Radiation-Induced Autophagy Contributes to Cell Death and Induces Apoptosis Partly in Malignant Glioma Cells
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Purpose Radiation-induced autophagy has been shown to play two different roles, in malignantglioma (MG) cells, cytocidal or cytoprotective. However, neither the role of radiation-inducedautophagy for cell death nor the existence of autophagy-induced apoptosis, a well-knowncell-death pathway after irradiation, has been verified yet. Materials and MethodsWe observed both temporal and dose-dependent response patterns of autophagy andapoptosis to radiation in MG cell lines. Additionally, we investigated the role of autophagyin apoptosis through knockdown of autophagy-related proteins. ResultsAutophagic activity measured by staining of acidic vesicle organelles and Western blottingof LC-3 protein increased in proportion to radiation dose from day 1 to 5 after irradiation. Apoptosis measured by annexin-V staining and Western blotting of cleaved poly(ADP-ribose)polymerase demonstrated relatively late appearance 3 days after irradiation thatincreased for up to 7 days. Blocking of pan-caspase (Z-VAD-FMK) did not affect apoptosisafter irradiation, but silencing of Atg5 effectively reduced radiation-induced autophagy, whichdecreased apoptosis significantly. Inhibition of autophagy in Atg5 knockdown cells wasshown to be beneficial for cell survival. Stable transfection of GFP-LC3 cells was observedafter irradiation. Annexin-V was localized in cells bearing GFP-LC3 punctuated spots, indicatingautophagy in immunofluorescence. Some of these punctuated GFP-LC3 bearing cellsformed conglomerated spots and died in final phase. ConclusionThese findings suggest that autophagy appears earlier than apoptosis after irradiation andthat a portion of the apoptotic population that appears later is autophagy-dependent. Thus,autophagy is a pathway to cell death after irradiation of MG cells.
Abstract
Purpose Radiation-induced autophagy has been shown to play two different roles, in malignantglioma (MG) cells, cytocidal or cytoprotective. However, neither the role of radiation-inducedautophagy for cell death nor the existence of autophagy-induced apoptosis, a well-knowncell-death pathway after irradiation, has been verified yet. Materials and MethodsWe observed both temporal and dose-dependent response patterns of autophagy andapoptosis to radiation in MG cell lines. Additionally, we investigated the role of autophagyin apoptosis through knockdown of autophagy-related proteins. ResultsAutophagic activity measured by staining of acidic vesicle organelles and Western blottingof LC-3 protein increased in proportion to radiation dose from day 1 to 5 after irradiation. Apoptosis measured by annexin-V staining and Western blotting of cleaved poly(ADP-ribose)polymerase demonstrated relatively late appearance 3 days after irradiation thatincreased for up to 7 days. Blocking of pan-caspase (Z-VAD-FMK) did not affect apoptosisafter irradiation, but silencing of Atg5 effectively reduced radiation-induced autophagy, whichdecreased apoptosis significantly. Inhibition of autophagy in Atg5 knockdown cells wasshown to be beneficial for cell survival. Stable transfection of GFP-LC3 cells was observedafter irradiation. Annexin-V was localized in cells bearing GFP-LC3 punctuated spots, indicatingautophagy in immunofluorescence. Some of these punctuated GFP-LC3 bearing cellsformed conglomerated spots and died in final phase. ConclusionThese findings suggest that autophagy appears earlier than apoptosis after irradiation andthat a portion of the apoptotic population that appears later is autophagy-dependent. Thus,autophagy is a pathway to cell death after irradiation of MG cells.
