The Water Extract of Juniperus communis L. Induces Cell Death and Sensitizes Cancer Cells to Cytostatic Drugs through p53 and PI3K/Akt Pathways

Juniper (Juniperus communis L.) is a northern coniferous plant generally used as a spice and for nutritional purposes in foods and drinks. It was previously reported that juniper extract (JE) affects p53 activity, cellular stress, and gene expression induced cell death in human neuroblastoma cells. Therefore, the effects of juniper on p53 and Akt signaling was examined further in A549 lung, 22RV1 and DU145 prostate, and HepG2 liver cancer cells using Western blot, confocal microscopy, and MTT analysis. We found that juniper simultaneously decreased cell viability, activated the p53 pathway, and inactivated the PI3K/Akt pathway. The p53 activation was associated with increased nuclear p53 level. Akt was dephosphorylated, and its inactivation was associated with increased levels of PHLPP1 and PHLPP2 phosphatases. Parallel increases of PARP suggest that JE decreased cell viability by activating cell death. In addition, JE potentiated the effects of gemcitabine and 5-fluorouracil anticancer drugs. Thus, JE can activate cell death in different cancer cell lines through p53 and Akt pathways

Clusterin enhances migration and invasion of prostate cancer cells through an isoform-specific Akt2/miR-190/PHLPP1 circuit

During prostate cancer progression cancer cells undergo a variety of molecular alterations that lead to the acquisition of uncontrolled growth properties. One such set of molecular alterations is mediated by the PI3K/Akt signaling pathway. Here, we describe a regulatory system that modulates the phosphoinosited 3-kinase/Akt (PI3K/Akt) pathway downstream of secreted Clusterin (sCLU) in normal and cancer prostate cells. The overexpression of sCLU is very frequent in prostate cancer, and can lead to Akt-activation. This prompted us to investigate how sCLU overexpression influences PI3K/Akt signaling network in a study model represented by human epithelial prostate PNT1A cells stably transfected with sCLU or with empty vector alone. We found that CLU cells show a marked differential phosphorylation of several members of the PI3K/Akt cascade, and in particular of Akt2. Moreover, we found that the phosphatase PHLPP1, known to dephosphorylate Akt2 at S473, is severely downregulated in CLU compared to MOCK cells. We thus investigated whether sCLU alters PHLPP1 protein stability or expression. Our results indicate that sCLU indeed stimulates PHLPP1 degradation by β-TrCP. Interestingly, we further demonstrated that sCLU alters also PHLPP1 through the negative regulator miR-190. Next, because sCLU has been reported to inhibit or to stimulate the aggressive behavior of cancer cells depending on the cell model, we investigated the effects of CLU overexpression or addition of recombinant Clusterin to the medium on cell migration and invasion in PNT1A cell line, which is not expected to display an invasive phenotype, and in the cancer prostate epithelial cell lines LNCaP and PC3. The result was extremely clear: not only CLU overexpression gives PNT1A cells the same behavior of wild-type PC3 cells, but also increases the migration and invasion index of all the above cell models by two to four times, compared to controls. As a confirmation, in the same model silencing of Clusterin abrogates migration of CLU cells. Next, the effect of Akt single-isoform silencing on cell migration was explored. While silencing of Akt1 affected migration only slightly, silencing of Akt2 prevented migration of both MOCK and CLU cells completely. The same result was obtained by pharmacological inhibition of Akt2. All together our results, clearly demonstrate for the first time that Clusterin can switch the low migration phenotype of normal prostate cells towards a high migration phenotype through the modulation of the expression of the PHLPP1 and, in turn, the activity of Akt2

Understanding the roles of the P2X7 receptor in solid tumour progression and therapeutic perspectives

P2X7 is an intriguing ionotropic receptor for which the activation by extracellular ATP induces rapid inward cationic currents and intracellular signalling pathways associated with numerous physiological processes such as the induction of the inflammatory cascade, the survival and proliferation of cells. In contrast, long-term stimulation of P2X7 is generally associated with membrane permeabilisation and cell death. Recently, P2X7 has attracted great attention in the cancer field, and particularly in the neoplastic transformation and the progression of solid tumours. A growing number of studies were published; however they often appeared contradictory in their results and conclusions. As such, the involvement of P2X7 in the oncogenic process remains unclear so far. The present review aims to discuss the current knowledge and hypotheses on the involvement of the P2X7 receptor in the development and progression of solid tumours, and highlight the different aspects that require further clarification in order to decipher whether P2X7 could be considered as a cancer biomarker or as a target for pharmacological intervention. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers

