Macrolide antibiotics enhance the antitumor effect of lansoprazole resulting in lysosomal membrane permeabilization‑associated cell death

Takeda,Atsuo; Takano,Naoharu; Kokuba,Hiroko; Hino,Hirotsugu; Moriya,Shota; Abe,Akihisa; Hiramoto,Masaki; Tsukahara,Kiyoaki; Miyazawa,Keisuke

doi:10.3892/ijo.2020.5138

Macrolide antibiotics enhance the antitumor effect of lansoprazole resulting in lysosomal membrane permeabilization‑associated cell death

Authors:
- Atsuo Takeda
- Naoharu Takano
- Hiroko Kokuba
- Hirotsugu Hino
- Shota Moriya
- Akihisa Abe
- Masaki Hiramoto
- Kiyoaki Tsukahara
- Keisuke Miyazawa
View Affiliations

Affiliations: Department of Otolaryngology (Head and Neck Surgery), Tokyo Medical University, Tokyo 160‑8402, Japan, Department of Biochemistry, Tokyo Medical University, Tokyo 160‑8402, Japan, Laboratory of Electron Microscopy, Tokyo Medical University, Tokyo 160‑8402, Japan
Published online on: October 21, 2020 https://doi.org/10.3892/ijo.2020.5138
Pages: 1280-1292
Copyright: © Takeda et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

The proton pump inhibitor lansoprazole (LPZ) inhibits the growth of several cancer cell lines, including A549 and CAL 27. We previously reported that macrolide antibiotics such as azithromycin (AZM) and clarithromycin (CAM) potently inhibit autophagic flux and that combining AZM or CAM with the epidermal growth factor receptor inhibitors enhanced their antitumor effect against various cancer cells. In the present study, we conducted the combination treatment with LPZ and macrolide antibiotics against A549 and CAL 27 cells and evaluated cytotoxicity and morphological changes using cell proliferation and viability assays, flow cytometric analysis, immunoblotting, and morphological assessment. Combination therapy with LPZ and AZM greatly enhanced LPZ‑induced cell death, whereas treatment with AZM alone exhibited negligible cytotoxicity. The observed cytotoxic effect was not mediated through apoptosis or necroptosis. Transmission electron microscopy of A549 cells treated with the LPZ + AZM combination revealed morphological changes associated with necrosis and accumulated autolysosomes with undigested contents. Furthermore, the A549 cell line with ATG5 knockout exhibited complete inhibition of autophagosome formation, which did not affect LPZ + AZM treatment‑induced cytotoxicity, thus excluding the involvement of autophagy‑dependent cell death in LPZ + AZM treatment‑induced cell death. A549 cells treated with LPZ + AZM combination therapy retained the endosomal Alexa‑dextran for extended duration as compared to untreated control cells, thus indicating impairment of lysosomal digestion. Notably, lysosomal galectin‑3 puncta expression induced due to lysosomal membrane permeabilization was increased in cells treated with LPZ + AZM combination as compared to the treatment by either agent alone. Collectively, the present results revealed AZM‑induced autolysosome accumulation, potentiated LPZ‑mediated necrosis, and lysosomal membrane permeabilization, thus suggesting the potential clinical application of LPZ + AZM combination therapy for cancer treatment.

