Rapamycin-sensitive pathway regulates mitochondrial membrane potential, autophagy, and survival in irradiated MCF-7 cells.
Radiation-induced inhibition of rapamycin-sensitive pathway and its effect on the cellular response to radiation were studied in the human breast cancer cell line MCF-7. Both radiation and rapamycin shared molecular targets and induced similar physiologic responses. Each of these treatments increased immunostaining of mammalian target of rapamycin (mTOR) in the nucleus, and radiation led to decreased phosphorylation of its autophosphorylation site Ser2481. In addition to dephosphorylation of established mTOR downstream effectors 4E-binding protein 1 and p70 ribosomal S6 kinase, both treatments decreased the level of eukaryotic initiation factor 4G. Experiments with the potentiometric dye, JC-1, revealed an oligomycin-dependent increase in mitochondrial membrane potential following radiation or rapamycin treatment, suggesting that both lead to reversal of F0F1ATPase activity. Both radiation and rapamycin induced sequestration of cytoplasmic material in autophagic vacuoles. In both cases, appearance of autophagic vacuoles involved the participation of microtubule-associated protein 1 light chain 3 (LC3). Transient cotransfection of green fluorescent protein-LC3 with either wild-type or dominant-negative mTOR further showed that inactivation of mTOR pathway is sufficient to induce autophagy in these cells. Finally, administration of rapamycin in combination with radiation led to enhanced mitochondria hyperpolarization, p53 phosphorylation, and increased cell death. Taken together, these experiments show that radiation-induced inhibition of rapamycin-sensitive pathway in MCF-7 cells causes changes in mitochondria metabolism, development of autophagy, and an overall decrease in cell survival.
Paglin
Shoshana
S
Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. paglins@mskcc.org
Lee
Na-Young
NY
Nakar
Charles
C
Fitzgerald
Megan
M
Plotkin
Jason
J
Deuel
Bethanne
B
Hackett
Nadia
N
McMahill
Melissa
M
Sphicas
Eleana
E
Lampen
Nina
N
Yahalom
Joachim
J
D000230:Adenocarcinoma / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology / Q000532:radiotherapy; D000903:Antibiotics, Antineoplastic / Q000037:antagonists & inhibitors / Q000494:pharmacology; D001343:Autophagy / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects*; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology / Q000532:radiotherapy*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D003593:Cytoplasm / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D047908:Intracellular Signaling Peptides and Proteins / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects*; D010766:Phosphorylation / Q000528:radiation effects; D011494:Protein Kinases / Q000378:metabolism*; D020123:Sirolimus / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D058570:TOR Serine-Threonine Kinases; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D014617:Vacuoles / Q000201:enzymology / Q000378:metabolism
D000903:Antibiotics, Antineoplastic; D047908:Intracellular Signaling Peptides and Proteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C015482:protein kinase modulator; D011494:Protein Kinases; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus