Advertisement

Apoptosis

, Volume 24, Issue 5–6, pp 414–433 | Cite as

Autophagy inhibition with chloroquine reverts paclitaxel resistance and attenuates metastatic potential in human nonsmall lung adenocarcinoma A549 cells via ROS mediated modulation of β-catenin pathway

  • Satabdi Datta
  • Diptiman Choudhury
  • Amlan Das
  • Dipanwita Das Mukherjee
  • Moumita Dasgupta
  • Shreya Bandopadhyay
  • Gopal ChakrabartiEmail author
Article

Abstract

Paclitaxel is one of the most commonly used drugs for the treatment of nonsmall cell lung cancer (NSCLC). However acquired resistance to paclitaxel, epithelial to mesenchymal transition and cancer stem cell formation are the major obstacles for successful chemotherapy with this drug. Some of the major reasons behind chemoresistance development include increased ability of the cancer cells to survive under stress conditions by autophagy, increased expression of drug efflux pumps, tubulin mutations etc. In this study we found that inhibition of autophagy with chloroquine prevented development of paclitaxel resistance in A549 cells with time and potentiated the effect of paclitaxel by increased accumulation of superoxide-producing damaged mitochondria, with elevated ROS generation, it also increased the apoptotic rate and sub G0/ G1 phase arrest with time in A549 cells treated with paclitaxel and attenuated the metastatic potential and cancer stem cell population of the paclitaxel-resistant cells by ROS mediated modulation of the Wnt/β-catenin signaling pathway, thereby increasing paclitaxel sensitivity. ROS here played a crucial role in modulating Akt activity when autophagy process was hindered by chloroquine, excessive ROS accumulation in the cell inhibited Akt activity. In addition, chloroquine pre-treatment followed by taxol (10 nM) treatment did not show significant toxicity towards non-carcinomas WI38 cells (lung fibroblast cells). Thus autophagy inhibition by CQ pre-treatment can be used as a fruitful strategy to combat the phenomenon of paclitaxel resistance development as well as metastasis in lung cancer.

Keywords

Paclitaxel-resistance Chloroquine Autophagy ROS EMT β-Catenin 

Abbreviations

NSCLC

Non small cell lung cancer

ROS

Reactive oxygen species

Tx

Paclitaxel/taxol

MDR1

Multidrug resistant protein 1

MRP1

Multidrug resistance-associated protein 1

Pgp

P-glycoprotein

EMT

Epithelial to mesenchymal transition

ROS

Reactive oxygen species

CQ

Chloroquine

DMEM

Dulbecco’s modified eagle’s Media

DMSO

Dimethyl sulfoxide

MDC

Monodancyl cadaverine

MSR

Mitosox red

OCR

Oxygen consumption rate

ECAR

Extracellular acidification rate

PE

Phycoerythrin

CSC

Cancer stem cell

NAC

N-acetyl-cysteine

Notes

Acknowledgements

The authors wish to thank Dr. Sib Sankar Roy, CSIR – Indian Institute of Chemical Biology, Kolkata, India for helping the use of Seahorse XFe24 Extracellular Flux Analyser facility. The work was supported by grants from Department of Science and Technology, Govt. of India (No. SR/SO/BB-14/2008) and Department of Biotechnology, Government of India (No. BT/ PR12889/AGR/36/624/2009) to G.Chakbarati. FACS and fluorescence microscope instruments facility were developed by grants from National Common Minimum Project, Government. of India. Confocal microscope instrument facility was developed by grants from DBT-IPLS facility. S Datta and D Das Mukherjee were supported by Senior Research fellowship from Council of Scientific and industrial research (CSIR), Government. of India. S. Bandopadhyay is supported by Junior research fellowship from Council of Scientific and industrial research (CSIR), India.

Compliance with Ethical Standards

Conflict of interest

All authors declare that they have no conflicts of interests.

