Investigational New Drugs

, Volume 29, Issue 4, pp 562–573

Cribrostatin 6 induces death in cancer cells through a reactive oxygen species (ROS)-mediated mechanism

  • Mirth T. Hoyt
  • Rahul Palchaudhuri
  • Paul J. Hergenrother


Cribrostatin 6 is a quinone-containing natural product that induces the death of cancer cell lines in culture, and its mechanism of action and scope of activity are unknown. Here we show that cribrostatin 6 has broad anticancer activity, potently inducing apoptotic cell death that is not preceded by any defined cell cycle arrest. Consistent with this data, we find that cribrostatin 6 treated cells have large amounts of reactive oxygen species (ROS) and, based on transcript profiling experiments and other data, this ROS generation is likely the primary mechanism by which cribrostatin 6 induces apoptosis. Given the success of certain ROS producers as anticancer agents, cribrostatin 6 has potential as a novel chemotherapeutic agent.


Cribrostatin 6 ROS Quiescent 3T3 Quinone HMOX1 Apoptosis Cancer stem cells Transcript profile Quiescent cells Drug-resistant cell lines 

Supplementary material

10637_2010_9390_MOESM1_ESM.pdf (2 mb)
Supporting Information AvailableMaterials and methods, spectral data for all new compounds, and pathway diagrams based on transcript profiling experiments. (PDF 2083 kb)


