Phosphatidylcholine-specific Phospholipase C Inhibitor Research Articles

D609 inhibition of phosphatidylcholine-specific phospholipase C attenuates prolonged insulin stimulation-mediated GLUT4 downregulation in 3T3-L1 adipocytes

Glucose uptake is stimulated by insulin via stimulation of glucose transporter 4 (GLUT4) translocation to the plasma membrane from intracellular compartments in adipose tissue and muscles. Insulin stimulation for prolonged periods depletes GLUT4 protein, particularly in highly insulin-responsive GLUT4 storage vesicles. This depletion mainly occurs via H2O2-mediated retromer inhibition. However, the post-receptor mechanism of insulin activation of oxidative stress remains unknown. Here, we show that phosphatidylcholine-specific phospholipase C (PC-PLC) plays an important role in insulin-mediated downregulation of GLUT4. In the study, 3T3-L1 adipocytes were exposed to a PC-PLC inhibitor, tricyclodecan-9-yl-xanthogenate (D609), for 30 min prior to the stimulation with 500 nM insulin for 4 h, weakening the depletion of GLUT4. D609 also prevents insulin-driven H2O2 generation in 3T3-L1 adipocytes. Exogenous PC-PLC and its product, phosphocholine (PCho), also caused GLUT4 depletion and promoted H2O2 generation in 3T3-L1 adipocytes. Furthermore, insulin-mediated the increase in the cellular membrane PC-PLC activity was observed in Amplex Red assays. These results suggested that PC-PLC plays an important role in insulin-mediated downregulation of GLUT4 and that PCho may serve as a signaling molecule.

Incorporation of a Nitric Oxide Donating Motif into Novel PC-PLC Inhibitors Provides Enhanced Anti-Proliferative Activity.

Inhibition of phosphatidylcholine-specific phospholipase C (PC-PLC) has previously been shown to be a potential target for novel cancer therapeutics. One downstream consequence of PC-PLC activity is the activation of NF-κB, a nuclear transcription factor responsible for transcribing genes related to oncogenic traits, such as proliferation, angiogenesis, metastasis, and cancer cell survival. Another biological pathway linked to NF-κB is the exogenous delivery of nitric oxide (NO), which decreases NF-κB activity through an apparent negative-feedback loop. In this study, we designed and synthesised 13 novel NO-releasing derivatives of our previously reported class of PC-PLC inhibitors, 2-morpholinobenzoic acids. These molecules contained a secondary benzylamine group, which was readily nitrosylated and subsequently confirmed to release NO in vitro using a DAF-FM fluorescence-based assay. It was then discovered that these NO-releasing derivatives possessed significantly improved anti-proliferative activity in both MDA-MB-231 and HCT116 cancer cell lines compared to their non-nitrosylated parent compounds. These results confirmed that the inclusion of an exogenous NO-releasing functional group onto a known PC-PLC inhibitor enhances anti-proliferative activity and that this relationship can be exploited in order to further improve the anti-proliferative activity of current/future PC-PLC inhibitors.

Development of 2-Morpholino-N-hydroxybenzamides as anti-proliferative PC-PLC inhibitors

Phosphatidylcholine-specific phospholipase C (PC-PLC) is a key enzyme involved in the metabolism of the mammalian phospholipid phosphatidylcholine into secondary messengers diacylglycerol (DAG) and phosphocholine. DAG and phosphocholine have been identified to amplify various cellular processes involved in oncogenesis such as proliferation, cell-cycle activation, differentiation and motility, therefore making PC-PLC a potential target for novel anti-cancer treatments. The current literature standard for PC-PLC inhibition, tricyclodecan-9-yl-potassium xanthate (D609), has been shown to arrest proliferation in multiple cancer cell lines, however, it is not drug-like resulting in low aqueous stability, making it a poor drug candidate. 2-Morpholinobenzoic acids have been shown to have improved PC-PLC inhibitory activity compared to D609, with molecular modelling identifying chelation of the carboxylic acid to catalytic Zn2+ ions in the PC-PLC active site being a key interaction. In this study, the carboxylic acid motif was replaced with a hydroxamic acid to strengthen the Zn2+ interaction. It was found that the hydroxamic acid derivatives displayed PC-PLC inhibitory activity similar, or better, than D609. Furthermore, these novel inhibitors had potent anti-proliferative activity in MDA-MB-231 and HCT-116 cancer cell lines, far greater than D609 and previous 2-morpholinobenzoic acids.

