Induction of nitric oxide synthase (NOS) by soluble glucocorticoid induced tumor necrosis factor receptor (sGITR) is modulated by IFN-gamma in murine macrophage

Earlier study showed that glucocorticoid induced tumor necrosis factor receptor (GITR), a new TNFR family, activated murine macrophages to express inducible nitric oxide synthase (iNOS) and to generate nitric oxide (NO). A possible involvement of pro-inflammatory cytokines on NO production by GITR was investigated in vitro systems and signaling molecules contributing to sGITR-induced iNOS production are determined in Raw 264.7 cells, a murine macrophage cell line. The result showed that the synergy was afforded by the combination of GITR with IFN-g in a dose-dependent manner but IFN-gamma alone was not able to induce NOS. No effects were observed with TNF-alpha, IL-1beta, or IL-6 co-treated with GITR. To determine signaling molecules contributing to sGITR-induced iNOS production, a specific inhibitor for signal pathway proteins tested showed that PDTC (NF-kappaB) and genistein (tyrosine kinase) inhibited NOS induction significantly, while sodium orthovanadate (tyrosine phosphatase) potentiated NOS expression. These results suggest that activations of NF-kappaB were involved in induction of iNOS by GITR and IFN-gamma priming caused earlier and stronger NF-kappaB activation.     

Saponins Extracted from Tea (Camellia Sinensis) Flowers Induces Autophagy in Ovarian Cancer Cells

Tea flower saponins (TFS) possess effective anticancer properties. The diversity and complexity of TFS increases the difficulty of their extraction and purification from tea flowers. Here, multiple methods including solvent extraction, microporous resin separation and preparative HPLC separation were used to obtain TFS with a yield of 0.34%. Furthermore, we revealed that TFS induced autophagy—as evidenced by an increase in MDC-positive cell populations and mCherry-LC3B-labeled autolysosomes and an upregulation of LC3II protein levels. 3-MA reversed the decrease in cell viability induced by TFS, showing that TFS induced autophagic cell death. TFS-induced autophagy was not dependent on the Akt/mTOR/p70S6K signaling pathway. TFS-induced autophagy in OVCAR-3 cells was accompanied by ERK pathway activation and reactive oxygen species (ROS) generation. This paper is the first report of TFS-mediated autophagy of ovarian cancer cells. These results provide new insights for future studies of the anti-cancer effects of TFS.     

Synergistic induction of cancer cell migration regulated by Gβγ and phosphatidylinositol 3-kinase

Phosphatidylinositol 3-kinase (PI3K) is essential for both G protein-coupled receptor (GPCR)- and receptor tyrosine kinase (RTK)-mediated cancer cell migration. Here, we have shown that maximum migration is achieved by full activation of phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 (P-Rex1) in the presence of Gβγ and PI3K signaling pathways. Lysophosphatidic acid (LPA)- induced migration was higher than that of epidermal growth factor (EGF)-induced migration; however, LPA-induced activation of Akt was lower than that stimulated by EGF. LPA-induced migration was partially blocked by either Gβγ or RTK inhibitor and completely blocked by both inhibitors. LPA-induced migration was synergistically increased in the presence of EGF and vice versa. In correlation with these results, sphingosine-1-phosphate (S1P)-induced migration was also synergistically induced in the presence of insulin-like growth factor-1 (IGF-1). Finally, silencing of P-Rex1 abolished the synergism in migration as well as in Rac activation. Moreover, synergistic activation of MMP-2 and cancer cell invasion was attenuated by silencing of P-Rex1. Given these results, we suggest that P-Rex1 requires both Gβγ and PI3K signaling pathways for synergistic activation of Rac, thereby inducing maximum cancer cell migration and invasion.     

Role of phospholipase D1 in glucose-induced insulin secretion in pancreatic �� cells

As glucose is known to induce insulin secretion in pancreatic β cells, this study investigated the role of a phospholipase D (PLD)-related signaling pathway in insulin secretion caused by high glucose in the pancreatic β-cell line MIN6N8. It was found that the PLD activity and PLD1 expression were both increased by high glucose (33.3 mM) treatment. The dominant negative PLD1 inhibited glucose-induced Beta2 expression, and glucose-induced insulin secretion was blocked by treatment with 1-butanol or PLD1-siRNA. These results suggest that high glucose increased insulin secretion through a PLD1-related pathway. High glucose induced the binding of Arf6 to PLD1. Pretreatment with brefeldin A (BFA), an Arf inhibitor, decreased the PLD activity as well as the insulin secretion. Furthermore, BFA blocked the glucose-induced mTOR and p70S6K activation, while mTOR inhibition with rapamycin attenuated the glucose induced Beta2 expression and insulin secretion. Thus, when taken together, PLD1 would appear to be an important regulator of glucose-induced insulin secretion through an Arf6/PLD1/mTOR/p70S6K/Beta2 pathway in MIN6N8 cells.     