  1. 1.
    Tewey KM, Rowe TC, Yang L, Halligan BD, Liu LF (1984) Adriamycin-induced DNA damage mediated by mammalian DNA topoisomerase II. Science 226:466–468PubMedCrossRefGoogle Scholar
  2. 2.
    Begleiter A (1983) Cytocidal action of the quinone group and its relationship to antitumor activity. Cancer Res 43:481–484PubMedGoogle Scholar
  3. 3.
    Bolzan AD, Bianchi MS (2001) Genotoxicity of streptonigrin: a review. Mutat Res 488:25–37PubMedCrossRefGoogle Scholar
  4. 4.
    Li V-S, Choi D, Tang M, Kohn H (1996) Concerning in vitro mitomycin-DNA alkylation. J Am Chem Soc 118:3765–3766CrossRefGoogle Scholar
  5. 5.
    Le SB, Hailer MK, Buhrow S et al (2007) Inhibition of mitochondrial respiration as a source of adaphostin-induced reactive oxygen species and cytotoxicity. J Biol Chem 282:8860–8872PubMedCrossRefGoogle Scholar
  6. 6.
    Lu HR, Zhu H, Huang M et al (2005) Reactive oxygen species elicit apoptosis by concurrently disrupting topoisomerase II and DNA-dependent protein kinase. Mol Pharmacol 68:983–994PubMedCrossRefGoogle Scholar
  7. 7.
    Pelicano H, Carney D, Huang P (2004) ROS stress in cancer cells and therapeutic implications. Drug Resist Updat 7:97–110PubMedCrossRefGoogle Scholar
  8. 8.
    Tsang WP, Chau SP, Kong SK, Fung KP, Kwok TT (2003) Reactive oxygen species mediate doxorubicin induced p53-independent apoptosis. Life Sci 73:2047–2058PubMedCrossRefGoogle Scholar
  9. 9.
    Swift LP, Rephaeli A, Nudelman A, Phillips DR, Cutts SM (2006) Doxorubicin-DNA adducts induce a non-topoisomerase II-mediated form of cell death. Cancer Res 66:4863–4871PubMedCrossRefGoogle Scholar
  10. 10.
    Coldwell KE, Cutts SM, Ognibene TJ, Henderson PT, Phillips DR (2008) Detection of adriamycin-DNA adducts by accelerator mass spectrometry at clinically relevant adriamycin concentrations. Nucleic Acids Res 36:e100PubMedCrossRefGoogle Scholar
  11. 11.
    Belcourt MF, Penketh PG, Hodnick WF et al (1999) Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA. Proc Natl Acad Sci USA 96:10489–10494PubMedCrossRefGoogle Scholar
  12. 12.
    Pettit GR, Collins JC, Herald DL et al (1992) Isolation and structure of cribrostatins 1 and 2 from the blue marine sponge Cribrochalina sp. Can J Chem 70:1170–1175CrossRefGoogle Scholar
  13. 13.
    Pettit GR, Collins JC, Knight JC et al (2003) Antineoplastic agents. 485. Isolation and structure of cribrostatin 6, a dark blue cancer cell growth inhibitor from the marine sponge Cribrochalina sp. J Nat Prod 66:544–547PubMedCrossRefGoogle Scholar
  14. 14.
    Pettit GR, Knight JC, Collins JC et al (2000) Antineoplastic agents 430. Isolation and structure of cribrostatins 3, 4, and 5 from the republic of maldives cribrochalina species. J Nat Prod 63:793–798PubMedCrossRefGoogle Scholar
  15. 15.
    Sandoval IT, Davis RA, Bugni TS, Concepcion GP, Harper MK, Ireland CM (2004) Cytotoxic isoquinoline quinones from sponges of the genus Petrosia. Nat Prod Res 18:89–93PubMedCrossRefGoogle Scholar
  16. 16.
    Nakahara S, Kubo A, Mikami Y, Ito J (2006) Synthesis of cribrostatin 6 and its related compounds. Heterocycles 68:515–520CrossRefGoogle Scholar
  17. 17.
    Pettit RK, Fakoury BR, Knight JC et al (2004) Antibacterial activity of the marine sponge constituent cribrostatin 6. J Med Microbiol 53:61–65PubMedCrossRefGoogle Scholar
  18. 18.
    Nakahara S, Kubo A (2004) Synthesis of cribrostatin 6. Heterocycles 63:2355–2362CrossRefGoogle Scholar
  19. 19.
    Nakahara S, Kubo A (2003) Catalytic hydrogenation of 8-acyloxy-1-cyanoisoquinoline and synthesis of 9-methoxy-9-deethoxy-cribrostatin 6. Heterocycles 60:2717–2725CrossRefGoogle Scholar
  20. 20.
    Markey MD, Kelly TR (2008) Synthesis of cribrostatin 6. J Org Chem 73:7441–7443PubMedCrossRefGoogle Scholar
  21. 21.