Supplementary material

10495_2019_1526_MOESM1_ESM.tif (3.5 mb)
Supplementary material 1 (TIF 3627 KB)
10495_2019_1526_MOESM2_ESM.tif (2.7 mb)
Supplementary material 2 (TIF 2726 KB)
10495_2019_1526_MOESM3_ESM.tif (3.7 mb)
Supplementary material 3 (TIF 3777 KB)
10495_2019_1526_MOESM4_ESM.tif (2.7 mb)
Supplementary material 4 (TIF 2719 KB)
10495_2019_1526_MOESM5_ESM.tif (3.9 mb)
Supplementary material 5 (TIF 3998 KB)

References

  1. 1.
    Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A (2015) Global cancer statistics, 2012. CA Cancer J Clin 65:87–108CrossRefGoogle Scholar
  2. 2.
    McGuire A, Martin M, Lenz C, Sollano JA (2015) Treatment cost of non-small cell lung cancer in three European countries: comparisons across France, Germany and England using administrative databases. J Med Econ 18(7):525–532CrossRefGoogle Scholar
  3. 3.
    Rowinsky EK, Donehower RC (1995) Paclitaxel (taxol). N Engl J Med 332:1004–1014CrossRefGoogle Scholar
  4. 4.
    Bharadwaj R, Yu H (2004) The spindle checkpoint, aneuploidy, and cancer. Oncogene 23:2016–2027CrossRefGoogle Scholar
  5. 5.
    Brito DA, Yang Z, Rieder CL (2008) Microtubules do not promote mitotic slippage when the spindle assembly checkpoint cannot be satisfied. J Cell Biol 182:623–629CrossRefGoogle Scholar
  6. 6.
    Gottesman MM, Pastan I (1993) Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu Rev Biochem 62:385–427CrossRefGoogle Scholar
  7. 7.
    Gottesman MM, Fojo T, Bates SE (2002) Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer 2:48–58CrossRefGoogle Scholar
  8. 8.
    Kavallaris M, Kuo DY, Burkhart CA et al (1997) Taxol-resistant epithelial ovarian tumors are associated with altered expression of specific beta-tubulin isotypes. J Clin Investig 100:1282–1293CrossRefGoogle Scholar
  9. 9.
    Singh A, Settleman J (2010) EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer. Oncogene 29:4741–4751CrossRefGoogle Scholar
  10. 10.
    Kriegenburg F, Ungermann C, Reggiori F (2018) Coordination of autophagosome-lysosome fusion by Atg8 family members. Curr Biol 28:R512–R518CrossRefGoogle Scholar
  11. 11.
    Mizushima N, Komatsu M (2011) Autophagy: renovation of cells and tissues. Cell 147:728–741CrossRefGoogle Scholar
  12. 12.
    Eskelinen EL (2011) The dual role of autophagy in cancer. Curr Opin Pharmacol 11:294–300CrossRefGoogle Scholar
  13. 13.
    Sui X, Chen R, Wang Z et al (2013) Autophagy and chemotherapy resistance: a promising therapeutic target for cancer treatment. Cell Death Dis 4:e838CrossRefGoogle Scholar
  14. 14.
    Yoon JH, Ahn SG, Lee BH, Jung SH, Oh SH (2012) Role of autophagy in chemoresistance: regulation of the ATM-mediated DNA-damage signaling pathway through activation of DNA-PKcs and PARP-1. Biochem Pharmacol 83:747–757CrossRefGoogle Scholar
  15. 15.
    Zhou Y, Sun K, Ma Y et al (2014) Autophagy inhibits chemotherapy-induced apoptosis through downregulating Bad and Bim in hepatocellular carcinoma cells. Sci Rep 4:5382CrossRefGoogle Scholar
  16. 16.
    Datta S, Choudhury D, Das A et al (2017) Paclitaxel resistance development is associated with biphasic changes in reactive oxygen species, mitochondrial membrane potential and autophagy with elevated energy production capacity in lung cancer cells: a chronological study. Tumour Biol 39:1010428317694314CrossRefGoogle Scholar
  17. 17.
    Li J, Hou N, Faried A, Tsutsumi S, Takeuchi T, Kuwano H (2009) Inhibition of autophagy by 3-MA enhances the effect of 5-FU-induced apoptosis in colon cancer cells. Ann Surg Oncol 16:761–771CrossRefGoogle Scholar