Design of Recombinant Stem Cell Factor–macrophage Colony Stimulating Factor Fusion Proteins and their Biological Activity <Emphasis Type="BoldItalic">In Vitro</Emphasis>

Summary Stem cell factor (SCF) and macrophage colony stimulating factor (M-CSF) can act in synergistic way to promote the growth of mononuclear phagocytes. SCF–M-CSF fusion proteins were designed on the computer using the Homology and Biopolymer modules of the software packages InsightII. Several existing crystal structures were used as templates to generate models of the complexes of receptor with fusion protein. The structure rationality of the fusion protein incorporated a series of flexible linker peptide was analyzed on InsightII system. Then, a suitable peptide GGGGSGGGGSGG was chosen for the fusion protein. Two recombinant SCF–M-CSF fusion proteins were generated by construction of a plasmid in which the coding regions of human SCF (1–165aa) and M-CSF (1–149aa) cDNA were connected by this linker peptide coding sequence followed by subsequent expression in insect cell. The results of Western blot and activity analysis showed that these two recombinant fusion proteins existed as a dimer with a molecular weight of ~84 KD under non-reducing conditions and a monomer of ~42 KD at reducing condition. The results of cell proliferation assays showed that each fusion protein induced a dose-dependent proliferative response. At equimolar concentration, SCF/M-CSF was about 20 times more potent than the standard monomeric SCF in stimulating TF-1 cell line growth, while M-CSF/SCF was 10 times of monomeric SCF. No activity difference of M-CSF/SCF or SCF/M-CSF to M-CSF (at same molar) was found in stimulating the HL-60 cell linear growth. The synergistic effect of SCF and M-CSF moieties in the fusion proteins was demonstrated by the result of clonogenic assay performed with human bone mononuclear, in which both SCF/M-CSF and M-CSF/SCF induced much higher number of CFU-M than equimolar amount of SCF or M-CSF or that of two cytokines mixture.     

Autophagic Activation and Decrease of Plasma Membrane Cholesterol Contribute to Anticancer Activities in Non-Small Cell Lung Cancer

Non-small cell lung cancer (NSCLC), an aggressive subtype of pulmonary carcinomas with high mortality, accounts for 85% of all lung cancers. Drug resistance and high recurrence rates impede the chemotherapeutic effect, making it urgent to develop new anti-NSCLC agents. Recently, we have demonstrated that para-toluenesulfonamide is a potential anti-tumor agent in human castration-resistant prostate cancer (CRPC) through inhibition of Akt/mTOR/p70S6 kinase pathway and lipid raft disruption. In the current study, we further addressed the critical role of cholesterol-enriched membrane microdomain and autophagic activation to para-toluenesulfonamide action in killing NSCLC. Similar in CRPC, para-toluenesulfonamide inhibited the Akt/mTOR/p70S6K pathway in NSCLC cell lines NCI-H460 and A549, leading to G1 arrest of the cell cycle and apoptosis. Para-toluenesulfonamide significantly decreased the cholesterol levels of plasma membrane. External cholesterol supplement rescued para-toluenesulfonamide-mediated effects. Para-toluenesulfonamide induced a profound increase of LC3-II protein expression and a significant decrease of p62 expression. Double staining of lysosomes and cellular cholesterol showed para-toluenesulfonamide-induced lysosomal transportation of cholesterol, which was validated using flow cytometric analysis of lysosome staining. Moreover, autophagy inhibitors could blunt para-toluenesulfonamide-induced effect, indicating autophagy induction. In conclusion, the data suggest that para-toluenesulfonamide is an effective anticancer agent against NSCLC through G1 checkpoint arrest and apoptotic cell death. The disturbance of membrane cholesterol levels and autophagic activation may play a crucial role to para-toluenesulfonamide action.     

