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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 -
Calcium-Prolactin Secretion Coupling in Rat Pituitary Lactotrophs Is Controlled by PI4-Kinase Alpha.Frontiers in Endocrinology 2021The role of calcium, but not of other intracellular signaling molecules, in the release of pituitary hormones by exocytosis is well established. Here, we analyzed the...
The role of calcium, but not of other intracellular signaling molecules, in the release of pituitary hormones by exocytosis is well established. Here, we analyzed the contribution of phosphatidylinositol kinases (PIKs) to calcium-driven prolactin (PRL) release in pituitary lactotrophs: PI4Ks - which control PI4P production, PIP5Ks - which synthesize PI(4, 5)P2 by phosphorylating the D-5 position of the inositol ring of PI4P, and PI3KCs - which phosphorylate PI(4, 5)P to generate PI(3, 4, 5)P. We used common and PIK-specific inhibitors to evaluate the strength of calcium-secretion coupling in rat lactotrophs. Gene expression was analyzed by single-cell RNA sequencing and qRT-PCR analysis; intracellular and released hormones were assessed by radioimmunoassay and ELISA; and single-cell calcium signaling was recorded by Fura 2 imaging. Single-cell RNA sequencing revealed the expression of and , as well as and , in lactotrophs. Wortmannin, a PI3K and PI4K inhibitor, but not LY294002, a PI3K inhibitor, blocked spontaneous action potential driven PRL release with a half-time of ~20 min when applied in 10 µM concentration, leading to accumulation of intracellular PRL content. Wortmannin also inhibited increase in PRL release by high potassium, the calcium channel agonist Bay K8644, and calcium mobilizing thyrotropin-releasing hormone without affecting accompanying calcium signaling. GSK-A1, a specific inhibitor of PI4KA, also inhibited calcium-driven PRL secretion without affecting calcium signaling and expression. In contrast, PIK93, a specific inhibitor of PI4KB, and ISA2011B and UNC3230, specific inhibitors of PIP5K1A and PIP5K1C, respectively, did not affect PRL release. These experiments revealed a key role of PI4KA in calcium-secretion coupling in pituitary lactotrophs downstream of voltage-gated and PI(4, 5)P2-dependent calcium signaling.
Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcium; Calcium Channel Agonists; Calcium Signaling; Exocytosis; Lactotrophs; Minor Histocompatibility Antigens; Phosphotransferases (Alcohol Group Acceptor); Prolactin; Protein Kinase Inhibitors; Rats; Sequence Analysis, RNA; Single-Cell Analysis; Wortmannin
PubMed: 35058881
DOI: 10.3389/fendo.2021.790441 -
Molecules (Basel, Switzerland) Jul 2018Wortmannin is a potent covalent inhibitor of PI3K that shows substantial in vivo toxicity and thus is unsuitable for systemic therapeutic applications. One possible...
Wortmannin is a potent covalent inhibitor of PI3K that shows substantial in vivo toxicity and thus is unsuitable for systemic therapeutic applications. One possible approach to minimize systemic toxicity is to generate a latent wortmannin pro-drug that will be selectively activated in target tissues. To test this approach, a wortmannin derivative with a leucine linker attached to C20 has been synthesized and tested for inhibition of PI3K activity in prostate cancer cells. Analysis of PI3K pathway inhibition by Wormannin-Leu (Wn-L) and intact Wortmannin (Wn) showed that attachment of Leu at C-20 decreased potency of PI3K pathway inhibition 10-fold compared to intact wortmannin, yet exceeded the potency of a competitive PI3K inhibitor LY294002.
Topics: Androstadienes; Calorimetry, Differential Scanning; Enzyme Inhibitors; Humans; Magnetic Resonance Spectroscopy; Molecular Structure; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Wortmannin
PubMed: 30036994
DOI: 10.3390/molecules23071791 -
Frontiers in Immunology 2018Interleukin-1β (IL-1β), a major pro-inflammatory cytokine, is a leaderless cytosolic protein whose secretion does not follow the classical endoplasmic...
