Combined Bcl-2/mammalian target of rapamycin inhibition leads to enhanced radiosensitization via induction of apoptosis and autophagy in non-small cell lung tumor xenograft model.
OBJECTIVE
Radiotherapy has a central role in the treatment of non-small cell lung cancer. Effectiveness of this modality, however, is often limited as resistance results from defects in cell death.


METHODS
We investigated whether simultaneous up-regulation of apoptosis, via Bcl-2 inhibitor ABT-737, and autophagy, via mammalian target of rapamycin inhibitor rapamycin, can be used to enhance radiosensitivity of H460 cells in vitro and growth delay in a xenograft model.


RESULTS
In vitro studies confirmed that ABT-737 and rapamycin induce apoptosis and autophagy, respectively. ABT-737 induced cleaved caspase-3, a marker of apoptosis, and rapamycin correlated with an increase in punctate localization of green fluorescent protein-LC3, characteristic of autophagy. The combination ABT-737/rapamycin markedly enhanced sensitivity of H460 cells to radiation (dose enhancement ratio = 2.47; P = 0.002) in clonogenic assay. In addition, the combination ABT-737/rapamycin/radiation showed a dramatic tumor growth delay in a mouse xenograft model. In vivo immunohistochemistry staining showed that combination therapy yielded over a 100% increase in caspase-3 activity (apoptosis) and a 6-fold decrease in p62 protein level (indicative of autophagic flux) compared with radiation alone control group. Moreover, cell proliferation (Ki-67 staining) was reduced by 77% (P = 0.001) and vascular density (von Willebrand factor staining) by 67.5% (P = 0.09) compared with radiation alone. Additional in vitro studies in human umbilical vein endothelial cells indicated that combined therapy also significantly decreases tubule formation.


CONCLUSIONS
These results suggest that concurrent induction of apoptosis and autophagy enhances radiation therapy both in vitro and in lung cancer xenograft models. Further investigations are warranted to assess the clinical potential of such strategy in lung cancer patients.
Department of Radiation Oncology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-5671, USA.
Kwang Woon
KW
Kim
Luigi
L
Moretti
Lauren Rhea
LR
Mitchell
Dae Kwang
DK
Jung
Bo
B
Lu
D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects*; D001713:Biphenyl Compounds / Q000008:administration & dosage*; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000473:pathology / Q000532:radiotherapy*; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000473:pathology / Q000532:radiotherapy*; D051379:Mice; D008819:Mice, Nude; D009596:Nitrophenols / Q000008:administration & dosage*; D010879:Piperazines / Q000008:administration & dosage; D011494:Protein Kinases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors; D011836:Radiation Tolerance / Q000187:drug effects*; D011838:Radiation-Sensitizing Agents / Q000627:therapeutic use; D020123:Sirolimus / Q000008:administration & dosage*; D013449:Sulfonamides / Q000008:administration & dosage*; D058570:TOR Serine-Threonine Kinases; D023041:Xenograft Model Antitumor Assays
C501332:ABT-737; D001713:Biphenyl Compounds; D009596:Nitrophenols; D010879:Piperazines; D019253:Proto-Oncogene Proteins c-bcl-2; D011838:Radiation-Sensitizing Agents; D013449:Sulfonamides; D011494:Protein Kinases; C546842:MTOR protein, human; C546843:mTOR protein, mouse; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus