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Investigational New Drugs

, Volume 30, Issue 2, pp 450–467 | Cite as

Putative mechanisms of antitumor activity of cyano-substituted heteroaryles in HeLa cells

  • Katja Ester
  • Fran Supek
  • Kristina Majsec
  • Marko Marjanović
  • David Lembo
  • Manuela Donalisio
  • Tomislav Šmuc
  • Ivana Jarak
  • Grace Karminski-Zamola
  • Marijeta Kralj
PRECLINICAL STUDIES

Summary

Six recently synthesized cyano-substituted heteroaryles, which do not bind to DNA but are highly cytotoxic against the human tumor cell line HeLa, were analyzed for their antitumor mechanisms of action (MOA). They did not interfere with the expression of human papillomavirus oncogenes integrated in the HeLa cell genome, but they did induce strong G1 arrest and result in the activation of caspase-3 and apoptosis. A computational analysis was performed that compared the antiproliferative activities of our compounds in 13 different tumor cell lines with those of compounds listed in the National Cancer Institute database. The results indicate that interference with cytoskeletal function and inhibition of mitosis are the likely antitumor MOA. Furthermore, a second in silico investigation revealed that the tumor cells that are sensitive to the cyano-substituted compounds show differences in their expression of locomotion genes compared with that of insensitive cell lines, thus corroborating the involvement of the cytoskeleton. This MOA was also confirmed experimentally: the cyano-substituted heteroaryles disrupted the actin and the tubulin networks in HeLa cells and inhibited cellular migration. However, further analysis indicated that multiple MOA may exist that depend on the position of the cyano-group; while cyano-substituted naphthiophene reduced the expression of cytoskeletal proteins, cyano-substituted thieno-thiophene-carboxanilide inhibited the formation of cellular reactive oxygen species.

Keywords

Cyano-substituted heteroaryles HeLa cells Random forest Apoptosis Cytoskeleton disruption Antioxidants 

Notes

Acknowledgments

We thank Dr. Jaganjac for assistance with measurements of intracellular ROS, and to Dr. Čimbora-Zovko who kindly provided antibodies for cytoskeletal-study. We also thank to Dr. Ragland for helpful comments.

This study was supported by grants from the Ministry of Science, Education and Sports of the Republic of Croatia to MK (098-0982464-2514) and to TŠ (098-0000000-3168) and from Ministero dell’Istruzione, dell’Università e della Ricerca, Italy (PRIN 2008) to DL.