  18. 18.
    Sasaki K, Tsuno NH, Sunami E et al (2010) Chloroquine potentiates the anti-cancer effect of 5-fluorouracil on colon cancer cells. BMC Cancer 10:370CrossRefGoogle Scholar
  19. 19.
    Yang YP, Hu LF, Zheng HF et al (2013) Application and interpretation of current autophagy inhibitors and activators. Acta pharmacol Sin 34:625–635CrossRefGoogle Scholar
  20. 20.
    Shingu T, Fujiwara K, Bogler O et al (2009) Inhibition of autophagy at a late stage enhances imatinib-induced cytotoxicity in human malignant glioma cells. Int J Cancer 124:1060–1071CrossRefGoogle Scholar
  21. 21.
    Kanzawa T, Germano IM, Komata T, Ito H, Kondo Y, Kondo S (2004) Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells. Cell Death Differ 11:448–457CrossRefGoogle Scholar
  22. 22.
    Solomon VR, Lee H (2009) Chloroquine and its analogs: a new promise of an old drug for effective and safe cancer therapies. Eur J Pharmacol 625:220–233CrossRefGoogle Scholar
  23. 23.
    Ganguli A, Choudhury D, Datta S, Bhattacharya S, Chakrabarti G (2014) Inhibition of autophagy by chloroquine potentiates synergistically anti-cancer property of artemisinin by promoting ROS dependent apoptosis. Biochimie 107:338–349CrossRefGoogle Scholar
  24. 24.
    Zheng Y, Zhao YL, Deng X et al (2009) Chloroquine inhibits colon cancer cell growth in vitro and tumor growth in vivo via induction of apoptosis. Cancer Investig 27:286–292CrossRefGoogle Scholar
  25. 25.
    Fan C, Wang W, Zhao B, Zhang S, Miao J (2006) Chloroquine inhibits cell growth and induces cell death in A549 lung cancer cells. Bioorgan Med Chem 14:3218–3222CrossRefGoogle Scholar
  26. 26.
    Qu X, Sheng J, Shen L et al (2017) Autophagy inhibitor chloroquine increases sensitivity to cisplatin in QBC939 cholangiocarcinoma cells by mitochondrial ROS. PloS ONE 12:e0173712CrossRefGoogle Scholar
  27. 27.
    Wang Y, Peng RQ, Li DD et al (2011) Chloroquine enhances the cytotoxicity of topotecan by inhibiting autophagy in lung cancer cells. Chin J Cancer 30:690–700CrossRefGoogle Scholar
  28. 28.
    Yang J, Zhang K, Wu J et al (2016) Wnt5a increases properties of lung cancer stem cells and resistance to Cisplatin through activation of Wnt5a/PKC signaling pathway. Stem Cells Int 2016:1690896Google Scholar
  29. 29.
    Deitrick J, Pruitt WM (2016) Wnt/beta Catenin-mediated signaling commonly altered in colorectal cancer. Prog Mol Biol Trans Sci 144:49–68CrossRefGoogle Scholar
  30. 30.
    MacDonald BT, Tamai K, He X (2009) Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell 17:9–26CrossRefGoogle Scholar
  31. 31.
    Anderson EC, Wong MH (2010) Caught in the Akt: regulation of Wnt signaling in the intestine. Gastroenterology 139:718–722CrossRefGoogle Scholar
  32. 32.
    Hart JR, Vogt PK (2011) Phosphorylation of AKT: a mutational analysis. Oncotarget 2:467–476CrossRefGoogle Scholar
  33. 33.
    Cao J, Xu D, Wang D et al (2009) ROS-driven Akt dephosphorylation at Ser-473 is involved in 4-HPR-mediated apoptosis in NB4 cells. Free Radic Biol Med 47:536–547CrossRefGoogle Scholar
  34. 34.
    Juan O, Albert A, Ordono F et al (2002) Low-dose weekly paclitaxel as second-line treatment for advanced non-small cell lung cancer: a phase II study. Jpn J Clin Oncol 32:449–454CrossRefGoogle Scholar
  35. 35.
    Strober W (2001) Trypan blue exclusion test of cell viability. Curr Protoc Immunol.  https://doi.org/10.1002/0471142735.ima03bs21 (Appendix 3:Appendix 3B)Google Scholar
  36. 36.
