Posted on May 20, 2021
The microenvironment in mantle cell lymphoma: cellular and molecular pathways and emerging targeted therapies
The microenvironment in mantle cell lymphoma: cellular and molecular pathways and emerging targeted therapies. and showing that this compound might be effective for the treatment of these patients . Regarding MCL, we previously reported that acadesine was cytotoxic for MCL cells alone or in combination with rituximab . However, the responses among the MCL samples were heterogeneous and the molecular mechanisms implicated in acadesine response were not fully characterized. In this manuscript, we provide insight around the signaling pathways implicated in the activity of the compound in MCL cells and explore a rational combination with ABT-199 to overcome acadesine resistance in MCL. RESULTS Acadesine induces apoptosis by a caspase-dependent mechanism and activates AMPK Ginsenoside Rh3 We previously reported that acadesine was able to induce cytotoxicity in MCL cell lines and primary MCL samples, although some differences in sensitivity were observed among them . With the aim to provide further evidence around the cell death mechanism triggered by the drug in these cells, we analyzed several apoptotic hallmarks. JEKO-1 and HBL-2 cell lines, with different sensitivity to the compound according to our previous results , and 3 primary MCL samples were incubated with acadesine (2 mM) for 24 hours and mitochondrial depolarization, caspase-3 activation and phosphatidylserine exposure were analyzed by flow cytometry. In all the samples studied, although at different magnitude, acadesine concomitantly decreased the mitochondrial membrane potential, activated the caspase-3 and increased the phospatidylserine exposure (Physique ?(Figure1A).1A). On the contrary, when the caspase inhibitor Q-VD-OPh was added, cells were rescued from caspase-3 activation and phosphatidylserine exposure but not from the loss of the mitochondrial membrane potential, indicating that the apoptosis induced by the nucleoside analogue was caspase-mediated (Physique ?(Figure1A1A). Open in a separate window Physique 1 Acadesine induces apoptosis and activates AMPKA. JEKO-1, HBL-2 and 3 primary MCL samples were preincubated for 1 hour with 10 M of the pan caspase inhibitor Q-VD-OPh and followed by a 24-hour exposure to acadesine 2 mM. Mitochondrial membrane potential (m), caspase-3 activation and phosphatidylserine exposure were evaluated by flow cytometry as detailed in Methods. B. MCL lines (JEKO-1 and HBL-2) and two representative primary MCL samples were cultured with acadesine 2 mM for 6 hours and protein levels of Bim, Puma and Noxa were determined by western blot. -tubulin was used as Ginsenoside Rh3 loading control. C. MCL lines (JEKO-1 and HBL-2) and two MCL primary samples were cultured with acadesine 2 mM for 6 hours. Phosphorylated and total levels of ACC were assessed by western blot using -tubulin as loading control. The ratio between the phosphorylated and unphosphorylated form was showed. Given that Rabbit Polyclonal to RPS19BP1 in CLL cells acadesine-induced apoptosis has been reported to be mediated by the up-regulation of the proapoptotic BH3-only proteins Bim, Noxa and Puma , we examined the levels of these proteins in our model. MCL cell lines and primary MCL cells were incubated with acadesine (2 mM) for 6 hours and BH3-only proteins were analyzed by western blot. As shown in Physique ?Physique1B,1B, no upregulation of any of these proteins in the samples studied was detected, suggesting a different mechanism of apoptosis induction in MCL cells. As previously reported, Bim expression was not detected in JEKO-1 cells due to a homozygous deletion at locus . Next, we verified whether acadesine Ginsenoside Rh3 was efficiently activating AMPK in MCL cells, as seen in the majority of cell types, including CLL . For this purpose, we assessed the levels of phosphorylation of the AMPK substrate, acetyl-CoA carboxylase (ACC), which is usually phosphorylated upon AMPK activation . Indeed, as shown in Physique ?Physique1C,1C, a 6-hour incubation with acadesine induced ACC phosphorylation in all MCL samples, indicating that acadesine activated the AMPK pathway. Acadesine induces VASP phosphorylation concomitantly to an inhibition of CXCL12-induced chemotaxis and cytoskeleton organization AMPK has been reported to regulate the phosphorylation of the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) . VASP phosphorylation results in inhibition of actin polymerization, cell adhesion and migration [22, 23]. Gene expression profile studies from our group suggested a potential role of acadesine in reducing the migration of MCL cell lines . In this context, we sought to explore whether the inhibition of.