SpringerLink
Log in

The microtubule depolymerizing agent naphthazarin induces both apoptosis and autophagy in A549 lung cancer cells

  • Original Paper
  • Published:
Apoptosis Aims and scope Submit manuscript

Abstract

Naphthazarin (DHNQ, 5,8-dihydroxy-l,4-naphthoquinone) is a naturally available 1,4-naphthoquinone derivatives. In this study, we focused on elucidating the cytotoxic mechanism of naphthazarin in A549 non-small cell lung carcinoma cells. Naphthazarin reduced the A549 cell viability considerably with an IC50 of 16.4 ± 1.6 μM. Naphthazarin induced cell death in a dose- and time-dependent manner by activating apoptosis and autophagy pathways. Specifically, we found naphthazarin inhibited the PI3K/Akt cell survival signalling pathway, measured by p53 and caspase-3 activation, and PARP cleavage. It also resulted in an increase in the ratio of Bax/Bcl2 protein levels, indicating activation of the mitochondrial apoptotic pathway. Similarly naphthazarin triggered LC3II expression and induced autophagic flux in A549 cells. We demonstrated further that naphthazarin is a microtubule inhibitor in cell-free system and in A549 cells. Naphthazarin treatment depolymerized interphase microtubules and disorganised spindle microtubules and the majority of cells arrested at the G2/M transition. Together, these data suggest that naphthazarin, a microtubule depolymerizer which activates dual cell death machineries, could be a potential novel chemotherapeutic agent.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11
Fig. 12

Similar content being viewed by others

References

  1. Papageorgiou VP, Assimopoulou AN, Couladouros EA, Hepworth D, Nicolaou KC (1998) The chemistry and biology of alkannin, shikonin and related naphthazarin natural products. Angew Chem Int Ed 38:270–300

    Article  Google Scholar 

  2. Assimopoulou AN, Boskou D, Papageorgiou VP (2004) Antioxidant activities of alkannin, shikonin and Alkanna tinctoria root extracts in oil substrates. Food Chem 87:433–438

    Article  CAS  Google Scholar 

  3. Chien Chang S, Wan S Jr, Shyh-Yuan L, Chia-Hung L, Gum-Hee L, Chang Ming S (2002) Antimicrobial activities of naphtazarins from Arnebia euchroma. J Nat Prod 65:1857–1862

    Google Scholar 

  4. Sasaki K, Abe H, Yoshizaki F (2002) In vitro antifungal activity of naphthoquinone derivatives. Biol Pharm Bull 25:669–670

    Article  PubMed  CAS  Google Scholar 

  5. Assimopoulou AN, Papageorgiou VP (2005) Radical scavenging activity of Alkanna tinctoria root extracts and their main constituents hydroxyl naphthoquinones. Phytother Res 19:141–147

    Article  PubMed  CAS  Google Scholar 

  6. Ozaki Y, Sakaguchi I, Tujimura M, Ikeda N, Nakayama M, Kato Y, Suzuki H, Satake M (1998) Study of the accelerating effect of granulation tissue in rats. Biol Pharm Bull 21:366–370

    PubMed  CAS  Google Scholar 

  7. Sanakawa U, Ebizuka Y, Miyazaki T, Isomura Y, Otsuka H, Shibata S, Inomata M, Fukuoka F (1977) Anti-tumor activity of shikonin and its derivatives. Chem Pharm Bull 25:2392–2395

    Google Scholar 

  8. Delgado MA, Elmaoued RA, Davis AS, George K, Deretic V (2008) Toll-like receptors control autophagy. EMBO J 27:1110–1121

    Article  PubMed  CAS  Google Scholar 

  9. Xu Y, Jagannath C, Liu XD, Sharafkhaneh A, Kolodziejska KE, Eissa NT (2007) Toll-like receptor 4 is a sensor for autophagy associated with innate immunity. Immunity 27:135–144

    Article  PubMed  CAS  Google Scholar 

  10. Eisenberg-Lerner A, Bialik S, Simon HU, Kimchi A (2009) Life and death partners: apoptosis, autophagy and the cross-talk between them. Cell Death Differ 16:966–975

    Article  PubMed  CAS  Google Scholar 

  11. Levine B, Yuan J (2005) Autophagy and cell death: an innocent convict? J Clin Invest 115:2679–2688

    Article  PubMed  CAS  Google Scholar 

  12. Maiuri MC, Zalckvar E, Kimchi A, Kroemer G (2007) Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol 8:741–752

    Article  PubMed  CAS  Google Scholar 

  13. Hickman JA (1992) Apoptosis induced by anticancer drugs. Cancer Metastasis Rev 11:121–139

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  15. Paz-Ares L, Blanco-Aparicio C, García-Carbonero R, Carnero A (2009) Inhibiting PI3K as a therapeutic strategy against cancer. Clin Transl Oncol 11:572–579

    Article  PubMed  CAS  Google Scholar 

  16. Earnshaw WC, Martins LM, Kaufmann SH (1999) Mammalian caspases: structure, activation, substrates and functions during apoptosis. Annu Rev Biochem 68:383–424