    Choudhury D, Ganguli A, Dastidar DG, Acharya BR, Das A, Chakrabarti G (2013) Apigenin shows synergistic anticancer activity with curcumin by binding at different sites of tubulin. Biochimie 95:1297–1309CrossRefGoogle Scholar
  37. 37.
    Choudhury D, Das A, Bhattacharya A, Chakrabarti G (2010) Aqueous extract of ginger shows antiproliferative activity through disruption of microtubule network of cancer cells. Food Chem Toxicol 48:2872–2880CrossRefGoogle Scholar
  38. 38.
    Acharya BR, Bhattacharyya S, Choudhury D, Chakrabarti G (2011) The microtubule depolymerizing agent naphthazarin induces both apoptosis and autophagy in A549 lung cancer cells. Apoptosis 16:924–939CrossRefGoogle Scholar
  39. 39.
    Das A, Chakrabarty S, Choudhury D, Chakrabarti G (2010) 1,4-Benzoquinone (PBQ) induced toxicity in lung epithelial cells is mediated by the disruption of the microtubule network and activation of caspase-3. Chem Res Toxicol 23:1054–1066CrossRefGoogle Scholar
  40. 40.
    Xia XY, Wu YM, Hou BS et al (2008) Evaluation of sperm mitochondrial membrane potential by JC-1 fluorescent staining and flow cytometry. Zhonghua nan ke xue 14:135–138Google Scholar
  41. 41.
    Biederbick A, Kern HF, Elsasser HP (1995) Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. Eur J Cell Biol 66:3–14Google Scholar
  42. 42.
    Jiang RD, Shen H, Piao YJ (2010) The morphometrical analysis on the ultrastructure of A549 cells. Rom J Morphol Embryol 51:663–667Google Scholar
  43. 43.
    Das Mukherjee D, Kumar NM, Tantak MP et al (2016) Development of novel Bis(indolyl)-hydrazide-hydrazone derivatives as potent microtubule-targeting cytotoxic agents against A549 lung cancer Cells. Biochemistry 55:3020–3035CrossRefGoogle Scholar
  44. 44.
    Yin JX, Yang RF, Li S et al (2010) Mitochondria-produced superoxide mediates angiotensin II-induced inhibition of neuronal potassium current. Am J Physiol Cell Physiol 298:C857–C865CrossRefGoogle Scholar
  45. 45.
    Zimmerman MC, Oberley LW, Flanagan SW (2007) Mutant SOD1-induced neuronal toxicity is mediated by increased mitochondrial superoxide levels. J Neurochem 102:609–618CrossRefGoogle Scholar
  46. 46.
    Dai T, Hu Y, Zheng H (2017) Hypoxia increases expression of CXC chemokine receptor 4 via activation of PI3K/Akt leading to enhanced migration of endothelial progenitor cells. Eur Rev Med Pharmacol Sci 21:1820–1827Google Scholar
  47. 47.
    Bertolini G, Roz L, Perego P et al (2009) Highly tumorigenic lung cancer CD133 + cells display stem-like features and are spared by cisplatin treatment. Proc Natl Acad Sci USA 106:16281–16286CrossRefGoogle Scholar
  48. 48.
    Wang P, Gao Q, Suo Z et al (2013) Identification and characterization of cells with cancer stem cell properties in human primary lung cancer cell lines. PloS ONE 8:e57020CrossRefGoogle Scholar
  49. 49.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  50. 50.
    Green K, Brand MD, Murphy MP (2004) Prevention of mitochondrial oxidative damage as a therapeutic strategy in diabetes. Diabetes 53(Suppl 1):S110–S118CrossRefGoogle Scholar
  51. 51.
    Kirkinezos IG, Moraes CT (2001) Reactive oxygen species and mitochondrial diseases. Semin Cell Dev Biol 12:449–457CrossRefGoogle Scholar
  52. 52.
    Verheyen EM, Gottardi CJ (2010) Regulation of Wnt/beta-catenin signaling by protein kinases. Dev Dyn 239:34–44Google Scholar
  53. 53.
    Hu Y, Qiao L, Wang S et al (2000) 3-(Hydroxymethyl)-bearing phosphatidylinositol ether lipid analogues and carbonate surrogates block PI3-K, Akt, and cancer cell growth. J Med Chem 43:3045–3051CrossRefGoogle Scholar
  54. 54.
