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Violacein induces death of RAS-mutated metastatic melanoma by impairing autophagy process

  • Original Article
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Tumor Biology

Abstract

Treatment of metastatic melanoma still remains a challenge, since in advanced stage it is refractory to conventional treatments. Most patients with melanoma have either B-RAF or N-RAS mutations, and these oncogenes lead to activation of the RAS-RAF-MEK-ERK and AKT signal pathway, keeping active the proliferation and survival pathways in the cell. Therefore, the identification of small molecules that block metastatic cell proliferation and induce cell death is needed. Violacein, a pigment produced by Chromobacterium violaceum found in Amazon River, has been used by our group as a biotool for scrutinizing signaling pathways associated with proliferation, survival, aggressiveness, and resistance of cancer cells. In the present study, we demonstrate that violacein diminished the viability of RAS- and RAF-mutated melanoma cells (IC50 value ∼500 nM), and more important, this effect was not abolished after treatment medium removal. Furthermore, violacein was able to reduce significantly the invasion capacity of metastatic melanoma cells in 3D culture. In the molecular context, we have shown for the first time that violacein causes a strong drop on histone deacetylase 6 expression, a proliferating activator, in melanoma cells. Besides, an inhibition of AXL and AKT was detected. All these molecular events propitiate an inhibition of autophagy, and consequently, melanoma cell death by apoptosis.

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Abbreviations

B-RAF:

B-RAF proto-oncogene, serine/threonine kinase

N-RAS:

N-RAS proto-oncogene, member of the RAS gene family

MEK:

Mitogen-activated protein kinase kinase

ERK:

Extracellular signal-regulated kinase

AKT:

Serine/threonine-specific protein kinase

AXL:

Tyrosine-protein kinase receptor UFO

LC3-I/II:

Microtubule-associated proteins 1A/1B light chains 3A/LC3A and 3B/LC3B

p62:

Sequestosome-1

p21:

Cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1

PARP-1:

Poly(ADP-ribose) polymerase-1

mTOR:

Mammalian target of rapamycin, Ser/Thr protein kinase

HSP90:

Heat shock protein 90 kDa

HDAC:

Histone deacetylase

SIRT:

NAD-dependent deacetylase sirtuin-1

References

  1. Ozben T. Mechanisms and strategies to overcome multiple drug resistance in cancer. FEBS Lett. 2006;580(12):2903–9. doi:10.1016/j.febslet.2006.02.020.

    Article  CAS  PubMed  Google Scholar 

  2. Eggermont AMM. Advances in systemic treatment of melanoma. Ann Oncol. 2010;21(7):vii339–44. doi:10.1093/annonc/mdq364.

    PubMed  Google Scholar 

  3. Eggermont AMM, Spatz A, Robert C. Cutaneous melanoma. Lancet. 2014;383(9919):816–27. doi:10.1016/S0140-6736(13)60802-8.

    Article  CAS  PubMed  Google Scholar 

  4. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, et al. Mutations of the BRAF gene in human cancer. Nature. 2002;417(6892):949–54. doi:10.1038/nature00766.

    Article  CAS  PubMed  Google Scholar 

  5. Banerji U, Affolter A, Judson I, Marais R, Workman P. BRAF and NRAS mutations in melanoma: potential relationships to clinical response to HSP90 inhibitors. Mol Cancer Ther. 2008;7(4):737–9. doi:10.1158/1535-7163.MCT-08-0145.

    Article  CAS  PubMed  Google Scholar 

  6. Melanoma BC. From melanocyte to genetic alterations and clinical options. Scientifica. 2013;2013:635203. doi:10.1155/2013/635203.

    Google Scholar 

  7. Kunz M. Oncogenes in melanoma: an update. Eur J Cell Biol. 2014;93(1–2):1–10. doi:10.1016/j.ejcb.2013.12.002.

    Article  CAS  PubMed  Google Scholar 

  8. Lazova R, Klump V, Pawelek J. Autophagy in cutaneous malignant melanoma. J Cutan Pathol. 2010;37(2):256–68. doi:10.1111/j.1600-0560.2009.01359.x.

    Article  PubMed  Google Scholar 

  9. Maes H, Agostinis P. Autophagy and mitophagy interplay in melanoma progression. Mitochondrion. 2014;19(Pt A):58–68. doi:10.1016/j.mito.2014.07.003.

    Article  CAS  PubMed  Google Scholar 

  10. Meng XX, Yao M, Zhang XD, Xu HX, Dong Q. ER stress-induced autophagy in melanoma. Clin Exp Pharmacol Physiol. 2015;42(8):811–6. doi:10.1111/1440-1681.12436.

