Cannabinoids Induce Cell Death in Leukemic Cells through Parthanatos and PARP-Related Metabolic Disruptions

BACKGROUND: We have previously described the antitumor effect of the cannabinoid WIN-55,212-2 (WIN-55) and a set of cannabinoid derivatives (CNB) specific for CB2 in multiple myeloma (Barbado et al, 2018). In AML, we also observed a potent and selective antileukemic effect, affecting signaling and metabolic pathways essential for the viability of tumor cells. Among them, we found an increased stress on the endoplasmic reticulum, mitochondrial damage, and alteration of the metabolism of ceramides, although none of these events turned out to be the main trigger of cell death, since the inhibition of each of them did not prevent the antileukemic effect of CNB. On the other hand, disruption of the mecanisms of DNA repair have been identified as a key oncogeneic event in different solid tumors, and some studies have also suggested that it might be involved in leukemogenesis. More specifically, PARP1 is involved in DNA damage repair. Other functions include the regulation of glycolysis enzymes through the addition of Poly ADP-Ribose (PAR) and the execution of Parthanatos, that occurs whenever PARP-1 becomes over-activated in response to extreme damage inducing nuclear translocation of AIF and depletion of NAD +. OBJECTIVES: In this study we set out to identify the ultimate mechanism that justifies the aforementioned pleiotropic effect of CNB on the metabolism of leukemic cells and their viability. METHODS: Cell viability was determined by MTT and flow cytometry. The mRNA and / or protein expression profile of AML samples or healthy progenitor cells were studied by qPCR and / or Western blot. Glycolytic flux was studied with the XF Glycolytic Rate Assay (Seahorse Biosciences). NAD + levels and glycolytic enzyme activity were measured using quantification kits. Parylation of different enzymes were confirmed by Co-IP using the corresponding antibodies. Finally, NOD/scid/IL-2R gammae null (NSG) mice were xenotransplanted with HL60-Luciferase cell line. Once the presence of leukemic cells was confirmed, treatment with vehicle, WIN-55 cannabinoid at a dose of 5 mg/kg/day or citarabine (ARA-C) at 50 mg/kg during 5 days was administered. Also we tested the effect of these compounds on normal hematopoiesis by treating healthy BALB-C mice. RESULTS: Pretreatment of leukemic cells with Olaparib, a PARP1 inhibitor, reversed WIN-55 induced apoptosis by almost 100%. WIN-55 affected the activity of most glycolysis enzymes, with a marked drop of the activity of GAPDH and pyruvate kinase which was reversed by Olaparib pretreatment. Also G6PDH activity was markedly affected upon culture with WIN-55. Co-IP confirmed parylation of these enzymes which was reversed with Olaparib. ECAR data detected by Seahorse also confirmed the drop in glycolytic capacity produced by WIN-55 in leukemic cells which again was reversed upon culture with Olaparib. In addition, the addition of nicotinamide mononucleotide (NAM), a precursor of NAD +, reversed the loss of viability produced by WIN-55. Next, we confirmed that PARP1 levels were significantly higher in leukemic cell lines and in a series of 40 AML patients as compared to healthy hematopoietic stem cells (HSC). Finally, we observed a translocation of AIF to the nucleus, confirming that WIN-55 produces PARTHANATOS. In a murine model we confirmed treatment with WIN-55,212-2 significantly prolonged survival in AML xenograft mice, with disappearance of the leukemic clone in a significant proportion of cases. By contrast, cannabinoids did not affect the viability of hematopoietic stem cells (HSC) in vivo, resulting in a lack of myeloid toxicity in healthy treated mice. CONCLUSIONS: WIN-55 exerts a selective antileukemic effect through the overactivation of PARP1, affecting the levels of parylation in enzymes involved in glycolysis and pentose phosphate pathways, leading to the translocation of AIF to the nucleus and to depletion of NAD +, which were reversed through PARP1 inhibition. These effects are not observed in normal HSC. These data are confirmed in murine models in which we confirmed the antileukemic effect of WIN-55 withouth hampering normal hematopoiesis. Figure 1 Figure 1. Disclosures Pérez-Simón: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Synthetic Cannabinoid Agonist WIN 55212-2 Targets Proliferation, Angiogenesis, and Apoptosis via MAPK/AKT Signaling in Human Endometriotic Cell Lines and a Murine Model of Endometriosis

Endometriosis (EM) is characterized by the growth of endometrium-like tissue outside the uterus, leading to chronic inflammation and pelvic pain. Lesion proliferation, vascularization, and associated inflammation are the hallmark features of EM lesions. The legalization of recreational cannabinoids has garnered interest in the patient community and is contributing to a greater incidence of self medication; however, it remains unknown if cannabinoids possess marked disease-modifying properties. In this study, we assess the effects of synthetic cannabinoid, WIN 55212-2 (WIN 55), in EM-representative in vitro and in vivo syngeneic mouse models. WIN 55 reduced proliferation and angiogenesis in vitro, via MAPK/Akt-mediated apoptosis. These findings were corroborated in a mouse model of EM, where we found reduced TRPV1 expression in the dorsal root ganglia of the EM mouse model exposed to WIN 55, suggesting reduced signaling of pain stimuli. Ultimately, these pieces of evidence support the use of cannabinoid receptor agonists as a potential therapeutic intervention for EM associated pain and inflammation.

