Inhibition Of Tyrosine Phosphorylation Research Articles

Antimicrobial cetylpyridinium chloride suppresses mast cell function by targeting tyrosine phosphorylation of Syk kinase.

Cetylpyridinium chloride (CPC) is a quaternary ammonium antimicrobial used in numerous personal care products, human food, cosmetic products, and cleaning solutions. Yet, there is minimal published data on CPC effects on eukaryotes, immune signaling, and human health. Previously, we showed that low-micromolar CPC inhibits rat mast cell function by inhibiting antigen (Ag)-stimulated Ca 2+ mobilization, microtubule polymerization, and degranulation. In this study, we extend the findings to human mast cells (LAD2) and present data indicating that CPC's mechanism of action centers on its positively-charged quaternary nitrogen in its pyridinium headgroup. CPC's inhibitory effect is independent of signaling platform receptor architecture. Tyrosine phosphorylation events are a trigger of Ca 2+ mobilization necessary for degranulation. CPC inhibits global tyrosine phosphorylation in Ag-stimulated mast cells. Specifically, CPC inhibits tyrosine phosphorylation of specific key players Syk kinase and LAT, a substrate of Syk. In contrast, CPC does not affect Lyn kinase phosphorylation. Thus, CPC's root mechanism is electrostatic disruption of particular tyrosine phosphorylation events essential for signaling. This work outlines the biochemical mechanisms underlying the effects of CPC on immune signaling and allows the prediction of CPC effects on cell types, like T cells, that share similar signaling elements.

Abstract B037: Utilizing GSK3β and integrin inhibitors to target STAT3 activity in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer known for its high rate of recurrence and metastatic ability. Patients face a poor prognosis and the five-year survival rate for those diagnosed with metastatic TNBC is very low. The transcription factor Signal Transducer and Activator of Transcription 3 (STAT3) is constitutively active in TNBC and functions to promote proliferation and metastasis. Therefore, it is a potential therapeutic target for TNBC. We used CLUE, an online bioinformatics tool from the Broad Institute, to identify potential STAT3 inhibitors. To do this, a STAT3 gene signature generated from TNBC cells was used to query the gene signatures from drug treatments in the CLUE dataset for compounds that caused a similar signature. The hypothesis is that a drug with a similar signature to the STAT3 inhibition signature would be a potential STAT3 inhibitor. Drugs were chosen based on high similarity rating and known function. One of the top drugs we chose to pursue was TWS-119, a GSK3β inhibitor. GSK3β is a serine/threonine kinase that regulates proliferation and cell survival. TWS-119 was found to decrease the viability of MDA-MB-231, MDA-MB-468, and Hs587T TNBC cell lines. To assess the ability of TWS-119 to inhibit STAT3 activity, we used a STAT3 responsive reporter and found that TWS-119 inhibited STAT3 activity in MDA-MB-468 cells. To determine the mechanism of inhibition of STAT3 activity, we analyzed the effects of TWS-119 on STAT3 tyrosine phosphorylation. Preliminary results suggest that TWS-119 inhibits STAT3 activity via inhibition of tyrosine phosphorylation. Having determined that TWS-119 inhibited STAT3 activity, we wanted to determine if TWS-119 had any effect on TNBC migration. We found that TWS-119 reduced the migratory ability of MDA-MB-231 cells. An additional drug identified from CLUE was Cilengitide, an inhibitor of integrin ⍺vβ3 which plays an important role in proliferation and migration. While Cilengitide only slightly decreased the viability of the three different TNBC cell lines (MDA-MB-231, MDA-MB-468, Hs587T), we found that combinations of Cilengitide and TWS-119 had synergistic effects on cell viability in the TNBC cells. Preliminary western blot analysis demonstrated that the combination of both drugs reduced the level of pY-STAT3. This suggests that this combination targeting STAT3 may be a viable treatment strategy for TNBC. However, the mechanism through which these drugs interact is unknown, and the cell signaling relationship between their known targets (GSK3β and integrin ⍺vβ3) and STAT3 is unclear. We are currently working to define the mechanism of action of these combination effects in TNBC cells. Using TWS-119 and Cilengitide, we can determine the connection between integrins, GSK3β, and STAT3 to better understand proliferation and metastasis and to identify new therapeutic options for TNBC patients. Citation Format: Emily A Pratt, Sarah R Walker. Utilizing GSK3β and integrin inhibitors to target STAT3 activity in triple negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B037.

TMEM16A activation for the fast block to polyspermy in the African clawed frog does not require conventional activation of egg PLCs.

