Tumor Biology

, Volume 37, Issue 10, pp 14049–14058 | Cite as

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

  • Paola R. Gonçalves
  • Karin J. P. Rocha-Brito
  • Maruska R. N. Fernandes
  • Julia L. Abrantes
  • Nelson Durán
  • Carmen V. Ferreira-HalderEmail author
Original Article


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.


Violacein Antitumor activity Melanoma Skin carcinoma Autophagy 



B-RAF proto-oncogene, serine/threonine kinase


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


Mitogen-activated protein kinase kinase


Extracellular signal-regulated kinase


Serine/threonine-specific protein kinase


Tyrosine-protein kinase receptor UFO


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




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


Poly(ADP-ribose) polymerase-1


Mammalian target of rapamycin, Ser/Thr protein kinase


Heat shock protein 90 kDa


Histone deacetylase


NAD-dependent deacetylase sirtuin-1



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.

Compliance with ethical standards

Conflicts of interest


Consent of publish

All authors read and approved the final manuscript.


  1. 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.CrossRefPubMedGoogle Scholar
  2. 2.
    Eggermont AMM. Advances in systemic treatment of melanoma. Ann Oncol. 2010;21(7):vii339–44. doi: 10.1093/annonc/mdq364.PubMedGoogle Scholar
  3. 3.
    Eggermont AMM, Spatz A, Robert C. Cutaneous melanoma. Lancet. 2014;383(9919):816–27. doi: 10.1016/S0140-6736(13)60802-8.CrossRefPubMedGoogle Scholar
  4. 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.CrossRefPubMedGoogle Scholar
  5. 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.CrossRefPubMedGoogle Scholar
  6. 6.
    Melanoma BC. From melanocyte to genetic alterations and clinical options. Scientifica. 2013;2013:635203. doi: 10.1155/2013/635203.Google Scholar
  7. 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.CrossRefPubMedGoogle Scholar
  8. 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.CrossRefPubMedGoogle Scholar
  9. 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.CrossRefPubMedGoogle Scholar
  10. 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.CrossRefPubMedGoogle Scholar
  11. 11.
    Baehrecke EH. Autophagy: dual roles in life and death? Nat Rev Mol Cell Biol. 2005;6(6):505–10. doi: 10.1038/nrm1666.CrossRefPubMedGoogle Scholar
  12. 12.
    Maycotte P, Thorburn A. Autophagy and cancer therapy. Cancer Biol Ther. 2011;11(2):127–37. doi: 10.4161/cbt.11.2.14627.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Rosenfeldt MT, Ryan KM. The multiple roles autophagy in cancer. Carcinogenesis. 2011;32(7):955–63. doi: 10.1093/carcin/bgr031.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 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.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 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.CrossRefPubMedGoogle Scholar
  16. 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.CrossRefPubMedGoogle Scholar
  17. 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.CrossRefPubMedGoogle Scholar
  18. 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.CrossRefPubMedGoogle Scholar
  19. 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.CrossRefPubMedGoogle Scholar
  20. 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.CrossRefGoogle Scholar
  21. 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.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 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.PubMedPubMedCentralGoogle Scholar
  23. 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. 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.CrossRefPubMedGoogle Scholar
  25. 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.CrossRefGoogle Scholar
  26. 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.CrossRefPubMedGoogle Scholar
  27. 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.CrossRefPubMedGoogle Scholar
  28. 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.CrossRefPubMedGoogle Scholar
  29. 29.
    Rusten TE, Stenmark H. p62, an autophagy hero or culprit? Nat Cell Biol. 2010;12:207–9. doi: 10.1038/ncb0310-207.CrossRefPubMedGoogle Scholar
  30. 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.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 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.CrossRefPubMedGoogle Scholar
  32. 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.CrossRefPubMedGoogle Scholar
  33. 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.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 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.CrossRefPubMedGoogle Scholar
  35. 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.CrossRefPubMedGoogle Scholar
  36. 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.CrossRefPubMedGoogle Scholar
  37. 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.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 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. CrossRefPubMedGoogle Scholar
  39. 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.CrossRefPubMedGoogle Scholar
  40. 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.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 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.CrossRefPubMedGoogle Scholar
  42. 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.CrossRefPubMedPubMedCentralGoogle Scholar
  43. 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.CrossRefPubMedGoogle Scholar
  44. 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.CrossRefPubMedGoogle Scholar
  45. 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.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 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.CrossRefPubMedGoogle Scholar
  47. 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.CrossRefPubMedPubMedCentralGoogle Scholar
  48. 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.CrossRefPubMedGoogle Scholar
  49. 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.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  • Paola R. Gonçalves
    • 1
    • 2
  • Karin J. P. Rocha-Brito
    • 2
  • Maruska R. N. Fernandes
    • 2
  • Julia L. Abrantes
    • 2
  • Nelson Durán
    • 3
    • 4
  • Carmen V. Ferreira-Halder
    • 2
    • 5
    Email author
  1. 1.Departamento de Ciências da Saúde, Centro Universitário Norte do Espírito SantoUniversidade Federal do Espírito SantoSão MateusBrazil
  2. 2.Departamento de Bioquímica e Biologia Tecidual, Instituto de BiologiaUniversidade Estadual de CampinasCampinasBrazil
  3. 3.Institute of ChemistryUniversidade Estadual de CampinasCampinasBrazil
  4. 4.Brazilian Nanotechnology National Laboratory (LNNano-CNPEM)CampinasBrazil
  5. 5.Unicamp. Rua Monteiro LobatoCidade Universitária ZeferinoCampinasBrazil

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