P2X7 in Cancer: From Molecular Mechanisms to Therapeutics

P2X7 is a transmembrane receptor expressed in multiple cell types including neurons, dendritic cells, macrophages, monocytes, B and T cells where it can drive a wide range of physiological responses from pain transduction to immune response. Upon activation by its main ligand, extracellular ATP, P2X7 can form a nonselective channel for cations to enter the cell. Prolonged activation of P2X7, via high levels of extracellular ATP over an extended time period can lead to the formation of a macropore, leading to depolarization of the plasma membrane and ultimately to cell death. Thus, dependent on its activation state, P2X7 can either drive cell survival and proliferation, or induce cell death. In cancer, P2X7 has been shown to have a broad range of functions, including playing key roles in the development and spread of tumor cells. It is therefore unsurprising that P2X7 has been reported to be upregulated in several malignancies. Critically, ATP is present at high extracellular concentrations in the tumor microenvironment (TME) compared to levels observed in normal tissues. These high levels of ATP should present a survival challenge for cancer cells, potentially leading to constitutive receptor activation, prolonged macropore formation and ultimately to cell death. Therefore, to deliver the proven advantages for P2X7 in driving tumor survival and metastatic potential, the P2X7 macropore must be tightly controlled while retaining other functions. Studies have shown that commonly expressed P2X7 splice variants, distinct SNPs and post-translational receptor modifications can impair the capacity of P2X7 to open the macropore. These receptor modifications and potentially others may ultimately protect cancer cells from the negative consequences associated with constitutive activation of P2X7. Significantly, the effects of both P2X7 agonists and antagonists in preclinical tumor models of cancer demonstrate the potential for agents modifying P2X7 function, to provide innovative cancer therapies. This review summarizes recent advances in understanding of the structure and functions of P2X7 and how these impact P2X7 roles in cancer progression. We also review potential therapeutic approaches directed against P2X7

Toluene diisocyanate exposure and autotaxin–lysophosphatidic acid signalling

Toluene diisocyanate (TDI) is a reactive chemical used in manufacturing plastics. TDI exposure adversely affects workers' health, causing occupational asthma, but individuals differ in susceptibility. We recently suggested a role for signalling mediated by the enzyme autotaxin (ATX) and its product, lysophosphatidic acid (LPA), in TDI toxicity. Here we genotyped 118 TDI-exposed workers for six single-nucleotide polymorphisms (SNPs) in genes encoding proteins implicated in ATX–LPA signalling: purinergic receptor P2X7 (P2RX7), C–C motif chemokine ligand 2 (CCL2), interleukin 1β (IL1B), and caveolin 1 (CAV1). Two P2RX7 SNPs (rs208294 and rs2230911) significantly modified the associations between a biomarker of TDI exposure (urinary 2,4-toluene diamine) and plasma LPA; two IL1B SNPs (rs16944 and rs1143634) did not. CAV1 rs3807989 modified the associations, but the effect was not statistically significant (p = 0.05–0.09). In vitro, TDI-exposed bronchial epithelial cells (16HBE14o-) rapidly released ATX and IL-1β. P2X7 inhibitors attenuated both responses, but confocal microscopy showed non-overlapping localizations of ATX and IL-1β, and down-regulation of CAV1 inhibited the ATX response but not the IL-1β response. This study indicates that P2X7 is pivotal for TDI-induced ATX–LPA signalling, which was modified by genetic variation in P2RX7. Furthermore, our data suggest that the TDI-induced ATX and IL-1β responses occur independently

Toluene diisocyanate: Induction of the autotaxin-lysophosphatidic acid axis and its association with airways symptoms.

Diisocyanates are industrial chemicals which have a wide range of applications in developed and developing countries. They are notorious lung toxicants and respiratory sensitizers. However, the mechanisms behind their adverse effects are not adequately characterized. Autotaxin (ATX) is an enzyme producing lysophosphatidic acid (LPA), and the ATX-LPA axis has been implicated in lung related inflammatory conditions and diseases, including allergic asthma, but not to toxicity of environmental low-molecular-weight chemicals. We investigated effects of toluene diisocyanate (TDI) on ATX induction in human lung epithelial cell models, and we correlated LPA-levels in plasma to biomarkers of TDI exposure in urine collected from workers exposed to <5ppb (parts per billion). Information on workers' symptoms was collected through interviews. One nanomolar TDI robustly induced ATX release within 10min in vitro. A P2X7- and P2X4-dependent microvesicle formation was implicated in a rapid ATX release and a subsequent protein synthesis. Co-localization between purinergic receptors and ATX was documented by immunofluorescence and confocal microscopy. The release was modulated by monocyte chemoattractant protein-1 (MCP-1) and by extracellular ATP. In workers, we found a dose-response relationship between TDI exposure biomarkers in urine and LPA levels in plasma. Among symptomatic workers reporting "sneezing", the LPA levels were higher than among non-symptomatic workers. This is the first report indicating induction of the ATX-LPA axis by an environmental low-molecular-weight chemical, and our data suggest a role for the ATX-LPA axis in TDI toxicity

