Triptolide-induced suppression of phospholipase D expression inhibits proliferation of MDA-MB-231 breast cancer cells

In spite of the importance of phospholipase D (PLD) in cell proliferation and tumorigenesis, little is known about the molecules regulating PLD expression. Thus, identification of small molecules inhibiting PLD expression would be an important advance for PLD-mediated physiology. We examined one such here, denoted "Triptolide", which was identified in a chemical screen for inhibitors of PLD expression using cell assay system based on measurement of PLD promoter activity. Triptolide significantly suppressed the expression of both PLD1 and PLD2 with sub-µM potency in MDA-MB-231 breast cancer cells as analyzed by promoter assay and RT-PCR. Moreover, triptolide abolished the protein level of PLD in a time and dose-dependent manner. Triptolide-induced PLD1 downregulation was also observed in all the cancer cells examined, suggesting a general phenomenon detected in various cancer cells. Decrease of PLD expression by triptolide suppressed both basal and PMA-induced PLD activity. In addition, triptolide inhibited activation of NFκB which increased PLD1 expression. Ultimately, downregulation of PLD by triptolide inhibited proliferation of breast cancer cells. Taken together, we demonstrate that triptolide suppresses the expression of PLD via inhibition of NFκB activation and then decreases cell proliferation.     

Phospholipase D activates HIF-1-VEGF pathway via phosphatidic acid

Growth factor-stimulated phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC), generating phosphatidic acid (PA) which may act as a second messenger during cell proliferation and survival. Therefore, PLD is believed to play an important role in tumorigenesis. In this study, a potential mechanism for PLD-mediated tumorigenesis was explored. Ectopic expression of PLD1 or PLD2 in human glioma U87 cells increased the expression of hypoxia-inducible factor-1α (HIF-1α) protein. PLD-induced HIF-1 activation led to the secretion of vascular endothelial growth factor (VEGF), a HIF-1 target gene involved in tumorigenesis. PLD induction of HIF-1α was significantly attenuated by 1-butanol which blocks PA production by PLD, and PA per se was able to elevate HIF-1α protein level. Inhibition of mTOR, a PA-responsive kinase, reduced the levels of HIF-1α and VEGF in PLD-overexpressed cells. Epidermal growth factor activated PLD and increased the levels of HIF-1α and VEGF in U87 cells. A specific PLD inhibitor abolished expression of HIF-1α and secretion of VEGF. PLD may utilize HIF-1-VEGF pathway for PLD-mediated tumor cell proliferation and survival.     

Phospholipase D activity is elevated in hepatitis C virus core protein-transformed NIH3T3 mouse fibroblast cells

Hepatitis C Virus (HCV) is associated with a severe liver disease and increased frequency in the development of hepatocellular carcinoma. Overexpression of HCV core protein is known to transform fibroblast cells. Phospholipase D (PLD) activity is commonly elevated in response to mitogenic signals, and has also been overexpressed and hyperactivated in some human cancer cells. The aim of this study was to understand how PLD was regulated in the HCV core protein-transformed NIH3T3 mouse fibroblast cells. We observed that PLD activity was elevated in the NIH3T3 cells overexpressing HCV core protein over the vector alone-transfected control cells, however, expression levels of PLD protein and protein kinase C (PKC) in the HCV core protein-transformed cells was similar to the control cells. Phorbol 12-myristate 13-acetate (PMA), which is known to activate PKC, stimulated PLD activity significantly more in the core protein-transformed cells, in comparison with that of the control cells. PLD activity assay using PKC isozyme-specific inhibitor and PKC translocation experiment showed that PKC-delta was mainly involved in the PMA- induced PLD activation in the core-transformed cells. Moreover, in cells overexpressing HCV core protein, PMA also stimulated p38 kinase more potently than that of the control cells, and an inhibitor of p38 kinase abolished PMA-induced PLD activation in cells overexpressing HCV core protein. Taken together, these results suggest that PLD might be implicated in core protein-induced transformation.     