References

  1. 1.
    Caleta I, Kralj M, Marjanovic M, Bertosa B, Tomic S, Pavlovic G, Pavelic K, Karminski-Zamola G (2009) Novel cyano- and amidinobenzothiazole derivatives: synthesis, antitumor evaluation, and X-ray and quantitative structure-activity relationship (QSAR) analysis. J Med Chem 52:1744–1756PubMedCrossRefGoogle Scholar
  2. 2.
    Hranjec M, Piantanida I, Kralj M, Suman L, Pavelic K, Karminski-Zamola G (2008) Novel amidino-substituted thienyl- and furylvinylbenzimidazole: derivatives and their photochemical conversion into corresponding diazacyclopenta[c]fluorenes. synthesis, interactions with DNA and RNA, and antitumor evaluation. 4. J Med Chem 51:4899–4910PubMedCrossRefGoogle Scholar
  3. 3.
    Kemnitzer W, Kuemmerle J, Jiang S, Sirisoma N, Kasibhatla S, Crogan-Grundy C, Tseng B, Drewe J, Cai SX (2009) Discovery of 1-benzoyl-3-cyanopyrrolo[1, 2-a]quinolines as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. 2: structure-activity relationships of the 4-, 5-, 6-, 7- and 8-positions. Bioorg Med Chem Lett 19:3481–3484PubMedCrossRefGoogle Scholar
  4. 4.
    Ester K, Hranjec M, Piantanida I, Caleta I, Jarak I, Pavelic K, Kralj M, Karminski-Zamola G (2009) Novel derivatives of pyridylbenzo[b]thiophene-2-carboxamides and benzo[b]thieno[2, 3-c]naphthyridin-2-ones: minor structural variations provoke major differences of antitumor action mechanisms. J Med Chem 52:2482–2492PubMedCrossRefGoogle Scholar
  5. 5.
    Jarak I, Kralj M, Suman L, Pavlovic G, Dogan J, Piantanida I, Zinic M, Pavelic K, Karminski-Zamola G (2005) Novel cyano- and N-isopropylamidino-substituted derivatives of benzo[b]thiophene-2-carboxanilides and benzo[b]thieno[2, 3-c]quinolones: synthesis, photochemical synthesis, crystal structure determination, and antitumor evaluation. 2. J Med Chem 48:2346–2360PubMedCrossRefGoogle Scholar
  6. 6.
    Jarak I, Kralj M, Piantanida I, Suman L, Zinić M, Pavelic K, Karminski-Zamola G (2006) Novel cyano- and amidino-substituted derivatives of thieno[2, 3-b]- and thieno[3, 2-b]thiophene-2-carboxanilides and thieno[3′, 2′:4, 5]thieno- and thieno[2′, 3′:4, 5]thieno [2, 3-c]quinolones: synthesis, photochemical synthesis, DNA binding, and antitumor evaluation. Bioorg Med Chem 14:2859–2868PubMedCrossRefGoogle Scholar
  7. 7.
    Starcević K, Kralj M, Piantanida I, Suman L, Pavelić K, Karminski-Zamola G (2006) Synthesis, photochemical synthesis, DNA binding and antitumor evaluation of novel cyano- and amidino-substituted derivatives of naphtho-furans, naphtho-thiophenes, thieno-benzofurans, benzo-dithiophenes and their acyclic precursors. Eur J Med Chem 41:925–939PubMedCrossRefGoogle Scholar
  8. 8.
    Milazzo S, Lejeune S, Ernst E (2007) Laetrile for cancer: a systematic review of the clinical evidence. Support Care Cancer 15:583–595PubMedCrossRefGoogle Scholar
  9. 9.
    Park HJ, Yoon SH, Han LS, Zheng LT, Jung KH, Uhm YK, Lee JH, Jeong JS, Joo WS, Yim SV, Chung JH, Hong SP (2005) Amygdalin inhibits genes related to cell cycle in SNU-C4 human colon cancer cells. World J Gastroenterol 11:5156–5161PubMedGoogle Scholar
  10. 10.
    Chang HK, Shin MS, Yang HY, Lee JW, Kim YS, Lee MH, Kim J, Kim KH, Kim CJ (2006) Amygdalin induces apoptosis through regulation of Bax and Bcl-2 expressions in human DU145 and LNCaP prostate cancer cells. Biol Pharm Bull 29:1597–1602PubMedCrossRefGoogle Scholar
  11. 11.
    Kemnitzer W, Kuemmerle J, Jiang S, Zhang HZ, Sirisoma N, Kasibhatla S, Crogan-Grundy C, Tseng B, Drewe J, Cai SX (2008) Discovery of 1-benzoyl-3-cyanopyrrolo[1, 2-a]quinolines as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. Part 1: structure-activity relationships of the 1- and 3-positions. Bioorg Med Chem Lett 18:6259–64PubMedCrossRefGoogle Scholar
  12. 12.
    Kemnitzer W, Jiang S, Wang Y, Kasibhatla S, Crogan-Grundy C, Bubenik M, Labrecque D, Denis R, Lamothe S, Attardo G, Gourdeau H, Tseng B, Drewe J, Cai SX (2008) Discovery of 4-aryl-4H-chromenes as a new series of apoptosis inducers using a cell- and caspase-based HTS assay. Part 5: modifications of the 2- and 3-positions. Bioorg Med Chem Lett 18(2):603–607PubMedCrossRefGoogle Scholar
  13. 13.
