Tuberc Respir Dis.  2013 Jul;75(1):9-17.

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

• Choi HS
• Jeong EH
• Lee TG
• Kim SY
• Kim HR
• Kim CH

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.

Keyword

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

MeSH Terms

Apoptosis
Apoptosis Regulatory Proteins
Autophagy
Blotting, Western
Carcinoma, Non-Small-Cell Lung
Caspase 3
Cell Cycle
Cell Cycle Checkpoints
Cell Death
DNA
Epidermal Growth Factor
Flow Cytometry
Lung
Lung Neoplasms
Lysosomes
Monensin
Phosphotransferases
Poly(ADP-ribose) Polymerases
Proteins
Quinazolines
Receptor, Epidermal Growth Factor
Receptor, erbB-2
Sirolimus
Stress, Physiological
TOR Serine-Threonine Kinases
Erlotinib Hydrochloride
Apoptosis Regulatory Proteins
Caspase 3
DNA
Epidermal Growth Factor
Monensin
Phosphotransferases
Poly(ADP-ribose) Polymerases
Proteins
Quinazolines
Receptor, Epidermal Growth Factor
Receptor, erbB-2
Sirolimus
TOR Serine-Threonine Kinases

Figure

• 

  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.

Reference

1. Marx J. Autophagy: is it cancer's friend or foe? Science. 2006; 312:1160–1161. PMID: 16728626.

2. Hippert MM, O'Toole PS, Thorburn A. Autophagy in cancer: good, bad, or both? Cancer Res. 2006; 66:9349–9351. PMID: 17018585.

3. Turcotte S, Giaccia AJ. Targeting cancer cells through autophagy for anticancer therapy. Curr Opin Cell Biol. 2010; 22:246–251. PMID: 20056398.

4. Kondo Y, Kanzawa T, Sawaya R, Kondo S. The role of autophagy in cancer development and response to therapy. Nat Rev Cancer. 2005; 5:726–734. PMID: 16148885.

5. Boya P, Gonzalez-Polo RA, Casares N, Perfettini JL, Dessen P, Larochette N, et al. Inhibition of macroautophagy triggers apoptosis. Mol Cell Biol. 2005; 25:1025–1040. PMID: 15657430.

6. Huczynski A, Stefanska J, Przybylski P, Brzezinski B, Bartl F. Synthesis and antimicrobial properties of monensin A esters. Bioorg Med Chem Lett. 2008; 18:2585–2589. PMID: 18375122.

7. Hirsch FR, Scagliotti GV, Langer CJ, Varella-Garcia M, Franklin WA. Epidermal growth factor family of receptors in preneoplasia and lung cancer: perspectives for targeted therapies. Lung Cancer. 2003; 41(Suppl 1):S29–S42. PMID: 12867060.

8. Scagliotti GV, Selvaggi G, Novello S, Hirsch FR. The biology of epidermal growth factor receptor in lung cancer. Clin Cancer Res. 2004; 10(12 Pt 2):4227s–4232s. PMID: 15217963.

9. Han W, Pan H, Chen Y, Sun J, Wang Y, Li J, et al. EGFR tyrosine kinase inhibitors activate autophagy as a cytoprotective response in human lung cancer cells. PLoS One. 2011; 6:e18691. PMID: 21655094.

10. Takezawa K, Okamoto I, Tanizaki J, Kuwata K, Yamaguchi H, Fukuoka M, et al. Enhanced anticancer effect of the combination of BIBW2992 and thymidylate synthase-targeted agents in non-small cell lung cancer with the T790M mutation of epidermal growth factor receptor. Mol Cancer Ther. 2010; 9:1647–1656. PMID: 20530710.

11. Sos ML, Rode HB, Heynck S, Peifer M, Fischer F, Kluter S, et al. Chemogenomic profiling provides insights into the limited activity of irreversible EGFR Inhibitors in tumor cells expressing the T790M EGFR resistance mutation. Cancer Res. 2010; 70:868–874. PMID: 20103621.

12. Meric-Bernstam F, Gonzalez-Angulo AM. Targeting the mTOR signaling network for cancer therapy. J Clin Oncol. 2009; 27:2278–2287. PMID: 19332717.