Influence of Cannabinoid on Interleukin-1β Treated Cartilage: An In Vitro Study

Category: Basic Sciences/Biologics Introduction/Purpose: Cannabinoids have been reported to possess the analgesic, immunomodulatory and anti-inflammatory properties. Recent studied further shown that cannabinoids attenuated joint damage in animal models of arthritis. However, the underlying mechanism has been completely understood. Interleukin-1β (IL-1β), a proinflammatory cytokine that can result in the degradation of cartilage, is associated with the pathogenesis of osteoarthritis. In this study, we hypothesize that cannabinoid can mitigate the detrimental effect of IL-1β on cartilage, thus reduce the progression of osteoarthritis. To test the hypothesis, we insulted human chondrocyte-derived cartilage with IL-1 β for 48 hours and then applied a synthetic cannabinoid agonist, Win- 55,212-2(Win-55), into the culture. The tissue phenotypes were assessed by real time polymerase chain reaction (PCR), histology and immunostaining. Methods: With the approval from CORID, human chondrocytes were isolated from healthy articular cartilage. P2 cells were used. MTS assay were employed to test the half-maximal (50%) inhibitory concentration (IC50). To generate cartilage in vitro, chondrocytes were pelleted and subjected to 14 days chondrogenic culture. The engineered cartilages were stimulated with 10 ng/ml IL-1β for 48 hours and then treated with different concentration of Win-55 (0.01, 0.1, or 1 µM) for another 48 hours. The tissue phenotype was assessed by glycosaminoglycan (GAG) assay, real-time PCR and histology. Results: We tested 10 doses, from 0.001µM up to 10 µM, and determined that the IC 50 of Win-55 on human chondrocytes for 2 days was ˜ 2 µM. Interestingly, this dose is significantly lower than the doses reported in similar studies. As shown in Figure 1, treatment with 2µM Win-55 causes the complete loss of GAG from engineer cartilage. In a relatively safe dose (<=1 µM), we did not observe obvious changes in all tested genes after the treatments of Win-55 (Figure 2). Conclusion: High dose of Win-55 may directly cause the degeneration of cartilage, while low dose of Win-55 doesn’t show beneficial influence on the phenotype of IL1-β-insulted cartilage. The reported anti-inflammatory effect of Win 55 on chondrocytes may due to the cytotoxicity or global inhibition of high dose Win 55 on cell activities. Therefore, if cannabinoid can be used to treat OA requires further investigation.

Treatment with Cannabinoids as a Promising Approach for Impairing Fibroblast Activation and Prostate Cancer Progression

Endo-, phyto- and synthetic cannabinoids have been proposed as promising anti-cancer agents able to impair cancer cells’ behavior without affecting their non-transformed counterparts. However, cancer outcome depends not only on cancer cells’ activity, but also on the stromal cells, which coevolve with cancer cells to sustain tumor progression. Here, we show for the first time that cannabinoid treatment impairs the activation and the reactivity of cancer-associated fibroblasts (CAFs), the most represented stromal component of prostate tumor microenvironment. Using prostate cancer-derived CAFs, we demonstrated that WIN 55-212.2 mesylate, a synthetic full agonist of cannabinoid receptors (CBs) 1 and 2, downregulates α-smooth muscle actin and matrix metalloprotease-2 expression, and it inhibits CAF migration, essential features to ensure the activated and reactive CAF phenotype. Furthermore, by impairing stromal reactivity, WIN 55-212.2 mesylate also negatively affects CAF-mediated cancer cells’ invasiveness. Using selective antagonists of CBs, we proved that CAFs response to WIN 55-212.2 mesylate is mainly mediated by CB2. Finally, we suggest that endocannabinoids self-sustain both prostate tumor cells migration and CAFs phenotype by an autocrine loop. Overall, our data strongly support the use of cannabinoids as anti-tumor agents in prostate cancer, since they are able to simultaneously strike both cancer and stromal cells.

Cannabinoids Derivatives Exert a Potent Antileukemic Effect By Modifying the Pattern of Membrane Sphingolipids