Discovery of novel phosphatidylcholine-specific phospholipase C drug-like inhibitors as potential anticancer agents

Phosphatidylcholine–specific phospholipase C (PC-PLC) is a promising target for new anticancer treatment. Herein, we report our work in the discovery of novel drug-like PC-PLC inhibitors. Virtual screening led to the identification of promising hits from four different structural series that contain the molecular scaffold of benzenesulphonamides (10), pyrido[3,4-b]indoles (22), morpholinobenzoic acid (84) and benzamidobenzoic acid (80). 164 structural analogues were tested to investigate the chemical space around the hit series and to generate preliminary structurally activity relationships (SAR). Two of the pyrido[3,4-b]indoles (22_10 and 22_15) had comparable or better potency as D609, an established but non-drug-like PC-PLC inhibitor. Furthermore, three morpholinobenzoic acids (84, 84_4 and 84_5) had superior potency than D609. Therefore, this study paves the way towards the development of drug-like PL-PLC inhibitors as potential anticancer agents.

Phosphatidylcholine-specific phospholipase C inhibition reduces HER2-overexpression, cell proliferation and in vivo tumor growth in a highly tumorigenic ovarian cancer model.

Antagonizing the oncogenic effects of human epidermal growth factor receptor 2 (HER2) with current anti-HER2 agents has not yet yielded major progress in the treatment of advanced HER2-positive epithelial ovarian cancer (EOC). Using preclinical models to explore alternative molecular mechanisms affecting HER2 overexpression and oncogenicity may lead to new strategies for EOC patient treatment. We previously reported that phosphatidylcholine-specific phospholipase C (PC-PLC) exerts a pivotal role in regulating HER2 overexpression in breast cancer cells. The present study, conducted on two human HER2-overexpressing EOC cell lines - SKOV3 and its in vivo-passaged SKOV3.ip cell variant characterized by enhanced in vivo tumorigenicity - and on SKOV3.ip xenografts implanted in SCID mice, showed: a) about 2-fold higher PC-PLC and HER2 protein expression levels in SKOV3.ip compared to SKOV3 cells; b) physical association of PC-PLC with HER2 in non-raft domains; c) HER2 internalization and ca. 50% reduction of HER2 mRNA and protein expression levels in SKOV3.ip cells exposed to the PC-PLC inhibitor tricyclodecan-9-yl-potassium xanthate (D609); d) differential effects of D609 and trastuzumab on HER2 protein expression and cell proliferation; e) decreased in vivo tumor growth in SKOV3.ip xenografts during in vivo treatment with D609; f) potential use of in vivo magnetic resonance spectroscopy (MRS) and imaging (MRI) parameters as biomarkers of EOC response to PC-PLC inhibition. Overall, these findings support the view that PC-PLC inhibition may represent an effective means to target the tumorigenic effects of HER2 overexpression in EOC and that in vivo MR approaches can efficiently monitor its effects.

Phosphatidylcholine-specific phospholipase C inhibition down- regulates CXCR4 expression and interferes with proliferation, invasion and glycolysis in glioma cells.

BackgroundThe chemokine receptor CXCR4 plays a crucial role in tumors, including glioblastoma multiforme (GBM), the most aggressive glioma.Phosphatidylcholine-specific phospholipase C (PC-PLC), a catabolic enzyme of PC metabolism, is involved in several aspects of cancer biology and its inhibition down-modulates the expression of growth factor membrane receptors interfering with their signaling pathways.In the present work we investigated the possible interplay between CXCR4 and PC-PLC in GBM cells.MethodsConfocal microscopy, immunoprecipitation, western blot analyses, and the evaluation of migration and invasion potential were performed on U87MG cells after PC-PLC inhibition with the xanthate D609. The intracellular metabolome was investigated by magnetic resonance spectroscopy; lactate levels and lactate dehydrogenase (LDH) activity were analyzed by colorimetric assay.ResultsOur studies demonstrated that CXCR4 and PC-PLC co-localize and are associated on U87MG cell membrane. D609 reduced CXCR4 expression, cell proliferation and invasion, interfering with AKT and EGFR activation and expression. Metabolic analyses showed a decrease in intracellular lactate concentration together with a decrement in LDH activity.ConclusionsOur data suggest that inhibition of PC-PLC could represent a new molecular approach in glioma biology not only for its ability in modulating cell metabolism, glioma growth and motility, but also for its inhibitory effect on crucial molecules involved in cancer progression.

Activation of Phosphatidylcholine-Specific Phospholipase C in Breast and Ovarian Cancer: Impact on MRS-Detected Choline Metabolic Profile and Perspectives for Targeted Therapy.

Elucidation of molecular mechanisms underlying the aberrant phosphatidylcholine cycle in cancer cells plays in favor of the use of metabolic imaging in oncology and opens the way for designing new targeted therapies. The anomalous choline metabolic profile detected in cancer by magnetic resonance spectroscopy and spectroscopic imaging provides molecular signatures of tumor progression and response to therapy. The increased level of intracellular phosphocholine (PCho) typically detected in cancer cells is mainly attributed to upregulation of choline kinase, responsible for choline phosphorylation in the biosynthetic Kennedy pathway, but can also be partly produced by activation of phosphatidylcholine-specific phospholipase C (PC-PLC). This hydrolytic enzyme, known for implications in bacterial infection and in plant survival to hostile environmental conditions, is reported to be activated in mitogen- and oncogene-induced phosphatidylcholine cycles in mammalian cells, with effects on cell signaling, cell cycle regulation, and cell proliferation. Recent investigations showed that PC-PLC activation could account for 20–50% of the intracellular PCho production in ovarian and breast cancer cells of different subtypes. Enzyme activation was associated with PC-PLC protein overexpression and subcellular redistribution in these cancer cells compared with non-tumoral counterparts. Moreover, PC-PLC coimmunoprecipitated with the human epidermal growth factor receptor-2 (HER2) and EGFR in HER2-overexpressing breast and ovarian cancer cells, while pharmacological PC-PLC inhibition resulted into long-lasting HER2 downregulation, retarded receptor re-expression on plasma membrane and antiproliferative effects. This body of evidence points to PC-PLC as a potential target for newly designed therapies, whose effects can be preclinically and clinically monitored by metabolic imaging methods.

Inhibition of Phosphatidylcholine-Specific Phospholipase C Interferes with Proliferation and Survival of Tumor Initiating Cells in Squamous Cell Carcinoma.

PurposeThe role of phosphatidylcholine-specific phospholipase C (PC-PLC), the enzyme involved in cell differentiation and proliferation, has not yet been explored in tumor initiating cells (TICs). We investigated PC-PLC expression and effects of PC-PLC inhibition in two adherent (AD) squamous carcinoma cell lines (A431 and CaSki), with different proliferative and stemness potential, and in TIC-enriched floating spheres (SPH) originated from them.ResultsCompared with immortalized non-tumoral keratinocytes (HaCaT) A431-AD cells showed 2.5-fold higher PC-PLC activity, nuclear localization of a 66-kDa PC-PLC isoform, but a similar distribution of the enzyme on plasma membrane and in cytoplasmic compartments. Compared with A431-AD, A431-SPH cells showed about 2.8-fold lower PC-PLC protein and activity levels, but similar nuclear content. Exposure of adherent cells to the PC-PLC inhibitor D609 (48h) induced a 50% reduction of cell proliferation at doses comprised between 33 and 50 μg/ml, without inducing any relevant cytotoxic effect (cell viability 95±5%). In A431-SPH and CaSki-SPH D609 induced both cytostatic and cytotoxic effects at about 20 to 30-fold lower doses (IC50 ranging between 1.2 and 1.6 μg/ml). Furthermore, D609 treatment of A431-AD and CaSki-AD cells affected the sphere-forming efficiency, which dropped in both cells, and induced down-modulation of stem-related markers mRNA levels (Oct4, Nestin, Nanog and ALDH1 in A431; Nestin and ALDH1 in CaSki cells).ConclusionsThese data suggest that the inhibition of PC-PLC activity may represent a new therapeutic approach to selectively target the most aggressive and tumor promoting sub-population of floating spheres originated from squamous cancer cells possessing different proliferative and stemness potential.

