The Preclinical Evaluation Of Triptonide(TN) As A Potential Anti-glioma Agent:A Mechanism Study

Objective:In the current study, we investigated the potential anti-glioma activity by a Traditional Chinese Medicine(TCM) monomer: Triptonide(TN). The underlying signaling mechanisms were also analyzed.Methods:1) MTT assay, trypan blue staining assay and clonogenicity assay were applied to test the potential role of TN on glioma cell survival, death and proliferation, respectively.2) Histone DNA ELISA assay, Caspase activity assay and Annexin V-PI FACS assay were performed to test glioma cell apoptosis after TN treatment.3) PI-FACS assay was performed to examine cell cycle progression in TN-treated glioma cells.4) Western blot assay was performed to test signaling changes in glioma cells after TN treatment.5) shRNA and mutation strategies were applied to neutralize AMP-activated protein kinase(AMPK) signaling, TN-induced in vitro anti-glioma cell activity was then analyzed.6) U87 MG xenograft glioma nude mice model was applied, the in vivo anti-glioma cell activity by TN was analyzed.Results:1) TN, at nM concentrations, potently inhibited survival and proliferation of human glioma cells(U87MG and U251MG). It was yet non-cytotoxic to the murine astrocytes.2) TN provoked significant apoptosis activation in glioma cells, whiling inducing glioma cell cycle arrest.3) TN induced significant AMPK signaling activation, causing subsequent degradation of multiple growth factor receptors(EGFR, PDGFR) in glioma cells.4) shRNA knockdown of AMPKα1 or AMPK mutation restored growth factor receptor(EGFR, PDGFR) expression, and attenuated TN-induced cytotoxicity in glioma cells.5) Intraperitoneal injection of TN(5-20mg/kg body weight) inhibited U87 MG xenograft growth in nude mice.Conclusions:TN inhibited human glioma cell survival and proliferation in vitro, whiling inducing cancer cell death and apoptosis. It also induced glioma cell G1-S cell cycle arrest. Intraperitoneal injection of TN at well-tolerated doses dramatically inhibited U87 MG xenograft growth in nude mice. AMPK activation and following growth factor receptor(i.e. EGFR and PGDFR) degradation may participate in TN’s anti-glioma cell activity.