Fertilization of an egg by more than one sperm, a condition known as polyspermy, leads to gross chromosomal abnormalities and is embryonic lethal for most animals. Consequently, eggs have evolved multiple processes to stop supernumerary sperm from entering the nascent zygote. For external fertilizers, such as frogs and sea urchins, fertilization signals a depolarization of the egg membrane, which serves as the fast block to polyspermy. Sperm can bind to, but will not enter, depolarized eggs. In eggs from the African clawed frog, Xenopus laevis, the fast block depolarization is mediated by the Ca2+-activated Cl- channel TMEM16A. To do so, fertilization activates phospholipase C, which generates IP3 to signal a Ca2+ release from the ER. Currently, the signaling pathway by which fertilization activates PLC during the fast block remains unknown. Here, we sought to uncover this pathway by targeting the canonical activation of the PLC isoforms present in the X. laevis egg: PLCγ and PLCβ. We observed no changes to the fast block in X. laevis eggs inseminated in inhibitors of tyrosine phosphorylation, used to stop activation of PLCγ, or inhibitors of Gαq/11 pathways, used to stop activation of PLCβ. These data suggest that the PLC that signals the fast block depolarization in X. laevis is activated by a novel mechanism.

Regulation of Syk activity by antiviral adaptor MAVS in FcεRI signaling pathway.

Mast cells are the major effector cell type for IgE-mediated allergic reactions. Recent studies revealed a role for mast cells in orchestrating the host response to viral infections. We studied the relationship between FcεRI (high-affinity IgE receptor) and RIG-I-like receptor (RLR)-mediated antiviral signaling pathways. Mast cells (BMMCs) were cultured from bone marrow cells from mice deficient in MAVS or other RLR signaling molecules. MAVS expression was restored by retroviral transduction of MAVS-deficient BMMCs. These cells were stimulated with IgE and antigen and their activation (degranulation and cytokine production/secretion) was quantified. FcεRI-mediated signaling events such as protein phosphorylation and Ca2+ flux were analyzed by western blotting and enzyme assays. WT and mutant mice as well as mast cell-deficient KitW-sh/W-sh mice engrafted with BMMCs were subjected to passive cutaneous anaphylaxis. Unexpectedly, we found that mast cells devoid of the adaptor molecule MAVS exhibit dramatically increased cytokine production upon FcεRI stimulation, despite near-normal degranulation. Consistent with these observations, MAVS inhibited tyrosine phosphorylation, thus catalytic activity of Syk kinase, the key signaling molecule for FcεRI-mediated mast cell activation. By contrast, mast cells deficient in RIG-I, MDA5 or IRF3, which are antiviral receptor and signaling molecules upstream or downstream of MAVS, exhibited reduced or normal mast cell activation. MAVS-deficient mice showed enhanced late-phase responses in passive cutaneous anaphylaxis. This study demonstrates that the adaptor MAVS in the RLR innate immune pathway uniquely intersects with the adaptive immune FcεRI signaling pathway.

A new role of H89: Reduces capacitation-like changes through inhibition of cholesterol efflux, calcium influx, and proteins tyrosine phosphorylation during sperm cryopreservation in buffalo

It is a known fact that cryopreservation initiates premature capacitation in spermatozoa during the cryopreservation process. Protein tyrosine phosphorylation is a landmark of cascade reaction accountable for capacitation or capacitation-like changes in spermatozoa. Therefore, our hypothesis was to test an inhibitor (H89) that reversibly inhibits the cascade reaction responsible for capacitation during the cryopreservation process but does not hamper normal capacitation and fertilizing ability of sperm. For this, sixteen ejaculates were collected from Murrah buffalo bulls (n = 4). Each ejaculate was divided into four equal aliquots and diluted in an egg yolk-based semen dilutor supplemented with 0, 2, 10, and 30 μM concentrations of H89 and cryopreserved. Interestingly, H89 reduces cholesterol efflux from spermatozoa and protects spermatozoa from membrane damage during the cryopreservation process. H89 did not prevent lipid peroxidation of the sperm membrane. H89 reduced intracellular calcium concentration in spermatozoa in a dose-dependent manner, but tyrosine phosphorylation reduction was observed in the 2 and 10 μM H89 groups. The CTC assay revealed that the percentage of uncapacitated spermatozoa in different treatment groups increases in a dose-dependent manner. In the in vitro capacitation medium, the effect of H89 is abolished and spermatozoa underwent normal capacitation, but H89-treated spermatozoa attached to zona pellucida in large numbers compared to untreated spermatozoa. In conclusion, H89 does not only inhibit tyrosine phosphorylation of spermatozoa but it reduces cholesterol efflux and calcium influx, and ultimately reduces capacitation-like changes during the cryopreservation process.

