Differential mechanisms involved in RG-7388 and Nutlin-3 induced cell death in SJSA-1 osteosarcoma cells

Abstract

Targeted therapy is becoming the mainstay of cancer treatment due to reduced side effects and enhanced tumor attack. In the last few decades, Murine Double Minute 2 (MDM2) protein has become one of the targets for developing cancer therapies. Blocking MDM2-p53 interaction has long been considered to offer a broad range of advantages during cancer treatment. In this study, we are reporting the differential mechanism of cell death induced by the two small-molecule inhibitors, named RG-7388 and Nutlin-3, that are specific for MDM2 in SJSA-1 Osteosarcoma cells (OS). Mechanistically, RG-7388 was able to enhance the phosphorylation of Mcl-1, which appears to significantly enhance its degradation, thereby relieving the pro-apoptotic protein Bak to execute the apoptosis mechanism. It was noted that the untreated SJSA-1 cells showed an accumulation of Mcl-1 levels, which was decreased following RG-7388 and to a lesser extent by Nutlin-3 and GSK-3β (glycogen synthase kinase 3β) inhibitor treatments. Additionally, we noted that CHIR-99021 (GSK-3β inhibitor) blocked the cytotoxicity exerted by RG-7388 on SJSA-1 cells by decreasing Bak levels. Since Bak is an important pro-apoptotic protein, we hypothesized that phosphorylation of Mcl-1 by GSK-3β could negatively impact the Mcl-1/Bak dimerization and relieve Bak to trigger the loss of mitochondrial membrane potential and thereby initiates apoptosis. We also observed that inhibition of GSK-3β mediated reduction in Bak levels had a protective effect on the mitochondrial membrane integrity, and thus, caused a significant inhibition of the caspase-3 activity and PARP cleavage. Nutlin-3, on the other hand, appears to increase the levels of Bax, leading to the inactivation of Bcl-2, consequently loss of mitochondrial membrane potential and release of Cytochrome c (Cyt c) and elevation of Apaf-1 triggering apoptosis. Thus, to the best of our knowledge, this is the first study that delineates the differences in the molecular mechanism involving two MDM2 inhibitors triggering apoptosis through parallel pathways in SJSA-1 cells. This study further opens new avenues for the use of RG-7388 in treating osteosarcomas that often becomes resistant to chemotherapy due to Bcl-2 overexpression.

Keywords: Apoptosis; GSK-3β; MDM2 inhibitors; Mcl-1 phosphorylation; Osteosarcoma.