Autophagy upregulation by inhibitors of caspase-3 and mTOR enhances radiotherapy in a mouse model of lung cancer.
Autophagy has been reported to be increased in irradiated cancer cells resistant to various apoptotic stimuli. We therefore hypothesized that induction of autophagy via mTOR inhibition could enhance radiosensitization in apoptosis-inhibited H460 lung cancer cells in vitro and in a lung cancer xenograft model. To test this hypothesis, combinations of Z-DEVD (caspase-3 inhibitor), RAD001 (mTOR inhibitor) and irradiation were tested in cell and mouse models. The combination of Z-DEVD and RAD001 more potently radiosensitized H460 cells than individual treatment alone. The enhancement in radiation response was not only evident in clonogenic survival assays, but also was demonstrated through markedly reduced tumor growth, cellular proliferation (Ki67 staining), apoptosis (TUNEL staining) and angiogenesis (vWF staining) in vivo. Additionally, upregulation of autophagy as measured by increased GFP-LC3-tagged autophagosome formation accompanied the noted radiosensitization in vitro and in vivo. The greatest induction of autophagy and associated radiation toxicity was exhibited in the tri-modality treatment group. Autophagy marker, LC-3-II, was reduced by 3-methyladenine (3-MA), a known inhibitor of autophagy, but further increased by the addition of lysosomal protease inhibitors (pepstatin A and E64d), demonstrating that there is autophagic induction through type III PI3 kinase during the combined therapy. Knocking down of ATG5 and beclin-1, two essential autophagic molecules, resulted in radiation resistance of lung cancer cells. Our report suggests that combined inhibition of apoptosis and mTOR during radiotherapy is a potential therapeutic strategy to enhance radiation therapy in patients with non-small cell lung cancer.
Kim
Kwang Woon
KW
Department of Radiation Oncology, Vanderbilt Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Hwang
Misun
M
Moretti
Luigi
L
Jaboin
Jerry J
JJ
Cha
Yong I
YI
Lu
Bo
B
D000818:Animals; D001343:Autophagy / Q000187:drug effects* / Q000502:physiology / Q000528:radiation effects; D053148:Caspase 3 / Q000502:physiology; D061945:Caspase Inhibitors; D002460:Cell Line; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D000068338:Everolimus; D005260:Female; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000532:radiotherapy*; D051379:Mice; D008819:Mice, Nude; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism* / Q000502:physiology; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology; D020123:Sirolimus / Q000031:analogs & derivatives / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D015854:Up-Regulation / Q000187:drug effects* / Q000528:radiation effects; D023041:Xenograft Model Antitumor Assays
D061945:Caspase Inhibitors; D047428:Protein Kinase Inhibitors; D011838:Radiation-Sensitizing Agents; D000068338:Everolimus; D011494:Protein Kinases; C546842:MTOR protein, human; C546843:mTOR protein, mouse; D058570:TOR Serine-Threonine Kinases; D053148:Caspase 3; D020123:Sirolimus