View Figures

View References

1	Kato Y, Ozawa S, Miyamoto C, Maehata Y, Suzuki A, Maeda T and Baba Y: Acidic extracellular microenvironment and cancer. Cancer Cell Int. 13:892013. View Article : Google Scholar : PubMed/NCBI
2	Luciani F, Spada M, De Milito A, Molinari A, Rivoltini L, Montinaro A, Marra M, Lugini L, Logozzi M, Lozupone F, et al: Effect of proton pump inhibitor pretreatment on resistance of solid tumors to cytotoxic drugs. J Natl Cancer Inst. 96:1702–1713. 2004. View Article : Google Scholar : PubMed/NCBI
3	Lu ZN, Tian B and Guo X: Repositioning of proton pump inhibitors in cancer therapy. Cancer Chemother Pharmacol. 80:925–937. 2017. View Article : Google Scholar : PubMed/NCBI
4	Spugnini EP and Fais S: Drug repurposing for anticancer therapies. A lesson from proton pump inhibitors. Expert Opin Ther Pat. 30:15–25. 2020. View Article : Google Scholar
5	Ihraiz WG, Ahram M and Bardaweel SK: Proton pump inhibitors enhance chemosensitivity, promote apoptosis, and suppress migration of breast cancer cells. Acta Pharm. 70:179–190. 2020. View Article : Google Scholar : PubMed/NCBI
6	Zhang B, Ling T, Zhaxi P, Cao Y, Qian L, Zhao D, Kang W, Zhang W, Wang L, Xu G and Zou X: Proton pump inhibitor pantoprazole inhibits gastric cancer metastasis via suppression of telomerase reverse transcriptase gene expression. Cancer Lett. 452:23–30. 2019. View Article : Google Scholar : PubMed/NCBI
7	Spugnini EP, Citro G and Fais S: Proton pump inhibitors as Anti-vacuolar-ATPases drugs: A novel anticancer strategy. J Exp Clin Cancer Res. 29:442010. View Article : Google Scholar
8	Geeviman K, Babu D and Prakash Babu P: Pantoprazole induces mitochondrial apoptosis and attenuates NF-κB signaling in glioma cells. Cell Mol Neurobiol. 38:1491–1504. 2018. View Article : Google Scholar : PubMed/NCBI
9	He J, Shi XY, Li ZM, Pan XH, Li ZL, Chen Y, Yan SJ and Xiao L: Proton pump inhibitors can reverse the YAP mediated paclitaxel resistance in epithelial ovarian cancer. BMC Mol Cell Biol. 20:492019. View Article : Google Scholar : PubMed/NCBI
10	Lu ZN, Shi ZY, Dang YF, Cheng YN, Guan YH, Hao ZJ, Tian B, He HW and Guo XL: Pantoprazole pretreatment elevates sensitivity to vincristine in drug-resistant oral epidermoid carcinoma in vitro and in vivo. Biomed Pharmacother. 120:1094782019. View Article : Google Scholar : PubMed/NCBI
11	Tan Q, Wang M, Yu M, Zhang J, Bristow RG, Hill RP and Tannock IF: Role of autophagy as a survival mechanism for hypoxic cells in tumors. Neoplasia. 18:347–355. 2016. View Article : Google Scholar : PubMed/NCBI
12	Tan Q, Joshua AM, Wang M, Bristow RG, Wouters BG, Allen CJ and Tannock IF: Up-regulation of autophagy is a mechanism of resistance to chemotherapy and can be inhibited by pantoprazole to increase drug sensitivity. Cancer Chemother Pharmacol. 79:959–969. 2017. View Article : Google Scholar : PubMed/NCBI
13	Tan Q, Joshua AM, Saggar JK, Yu M, Wang M, Kanga N, Zhang JY, Chen X, Wouters BG and Tannock IF: Effect of pantoprazole to enhance activity of docetaxel against human tumour xenografts by inhibiting autophagy. Br J Cancer. 112:832–840. 2015. View Article : Google Scholar : PubMed/NCBI
14	Marino ML, Fais S, Djavaheri-Mergny M, Villa A, Meschini S, Lozupone F, Venturi G, Della Mina P, Pattingre S, Rivoltini L, et al: Proton pump inhibition induces autophagy as a survival mechanism following oxidative stress in human melanoma cells. Cell Death Dis. 1:e872010. View Article : Google Scholar
15	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. View Article : Google Scholar : PubMed/NCBI
16	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
17	Hirasawa K, Moriya S, Miyahara K, Kazama H, Hirota A, Takemura J, Abe A, Inazu M, Hiramoto M, Tsukahara K and Miyazawa K: Macrolide antibiotics exhibit cytotoxic effect under amino Acid-depleted culture condition by blocking autophagy flux in head and neck squamous cell carcinoma cell lines. PLoS One. 11:e01645292016. View Article : Google Scholar : PubMed/NCBI