Ox-LDL suppresses PMA-induced MMP-9 expression and activity through CD36-mediated activation of PPAR-g

During chronic inflammatory response, mono- cytes/macrophages produce 92-kDa matrix metalloproteinase-9 (MMP-9), which may contribute to their extravasation, migration and tissue remodeling. Activation of peroxisome proliferator- activated factor receptor-g (PPAR-g) has been shown to inhibit MMP-9 activity. To evaluate whether ox-LDL, a PPAR-g activator, inhibits PMA-induced MMP-9 expression and activity, and if so, whether CD36 and PPAR-g are involved in this process, we investigated the effect of ox-LDL on MMP-9 expression and activity in PMA-activated human monocytic cell line U937. PMA-induced MMP-9 expression and activity were suppressed by the treatment with ox-LDL (50 mg/ml) or PPAR-g activators such as troglitazone (5 mM), ciglitazone (5 mM), and 15d- PGJ2 (1 mM) for 24 h. This ox-LDL or PPAR-g activator-mediated inhibition of MMP-9 activity was diminished by the pre-treatment of cells with a blocking antibody to CD36, or PGF2a (0.3 mM), which is a PPAR-g inhibitor, as well as overexpression of a dominant-negative form of CD36. Taken together, these results suggest that ox-LDL suppresses PMA-induced MMP-9 expression and activity through CD36-mediated activation of PPAR-g.     

Trp-Lys-Tyr-Met-Val-Met stimulates phagocytosis via phospho-lipase D-dependent signaling in mouse dendritic cells

Dendritic cells (DCs) play a key role in activating the immune response against invading pathogens as well as dying cells or tumors. Although the immune response can be initiated by the phagocytic activity by DCs, the molecular mechanism involved in this process has not been fully investigated. Trp-Lys-Tyr-Met-Val-Met-NH(2) (WKYMVM) stimulates the activation of phospholipase D (PLD) via Ca(2+) increase and protein kinase C activation in mouse DC cell line, DC2.4. WKYMVM stimulates the phagocytic activity, which is inhibited in the presence of N-butanol but not t-butanol in DC2.4 cells. Furthermore, the addition of phosphatidic acid, an enzymatic product of PLD activity, enhanced the phagocytic activity in DC2.4 cells. Since at least two of formyl peptide receptor (FPR) family (FPR1 and FPR2) are expressed in DC2.4 as well as in mouse bone marrow-derived dendritic cells, this study suggests that the activation of FPR family by WKYMVM stimulates the PLD activity resulting in phagocytic activity in DC2.4 cells.     

Involvement of protein kinase C pathway in UVC-stimulated phospholipase D2 activity in Vero 76 cells

Phospholipase D (PLD) activity is known to be related to oxidant-induced cellular signaling and membrane disturbance. Previously, an induction of PLD activity in various cell lines by X-ray irradiation was observed. In this study, we examined the effect of UVC radiation on the PLD activity in Vero 76 cells. At a dose of 10 kJ/m2 of UVC irradiation, the PLD activity was stimulated approximately 10-fold over the basal activity. This UVC-induced PLD activity was found to be dependent on the presence of extracellular calcium and was inhibited by catalase as well as amifostine-an intracellular thiol antioxidant. Pretreatments with Ro32-0432-a selective inhibitor of protein kinase C (PKC)-and downregulation of PKC by preincubation of phorbol 12-myristate 13-acetate significantly inhibited the UVC-induced PLD activity. UVC-stimulated PLD activity was observed only in murine PLD2 (mPLD2)-transfected Vero 76 cells and not in human PLD1 (hPLD1)-transfected cells. Transient incorporation of PKC with mPLD2 and the phosphorylation of mPLD2 by a and b forms of PKC by UVC irradiation were observed. These results suggest that the UVC-stimulated PLD activity in Vero 76 cells is mediated through transient phosphorylation of PLD2 by the translocation of PKC to PLD2.     

ATP-induced focal adhesion kinase activity is negatively modulated by phospholipase D2 in PC12 cells

Extracellular ATP has been known to modulate various cellular responses including mitogenesis, secretion and morphogenic activity in neuronal cells. In the ATP-induced morphogenic activity, focal adhesion kinase(s) such as Fak have been suggested to play a critical role. Binding of ATP to its specific cell surface receptor in PC12 cells induces phospholipase D (PLD) activity. However, the role of PLD on ATP-induced Fak activation in PC12 cells remains unclear. In this study, we investigated the role of PLD on the ATP-induced Fak activation and paxillin phosphorylation using two established cell lines: wild type PLD2- and lipase-inactive mutant PLD2-inducible PC12 cells. Stimulation of cells with ATP caused PLD2 activation via classical protein kinase C activation. ATP also induced Fak activation, and paxillin phosphorylation, and were dramatically reduced by wild type PLD2 overexpression but not by lipase-inactive mutant PLD2 overexpression. When the PC12 cells were pretreated with propranolol, a specific inhibitor for phosphatidic acid phosphohydrolase resulting in the accumulation of PA, ATP-induced Fak activation and paxillin phosphorylation were also reduced. We found that inhibition of tyrosine phosphatases by pervanadate completely blocked PLD2-dependent Fak and paxillin dephosphorylation. Taken together, we suggest that PLD2 activity might play a negative role in ATP-induced Fak and paxillin phosphorylation possibly through tyrosine phosphatases.     

