Skip to main content

Advertisement

SpringerLink
Log in

Autophagy, cell death and sustained senescence arrest in B16/F10 melanoma cells and HCT-116 colon carcinoma cells in response to the novel microtubule poison, JG-03-14

  • Original Article
  • Published:
Cancer Chemotherapy and Pharmacology Aims and scope Submit manuscript

Abstract

Purpose

Previous studies have shown that the novel microtubule poison, JG-03-14, which binds to the colchicine binding site of tubulin, has the capacity to kill breast tumor cells primarily through the promotion of autophagy. The current work was designed to determine whether autophagy was, in fact, the primary mode of action as well as susceptibility to JG-03-14 in two additional tumor cell models, the B16/F10 murine melanoma cell line and the HCT-116 human colon cancer cell line.

Methods

Drug cytotoxicity was monitored based on viable cell number and clonogenic survival. Apoptosis was assessed by DAPI staining, the TUNEL assay and/or FACS analysis. Autophagy was monitored based on staining with acridine orange, redistribution and punctuation of RFP-LC3 and electron microscopy as well as p62 degradation. Senescence was evaluated based on β-galactosidase staining and alterations in cell morphology. Drug effects were also evaluated in a murine model of B16/F10 cells that localizes to the lungs while peripheral neuropathy was assessed by three complementary behavioral assays.

Results

Both HCT-116 colon cancer cells and B16/F10 melanoma cells were sensitive to JG-03-14 in that the drug demonstrated tumor cell killing. However, there was minimal induction of apoptosis. In contrast, there was clear evidence for autophagy and autophagic flux while the residual surviving cells appeared to be in a state of irreversible senescence. Inhibition of drug-induced autophagy in either the melanoma cells or the colon carcinoma cells was only slightly protective as the cells instead died by apoptosis. JG-03-14 reduced the size of tumor nodules in mice lungs; furthermore, the drug did not promote peripheral neuropathy.

Conclusions

Taken together with evidence for its actions as a vascular disrupting agent, these observations support the potential utility of JG-03-14 to effectively treat malignancies that might be resistant to conventional chemotherapy through evasion of apoptosis.

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

Access this article

Log in via an institution

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

Similar content being viewed by others

Notes

  1. Prior to the combination treatment studies, the toxicity of 3-methyladenine (3-MA), wortmannin, LY-249004, bafilomycin A-1 and chloroquine, all of which have been shown to inhibit autophagy in a variety of experimental systems [44–46], was examined in the B16/F10 and HCT-116 cell lines. Bafilomycin was chosen for the studies in the B16/F10 cells, and chloroquine was chosen for studies in the HCT-116 cells based on the fact that these agents exhibited minimal toxicity at concentrations where autophagy inhibition was evident.

References

  1. Mooberry SL, Weiderhold KN, Dakshanamurthy S, Hamel E, Banner EJ, Kharlamova A et al (2007) Identification and characterization of a new tubulin-binding tetrasubstituted brominated pyrrole. Mol Pharm 72:132–140

    Article  CAS  Google Scholar 

  2. Arthur CR, Gupton JT, Kellogg GE, Yeudall WA, Cabot MC, Newsham IF et al (2007) Autophagic cell death, polyploidy and senescence induced in breast tumor cells by the substituted pyrrole JG-03-14, a novel microtubule poison. Biochem Pharm 74:981–991

    Article  PubMed  CAS  Google Scholar 

  3. Gewirtz D, Holt SE, Elmore LW (2008) Accelerated senescence: an emerging role in tumor cell response to chemotherapy and radiation. Biochem Pharmacol 76:947–957

    Article  PubMed  CAS  Google Scholar 

  4. Daylot-Herman N, Delgago-Lopez F, Gewirtz DA, Gupton JT, Schwartz EL (2009) Interference with endothelial cell function by JG-03-14, an agent that binds to the colchicine site on microtubules. Biochem Pharm 79:1167–1177