Interleukin-1β (IL-1β), a major pro-inflammatory cytokine, is a leaderless cytosolic protein whose secretion does not follow the classical endoplasmic reticulum-to-Golgi pathway, and for which a canonical mechanism of secretion remains to be established. Neutrophils are essential players against bacterial and fungi infections. These cells are rapidly and massively recruited from the circulation into infected tissues and, beyond of displaying an impressive arsenal of toxic weapons effective to kill pathogens, are also an important source of IL-1β in infectious conditions. Here, we analyzed if an unconventional secretory autophagy mechanism is involved in the exportation of IL-1β by these cells. Our findings indicated that inhibition of autophagy with 3-methyladenine and Wortmannin markedly reduced IL-1β secretion induced by LPS + ATP, as did the disruption of the autophagic flux with Bafilomycin A1 and E64d. These compounds did not noticeable affect neutrophil viability ruling out that the effects on IL-1β secretion were due to cell death. Furthermore, VPS34IN-1, a specific autophagy inhibitor, was still able to reduce IL-1β secretion when added after it was synthesized. Moreover, siRNA-mediated knockdown of ATG5 markedly reduced IL-1β secretion in neutrophil-differentiated PLB985 cells. Upon LPS + ATP stimulation, IL-1β was incorporated to an autophagic compartment, as was revealed by its colocalization with LC3B by confocal microscopy. Overlapping of IL-1β-LC3B in a vesicular compartment peaked before IL-1β increased in culture supernatants. On the other hand, stimulation of autophagy by cell starvation augmented the colocalization of IL-1β and LC3B and then promoted neutrophil IL-1β secretion. In addition, specific ELISAs indicated that although both IL-1β and pro-IL-1β are released to culture supernatants upon neutrophil stimulation, autophagy only promotes IL-1β secretion. Furthermore, the serine proteases inhibitor AEBSF reduced IL-1β secretion. Moreover, IL-1β could be also found colocalizing with elastase, suggesting both some vesicles containing IL-1β intersect azurophil granules content and that serine proteases also regulate IL-1β secretion. Altogether, our findings indicate that an unconventional autophagy-mediated secretory pathway mediates IL-1β secretion in human neutrophils.
Topics: Adenine; Adenosine Triphosphate; Autophagy; Autophagy-Related Protein 5; Cell Line; Humans; Inflammation Mediators; Interleukin-1beta; Lipopolysaccharides; Macrolides; Microtubule-Associated Proteins; Neutrophils; Protein Transport; RNA, Small Interfering; Secretory Pathway; Serine Proteases; Wortmannin
PubMed: 29515581
DOI: 10.3389/fimmu.2018.00269 -
Cellular and Molecular Life Sciences :... Oct 2022Astrocytes, an abundant type of glial cells, are the key cells providing homeostasis in the central nervous system. Due to their susceptibility to infection, combined...
Astrocytes, an abundant type of glial cells, are the key cells providing homeostasis in the central nervous system. Due to their susceptibility to infection, combined with high resilience to virus-induced cell death, astrocytes are now considered one of the principal types of cells, responsible for virus retention and dissemination within the brain. Autophagy plays an important role in elimination of intracellular components and in maintaining cellular homeostasis and is also intertwined with the life cycle of viruses. The physiological significance of autophagy in astrocytes, in connection with the life cycle and transmission of viruses, remains poorly investigated. In the present study, we investigated flavivirus-induced modulation of autophagy in human astrocytes by monitoring a tandem fluorescent-tagged LC3 probe (mRFP-EGFP-LC3) with confocal and super-resolution fluorescence microscopy. Astrocytes were infected with tick-borne encephalitis virus (TBEV) or West Nile virus (WNV), both pathogenic flaviviruses, and with mosquito-only flavivirus (MOF), which is considered non-pathogenic. The results revealed that human astrocytes are susceptible to infection with TBEV, WNV and to a much lower extent also to MOF. Infection and replication rates of TBEV and WNV are paralleled by increased rate of autophagy, whereas autophagosome maturation and the size of autophagic compartments are not affected. Modulation of autophagy by rapamycin and wortmannin does not influence TBEV and WNV replication rate, whereas bafilomycin A1 attenuates their replication and infectivity. In human astrocytes infected with MOF, the low infectivity and the lack of efficient replication of this flavivirus are mirrored by the absence of an autophagic response.
