The G-protein-coupled central cannabinoid receptor (CB1) has been shown to be functionally associated with several biological responses including inhibition of adenylate cyclase, modulation of ion channels and induction of the immediate-early gene Krox-24. Using stably transfected Chinese Hamster Ovary cells expressing human CB1 we show here that cannabinoid treatment induces both phosphorylation and activation of mitogen-activated protein (MAP) kinases, and that these effects are inhibited by SR 141716A, a selective CB1 antagonist. The two p42 and p44 kDa MAP kinases are activated in a time- and dose-dependent manner. The rank order of potency for the activation of MAP kinases with various cannabinoid agonists is CP-55940 > delta 9-tetrahydrocannabinol > WIN 55212.2, in agreement with the pharmacological profile of CB1. The activation of MAP kinases is blocked by pertussis toxin but not by treatment with hydrolysis-resistant cyclic AMP analogues. This suggests that the signal transduction pathway between CB1 and MAP kinases involves a pertussis-toxin-sensitive GTP-binding protein and is independent of cyclic AMP metabolism. This coupling of CB1 subtype and mitogenic signal pathway, also observed in the human astrocytoma cell line U373 MG, may explain the mechanism of action underlying cannabinoid-induced Krox-24 induction.

Cannabinoids, known for their psychoactive effects, also possess iminunomodulatory properties. The recent isolation and cloning of the G-protein-coupled peripheral cannabinoid receptor (CB2), mainly expressed in immune tissues, have provided molecular tools to determine how cannabinoid compounds may mediate immunomodulation. We here investigated the CB2 signaling properties using stably transfected Chinese hamster ovary cells expressing human CB2. First, we showed that stimulation by a cannabinoid agonist activated mitogen-activated protein (MAP) kinase in time-and dose-dependent manners. The rank order of potency for MAP kinase activation of cannabinoid agonists correlated well with their binding capacities. Second, we demonstrated that, following MAP kinase activation, cannabinoids induced the expression of the growth-related gene Krox-24, also known as NGFI-A, 28268, and egr-1 .Pertussis toxin completely prevented both MAP kinase activation and Krox-24 induction, even more these responses appeared to be dependent of specific proteine kinase C isoforms and independent of inhibition of adenylyl cyclase. A similar coupling of CB2 to a mitogenic pathway and to the regulation of Krox-24 expression was also observed in human promyelocytic cells HL60. Taken together, these findings provide evidence for a functional role of the CB2 receptor in gene induction mediated by the MAP kinase network.Keywords: peripheral cannabinoid receptor; cannabinoid receptor CB2 ; mitogen-activated protein kinase ; Krox-24: cannabinoid.A'-Tetrahydrocannabinol, the major active component of marijuana as well as other cannabinoids, is known to exert a wide range of physiological effects : drowsiness, alterations in cognition and memory, analgesia, as well as anti-inflammatory and immunomodulatory effects [I, 21. Many studies have been conducted to decipher the complexity of the cannabinoid system. First attributed to nonspecific cell membrane alterations, the cannabinoid effects are now known to be mediated through cannabinoid receptors. Two proteins with seven transmembranespanning domains typical of G-protein-coupled receptors have been identified as tetrahydrocannabinol receptors and referred to as CBI and CB2.The CBI receptor is predominantly expressed in the brain [3, 41 and could account for the psychoactive effects of cannabinoids. This receptor is also found in the periphery but at a much lower abundance [5-71. Several signaling pathways triggered by the stimulation of CBI have already been described, all being sensitive to pertussis toxin (PTX). Activation of CBI inhibits adenylyl cyclase activity [8J as well as voltage-depenCorrespondence to P. Casellas, Sanofi Recherche, 371 rue du Pr. Joseph Blayac, F-34184 Montpellier cedex 04, France.Abbrevintions. MAP, mitogen-activated protein; MBP, myelin basic protein; CHO, Chinese hamster ovary; CB1, central cannabinoid receptor; CB2, peripheral cannabinoid receptor; Br*cAMP, 8-bromoadenosine 3',S'-monophosphate; Bt,cAMP, P,2'-dibutyryl-adenosine 3',S'-monophosphate; EMSA, electrophoret...

The recent isolation and cloning of the G protein-coupled central cannabinoid receptor (CB1) from brain tissue has provided a molecular basis to elucidate how cannabinoid compounds may mediate their psychoactive effects. Here we report the high expression of cannabinoid receptors in human astrocytoma tumors of different grades, in the astrocytoma cell lines U373 MG and GL-15, as well as in normal astrocytes. From an analysis of the coupling mechanisms of functional CB1 receptors in U373 MG, we show that, in addition to the inhibition of adenylyl cyclase, activation by the cannabinoid agonist CP-55940 induces the expression of the immediate-early gene krox-24, also known as NGFI-A, zif/268, egr-1, and TIS8. The amount of Krox-24 protein and the level of Krox-24 DNA binding activity, as measured by Western blot and electrophoretic mobility shift assay, respectively, were also increased by the addition of CP-55940. These effects were blocked by incubation with pertussis toxin but not by treatment with hydrolysis-resistant cAMP analogues, suggesting that the transduction pathway between the cannabinoid receptor and krox-24 involves a pertussis toxin-sensitive GTP-binding protein and is independent of cAMP metabolism. The specific involvement of CB1 in Krox-24 induction was demonstrated in Chinese hamster ovary cells transfected with the human CB1 receptor and also in experiments using the CB1-selective cannabinoid antagonist SR 141716A.

N-Heterocyclic carbene (NHC) platinum complexes have been highlighted as a promising and original platform for building new cytotoxic drugs of the cisplatin series. Mixed NHC-amine Pt(II) complexes have been prepared via a facile and modular two step sequence leading to trans-configured square planar species. They have been characterized by spectroscopic methods and X-ray diffraction studies. Their efficiency against both cisplatin sensitive (CEM and H460) and resistant (A2780/DDP, CH1/DDP, and SK-OV-3) cell lines has been demonstrated by in vitro experiments.

No abstract