-Dawley rats were randomized into four groups (6 in each) and received the following regimens: saline, intravenous DOX (5mg/kg) alone, and DOX co-administrated with either 20 or 40mg/kg nilotinib. Blood was withdrawn at 12 time points till 72h after DOX injection and the concentrations of DOX and its metabolite doxorubicinol (DOXol) in serum and cardiac tissue were assayed by LC-MS-MS method. To determine the cardiotoxicity, the following parameters were investigated: creatine kinase, lactate dehydrogenase, malondialdehyde, and superoxide dismutase. Histopathological examination of heart section was carried out to evaluate the extent of cardiotoxicity after treatments. The results showed that pretreatment of 40mg/kg nilotinib increased the AUC0-t and Cmax of DOX and DOXol. However, their accumulation in cardiac tissue was significantly decreased when compared with the group that received DOX alone. In addition, biochemical and histopathological results showed that 40mg/kg nilotinib reduced the cardiotoxicity induced by DOX administration. In conclusion, co-administration of nilotinib increased serum exposure, but significantly decreased the accumulation of DOX in cardiac tissue. Consistent with in vitro profile, oral dose of 40mg/kg nilotinib significantly decreased the cardiotoxicity of DOX in rat by enhancing P-gp activity in the heart.

ABCB1-overexpressing MG63/DOX cell xenograft model: maintain the MDR phenotype in vivo.

CONTEXT: Multi-drug resistance (MDR) constitutes a major obstacle in the effectiveness of chemotherapy. P-Glycoprotein (P-gp), the product of ABCB1 gene, is a transmembrane transporter that actively pumps cytotoxic drugs out of tumor cells resulting in MDR. OBJECTIVE: We sought to establish an MG63/DOX cell xenografts model that maintained the MDR phenotype and molecular properties in vivo in order to screen for new P-gp inhibitors. MATERIALS AND METHODS: The cytotoxicities of doxorubicin, paclitaxel and cytarabine were evaluated by MTT assays. P-gp activity was measured by rhodamine 123 accumulation using flow cytometry. P-gp expression in MG63/DOX cells and tumor tissues was detected by western blotting and immunohistochemistry. RESULTS: Our results showed that MG63/DOX cells exhibited 70-fold resistance to doxorubicin and more than 150-fold resistance to paclitaxel compared with parent MG63 cells. Furthermore, the ABCB1 inhibitor verapamil (10 µM) effectively reversed doxorubicin and paclitaxel resistance by 90- and 200-fold, respectively. The intracellular accumulation of rhodamine 123 was significantly increased (8.35-fold) in MG63/DOX cell, as compared to MG63 cells, in the presence of 10 µM verapamil. MG63/DOX tumor chunk xenografts had a high formation rate (88%). Finally, we found that the ABCB1 gene was overexpressed in different generations of solid tumors. DISCUSSION AND CONCLUSION: These data demonstrated that MG63/DOX tumor chunk subculture in vivo retained their molecular properties. This model could serve as a convenient system for the preclinical investigation of drug combinations and the screening of new agents to reverse ABCB1-mediated MDR.

Costunolide alleviated DDC induced ductular reaction and inflammatory response in murine model of cholestatic liver disease.

Purpose: Cholestatic liver diseases are groups of hepatobiliary diseases without curative drug-based therapy options. Regulation of bile acid (BA) metabolism, hepatoperiductal fibrosis, and inflammatory response indicated present novel methods for the treatment of cholestatic liver disease. Costunolide (COS) from herb Saussurea lappa exerts a pharmacological effect of regulation of BA metabolism, liver fbrosis and inflammatory response. The present study aimed to clarify the pharmacodynamic effects of COS against the murine model of cholestatic liver disease. Methods: We established a murine model of cholestatic liver disease through chronic feeding of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet for 28 days. Two independent in vivo experiments were designed to reveal the pharmacological effect of COS against cholestatic liver disease. In the first experiment, two dosages of COS (10 and 30 mg/kg) were intraperitoneally injected into model mice daily for 14 days. In the second experiment, high dosage of COS (30 mg/kg) was intraperitoneally injected into control and model mice daily for 28 days. Results: In the evaluation of the hepatoprotective effect of COS, COS showed dosage-dependent improvement of cholestatic liver disease, including ductular reaction, hepatoperiductal fibrosis, and inflammatory response. The mechanism of COS-mediated hepatoprotective effects mainly relies on the regulation of BA metabolism, and the inflammatory response. DDC diet feed induced hepatic BA metabolism, transport and circulation dysfunction. COS treatment not only regulated the BA metabolism and transport gene, but also reprogrammed hepatic primary and secondary BA concentrations. DDC induced hepatic infiltrated monocytes derived macrophages and lymphocytes were inhibited, while Kupffer cells were preserved by COS treatment. The liver elevating inflammatory cytokines of DDC diet feed were alleviated by COS. Moreover, high dosage of 30 mg/kg COS treatment for 28 days resulted in no significant serological changes and no obvious hepatic histopathological changes when compared with control mice. Conclusion: COS protected against DDC diet feeding-induced cholestatic liver disease since COS regulated BA metabolism, ductular reaction, hepatoperiductal fibrosis and inflammatory response. COS is suggested as a potential natural product for the treatment of cholestatic liver disease.