Mesenchymal Stem Cells and Extracellular Vesicles Derived from Canine Adipose Tissue Ameliorates Inflammation, Skin Barrier Function and Pruritus by Reducing JAK/STAT Signaling in Atopic Dermatitis.

Canine atopic dermatitis (AD) is a common chronic inflammatory skin disorder resulting from imbalance between T lymphocytes. Current canine AD treatments use immunomodulatory drugs, but some of the dogs have limitations that do not respond to standard treatment, or relapse after a period of time. Thus, the purpose of this study was to evaluate the immunomodulatory effect of mesenchymal stem cells derived from canine adipose tissue (cASCs) and cASCs-derived extracellular vesicles (cASC-EVs) on AD. First, we isolated and characterized cASCs and cASCs-EVs to use for the improvement of canine atopic dermatitis. Here, we investigated the effect of cASCs or cASC-EVs on DNCB-induced AD in mice, before using for canine AD. Interestingly, we found that cASCs and cASC-EVs improved AD-like dermatitis, and markedly decreased levels of serum IgE, (49.6%, p = 0.002 and 32.1%, p = 0.016 respectively) epidermal inflammatory cytokines and chemokines, such as IL-4 (32%, p = 0.197 and 44%, p = 0.094 respectively), IL-13 (47.4%, p = 0.163, and 50.0%, p = 0.039 respectively), IL-31 (64.3%, p = 0.030 and 76.2%, p = 0.016 respectively), RANTES (66.7%, p = 0.002 and 55.6%, p = 0.007) and TARC (64%, p = 0.016 and 86%, p = 0.010 respectively). In addition, cASCs or cASC-EVs promoted skin barrier repair by restoring transepidermal water loss, enhancing stratum corneum hydration and upregulating the expression levels of epidermal differentiation proteins. Moreover, cASCs or cASC-EVs reduced IL-31/TRPA1-mediated pruritus and activation of JAK/STAT signaling pathway. Taken together, these results suggest the potential of cASCs or cASC-EVs for the treatment of chronic inflammation and damaged skin barrier in AD or canine AD.

Mealworm Oil (MWO) Enhances Wound Healing Potential through the Activation of Fibroblast and Endothelial Cells.

Mealworm and mealworm oil (MWO) have been reported to affect antioxidant, anti-coagulation, anti-adipogenic and anti-inflammatory activities. However, the function of MWO in wound healing is still unclear. In this study, we found that MWO induced the migration of fibroblast cells and mRNA expressions of wound healing factors such as alpha-smooth muscle actin (α-SMA), collagen-1 (COL-1) and vascular endothelial growth factor (VEGF) in fibroblast cells. The tube formation and migration of endothelial cells were promoted through the activation of VEGF/VEGF receptor-2 (VEGFR-2)-mediated downstream signals including AKT, extracellular signal-regulated kinase (ERK) and p38 by MWO-stimulated fibroblasts for angiogenesis. Moreover, we confirmed that MWO promoted skin wound repair by collagen synthesis, re-epithelialization and angiogenesis in an in vivo excisional wound model. These results demonstrate that MWO might have potential as a therapeutic agent for the treatment of skin wounds.

3'-sialyllactose targets cell surface protein, SIGLEC-3, and induces megakaryocyte differentiation and apoptosis by lipid raft-dependent endocytosis.

3'-sialyllactose is one of the abundant components in human milk oligosaccharides (HMOs) that protect infants from various viral infections in early stages of immune system development. 3SL is a combination of lactose and sialic acid. Most sialic acids are widely expressed in animal cells and they bind to siglec proteins. In this study, we demonstrate that 3SL specifically binds to CD33. It induces megakaryocyte differentiation and subsequent apoptosis by targeting cell surface protein siglec-3 (CD33) in human chronic myeloid leukemia K562 cells. The 3SL-bound CD33 was internalized to the cytosol via caveolae-dependent endocytosis. At the molecular level, 3SL-bound CD33 recruits the suppressor of cytokine signaling 3 (SOCS3) and SH2 domain-containing protein tyrosine phosphatase 1 (SHP1). SOCS3 is degraded with CD33 by proteasome degradation, while SHP-1 activates extracellular signal-regulated kinase (ERK) to induce megakaryocytic differentiation and subsequent apoptosis. The present study, therefore, suggests that 3SL is a potential anti-leukemia agent affecting differentiation and apoptosis.

