Macrolides sensitize EGFR-TKI-induced non-apoptotic cell death via blocking autophagy flux in pancreatic cancer cell lines

Mukai,Shuntaro; Moriya,Shota; Hiramoto,Masaki; Kazama,Hiromi; Kokuba,Hiroko; Che,Xiao-Fang; Yokoyama,Tomohisa; Sakamoto,Satoshi; Sugawara,Akihiro; Sunazuka,Toshiaki; Ōmura,Satoshi; Handa,Hiroshi; Itoi,Takao; Miyazawa,Keisuke

doi:10.3892/ijo.2015.3237

Macrolides sensitize EGFR-TKI-induced non-apoptotic cell death via blocking autophagy flux in pancreatic cancer cell lines

Authors:
- Shuntaro Mukai
- Shota Moriya
- Masaki Hiramoto
- Hiromi Kazama
- Hiroko Kokuba
- Xiao-Fang Che
- Tomohisa Yokoyama
- Satoshi Sakamoto
- Akihiro Sugawara
- Toshiaki Sunazuka
- Satoshi Ōmura
- Hiroshi Handa
- Takao Itoi
- Keisuke Miyazawa
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Affiliations: Department of Gastroenterology and Hepatology, Tokyo Medical University, Tokyo, Japan, Department of Biochemistry, Tokyo Medical University, Tokyo, Japan, Laboratory of Electron Microscopy, Tokyo Medical University, Tokyo, Japan, Department of Clinical Oncology, Tokyo Medical University, Tokyo, Japan, Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan, Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato University, Tokyo, Japan, Department of Nanoparticle Translational Research, Tokyo Medical University, Tokyo, Japan
Published online on: November 9, 2015 https://doi.org/10.3892/ijo.2015.3237
Pages: 45-54
Copyright: © Mukai et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Pancreatic cancer is one of the most difficult types of cancer to treat because of its high mortality rate due to chemotherapy resistance. We previously reported that combined treatment with gefitinib (GEF) and clarithromycin (CAM) results in enhanced cytotoxicity of GEF along with endoplasmic reticulum (ER) stress loading in non-small cell lung cancer cell lines. An epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) such as GEF induces autophagy in a pro-survival role, whereas CAM inhibits autophagy flux in various cell lines. Pronounced GEF-induced cytotoxicity therefore appears to depend on the efficacy of autophagy inhibition. In the present study, we compared the effect on autophagy inhibition among such macrolides as CAM, azithromycin (AZM), and EM900, a novel 12-membered non-antibiotic macrolide. We then assessed the enhanced GEF-induced cytotoxic effect on pancreatic cancer cell lines BxPC-3 and PANC-1. Autophagy flux analysis indicated that AZM is the most effective autophagy inhibitor of the three macrolides. CAM exhibits an inhibitory effect but less than AZM and EM900. Notably, the enhancing effect of GEF-induced cytotoxicity by combining macrolides correlated well with their efficient autophagy inhibition. However, this pronounced cytotoxicity was not due to upregulation of apoptosis induction, but was at least partially mediated through necroptosis. Our data suggest the possibility of using macrolides as ‘chemosensitizers’ for EGFR-TKI therapy in pancreatic cancer patients to enhance non-apoptotic tumor cell death induction.