13. Zou ZQ, Zhang XH, Wang F, Shen QJ, Xu J, Zhang LN, et al. A novel dual PI3Kalpha/mTOR inhibitor PI-103 with high antitumor activity in non-small cell lung cancer cells. Int J Mol Med. 2009; 24:97–101. PMID: 19513541.

14. Sheppard K, Kinross KM, Solomon B, Pearson RB, Phillips WA. Targeting PI3 kinase/AKT/mTOR signaling in cancer. Crit Rev Oncog. 2012; 17:69–95. PMID: 22471665.

15. Mathew R, Karantza-Wadsworth V, White E. Role of autophagy in cancer. Nat Rev Cancer. 2007; 7:961–967. PMID: 17972889.

16. Degenhardt K, Mathew R, Beaudoin B, Bray K, Anderson D, Chen G, et al. Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell. 2006; 10:51–64. PMID: 16843265.

17. Amaravadi RK, Yu D, Lum JJ, Bui T, Christophorou MA, Evan GI, et al. Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma. J Clin Invest. 2007; 117:326–336. PMID: 17235397.

18. Amaravadi RK, Lippincott-Schwartz J, Yin XM, Weiss WA, Takebe N, Timmer W, et al. Principles and current strategies for targeting autophagy for cancer treatment. Clin Cancer Res. 2011; 17:654–666. PMID: 21325294.

19. Svensson JP, Fry RC, Wang E, Somoza LA, Samson LD. Identification of novel human damage response proteins targeted through yeast orthology. PLoS One. 2012; 7:e37368. PMID: 22615993.

20. Hou W, Han J, Lu C, Goldstein LA, Rabinowich H. Autophagic degradation of active caspase-8: a crosstalk mechanism between autophagy and apoptosis. Autophagy. 2010; 6:891–900. PMID: 20724831.

21. Li X, Lu Y, Pan T, Fan Z. Roles of autophagy in cetuximab-mediated cancer therapy against EGFR. Autophagy. 2010; 6:1066–1077. PMID: 20864811.

22. Wu YT, Tan HL, Shui G, Bauvy C, Huang Q, Wenk MR, et al. Dual role of 3-methyladenine in modulation of autophagy via different temporal patterns of inhibition on class I and III phosphoinositide 3-kinase. J Biol Chem. 2010; 285:10850–10861. PMID: 20123989.

23. Powis G, Bonjouklian R, Berggren MM, Gallegos A, Abraham R, Ashendel C, et al. Wortmannin, a potent and selective inhibitor of phosphatidylinositol-3-kinase. Cancer Res. 1994; 54:2419–2423. PMID: 8162590.

24. Blommaart EF, Krause U, Schellens JP, Vreeling-Sindelarova H, Meijer AJ. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur J Biochem. 1997; 243:240–246. PMID: 9030745.

25. Kaini RR, Hu CA. Synergistic killing effect of chloroquine and androgen deprivation in LNCaP cells. Biochem Biophys Res Commun. 2012; 425:150–156. PMID: 22819840.

26. Rahim R, Strobl JS. Hydroxychloroquine, chloroquine, and all-trans retinoic acid regulate growth, survival, and histone acetylation in breast cancer cells. Anticancer Drugs. 2009; 20:736–745. PMID: 19584707.

27. Klionsky DJ, Elazar Z, Seglen PO, Rubinsztein DC. Does bafilomycin A1 block the fusion of autophagosomes with lysosomes? Autophagy. 2008; 4:849–950. PMID: 18758232.

28. Swampillai AL, Salomoni P, Short SC. The role of autophagy in clinical practice. Clin Oncol (R Coll Radiol). 2012; 24:387–395. PMID: 22032864.

29. Butaye P, Devriese LA, Haesebrouck F. Antimicrobial growth promoters used in animal feed: effects of less well known antibiotics on gram-positive bacteria. Clin Microbiol Rev. 2003; 16:175–188. PMID: 12692092.

30. Ketola K, Vainio P, Fey V, Kallioniemi O, Iljin K. Monensin is a potent inducer of oxidative stress and inhibitor of androgen signaling leading to apoptosis in prostate cancer cells. Mol Cancer Ther. 2010; 9:3175–3185. PMID: 21159605.