Ilimaquinone Induces the Apoptotic Cell Death of Cancer Cells by Reducing Pyruvate Dehydrogenase Kinase 1 Activity.

In cancer cells, aerobic glycolysis rather than oxidative phosphorylation (OxPhos) is generally preferred for the production of ATP. In many cancers, highly expressed pyruvate dehydrogenase kinase 1 (PDK1) reduces the activity of pyruvate dehydrogenase (PDH) by inducing the phosphorylation of its E1α subunit (PDHA1) and subsequently, shifts the energy metabolism from OxPhos to aerobic glycolysis. Thus, PDK1 has been regarded as a target for anticancer treatment. Here, we report that ilimaquinone (IQ), a sesquiterpene quinone isolated from the marine sponge Smenospongia cerebriformis, might be a novel PDK1 inhibitor. IQ decreased the cell viability of human and murine cancer cells, such as A549, DLD-1, RKO, and LLC cells. The phosphorylation of PDHA1, the substrate of PDK1, was reduced by IQ in the A549 cells. IQ decreased the levels of secretory lactate and increased oxygen consumption. The anticancer effect of IQ was markedly reduced in PDHA1-knockout cells. Computational simulation and biochemical assay revealed that IQ interfered with the ATP binding pocket of PDK1 without affecting the interaction of PDK1 and the E2 subunit of the PDH complex. In addition, similar to other pyruvate dehydrogenase kinase inhibitors, IQ induced the generation of mitochondrial reactive oxygen species (ROS) and depolarized the mitochondrial membrane potential in the A549 cells. The apoptotic cell death induced by IQ treatment was rescued in the presence of MitoTEMPO, a mitochondrial ROS inhibitor. In conclusion, we suggest that IQ might be a novel candidate for anticancer therapeutics that act via the inhibition of PDK1 activity.

Esculentoside H inhibits colon cancer cell migration and growth through suppression of MMP-9 gene expression via NF-kB signaling pathway.

A water-soluble saponin, Esculentoside H (EsH), 3-O-(O-ß-d-glucopyranosyl-(1→4)-ß-d-xylopyranosyl)-28-ß-d-glucopyranosylphytolaccagenin has been isolated and purified from the root extract of perennial plant Phytolacca esculenta. EsH is known to be an anticancer compound, having a capacity for TNF-α release. However, the effects of EsH on migration and growth in tumor cells have not yet been reported. In the current study, the suppressive effects of EsH on phorbol 12-myristate 13-acetate (PMA)-induced cell migration were examined in murine colon cancer CT26 cells and human colon cancer HCT116 cells. Interestingly, the transwell assay and wound healing show that EsH suppresses the PMA-induced migration and growth potential of HCT116 and CT26 colon cancer cells, respectively. EsH dose-dependently suppressed matrix metalloproteinases-9 (MMP-9) expression that was upregulated upon PMA treatment in messenger RNA levels and protein secretion. Since the expression of MMP-9 is correlated with nuclear factor-κB (NF-κB) signaling, it has been examined whether EsH inhibits PMA-induced IκB phosphorylation that leads to the suppression of NK-κB nuclear translocation. EsH repressed the phosphorylation level of JNK, but not extracellular signal-regulated kinase and p38 signaling when the cells were treated with PMA. Overall, these results demonstrated that EsH could suppress cancer migration through blockage of the JNK1/2 and NF-κB signaling-mediated MMP-9 expression.

Tyrosine phosphorylation of mitochondrial pyruvate dehydrogenase kinase 1 is important for cancer metabolism.

Many tumor cells rely on aerobic glycolysis instead of oxidative phosphorylation for their continued proliferation and survival. Myc and HIF-1 are believed to promote such a metabolic switch by, in part, upregulating gene expression of pyruvate dehydrogenase (PDH) kinase 1 (PDHK1), which phosphorylates and inactivates mitochondrial PDH and consequently pyruvate dehydrogenase complex (PDC). Here we report that tyrosine phosphorylation enhances PDHK1 kinase activity by promoting ATP and PDC binding. Functional PDC can form in mitochondria outside of the matrix in some cancer cells and PDHK1 is commonly tyrosine phosphorylated in human cancers by diverse oncogenic tyrosine kinases localized to different mitochondrial compartments. Expression of phosphorylation-deficient, catalytic hypomorph PDHK1 mutants in cancer cells leads to decreased cell proliferation under hypoxia and increased oxidative phosphorylation with enhanced mitochondrial utilization of pyruvate and reduced tumor growth in xenograft nude mice. Together, tyrosine phosphorylation activates PDHK1 to promote the Warburg effect and tumor growth.