참고문헌 (25)
- Walker MD, "An analysis of dose-effect relationship in the radiotherapy of malignant gliomas", Int J Radiat Oncol Biol Phys, 5, pp.1725~1731 (1979)
- Hochberg FH, "Assumptions in the radiotherapy of glioblastoma", Neurology, 30, pp.907~911 (1980)
- Olive PL, "Apoptosis: an indicator of radiosensitivity in vitro?", Int J Radiat Biol, 71, pp.695~707 (1997)
- Shinomiya N, "New concepts in radiation-induced apoptosis : 'premitotic apoptosis' and 'postmitotic apoptosis'", J Cell Mol Med, 5, pp.240~253 (2001)
- Ning S, "G2", M-phase arrest and death by apoptosis of HL60 cells irradiated with exponentially decreasing low-doserate gamma radiation, 1999, pp.151, 659~669
- Kondo Y, "The role of autophagy in cancer development and response to therapy", Nat Rev Cancer, 5, pp.726~734 (2005)
- Shintani T, "Autophagy in health and disease : a double-edged sword", Science, 306, pp.990~995 (2004)
- Lambert LA, "Autophagy : a novel mechanism of synergistic cytotoxicity between doxorubicin and roscovitine in a sarcoma model", Cancer Res, 68, pp.7966~7974 (2008)
- Paglin S, "A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles", Cancer Res, 61, pp.439~444 (2001)
- Yao KC, "Molecular response of human glioblastoma multiforme cells to ionizing radiation : cell cycle arrest, modulation of the expression of cyclin-dependent kinase inhibitors, and autophagy", J Neurosurg, 98, pp.378~384 (2003)
- Ito H, "Radiationinduced autophagy is associated with LC3 and its inhibition sensitizes malignant glioma cells", Int J Oncol, 26, pp.1401~1410 (2005)
- Yousefi S, "Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis", Nat Cell Biol, 8, pp.1124~1132 (2006)
- Luo S, "Atg5 and Bcl-2 provide novel insights into the interplay between apoptosis and autophagy", Cell Death Differ, 14, pp.1247~1250 (2007)
- Kanzawa T, "Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells", Cell Death Differ, 11, pp.448~457 (2004)
- Fowler JF, "The linear-quadratic formula and progress in fractionated radiotherapy", Br J Radiol, 62, pp.679~694 (1989)
- Shinomiya N, "Different mechanisms between premitotic apoptosis and postmitotic apoptosis in X-irradiated U937 cells", Int J Radiat Oncol Biol Phys, 47, pp.767~777 (2000)
- Su LN, "Prolonged cell cycle delay in radioresistant human cell lines transfected with activated ras oncogene and", or simian virus 40 T-antigen, 1993, pp.133, 73~79
- Stuschke M, "Radiation-induced apoptosis in human non-small-cell lung cancer cell lines is secondary to cell-cycle progression beyond the G2-phase checkpoint", Int J Radiat Biol, 78, pp.807~819 (2002)
- Broker LE, "Late activation of apoptotic pathways plays a negligible role in mediating the cytotoxic effects of discodermolide and epothilone B in non-small cell lung cancer cells", Cancer Res, 62, pp.4081~4088 (2002)
- Cummings BS, "Identification of caspase-independent apoptosis in epithelial and cancer cells", J Pharmacol Exp Ther, 310, pp.126~134 (2004)
- Chaitanya GV, "Differential PARP cleavage : an indication of heterogeneous forms of cell death and involvement of multiple proteases in the infarct of focal cerebral ischemia in rat", Cell Mol Neurobiol, 29, pp.563~573 (2009)
- Gwak HS, "Combined action of the dinuclear platinum compound BBR3610 with the PI3-K inhibitor PX-866 in glioblastoma", Int J Cancer, 128, pp.787~796 (2011)
- Codogno P, "Atg5 : more than an autophagy factor", Nat Cell Biol, 8, pp.1045~1047 (2006)
- Shimizu S, "Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes", Nat Cell Biol, 6, pp.1221~1228 (2004)
- Kim KW, "Autophagy for cancer therapy through inhibition of pro-apoptotic proteins and mammalian target of rapamycin signaling", J Biol Chem, 281, pp.36883~36890 (2006)