    Liou GY, Storz P (2010) Reactive oxygen species in cancer. Free Radic Res 44:479–496CrossRefGoogle Scholar
  55. 55.
    Yusuf RZ, Duan Z, Lamendola DE, Penson RT, Seiden MV (2003) Paclitaxel resistance: molecular mechanisms and pharmacologic manipulation. Curr Cancer Drug Targets 3:1–19CrossRefGoogle Scholar
  56. 56.
    Fan QW, Cheng C, Hackett C et al (2010) Akt and autophagy cooperate to promote survival of drug-resistant glioma. Sci Signal 3:ra81CrossRefGoogle Scholar
  57. 57.
    Liang X, Tang J, Liang Y, Jin R, Cai X (2014) Suppression of autophagy by chloroquine sensitizes 5-fluorouracil-mediated cell death in gallbladder carcinoma cells. Cell Biosci 4:10CrossRefGoogle Scholar
  58. 58.
    Verschooten L, Barrette K, Van Kelst S et al (2012) Autophagy inhibitor chloroquine enhanced the cell death inducing effect of the flavonoid luteolin in metastatic squamous cell carcinoma cells. PLoS ONE 7:e48264CrossRefGoogle Scholar
  59. 59.
    Peng X, Gong F, Chen Y et al (2014) Autophagy promotes paclitaxel resistance of cervical cancer cells: involvement of Warburg effect activated hypoxia-induced factor 1-alpha-mediated signaling. Cell Death Dis 5:e1367CrossRefGoogle Scholar
  60. 60.
    Korolchuk VI, Mansilla A, Menzies FM, Rubinsztein DC (2009) Autophagy inhibition compromises degradation of ubiquitin-proteasome pathway substrates. Mol Cell 33:517–527CrossRefGoogle Scholar
  61. 61.
    Suski JM, Lebiedzinska M, Bonora M, Pinton P, Duszynski J, Wieckowski MR (2012) Relation between mitochondrial membrane potential and ROS formation. Methods Mol Biol 810:183–205CrossRefGoogle Scholar
  62. 62.
    Andre N, Braguer D, Brasseur G et al (2000) Paclitaxel induces release of cytochrome c from mitochondria isolated from human neuroblastoma cells’. Cancer Res 60:5349–5353Google Scholar
  63. 63.
    Redmann M, Benavides GA, Berryhill TF et al (2017) Inhibition of autophagy with bafilomycin and chloroquine decreases mitochondrial quality and bioenergetic function in primary neurons. Redox Biol 11:73–81CrossRefGoogle Scholar
  64. 64.
    Mullarky E, Cantley LC (2015) Diverting Glycolysis to Combat Oxidative Stress. In: Nakao K, Minato N, Uemoto S (eds) Innovative Medicine, Basic Research and Development, Tokyo, pp 3–23CrossRefGoogle Scholar
  65. 65.
    Wang J, Wei Q, Wang X et al (2016) Transition to resistance: an unexpected role of the EMT in cancer chemoresistance. Genes Dis 3:3–6CrossRefGoogle Scholar
  66. 66.
    Leslie NR, Bennett D, Lindsay YE, Stewart H, Gray A, Downes CP (2003) Redox regulation of PI 3-kinase signalling via inactivation of PTEN. EMBO J 22:5501–5510CrossRefGoogle Scholar
  67. 67.
    Liu C, Gong K, Mao X, Li W (2011) Tetrandrine induces apoptosis by activating reactive oxygen species and repressing Akt activity in human hepatocellular carcinoma. Int J Cancer 129:1519–1531CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Satabdi Datta
    • 1
  • Diptiman Choudhury
    • 2
  • Amlan Das
    • 3
  • Dipanwita Das Mukherjee
    • 1
  • Moumita Dasgupta
    • 1
  • Shreya Bandopadhyay
    • 4
  • Gopal Chakrabarti
    • 1
    Email author
  1. 1.Department of Biotechnology and Dr. B. C. Guha Centre for Genetic Engineering and BiotechnologyUniversity of CalcuttaKolkataIndia
  2. 2.School of Chemistry and BiochemistryThapar Institute of Engineering and TechnologyPatialaIndia
  3. 3.Department of ChemistryNational Institute of TechnologyRavanglaIndia
  4. 4.Cell Biology & Physiology DivisionCSIR – Indian Institute of Chemical BiologyKolkataIndia

Personalised recommendations