    Knueppel D, Martin SF (2009) Total synthesis of cribrostatin 6. Angew Chem Int Ed Engl 48:2569–2571PubMedCrossRefGoogle Scholar
  22. 22.
    Vichai V, Kirtikara K (2006) Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc 1:1112–1116PubMedCrossRefGoogle Scholar
  23. 23.
    McGrath T, Center MS (1988) Mechanisms of multidrug resistance in HL60 cells: evidence that a surface membrane protein distinct from P-glycoprotein contributes to reduced cellular accumulation of drug. Cancer Res 48:3959–3963PubMedGoogle Scholar
  24. 24.
    Jackson RC (1989) The problem of the quiescent cancer cell. Adv Enzyme Regul 29:27–46PubMedCrossRefGoogle Scholar
  25. 25.
    Jordan CT, Guzman ML, Noble M (2006) Cancer stem cells. N Engl J Med 355:1253–1261PubMedCrossRefGoogle Scholar
  26. 26.
    Dean M, Fojo T, Bates S (2005) Tumour stem cells and drug resistance. Nat Rev Cancer 5:275–284PubMedCrossRefGoogle Scholar
  27. 27.
    Horiatis D, Wang Q, Pinski J (2004) A new screening system for proliferation-independent anticancer agents. Cancer Lett 210:119–124PubMedCrossRefGoogle Scholar
  28. 28.
    Visvader JE, Lindeman GJ (2008) Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer 8:755–768PubMedCrossRefGoogle Scholar
  29. 29.
    Siu WY, Arooz T, Poon RY (1999) Differential responses of proliferating versus quiescent cells to adriamycin. Exp Cell Res 250:131–141PubMedCrossRefGoogle Scholar
  30. 30.
    Jainchill JL, Todaro GJ (1970) Stimulation of cell growth in vitro by serum with and without growth factor relation to contact inhibition and viral transformation. Exp Cell Res 59:137–146PubMedCrossRefGoogle Scholar
  31. 31.
    Baguley BC, Falkenhaug EM (1978) The interaction of ethidium with synthetic double-stranded polynucleotides at low ionic strength. Nucleic Acids Res 5:161–171PubMedCrossRefGoogle Scholar
  32. 32.
    Nitiss JL (2009) Targeting DNA topoisomerase II in cancer chemotherapy. Nat Rev Cancer 9:338–350PubMedCrossRefGoogle Scholar
  33. 33.
    Palchaudhuri R, Hergenrother PJ (2007) DNA as a target for anticancer compounds: methods to determine the mode of binding and the mechanism of action. Curr Opin Biotechnol 18:497–503PubMedCrossRefGoogle Scholar
  34. 34.
    Pommier Y (2006) Topoisomerase I inhibitors: camptothecins and beyond. Nat Rev Cancer 6:789–802PubMedCrossRefGoogle Scholar
  35. 35.
    Hande KR (1998) Clinical applications of anticancer drugs targeted to topoisomerase II. Biochim Biophys Acta 1400:173–184PubMedGoogle Scholar
  36. 36.
    Bolognese A, Correale G, Manfra M, Esposito A, Novellino E, Lavecchia A (2008) Antitumor agents 6. Synthesis, structure-activity relationships, and biological evaluation of spiro[imidazolidine-4, 3′-thieno[2, 3-g]quinoline]-tetraones and spiro[thieno[2, 3-g]quinoline-3, 5′-[1, 2, 4]triazinane]-tetraones with potent antiproliferative activity. J Med Chem 51:8148–8157PubMedCrossRefGoogle Scholar
  37. 37.
    Suzuki K, Yahara S, Kido Y, Nagao K, Hatano Y, Uyeda M (1998) Topostatin, a novel inhibitor of topoisomerases I and II produced by Thermomonospora alba strain No. 1520. II. Physico-chemical properties and structure elucidation. J Antibiot (Tokyo) 51:999–1003Google Scholar
  38. 38.
    Yoshinari T, Yamada A, Uemura D et al (1993) Induction of topoisomerase I-mediated DNA cleavage by a new indolocarbazole, ED-110. Cancer Res 53:490–494PubMedGoogle Scholar
  39. 39.
    Chene P, Rudloff J, Schoepfer J et al (2009) Catalytic inhibition of topoisomerase II by a novel rationally designed ATP-competitive purine analogue. BMC Chem Biol 9:1PubMedCrossRefGoogle Scholar
  40. 40.
    Meng LH, Zhang JS, Ding J (2001) Salvicine, a novel DNA topoisomerase II inhibitor, exerting its effects by trapping enzyme-DNA cleavage complexes. Biochem Pharmacol 62:733–741PubMedCrossRefGoogle Scholar