The microtubule network enables Src kinase interaction with the Na,K-ATPase to generate Ca2+ flashes in smooth muscle cells.

Background: Several local Ca2+ events are characterized in smooth muscle cells. We have previously shown that an inhibitor of the Na,K-ATPase, ouabain induces spatially restricted intracellular Ca2+ transients near the plasma membrane, and suggested the importance of this signaling for regulation of intercellular coupling and smooth muscle cell contraction. The mechanism behind these Na,K-ATPase-dependent “Ca2+ flashes” remains to be elucidated. In addition to its conventional ion transport function, the Na,K-ATPase is proposed to contribute to intracellular pathways, including Src kinase activation. The microtubule network is important for intracellular signaling, but its role in the Na,K-ATPase-Src kinase interaction is not known. We hypothesized the microtubule network was responsible for maintaining the Na,K-ATPase-Src kinase interaction, which enables Ca2+ flashes. Methods: We characterized Ca2+ flashes in cultured smooth muscle cells, A7r5, and freshly isolated smooth muscle cells from rat mesenteric artery. Cells were loaded with Ca2+-sensitive fluorescent dyes, Calcium Green-1/AM and Fura Red/AM, for ratiometric measurements of intracellular Ca2+. The Na,K-ATPase α2 isoform was knocked down with siRNA and the microtubule network was disrupted with nocodazole. An involvement of the Src signaling was tested pharmacologically and with Western blot. Protein interactions were validated with proximity ligation assays. Results: The Ca2+ flashes were induced by micromolar concentrations of ouabain. Knockdown of the α2 isoform Na,K-ATPase abolished Ca2+ flashes, as did inhibition of tyrosine phosphorylation with genistein and PP2, and the inhibitor of the Na,K-ATPase-dependent Src activation, pNaKtide. Ouabain-induced Ca2+ flashes were associated with Src kinase activation by phosphorylation. The α2 isoform Na,K-ATPase and Src kinase colocalized in the cells. Disruption of microtubule with nocodazole inhibited Ca2+ flashes, reduced Na,K-ATPase/Src interaction and Src activation. Conclusion: We demonstrate that the Na,K-ATPase-dependent Ca2+ flashes in smooth muscle cells require an interaction between the α2 isoform Na, K-ATPase and Src kinase, which is maintained by the microtubule network.

Asialoglycoprotein Receptor 1 Functions as a Tumor Suppressor in Liver Cancer via Inhibition of STAT3.

ASGR1 downregulation by DNA methylation facilitates liver tumorigenesis by increasing STAT3 phosphorylation.

822-P: The Harmine and Exendin4 Combination Markedly Expands Human Beta-Cell Mass In Vivo in Diabetic Mice

Diabetes results from diminished functional β-cell mass. Small molecule inhibitors of Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A (DYRK1A) markedly increase human β-cell replication in vitro and in vivo. Furthermore, combination of DYRK1A inhibitors such as Harmine (H) with widely clinically used glucagon-like peptide 1 receptor agonists such as Exendin-4 (E) further enhance human β-cell proliferation. However, whether H+E combination increases human β-cell mass and improves glucose homeostasis in diabetic immunosuppressed mice transplanted with a marginal number of human islets is unknown. Human islets from 5-6 different adult healthy donors were transplanted under the kidney capsule (200 islets per kidney) of streptozotocin-induced diabetic immunodeficient Rag1-/- mice. At the time of transplantation, osmotic Alzet minipumps were implanted for continuous delivery of vehicle V, H, E and H+E for a period of 12 weeks. At the end of the study, human islet-bearing kidneys were harvested, labeled with insulin antisera and cleared using a modification of the iDISCO+ method. Imaging and analysis of the β-cell volume was done using the Light-sheet Ultramicroscope II and Imaris software. Transplanted diabetic mice who received the combination of H+E showed a significant increase of human β-cell volume up to 600% compared with mice treated with V or the drugs alone. This increase correlated with a significant increase in plasma human insulin levels, sustained normalization of blood glucose levels and significantly improved glucose tolerance. Our studies provide unequivocal documentation that treatment with the harmine and exendin-4 combination can lead to actual increases in human β-cell mass in diabetic conditions suggesting that β-cell replenishment might be possible in humans with diabetes. Disclosure K.A.Beliard: None. S.Stanley: None. A.Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. C.Rosselot: None. Y.Li: None. A.Alvarsson: None. P.Wang: None. K.Thakkar: None. D.Guevara: None. R.J.Devita: None. A.F.Stewart: None. Funding NIH/NIDDK 1R01DK105015-05.

