High let radiation modulates autophagic flux in glioblastoma cells

Advisors: Linda S. Yasui.Committee members: Barry Bode; Sherine Elsawa; Linda S. Yasui.Includes bibliographical references.Includes illustrations.Glioblastoma (GBM) are highly malignant brain cancers that arise from the astrocytes of the brain. A diagnosis of GBM forecasts a poor prognosis even with the prescription of aggressive therapy consisting of surgery and radiation therapy with temozolomide. The average survival time with therapy is only 15 months with near universal recurrence. New therapeutic approaches are urgently needed to overcome recurrence. Radiation therapy is a highly effective treatment modality prescribed for brain cancer. Energy metabolism is altered in cancer cells and GBM in particular compared to normal surrounding cells. A major adaptation to the altered metabolism in GBM cells is an adjusted cellular recycling program called autophagy. Thus to target cancer cells and spare normal surrounding cells, autophagy has been identified as a prospective therapeutic target. Further, since the ability of autophagy to run to completion (termed autophagic flux), is a key feature of autophagy, we proposed focusing on this aspect of autophagy for therapeutic targeting. To test this hypothesis, several methods to assess autophagic flux were performed at 0, 3, 5 and 7 days after irradiation with conventional photon irradiation or higher linear energy transfer (LET) fast neutron irradiation. Autophagic flux was assayed in cells engineered to stably express a tandem autophagy marker protein, LC3B covalently linked to mCherry and eGFP. Other methods used to measure autophagic flux included ultrastructural analysis of autophagosomes and both gene and protein expression of another biomarker of autophagy, p62. Greater success at killing GBM cells was achieved using high LET radiation therapy. The higher level of cell killing corresponded with a greater disruption of autophagic flux in U251 GBM cells. On day 7 after fast neutron irradiation, a 3-fold increase in p62 expression and an aberrant ultrastructure of autophagosomes were found in U251 cells, and a significant decrease in p62 protein level was found in U87. These results strongly indicated that there is an important role for autophagy (in particular autophagic flux) in radiation induced cell death, especially in response to high LET irradiation.M.S. (Master of Science