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Journal of Experimental & Clinical... May 2022Previous in vitro hepatocyte differentiation model showed that TROY was specifically expressed in liver progenitor cells and a small proportion of hepatocellular...
BACKGROUND
Previous in vitro hepatocyte differentiation model showed that TROY was specifically expressed in liver progenitor cells and a small proportion of hepatocellular carcinoma cells, suggesting that TROY may participate in hepatocellular carcinoma (HCC) stemness regulation. Here, we aim to investigate the role and mechanism of TROY in HCC pathogenesis.
METHOD
Bioinformatics analysis of the TCGA dataset has been used to identify the function and mechanism of TROY. Spheroid, apoptosis, and ALDH assay were performed to evaluate the stemness functions. Validation of the downstream pathway was based on Western blot, co-immunoprecipitation, and double immunofluorescence.
RESULTS
HCC tissue microarray study found that a high frequency of TROY-positive cells was detected in 53/130 (40.8%) of HCC cases, which was significantly associated with poor prognosis and tumor metastasis. Functional studies revealed that TROY could promote self-renewal, drug resistance, tumorigenicity, and metastasis of HCC cells. Mechanism study found that TROY could interact with PI3K subunit p85α, inducing its polyubiquitylation and degradation. The degradation of p85α subsequently activate PI3K/AKT/TBX3 signaling and upregulated pluripotent genes expression including SOX2, NANOG, and OCT4, and promoted EMT in HCC cells. Interestingly, immune cell infiltration analysis found that upregulation of TROY in HCC tissues was induced by TGF-β1 secreted from CAFs. PI3K inhibitor wortmannin could effectively impair tumor stemness to sorafenib.
CONCLUSION
We demonstrated that TROY is an HCC CSC marker and plays an important role in HCC stemness regulation. Targeting TROY-positive CSCs with PI3K inhibitor wortmannin combined with chemo- or targeted drugs might be a novel therapeutic strategy for HCC patients.
Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Class Ia Phosphatidylinositol 3-Kinase; Humans; Liver Neoplasms; Molecular Targeted Therapy; Neoplastic Stem Cells; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Receptors, Tumor Necrosis Factor; Signal Transduction; T-Box Domain Proteins; Wortmannin
PubMed: 35610614
DOI: 10.1186/s13046-022-02401-6 -
Biomolecules Jun 2022HSPA1A is a molecular chaperone that regulates the survival of stressed and cancer cells. In addition to its cytosolic pro-survival functions, HSPA1A also localizes and...
HSPA1A is a molecular chaperone that regulates the survival of stressed and cancer cells. In addition to its cytosolic pro-survival functions, HSPA1A also localizes and embeds in the plasma membrane (PM) of stressed and tumor cells. Membrane-associated HSPA1A exerts immunomodulatory functions and renders tumors resistant to standard therapies. Therefore, understanding and manipulating HSPA1A's surface presentation is a promising therapeutic. However, HSPA1A's pathway to the cell surface remains enigmatic because this protein lacks known membrane localization signals. Considering that HSPA1A binds to lipids, like phosphatidylserine (PS) and monophosphorylated phosphoinositides (PIPs), we hypothesized that this interaction regulates HSPA1A's PM localization and anchorage. To test this hypothesis, we subjected human cell lines to heat shock, depleted specific lipid targets, and quantified HSPA1A's PM localization using confocal microscopy and cell surface biotinylation. These experiments revealed that co-transfection of HSPA1A with lipid-biosensors masking PI(4)P and PI(3)P significantly reduced HSPA1A's heat-induced surface presentation. Next, we manipulated the cellular lipid content using ionomycin, phenyl arsine oxide (PAO), GSK-A1, and wortmannin. These experiments revealed that HSPA1A's PM localization was unaffected by ionomycin but was significantly reduced by PAO, GSK-A1, and wortmannin, corroborating the findings obtained by the co-transfection experiments. We verified these results by selectively depleting PI(4)P and PI(4,5)P using a rapamycin-induced phosphatase system. Our findings strongly support the notion that HSPA1A's surface presentation is a multifaceted lipid-driven phenomenon controlled by the binding of the chaperone to specific endosomal and PM lipids.
Topics: Cell Membrane; HSP70 Heat-Shock Proteins; Humans; Ionomycin; Phosphatidylinositol Phosphates; Wortmannin
PubMed: 35740982
DOI: 10.3390/biom12060856 -
Journal of Natural Products Mar 2019In the course of our studies of coprophilous fungi as sources of antifungal agents, a strain of an undescribed species in the genus Niesslia (TTI-0426) was isolated from...