  41. 41.
    Park HJ, Lee HJ, Lee EJ et al (2003) Cytotoxicity and DNA topoisomerase inhibitory activity of benz[f]indole-4, 9-dione analogs. Biosci Biotechnol Biochem 67:1944–1949PubMedCrossRefGoogle Scholar
  42. 42.
    Tzu-Hao W, Hsin-Shih W, Yung-Kwei S (2000) Paclitaxel-induced cell death. Cancer 88:2619–2628CrossRefGoogle Scholar
  43. 43.
    Jordan P, Carmo-Fonseca M (2000) Molecular mechanisms involved in cisplatin cytotoxicity. Cell Mol Life Sci 57:1229–1235PubMedCrossRefGoogle Scholar
  44. 44.
    Tsao YP, D’Arpa P, Liu LF (1992) The involvement of active DNA synthesis in camptothecin-induced G2 arrest: altered regulation of p34cdc2/cyclin B. Cancer Res 52:1823–1829PubMedGoogle Scholar
  45. 45.
    Hsiang YH, Hertzberg R, Hecht S, Liu LF (1985) Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem 260:14873–14878PubMedGoogle Scholar
  46. 46.
    Ling YH, el-Naggar AK, Priebe W, Perez-Soler R (1996) Cell cycle-dependent cytotoxicity, G2/M phase arrest, and disruption of p34cdc2/cyclin B1 activity induced by doxorubicin in synchronized P388 cells. Mol Pharmacol 49:832–841PubMedGoogle Scholar
  47. 47.
    Kletsas D, Barbieri D, Stathakos D et al (1998) The highly reducing sugar 2-deoxy-D-ribose induces apoptosis in human fibroblasts by reduced glutathione depletion and cytoskeletal disruption. Biochem Biophys Res Commun 243:416–425PubMedCrossRefGoogle Scholar
  48. 48.
    Sheikh MS, Garcia M, Zhan Q, Liu Y, Fornace AJ Jr (1996) Cell cycle-independent regulation of p21Waf1/Cip1 and retinoblastoma protein during okadaic acid-induced apoptosis is coupled with induction of Bax protein in human breast carcinoma cells. Cell Growth Differ 7:1599–1607PubMedGoogle Scholar
  49. 49.
    Durrant D, Richards JE, Walker WT, Baker KA, Simoni D, Lee RM (2008) Mechanism of cell death induced by cis-3, 4′, 5-trimethoxy-3′-aminostilbene in ovarian cancer. Gynecol Oncol 110:110–117PubMedCrossRefGoogle Scholar
  50. 50.
    Criddle DN, Gillies S, Baumgartner-Wilson HK et al (2006) Menadione-induced reactive oxygen species generation via redox cycling promotes apoptosis of murine pancreatic acinar cells. J Biol Chem 281:40485–40492PubMedCrossRefGoogle Scholar
  51. 51.
    Kirshner JR, He S, Balasubramanyam V et al (2008) Elesclomol induces cancer cell apoptosis through oxidative stress. Mol Cancer Ther 7:2319–2327PubMedCrossRefGoogle Scholar
  52. 52.
    Place AE, Suh N, Williams CR et al (2003) The novel synthetic triterpenoid, CDDO-imidazolide, inhibits inflammatory response and tumor growth in vivo. Clin Cancer Res 9:2798–2806PubMedGoogle Scholar
  53. 53.
    Cervantes A, Pinedo HM, Lankelma J, Schuurhuis GJ (1988) The role of oxygen-derived free radicals in the cytotoxicity of doxorubicin in multidrug resistant and sensitive human ovarian cancer cells. Cancer Lett 41:169–177PubMedCrossRefGoogle Scholar
  54. 54.
    Cathcart R, Schwiers E, Ames BN (1983) Detection of picomole levels of hydroperoxides using a fluorescent dichlorofluorescein assay. Anal Biochem 134:111–116PubMedCrossRefGoogle Scholar
  55. 55.
    Lamb J (2007) The connectivity map: a new tool for biomedical research. Nat Rev Cancer 7:54–60PubMedCrossRefGoogle Scholar
  56. 56.
    Hieronymus H, Lamb J, Ross KN et al (2006) Gene expression signature-based chemical genomic prediction identifies a novel class of HSP90 pathway modulators. Cancer Cell 10:321–330PubMedCrossRefGoogle Scholar
  57. 57.
    Lamb J, Crawford ED, Peck D et al (2006) The connectivity map: using gene-expression signatures to connect small molecules, genes, and disease. Science 313:1929–1935PubMedCrossRefGoogle Scholar
  58. 58.
    Shibahara S (2003) The heme oxygenase dilemma in cellular homeostasis: new insights for the feedback regulation of heme catabolism. Tohoku J Exp Med 200:167–186PubMedCrossRefGoogle Scholar