Evidence for the role of phosphatidylcholine-specific phospholipase in experimental subarachnoid hemorrhage in rats

Neuron apoptosis and inflammatory responses contribute to subarachnoid hemorrhage (SAH)-induced early brain injury (EBI), which is the main aspect that affects patients' outcome. Previous research has demonstrated that phosphatidylcholine-specific phospholipase C (PC-PLC) plays critical roles in cell apoptosis and various inflammatory responses, and that tricyclodecan-9-yl-xanthogenate (D609), a well known PC-PLC inhibitor, is a powerful agent to protect brain from cerebral ischemic injury and SAH-induced cerebral vasospasm. However, the association between PC-PLC and SAH-induced EBI is undetermined. Therefore, we sought to investigate whether PC-PLC was implicated in SAH-induced EBI. Compared with sham group, an upregulation of PC-PLC activity was detected in the brain tissue and serum of SAH group. Pharmacological blockade of PC-PLC by D609 attenuated neurological behavior impairment, brain edema and blood–brain barrier (BBB) damage induced by SAH. In addition, D609 treatment significantly inhibited SAH-induced inflammatory response and neuron apoptosis. Furthermore, inhibition of PC-PLC in primary-cultured rat cortical neurons attenuated oxyhemoglobin (OxyHb)-induced apoptosis morphology and decrease in survival rate. In conclusion, our data suggest that PC-PLC participates in SAH-induced EBI.

Phosphatidylethanolamine binding protein 1 in vacular endothelial cell autophagy and atherosclerosis

We previously found that phosphatidylcholine-specific phospholipase C (PC-PLC) was a key inducing element of atherosclerosis, and might negatively regulate human umbilical vein endothelial cell (HUVEC) autophagy. To further investigate the mechanism of PC-PLC action, we initially identified phosphatidylethanolamine binding protein 1 (PEBP1) as a binding partner of PC-PLC by using mass spectrometry (MS, MALDI-TOF/TOF). We found that PEBP1 positively regulated PC-PLC activity in HUVECs, and inhibition of PC-PLC by its inhibitor D609 suppressed PEBP1 expression dramatically. Moreover, both PC-PLC and PEBP1 negatively regulated HUVEC autophagy independently of mammalian target of rapamycin (mTOR). Furthermore, the PEBP1 level was elevated during the development of atherosclerosis, while D609 significantly decreased the upregulated PEBP1 level in apoE(-/-) mice.

Evidence for the role of phosphatidylcholine-specific phospholipase C in sustained hypoxic pulmonary vasoconstriction

The aim of the study was to investigate the role of phosphatidylcholine-specific phospholipase C (PC-PLC) in hypoxic pulmonary vasoconstriction (HPV) and elucidate its possible interactions within HPV mechanism. Inhibition of PC-PLC with D609 (30μM) resulted in partial reduction of the transient phase and almost complete abolition of the sustained phase of HPV in isolated rat intrapulmonary arteries (IPAs). Intravenous injection of D609 (5mg/kg) 30min before the onset of hypoxia prevented the development of acute hypoxic pulmonary hypertension (AHPH) in rats. D609 also inhibited pulmonary vasoconstriction induced with a generator of superoxide anions LY83583, but not the one induced with hydrogen peroxide. Protein kinase C (PKC) inhibition with Ro-31-8220 partially diminished the transient phase of hypoxic contraction in IPA while the sustained phase remained unchanged. Phosphocholine, known to be released due to phosphatidylcholine breakdown by PC-PLC, induced sustained contraction in isolated IPA and also transient pulmonary and systemic hypertension if administered intravenously (70mg/kg). We conclude that PC-PLC plays an important role in sustained HPV possibly through the activation of PKC-independent mechanism, which may be coupled with phosphocholine release.