    Saczewski F, Reszka P, Gdaniec M, Grünert R, Bednarski PJ (2004) Synthesis, X-ray crystal structures, stabilities, and in vitro cytotoxic activities of new heteroarylacrylonitriles. J Med Chem 47:3438–3449PubMedCrossRefGoogle Scholar
  14. 14.
    Saczewski F, Stencel A, Bieńczak AM, Langowska KA, Michaelis M, Werel W, Hałasa R, Reszka P, Bednarski PJ (2008) Structure-activity relationships of novel heteroaryl-acrylonitriles as cytotoxic and antibacterial agents. Eur J Med Chem 43:1847–1857PubMedCrossRefGoogle Scholar
  15. 15.
    Hranjec M, Pavlovic G, Marjanovic M, Kralj M, Karminski-Zamola G (2010) Benzimidazole derivatives related to 2, 3-acrylonitriles, benzimidazo[1, 2-a]quinolines and fluorenes: synthesis, antitumor evaluation in vitro and crystal structure determination. Eur J Med Chem 45:2405–2417PubMedCrossRefGoogle Scholar
  16. 16.
    Supek F, Kralj M, Marjanovic M, Suman L, Smuc T, Krizmanic I, Zinic B (2008) Atypical cytostatic mechanism of N-1-sulfonylcytosine derivatives determined by in vitro screening and computational analysis. Invest New Drugs 26:97–110PubMedCrossRefGoogle Scholar
  17. 17.
    Breiman L (2001) Random forests. Mach Learn 45:5–32CrossRefGoogle Scholar
  18. 18.
    Díaz-Uriarte R, Alvarez de Andrés S (2006) Gene selection and classification of microarray data using random forest. BMC Bioinform 7:3CrossRefGoogle Scholar
  19. 19.
    Strobl C, Boulesteix AL, Kneib T, Augustin T, Zeileis A (2008) Conditional variable importance for random forests. BMC Bioinform 9:307CrossRefGoogle Scholar
  20. 20.
    Segal E, Friedman N, Koller D, Regev A (2004) A module map showing conditional activity of expression modules in cancer. Nat Genet 36:1090–1098PubMedCrossRefGoogle Scholar
  21. 21.
    Rubin E (2006) List mania: interpreting microarray results with the L2L server. Brief Bioinform 7:121–122CrossRefGoogle Scholar
  22. 22.
    Aldridge GM, Podrebarac DM, Greenough WT, Weiler IJ (2008) The use of total protein stains as loading controls: an alternative to high-abundance single-protein controls in semi-quantitative immunoblotting. J Neurosci Methods 172:250–254PubMedCrossRefGoogle Scholar
  23. 23.
    Rosenkranz AR, Schmaldienst S, Stuhlmeier KM, Chen W, Knapp W, Zlabinger GJ (1992) A microplate assay for the detection of oxidative products using 2′, 7′-dichlorofluorescin-diacetate. J Immunol Methods 156:39–45PubMedCrossRefGoogle Scholar
  24. 24.
    Lacroix M (2008) Persistent use of “false” cell lines. Int J Cancer 122:1–4PubMedCrossRefGoogle Scholar
  25. 25.
    Joseph JP, Grierson I, Hitchings RA (1989) Taxol, cytochalasin B and colchicine effects on fibroblast migration and contraction: a role in glaucoma filtration surgery? Curr Eye Res 8:203–215PubMedCrossRefGoogle Scholar
  26. 26.
    DeFilippis RA, Goodwin EC, Wu L, DiMaio D (2003) Endogenous human papillomavirus E6 and E7 proteins differentially regulate proliferation, senescence, and apoptosis in HeLa cervical carcinoma cells. J Virol 77:1551–1163PubMedCrossRefGoogle Scholar
  27. 27.
    Pontano LL, Diehl JA (2009) DNA damage-dependent cyclin D1 proteolysis: GSK3beta holds the smoking gun. Cell Cycle 8:824–827PubMedCrossRefGoogle Scholar
  28. 28.
    Yazbeck VY, Buglio D, Georgakis GV, Li Y, Iwado E, Romaguera JE, Kondo S, Younes A (2008) Temsirolimus downregulates p21 without altering cyclin D1 expression and induces autophagy and synergizes with vorinostat in mantle cell lymphoma. Exp Hematol 36:443–450PubMedCrossRefGoogle Scholar
  29. 29.
    Jin YH, Yoo KJ, Lee YH, Lee SK (2000) Caspase 3-mediated cleavage of p21WAF1/CIP1 associated with the cyclin A-cyclin-dependent kinase 2 complex is a prerequisite for apoptosis in SK-HEP-1 cells. J Biol Chem 275:30256–30263PubMedCrossRefGoogle Scholar
  30. 30.
    Porter AG, Jänicke RU (1999) Emerging roles of caspase-3 in apoptosis. Cell Death Differ 6:99–104PubMedCrossRefGoogle Scholar
  31. 31.
    Soldani C, Scovassi AI (2002) Poly(ADP-ribose) polymerase-1 cleavage during apoptosis: an update. Apoptosis 7:321–328PubMedCrossRefGoogle Scholar
  32. 32.
    Friedl P, Wolf K (2003) Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer 3:362–374PubMedCrossRefGoogle Scholar