Differential effects of Fas cross-linking on phospholipase D activation and related lipid metabolism in Fas-resistant A20 cells

A20 murine lymphoma cells undergoing Fas-mediated apoptosis showed increase in the activity of phospholipase D (PLD), which is involved in proliferative or mitogenic cellular responses. Using A20 cell lines that were resistant to Fas-induced apoptosis, we investigated the differential effects of Fas cross-linking on PLD activity and sphingolipid metabolism. The basal PLD activities in all of the selected three Fas-resistant clones (#5, #8, and #11) were about 2~4 folds higher than that of wild type A20 cells. Among the PLD isoforms, PLD2 expression was increased in all of the selected Fas-resistant clones. The Fas downstream signaling events triggered by Fas cross-linking, including the activations of PLD, phosphatidylcholine-specific phospholipase C (PC-PLC), sphingomyelinase (SMase), the increase in diacylglycerol (DAG) and protein phosphorylation levels, and the translocation of protein kinase C to membrane were not changed in both of Fas-resistant clone #5 and #8. In contrast, Fas cross-linking stimulated the activity of PLD, PC-PLC, and SMase, translocation of PKC, and protein phosphorylation in Fas-resistant clone #11, similar to that of wild type cells. We also found that clone #11 had a different Fas sequence encoding Fas B which has been known to inhibit Fas-induced apoptosis. These findings suggest that increased PLD2 expression resulting in increased basal PLD activity and the blockade of Fas downstream signaling cascades may be involved to limit apoptosis induced by Fas cross-linking.     

Phospholipase D inhibitor enhances radiosensitivity of breast cancer cells

Radiation and drug resistance remain the major challenges and causes of mortality in the treatment of locally advanced, recurrent and metastatic breast cancer. Dysregulation of phospholipase D (PLD) has been found in several human cancers and is associated with resistance to anticancer drugs. In the present study, we evaluated the effects of PLD inhibition on cell survival, cell death and DNA damage after exposure to ionizing radiation (IR). Combined IR treatment and PLD inhibition led to an increase in the radiation-induced apoptosis of MDA-MB-231 metastatic breast cancer cells. The selective inhibition of PLD1 and PLD2 led to a significant decrease in the IR-induced colony formation of breast cancer cells. Moreover, PLD inhibition suppressed the radiation-induced activation of extracellular signal-regulated kinase and enhanced the radiation-stimulated phosphorylation of the mitogen-activated protein kinases p38 and c-Jun N-terminal kinase. Furthermore, PLD inhibition, in combination with radiation, was very effective at inducing DNA damage, when compared with radiation alone. Taken together, these results suggest that PLD may be a useful target molecule for the enhancement of the radiotherapy effect.     

Phospholipase D is involved in oxidative stress-induced migration of vascular smooth muscle cells via tyrosine phosphorylation and protein kinase C

Oxidative stress has been implicated in mediation of vascular disorders. In the presence of vanadate, H(2)O(2) induced tyrosine phosphorylation of PLD1, protein kinase C-alpha (PKC-alpha), and other unidentified proteins in rat vascular smooth muscle cells (VSMCs). Interestingly, PLD1 was found to be constitutively associated with PKC-alpha in VSMCs. Stimulation of the cells by H(2)O(2) and vanadate showed a concentration-dependent tyrosine phosphorylation of the proteins in PLD1 immunoprecipitates and activation of PLD. Pretreatment of the cells with the protein tyrosine kinase inhibitor, genistein resulted in a dose-dependent inhibition of H(2)O(2)-induced PLD activation. PKC inhibitor and down-regulation of PKC abolished H(2)O(2)-stimulated PLD activation. The cells stimulated by oxidative stress (H(2)O(2)) caused increased cell migration. This effect was prevented by the pretreatment of cells with tyrosine kinase inhibitors, PKC inhibitors, and 1-butanol, but not 3-butanol. Taken together, these results suggest that PLD might be involved in oxidative stress-induced migration of VSMCs, possibly via tyrosine phosphorylation and PKC activation.     