SS-4 is a highly selective small molecule inhibitor of STAT3 tyrosine phosphorylation that potently inhibits GBM tumorigenesis in vitro and in vivo

Glioblastoma (GBM) is a highly aggressive cancer with a dismal prognosis. Constitutively active STAT3 has a causal role in GBM progression and is associated with poor patient survival. We rationally designed a novel small molecule, SS-4, by computational modeling to specifically interact with STAT3. SS-4 strongly and selectively inhibited STAT3 tyrosine (Y)-705 phosphorylation in MT330 and LN229 GBM cells and inhibited their proliferation and induced apoptosis with an IC50 of ∼100 nM. The antiproliferative and apoptotic actions of SS-4 were Y-705 phosphorylation dependent, as evidenced by its lack of effects on STAT3 knockout (STAT3KO) cells or STAT3KO cells that overexpressed a phospho-Y705 deficient (STAT3Y705F) mutant, and the recovery of effects when wild-type STAT3 or a phospho-serine (S)727 deficient mutant was expressed in STAT3KO cells. SS-4 increased the expression of STAT3 repressed genes, while decreasing the expression of STAT3 promoted genes. Importantly, SS-4 markedly reduced the growth of GBM intracranial tumor xenografts. These data together identify SS-4 as a potent STAT3 inhibitor that selectively blocks Y705-phosphorylation, induces apoptosis, and inhibits growth of human GBM models in vitro and in vivo.

Vitamin D-Binding Protein is Involved in the Pathogenesis of Preeclampsia by Inhibiting the Tyrosine Phosphorylation of Vascular Endothelial Growth Factor Receptor-2 in Endothelial Cells

Objective: The role of Vitamin D-binding protein (DBP) in preeclampsia (PE) pathogenesis is unknown. In this study, we compared the expression of DBP in the placentas of PE patients with the placentas of normotensive pregnant women with placenta previa controls, and aimed to explore the effect of DBP on endothelial cells (ECs) and the underlying mechanism. Methods: DBP expression in placental tissues collected from PE patients and controls was evaluated by immunohistochemistry. The downregulation and upregulation of DBP expression in HTR-8/SVneo cells were examined using DBP-targeting small interfering RNA (siRNA) and DBP-expression vector, respectively. The conditioned media of these DBP-overexpressing and DBP-siRNA HTR-8/SVneo cells were collected and added to human umbilical vein EC (HUVEC) cultures. Angiogenic effects on HUVECs were assessed by tube formation assays, and the proliferation and migration of HUVECs were examined using the Real-Time Cell Analyzer. The expression of vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR)-2, as well as the phosphorylation of different residues of VEGFR-2 in HUVECs, were determined by western blotting. Results: DBP expression was significantly increased in the placental tissues collected from PE patients. The conditioned medium of DBP-overexpressing HTR-8/SVneo cells potently inhibited tube formation by HUVECs, in addition to their proliferation and migration. Furthermore, treatment of HUVECs with the conditioned medium of DBP-overexpressing HTR-8/SVneo cells decreased the phosphorylation of VEGFR-2 at tyrosine 996, whereas the treatment of these cells with the conditioned medium of DBP-siRNA HTR-8/SVneo cells increased the phosphorylation of VEGFR-2 at tyrosine 951, 996, and 1,175. Conclusions: The expression of DBP is increased in the placentas of PE patients. DBP plays potential roles in endothelial dysfunction, which contributes to PE development, by inhibiting tyrosine phosphorylation of VEGFR-2 in ECs.