Topics: Animals; Humans; Astrocytes; Wortmannin; Encephalitis Viruses, Tick-Borne; Autophagy; Sirolimus; Virus Replication
PubMed: 36283999
DOI: 10.1007/s00018-022-04578-7 -
American Journal of Physiology.... Mar 2020Excess androgen-induced obesity has become a public health problem, and its prevalence has increased substantially in recent years. Chemokine-like receptor 1 (CMKLR1), a...
Excess androgen-induced obesity has become a public health problem, and its prevalence has increased substantially in recent years. Chemokine-like receptor 1 (CMKLR1), a receptor of chemerin secreted by adipose tissue, is linked to adipocyte differentiation, adipose tissue development, and obesity. However, the effect of CMKLR1 signaling on androgen-mediated adiposity in vivo remains unclear. Using CMKLR1-knockout mice, we constructed an androgen-excess female mouse model through 5α-dihydrotestosterone (DHT) treatment and an androgen-deficient male mouse model by orchidectomy (ORX). For mechanism investigation, we used 2-(α-Naphthoyl) ethyltrimethylammonium iodide (α-NETA), an antagonist of CMKLR1, to suppress CMKLR1 in vivo and wortmannin, a PI3K signaling antagonist, to treat brown adipose tissue (BAT) explant cultures in vitro Furthermore, we used histological examination and quantitative PCR, as well as Western blot analysis, glucose tolerance tests, and biochemical analysis of serum, to describe the phenotypes and the changes in gene expression. We demonstrated that excess androgen in the female mice resulted in larger cells in the white adipose tissue (WAT) and the BAT, whereas androgen deprivation in the male mice induced a reduction in cell size. Both of these adipocyte size effects could be attenuated in the CMKLR1-knockout mice. CMKLR1 deficiency influenced the effect of androgen treatment on adipose tissue by regulating the mRNA expression of the androgen receptor (AR) and adipocyte markers (such as Fabp4 and Cidea). Moreover, suppression of CMKLR1 by α-NETA could also reduce the extent of the adipocyte cell enlargement caused by DHT. Furthermore, we found that DHT could reduce the levels of phosphorylated ERK (pERK) in the BAT, while CMKLR1 inactivation inhibited this effect, which had been induced by DHT, through the PI3K signaling pathway. These findings reveal an antiobesity role of CMKLR1 deficiency in regulating lipid accumulation, highlighting the scientific importance for the further development of small-molecule CMKLR1 antagonists as fundamental research tools and/or as potential drugs for use in the treatment of adiposity.
Topics: Adipocytes; Adipose Tissue, Brown; Androgens; Animals; Body Weight; Cell Size; Dihydrotestosterone; Female; Lipid Metabolism; MAP Kinase Signaling System; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Naphthalenes; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Quaternary Ammonium Compounds; Receptors, Chemokine; Wortmannin
PubMed: 31910029
DOI: 10.1152/ajpendo.00176.2019 -
Cell Communication and Signaling : CCS May 2021Cells adapt their metabolism and activities in response to signals from their surroundings, and this ability is essential for their survival in the face of...
BACKGROUND
Cells adapt their metabolism and activities in response to signals from their surroundings, and this ability is essential for their survival in the face of perturbations. In tissues a deficit of these mechanisms is commonly associated with cellular aging and diseases, such as cardiovascular disease, cancer, immune system decline, and neurological pathologies. Several proteins have been identified as being able to respond directly to energy, nutrient, and growth factor levels and stress stimuli in order to mediate adaptations in the cell. In particular, mTOR, AMPK, and sirtuins are known to play an essential role in the management of metabolic stress and energy balance in mammals.