Ganglioside GM3 suppresses lipopolysaccharide-induced inflammatory responses in rAW 264.7 macrophage cells through NF-κB, AP-1, and MAPKs signaling.

Gangliosides are known to specifically inhibit vascular leukocyte recruitment and consequent interaction with the injured endothelium, the basic inflammatory process. In this study, we have found that the production of nitric oxide (NO), a main regulator of inflammation, is suppressed by GM3 on murine macrophage RAW 264.7 cells, when induced by LPS. In addition, GM3 attenuated the increase in cyclooxyenase-2 (COX-2) protein and mRNA levels in lipopolysaccharide (LPS)-activated RAW 264.7 cells in a dose-dependent manner. Moreover, GM3 inhibited the expression and release of pro-inflammatory cytokines of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß) in RAW 264.7 macrophages. At the intracellular level, GM3 inhibited LPS-induced nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activator protein (AP)-1 in RAW 264.7 macrophages. We, therefore, investigated whether GM3 affects mitogen-activated protein kinase (MAPK) phosphorylation, a process known as the upstream signaling regulator. GM3 dramatically reduced the expression levels of the phosphorylated forms of ERK, JNK, and p38 in LPS-activated RAW 264.7 cells. These results indicate that GM3 is a promising suppressor of the vascular inflammatory responses and ganglioside GM3 suppresses the LPS-induced inflammatory response in RAW 264.7 macrophages by suppression of NF-κB, AP-1, and MAPKs signaling. Accordingly, GM3 is suggested as a beneficial agent for the treatment of diseases that are associated with inflammation.

Water-extracted branch of Cinnamomum cassia promotes lung cancer cell apoptosis by inhibiting pyruvate dehydrogenase kinase activity.

Cinnamomum cassia Blume has been widely reported as the anti-tumor agent. However, the precise mechanism underlying its pro-apoptotic action is still not clear. Restraining aerobic glycolysis through suppression of pyruvate dehydrogenase kinase (PDHK) is a promising strategy for cancer inhibition. In this study, we performed to investigate the anti-tumor action of C. cassia is mediated by PDHK inhibition. The inhibition of water-extracted branch of C. cassia (WBCC) on the activity of PDHK using both in vitro and cell-based kinase assay were examined in several lung cancer cells. WBCC reduced viabilities of several lung cancer cells with minimal cytotoxicity on normal bronchial epithelial cells. WBCC decreased lactate production through inhibiting activity of PDHK. In consequence of PDHK inhibition, WBCC increased ROS production, which damage mitochondria membrane stability. In addition, WBCC induced ROS- and mitochondria-dependent apoptotic cell death. Among the components of WBCC, cinnamic acid was founded as a major inhibitor on PDHK activity. This is first report that WBCC induces apoptosis of lung cancer cells through inhibiting PDHK activity. Our findings suggest that WBCC and cinnamic acid can be potential candidates for developing novel anti-cancer drugs through glycolysis metabolism.

Monosialyl Ganglioside GM3 Decreases Apolipoprotein B-100 Secretion in Liver Cells.

