?(Fig
?(Fig.5b).5b). apoptotic signalling was analyzed following soluble-TRAIL or anti-TRAIL-R2 agonist antibody treatment. Lipid rafts were isolated by Triton X-100 fractionation. Preclinical ex vivo studies were performed in OC cells derived from individuals ascites using autologous PBLs as effectors and a bispecific anti-TRAIL-R2/anti-CD3 antibody as triggering agent. Results Here we demonstrate that siCHKA specifically overcomes resistance to TRAIL-mediated apoptosis in OC cells. Upon siCHKA we recognized: a significant sensitization to caspase-dependent apoptosis induced by both soluble TRAIL and anti-TRAIL-R2 agonist antibody, a specific increase of TRAIL-R2 manifestation and TRAIL-R2 relocation into lipid rafts. In siCHKA-OC cells the acquired TRAIL level of sensitivity was completely reverted upon recovery (S)-(-)-Perillyl alcohol of ChoK manifestation but, at variance of additional tumor cell types, TRAIL level of sensitivity was not efficiently phenocopied by methionine deprivation. Of notice, we were also able to display that siCHKA sensitized tumor cells derived ex lover STAT6 vivo from OC individuals ascites to the cytotoxic activity of autologous lymphocytes redirected by a bispecific anti-TRAIL-R2/anti-CD3 antibody. Conclusions Our findings suggest that ChoK/impairment, by repairing drug-induced or receptor-mediated cell death, could be a suitable restorative strategy to be applied in combination with chemotherapeutics or immunomodulators to improve OC individuals outcome. Drug resistance is a complex phenomenon associated with several alterations influencing multiple pathways [3]. Malignancy cells frequently acquire (S)-(-)-Perillyl alcohol the ability to resist to apoptotic stimuli induced by chemotherapeutic providers or by death receptors (S)-(-)-Perillyl alcohol triggering. With this scenario, we contributed to define the mechanisms of OC escape from the death receptor CD95/Fas signaling [4C6]. TNF-related apoptosis-inducing ligand (TRAIL) receptor 2 (R2) is one of the death-inducing receptors induced by TRAIL, a member of the TNF ligand super-family of cytokines, produced and secreted by most immune cells [7] and regarded as a encouraging anticancer agent due to its remarkable ability to selectively destroy neoplastic cells sparing normal ones. However, the majority of solid tumors including OC are known to develop resistance to TRAIL-induced apoptosis. Overcoming this resistance still remains an open challenge [8, 9] and in this perspective, we recently showed that bispecific antibodies directed to TRAIL-R2 as tumor-associated antigen (TAA) and to CD3 as triggering molecule were able to redirect polyclonal MHC-unrestricted T lymphocytes cytotoxicity toward OC cells [10]. Among the complex alterations responsible for acquired resistance to death stimuli, there is also the metabolic reprogramming of tumor cells [11]. Interestingly, it was recently reported that methionine deprivation in different cancer models could induce a targetable vulnerability by increasing TRAIL-R2 manifestation [12C14]. Like many other malignancy types, OC shows alterations in the main metabolic pathways [15, 16] and the We shown that the increase in PCho content material in OC cells is mainly maintained from the overexpression and activity of choline kinase alpha (ChoKgene silencing generally hampered OC aggressiveness both in vitro and in vivo by reducing cell proliferation, migration and invasion capabilities [23]. Furthermore, following ChoK impairment we observed a decreased intracellular content material of metabolites other than PCho such as cysteine, serine, methionine and glutathione suggesting deep alterations in circuitry related to one-carbon cycles [24, 25]. In particular we found that the decrease in glutathione level impaired oxidative (S)-(-)-Perillyl alcohol stress redox status, therefore raising intracellular reactive oxygen species that improved in vitro level of sensitivity to standard chemotherapeutics [24]. Here we aimed to investigate in in vitro and ex lover (S)-(-)-Perillyl alcohol vivo OC models whether the ChoK pro-tumorigenic functions could also impact susceptibility to TRAIL-dependent apoptosis and in particular if the inactivation of ChoK/manifestation and function could be a suitable strategy to conquer TRAIL resistance. Methods Cell ethnicities The following OC cell lines were used: SKOV3, OVCAR5, A2780 (from ATCC), INTOV11 (acquired in our laboratory from a high-grade serous OC [23]), A2774 (offered from dr. S. Ferrini, IRCCS Ospedale Policlinico S. Martino, Italy) were managed in RPMI1640.