    Article  Google Scholar 

  5. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63

    Article  PubMed  CAS  Google Scholar 

  6. Demasters G, Di X, Newsham I, Shiu R, Gewirtz DA (2006) Potentiation of radiation sensitivity in breast tumor cells by the vitamin D3 analogue, EB 1089, through promotion of autophagy and interference with proliferative recovery. Molec Cancer Ther 5:2786–2797

    Article  CAS  Google Scholar 

  7. Negoescu A, Lorimier P, Labat-Moleur F, Drouet C, Robert C, Guillermet C et al (1996) In situ apoptotic cell labeling by the TUNEL method: improvement and evaluation on cell preparations. Histochem Soc 44:959–968

    CAS  Google Scholar 

  8. Vakifahmetoglu H, Olsson M, Zhivotovsky B (2008) Death through a tragedy: mitotic catastrophe. Cell Death Diff 15:1153–1162

    Article  CAS  Google Scholar 

  9. Paglin S, Hollister T, Delohery T, Hackett N, McMahill M, Sphicas E et al (2001) A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer Res 61:439–444

    PubMed  CAS  Google Scholar 

  10. Bampton ET, Goemans CG, Niranjan D, Mizushima N, Tolkovsky AM (2005) The dynamics of autophagy visualized in live cells: from autophagosome formation to fusion with endo/lysosomes. Autophagy 1:23–36

    Article  PubMed  CAS  Google Scholar 

  11. Ylä-Anttila P, Vihinen H, Jokitalo E, Eskelinen EL (2009) Monitoring autophagy by electron microscopy in mammalian cells. Methods Enzymol 452:143–164

    Article  PubMed  Google Scholar 

  12. Campisi J, d’Adda di Fagagna F (2007) Cellular senescence: when bad things happen to good cells. Nat Rev Molec Cell Biol 8:729–740

    Article  CAS  Google Scholar 

  13. Song YS, Lee BY, Hwang ES (2005) Distinct ROS and biochemical profiles in cells undergoing DNA damage-induced senescence and apoptosis. Mech Ageing Dev 126:580–590

    Article  PubMed  CAS  Google Scholar 

  14. Callizot N, Andriambeloson E, Glass J, Revel M, Ferro P, Cirillo R et al (2008) Interleukin-6 protects against paclitaxel, cisplatin and vincristine-induced neuropathies without impairing chemotherapeutic activity. Cancer Chemother Pharmacol 62:995–1007

    Article  PubMed  CAS  Google Scholar 

  15. Sills RC, Valentine WM, Moser V, Graham DG, Morgan DL (2000) Characterization of carbon disulfide neurotoxicity in C57BL6 mice: behavioral, morphologic, and molecular effects. Toxicol Pathol 28:142–148

    Article  PubMed  CAS  Google Scholar 

  16. Tormo D, Checinska A, Alonso-Curbelo D, Perez-Guijarro E, Canon E, Riveiero-Falkenbach E et al (2009) Targeted activation of innate immunity for therapeutic induction of autophagy and apoptosis in melanoma cells. Cancer Cell 16:103–114

    Article  PubMed  CAS  Google Scholar 

  17. Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T et al (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19:5720–5728

    Article  PubMed  CAS  Google Scholar 

  18. Bjørkøy G, Lamark T, Pankiv S, Øvervatn A, Brech A, Johansen T (2009) Monitoring autophagic degradation of p62/SQSTM1. Methods Enzymol 452:181–197

    Article  PubMed  Google Scholar 

  19. Shintani T, Klionsky DJ (2004) Autophagy in health and disease: a double-edged sword. Science 30:990–995

    Article  Google Scholar 

  20. Scarlatti F, Granata R, Meijer AJ, Codogno P (2009) Does autophagy have a license to kill mammalian cells? Cell Death Diff 16:12–20

    Article  CAS  Google Scholar 

  21. Elmore LW, Di X, Di Y-D, Holt SE, Gewirtz DA (2005) Evasion of chemotherapy-induced senescence in breast cancer cell: implications for treatment response. Clin Cancer Res 11:2637–2643

    Article  PubMed  CAS  Google Scholar 

  22. Jones KR, Elmore LW, Jackson-Cook C, Demasters G, Povirk LF, Holt SE et al (2005) P53-dependent accelerated senescence induced by ionizing radiation in breast tumour cells. Int J Rad Biol 81:445–458