Activation of epidermal growth factor receptor is responsible for pervanadate-induced phospholipase D activation

Pervanadate, a complex of vanadate and H(2)O(2), has an insulin mimetic effect, and acts as an inhibitor of protein tyrosine phosphatase. Pervanadate-induced phospholipase D (PLD) activation is known to be dependent on the tyrosine phosphorylation of cellular proteins and protein kinase C (PKC) activation, and yet underlying molecular mechanisms are not clearly understood. Here, we investigated the signaling pathway of pervanadate-induced PLD activation in Rat2 fibroblasts. Pervanadate increased PLD activity in dose- and time- dependent manner. Protein tyrosine kinase inhibitor, genistein, blocked PLD activation. Interestingly, AG-1478, a specific inhibitor of the tyrosine kinase activity of epidermal growth factor receptor (EGFR) blocked not only the PLD activation completely but also phosphorylation of p38 mitogen-activated protein kinase (MAPK). However, AG-1295, an inhibitor specific for the tyrosine kinase activity of pletlet drived growth factor receptor (PDGFR) did not show any effect on the PLD activation by pervanadate. We further found that pervanadate increased phosphorylation levels of p38, extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK). SB203580, a p38 MAPK inhibitor, blocked the PLD activation completely. However, the inhibitions of ERK by the treatment of PD98059 or of JNK by the overexpression of JNK interacting peptide JBD did not show any effect on pervanadate-induced PLD activation. Inhibition or down-regulation of PKC did not alter the pervanadate-induced PLD activation in Rat2 cells. Thus, these results suggest that pervanadate-induced PLD activation is coupled to the transactivation of EGFR by pervanadate resulting in the activation of p38 MAP kinase.     

NADPH oxidase activation contributes to native low-density lipoprotein-induced proliferation of human aortic smooth muscle cells

Elevated plasma concentration of native low-density lipoprotein (nLDL) is associated with vascular smooth muscle cell (VSMC) activation and cardiovascular disease. We investigated the mechanisms of superoxide generation and its contribution to pathophysiological cell proliferation in response to nLDL stimulation. Lucigenin-induced chemiluminescence was used to measure nLDL-induced superoxide production in human aortic smooth muscle cells (hAoSMCs). Superoxide production was increased by nicotinamide adenine dinucleotide phosphate (NADPH) and decreased by NADPH oxidase inhibitors in nLDL-stimulated hAoSMC and hAoSMC homogenates, as well as in prepared membrane fractions. Extracellular signal-regulated kinase 1/2 (Erk1/2), protein kinase C-θ (PKCθ) and protein kinase C-β (PKCβ) were phosphorylated and maximally activated within 3 min of nLDL stimulation. Phosphorylated Erk1/2 mitogen-activated protein kinase, PKCθ and PKCβ stimulated interactions between p47phox and p22phox; these interactions were prevented by MEK and PKC inhibitors (PD98059 and calphostin C, respectively). These inhibitors decreased nLDL-dependent superoxide production and blocked translocation of p47phox to the membrane, as shown by epifluorescence imaging and cellular fractionation experiments. Proliferation assays showed that a small interfering RNA against p47phox, as well as superoxide scavenger and NADPH oxidase inhibitors, blocked nLDL-induced hAoSMC proliferation. The nLDL stimulation in deendothelialized aortic rings from C57BL/6J mice increased dihydroethidine fluorescence and induced p47phox translocation that was blocked by PD98059 or calphostin C. Isolated aortic SMCs from p47phox^−/− mice (mAoSMCs) did not respond to nLDL stimulation. Furthermore, NADPH oxidase 1 (Nox1) was responsible for superoxide generation and cell proliferation in nLDL-stimulated hAoSMCs. These data demonstrated that NADPH oxidase activation contributed to cell proliferation in nLDL-stimulated hAoSMCs.     

The p38 mitogen-activated protein kinase is involved in stress-induced phospholipase D activation in vascular smooth muscle cells

Oxidative stress has been implicated in mediation of vascular disorders. Earlier study showed that the exposure of vascular smooth muscle cells (VSMC) to pervanadate (hydrogen peroxide plus orthovanadate) resulted in the accumulation of [3H]phosphatidylbutanol. In this study, the effect of pervanadate on the activation of p38 mitogen-activated protein kinase (p38 MAPK) was studied in the VSMC. Pervanadate treatment activated p38 MAPK in a dose-and time-dependent manner. Interestingly, specific inhibition of p38 MAPK with SB203580 attenuated pervanadate-induced PLD activation. This correlates with the finding that expression of dominant negative mutants of MKK3/6 inhibited the PLD activation. SB203580 pretreatment also inhibited other cellular stressors (i.e. high osmolarity and UV light)-induced PLD activation. The possible correlationship of p38 MAPK activation with PKC was examined since PKC is reported to be involved in the pervanadate-induced PLD activation. Calphostin C, a PKC inhibitor, suppressed pervanadate-induced p38 MAPK and PLD activation in a dose-dependent manner. These results suggest that PKC-p38 MAPK may represent an upstream pathway of PLD in the signal transduction of cellular stress.     