    Article  CAS  PubMed  Google Scholar 

  11. Baehrecke EH. Autophagy: dual roles in life and death? Nat Rev Mol Cell Biol. 2005;6(6):505–10. doi:10.1038/nrm1666.

    Article  CAS  PubMed  Google Scholar 

  12. Maycotte P, Thorburn A. Autophagy and cancer therapy. Cancer Biol Ther. 2011;11(2):127–37. doi:10.4161/cbt.11.2.14627.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Rosenfeldt MT, Ryan KM. The multiple roles autophagy in cancer. Carcinogenesis. 2011;32(7):955–63. doi:10.1093/carcin/bgr031.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Villar VH, Merhi F, Djavaheri-Mergny M, Durán RV. Glutaminolysis and autophagy in cancer. Autophagy. 2015;11(8):1198–208. doi:10.1080/15548627.2015.1053680.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Melo PS, Maria SS, de Campos Vidal B, Haun M, Durán N. Violacein cytotoxicity and induction of apoptosis in V79 cells. In Vitro Cell Dev Biol Anim. 2000;36(8):539–43. doi:10.1290/1071-2690(2000)036<0539:VCAIOA>2.0.CO;2.

    Article  CAS  PubMed  Google Scholar 

  16. Durán N, Menck CFM. Chromobacterium violaceum: a review of pharmacological and industrial perspectives. Crit Rev Microbiol. 2001;27(3):201–22. doi:10.1080/20014091096747.

    Article  PubMed  Google Scholar 

  17. Ferreira CV, Bos CL, Versteeg HH, Justo GZ, Durán N, Peppelenbosch MP. Molecular mechanism of violacein-mediated human leukemia cell death. Blood. 2004;104(5):1459–64. doi:10.1182/blood-2004-02-0594.

    Article  CAS  PubMed  Google Scholar 

  18. de Carvalho DD, Costa FTM, Duran N, Haun M. Cytotoxic activity of violacein in human colon cancer cells. Toxicol in Vitro. 2006;20(8):1514–21. doi:10.1016/j.tiv.2006.06.007.

    Article  PubMed  Google Scholar 

  19. Bromberg N, Dreyfuss JL, Regatieri CV, Palladino MV, Durán N, Nader HB, et al. Growth inhibition and pro-apoptotic activity of violacein in Ehrlich ascites tumor. Chem Biol Interact. 2010;186(1):43–52. doi:10.1016/j.cbi.2010.04.016.

    Article  CAS  PubMed  Google Scholar 

  20. Durán M, Faljoni-Alario A, Durán N. Chromobacterium violaceum and its important metabolites—review. Folia Microbiol. 2010;55(6):535–47. doi:10.1007/s12223-010-0088-4.

    Article  Google Scholar 

  21. Queiroz KC, Milani R, Ruela-de-Sousa RR, Fuhler GM, Justo GZ, Zambuzzi WF, et al. Violacein induces death of resistant leukaemia cells via kinome reprogramming, endoplasmic reticulum stress and Golgi apparatus collapse. PLoS One. 2012;7(10):e45362. doi:10.1371/journal.pone.0045362.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Mehta T, Vercruysse K, Johnson T, Ejiofor AO, Myles E, Quick QA. Violacein induces p44/42 mitogen-activated protein kinase-mediated solid tumor cell death and inhibits tumor cell migration. Mol Med Rep. 2015;12(1):1443–8. doi:10.3892/mmr.2015.3525.

    PubMed  PubMed Central  Google Scholar 

  23. Masuelli L, Pantanella F, La Regina G, Benvenuto M, Fantini M, Mattera R, et al. Violacein, an indole-derived purple-colored natural pigment produced by Janthinobacterium lividum, inhibits the growth of head and neck carcinoma cell lines both in vitro and in vivo. Tumour Biol. 2015;1–13. doi 10.1007/s13277-015-4207-3.

  24. Alshatwi AA, Subash-Babu P, Antonisamy P. Violacein induces apoptosis in human breast cancer cells through up regulation of BAX, p53 and down regulation of MDM2. Exp Toxicol Pathol. 2016;68(1):89–97. doi:10.1016/j.etp.2015.10.002.

    Article  CAS  PubMed  Google Scholar 

  25. Rettori D, Duran N. Production, extraction and purification of violacein: an antibiotic pigment produced by Chromobacterium violaceum. World J Microbiol Biotechnol. 1998;14:685–8.

    Article  CAS  Google Scholar 

  26. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and citotoxicity assays. J Immunol Methods. 1983;65(1–2):55–63. doi:10.1016/0022-1759(83)90303-4.

    Article  CAS  PubMed  Google Scholar 

  27. Smalley KS, Haass NK, Brafford PA, Lioni M, Flaherty KT, Herlyn M. Multiple signaling pathways must be targeted to overcome drug resistance in cell lines derived from melanoma metastases. Mol Cancer Ther. 2006;5:1136–44. doi:10.1158/1535-7163.MCT-06-0084.