Comparative studies of the colony-promoting activity of porcine kidney extract with several interleukins and colony-stimulating factors.

Porcine kidney extracts (PKE) possess colony-promoting activity (CPA) which stimulates primitive hematopoietic cells in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), but PKE itself does not stimulate colony formation on murine bone marrow cells. We have compared the CPA of PKE with that of recombinant cytokines or CSFs such as interleukin-1 alpha (IL-1 alpha), IL-3, IL-6, granulocyte colony-stimulating factor (G-CSF), GM-CSF and macrophage colony-stimulating factor (CSF-1). All of these factors were less potent than PKE. Furthermore, the combinations of IL-1 alpha or PKE with G-CSF, GM-CSF, IL-3 or IL-6 were examined in the presence of one of these factors such as CSF. It is found that PKE acts synergistically with G-CSF, GM-CSF, IL-3 and IL-6, showing enhancement ratios of 10, 2.5, 4.2 and 30, respectively. The combination of IL-1 alpha resulted in poor colony formation in contrast with those of PKE, except for CSF-1. These results suggest that the CPA of the factor(s) in PKE differ from the cytokines and CSFs tested in this study, and is significantly affected by various types of CSF.     

Involvement of DPY19L3 in Myogenic Differentiation of C2C12 Myoblasts

DPY19L3 has been identified as a C-mannosyltransferase for thrombospondin type-1 repeat domain-containing proteins. In this study, we focused on the role of DPY19L3 in the myogenic differentiation of C2C12 mouse myoblast cells. We carried out DPY19L3 gene depletion using the CRISPR/Cas9 system. The result showed that these DPY19L3-knockout cells could not be induced for differentiation. Moreover, the phosphorylation levels of MEK/ERK and p70S6K were suppressed in the DPY19L3-knockout cells compared with that of parent cells, suggesting that the protein(s) that is(are) DPY19L3-mediated C-mannosylated and regulate(s) MEK/ERK or p70S6K signaling is(are) required for the differentiation.     

Fisetin Inhibits UVB‐induced Cutaneous Inflammation and Activation of PI3K/AKT/NFκB Signaling Pathways in SKH‐1 Hairless Mice

Solar ultraviolet B (UVB) radiation has been shown to induce inflammation, DNA damage, p53 mutations and alterations in signaling pathways eventually leading to skin cancer. In this study, we investigated whether fisetin reduces inflammatory responses and modulates PI3K/AKT/NFκB cell survival signaling pathways in UVB‐exposed SKH‐1 hairless mouse skin. Mice were exposed to 180 mJ cm^?2 of UVB radiation on alternate days for a total of seven exposures, and fisetin (250 and 500 nmol) was applied topically after 15 min of each UVB exposure. Fisetin treatment to UVB‐exposed mice resulted in decreased hyperplasia and reduced infiltration of inflammatory cells. Fisetin treatment also reduced inflammatory mediators such as COX‐2, PGE₂ as well as its receptors (EP1–EP4) and MPO activity. Furthermore, fisetin reduced the level of inflammatory cytokines TNFα, IL‐1β and IL‐6 in UVB‐exposed skin. Fisetin treatment also reduced cell proliferation markers as well as DNA damage as evidenced by increased expression of p53 and p21 proteins. Further studies revealed that fisetin inhibited UVB‐induced expression of PI3K, phosphorylation of AKT and activation of the NFκB signaling pathway in mouse skin. Overall, these data suggest that fisetin may be useful against UVB‐induced cutaneous inflammation and DNA damage.     

UVA-activated 8-methoxypsoralen (PUVA) causes G2/M cell cycle arrest in Karpas 299 T-lymphoma cells

We investigated the effect of UVA-activated 8-methoxypsoralen (PUVA) on the cell line Karpas 299 derived from anaplastic large-cell lymphoma (ALCL) expressing chimeric fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM/ALK). NPM/ALK activates phosphatidylinositol 3 kinase (PI3K)/Akt pathway responsible for the cell protection from apoptosis. We found that PUVA treatment first induced G2/M cell cycle arrest resulting in a decrease in the cell proliferation rate. The mitochondrial apoptosis was triggered immediately following PUVA treatment, as we judged from the unmasking of mitochondrial membrane antigen 7A6. However, the mitochondrial membrane depolarization was not observed and caspase-3 was only slightly activated. The late apoptotic events were lacking: neither translocation of phosphatidylserine to the outer side of plasma membrane nor DNA fragmentation occurred. We revealed that PUVA enhanced the expression of peroxiredoxin, stress protein endoplasmin and galectin-3. Galectin-3 has been shown to protect mitochondrial membrane integrity and prevent cytochrome c release thereby blocking the effector stage of apoptosis. We suggest that the elevated level of this protein following PUVA treatment acts in synergy with the constitutively expressed chimeric kinase NPM/ALK to block the apoptosis.     