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1	Gresham GK, Wells GA, Gill S, Cameron C and Jonker DJ: Chemotherapy regimens for advanced pancreatic cancer: A systematic review and network meta-analysis. BMC Cancer. 14:4712014. View Article : Google Scholar : PubMed/NCBI
2	Von Hoff DD, Ervin T, Arena FP, Chiorean EG, Infante J, Moore M, Seay T, Tjulandin SA, Ma WW, Saleh MN, et al: Increased survival in pancreatic cancer with nab-paclitaxel plus gemcitabine. N Engl J Med. 369:1691–1703. 2013. View Article : Google Scholar : PubMed/NCBI
3	Ueda S, Ogata S, Tsuda H, Kawarabayashi N, Kimura M, Sugiura Y, Tamai S, Matsubara O, Hatsuse K and Mochizuki H: The correlation between cytoplasmic overexpression of epidermal growth factor receptor and tumor aggressiveness: Poor prognosis in patients with pancreatic ductal adenocarcinoma. Pancreas. 29:e1–e8. 2004. View Article : Google Scholar : PubMed/NCBI
4	Moore MJ, Goldstein D, Hamm J, Figer A, Hecht JR, Gallinger S, Au HJ, Murawa P, Walde D, Wolff RA, et al; National Cancer Institute of Canada Clinical Trials Group. Erlotinib plus gemcitabine compared with gemcitabine alone in patients with advanced pancreatic cancer: A phase III trial of the National Cancer Institute of Canada Clinical Trials Group. J Clin Oncol. 25:1960–1966. 2007. View Article : Google Scholar : PubMed/NCBI
5	Yang ZY, Yuan JQ, Di MY, Zheng DY, Chen JZ, Ding H, Wu XY, Huang YF, Mao C and Tang JL: Gemcitabine plus erlotinib for advanced pancreatic cancer: A systematic review with meta-analysis. PLoS One. 8:e575282013. View Article : Google Scholar : PubMed/NCBI
6	Diaz Beveridge R, Alcolea V, Aparicio J, Segura Á, García J, Corbellas M, Fonfría M, Giménez A and Montalar J: Management of advanced pancreatic cancer with gemcitabine plus erlotinib: Efficacy and safety results in clinical practice. JOP. 15:19–24. 2014.PubMed/NCBI
7	Mizushima N, Levine B, Cuervo AM and Klionsky DJ: Autophagy fights disease through cellular self-digestion. Nature. 451:1069–1075. 2008. View Article : Google Scholar : PubMed/NCBI
8	Galluzzi L, Pietrocola F, Levine B and Kroemer G: Metabolic control of autophagy. Cell. 159:1263–1276. 2014. View Article : Google Scholar : PubMed/NCBI
9	White E: Deconvoluting the context-dependent role for autophagy in cancer. Nat Rev Cancer. 12:401–410. 2012. View Article : Google Scholar : PubMed/NCBI
10	Ciardiello F and Tortora G: EGFR antagonists in cancer treatment. N Engl J Med. 358:1160–1174. 2008. View Article : Google Scholar : PubMed/NCBI
11	Han W, Pan H, Chen Y, Sun J, Wang Y, Li J, Ge W, Feng L, Lin X, Wang X, et al: EGFR tyrosine kinase inhibitors activate autophagy as a cytoprotective response in human lung cancer cells. PLoS One. 6:e186912011. View Article : Google Scholar : PubMed/NCBI
12	Jutten B and Rouschop KM: EGFR signaling and autophagy dependence for growth, survival, and therapy resistance. Cell Cycle. 13:42–51. 2014. View Article : Google Scholar :
13	Fung C, Chen X, Grandis JR and Duvvuri U: EGFR tyrosine kinase inhibition induces autophagy in cancer cells. Cancer Biol Ther. 13:1417–1424. 2012. View Article : Google Scholar : PubMed/NCBI
14	Kim YC and Guan KL: mTOR: A pharmacologic target for autophagy regulation. J Clin Invest. 125:25–32. 2015. View Article : Google Scholar : PubMed/NCBI
15	Wei Y, Zou Z, Becker N, Anderson M, Sumpter R, Xiao G, Kinch L, Koduru P, Christudass CS, Veltri RW, et al: EGFR-mediated Beclin 1 phosphorylation in autophagy suppression, tumor progression, and tumor chemoresistance. Cell. 154:1269–1284. 2013. View Article : Google Scholar : PubMed/NCBI
16	Tan X, Thapa N, Sun Y and Anderson RA: A kinase-independent role for EGF receptor in autophagy initiation. Cell. 160:145–160. 2015. View Article : Google Scholar : PubMed/NCBI
17	Nakamura M, Kikukawa Y, Takeya M, Mitsuya H and Hata H: Clarithromycin attenuates autophagy in myeloma cells. Int J Oncol. 37:815–820. 2010.PubMed/NCBI
18	Renna M, Schaffner C, Brown K, Shang S, Tamayo MH, Hegyi K, Grimsey NJ, Cusens D, Coulter S, Cooper J, et al: Azithromycin blocks autophagy and may predispose cystic fibrosis patients to mycobacterial infection. J Clin Invest. 121:3554–3563. 2011. View Article : Google Scholar : PubMed/NCBI
19	Moriya S, Che XF, Komatsu S, Abe A, Kawaguchi T, Gotoh A, Inazu M, Tomoda A and Miyazawa K: Macrolide antibiotics block autophagy flux and sensitize to bortezomib via endoplasmic reticulum stress-mediated CHOP induction in myeloma cells. Int J Oncol. 42:1541–1550. 2013.PubMed/NCBI
20	Moriya S, Komatsu S, Yamasaki K, Kawai Y, Kokuba H, Hirota A, Che XF, Inazu M, Gotoh A, Hiramoto M, et al: Targeting the integrated networks of aggresome formation, proteasome, and autophagy potentiates ER stress-mediated cell death in multiple myeloma cells. Int J Oncol. 46:474–486. 2015.