    Article  CAS  PubMed  Google Scholar 

  28. Alonso-Curbelo D, Riveiro-Falkenbach E, Pérez-Guijarro E, Cifdaloz M, Karras P, Osterloh L, Megías D, Cañón E, Calvo TG, Olmeda D, Gómez-López G, Graña O, Sánchez-Arévalo Lobo VJ, Pisano DG, Wang HW, Ortiz-Romero P, Tormo D, Hoek K, Rodríguez-Peralto JL, Joyce JA, Soengas MS. RAB7 controls melanoma progression by exploiting a lineage-specific wiring of the endolysosomal pathway. Cancer Cell. 2014;26(1):61–76. doi:10.1016/j.ccr.2014.04.030.

    Article  CAS  PubMed  Google Scholar 

  29. Rusten TE, Stenmark H. p62, an autophagy hero or culprit? Nat Cell Biol. 2010;12:207–9. doi:10.1038/ncb0310-207.

    Article  CAS  PubMed  Google Scholar 

  30. Niezgoda A, Niezgoda P, Czajkowski R. Novel approaches to treatment of advanced melanoma: a review on targeted therapy and immunotherapy. Biomed Res Int. 2015;2015:851387. doi:10.1155/2015/851387.

    Article  PubMed  PubMed Central  Google Scholar 

  31. de Fátima A, Zambuzzi WF, Modolo LV, Tarsitano CA, Gadelha FR, Hyslop S, et al. Cytotoxicity of goniothalamin enantiomers in renal cancer cells: involvement of nitric oxide, apoptosis and autophagy. Chem Biol Interact. 2008;176(2–3):143–50. doi:10.1016/j.cbi.2008.08.003.

    Article  PubMed  Google Scholar 

  32. Bispo-de-Jesus M, Zambuzzi WF, Ruela de Sousa RR, Areche C, Santos de Souza AC, Aoyama H, et al. Ferruginol suppresses survival signaling pathways in androgen-independent human prostate cancer cells. Biochimie. 2008;90(6):843–54. doi:10.1016/j.biochi.2008.01.011.

    Article  CAS  PubMed  Google Scholar 

  33. Ruela-de-Sousa RR, Fuhler GM, Blom N, Ferreira CV, Aoyama H, Peppelenbosch MP. Cytotoxicity of apigenin on leukemia cell lines: implications for prevention and therapy. Cell Death Dis. 2010;1:e19. doi:10.1038/cddis.2009.18.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Pelizzaro-Rocha KJ, de Jesus MB, Ruela-de-Sousa RR, Nakamura CV, Reis FS, de Fátima A, et al. Calix[6]arene bypasses human pancreatic cancer aggressiveness: downregulation of receptor tyrosine kinases and induction of cell death by reticulum stress and autophagy. Biochim Biophys Acta. 2013;1833(12):2856–65. doi:10.1016/j.bbamcr.2013.07.010.

    Article  CAS  PubMed  Google Scholar 

  35. Barcelos RC, Pelizzaro-Rocha KJ, Pastre JC, Dias MP, Ferreira-Halder CV, Pilli RA. A new goniothalamin N-acylated aza-derivative strongly downregulates mediators of signaling transduction associated with pancreatic cancer aggressiveness. Eur J Med Chem. 2014;87:745–58. doi:10.1016/j.ejmech.2014.09.085.

    Article  CAS  PubMed  Google Scholar 

  36. Kodach LL, Bos CL, Durán N, Peppelenbosch MP, Ferreira CV, Hardwick JC. Violacein synergistically increases 5-fluorouracil cytotoxicity, induces apoptosis and inhibits Akt-mediated signal transduction in human colorectal cancer cells. Carcinogenesis. 2006;27(3):508–16. doi:10.1093/carcin/bgi307.

    Article  CAS  PubMed  Google Scholar 

  37. Woan KV, Lienlaf M, Perez-Villaroel P, Lee C, Cheng F, Knox T, et al. Targeting histone deacetylase 6 mediates a dual anti-melanoma effect: enhanced antitumor immunity and impaired cell proliferation. Mol Oncol. 2015;9(7):1447–57. doi:10.1016/j.molonc.2015.04.002.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Kreis NN, Louwen F, Yuan J. Less understood issues: p21(Cip1) in mitosis and its therapeutic potential. Oncogene. 2015;34(14):1758–67. doi:10.1038/onc.2014.133.

    Article  CAS  PubMed  Google Scholar 

  39. Corazzari M, Fimia GM, Lovat P, Piacentini M. Why is autophagy important for melanoma? Molecular mechanisms and therapeutic implications. Semin Cancer Biol. 2013;23(5):337–43. doi:10.1016/j.semcancer.2013.07.001.