In the course of our studies of coprophilous fungi as sources of antifungal agents, a strain of an undescribed species in the genus Niesslia (TTI-0426) was isolated from horse dung collected in Texas. An extract from fermentation cultures of this strain afforded two new antifungal wortmannin derivatives, wortmannins C and D (1 and 2), as well as four additional new related compounds, wortmannines B1-B4 (3-6), containing an unusual ring system. The structures of these metabolites were established mainly by analysis of HRESIMS and 2D NMR data. Relative configurations were assigned using NOESY data, and the structure assignments were supported by NMR comparison with similar compounds. Wortmannins C and D showed activity against Cryptococcus neoformans and Candida albicans in disk assays, but low MIC potency observed for 1 was suggested to be due in part to efflux processes on the basis of assay results for a Schizosaccharomyces pombe efflux mutant in comparison to wild-type.
Topics: Candida albicans; Cryptococcus neoformans; Fermentation; Hypocreales; Microbial Sensitivity Tests; Molecular Structure; Schizosaccharomyces; Spectrum Analysis; Wortmannin
PubMed: 30844268
DOI: 10.1021/acs.jnatprod.8b00923 -
Medicinal Research Reviews Sep 2015Although fungi produce highly structurally diverse metabolites, many of which have served as excellent sources of pharmaceuticals, no fungi-derived agent has been... (Review)
Review
Although fungi produce highly structurally diverse metabolites, many of which have served as excellent sources of pharmaceuticals, no fungi-derived agent has been approved as a cancer drug so far. This is despite a tremendous amount of research being aimed at the identification of fungal metabolites with promising anticancer activities. This review discusses the results of clinical testing of fungal metabolites and their synthetic derivatives, with the goal to evaluate how far we are from an approved cancer drug of fungal origin. Also, because in vivo studies in animal models are predictive of the efficacy and toxicity of a given compound in a clinical situation, literature describing animal cancer testing of compounds of fungal origin is reviewed as well. Agents showing the potential to advance to clinical trials are also identified. Finally, the technological challenges involved in the exploitation of fungal biodiversity and procurement of sufficient quantities of clinical candidates are discussed, and potential solutions that could be pursued by researchers are highlighted.
Topics: Androstadienes; Animals; Antineoplastic Agents; Aphidicolin; Biological Products; Clinical Trials as Topic; Cyclohexanes; Diketopiperazines; Disease Models, Animal; Drug Design; Drug Resistance, Neoplasm; Fatty Acids, Unsaturated; Female; Fungi; Humans; Macrolides; Male; Mice; Neoplasms; Polycyclic Sesquiterpenes; Sesquiterpenes; Trichothecenes; Wortmannin
PubMed: 25850821
DOI: 10.1002/med.21348 -
Endocrinology Jan 202217β-estradiol (E2) treatment of ovariectomized adult mice stimulates the uterine PI3K-AKT signaling pathway and epithelial proliferation through estrogen receptor 1...
17β-estradiol (E2) treatment of ovariectomized adult mice stimulates the uterine PI3K-AKT signaling pathway and epithelial proliferation through estrogen receptor 1 (ESR1). However, epithelial proliferation occurs independently of E2/ESR1 signaling in neonatal uteri. Similarly, estrogen-independent uterine epithelial proliferation is seen in adulthood in mice lacking Ezh2, critical for histone methylation, and in wild-type (WT) mice treated neonatally with estrogen. The role of AKT in estrogen-independent uterine epithelial proliferation was the focus of this study. Expression of the catalytically active phosphorylated form of AKT (p-AKT) and epithelial proliferation were high in estrogen receptor 1 knockout and WT mice at postnatal day 6, when E2 concentrations were low, indicating that neither ESR1 nor E2 are essential for p-AKT expression and epithelial proliferation in these mice. However, p-AKT levels and proliferation remained estrogen responsive in preweaning WT mice. Expression of p-AKT and proliferation were both high in uterine luminal epithelium of mice estrogenized neonatally and ovariectomized during adulthood. Increased expression of phosphorylated (inactive) EZH2 was also observed. Consistent with this, Ezh2 conditional knockout mice show ovary-independent uterine epithelial proliferation and high epithelial p-AKT. Thus, adult p-AKT expression is constitutive and E2/ESR1 independent in both model systems. Finally, E2-induced p-AKT expression and normal uterine proliferation did not occur in mice lacking membrane (m)ESR1, indicating a key role for membrane ESR1 in AKT activation. These findings emphasize the importance of AKT activation in promoting uterine epithelial proliferation even when that proliferation is not E2/ESR1 dependent and further indicate that p-AKT can be uncoupled from E2/ESR1 signaling in several experimental scenarios.