    Article  PubMed  CAS  Google Scholar 

  23. Robeson RS, Kussick SJ, Vallieres E, Chen SJ, Wu DY (2005) Escape from therapy-induced accelerated cellular senescence in p53-null lung cancer cells and in human lung cancers. Cancer Res 65:2795–2803

    Article  Google Scholar 

  24. Chao SK, Lin J, Brouwer-Visser J, Smith AB 3rd, Horwitz SB, McDaid HM (2011) Resistance to discodermolide, a microtubule-stabilizing agent and senescence inducer, is 4E-BP1-dependent. Proc Natl Acad Sci USA 108:391–396

    Article  PubMed  CAS  Google Scholar 

  25. Carlson K, Ocean AJ (2011) Peripheral neuropathy with microtubule-targeting agents: occurrence and management approach. Clin Breast Cancer 11:73–81

    Article  PubMed  CAS  Google Scholar 

  26. Swain SM, Arezzo JC (2008) Neuropathy associated with microtubule inhibitors: diagnosis, incidence, and management. Clin Adv Hematol Oncol 6:455–467

    Google Scholar 

  27. Gray-Schopfer V, Wellbrock C, Marais R (2007) Melanoma biology and new targeted therapy. Nature 445(851–7):28

    Google Scholar 

  28. Chin L, Garraway LA, Fisher DE (2006) Malignant melanoma: genetics and therapeutics in the genomic era. Genes Dev 20:2149–2182

    Article  PubMed  CAS  Google Scholar 

  29. Bajetta E, Del Vecchio M, Bernard-Marty C, Vitali M, Buzzoni R, Rixe O et al (2006) Metastatic melanoma: chemotherapy. Semin Oncol 29:427–445

    Article  Google Scholar 

  30. Sorensen NM, Schrohl AS, Jensen V, Christensen IJ, Nielsen HJ, Brunner N (2008) Comparative studies of tissue inhibitor of metalloproteinases-1 in plasma, serum and tumour tissue extracts from patients with primary colorectal cancer. Scan J Gastroenterol 43:186–191

    Article  CAS  Google Scholar 

  31. Schwartz RN (2008) Management of early and advanced colorectal cancer: therapeutic issues. Am J Health Syst Pharm 65(11 Suppl 4):S8–S14

    Article  PubMed  CAS  Google Scholar 

  32. Greene LM, O’Boyle NM, Nolan DP, Meegan MJ, Zisterer DM (2012) The vascular targeting agent Combretastatin-A4 directly induces autophagy in adenocarcinoma-derived colon cancer cells. Biochem Pharmacol 84:612–624

    Article  PubMed  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  34. Amaravadi RK, Duonan Y, Lum JJ, 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 

  35. Boya P, González-Polo R, Casares N, Perfettini J, Dessen P, Larochette N et al (2005) Inhibition of macroautophagy triggers apoptosis. Mol Cell Biol 25:1025–1040

    Article  PubMed  CAS  Google Scholar 

  36. González-Polo RA, Niso-Santano M, Ortiz-Ortiz MA, Gómez-Martín A, Morán JM, Garcia-Rubio L et al (2007) Inhibition of paraquat-induced autophagy accelerates the apoptotic cell death in neuroblastoma SH-SY5Y cells. Tox Sci 97:448–458

    Article  Google Scholar 

  37. Prabhudesai SG, Rekhraj S, Roberts G, Darzi AW, Ziprin P (2007) Apoptosis and chemo-resistance in colorectal cancer. J Surg Onc 96:77–88

    Article  CAS  Google Scholar 

  38. Fulda S (2008) Tumor resistance to apoptosis. Int J Cancer 124:511–515

    Article  Google Scholar 

  39. Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57–70

    Article  PubMed  CAS  Google Scholar 

  40. Haugstetter AM, Loddenkemper C, Lenz D, Grone J, Standfub C, Dorken B et al (2010) Cellular senescence predicts treatment outcome in metastasised colon cancer. Br J Cancer 103:505–509