18	Mukai S, Moriya S, Hiramoto M, Kazama H, Kokuba H, Che XF, Yokoyama T, Sakamoto S, Sugawara A, Sunazuka T, et al: Macrolides sensitize EGFR-TKI-induced non-apoptotic cell death via blocking autophagy flux in pancreatic cancer cell lines. Int J Oncol. 48:45–54. 2016. View Article : Google Scholar : PubMed/NCBI
19	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
20	Komatsu S, Miyazawa K, Moriya S, Takase A, Naito M, Inazu M, Kohno N, Itoh M and Tomoda A: Clarithromycin enhances Bortezomib-induced cytotoxicity via endoplasmic reticulum Stress-mediated CHOP (GADD153) induction and autophagy in breast cancer cells. Int J Oncol. 40:1029–1039. 2012. View Article : Google Scholar
21	Yang JC, Lu CW and Lin CJ: Treatment of Helicobacter pylori infection: Current status and future concepts. World J Gastroenterol. 20:5283–5293. 2014. View Article : Google Scholar : PubMed/NCBI
22	Saito Y, Moriya S, Kazama H, Hirasawa K, Miyahara K, Kokuba H, Hino H, Kikuchi H, Takano N, Hiramoto M, et al: Amino acid starvation culture condition sensitizes EGFR-expressing cancer cell lines to gefitinib-mediated cytotoxicity by inducing atypical necroptosis. Int J Oncol. 52:1165–1177. 2018.PubMed/NCBI
23	O'Prey J, Sakamaki J, Baudot AD, New M, Van Acker T, Tooze SA, Long JS and Ryan KM: Application of CRISPR/Cas9 to autophagy research. Methods Enzymol. 588:79–108. 2017. View Article : Google Scholar : PubMed/NCBI
24	Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA and Zhang F: Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 11:2281–2308. 2013. View Article : Google Scholar
25	Aits S, Kricker J, Liu B, Ellegaard AM, Hämälistö S, Tvingsholm S, Corcelle-Termeau E, Høgh S, Farkas T, Holm Jonassen A, et al: Sensitive detection of lysosomal membrane permeabilization by lysosomal galectin puncta assay. Autophagy. 11:1408–1424. 2015. View Article : Google Scholar :
26	Galluzzi L, Vitale I, Aaronson SA, Abrams JM, Adam D, Agostinis P, Alnemri ES, Altucci L, Amelio I, Andrews DW, et al: Molecular mechanisms of cell death: Recommendations of the nomenclature committee on cell death 2018. Cell Death Differ. 25:486–541. 2018. 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	Wang F, Gómez-Sintes R and Boya P: Lysosomal membrane permeabilization and cell death. Traffic. 19:918–931. 2018. View Article : Google Scholar : PubMed/NCBI
29	Aits S and Jäättelä M: Lysosomal cell death at a glance. J Cell Sci. 126:1905–1912. 2013. View Article : Google Scholar : PubMed/NCBI
30	Andrea B and Juan SB: Lysosomes as dynamic regulators of cell and organismal homeostasis. Nat Rev Mol Cell Biol. 21:101–118. 2020. View Article : Google Scholar
31	Zhang S, Wang Y and Li SJ: Lansoprazole induces apoptosis of breast cancer cells through inhibition of intracellular proton extrusion. Biochem Biophys Res Commun. 448:424–429. 2014. View Article : Google Scholar : PubMed/NCBI
32	Liu W, Baker SS, Trinidad J, Burlingame AL, Baker RD, Forte JG, Virtuoso LP, Egilmez NK and Zhu L: Inhibition of lysosomal enzyme activities by proton pump inhibitors. J Gastroenterol. 48:1343–1352. 2013. View Article : Google Scholar : PubMed/NCBI
33	Česen MH, Pegan K, Spes A and Turk B: Lysosomal pathways to cell death and their therapeutic applications. Exp Cell Res. 318:1245–1251. 2012. View Article : Google Scholar : PubMed/NCBI
34	Zhitomirsky B, Yunaev A, Kreiserman R, Kaplan A, Stark M and Assaraf YG: Lysosomotropic drugs activate TFEB via lysosomal membrane fluidization and consequent inhibition of mTORC1 activity. Cell Death Dis. 9:11912018. View Article : Google Scholar : PubMed/NCBI
35	Ballabio A and Bonifacino JS: Lysosomes as dynamic regulators of cell and organismal homeostasis. Nat Rev Mol Cell Biol. 21:101–118. 2020. View Article : Google Scholar
36	Papadopoulos C, Kravic B and Meyer H: Repair or lysophagy: Dealing with damaged lysosomes. J Mol Biol. 432:231–239. 2020. View Article : Google Scholar