Some sialic acid-containing glycolipids are known to regulate development of atherosclerosis with accumulated plasma apolipoprotein B-100 (Apo-B)-containing lipoproteins, because Apo-B as an atherogenic apolipoprotein is assembled mainly in VLDL and LDL. Previously, we have elucidated that disialyl GD3 promotes the microsomal triglyceride transfer protein (MTP) gene expression and secretion of triglyceride (TG)-assembled ApoB, claiming the GD3 role in ApoB lipoprotein secretion in liver cells. In the synthetic pathway of gangliosides, GD3 is synthesized by addition of a sialic acid residue to GM3. Thus, there should be some regulatory links between GM3 and GD3. In this study, exogenous and endogenous monosialyl GM3 has been examined how GM3 plays a role in ApoB secretion in Chang liver cells in a view point of MTP and ApoB degradation in the same cells. The level of GM3 ganglioside in the GM3 synthase gene-transfected cells was increased in the cell extract, but not in the medium. In addition, GM3 synthase gene-transfected cells showed a diminished secretion of TG-enriched ApoB with a lower content of TG in the medium. Exogenous GM3 treatment for 24 h exerted a dose dependent inhibitory effect on ApoB secretion together with TG, while a liver-specific albumin was unchanged, indicating that GM3 effect is limited to ApoB secretion. GM3 decreased the mRNA level of MTP gene, too. ApoB protein assembly dysregulated by GM3 indicates the impaired ApoB secretion is caused by a proteasome-dependent pathway. Treatment with small interfering RNAs (siRNAs) decreased ApoB secretion, but GM3-specific antibody did not. These results indicate that plasma membrane associated GM3 inhibits ApoB secretion, lowers development of atherosclerosis by decreasing the secretion of TG-enriched ApoB containing lipoproteins, suggesting that GM3 is an inhibitor of ApoB and TG secretion in liver cells. J. Cell. Biochem. 118: 2168-2181, 2017. © 2017 Wiley Periodicals, Inc.

Anti-Inflammatory Effect of Ascochlorin in LPS-Stimulated RAW 264.7 Macrophage Cells Is Accompanied With the Down-Regulation of iNOS, COX-2 and Proinflammatory Cytokines Through NF-κB, ERK1/2, and p38 Signaling Pathway.

A natural compound C23 H32 O4 Cl, ascochlorin (ASC) isolated from an incomplete fungus, Ascochyta viciae has been known to have several biological activities as an antibiotic, antifungal, anti-cancer, anti-hypolipidemic, and anti-hypertension agent. In this study, anti-inflammatory activity has been investigated in lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells, since ASC has not been observed on the inflammatory events. The present study has clearly shown that ASC (1-50 µM) significantly suppressed the production of nitric oxide (NO) and prostaglandin E2 (PGE2 ) and decreased the gene expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in a dose-dependent manner. Moreover, ASC inhibited the mRNA expression and the protein secretion of interleukin (IL)-1ß and IL-6 but not tumor necrosis factor (TNF)-α in LPS-stimulated RAW 264.7 macrophage cells. In addition, ASC suppressed nuclear translocation and DNA binding affinity of nuclear factor-κB (NF-κB). Furthermore, ASC down-regulated phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2) and p-p38. These results demonstrate that ASC exhibits anti-inflammatory effects in RAW 264.7 macrophage cells.

Luteolin inhibits recruitment of monocytes and migration of Lewis lung carcinoma cells by suppressing chemokine (C-C motif) ligand 2 expression in tumor-associated macrophage.

Tumor-associated macrophages (TAMs) play pivotal roles in the progression of cancer. In order to investigate a novel candidate that inhibits the tumor-supporting M2-like phenotype of TAMs, a murine macrophage cell line RAW 264.7 cells were treated with interleukin (IL)-4. Luteolin inhibited phosphorylation of signal transducer and activator of transcription 6 (STAT6), a main downstream signal of IL-4, and reduced the expression of the M2-associated genes. In addition, Luminex multiplex analysis for secreted cytokines revealed that IL-4-enhanced secretion of chemokine (C-C motif) ligand 2 (CCL2) was reduced by luteolin treatment. IL-4-stimulated migration of monocyte, THP-1 cells, was inhibited by luteolin treatment and recovered by recombinant CCL2 supplement. Moreover, luteolin decreased migration of Lewis lung carcinoma cells in a CCL2-dependent manner. Given the important role of the TAM phenotype in the tumor microenvironment, inhibitory effect of luteolin on the monocyte recruitment and cancer migration via suppression of the TAM-secreted CCL2 may suggest a novel therapeutic approach to treat malignant tumors.

Integrin αVß3 and αVß5 are required for leukemia inhibitory factor-mediated the adhesion of trophoblast cells to the endometrial cells.