Topics: Animals; Animals, Newborn; Catalysis; Cell Proliferation; Epithelium; Estrogens; Female; Genotype; Histones; Male; Methylation; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Uterus; Wortmannin
PubMed: 34791100
DOI: 10.1210/endocr/bqab233 -
Journal of Leukocyte Biology Apr 2021Extracellular histones have been implicated as a cause of tissue inflammatory injury in a variety of disorders including sepsis, lung, and liver diseases. However,...
Extracellular histones have been implicated as a cause of tissue inflammatory injury in a variety of disorders including sepsis, lung, and liver diseases. However, little is known about their interactions with neutrophils and how this might contribute to injury. Here, it is shown that histone H4 acts as neutrophil activator by inducing hydrogen peroxide production, degranulation, cell adhesion, and IL-8 generation. Histone H4 caused permeabilization of the neutrophil membrane (a phenomenon described in other cell types) leading to accelerated cell death. H4 caused sustained rise in neutrophil intracellular calcium that is necessary for respiratory burst activation and degranulation. Convincing evidence was not found for TLRs or ATP receptors in H4 mediated activation. However, pertussis toxin and wortmannin (inhibitors of G protein and PI3K) inhibited H4-induced hydrogen peroxide production and degranulation. These studies suggest that release of histone H4 in sites of infection or inflammation may potentiate neutrophil activation and promote additional inflammatory responses. These studies may provide a better basis for developing novel therapeutic strategies to block neutrophil extracellular trap (NET) and H4-related pathology in sepsis and various forms of lung injury including that induced by viruses like influenza or SAR-CoV2.
Topics: Calcium; Cell Adhesion; Cell Degranulation; Cell Membrane; Cell Membrane Permeability; Histones; Humans; Hydrogen Peroxide; Integrins; Monocytes; Neutrophil Activation; Neutrophils; Peroxidase; Pertussis Toxin; Respiratory Burst; Signal Transduction; Wortmannin
PubMed: 32803840
DOI: 10.1002/JLB.3A0620-342R -
Annals of Hepatology 2022Type 2 diabetes mellitus (T2DM) increases the occurrence and mortality of liver cancer. Insulin growth factor (IGF) plays a crucial role in the development of diabetes...
INTRODUCTION AND OBJECTIVES
Type 2 diabetes mellitus (T2DM) increases the occurrence and mortality of liver cancer. Insulin growth factor (IGF) plays a crucial role in the development of diabetes and liver cancer, and specifically, IGF-1 may be involved in the development of liver cancer with preexisting T2DM. Autophagy contributes to cancer cell survival and apoptosis. However, the relationship between IGF-1 and autophagy has rarely been evaluated. The purpose of this study was to investigate whether IGF-1 promotes the development of liver cancer in T2DM patients by promoting autophagy.
MATERIALS AND METHODS
Thirty-three hepatocellular carcinoma (HCC) patients with T2DM and 33 age-matched patients with HCC without T2DM were included in this study. We analyzed the expression of IGF-1 and autophagy-related LC3 and p62 mRNA and the prognosis of two groups. In vitro, we stimulated HepG2 cells with IGF-1 and then detected changes in autophagy and cell proliferation, apoptosis, and migration in the presence/absence of wortmannin, an autophagy inhibitor.
RESULTS
IGF-1 promoted autophagy, resulting in inhibition of apoptosis and induction of growth and migration of HepG2 cells. Inhibition of autophagy by wortmannin impaired IGF-1 function. Higher expression of IGF-1 was detected in HCC patients with T2DM. IGF-1 expression was higher in liver cancer tissue compared to paracancerous tissue. Elevated IGF-1 was associated with a poor prognosis in patients with HCC.
CONCLUSIONS
IGF-1 participates in the development of liver cancer by inducing autophagy. Elevated IGF-1 was a prognostic factor for patients with HCC, especially when accompanied by T2DM.
Topics: Autophagy; Carcinoma, Hepatocellular; Cell Line, Tumor; Diabetes Mellitus, Type 2; Humans; Insulin; Insulin-Like Growth Factor I; Liver Neoplasms; Wortmannin
PubMed: 35297369
DOI: 10.1016/j.aohep.2022.100697 -
Journal of Cellular and Molecular... Dec 2023Neonatal respiratory system disease is closely associated with embryonic lung development. Our group found that integrin β4 (ITGB4) is downregulated in the airway...