METHODS
To understand the complex interactions of these signalling pathways and environmental signals, and how those interactions may impact lifespan and health-span, we have developed a computational model of metabolic signalling pathways. Specifically, the model includes (i) the insulin/IGF-1 pathway, which couples energy and nutrient abundance to the execution of cell growth and division, (ii) mTORC1 and the amino acid sensors such as sestrin, (iii) the Preiss-Handler and salvage pathways, which regulate the metabolism of NAD+ and the NAD+ -consuming factor SIRT1, (iv) the energy sensor AMPK, and (v) transcription factors FOXO and PGC-1α.
RESULTS
The model simulates the interactions among key regulators such as AKT, mTORC1, AMPK, NAD+ , and SIRT, and predicts their dynamics. Key findings include the clinically important role of PRAS40 and diet in mTORC1 inhibition, and a potential link between SIRT1-activating compounds and premature autophagy. Moreover, the model captures the exquisite interactions of leucine, sestrin2, and arginine, and the resulting signal to the mTORC1 pathway. These results can be leveraged in the development of novel treatment of cancers and other diseases.
CONCLUSIONS
This study presents a state-of-the-art computational model for investigating the interactions among signaling pathways and environmental stimuli in growth, ageing, metabolism, and diseases. The model can be used as an essential component to simulate gene manipulation, therapies (e.g., rapamycin and wortmannin), calorie restrictions, and chronic stress, and assess their functional implications on longevity and ageing-related diseases. Video Abstract.
Topics: AMP-Activated Protein Kinases; Amino Acids; Animals; Biological Transport; Cell Line; Energy Metabolism; Glucose; Humans; Insulin; Mechanistic Target of Rapamycin Complex 1; Mice; Models, Biological; Protein Binding; Signal Transduction; Sirolimus; Sirtuins; Wortmannin
PubMed: 34016143
DOI: 10.1186/s12964-021-00706-1 -
Scientific Reports Jun 2022Insulin exerts positive inotropic effects on cardiac muscle; however, the relationship between cardiac contractility and phosphoinositol-3-kinase/Akt (PI3K/Akt)...
Insulin exerts positive inotropic effects on cardiac muscle; however, the relationship between cardiac contractility and phosphoinositol-3-kinase/Akt (PI3K/Akt) activation remains unclear. We hypothesized that the positive inotropic effects of insulin are dose-dependent and mediated via the PI3K/Akt pathway in isolated normal rat hearts. The Institutional Animal Investigation Committee approved the use of hearts excised from rats under pentobarbital anesthesia. The hearts were perfused at a constant pressure using the Langendorff technique. After stabilization (baseline), the hearts were randomly divided into the following four insulin (Ins) groups: 1) Ins0 (0 IU/L), 2) Ins0.5 (0.5 IU/L), 3) Ins5 (5 IU/L), and 4) Ins50 (50 IU/L) (n = 8 in each group). To clarify the role of the PI3K/Akt pathway in insulin-dependent inotropic effects, we also treated the insulin groups with the PI3K inhibitor wortmannin (InsW): 5) InsW0 (0 IU/L), 6) InsW0.5 (0.5 IU/L), 7) InsW5 (5 IU/L), and 8) InsW50 (50 IU/L). Hearts were perfused with Krebs-Henseleit buffer solution with or without wortmannin for 10 min, followed by 20 min perfusion with the solution containing each concentration of insulin. The data were recorded as the maximum left ventricular derivative of pressure development (LV dP/dt max). Myocardial p-Akt levels were measured at 3 min, 5 min, and at the end of the perfusion. In the Ins groups, LV dP/dt max in Ins5 and Ins50 increased by 14% and 48%, respectively, 3 min after insulin perfusion compared with the baseline. Tachyphylaxis was observed after 10 min in the Ins5 and Ins50 treatment groups. Wortmannin partially inhibited the positive inotropic effect of insulin; although insulin enhanced p-Akt levels at all time points compared with the control group, this increase was suppressed in the presence of wortmannin. The positive inotropic effect of insulin is dose-dependent and consistent with Akt activation. This effect mediated by high doses of insulin on cardiac tissue was temporary and caused tachyphylaxis, potentially triggered by Akt overactivation, which leads beta 1 deactivation.