    Article  CAS  PubMed  Google Scholar 

  40. Hassan M, Selimovic D, Hannig M, Haikel Y, Brodell RT, Megahed M. Endoplasmic reticulum stress-mediated pathways to both apoptosis and autophagy: significance for melanoma treatment. World J Exp Med. 2015;5(4):206–17. doi:10.5493/wjem.v5.i4.206.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Wang C, Hu Q, Shen HM. Pharmacological inhibitors of autophagy as novel cancer therapeutic agents. Pharmacol Res. 2016;105:164–75. doi:10.1016/j.phrs.2016.01.028.

    Article  CAS  PubMed  Google Scholar 

  42. Ma XH, Piao S, Wang D, Mcafee QW, Nathanson KL, Lum JJ, Li LZ, Amaravadi RK. Measurements of tumor cell autophagy predict invasiveness, resistance to chemotherapy, and survival in melanoma. Clin Cancer Res. 2011;17:3478–89. doi:10.1158/1078-0432.CCR-10-2372.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Verma A, Warner SL, Vankayalapati H, Bearss DJ, Sharma S. Targeting Axl and Mer kinases in cancer. Mol Cancer Ther. 2011;10(10):1763–73. doi:10.1158/1535-7163.MCT-11-0116.

    Article  CAS  PubMed  Google Scholar 

  44. Linger RM, Cohen RA, Cummings CT, Sather S, Migdall-Wilson J, Middleton DH, et al. Mer or Axl receptor tyrosine kinase inhibition promotes apoptosis, blocks growth and enhances chemosensitivity of human non-small cell lung cancer. Oncogene. 2013;32(29):3420–31. doi:10.1038/onc.2012.355.

    Article  CAS  PubMed  Google Scholar 

  45. Krishnamoorthy GP, Guida T, Alfano L, Avilla E, Santoro M, Carlomagno F, et al. Molecular mechanism of 17-allylamino-17-demethoxygeldanamycin (17-AAG)-induced AXL receptor tyrosine kinase degradation. J Biol Chem. 2013;288(24):17481–94. doi:10.1074/jbc.M112.439422.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Risso G, Blaustein M, Pozzi B, Mammi P, Srebrow A. Akt/PKB: one kinase, many modifications. Biochem J. 2015;468(2):203–14. doi:10.1042/BJ20150041.

    Article  CAS  PubMed  Google Scholar 

  47. Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H, Arozena AA, et al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy. 2016;12(1):1–222. doi:10.1080/15548627.2015.1100356.

    Article  PubMed  PubMed Central  Google Scholar 

  48. Komatsu M, Ichimura Y. Physiological significance of selective degradation of p62 by autophagy. FEBS Lett. 2010;584(7):1374–8. doi:10.1016/j.febslet.2010.02.017.

    Article  CAS  PubMed  Google Scholar 

  49. Bitto A, Lerner CA, Nacarelli T, Crowe E, Torres C. Sell C. P62/SQSTM1 at the interface of aging, autophagy, and disease. Age. 2014;36(3):9626. doi:10.1007/s11357-014-9626-3.

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

This study was supported by São Paulo Research Foundation (FAPESP) and The National Council for Scientific and Technological Development (CNPq). Postdoctoral fellowship for K.J.P.R-B (Proc. 2013/08896-3) was provided by FAPESP, and C.V.F-H. was supported by research fellowships from CNPq.

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Authors and Affiliations

  1. Departamento de Ciências da Saúde, Centro Universitário Norte do Espírito Santo, Universidade Federal do Espírito Santo, São Mateus, Espírito Santo, Brazil

    Paola R. Gonçalves

  2. Departamento de Bioquímica e Biologia Tecidual, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil

    Paola R. Gonçalves, Karin J. P. Rocha-Brito, Maruska R. N. Fernandes, Julia L. Abrantes & Carmen V. Ferreira-Halder

  3. Institute of Chemistry, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil

    Nelson Durán

  4. Brazilian Nanotechnology National Laboratory (LNNano-CNPEM), Campinas, SP, Brazil

    Nelson Durán

  5. Unicamp. Rua Monteiro Lobato, Cidade Universitária Zeferino, 255/ Bloco E1/ Lab 01/ CEP 13083-862, Campinas, São Paulo, Brazil

    Carmen V. Ferreira-Halder

Authors
  1. Paola R. Gonçalves
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  2. Karin J. P. Rocha-Brito
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  3. Maruska R. N. Fernandes
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  5. Nelson Durán
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  6. Carmen V. Ferreira-Halder
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Correspondence to Carmen V. Ferreira-Halder.

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Paola R. Gonçalves and Karin J. P. Rocha-Brito contributed equally to this study.

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Gonçalves, P.R., Rocha-Brito, K.J.P., Fernandes, M.R.N. et al. Violacein induces death of RAS-mutated metastatic melanoma by impairing autophagy process. Tumor Biol. 37, 14049–14058 (2016). https://doi.org/10.1007/s13277-016-5265-x

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