    Article  PubMed  CAS  Google Scholar 

  41. Goehe RW, Di X, Sharma K, Bristol M, Henderson S, Valerie K, Rodier F, Davalos A, Gewirtz D (2012) The autophagy-senescence connection in chemotherapy; must tumor cells (self) eat before they sleep? J Pharmacol Exp Ther (Epub ahead of print)

  42. Wachsberger PR, Burd R, Marero N, Daskalakis C, Ryan A, McCue P et al (2005) Effect of the tumor vascular-damaging Agent, ZD6126, on the radioresponse of U87 glioblastoma. Clin Cancer Res 11:835–842

    PubMed  CAS  Google Scholar 

  43. El-Emir E, Boxer GM, Petrie IA, Boden RW, Dearling JL, Begent RH et al (2005) Tumour parameters affected by combretastatin A-4 phosphate therapy in a human colorectal xenograft model in nude mice. Eur J Cancer 41:799–806

    Article  PubMed  CAS  Google Scholar 

  44. Seglen PO, Gordon PB (1982) 3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Nat Acad Sci 79:1889–1892

    Article  PubMed  CAS  Google Scholar 

  45. Meschini S, Condello M, Calcabrini A, Marra M, Formisano G, Lista P (2008) The plant alkaloid voacamine induces apoptosis-independent autophagic cell death on both sensitive and multidrug resistant human osteosarcoma cells. Autophagy 4:1020–1033

    PubMed  CAS  Google Scholar 

  46. Shacka JJ, Klocke BJ, Shibata M, Uchiyama Y, Datta G, Schmidt RE et al (2006) Bafilomycin A1 inhibits chloroquine-induced death of cerebellar granule neurons. Molec Pharm 69:1125–1136

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported, in part, by NIH Grant # 2R15 CA067236-04 to Dr. John Gupton and NIH Grant # RO1 CA135043-01A1 to Dr. David Gewirtz. Electron microscopy was performed at the VCU—Department of Neurobiology and Anatomy Microscopy Facility, supported, in part, with funding from NIH-NINDS Center core grant P30NS047463. We acknowledge the support of the Flow cytometry shared resource facility supported in part by NIH Grant P30CA16059. We are grateful to the Anatomic Pathology Research Services for the histology work. The RFP-LC3 vector was generously provided by Dr. Keith Miskimins at the University of South Dakota and was originally developed by the laboratory of Dr. A.M. Tolkovsky. We are grateful to Dr. Fiorenza Ianzini at the University of Iowa for guidance with the assessment of mitotic catastrophe.

Author information

Authors and Affiliations

  1. Department of Pharmacology and Pharmacology, Massey Cancer Center, Virginia Commonwealth University, P.O. Box 980035, Richmond, VA, 23298, USA

    Jonathan W. Biggers, Tuyen Nguyen, Xu Di, Sean M. Emery, Moureq Alotaibi, Kimber L. White Jr., Ronetta Brown & David A. Gewirtz

  2. Department of Chemistry, University of Richmond, Richmond, VA, 23173, USA

    John T. Gupton

  3. Department of Anatomy and Neurobiology, Virginia Commonwealth University, Richmond, VA, 23173, USA

    Scott C. Henderson

  4. Department of Anatomic Pathology, Virginia Commonwealth University, Richmond, VA, 23173, USA

    Jorge Almenara

Authors
  1. Jonathan W. Biggers
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tuyen Nguyen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xu Di
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. John T. Gupton
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Scott C. Henderson
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sean M. Emery
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Moureq Alotaibi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Kimber L. White Jr.
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Ronetta Brown
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jorge Almenara
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. David A. Gewirtz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to David A. Gewirtz.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Biggers, J.W., Nguyen, T., Di, X. et al. Autophagy, cell death and sustained senescence arrest in B16/F10 melanoma cells and HCT-116 colon carcinoma cells in response to the novel microtubule poison, JG-03-14. Cancer Chemother Pharmacol 71, 441–455 (2013). https://doi.org/10.1007/s00280-012-2024-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00280-012-2024-6

Keywords

Search

Navigation