Clusterin enhances migration and invasion of prostate cancer cells through an isoform-specific Akt2/miR-190/PHLPP1 circuit.

During prostate cancer progression cancer cells undergo a variety of molecular alterations that lead to the acquisition of uncontrolled growth properties. One such set of molecular alterations is mediated by the PI3K/Akt signaling pathway. Here, we describe a regulatory system that modulates the phosphoinosited 3-kinase/Akt (PI3K/Akt) pathway downstream of secreted Clusterin (sCLU) in normal and cancer prostate cells. The overexpression of sCLU is very frequent in prostate cancer, and can lead to Akt-activation. This prompted us to investigate how sCLU overexpression influences PI3K/Akt signaling network in a study model represented by human epithelial prostate PNT1A cells stably transfected with sCLU or with empty vector alone. We found that CLU cells show a marked differential phosphorylation of several members of the PI3K/Akt cascade, and in particular of Akt2. Moreover, we found that the phosphatase PHLPP1, known to dephosphorylate Akt2 at S473, is severely downregulated in CLU compared to MOCK cells. We thus investigated whether sCLU alters PHLPP1 protein stability or expression. Our results indicate that sCLU indeed stimulates PHLPP1 degradation by β-TrCP. Interestingly, we further demonstrated that sCLU alters also PHLPP1 through the negative regulator miR-190. Next, because sCLU has been reported to inhibit or to stimulate the aggressive behavior of cancer cells depending on the cell model, we investigated the effects of CLU overexpression or addition of recombinant Clusterin to the medium on cell migration and invasion in PNT1A cell line, which is not expected to display an invasive phenotype, and in the cancer prostate epithelial cell lines LNCaP and PC3. The result was extremely clear: not only CLU overexpression gives PNT1A cells the same behavior of wild-type PC3 cells, but also increases the migration and invasion index of all the above cell models by two to four times, compared to controls. As a confirmation, in the same model silencing of Clusterin abrogates migration of CLU cells. Next, the effect of Akt single-isoform silencing on cell migration was explored. While silencing of Akt1 affected migration only slightly, silencing of Akt2 prevented migration of both MOCK and CLU cells completely. The same result was obtained by pharmacological inhibition of Akt2. All together our results, clearly demonstrate for the first time that Clusterin can switch the low migration phenotype of normal prostate cells towards a high migration phenotype through the modulation of the expression of the PHLPP1 and, in turn, the activity of Akt2

Human Peripheral Blood Eosinophils Express High Levels of the Purinergic Receptor P2X4

International audienceExtracellular nucleotides are important mediators of cell activation and trigger multiple responses via membrane receptors known as purinergic receptors (P2). P2X receptors are ligand-gated ion channels, activated by extracellular ATP. P2X4 is one of the most sensitive purinergic receptors, that is typically expressed by neurons, microglia, and some epithelial and endothelial cells. P2X4 mediates neuropathic pain via brain-derived neurotrophic factor and is also involved in inflammation in response to high ATP release. It is therefore involved in multiple inflammatory pathologies as well as neurodegenerative diseases. We have produced monoclonal antibodies (mAb) directed against this important human P2X4 receptor. Focusing on two mAbs, we showed that they also recognize mouse and rat P2X4. We demonstrated that these mAbs can be used in flow cytometry, immunoprecipitation and immunohistochemistry, but not in Western blot assays, indicating that they target conformational epitopes. We also characterised the expression of P2X4 receptor on mouse and human peripheral blood lymphocytes (PBL). We showed that P2X4 is expressed at the surface of several leukocyte cell types, with the highest expression level on eosinophils, making them potentially sensitive to adenosine triphosphate (ATP). P2X4 is expressed by leucocytes, in human and mouse, with a significant gender difference, males having higher surface expression levels than females. Our findings reveal that PBL express significant levels of P2X4 receptor, and suggest an important role of this receptor in leukocyte activation by ATP, particularly in P2X4high expressing eosinophils