21	Verfaillie T, Salazar M, Velasco G and Agostinis P: Linking ER stress to autophagy: Potential implications for cancer therapy. Int J Cell Biol. 2010:9305092010. View Article : Google Scholar : PubMed/NCBI
22	Wang M and Kaufman RJ: The impact of the endoplasmic reticulum protein-folding environment on cancer development. Nat Rev Cancer. 14:581–597. 2014. View Article : Google Scholar : PubMed/NCBI
23	Tabas I and Ron D: Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress. Nat Cell Biol. 13:184–190. 2011. View Article : Google Scholar : PubMed/NCBI
24	Sugita S, Ito K, Yamashiro Y, Moriya S, Che XF, Yokoyama T, Hiramoto M and Miyazawa K: EGFR-independent autophagy induction with gefitinib and enhancement of its cytotoxic effect by targeting autophagy with clarithromycin in non-small cell lung cancer cells. Biochem Biophys Res Commun. 461:28–34. 2015. View Article : Google Scholar : PubMed/NCBI
25	Sugawara A, Sueki A, Hirose T, Nagai K, Gouda H, Hirono S, Shima H, Akagawa KS, Omura S and Sunazuka T: Novel 12-membered non-antibiotic macrolides from erythromycin A; EM900 series as novel leads for anti-inflammatory and/or immunomodulatory agents. Bioorg Med Chem Lett. 21:3373–3376. 2011. View Article : Google Scholar : PubMed/NCBI
26	Dragowska WH, Weppler SA, Wang JC, Wong LY, Kapanen AI, Rawji JS, Warburton C, Qadir MA, Donohue E, Roberge M, et al: Induction of autophagy is an early response to gefitinib and a potential therapeutic target in breast cancer. PLoS One. 8:e765032013. View Article : Google Scholar : PubMed/NCBI
27	Mizushima N and Yoshimori T: How to interpret LC3 immunoblotting. Autophagy. 3:542–545. 2007. View Article : Google Scholar : PubMed/NCBI
28	Jiang P and Mizushima N: LC3- and p62-based biochemical methods for the analysis of autophagy progression in mammalian cells. Methods. 75:13–18. 2015. View Article : Google Scholar
29	Degterev A, Hitomi J, Germscheid M, Ch'en IL, Korkina O, Teng X, Abbott D, Cuny GD, Yuan C, Wagner G, et al: Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol. 4:313–321. 2008. View Article : Google Scholar : PubMed/NCBI
30	Sui X, Kong N, Zhu M, Wang X, Lou F, Han W and Pan H: Cotargeting EGFR and autophagy signaling: A novel therapeutic strategy for non-small-cell lung cancer. Mol Clin Oncol. 2:8–12. 2014.PubMed/NCBI
31	Mindell JA: Lysosomal acidification mechanisms. Annu Rev Physiol. 74:69–86. 2012. View Article : Google Scholar : PubMed/NCBI
32	Harada M, Sakisaka S, Yoshitake M, Kin M, Ohishi M, Shakado S, Mimura Y, Noguchi K, Sata M and Tanikawa K: Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPases, inhibits the receptor-mediated endocytosis of asialoglycoproteins in isolated rat hepatocytes. J Hepatol. 24:594–603. 1996. View Article : Google Scholar : PubMed/NCBI
33	Nujić K, Smith M, Lee M, Belamarić D, Tomašković L, Alihodžić S, Malnar I, Polančec D, Schneider K and Eraković Haber V: Valosin containing protein (VCP) interacts with macrolide antibiotics without mediating their anti-inflammatory activities. Eur J Pharmacol. 677:163–172. 2012. View Article : Google Scholar
34	Watts GD, Wymer J, Kovach MJ, Mehta SG, Mumm S, Darvish D, Pestronk A, Whyte MP and Kimonis VE: Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein. Nat Genet. 36:377–381. 2004. View Article : Google Scholar : PubMed/NCBI
35	Meyer H and Weihl CC: The VCP/p97 system at a glance: Connecting cellular function to disease pathogenesis. J Cell Sci. 127:3877–3883. 2014. View Article : Google Scholar : PubMed/NCBI
36	Meyer H, Bug M and Bremer S: Emerging functions of the VCP/p97 AAA-ATPase in the ubiquitin system. Nat Cell Biol. 14:117–123. 2012. View Article : Google Scholar : PubMed/NCBI
37	Gonzalez MA, Feely SM, Speziani F, Strickland AV, Danzi M, Bacon C, Lee Y, Chou TF, Blanton SH, Weihl CC, et al: A novel mutation in VCP causes Charcot-Marie-Tooth Type 2 disease. Brain. 137:2897–2902. 2014. View Article : Google Scholar : PubMed/NCBI
38	Nikoletopoulou V, Markaki M, Palikaras K and Tavernarakis N: Crosstalk between apoptosis, necrosis and autophagy. Biochim Biophys Acta. 1833.3448–3459. 2013.
39	Vanden Berghe T, Linkermann A, Jouan-Lanhouet S, Walczak H and Vandenabeele P: Regulated necrosis: The expanding network of non-apoptotic cell death pathways. Nat Rev Mol Cell Biol. 15:135–147. 2014. View Article : Google Scholar : PubMed/NCBI
40	Feoktistova M and Leverkus M: Programmed necrosis and necroptosis signalling. FEBS J. 282:19–31. 2015. View Article : Google Scholar
41	Bray K, Mathew R, Lau A, Kamphorst JJ, Fan J, Chen J, Chen HY, Ghavami A, Stein M, DiPaola RS, et al: Autophagy suppresses RIP kinase-dependent necrosis enabling survival to mTOR inhibition. PLoS One. 7:e418312012. View Article : Google Scholar : PubMed/NCBI