Pyruvate Dehydrogenase Kinase Modulates Platelet Function and Thrombosis

Background and objective: Pyruvate dehydrogenase kinases (PDK 1-4) are mitochondrial enzymes that regulate the activity of pyruvate dehydrogenase (PDH) complex, which converts pyruvate (end product of glycolysis in cytosol) to acetyl coenzyme A that is further oxidized to produce energy (oxidative phosphorylation; OXPHOS). Like most normal cells, resting platelets rely primarily on OXPHOS to generate ATP, whereas activated platelets exhibit a high level of aerobic glycolysis (conversion of glucose to lactate in the presence of oxygen, a phenomenon referred to as the Warburg effect in tumor cells). It seems paradoxical that why platelets prefer aerobic glycolysis instead of OXPHOS. The rate of ATP generation is faster in aerobic glycolysis compared to OXPHOS, which is well suited for high-energy demand of activated platelets to form blood clot. We tested the hypothesis that manipulating aerobic glycolysis (metabolic reprogramming), by down regulating PDK activity in the mitochondria of platelets, will inhibit platelet function and thereby thrombosis.Methods: Global knock out or floxed PDK mice does not exists, therefore we used dichloroacetic acid (DCA), a potent inhibitor of PDKs, known to divert metabolism back from aerobic glycolysis to OXPHOS. DCA has been successfully used in clinical trials to treat several type of cancer. In human and murine platelets, using standardized platelet in vitro assays, we determined the mechanistic role of PDK in regulating platelet function. Rate of glycolysis and mitochondrial respiration was measured using the Seahorse XF24 extracellular flux analyzer. Using intravital microscopy, thrombus growth was evaluated in vitro (microfluidics flow chamber) and in vivo (FeCl3-induced carotid artery thrombosis) models.Results: Using, extracellular flux analysis, we first confirmed that activated platelets have higher aerobic glycolysis. With both human and mice platelets, we found that DCA (10 and 25 mM) significantly inhibited in vitro platelet aggregation to collagen and convulxin-induced signaling, but not thrombin or ADP-induced signaling in a dose-dependent manner (P<0.05 vs. vehicle treated). DCA also inhibited static adhesion of washed platelets to collagen in dose-dependent manner. Furthermore, we found that DCA significantly inhibited tyrosine phosphorylation of Syk and PLCγ2 in the GPVI signaling pathway with both mice and human platelets (P<0.05 vs. vehicle treated). Metabolic profiling, using extracellular flux analysis, showed that DCA significantly inhibited extracellular acidification rate in convulxin-stimulated platelets in dose-dependent manner. Using microfluidics flow chamber, we found that both mouse and human whole blood treated with DCA formed small thrombi when perfused over collagen for 10 min at a arterial shear rate of 1500s-1 (P<0.05 vs. vehicle control), suggesting that inhibiting PDK with DCA limits thrombus growth. Finally, wild-type mice pre-treated with DCA (600 mg/Kg body weight) were less susceptible to thrombosis in FeCl3-induced arterial thrombosis model (P<0.05 vs. control, N=10 mice/group), without altered hemostasis (tail-bleeding assay). The precise mechanism how aerobic glycolysis modulates GPVI signaling pathway is unclear and is currently under investigation.Conclusion: Our results suggest that PDK present in mitochondria regulates platelet function and thrombus growth, most likely via GPVI signaling pathway. DisclosuresNo relevant conflicts of interest to declare.

CDK control pathways integrate cell size and ploidy information to control cell division.

Maintenance of cell size homeostasis is a property that is conserved throughout eukaryotes. Cell size homeostasis is brought about by the co-ordination of cell division with cell growth and requires restriction of smaller cells from undergoing mitosis and cell division, whilst allowing larger cells to do so. Cyclin-CDK is the fundamental driver of mitosis and therefore ultimately ensures size homeostasis. Here we dissect determinants of CDK activity in vivo to investigate how cell size information is processed by the cell cycle network in fission yeast. We develop a high-throughput single-cell assay system of CDK activity in vivo and show that inhibitory tyrosine phosphorylation of CDK encodes cell size information, with the phosphatase PP2A aiding to set a size threshold for division. CDK inhibitory phosphorylation works synergistically with PP2A to prevent mitosis in smaller cells. Finally, we find that diploid cells of equivalent size to haploid cells exhibit lower CDK activity in response to equal cyclin-CDK enzyme concentrations, suggesting that CDK activity is reduced by increased DNA levels. Therefore, scaling of cyclin-CDK levels with cell size, CDK inhibitory phosphorylation, PP2A, and DNA-dependent inhibition of CDK activity, all inform the cell cycle network of cell size, thus contributing to cell size homeostasis.