  59. 59.
    Pietsch EC, Chan JY, Torti FM, Torti SV (2003) Nrf2 mediates the induction of ferritin H in response to xenobiotics and cancer chemopreventive dithiolethiones. J Biol Chem 278:2361–2369PubMedCrossRefGoogle Scholar
  60. 60.
    Ferris CD, Jaffrey SR, Sawa A et al (1999) Haem oxygenase-1 prevents cell death by regulating cellular iron. Nat Cell Biol 1:152–157PubMedCrossRefGoogle Scholar
  61. 61.
    Nagai T, Kikuchi S, Ohmine K et al (2008) Hemin reduces cellular sensitivity to imatinib and anthracyclins via Nrf2. J Cell Biochem 104:680–691PubMedCrossRefGoogle Scholar
  62. 62.
    Brown JM (1993) SR 4233 (tirapazamine): a new anticancer drug exploiting hypoxia in solid tumours. Br J Cancer 67:1163–1170PubMedCrossRefGoogle Scholar
  63. 63.
    Marcu L, Olver I (2006) Tirapazamine: from bench to clinical trials. Curr Clin Pharmacol 1:71–79PubMedCrossRefGoogle Scholar
  64. 64.
    Zafarullah M, Li WQ, Sylvester J, Ahmad M (2003) Molecular mechanisms of N-acetylcysteine actions. Cell Mol Life Sci 60:6–20PubMedCrossRefGoogle Scholar
  65. 65.
    Matsunaga, T, Tsuji, Y, Kaai, K, et al (2010) Toxicity against gastric cancer cells by combined treatment with 5-fluorouracil and mitomycin c: implication in oxidative stress. Cancer Chemother Pharmacol. doi:10.1007/s00280-009-1292-5
  66. 66.
    Laux I, Nel A (2001) Evidence that oxidative stress-induced apoptosis by menadione involves Fas-dependent and Fas-independent pathways. Clin Immunol 101:335–344PubMedCrossRefGoogle Scholar
  67. 67.
    Kang YH, Yi MJ, Kim MJ et al (2004) Caspase-independent cell death by arsenic trioxide in human cervical cancer cells: reactive oxygen species-mediated poly(ADP-ribose) polymerase-1 activation signals apoptosis-inducing factor release from mitochondria. Cancer Res 64:8960–8967PubMedCrossRefGoogle Scholar
  68. 68.
    Lee CS, Park SY, Ko HH, Han ES (2004) Effect of change in cellular GSH levels on mitochondrial damage and cell viability loss due to mitomycin c in small cell lung cancer cells. Biochem Pharmacol 68:1857–1867PubMedCrossRefGoogle Scholar
  69. 69.
    Siu WY, Yam CH, Poon RYC (1999) G1 versus G2 cell cycle arrest after adriamycin-induced damage in mouse Swiss3T3 cells. FEBS Lett 461:299–305PubMedCrossRefGoogle Scholar
  70. 70.
    Matzno S, Yamaguchi Y, Akiyoshi T, Nakabayashi T, Matsuyama K (2008) An attempt to evaluate the effect of vitamin K3 using as an enhancer of anticancer agents. Biol Pharm Bull 31:1270–1273PubMedCrossRefGoogle Scholar
  71. 71.
    Emil Mladenov ITBA (2007) Activation of the S phase DNA damage checkpoint by mitomycin C. J Cell Physiol 211:468–476PubMedCrossRefGoogle Scholar
  72. 72.
    Trachootham D, Alexandre J, Huang P (2009) Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat Rev Drug Discov 8:579–591PubMedCrossRefGoogle Scholar
  73. 73.
    Berkenblit A, Eder JP Jr, Ryan DP et al (2007) Phase I clinical trial of STA-4783 in combination with paclitaxel in patients with refractory solid tumors. Clin Cancer Res 13:584–590PubMedCrossRefGoogle Scholar
  74. 74.
    Tuma RS (2008) Reactive oxygen species may have antitumor activity in metastatic melanoma. J Natl Cancer Inst 100:11–12PubMedCrossRefGoogle Scholar
  75. 75.
    Hauschild A, Eggermont AM, Jacobson E, O’Day SJ (2009) Phase III, randomized, double-blind study of elesclomol and paclitaxel versus paclitaxel alone in stage IV metastatic melanoma (MM). J Clin Oncol (Meeting Abstracts) 27:LBA9012Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Mirth T. Hoyt
    • 1
  • Rahul Palchaudhuri
    • 1
  • Paul J. Hergenrother
    • 1
  1. 1.Department of Chemistry, Roger Adams LaboratoryUniversity of IllinoisUrbanaUSA

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