The embryo implantation including adhesion between trophoblast and endometrium is a crucial process for the successful pregnancy. LIF and adhesion molecules including integrins are known as significant factors for embryo implantation. However, the function of LIF on the regulation of adhesion molecule expression and promotion of trophoblast adhesion to endometrial cells has not been fully elucidated. Here we show that LIF significantly induced mRNA expression of ITGAV, ITGB3, and ITGB5 in endometrial cells, as evidenced by RT-PCR and qRT-PCR analysis. Based on the results from treatment of antagonist for LIF receptor (hLA), LIF positively regulates expression of integrin αV, ß3, and ß5, and adhesion of the human trophectoderm-derived JAr cells to endometrial Ishikawa cells. Furthermore, the adhesion between trophoblastic cells and LIF-stimulated endometrial cells was significantly reduced by neutralization of LIF-mediated integrin ß3 and ß5 expression on endometrial cell surface with integrin subunit ß3 and ß5 antibodies. Taken together, we firstly demonstrate that LIF enhances the adhesion of trophoblastic cells to endometrial cells by up-regulating expression of integrin heterodimer αVß3 and αVß5, indicating the promotion of endometrial receptivity for embryo implantation.

Abiotic stress of ambient cold temperature regulates the host receptivity to pathogens by cell surfaced sialic acids.

Ambient cold temperature, as an abiotic stress, regulates the survival, stability, transmission, and infection of pathogens. However, the effect of cold temperature on the host receptivity to the pathogens has not been fully studied. In this study, the expression of terminal α-2,3- and α-2,6-sialic acids were increased in murine lung tissues, especially bronchial epithelium, by exposure to cold condition. The expression of several sialyltransferases were also increased by exposure to cold temperature. Furthermore, in human bronchial epithelial BEAS-2B cells, the expressions of α-2,3- and α-2,6-sialic acids, and mRNA levels of sialyltransferases were increased in the low temperature condition at 33 °C. On the other hand, the treatment of Lith-Gly, a sialyltransferase inhibitor, blocked the cold-induced expression of sialic acids on surface of BEAS-2B cells. The binding of influenza H1N1 hemagglutinin (HA) toward BEAS-2B cells cultured at low temperature condition was increased, compared to 37 °C. In contrast, the cold-increased HA binding was blocked by treatment of lithocholicglycine and sialyl-N-acetyl-D-lactosamines harboring α-2,3- and α-2,6-sialyl motive. These results suggest that the host receptivity to virus at cold temperature results from the expressions of α-2,3- and α-2,6-sialic acids through the regulation of sialyltransferase expression.

Housekeeping promoter 5'pcmah-2 of pig CMP-N-acetylneuraminic acid hydroxylase gene for NeuGc expression.

In the present study, we isolated pCMAH house-keeping promoter regions (Ph), which are responsible for transcriptional regulation and which are located upstream of the alternative transcript pcmah-2. Luciferase reporter assays using serial construction of each deleted promoter demonstrated that the Ph promoter was highly active in pig-derived kidney PK15. Ph promoter of pcmah lacked a TATA box, but contained three putative Sp1 binding sites. Mutations of these Sp1 binding sites always resulted in the reduction of luciferase activities in Ph-334. In addition, treatment with mithramycin A (25-100 nM) decreased the luciferase activities of the Ph promoters and NeuGc expression in a dose-dependent manner. Electrophoretic mobility shift assay analysis revealed that the probes containing each Sp1 binding site bound to Sp1. Taken together, the results indicate that Sp1 bind to their putative binding sites on the Ph promoter regions of pcmah and positively regulate the promoter activity in pig kidney cells. Interspecies comparison of 5'UTRs and 5'flanking regions shows high homology between pig and cattle, and Sp1 binding sites existing in genomic regions corresponding Ph region are evolutionally conserved.

Water-extracted Perilla frutescens increases endometrial receptivity though leukemia inhibitory factor-dependent expression of integrins.

The leaves and stems of Perilla frutescens var. acuta Kudo (PF) have been used to prevent threatened abortion in traditional medicine in the East Asian countries. Because reduced receptivity of endometrium is a cause of abortion, we analyzed the action of PF on the endometrial receptivity. PF increased the level of leukemia inhibitory factor (LIF), a major cytokine regulating endometrial receptivity, and LIF receptor in human endometrial Ishikawa cells. The PF-induced LIF expression was mediated by c-jun N-terminal kinase (JNK) and p38 pathways. Adhesion between Ishikawa cells and trophoblastic JAr cells stimulated by PF treatment was abolished by knock down of LIF expression or antagonism of LIFR. In addition, the expressions of integrin ß3 and ß5 were increased by PF treatment in Ishikawa cells. The PF-induced expression of integrin ß3 and ß5 was reduced with an LIFR antagonist. Neutralization of both integrins successfully blocked PF-stimulated adhesion of JAr cells and Ishikawa cells. These results suggest that PF enhanced the adhesion between Ishikawa cells and JAr cells by increasing the expression of integrin ß3 and ß5 via an LIF-dependent pathway. Given the importance of endometrial receptivity in successful pregnancy, PF can be a novel and effective candidate for improving pregnancy rate.

Cold stress aggravates inflammatory responses in an LPS-induced mouse model of acute lung injury.

Although the relationship between environmental cold temperature and susceptibility to respiratory infection is generally accepted, the effect of ambient cold temperature on host reactivity in lung inflammation has not been fully studied. To examine the function of ambient cold temperature on lung inflammation, mice were exposed to 4 °C for 8 h each day for 14 days. In the lungs of mice exposed to cold stress, inflammatory cells in bronchoalveolar lavage (BAL) fluid and lung tissues were slightly increased by about twofold. However, the structures of pulmonary epithelial cells were kept within normal limits. Next, we examined the effect of cold stress on the inflammatory responses in a lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model. The infiltration of neutrophils and inflammation of lung tissue determined by histology were significantly increased by exposure to ambient cold temperature. In addition, the production of pro-inflammatory cytokines including interleukin (IL)-12, IL-17, and monokine induced by gamma interferon (MIG) was elevated by exposure to cold stress. Therefore, we suggest that cold stress is a factor that exacerbates lung inflammation including ALI. To our knowledge, this is the first report on the relationship between cold stress and severity of lung inflammation.

Exogenous and Endogeneous Disialosyl Ganglioside GD1b Induces Apoptosis of MCF-7 Human Breast Cancer Cells.

Gangliosides have been known to play a role in the regulation of apoptosis in cancer cells. This study has employed disialyl-ganglioside GD1b to apoptosis in human breast cancer MCF-7 cells using exogenous treatment of the cells with GD1b and endogenous expression of GD1b in MCF-7 cells. First, apoptosis in MCF-7 cells was observed after treatment of GD1b. Treatment of MCF-7 cells with GD1b reduced cell growth rates in a dose and time dependent manner during GD1b treatment, as determined by XTT assay. Among the various gangliosides, GD1b specifically induced apoptosis of the MCF-7 cells. Flow cytometry and immunofluorescence assays showed that GD1b specifically induces apoptosis in the MCF-7 cells with Annexin V binding for apoptotic actions in early stage and propidium iodide (PI) staining the nucleus of the MCF-7 cells. Treatment of MCF-7 cells with GD1b activated apoptotic molecules such as processed forms of caspase-8, -7 and PARP (Poly(ADP-ribose) polymerase), without any change in the expression of mitochondria-mediated apoptosis molecules such as Bax and Bcl-2. Second, to investigate the effect of endogenously produced GD1b on the regulation of cell function, UDP-gal: ß1,3-galactosyltransferase-2 (GD1b synthase, Gal-T2) gene has been transfected into the MCF-7 cells. Using the GD1b synthase-transfectants, apoptosis-related signal proteins linked to phenotype changes were examined. Similar to the exogenous GD1b treatment, the cell growth of the GD1b synthase gene-transfectants was significantly suppressed compared with the vector-transfectant cell lines and transfection activated the apoptotic molecules such as processed forms of caspase-8, -7 and PARP, but not the levels of expression of Bax and Bcl-2. GD1b-induced apoptosis was blocked by caspase inhibitor, Z-VAD. Therefore, taken together, it was concluded that GD1b could play an important role in the regulation of breast cancer apoptosis.

Tyr-94 phosphorylation inhibits pyruvate dehydrogenase phosphatase 1 and promotes tumor growth.