Topics: Animals; Heart; Insulin; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Wortmannin
PubMed: 35697740
DOI: 10.1038/s41598-022-14092-2 -
Journal of Controlled Release :... Apr 2018Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States; the predominant cause for mortality is metastasis to distant organs (e.g.,...
Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States; the predominant cause for mortality is metastasis to distant organs (e.g., lung). A major problem limiting the success of chemotherapy in metastatic CRC is the inability to target tumor tissues selectively and avoid severe side effects to normal tissues and organs. Here, we demonstrate polymeric nanoparticles (PNPs) entrapping chemotherapeutic agents provide a new therapeutic option for treating CRC that has metastasized to the lung. PNPs assembled from FDA approved biocompatible block copolymer accumulated predominantly in lung tissue. PNPs showed negligible accumulation in liver, spleen and kidneys, which was confirmed by fluorescent nanoparticle imaging and analysis of PI3K inhibition in the organs. PNPs entrapping PI3K inhibitors (i.e., wortmannin and PX866) suppressed CRC lung metastasis growth, and SN-38-loaded PNPs completely eliminated CRC lung metastasis. Our results demonstrate that polymer-drug nanoparticles offer a new approach to reduce toxicity of cancer therapy and has the potential to improve outcomes for patients with lung metastasis.
Topics: Animals; Colorectal Neoplasms; Drug Carriers; Gonanes; HT29 Cells; Humans; Irinotecan; Lung Neoplasms; Male; Mice, Inbred BALB C; Mice, Nude; Mice, SCID; Nanoparticles; Phosphoinositide-3 Kinase Inhibitors; Polymers; Topoisomerase I Inhibitors; Wortmannin
PubMed: 29421609
DOI: 10.1016/j.jconrel.2018.02.008 -
Nutrients Dec 2022Hair loss remains a significant problem that is difficult to treat; therefore, there is a need to identify safe natural materials that can help patients with hair loss....
Hair loss remains a significant problem that is difficult to treat; therefore, there is a need to identify safe natural materials that can help patients with hair loss. We evaluated the hair anagen activation effects of limonin, which is abundant in immature citrus fruits. Limonin increased the proliferation of rat dermal papilla cells (rDPC) by changing the levels of cyclin D1 and p27, and increasing the number of BrdU-positive cells. Limonin increased autophagy by decreasing phosphorylated mammalian target of rapamycin levels and increasing the phospho-Raptor, ATG7 and LC3B. Limonin also activated the Wnt/β-catenin pathway by increasing phospho-β-catenin levels. XAV939, a Wnt/β-catenin inhibitor, inhibited these limonin-induced changes, including induced autophagy, BrdU-positive cells, and cell proliferation. Limonin increased the phosphorylated AKT levels in both two-dimensional cultured rDPC and three-dimensional spheroids. Treatment with the PI3K inhibitor wortmannin inhibited limonin-induced proliferation, and disrupted other limonin-mediated changes, including decreased p27, increased BrdU-positive cells, induced autophagy, and increased ATG7 and LC3B levels. Wortmannin also inhibited limonin-induced cyclin D1 and LC3 expression in spheroids. Collectively, these results indicate that limonin can enhance anagen signaling by activating autophagy via targeting the Wnt/β-catenin and/or PI3K/AKT pathways in rDPC, highlighting a candidate nutrient for hair loss treatment.
Topics: Animals; Rats; Alopecia; beta Catenin; Bromodeoxyuridine; Cell Proliferation; Cells, Cultured; Cyclin D1; Fruit; Hair Follicle; Limonins; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Wnt Signaling Pathway; Wortmannin
PubMed: 36558517
DOI: 10.3390/nu14245358