Tuberc Respir Dis.  2012 Apr;72(4):343-351.

Dual Inhibition of PI3K/Akt/mTOR Pathway and Role of Autophagy in Non-Small Cell Lung Cancer Cells

Affiliations
  • 1Division of Pulmonology, Department of Internal Medicine, Korea Cancer Center Hospital, Seoul, Korea. cheol@kcch.re.kr

Abstract

BACKGROUND
The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling axis has emerged as a novel target for cancer therapy. Agents that inhibit this pathway are currently under development for lung cancer treatment. In the present study, we have tested whether dual inhibition of PI3K/Akt/mTOR signaling can lead to enahnced antitumor effects. We have also examined the role of autophagy during this process.
METHODS
We analyzed the combination effect of the mTOR inhibitor, temsirolimus, and the Akt inhibitor, GSK690693, on the survival of NCI-H460 and A549 non-small cell lung cancer cells. Cell proliferation was determined by MTT assay and apoptosis induction was evaluated by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Autophagy induction was also evaluated by acridine orange staining. Changes of apoptosis or autophagy-related proteins were evaluated by western blot analysis.
RESULTS
Combination treatment with temsirolimus and GSK690693 caused synergistically increased cell death in NCI-H460 and A549 cells. This was attributable to increased induction of apoptosis. Caspase 3 activation and poly(ADP-ribose) polymerase cleavage accompanied these findings. Autophagy also increased and inhibition of autophagy resulted in increased cell death, suggesting its cytoprotective role during this process.
CONCLUSION
Taken together, our results suggest that the combination of temsirolimus and GSK690693 could be a novel strategy for lung cancer therapy. Inhibition of autophagy could also be a promising method of enhancing the combination effect of these drugs.

Keyword

Phosphatidylinositol 3-Kinases; TOR Serine-Threonine Kinases; Carcinoma, Non-Small Cell Lung

MeSH Terms

Acridine Orange
Apoptosis
Autophagy
Axis, Cervical Vertebra
Blotting, Western
Carcinoma, Non-Small-Cell Lung
Caspase 3
Cell Death
Cell Proliferation
DNA Nucleotidylexotransferase
Flow Cytometry
Lung Neoplasms
Oxadiazoles
Phosphatidylinositol 3-Kinases
Poly(ADP-ribose) Polymerases
Proteins
Sirolimus
TOR Serine-Threonine Kinases
Acridine Orange
Caspase 3
DNA Nucleotidylexotransferase
Oxadiazoles
Phosphatidylinositol 3-Kinases
Poly(ADP-ribose) Polymerases
Proteins
Sirolimus
TOR Serine-Threonine Kinases

Figure

  • Figure 1 (A) Combination effect of temsirolimus and GSK690693 on cell proliferation determined by using the MTT assay. Cells were treated with the indicated concentrations of temsirolimus and GSK690693, and proliferation was determined 72 hours later. (B) Combination indices were calculated as described in Materials and Methods. Synergistic combination effect was found in higher concentrations of temsirolimus and GSK690693.

  • Figure 2 Enhanced apoptosis by combination treatment with GSK690693 and temsirolimus in NCI-H460 and A549 cells. Cells were treated with GSK690693 and temsirolimus for 48 hours. After staining with annexin V-FITC, cells were analyzed by flow cytometry to analyze apoptosis.

  • Figure 3 Enhanced apoptosis by combination treatment with GSK690693 and temsirolimus in NCI-H460 and A549 cells. Cells were treated with GSK690693 and temsirolimus for 48 hours. After staining with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), cells were examined with confocal microscope to analyze apoptosis. Red: propidium iodide; green: TUNEL.

  • Figure 4 Enhanced autophagy by combination treatment with GSK690693 and temsirolimus in NCI-H460 and A549 cells. Cells were treated with GSK690693 and temsirolimus for 48 hours. After staining with acridine orange, cells were examined with confocal microscope to analyze autophagolysome formation. Red: acidic vesicular organelle.

  • Figure 5 Western blot analysis of cleaved caspase 3, poly(ADP-ribose) polymerase (PARP), beclin 1, and light chain 3 (LC3). NCI-H460 cells were exposed to GSK690693 and temsirolimus for 24 or 48 hours and harvested for the results. Actin served as a loading control. C: control; G: GSK690693; T: temsirolimus; GT: GSK690693 and temsirolimus.

  • Figure 6 Inhibition of apoptosis or autophagy and its influence on the combination effect of GSK690693 and temsirolimus. Cells were pretreated with a pancaspase inhibitor, Z-VAD-FMK (50µM) and then treated with GSK690693 and temsirolimus for MTT analysis. On the other hand, cells were treated with GSK690693, temsirolimus, and an autophagy inhibitor, 3-methyladenine (3-MA; 250µM) for MTT analysis. *p<0.05, compared with non-treated control.


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