Differential modulation of zinc-stimulated p21(Cip/WAF1) and cyclin D1 induction by inhibition of PI3 kinase in HT-29 colorectal cancer cells

Activation of the extra cellular signal regulated kinase (ERK) pathway is involved in both proliferation and growth arrest of cells depending on intensity and duration of stimuli. In this study, we have elucidated differential regulation of the zinc-stimulated p21(CiP/WAF1) and cyclin D1 activation by inhibition of phosphoinositide 3-kinase (PI3K). In HT-29 colorectal cancer cells, the ERK activities were increased by zinc, which was accompanied by the induction of p21(Cip/WAF1) and cyclin D1. However, in the HT-29 cells pre-treated with PI3K inhibitor, LY294002, zinc induced further the p21(CiP/WAF) induction whereas abrogated cyclin D1 induction. In addition, the induction of p21(Cip/WAF1) expression completely inhibited the incorporation of bromodeoxyuridine (BrdU) into the nucleus, indicating that p21(CiP/WAF1) is an important indicator for ERK-dependent growth arrest. These studies suggest presence of an inter-related regulatory mechanism of cell proliferation by ERK and PI3K pathways.     

Theasaponin E1 Inhibits Platinum-Resistant Ovarian Cancer Cells through Activating Apoptosis and Suppressing Angiogenesis

Novel therapeutic strategies for ovarian cancer treatment are in critical need due to the chemoresistance and adverse side effects of platinum-based chemotherapy. Theasaponin E₁ (TSE1) is an oleanane-type saponin from Camellia sinensis seeds. Its apoptosis-inducing, cell cycle arresting and antiangiogenesis activities against platinum-resistant ovarian cancer cells were elucidated in vitro and using the chicken chorioallantoic membrane (CAM) assay. The results showed that TSE1 had more potent cell growth inhibitory effects on ovarian cancer OVCAR-3 and A2780/CP70 cells than cisplatin and was lower in cytotoxicity to normal ovarian IOSE-364 cells. TSE1 significantly induced OVCAR-3 cell apoptosis via the intrinsic and extrinsic apoptotic pathways, slightly arresting cell cycle at the G2/M phase, and obviously inhibited OVCAR-3 cell migration and angiogenesis with reducing the protein secretion and expression of vascular endothelial growth factor (VEGF). Western bolt assay showed that Serine/threonine Kinase (Akt) signaling related proteins including Ataxia telangiectasia mutated kinase (ATM), Phosphatase and tensin homolog (PTEN), Akt, Mammalian target of rapamycin (mTOR), Ribosome S6 protein kinase (p70S6K) and e IF4E-binding protein 1(4E-BP1) were regulated, and Hypoxia inducible factor-1α (HIF-1α) protein expression was decreased by TSE1 in OVCAR-3 cells. Moreover, TSE1 treatment potently downregulated protein expression of the Notch ligands including Delta-like protein 4 (Dll4) and Jagged1, and reduced the protein level of the intracellular domain (NICD) of Notch1. Combination treatment of TSE1 with the Notch1 signaling inhibitor tert-butyl (2S)-2-[[(2S)-2-[[2-(3,5-difluorophenyl)acetyl]amino]propanoyl]amino]-2-phenylacetate (DAPT), or the Akt signaling inhibitor wortmannin, showed a stronger inhibition toward HIF-1α activation compared with single compound treatment. Taken together, TSE1 might be a potential candidate compound for improving platinum-resistant ovarian cancer treatment via Dll4/Jagged1-Notch1-Akt-HIF-1α axis.     

