Autophagy Inhibition with Monensin Enhances Cell Cycle Arrest and Apoptosis Induced by mTOR or Epidermal Growth Factor Receptor Inhibitors in Lung Cancer Cells

Abstract

Background: In cancer cells, autophagy is generally induced as a pro-survival mechanism in response to treatment-associated genotoxic and metabolic stress. Thus, concurrent autophagy inhibition can be expected to have a synergistic effect with chemotherapy on cancer cell death. Monensin, a polyether antibiotic, is known as an autophagy inhibitor, which interferes with the fusion of autophagosome and lysosome. There have been a few reports of its effect in combination with anticancer drugs. We performed this study to investigate whether erlotinib, an epidermal growth factor receptor inhibitor, or rapamycin, an mammalian target of rapamycin (mTOR) inhibitor, is effective in combination therapy with monensin in non-small cell lung cancer cells.

Methods: NCI-H1299 cells were treated with rapamycin or erlotinib, with or without monensin pretreatment, and then subjected to growth inhibition assay, apoptosis analysis by flow cytometry, and cell cycle analysis on the basis of the DNA contents histogram. Finally, a Western blot analysis was done to examine the changes of proteins related to apoptosis and cell cycle control.

Results: Monensin synergistically increases growth inhibition and apoptosis induced by rapamycin or erlotinib. The number of cells in the sub-G1 phase increases noticeably after the combination treatment. Increase of proapoptotic proteins, including bax, cleaved caspase 3, and cleaved poly(ADP-ribose) polymerase, and decrease of anti-apoptotic proteins, bcl-2 and bcl-xL, are augmented by the combination treatment with monensin. The promoters of cell cycle progression, notch3 and skp2, decrease and p21, a cyclin-dependent kinase inhibitor, accumulates within the cell during this process.

Conclusion: Our findings suggest that concurrent autophagy inhibition could have a role in lung cancer treatment.

Keywords: Apoptosis; Autophagy; Cell Cycle; Epidermal Growth Factor Receptor-Neu Receptor; Monensin; TOR Serine-Threonine Kinases.

Figure 1

Enhancement of growth inhibition by combination treatment with monensin and rapamycin or erlotinib in NCI-H1299 cells. NCI-H1299 cells were pretreated with 50 nM monensin for 4 hours before treatment with 10 µM of rapamycin or erlotinib for 48 hours. (A) Morphology of cells was observed by optical microscopy. Monensin accelerates morphologic changes induced by rapamycin or erlotinib. (B) Cellular growth inhibition was analyzed by MTT assay. Monensin increases the growth inhibition induced by rapamycin or erlotinib. The data shown are representative of three independent experiments with similar results; error bars are mean±standard deviation. Mann-Whitney U-test was used for comparisons. ^*p<0.01, single versus combination treatment. (C) Effects of monensin in combination with rapamycin or erlotinib on growth inhibition was calculated on the basis of the combination index (CI) of each concentration. CI values< 0.7 indicate synergism. Rapa: rapamycin; Erl: erlotinib; Mon: monensin.

Figure 2

Increased apoptosis by treatment with monensin in combination with rapamycin or erlotinib in NCI-H1299 cells. (A) Inhibition of autophagy was detected by acridine orange staining. NCI-H1299 cells were treated with rapamycin or erlotinib for 48 hours with or without monensin pretreatment, stained with acridine orange (1 µg/mL), and then visualized under a red-filtered confocal microscope. Rapamycin- and erlotinib-induced formation of acidic vesicular organelles (white arrows) is effectively inhibited by monensin treatment. (B) Apoptosis was analyzed by flow cytometry with annexin V staining after each treatment described above. Numbers of annexin V-positive apoptotic cells increase after combination treatment with monensin, compared to treatment with rapamycin or erlotinib alone (left). The percentage of annexin V-positive apoptotic cells was calculated on the basis of the results shown in the histogram (right). The data shown are representative of three independent experiments with similar results; error bars are mean±standard deviation (SD). Mann-Whitney U-test was used for comparisons. ^*p<0.05, single versus combination. (C) Percentage of cells in the sub-G₁ phase was determined on the basis of the DNA contents histogram from propidium iodide-stained cells. When rapamycin- or erlotinib-treated cells are co-treated with monensin, the number of cells in the sub-G₁ phase noticeably increases. The data shown are representative of three independent experiments with similar results; error bars are mean±SD. Mann-Whitney U-test was used for comparisons. ^*p<0.01, single versus combination. Con: control; Rapa: rapamycin; Erl: erlotinib; Mon: monensin.

Figure 3

The combination effect is mediated by apoptotic pathways in NCI-H1299 cells. NCI-H1299 cells were exposed to monensin (50 nM, 4 hours), followed by rapamycin or erlotinib (10 µM, 48 hours), and harvested for western blot analysis. Western blotting was performed with specific antibodies, and β-actin served as a loading control. (A) p-p70S6K expression decreases after treatment with rapamycin or erlotinib. The type II form of LC3 accumulates and degradation of p62 is blocked after treatment with monensin, which blocks fusion of autophagosome and lysosome. (B) After combination treatment, increases of proapoptotic proteins, including bax, cleaved caspase 3, and cleaved poly(ADP-ribose) polymerase (PARP) are enhanced. By contrast, decreases of anti-apoptotic proteins, bcl-2 and bcl-xL, are also augmented. Con: control; Rapa: rapamycin; Erl: erlotinib; Mon: monensin.

Figure 4

Effects of monensin combination treatment on the cell cycle. Cells were treated with rapamycin or erlotinib for 48 hours, with or without monensin pretreatment, and then subjected to cell cycle analysis. (A) The percentage of cells in the G₁ phase increases after combination treatment with monensin compared to that with rapamycin or erlotinib alone. The data shown are representative of three independent experiments with similar results; error bars are mean±standard deviation (SD). Mann-Whitney U-test was used for comparisons. ^*p<0.05, single versus combination. (B) Cell lysates were collected and subjected to western blotting with specific antibodies. The levels of the regulators of the G₁ cell cycle phase, notch3 and skp2, decrease and that of the cyclin-dependent kinase inhibitor, p21 increases on co-treatment with monensin. The results indicate that monensin leads to cell cycle arrest in G₁ phase. Con: control; Rapa: rapamycin; Erl: erlotinib; Mon: monensin.