Inhibition of phosphatidylcholine-specific phospholipase C results in loss of mesenchymal traits in metastatic breast cancer cells

IntroductionAcquisition of mesenchymal characteristics confers to breast cancer (BC) cells the capability of invading tissues different from primary tumor site, allowing cell migration and metastasis. Regulators of the mesenchymal-epithelial transition (MET) may represent targets for anticancer agents. Accruing evidence supports functional implications of choline phospholipid metabolism in oncogene-activated cell signaling and differentiation. We investigated the effects of D609, a xanthate inhibiting phosphatidylcholine-specific phospholipase C (PC-PLC) and sphingomyelin synthase (SMS), as a candidate regulator of cell differentiation and MET in the highly metastatic BC cell line MDA-MB-231.MethodsPC-PLC expression and activity were investigated using confocal laser scanning microscopy (CLSM), immunoblotting and enzymatic assay on human MDA-MB-231 compared with MCF-7 and SKBr3 BC cells and a nontumoral immortalized counterpart (MCF-10A). The effects of D609 on PC-PLC and SMS activity, loss of mesenchymal markers and changes in migration and invasion potential were monitored in MDA-MB-231 cells by enzymatic assays, CLSM, immunoblotting and transwell chamber invasion combined with scanning electron microscopy examinations. Cell proliferation, formation and composition of lipid bodies and cell morphology were investigated in D609-treated BC cells by cell count, CLSM, flow-cytometry of BODIPY-stained cells, nuclear magnetic resonance and thin-layer chromatography.ResultsPC-PLC (but not phospholipase D) showed 2- to 6-fold activation in BC compared with nontumoral cells, the highest activity (up to 0.4 pmol/μg protein/min) being detected in the poorly-differentiated MDA-MB-231 cells. Exposure of the latter cells to D609 (50 μg/mL, 24-72 h) resulted into 60-80% PC-PLC inhibition, while SMS was transiently inhibited by a maximum of 21%. These features were associated with progressive decreases of mesenchymal traits such as vimentin and N-cadherin expression, reduced galectin-3 and milk fat globule EGF-factor 8 levels, β-casein formation and decreased in vitro cell migration and invasion. Moreover, proliferation arrest, changes in cell morphology and formation of cytosolic lipid bodies typical of cell differentiation were induced by D609 in all investigated BC cells.ConclusionsThese results support a critical involvement of PC-PLC in controlling molecular pathways responsible for maintaining a mesenchymal-like phenotype in metastatic BC cells and suggests PC-PLC deactivation as a means to promote BC cell differentiation and possibly enhance the effectiveness of antitumor treatments.

In vivo detection of phospholipase C by enzyme-activated near-infrared probes.

In this article, the characterization of the first near-infrared (NIR) phospholipase-activated molecular beacon is reported, and its utility for in vivo cancer imaging is demonstrated. The probe consists of three elements: a phospholipid (PL) backbone to which the NIR fluorophore, pyropheophorbide a (Pyro), and the NIR Black Hole Quencher 3 (BHQ) were conjugated. Because of the close proximity of BHQ to Pyro, the Pyro-PtdEtn-BHQ probe is self-quenched until enzyme hydrolysis releases the fluorophore. The Pyro-PtdEtn-BHQ probe is highly specific to one isoform of phospholipase C, phosphatidylcholine-specific phospholipase C (PC-PLC), responsible for catabolizing phosphatidylcholine directly to phosphocholine. Incubation of Pyro-PtdEtn-BHQ in vitro with PC-PLC demonstrated a 150-fold increase in fluorescence that could be inhibited by the specific PC-PLC inhibitor tricyclodecan-9-yl xanthogenate (D609) with an IC(50) of 34 ± 8 μM. Since elevations in phosphocholine have been consistently observed by magnetic resonance spectroscopy in a wide array of cancer cells and solid tumors, we assessed the utility of Pyro-PtdEtn-BHQ as a probe for targeted tumor imaging. Injection of Pyro-PtdEtn-BHQ into mice bearing DU145 human prostate tumor xenografts followed by in vivo NIR imaging resulted in a 4-fold increase in tumor radiance over background and a 2 fold increase in the tumor/muscle ratio. Tumor fluorescence enhancement was inhibited with the administration of D609. The ability to image PC-PLC activity in vivo provides a unique and sensitive method of monitoring one of the critical phospholipase signaling pathways activated in cancer, as well as the phospholipase activities that are altered in response to cancer treatment.

Tricyclodecan-9-yl-Xanthogenate (D609) Mechanism of Actions: A Mini-Review of Literature

Tricyclodecan-9-yl-xanthogenate (D609) is known for its antiviral and antitumor properties. D609 actions are widely attributed to inhibiting phosphatidylcholine (PC)-specific phospholipase C (PC-PLC). D609 also inhibits sphingomyelin synthase (SMS). PC-PLC and/or SMS inhibition will affect lipid second messengers 1,2-diacylglycerol (DAG) and/or ceramide. Evidence indicates either PC-PLC and/or SMS inhibition affected the cell cycle and arrested proliferation, and stimulated differentiation in various in vitro and in vivo studies. Xanthogenate compounds are also potent antioxidants and D609 reduced Aß-induced toxicity, attributed to its antioxidant properties. Zn²⁺ is necessary for PC-PLC enzymatic activity; inhibition by D609 might be attributed to its Zn²⁺ chelation. D609 has also been proposed to inhibit acidic sphingomyelinase or down-regulate hypoxia inducible factor-1α; however these are down-stream events related to PC-PLC inhibition. Characterization of the mammalian PC-PLC is limited to inhibition of enzymatic activity (frequently measured using Amplex red assay with bacterial PC-PLC as a standard). The mammalian PC-PLC has not been cloned; sequenced and structural information is unavailable. D609 showed promise in cancer studies, reduced atherosclerotic plaques (inhibition of PC-PLC) and cerebral infarction after stroke (PC-PLC or SMS). D609 actions as an antagonist to pro-inflammatory cytokines have been attributed to PC-PLC. The purpose of this review is to comprehensively evaluate the literature and summarize the findings and relevance to cell cycle and CNS pathologies.

Lipopolysaccharide activated phosphatidylcholine‐specific phospholipase C and induced IL‐8 and MCP‐1 production in vascular endothelial cells

Secretion of proinflammatory cytokines by lipopolysaccharide (LPS) activated vascular endothelial cells (VECs) contributes substantially to the pathogenesis of several inflammatory diseases such as atherosclerosis and septic shock. However, the mechanisms involved in this process are not well understood. Here, we investigated the role of phosphatidylcholine-specific phospholipase C (PC-PLC) in LPS-induced IL-8 and MCP-1 production in VECs. The results showed that LPS elevated the level of PC-PLC and the production of IL-8 and MCP-1 in Human umbilical vein vascular endothelial cells (HUVECs). Blocking the function of PC-PLC by exploiting the neutralization antibody of PC-PLC or tricyclodecan-9-yl-xanthogenate (D609), an inhibitor of PC-PLC, significantly inhibited LPS-induced production of IL-8 and MCP-1 in HUVECs. Furthermore, the in vivo experimental results showed that the levels of PC-PLC, IL-8, and MCP-1 in the aortic endothelium and serum were increased in mice injected with LPS. The increased levels of these molecules were also inhibited by the treatment with D609. The data suggested that blocking PC-PLC function significantly inhibited LPS-induced IL-8 and MCP-1 production in cultured HUVECs and in vivo. PC-PLC might be a potential target for therapy in inflammation associated-diseases such as atherosclerosis.

Cerebrospinal fluid phospholipase C activity increases in migraine

Adrenaline, serotonin, cannabinoid and estrogen receptors are involved in migraine pathophysiology. The signaling of these receptors change phosphatidylcholine-specific phospholipase C (PC-PLC) activity, but there have been no reported PC-PLC studies in migraine. We identified PC-PLC activity in blood and cerebrospinal fluid (CSF), and quantified it in samples from ictal and interictal migraineurs without aura and healthy controls. Pre-incubation with a specific PC-PLC inhibitor, D609, inhibited enzyme activity (p < .0001) and confirms its presence in CSF. PC-PLC activity was higher in the CSF from ictal migraineurs compared to controls (mean relative fluorescence unit [RFU]/µg/min [standard deviation, SD] 13.1 [3.07] vs. 9.3 [1.97]; p = .002) and, in a paired analysis, in migraineurs during ictal compared to interictal states (11.7 [1.6] vs. 7.9 [1.5]; p = .02). CSF PC-PLC activity in the ictal state correlated negatively with migraine frequency (r = -0.82). Plasma PC-PLC activity was 250-300 times less than in CSF and did not increase in migraine, implicating the brain as the source of the CSF enzyme changes. This is the first report of PC-PLC activity in CSF and of its alteration in migraine. We propose that these PC-PLC changes in CSF reflect the overall receptor fluctuations in migraine.

Inhibition of phosphatidylcholine-specific phospholipase C downregulates HER2 overexpression on plasma membrane of breast cancer cells

IntroductionOverexpression on plasma membrane of human epidermal growth factor receptor 2 (HER2) is reported in 25% to 30% of breast cancers. Heterodimer formation with cognate members of the epidermal growth factor receptor (EGFR) family, such as HER3 and EGFR, activates abnormal cell-signalling cascades responsible for tumorigenesis and further transcriptional HER2 gene upregulation. Targeting the molecular mechanisms controlling HER2 overexpression and recycling may effectively deactivate this feedback-amplification loop. We recently showed that inactivation of phosphatidylcholine-specific phospholipase C (PC-PLC) may exert a pivotal role in selectively modulating the expression on the membrane of specific receptors or proteins relevant to cell function. In the present study, we investigated the capability of PC-PLC inhibition to target the molecular mechanisms controlling HER2 overexpression on the membrane of breast cancer cells by altering the rates of its endocytosis and lysosomal degradation.MethodsLocalization on the membrane and interaction of PC-PLC with HER2, EGFR, and HER3 were investigated on HER2-overexpressing and HER2-low breast cancer cell lines, by using confocal laser scanning microscopy, flow cytometry, cell-surface biotinylation, isolation of lipid rafts, and immunoprecipitation experiments. The effects of the PC-PLC inhibitor tricyclodecan-9-yl-potassium xanthate (D609) on HER2 expression on the membrane and on the levels of overall HER2, HER2-HER3, and HER2-EGFR contents were monitored in the HER2-overexpressing SKBr3 cells, after either transient or continuous receptor engagement with anti-HER2 monoclonal antibodies, including trastuzumab. Changes of HER2 expression and cell proliferation were examined in SKBr3, BT-474, and MDA-MB-453 cells continuously exposed to D609 alone or combined with trastuzumab.ResultsPC-PLC selectively accumulates on the plasma membrane of HER2-overexpressing cells, where it colocalizes and associates with HER2 in raft domains. PC-PLC inhibition resulted in enhanced HER2 internalization and lysosomal degradation, inducing downmodulation of HER2 expression on the membrane. Moreover, PC-PLC inhibition resulted in strong retardation of HER2 reexpression on the membrane and a decrease in the overall cellular contents of HER2, HER2-HER3, and HER2-EGFR heterodimers. The PC-PLC inhibitor also induced antiproliferative effects, especially in trastuzumab-resistant cells.ConclusionsThe results pointed to PC-PLC inhibition as a potential means to counteract the tumorigenic effects of HER2 amplification and complement the effectiveness of current HER2-targeting therapies.