    Article  PubMed  CAS  Google Scholar 

  17. Vivanco I, Sawyers CL (2002) The phosphatidylinositol 3-kinase-Akt pathway in human cancer. Nat Rev Cancer 2:489–501

    Article  PubMed  CAS  Google Scholar 

  18. Lodish H, Baltimore D, Berk A, Zipursky SL, Matsudaira P, Darnell J (1999) Molecular cell biology, 5th edn. W. H. Freeman, New York, pp 806–827

    Google Scholar 

  19. Jordan MA, Wilson L (2004) Microtubules as a target for anticancer drugs. Nat Rev Cancer 4:253–265

    Article  PubMed  CAS  Google Scholar 

  20. Jordan MA (2002) Mechanism of action of antitumor drugs that interacts with microtubules and tubulin. Curr Med Chem Anticancer Agents 2:1–17

    Article  PubMed  CAS  Google Scholar 

  21. Acharya BR, Bhattacharyya B, Chakrabarti G (2008) The natural naphthoquinone plumbagin exhibits antiproliferative activity and disrupts the microtubule network through tubulin binding. Biochemistry 47:7838–7845

    Article  PubMed  CAS  Google Scholar 

  22. Acharya BR, Choudhury D, Das A, Chakrabarti G (2009) Vitamin K3 disrupts the microtubule networks by binding to tubulin: a novel mechanism of its antiproliferative activity. Biochemistry 48:6963–6974

    Article  PubMed  CAS  Google Scholar 

  23. Bonfoco E, Ceccatelli S, Manzo L, Nicotera P (1995) Colchicine induces apoptosis in cerebellar granule cells. Exp Cell Res 218:189–200

    Article  PubMed  CAS  Google Scholar 

  24. Liu XM, Wang LG, Kreis W, Budman DR, Adams LM (2001) Differential effect of vinorelbine versus paclitaxel on ERK2 kinase activity during apoptosis in MCF-7 cells. Br J Cancer 85:1403–1411

    Article  PubMed  CAS  Google Scholar 

  25. Levine B, Klionsky DJ (2004) Development by self-digestion. Dev Cell 6(4):463–477

    Article  PubMed  CAS  Google Scholar 

  26. Alva AS, Gultekin SH, Baehrecke EH (2004) Autophagy in human tumors: cell survival or death? Cell Death Differ 11:1046–1048

    Article  PubMed  CAS  Google Scholar 

  27. Gozuacik D, Kimchi A (2004) Autophagy as a cell death and tumor suppressor mechanism. Oncogene 23:2891–2906

    Article  PubMed  CAS  Google Scholar 

  28. Kochl R, Tooze SA (2006) Microtubules facilitate autophagosome formation and fusion of autophagosomes with endosomes. Traffic 7:129–145

    Article  PubMed  CAS  Google Scholar 

  29. Reunanen H, Marttinen M, Hirsimaki P (1988) Effects of griseofulvin and nocodazole on the accumulation of autophagic vacuoles in Ehrlich ascites tumor cells. Exp Mol Pathol 48:97–102

    Article  PubMed  CAS  Google Scholar 

  30. Fengsrud M, Roos N, Berg T, Liou W, Slot JW, Seglen PO (1995) Ultrastructural and immunocytochemical characterization of autophagic vacuoles in isolated hepatocytes: effects of Vinblastine and Asparagine on vacuole distributions. Exp Cell Res 221:504–519

    Article  PubMed  CAS  Google Scholar 

  31. Po-Lin K, Ya-Ling H, Chien-Yu C (2006) Plumbagin induces G2-M arrest and autophagy by inhibiting the AKT/mammalian target of rapamycin pathway in breast cancer cells. Mol Cancer Ther 5(12):3209–3221

    Article  Google Scholar 

  32. Amaravadi RK, Yu D, Lum JJ, Bui T, Christophorou MA, Evan GI, Thomas-Tikhonenko A, Thompson CB (2007) Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma. J Clin Invest 117:326–336

    Article  PubMed  CAS  Google Scholar 

  33. Boya P, Gonzalez-Polo RA, Casares N, Perfettini JL, Dessen P, Larochette N, Metivier D, Meley D, Souquere S, Yoshimori T, Pierron G, Codogno P, Kroemer G (2005) Inhibition of macroautophagy triggers apoptosis. Mol Cell Biol 25:1025–1040

    Article  PubMed  CAS  Google Scholar 

  34. Ci-Hui Y, Zhong-Qin L, Zhen-Lun G, Ya-** Y, Paul R, Zheng-Hong Q (2006) Contributions of autophagic and apoptotic mechanisms to CrTX-induced death of K562 cells. Toxicon 47:521–530

    Article  Google Scholar 

  35. Cheng Y, Feng Q, Takashi I (2009) Molecular mechanisms of oridonin-induced apoptosis and autophagy in murine fibrosarcoma L929 cells. Autophagy 5(3):430–431