Many cancer cells rely more on aerobic glycolysis (the Warburg effect) than mitochondrial oxidative phosphorylation and catabolize glucose at a high rate. Such a metabolic switch is suggested to be due in part to functional attenuation of mitochondria in cancer cells. However, how oncogenic signals attenuate mitochondrial function and promote the switch to glycolysis remains unclear. We previously reported that tyrosine phosphorylation activates and inhibits mitochondrial pyruvate dehydrogenase kinase (PDK) and phosphatase (PDP), respectively, leading to enhanced inhibitory serine phosphorylation of pyruvate dehydrogenase (PDH) and consequently inhibition of pyruvate dehydrogenase complex (PDC) in cancer cells. In particular, Tyr-381 phosphorylation of PDP1 dissociates deacetylase SIRT3 and recruits acetyltransferase ACAT1 to PDC, resulting in increased inhibitory lysine acetylation of PDHA1 and PDP1. Here we report that phosphorylation at another tyrosine residue, Tyr-94, inhibits PDP1 by reducing the binding ability of PDP1 to lipoic acid, which is covalently attached to the L2 domain of dihydrolipoyl acetyltransferase (E2) to recruit PDP1 to PDC. We found that multiple oncogenic tyrosine kinases directly phosphorylated PDP1 at Tyr-94, and Tyr-94 phosphorylation of PDP1 was common in diverse human cancer cells and primary leukemia cells from patients. Moreover, expression of a phosphorylation-deficient PDP1 Y94F mutant in cancer cells resulted in increased oxidative phosphorylation, decreased cell proliferation under hypoxia, and reduced tumor growth in mice. Together, our findings suggest that phosphorylation at different tyrosine residues inhibits PDP1 through independent mechanisms, which act in concert to regulate PDC activity and promote the Warburg effect.

Tyr-301 phosphorylation inhibits pyruvate dehydrogenase by blocking substrate binding and promotes the Warburg effect.

The mitochondrial pyruvate dehydrogenase complex (PDC) plays a crucial role in regulation of glucose homoeostasis in mammalian cells. PDC flux depends on catalytic activity of the most important enzyme component pyruvate dehydrogenase (PDH). PDH kinase inactivates PDC by phosphorylating PDH at specific serine residues, including Ser-293, whereas dephosphorylation of PDH by PDH phosphatase restores PDC activity. The current understanding suggests that Ser-293 phosphorylation of PDH impedes active site accessibility to its substrate pyruvate. Here, we report that phosphorylation of a tyrosine residue Tyr-301 also inhibits PDH α 1 (PDHA1) by blocking pyruvate binding through a novel mechanism in addition to Ser-293 phosphorylation. In addition, we found that multiple oncogenic tyrosine kinases directly phosphorylate PDHA1 at Tyr-301, and Tyr-301 phosphorylation of PDHA1 is common in EGF-stimulated cells as well as diverse human cancer cells and primary leukemia cells from human patients. Moreover, expression of a phosphorylation-deficient PDHA1 Y301F mutant in cancer cells resulted in increased oxidative phosphorylation, decreased cell proliferation under hypoxia, and reduced tumor growth in mice. Together, our findings suggest that phosphorylation at distinct serine and tyrosine residues inhibits PDHA1 through distinct mechanisms to impact active site accessibility, which act in concert to regulate PDC activity and promote the Warburg effect.

Induction of Apoptosis and Antitumor Activity of Eel Skin Mucus, Containing Lactose-Binding Molecules, on Human Leukemic K562 Cells.

For innate immune defense, lower animals such as fish and amphibian are covered with skin mucus, which acts as both a mechanical and biochemical barrier. Although several mucus sources have been isolated and studied for their biochemical and immunological functions, the precise mechanism(s) of action remains unknown. In the present study, we additionally found the eel skin mucus (ESM) to be a promising candidate for use in anti-tumor therapy. Our results showed that the viability of K562 cells was decreased in a dose-dependent manner by treatment with the isolated ESM. The cleaved forms of caspase-9, caspase-3 and poly adenosine diphosphate-ribose polymerase were increased by ESM. The levels of Bax expression and released cytochrome C were also increased after treatment with ESM. Furthermore, during the ESM mediated-apoptosis, phosphorylation levels of ERK1/2 and p38 but not JNK were increased and cell viabilities of the co-treated cells with ESM and inhibitors of ERK 1/2 or p38 were also increased. In addition, treatment with lactose rescued the ESM-mediated decrease in cell viability, indicating lactose-containing glycans in the leukemia cells acted as a counterpart of the ESM for interaction. Taken together, these results suggest that ESM could induce mitochondria-mediated apoptosis through membrane interaction of the K562 human leukemia cells. To the best of our knowledge, this is the first observation that ESM has anti-tumor activity in human cells.