Neonatal respiratory system disease is closely associated with embryonic lung development. Our group found that integrin β4 (ITGB4) is downregulated in the airway epithelium of asthma patients. Asthma is the most common chronic respiratory illness in childhood. Therefore, we suspect whether the deletion of ITGB4 would affect fetal lung development. In this study, we characterized the role of ITGB4 deficiency in bronchopulmonary dysplasia (BPD). ITGB4 was conditionally knocked out in CCSP-rtTA, Tet-O-Cre and ITGB4 triple transgenic mice. Lung tissues at different developmental stages were collected for experimental detection and transcriptome sequencing. The effects of ITGB4 deficiency on lung branching morphogenesis were observed by fetal mouse lung explant culture. Deleting ITGB4 from the airway epithelial cells results in enlargement of alveolar airspaces, inhibition of branching, the abnormal structure of epithelium cells and the impairment of cilia growth during lung development. Scanning electron microscopy showed that the airway epithelial cilia of the β4 group appear to be sparse, shortened and lodging. Lung-development-relevant factors such as SftpC and SOX2 significantly decreased both mRNA and protein levels. KEGG pathway analysis indicated that multiple ontogenesis-regulating-relevant pathways converge to FAK. Accordingly, ITGB4 deletion decreased phospho-FAK, phospho-GSK3β and SOX2 levels, and the correspondingly contrary consequence was detected after treatment with GSK3β agonist (wortmannin). Airway branching defect of β4 mice lung explants was also partly recovered after wortmannin treatment. Airway epithelial-specific deletion of ITGB4 contributes to lung developmental defect, which could be achieved through the FAK/GSK3β/SOX2 signal pathway.
Topics: Animals; Humans; Infant, Newborn; Mice; Asthma; Bronchopulmonary Dysplasia; Epithelial Cells; Glycogen Synthase Kinase 3 beta; Integrin beta4; Lung; Mice, Transgenic; Wortmannin
PubMed: 37698050
DOI: 10.1111/jcmm.17948 -
Autophagy Apr 2023Macroautophagy/autophagy is a multistep degradative process that is essential for maintaining cellular homeostasis and is often dysregulated during disease....
Macroautophagy/autophagy is a multistep degradative process that is essential for maintaining cellular homeostasis and is often dysregulated during disease. Systematically quantifying flux through this pathway is critical for gaining fundamental insights and effectively modulating this process. Established methods to quantify flux use steady-state measurements, which provide limited information about the perturbation and the cellular response. We present a theoretical and experimental framework to measure autophagic steps in the form of rates under non-steady-state conditions. We use this approach to measure temporal responses to rapamycin and wortmannin treatments, two commonly used autophagy modulators. We quantified changes in autophagy rates in as little as 10 min, which can establish direct mechanisms for autophagy perturbation before feedback begins. We identified concentration-dependent effects of rapamycin on the initial and temporal progression of autophagy rates. We also found variable recovery time from wortmannin's inhibition of autophagy, which is further accelerated by rapamycin. Furthermore, we applied this approach to study the effect of serum and glutamine starvation on autophagy. Serum starvation led to a rapid and transient increase in all the rates. Glutamine starvation led to a decrease in the rates on a longer timescale. In summary, this new approach enables the quantification of autophagy flux with high sensitivity and temporal resolution and facilitates a comprehensive understanding of this process.
Topics: Humans; Autophagy; Glutamine; Wortmannin; Lysosomes; Sirolimus
PubMed: 36026492
DOI: 10.1080/15548627.2022.2117515 -
Aging Aug 2021Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer with a poor prognosis and a high recurrence rate. gene is frequently mutated in...
Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer with a poor prognosis and a high recurrence rate. gene is frequently mutated in breast cancer, with as the hotspot mutation reported in TNBC. We used the ZINC database to screen natural compounds that could be structurally modified to develop drugs targeting the PIK3CA H1047R mutant protein in the PI3K pathway. The LibDock module showed that 2,749 compounds could strongly bind to the PIK3CA H1047R protein. Ultimately, the top 20 natural ligands with high LibDock scores were used for further analyses including assessment of ADME (absorption, distribution, metabolism, and excretion), toxicity, stability, and binding affinity. ZINC000004098448 and ZINC000014715656 were selected as the safest drug candidates with strong binding affinity to PIK3CA H1047R, no hepatotoxicity, less carcinogenicity, better plasma protein binding (PPB) properties, and enhanced intestinal permeability and absorption than the two reference drugs, PKI-402 and wortmannin. Moreover, their lower potential energies than those of PIK3CA H1047R confirmed the stability of the ligand-receptor complex under physiological conditions. ZINC000004098448 and ZINC000014715656 are thus safe and stable leads for designing drugs against PIK3CA H1047R as part of a targeted therapeutic approach for patients with TNBC.
Topics: Binding Sites; Class I Phosphatidylinositol 3-Kinases; Computational Biology; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Female; Humans; Ligands; Models, Molecular; Mutation, Missense; Phenylurea Compounds; Pyrimidines; Signal Transduction; Triple Negative Breast Neoplasms; Wortmannin
PubMed: 34415239
DOI: 10.18632/aging.203409