Important role of annexin A2 (ANXA2) in new blood vessel development in vivo and human triple negative breast cancer (TNBC) growth

Development of new blood vessels in the tumor microenvironment is an essential component of tumor progression during which newly formed blood vessels nourish tumor cells and play a critical role in rapid tumor growth, invasion and metastasis. Nevertheless, how tumor cells develop new blood vessels in the tumor microenvironment (TME) have been enigmatic. Previously, we have shown specific overexpression of ANX A2 in TNBC cells regulates plasmin generation and suspected a role in neoangiogenesis. In this report, we used Matrigel plug model of in vivo angiogenesis and confirmed its role in new blood vessel development. Next, we tested if blocking of ANX A2 in aggressive human breast TME can inhibit angiogenesis and tumor growth in vivo. We showed that aggressive human breast tumor cells growing in nude mice can induce intense neoangiogenesis in the tumor mass. Blocking of ANXA2 significantly inhibited neoangiogenesis and resulted in inhibition of tumor growth. Interestingly, we identified that blocking of ANXA2 significantly inhibited tyrosine phosphorylation (Tyr-P) of ANXA2 implying its involvement in tyrosine signaling pathway and suggesting it may regulate angiogenesis. Taken together, our experimental evidence suggests that ANX A2 could be a novel strategy for disruption of tyrosine signaling and inhibition of neoangiogenesis in breast tumor.

Na+/K+-ATPase α4 regulates sperm hyperactivation while Na+/K+-ATPase α1 regulates basal motility in hamster spermatozoa

Recently, it was reported that hamster sperm hyperactivation is regulated by extracellular Na+. Two types of catalytic Na+/K+-ATPase (NKA) α subunits (α1 and α4) are present in spermatozoa. In this work, the contribution of these NKA subunits to the regulation of hamster sperm hyperactivation was investigated using the specific inhibitor ouabain. When 10−6 M ouabain was added to the modified Tyrode’s albumin lactate pyruvate medium (mTALP) medium, hyperactivation was significantly inhibited, whereas 10−5-10−4 M ouabain was needed to significantly reduce the amount of motile spermatozoa. When a more detailed analysis of flagellar movement was performed, 10−6 M ouabain suppressed the hyperactivation-associated change in the patterns of flagellar motion without affecting the sliding velocity of microtubules. Since 10−6 M ouabain specifically inhibits the α4 subunit while 10−5-10−4 M ouabain inhibits both the α1 and α4 subunits, these results suggest the α1 subunit is necessary for the maintenance of motility while the α4 subunit is necessary for the hyperactivation-associated change in flagellar movement. Ouabain did not inhibit tyrosine phosphorylation, and activation of tyrosine phosphorylation-dependent signaling had no effect on the inhibition of hyperactivation by ouabain. The immediate recovery of hyperactivation was observed when ouabain was washed out after a 3-h incubation. whereas the administration of ouabain after the onset of hyperactivation significantly inhibited hyperactivation. These results suggest ouabain inhibited hyperactivation in a manner that was independent of time-requiring phosphorylation-mediated signaling.

Anti-allergic action of O-methylated EGCG in green tea cultivar Benifuuki

The anti-allergic effect of epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3″Me) and epigallocatechin-3-O-(4-O-methyl) gallate (EGCG4″Me) isolated from Japanese or Taiwanese tea (Camellia sinensis L.) leaves. These catechins strongly inhibited mast cell activation and histamine release after FcepsilonRI cross-linking through the suppression of tyrosine phosphorylation (Lyn) of cellular protein kinase, and the suppression of myosin light chain phosphorylation and high-affinity IgE receptor expression via the binding to 67kDa laminin receptor. A double blind clinical study on subjects with Japanese cedar pollinosis was carried out. At the eleventh weeks after starting to intake, the most severe cedar pollen scattering period, symptoms i.e. blowing nose, itch of eyes were significantly relieved in Benifuuki group compared with placebo group. Over one consecutive month intake of Benifuuki green containing O-methylated catechin tea was useful for reduction of some symptoms derived from Japanese cedar pollinosis, and did not affect any normal immune response in the subjects with Japanese cedar-pollinosis. From the investigation that the effects of cultivars, tea seasons of crops and manufacturing methods, green or semi-fermented teas, made from fully-matured Benifuuki in second crop season, should be consumed. It is possible to develop functional articles such as beverage or food with this Benifuuki green tea.

Mercury-Induced Inhibition of Tyrosine Phosphorylation of Sperm Proteins and Altered Functional Dynamics of Buck Spermatozoa: an In Vitro Study.

