.20 (s) - Protopanaxadiol The Adjustment Mechanism Of The Different Cell Types And The Akt Signaling Pathway Associated With Lipid Rafts

1. BackgroundGlioblastoma accounts for approximately 40%-53% of primary central nervous system tumors. In clinic, surgery is the first choice, but it is difficult to completely excision because of its invasive growth. Survival rates are still low with radiotherapy and chemotherapy. In present, most of the antitumor drugs clinically have strong side effects and bad damage to patients who finally give up the treatment. Addtionally, it is easy to produce resistance to cytotoxic drugs during chemotherapy and there is no significant trend that morbidity and mortality of brain tumors are decreased. Therefore, searching for safe, effective, low toxicity and reversing resistance to antitumor drugs especially from the natural compounds has become more and more important.20S-protopanaxadiol (aPPD) is a metabolite of ginseng saponins which is reported to be pro-apoptotic in some cells but anti-apoptotic in neuronal cells by regulating Akt signalling. The plasma membrane (PM), as a major site of Akt activation, contains multiple microdomains, and among these, a cholesterol-rich, detergent-resistant type of microdomain known as the lipid raft has been suggested as a critical platform for cell signalling. Akt signalling in lipid rafts has only recently been examined as an important oncogenic pathway. Due to its cholesterol-like structure, we hypothesized that aPPD may regulate Akt signalling by interacting with lipid rafts.2. ObjectiveTo investigate that aPPD can inhibit raft-associated Akt signalling pathway and induce the apoptotic mechanisms.3. MethodsIn our study, we compared the changes of Akt signalling pathway and the pharmacological effects after aPPD treatment between glioblastoma U87MG cells and neuroblastoma N2a cells. (1) The microscope was performed to identify morphology of U87MG cells apoptosis. (2) MTT assay was performed to determine IC50 value and apoptosis rate. (3) Immunofluorescence and Co-immunoprecipitation were performed to determine the effect of lipid rafts and the location of PDK1 and Akt after aPPD treatment. (4) Total Akt and phosphorylation, Akt upstream of PI3K (p85 and p110), PDK1, ILK1, mTORC2, PHLPP and Akt downstream of total Bad and phosphorylation were detected by western blotting. (5) MTT assay and western blotting were performed to determine the cellular response to chemotherapeutic and excitotoxic agents after aPPD treatment. (6) In vitro scratch assay was performed to measure 24h cell migration rates of both U87MG and N2a cells treated with aPPD.4. Results(1) aPPD is highly effective in interfering with the protein composition of the lipid rafts without altering the level of cholesterol; (2) The effects of aPPD on the raft resident proteins are highly cell type-dependent; (3) aPPD suppresses the activity of the Akt pathway in the lipid rafts of glioma cells but increases it in neuronal cells without affecting the Akt activity in the total plasma membrane of both types of cells; (4) The raft-associated Akt activity is regulated by aPPD by altering the levels of phosphatases in the raft; (5) The difference in the effect of aPPD on the activity of the raft-associated Akt pathway results in inhibition on cell migration and enhanced cytotoxicity with TAXOL or Vinblastine in U87MG cells but attenuated excitotoxity with NMDA in N2a cells.5. ConclusionThe activity of raft-associated but not total membrane Akt determines its cellular functions. Lipid rafts differ in different types of cells, which allows for the possibility of cell-type-specific targeting for which aPPD might prove to be a useful agent.