Functional roles of PC‐PLC and Cdc20 in the cell cycle, proliferation, and apoptosis

Phosphatidylcholine-specific phospholipase C (PC-PLC) is the major enzyme in the Phosphatidylcholine (PC) cycle and is involved in many long-term cellular responses such as activation, proliferation, and differentiation events. Cell division cycle 20 homolog (Cdc20) is an essential cell-cycle regulator required for the completion of mitosis. Our previous studies identified the interaction between PC-PLC and Cdc20. Through the interaction, Cdc20 could mediate the degradation of PC-PLC by Cdc20-mediated ubiquitin proteasome pathway (UPP). In this study, we found that PC-PLC might not be involved in cancer metastasis. Inhibition of PC-PLC by D609 could cause cell proliferation inhibition and apoptosis inhibition in CBRH-7919 cells. Inhibition of PC-PLC could also influence the cell cycle by arresting the cells in G1 phase, and Cdc20 might be involved in these processes. Taken together, in this report, we provided new evidence for the functional roles of PC-PLC and Cdc20 in the cell cycle, proliferation, and apoptosis in CBRH-7919 cells.

D609 Inhibits Progression of Preexisting Atheroma and Promotes Lesion Stability in Apolipoprotein E −/− Mice

Atherosclerosis is considered to be a chronic inflammatory disease. Previous research has demonstrated that phosphatidylcholine-specific phospholipase C (PC-PLC) plays critical roles in various inflammatory responses. However, the association between PC-PLC and atherosclerosis is undetermined. Therefore, we sought to investigate whether PC-PLC was implicated in atherosclerosis. Immunofluorescence analysis revealed an upregulation of PC-PLC in the aortic endothelium from apolipoprotein E-deficient (apoE(-/-)) mice. PC-PLC level and activity were also increased in human umbilical vein endothelial cells in response to oxidized low-density lipoprotein treatment. Pharmacological blockade of PC-PLC by D609 inhibited the progression of preexisting atherosclerotic lesions in apoE(-/-) mice and changed the lesion composition into a more stable phenotype. Using a combination of pharmacological inhibition, polyclonal antibodies, confocal laser scanning microscopy and Western blotting, we demonstrated that PC-PLC was required for endothelial expression of lectin-like oxidized low-density lipoprotein receptor-1. In addition, D609 treatment significantly decreased the aortic endothelial expression of the vascular cell adhesion molecule-1 and the intercellular adhesion molecule-1. Furthermore, inhibition of PC-PLC in human umbilical vein endothelial cells reduced the oxidized low-density lipoprotein induced expression of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and monocyte chemotactic protein-1. Our data suggest that PC-PLC contributes to the progression of atherosclerosis.

Functional role of phosphatidylcholine‐specific phospholipase C in regulating CD16 membrane expression in natural killer cells

CD16, the low-affinity FcIgG receptor (FcgammaRIIIA), is predominantly expressed in human NK cells. Our recent findings indicate that CD16 expression on the outer membrane surface of NK cells is correlated with the membrane expression of phosphatidylcholine-specific phospholipase C (PC-PLC). In the present study we analyzed the trafficking of CD16 from the plasma membrane to cytoplasmic regions, after stimulation with specific mAb. The CD16 receptor is internalized, likely degraded and newly synthesized; its endocytosis is independent of ATP, but requires an integral and functional actin cytoskeleton. Antibody-mediated CD16 cross-linking results in an approximately twofold increase in PC-PLC enzymatic activity within 10 min. Analysis of PC-PLC and CD16 distribution in NK cell plasma membrane demonstrates that the proteins are physically associated and partially accumulated in lipid rafts. Pre-incubation of NK cells with a PC-PLC inhibitor, D609, causes a dramatic decrease both in CD16 receptor and PC-PLC enzyme expression on the plasma membrane. Interestingly, among phenotype PBL markers, only CD16 is strongly down-modulated by D609 treatment. CD16-mediated cytotoxicity is also reduced after D609 incubation. Taken together, these data suggest that the PC-PLC enzyme could play an important role in regulating CD16 membrane expression, the CD16-mediated cytolytic mechanism and CD16-triggered signal transduction.