Present study was undertaken on buck spermatozoa to investigate the effect of mercuric chloride on functional dynamics of buck spermatozoa. Four different concentrations (0.031, 0.125, 0.25 and 1.25μg/mL) of mercuric chloride, which were 1/40th, 1/10th, 1/5th and equivalent to the LC50 value of HgCl2, were selected for studying their effect following in vitro exposure for 15min and 3h. Exposure of spermatozoa to 0.031μg/mL mercuric chloride for 3h resulted in significant (p < 0.05) decrease in sperm motility, sperm having intact membrane, intact acrosome and high mitochondrial trans-membrane potential. However, following exposure to higher concentrations (0.25, 1.25μg/mL), similar results were observed even after 15min of exposure. HgCl2 significantly (p < 0.05) increased the levels of malondialdehyde and reactive oxygen species and significantly (p < 0.05) decreased total antioxidant capacity and superoxide dismutase activity in spermatozoa within 15min of exposure. Mercuric chloride-treated spermatozoa did not show capacitation, rather exhibited spontaneous acrosome reaction along with significant increase in intracellular Ca2+ and cAMP levels. Immuno-blotting of semen samples of control and 0.031μg/mL mercury-treated groups showed low intensity bands of p55, p70, p80, p105 and p190kDa tyrosine phosphorylation proteins while higher concentration-treated groups showed no such bands. Our findings evidently suggest that mercuric chloride even at 0.031μg/mL adversely affected sperm functions, inhibited tyrosine phosphorylation proteins and capacitation due to oxidative stress. Spontaneous acrosome reaction (AR) in mercury-treated spermatozoa may possibly be due to increase in intracellular Ca2+ and cAMP levels, and capacitation failure may be due to inhibition of tyrosine phosphorylation of proteins.

C1GALT1 is associated with poor survival and promotes soluble Ephrin A1-mediated cell migration through activation of EPHA2 in gastric cancer

C1GALT1 controls the crucial step of GalNAc-type O-glycosylation and is associated with both physiologic and pathologic conditions, including cancers. EPH receptors comprise the largest family of receptor tyrosine kinases (RTKs) and modulate a diverse range of developmental processes and human diseases. However, the role of C1GALT1 in the signaling of EPH receptors remains largely overlooked. Here, we showed that C1GALT1 high expression in gastric adenocarcinomas correlated with adverse clinicopathologic features and is an independent prognostic factor for poor overall survival. Silencing or loss of C1GALT1 inhibited cell viability, migration, invasion, tumor growth and metastasis, as well as increased apoptosis and cytotoxicity of 5-fluorouracil in AGS and MKN45 cells. Phospho-RTK array and western blot analysis showed that C1GALT1 depletion suppressed tyrosine phosphorylation of EPHA2 induced by soluble Ephrin A1-Fc. O-glycans on EPHA2 were modified by C1GALT1 and both S277A and T429A mutants, which are O-glycosites on EPHA2, dramatically enhanced phosphorylation of Y588, suggesting that not only overall O-glycan structures but also site-specific O-glycosylation can regulate EPHA2 activity. Furthermore, depletion of C1GALT1 decreased Ephrin A1-Fc induced migration and reduced Ephrin A1 binding to cell surfaces. The effects of C1GALT1 knockdown or knockout on cell invasiveness in vitro and in vivo were phenocopied by EPHA2 knockdown in gastric cancer cells. These results suggest that C1GALT1 promotes phosphorylation of EPHA2 and enhances soluble Ephrin A1-mediated migration primarily by modifying EPHA2 O-glycosylation. Our study highlights the importance of GalNAc-type O-glycosylation in EPH receptor-regulated diseases and identifies C1GALT1 as a potential therapeutic target for gastric cancer.

Anethole compromises human sperm function by affecting the sperm intracellular calcium concentration and tyrosine phosphorylation

Anethole is a natural anisole derivative that has been widely used in food and daily chemical industries, agricultural applications and the traditional medicine. It is closely related to aspects of daily life, and humans can easily be exposed to it. Although the reproductive toxicity of anethole was shown in the rat, its effect on human reproduction remains unknown. In this study, we examined the effect of anethole on human sperm in vitro. Different anethole doses (0.1, 1, 10, and 100 μM) were applied to ejaculated human sperm. Fertilization-essential functions, as well as the intracellular calcium concentration ([Ca2+]i) and tyrosine phosphorylation, two vital factors for regulating sperm function, were measured. The results indicated that 10 and 100 μM anethole significantly reduced the motility, hyperactivation, and penetration ability of human sperm (P < 0.05) and inhibited the increase in human sperm functions induced by progesterone, a hormone essential for sperm function activation. Additionally, 10 and 100 μM anethole decreased both basal and progesterone-increased tyrosine phosphorylation, [Ca2+]i, and the current of CATSPER, a cation channel of sperm predominant for Ca2+ influx. These results suggest that anethole inhibits human sperm functions by reducing sperm [Ca2+]i through CATSPER and suppressing tyrosine phosphorylation in vitro, raising the fact that the caution is needed when overtaking anethole.

