122 - Plumbagin: Putative Mechanisms of Action mediating Cells Cycle, Metabolism, and Apoptosisin Glioblastoma cells

Abstract

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor characterized by a highly infiltrative growth pattern and resistance to chemotherapy. Phytochemicals due to their specificity and low cytotoxicity have drawn major attention as a potent and safer alternative to conventional chemotherapy in treating cancer. In this study, we have investigated the potential anti-cancerous effects of a napthaquinone, plumbagin (PLU), using GBM cells (U87 MG) both in vitro and in vivo. Cytotoxicity studies done with MTT assay showed that PLU decreased viability and growth of these cells in a dose-dependent manner. Further, PLU facilitated cell cycle arrest at G2/M checkpoint that was also confirmed by down regulation of cell cycle marker proteins CDK2 and CDK-4. Similarly, cell apoptosis, generation of reactive oxygen species (ROS), reactive nitrogen species (RNS), and the change in mitochondrial membrane potential (ΔΨm) were studied using flow cytometry. PLU increased ROS generation in a dose dependent manner, which led to increased mitochondrial depolarization as well as activation of caspase-3/7 confirming involvement of mitochondrial pathway of apoptosis in the exposed cells. These effects were further substantiated by noting increased DNA fragmentation, chromatin condensation and apoptotic DNA content and also upregulation of USP3, a deubiquitinase, and Caspase 3 in PLU treated cells. We also investigated its effect on the subpopulation of brain cancer cells exhibiting cancer stem cells (CSC) like characteristics. GBM CSCs show a characteristic enhanced ALDH activity and possess a capability to form neurosperes in non-adherent cultures. A reduced ALDH activity was also noted in these cells after treatment with PLU suggesting its potential effect on CSCs. Molecular docking has revealed Akt/mTOR signaling as the potential target of PLU. Accordingly, ongoing studies are underway to validate this further along with in vivo assessment of PLU efficacy using orthotopic xenograft tumor model in Wistar rat. Together, our findings have demonstrated plumbagin, as a promising natural chemotherapeutic agent, having potential implication in treating GBM.