    Article  PubMed  Google Scholar 

  36. 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 72:248–254

    Article  PubMed  CAS  Google Scholar 

  37. Sen T, Moulik S, Dutta A, Choudhury PR, Banerji A, Das S, Roy M, Chatterjee A (2009) Multifunctional effect of epigallocatechin-3-gallate (EGCG) in down regulation of gelatinase-A (MMP-2) in human breast cancer cell line MCF-7. Life Sci 84:194–204

    Article  PubMed  CAS  Google Scholar 

  38. Minnoti AM, Barlow SB, Cabral F (1991) Resistance to antimitotic drugs in Chinese hamster ovary cells correlates with changes in the level of polymerized tubulin. J Biol Chem 266:3987–3994

    Google Scholar 

  39. Hamel E, Lin CM (1981) Glutamate-induced polymerization of tubulin: characteristics of the reaction and application to the large scale purification of tubulin. Arch Biochem Biophys 209:29–40

    Article  PubMed  CAS  Google Scholar 

  40. Gaskin F, Cantor CR, Shelanski ML (1974) Turbidimetric studies of the in vitro assembly and disassembly of porcine neurotubules. J Mol Biol 89:737–755

    Article  PubMed  CAS  Google Scholar 

  41. Kanzawa T, Kondo Y, Hideki I, Germano I (2003) Induction of autophagic cell death in malignant glioma cells by arsenic trioxide. Cancer Res 63:2103–2108

    PubMed  CAS  Google Scholar 

  42. Liu B, Cheng Y, Zhang B, He-jiao B, **-Ku B (2009) Polygonatum cyrtonema lectin induces apoptosis and autophagy in human melanoma A375 cells through a mitochondria-mediated ROS–p38–p53 pathway. Cancer Lett 275:54–60

    Article  PubMed  CAS  Google Scholar 

  43. Munafo DB, Colombo MI (2001) A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation. J Cell Sci 114:3619–3629

    PubMed  CAS  Google Scholar 

  44. Mizushima N, Yoshimori T (2007) How to interpret LC3 immuno-blotting. Autophagy 3(6):542–545

    PubMed  CAS  Google Scholar 

  45. Jeong-Sun J, Varadhachary AS, Miller SE, Weihl CC (2010) Quantitation of “autophagic flux” in mature skeletal muscle. Autophagy 6(7):929–935

    Article  Google Scholar 

  46. **ao H, Jian-xun L, **n-zhi L (2011) Salvianolic acid B inhibits autophagy and protects starving cardiac myocytes. Acta Pharmacol Sin 32:38–44

    Article  Google Scholar 

  47. Huang SW, Liu KT, Chang CC, Chen YJ, Wu CY, Tsai JJ, Lu WC, Wang YT, Liu CM, Shieh JJ (2010) Imiquimod simultaneously induces autophagy and apoptosis in human basal cell carcinoma cells. Br J Dermatol 163(2):310–320

    Article  PubMed  CAS  Google Scholar 

  48. Fujiwara Y, Hosokawa Y, Watanabe K, Tanimura S, Ozaki K, Kohno M (2007) Blockade of the phosphatidylinositol-3-kinase-Akt signaling pathway enhances the induction of apoptosis by microtubule-destabilizing agents in tumor cells in which the pathway is constitutively activated. Mol Cancer Ther 6:1133–1142

    Article  PubMed  CAS  Google Scholar 

  49. Kapeller R, Toker A, Cantley LC, Carpenter CL (1995) Phospho- inositide 3-kinase binds constitutively to alpha/beta-tubulin and binds to gamma-tubulin in response to insulin. J Biol Chem 270:25985–25991

    Article  PubMed  CAS  Google Scholar 

  50. Petiot A, Ogier-Denis E, Blommaart EF, Meijer AJ, Codogno P (2000) Distinct classes of phosphatidylinositol 3-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J Biol Chem 275:992–998

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

Authors like to thank Prof. Geri Kreitzer (Department of Cell and Developmental Biology, Weill Cornell Medical College, Cornell University, USA) for her critical reading of the manuscript. The work was supported by grants from DST, Govt. of India (No. SR/SO/BB-14/2008) and DBT, Govt. of India (No. BT/PR12889/AGR/36/624/2009) to GC. Confocal Microscope and FACS facilities are developed by the grant from National Common Minimum Program, Govt. of India.

Conflict of interest

The authors declare that there are no conflicts of interest.

Author information

Author notes

  1. Bipul R. Acharya

    Present address: Department of Cell and Developmental Biology, Weill Cornell Medical College, Cornell University, New York, NY, 10065, USA

Authors and Affiliations

  1. Department of Biotechnology and Dr. B.C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, WB, 700019, India

    Bipul R. Acharya, Surela Bhattacharyya, Diptiman Choudhury & Gopal Chakrabarti

Authors
  1. Bipul R. Acharya
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Surela Bhattacharyya
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Diptiman Choudhury
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gopal Chakrabarti
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Gopal Chakrabarti.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Acharya, B.R., Bhattacharyya, S., Choudhury, D. et al. The microtubule depolymerizing agent naphthazarin induces both apoptosis and autophagy in A549 lung cancer cells. Apoptosis 16, 924–939 (2011). https://doi.org/10.1007/s10495-011-0613-1

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10495-011-0613-1

Keywords

Search

Navigation