Pre-metastatic niche triggers SDF-1/CXCR4 axis and promotes organ colonisation by hepatocellular circulating tumour cells via downregulation of Prrx1

BackgroundCirculating tumour cells (CTCs), especially mesenchymal CTCs, are important determinants of metastasis, which leads to most recurrence and mortality in hepatocellular carcinoma (HCC). However, little is known about the underlying mechanisms of CTC colonisation in pre-metastatic niches.MethodsDetection and classification of CTCs in patients were performed using the CanPatrol™ system. A lentiviral vector expressing Prrx1-targeting shRNA was constructed to generate a stable HCC cell line with low expression of Prrx1. The effect of Prrx1 knockdown on stemness, migration, and drug resistance of the cell line was assessed, including involvement of SDF-1/CXCR4 signalling. Promising clinical applications of an inhibitor of STAT3 tyrosine phosphorylation, C188–9, and specific blockade with CXCR4 antibody were explored.ResultsThe number of mesenchymal CTCs in blood was closely associated with tumour recurrence or metastasis. Pre-metastatic niche-derived SDF-1 could downregulate Prrx1, which induced the stemness, drug resistance, and increased expression of CXCR4 in HCC cells through the STAT3 pathway in vitro. In vivo, mice bearing tumours of Prrx1 low-expressing cells had significantly shorter survival. In xenograft tumours and clinical samples, loss of Prrx1 was negatively correlated with increased expression of CXCR4 in lung metastatic sites compared with that in the primary foci.ConclusionsThese findings demonstrate that decreased expression of Prrx1 stimulates SDF-1/CXCR4 signalling and contributes to organ colonisation with blood CTCs in HCC. STAT3 inhibition and specific blockade of CXCR4 have clinical potential as therapeutics for eliminating organ metastasis in advanced HCC.

PAQR3 suppresses the growth of non-small cell lung cancer cells via modulation of EGFR-mediated autophagy

ABSTRACT Macroautophagy/autophagy is an evolutionarily conserved intracellular process that recycles and degrades intracellular components to sustain homeostasis in response to deficiency of nutrients or growth factors. PAQR3 is a newly discovered tumor suppressor that also regulates autophagy induced by nutrient starvation via AMPK and MTORC1 signaling pathways. In this study, we investigated whether PAQR3 modulates EGFR-mediated autophagy and whether such regulation is associated with the tumor suppressive activity of PAQR3. PAQR3 is able to inhibit the in vitro and in vivo growth of non-small cell lung cancer (NSCLC) cells. PAQR3 potentiates autophagy induced by EGFR inhibitor erlotinib. Knockdown of PAQR3 abrogates erlotinib-mediated reduction of BECN1 interaction with autophagy inhibitory proteins RUBCN/Rubicon and BCL2. PAQR3 blocks the interaction of BECN1 with the activated form of EGFR and inhibits tyrosine phosphorylation of BECN1. Furthermore, inhibition of autophagy by knocking down ATG7 abrogates the tumor suppressive activity of PAQR3 in NSCLC cells. Collectively, these data indicate that PAQR3 suppresses tumor progression of NSCLC cells through modulating EGFR-regulated autophagy. Abbreviations AKT: thymoma viral proto-oncogene; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG14: autophagy related 14; BCL2: B cell leukemia/lymphoma 2; BECN1: beclin 1; CCK-8: cell counting kit-8; CQ: chloroquine diphosphate; DMEM: Dulbecco’s modified Eagle’s medium; EdU: 5-ethynyl-2ʹ-deoxyuridine; EGFR: epidermal growth factor receptor; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IgG: Immunoglobulin G; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; MTT: thiazolyl blue tetrazolium bromide; NSCLC: Non-small cell lung cancer; MAP2K/MEK: mitogen-activated protein kinase kinase; MAPK/ERK: mitogen-activated protein kinase; PAQR3: progestin and adipoQ receptor family member 3; PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; PRKAA/AMPK: protein kinase, AMP-activated alpha catalytic; RUBCN: rubicon autophagy regulator; RPS6: ribosomal protein S6; RAS: Ras proto-oncogene; RAF: Raf proto-oncogene; TKI: tyrosine kinase inhibitor; TUBA4A: tubulin alpha 4a; UVRAG: UV radiation resistance associated.