  33. 33.
    Hall A (2009) The cytoskeleton and cancer. Cancer Metastasis Rev 28:5–14PubMedCrossRefGoogle Scholar
  34. 34.
    Mani SA, Yang J, Brooks M, Schwaninger G, Zhou A, Miura N, Kutok JL, Hartwell K, Richardson AL, Weinberg RA (2007) Mesenchyme Forkhead 1 (FOXC2) plays a key role in metastasis and is associated with aggressive basal-like breast cancers. Proc Natl Acad Sci USA 104:10069–10074PubMedCrossRefGoogle Scholar
  35. 35.
    Guarino M, Rubino B, Ballabio G (2007) The role of epithelial-mesenchymal transition in cancer pathology. Pathology 39:305–318PubMedCrossRefGoogle Scholar
  36. 36.
    Humbert PO, Grzeschik NA, Brumby AM, Galea R, Elsum I, Richardson HE (2008) Control of tumourigenesis by the Scribble/Dlg/Lgl polarity module. Oncogene 27:6888–6907PubMedCrossRefGoogle Scholar
  37. 37.
    Thomas M, Narayan N, Pim D, Tomaić V, Massimi P, Nagasaka K, Kranjec C, Gammoh N, Banks L (2008) Human papillomaviruses, cervical cancer and cell polarity. Oncogene 27:7018–7030PubMedCrossRefGoogle Scholar
  38. 38.
    Circu ML, Aw TY (2010) Reactive oxygen species, cellular redox systems, and apoptosis. Free Radic Biol Med 48:749–762PubMedCrossRefGoogle Scholar
  39. 39.
    Jang JH, Lee TJ, Yang ES, Min DS, Kim YH, Kim SH, Choi YH, Park JW, Choi KS, Kwon TK (2010) Compound C sensitizes Caki renal cancer cells to TRAIL-induced apoptosis through reactive oxygen species-mediated down-regulation of c-FLIP(L) and Mcl-1. Exp Cell Res 316:2194–2203PubMedCrossRefGoogle Scholar
  40. 40.
    Foti MC, Sharma SK, Shakya G, Prasad AK, Nicolosi G, Bovicelli P, Ghosh B, Raj HG, Rastogi RC, Parmar VS (2005) Biopolyphenolics as antioxidants: studies under an Indo-Italian CSIR-CNR project. Pure Appl Chem 77:91–101CrossRefGoogle Scholar
  41. 41.
    Pinto-Basto D, Silva JP, Queiroz MJ, Moreno AJ, Coutinho OP (2009) Antioxidant activity of synthetic diarylamines: a mitochondrial and cellular approach. Mitochondrion 9:17–26PubMedCrossRefGoogle Scholar
  42. 42.
    Ferreira IC, Queiroz MJ, Vilas-Boas M, Estevinho LM, Begouinb A, Kirsch G (2006) Evaluation of the antioxidant properties of diarylamines in the benzo[b]thiophene series by free radical scavenging activity and reducing power. Bioorg Med Chem Lett 16:1384–1387PubMedCrossRefGoogle Scholar
  43. 43.
    Brash DE, Havre PA (2002) New careers for antioxidants. Proc Natl Acad Sci USA 99:13969–13971PubMedCrossRefGoogle Scholar
  44. 44.
    Havre PA, O’Reilly S, McCormick J, Brash DE (2002) Transformed and tumor-derived human cells exhibit preferential sensitivity to the thiol antioxidants, N-Acetyl cysteine and penicillamine. Cancer Res 62:1443–1449PubMedGoogle Scholar
  45. 45.
    Mukhtar H, Ahmad N (1999) Green tea in chemoprevention of cancer. Toxicol Sci 52:111–117PubMedGoogle Scholar
  46. 46.
    Leavesley HB, Li L, Prabhakaran K, Borowitz JL, Isom GE (2008) Interaction of cyanide and nitric oxide with cytochrome c oxidase: implications for acute cyanide toxicity. Toxicol Sci 101:101–111PubMedCrossRefGoogle Scholar
  47. 47.
    Li M, Wang A-J, Xu J-X (2008) Redox state of cytochrome c regulates cellular ROS and caspase cascade in permeablized cell model. Protein Pept Lett 15:200–205PubMedCrossRefGoogle Scholar
  48. 48.
    Sabelli R, Iorio E, De Martino A, Podo F, Ricci A (2008) Rhodanese–thioredoxin system and allyl sulfur compounds: implications in apoptosis induction. FEBS J 275:3884–3899PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Katja Ester
    • 1
  • Fran Supek
    • 2
  • Kristina Majsec
    • 1
  • Marko Marjanović
    • 1
  • David Lembo
    • 3
  • Manuela Donalisio
    • 3
  • Tomislav Šmuc
    • 2
  • Ivana Jarak
    • 4
  • Grace Karminski-Zamola
    • 4
  • Marijeta Kralj
    • 1
  1. 1.Division of Molecular MedicineRuđer Bošković InstituteZagrebCroatia
  2. 2.Division of ElectronicsRuđer Bošković InstituteZagrebCroatia
  3. 3.Department of Clinical and Biological Sciences, S. Luigi Gonzaga Medical SchoolUniversity of TurinTurinItaly
  4. 4.Department of Organic Chemistry, Faculty of Chemical Engineering and TechnologyUniversity of ZagrebZagrebCroatia

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