Endocannabinoid system is a set of ligands, receptors and endogenous enzymes which modulate a variety of physiological effects. There are two well-characterized cannabinoid receptors, CB1 (mainly expressed in Central Nervous System) and CB2 (mainly in hematopoietic cells). Here, we tested the effect of the cannabinoid WIN-55 212-2 in acute myeloid leukemia (AML) in vitro, ex vivo and in vivo and studied the molecular signaling pathways involved in this effect. Moreover, we synthesized a new family of twelve cannabinoids that are specific to CB2 receptor. For their design and synthesis, computational techniques of docking, analytical and spectroscopic techniques such as mass spectrometry (MS) were used. To assess the anti-leukemia effect of the different cannabinoids, we analyzed cell viability by MTT and flow cytometry using six human AML cell lines, primary cells from healthy donors (hematopoietic progenitor cells (HPC) and lymphocytes) and blasts from AML patients. Cannabinoids induced a potent proapoptotic effect on AML cell lines and on primary leukemic cells, which was not observed in normal HPC and lymphocytes from healthy donors. Fragmentation of PARP and activation of caspases 2, 3, 8 and 9 were confirmed by western-blot. Other proteins involved in the effect of cannabinoids were p-AKT, p-ERK 1/2, p-38 and p- JNK. Also studies on p-PERK, p-IRE1 and CHOP confirmed an increased endoplasmic reticulum stress upon exposure to cannabinoids. Mitochondrial damage was analyzed by flow cytometry using TMRE and by MitoSOXTMRed. These assays confirmed a very early mitochondrial damage in leukemic cells which was not observed in normal hematopoietic progenitor cells. Moreover, we analyzed the ceramide levels, a membrane lipid associated with death/survival cell processes by HPLC and immunohistochemistry. Remarkably, we observed significant differences in the amounts of certain subtypes of ceramides in untreated versus treated leukemic cells. The proapoptotic effect of cannabinoids on AML cells was abolished upon co-culture with either CB2 receptor antagonists or with pancaspase inhibitors. Finally, NOD/scid/IL-2R gammae null (NSG) mice were xenotransplanted with HL60 cell line. We confirmed disease infiltration in bone marrow (BM) by BM aspirates and flow cytometry assays. Once the presence of leukemic cells was confirmed, treatment with vehicle, WIN-55 cannabinoid at a dose of 5 mg/kg/day or citarabine (ARA-C) at 50 mg/kg during 5 days was administered. We observed a significantly increased survival among mice treated with WIN-55 cannabinoid as compared to both the control group and the group treated with ARA-C. In addition, we tested in vivo the effect of these compounds on normal hematopoiesis by treating healthy BALB-C mice. We confirmed that cannabinoids did not affect the viability of the different populations of hematopoietic progenitors (LK, GMP, CMP) and, moreover, an increased platelet count was observed in treated mice. Our findings indicate that cannabinoids display a highly selective proapoptotic effect against leukemic cells. Several pathways are involved in this effect, the modification in the ceramide pattern playing a main role. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

Ο ρόλος του ενδογενούς συστήματος των κανναβινοειδών σε συμπεριφορικές και νευροβιολογικές παραμέτρους που σχετίζονται με τις γνωστικές λειτουργίες

Τα εξωγενώς χορηγούμενα κανναβινοειδή και το ενδογενέςκανναβινοειδές σύστημα εμπλέκονται σε αρκετές φυσιολογικέςλειτουργίες και διαταραχές του ΚΝΣ. Ο σκοπός της παρούσας εργασίαςαφορούσε στη μελέτη συγκεκριμένων συμπεριφορικών καινευροχημικών μεταβλητών μετά από τη χορήγηση κανναβινοειδών.Ειδικότερα, μελετήθηκαν οι επιδράσεις των αγωνιστών των CB1υποδοχέων των κανναβινοιεδών σε λειτουργίες μνήμης και μάθησης καιστο γλουταματεργικό σύστημα. Για το σκοπό της μελέτης αυτήςχρησιμοποιήθηκαν το WIN 55-212,2 και η Δ9-THC, το κύριοψυχοδραστικό συστατικό της κάνναβης. Τα αποτελέσματα της μελέτης έδειξαν ότι μικρές δόσεις του WIN55,212-2, οι οποίες δεν επηρεάζουν σημαντικά την κινητικότητα,προσβάλουν τη μη συνειρμική μνήμη, τις διαφορετικές εκφάνσεις τηςαναγνωριστικής μνήμης και τη χωρική μνήμη. Σχετικά με τη γλουταματεργική επίδραση, οι χαμηλές μηκατασταλτικές και υψηλότερες κατασταλτικές δόσεις του WIN 55,212-2και Δ9-THC μετέβαλαν την ιστική συγκέντρωση του γλουταμικού, σεαρκετές εγκεφαλικές περιοχές. Ειδικότερα η χορήγηση του WIN 55,212-2 προκάλεσε αύξηση των εξωκυττάριων συγκεντρώσεων του γλουταμικούστον προμετωπιαίο φλοιό σε αντίθεση με την μείωση των επίπεδων σευποφλοιώδεις περιοχές.Οι συγκεκριμένες ουσίες (Δ9-THC και WIN 55-212,2) μετά από οξείασυστηματική χορήγηση προκάλεσαν σημαντικές μεταβολές στιςσυμπεριφορικές δοκιμασίες μνήμης και μάθησης και επίσης ex vivo καιin vivo μεταβολές στη γλουταματεργική λειτουργία σε διαφορετικέςεγκεφαλικές περιοχές που σχετίζονται με κινητικές λειτουργίες, τοκύκλωμα ανταμοιβής, τη νευροπλαστικότητα και τις γνωστικέςλειτουργίες.