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.     

Wnt/GSK3β/β-catenin signaling pathway regulates calycosin-mediated anticancer effects in glioblastoma multiforme cells

Calycosin, an O-methylated isoflavone, has been widely reported to induce anticancer activity in different cancer cells in vitro. Nonetheless, the associated mechanism of calycosin in glioblastoma multiforme cells (U87) still remains unknown. To explore the anticancer effects, the apoptotic mechanism of calycosin via Wnt/GSK3β/β-catenin signaling was explored in U87 cells. Different assays including: cytotoxicity, free radical determination, SOD and CAT activity, GSH content, qPCR, mitochondrial membrane potential, caspase activity, and western blotting assays were performed. It was shown that calycosin mitigated cell viability in U87 cells, whereas it showed no apparent effect on BV2 microglial cells. Calycosin triggered apoptosis via upregulating the mitochondria-associated caspase pathway in U87 cells. Calycosin induced the reduction of the mitochondrial membrane potential, overexpression of Bax, downexpression of Bcl-2, and activation of caspase-9 and caspase-3. Calycosin-stimulated apoptosis was associated with the upregulation of free radical scavenging through the modulation of antioxidant enzymes, such as SOD and CAT as well as the level of GSH. The apoptotic activity of calycosin was mediated by suppression of pGSK-3βser9, β-catenin, and c-Myc at protein level. The present study suggested that calycosin triggers U87 cell death through an antioxidant effect mediated by Wnt/GSK3β/β-catenin signaling pathway.     

Diketoacetonylphenalenone,Derived from Hawaiian Volcanic Soil-Associated Fungus Penicillium herquei FT729, Regulates T Cell Activation via Nuclear Factor-κB and Mitogen-Activated Protein Kinase Pathway

In immunological responses, controlling excessive T cell activity is critical for immunological homeostasis maintenance. Diketoacetonylphenalenone, derived from Hawaiian volcanic soil-associated fungus Penicillium herquei FT729, possesses moderate anti-inflammatory activity in RAW 264.7 cells but its immunosuppressive effect on T cell activation is unknown. In the present study, diketoacetonylphenalenone (up to 40 μM) did not show cytotoxicity in T cells. Western blot analysis showed treatment with diketoacetonylphenalenone did not alter the expression of anti-apoptotic proteins. Pretreatment with diketoacetonylphenalenone suppressed the interleukin-2 production in activated T cells induced by T cell receptor-mediated stimulation and PMA/A23187. The CFSE-proliferation assay revealed the inhibitory effect of diketoacetonylphenalenone on the proliferation of T cells. The expression of surface molecules on activated T cells was also reduced. We discovered the suppression of the TAK1-IKKα-NF-κB pathway by pretreatment with diketoacetonylphenalenone abrogated mitogen-activated protein kinase (MAPK) signaling in activated T cells. These results suggest that diketoacetonylphenalenone effectively downregulates T cell activity via the MAPK pathway and provides insight into the therapeutic potential of immunosuppressive reagents.     

Human telomerase catalytic subunit (hTERT) suppresses p53-mediated anti-apoptotic response via induction of basic fibroblast growth factor

Although human telomerase catalytic subunit (TERT) has several cellular functions including telomere homeostasis, genomic stability, cell proliferation, and tumorigenesis, the molecular mechanism underlying anti-apoptosis regulated by TERT remains to be elucidated. Here, we show that ectopic expression of TERT in spontaneously immortalized human fetal fibroblast (HFFS) cells, which are a telomerase- and p53-positive, leads to increases of cell proliferation and transformation, as well as a resistance to DNA damage response and inactivation of p53 function. We found that TERT and a mutant TERT (no telomerase activity) induce expression of basic fibroblast growth factor (bFGF), and ectopic expression of bFGF also allows cells to be resistant to DNA-damaging response and to suppress activation of p53 function under DNA-damaging induction. Furthermore, loss of TERT or bFGF markedly increases a p53 activity and DNA-damage sensitivity in HFFS, HeLa and U87MG cells. Therefore, our findings indicate that a novel TERT-bFGF axis accelerates the inactivation of p53 and consequent increase of resistance to DNA-damage response.