Combined Treatment with PI3K Inhibitors BYL-719 and CAL-101 Is a Promising Antiproliferative Strategy in Human Rhabdomyosarcoma Cells

Rhabdomyosarcoma (RMS) is a highly malignant and metastatic pediatric cancer arising from skeletal muscle myogenic progenitors. Recent studies have shown an important role for AKT signaling in RMS progression. Aberrant activation of the PI3K/AKT axis is one of the most frequent events occurring in human cancers and serves to disconnect the control of cell growth, survival, and metabolism from exogenous growth stimuli. In the study reported here, a panel of five compounds targeting the catalytic subunits of the four class I PI3K isoforms (p110α, BYL-719 inhibitor; p110β, TGX-221 inhibitor; p110γ, {"type":"entrez-protein","attrs":{"text":"CZC24832","term_id":"994587862","term_text":"CZC24832"}}CZC24832; p110δ, CAL-101 inhibitor) and the dual p110α/p110δ, AZD8835 inhibitor, were tested on the RMS cell lines RD, A204, and SJCRH30. Cytotoxicity, cell cycle, apoptosis, and the activation of downstream targets were analyzed. Of the individual inhibitors, BYL-719 demonstrated the most anti-tumorgenic properties. BYL-719 treatment resulted in G1/G0 phase cell cycle arrest and apoptosis. When combined with CAL-101, BYL-719 decreased cell viability and induced apoptosis in a synergistic manner, equaling or surpassing results achieved with AZD8835. In conclusion, our findings indicate that BYL-719, either alone or in combination with the p110δ inhibitor, CAL-101, could represent an efficient treatment for human rhabdomyosarcoma presenting with aberrant upregulation of the PI3K signaling pathway.     

Activation of B cells using Schneider 2 cells expressing CD40 ligand for the enhancement of antigen presentation in vitro

CD40 ligand (CD40L) expressed by activated CD4+ T cells is a family member of membrane bound TNF family ligand and its interaction with CD40 expressed in APC has been shown to contribute in enhancing immune response. Exogenous stimulation through CD40 has been performed using soluble trimeric CD40L, anti-CD40 monoclonal antibody and cells expressing CD40L. Schneider 2 (S2) cells, a cell line derived from Drosophila melanogaster, was transfected with a plasmid vector, pAc5.1/V5-HisA, for the constitutive expression of CD40L (S2-CD40L). Upon incubation of S2-CD40L with B-lymphocytes for 6 days, activated B cells were examined by counting B cell numbers and for activation markers including CD86 and HLA Class II molecules. The activated B cells were tested for its efficient APC function by mixed lymphocyte reactions (MLR) and enzyme-linked Immunospot (ELISPOT) assay. S2-CD40L was cultured for a year and maintained CD40L expression (>90%). S2-CD40L induced B cell activation as demonstrated by increment of total B cells and up-regulation of CD86 and MHC Class II molecules. Activated B cells pulsed with peptide from human cytomegalovirus pp65 antigen efficiently induced both proliferation and IFN-gamma secretion of T cells. Our result suggests that S2-CD40L can efficiently and conveniently generate B cells as a functional APC and represents a potential role for B-cell mediated cancer immunotherapy.     

Activation of heat shock protein 70 promoter with meso-tetrahydroxyphenyl chlorin photodynamic therapy reported by green fluorescent protein in vitro and in vivo

Cellular responses to photodynamic therapy (PDT) include induction of heat shock proteins (HSP). We examined meso-tetrahydroxyphenyl chlorin (mTHPC) PDT-mediated HSP activation in EMT6 cells stably transfected with a plasmid containing the gene for green fluorescent protein (GFP) driven by an hsp70 promoter. mTHPC incubation induced concentration-dependent GFP expression. Irradiation of cells exposed to a sensitizer concentration that induced a slight increase in GFP and no loss of cell viability resulted in fluence-dependent GFP accumulation. In response to drug only and to PDT, GFP levels increased to a maximum of four- to five-fold above control levels with increasing drug or fluence and then decreased at higher doses. A trypan blue-exclusion assay confirmed that decreased GFP levels in both cases were due to a loss of cell viability. For initial evaluation in vivo, HSP70/ GFP-transfected EMT6 tumors were grown in BALB/c mice and subjected to mTHPC-PDT with a fluence of 1 J/cm2. Six hours after PDT, GFP fluorescence was imaged in these tumors through the intact skin in vivo. These results indicate that sublethal doses of mTHPC-PDT stimulate GFP expression under the control of an hsp70 promoter and illustrate the potential of noninvasively monitoring reporter protein fluorescence as a measure of molecular response to PDT.