title: The endocannabinoid anandamide induces apoptosis in cytotrophoblast cells: involvement of both mitochondrial and death receptor pathways.
abstract: BACKGROUND
  A balanced proliferation, apoptosis and differentiation in trophoblast cells of the human placenta is crucial for a proper placental development. Alteration in trophoblast apoptosis and differentiation are associated with gestational-related complications, such as preeclampsia, intrauterine growth restriction or miscarriages. The endocannabinoids (eCBs) have been recognized as new interveners in pregnancy events such as implantation and decidualization. However, their importance in placentation is poorly understood. We hypothesise that these novel lipid mediators may intervene in cytotrophoblast apoptosis and, concomitantly, have a role during placental development.
  
  
  METHODS
  primary human cytotrophoblasts (hCTs) and the human trophoblast-like choriocarcinoma cell line BeWo cells were exposed to Anandamide (AEA). It was investigated the cellular pathways involved in cell death, by the assessment of cell morphology, caspases activity, mitochondrial membrane potential (Δψm), reactive oxygen/nitrogen species (ROS/RNS) and western blot of cleaved Poly (ADP-ribose) polymerase 1 (PARP-1), truncated Bid (t-Bid) and IκB-α.
  
  
  RESULTS
  AEA decreased hCTs viability and induced morphological features of apoptosis (chromatin condensation and fragmentation), caspase-3/7 activation and PARP-1 cleavage. In BeWo, AEA also increased the activities of caspase-3/7 and 9, induced loss in Δψm and production of ROS/RNS. These effects were reversed by either CB1 or CB2 antagonists, whereas the increase in caspase-3/7 activity was only reversed with CB2 blockage. AEA-treated cells showed increased caspase-8 activation and formation of t-Bid, suggesting the interplay between intrinsic and extrinsic apoptotic pathways. AEA also increased IκB-α expression, a NF-κB regulatory protein.
  
  
  CONCLUSIONS
  Our results highlight the importance of eCBs in cytotrophoblast cell apoptosis and indicate that a crosstalk between intrinsic and extrinsic apoptotic pathways is involved in AEA-induced effects.
authors.affiliation: Departamento de Ciências Biológicas, Laboratório de Bioquímica, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal; Instituto de Biologia Molecular e Celular da Universidade do Porto (IBMC), Porto, Portugal.
authors.forename: M A
authors.initials: MA
authors.lastname: Costa
authors.affiliation: Departamento de Ciências Biológicas, Laboratório de Bioquímica, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal; Instituto de Biologia Molecular e Celular da Universidade do Porto (IBMC), Porto, Portugal.
authors.forename: B M
authors.initials: BM
authors.lastname: Fonseca
authors.affiliation: Departamento de Ciências Biológicas, Laboratório de Bioquímica, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal; Instituto de Biologia Molecular e Celular da Universidade do Porto (IBMC), Porto, Portugal.
authors.forename: N A
authors.initials: NA
authors.lastname: Teixeira
authors.affiliation: Departamento de Ciências Biológicas, Laboratório de Bioquímica, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal; Instituto de Biologia Molecular e Celular da Universidade do Porto (IBMC), Porto, Portugal. Electronic address: george@ff.up.pt.
authors.forename: G
authors.initials: G
authors.lastname: Correia-da-Silva
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D001095:Arachidonic Acids / Q000494:pharmacology*; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D063388:Endocannabinoids / Q000494:pharmacology*; D005260:Female; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000378:metabolism*; D010920:Placenta / Q000187:drug effects / Q000378:metabolism; D053284:Polyunsaturated Alkamides / Q000494:pharmacology*; D011247:Pregnancy; D043884:Receptor, Cannabinoid, CB1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D043885:Receptor, Cannabinoid, CB2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D053218:Receptors, Death Domain / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D014327:Trophoblasts / Q000187:drug effects* / Q000378:metabolism
chemical_list: D001095:Arachidonic Acids; D063388:Endocannabinoids; D053284:Polyunsaturated Alkamides; D043884:Receptor, Cannabinoid, CB1; D043885:Receptor, Cannabinoid, CB2; D053218:Receptors, Death Domain; D053148:Caspase 3; C078814:anandamide
keywords: Anandamide; Apoptosis; Cytotrophoblasts; Endocannabinoid signalling; Placenta