Lipophilic aroylhydrazone chelator HNTMB and its multiple effects on ovarian cancer cells

BMC Cancer

Volumn 10, Issue , 2010, Pages

  Kim, Kyu K ^a   Lange, Thilo S ^a,b   Singh, Rakesh K ^a   Brard, Laurent ^a  

^a BROWN UNIVERSITY   (United States)

^b BROWN UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

2 HYDROXY 1 NAPHTHYLALDEHYDE 2 THIOPHENECARBOXYHYDRAZONE; 2 HYDROXY 1 NAPHTHYLALDEHYDE 3,4,5 TRIMETHOXYBENZOYLHYDRAZONE; 2 HYDROXY 1 NAPHTHYLALDEHYDE 4 T BUTYLBENZOYLHYDRAZONE; 2 HYDROXY 1 NAPHTHYLALDEHYDE ISONICOTINOYLHYDRAZONE; 3 METHOXYSALICYLALDEHYDE 4 T BUTYLBENZOYLHYDRAZONE; ANTINEOPLASTIC AGENT; CHELATING AGENT; DEFEROXAMINE; REACTIVE OXYGEN METABOLITE; UNCLASSIFIED DRUG; COPPER; HYDRAZONE DERIVATIVE; IRON DERIVATIVE;

APOPTOSIS; ARTICLE; CANCER CELL CULTURE; CANCER INHIBITION; CELL CYCLE ARREST; CELL CYCLE G2 PHASE; CELL CYCLE M PHASE; CELL CYCLE REGULATION; CELL VIABILITY; CHROMATIN ASSEMBLY AND DISASSEMBLY; COMPLEX FORMATION; CONTROLLED STUDY; DRUG CYTOTOXICITY; DRUG STRUCTURE; FEMALE; FLUORESCENCE ACTIVATED CELL SORTING; FLUORESCENCE MICROSCOPY; HUMAN; HUMAN CELL; IC 50; LIPOPHILICITY; NICK END LABELING; OVARY CANCER; OXIDATION; SIGNAL TRANSDUCTION; WESTERN BLOTTING; ADENOCARCINOMA; ANIMAL; CELL DIVISION; CELL GROWTH; DNA FRAGMENTATION; DRUG EFFECT; DRUG SCREENING; METABOLISM; OVARY TUMOR; PATHOLOGY; RAT; TUMOR CELL LINE;

ADENOCARCINOMA; ANIMALS; APOPTOSIS; CELL DIVISION; CELL GROWTH PROCESSES; CELL LINE, TUMOR; CHELATING AGENTS; COPPER; DNA FRAGMENTATION; DRUG SCREENING ASSAYS, ANTITUMOR; FEMALE; G2 PHASE; HUMANS; HYDRAZONES; IRON COMPOUNDS; OVARIAN NEOPLASMS; RATS; REACTIVE OXYGEN SPECIES;

EID: 77949911259     PISSN: None     EISSN: 14712407     Source Type: Journal    
DOI: 10.1186/1471-2407-10-72     Document Type: Article

References (82)

1. Heintz APM, Odicino F, Maisonneuve P, Beller U, Benedet JL, Creasman WT. International federation of gynecology and obstetrics 25th annual report. carcinoma of the ovary. Int J Gyn Obst 2003, 83:135-137.
2. American Cancer Society. Cancer Facts and Figures 2008. , http://www.cancer.org
3. McGuire WP, Hoskins WJ, Brady MF, Kucera PR, Partridge EE, Look KY, Clarke-Pearson DL, Davidson M. Cyclophosphamide and cisplatin compared with paclitaxel and cisplatin in patients with stage III and stage IV ovarian cancer. N Engl J Med 1996, 334:1-6. 10.1056/NEJM199601043340101, 7494563.
4. Piccart MJ, Bertelsen K, James K, Cassidy J, Mangioni C, Simonsen E, Stuart G, Kaye S, Vergote I, Blom R, Grimshaw R, Atkinson RJ, Swenerton KD, Trope C, Nardi M, Kaern J, Tumolo S, Timmers P, Roy JA, Lhoas F, Lindvall B, Bacon M, Birt A, Andersen JE, Zee B, Paul J, Baron B, Pecorelli S. Randomized intergroup trial of cisplatin-paclitaxel versus cisplatin-cyclophosphamide in women with advanced epithelial ovarian cancer: three-year results. J Natl Cancer Inst 2000, 92:699-708. 10.1093/jnci/92.9.699, 10793106.
5. Leitao MM, Hummer A, Dizon DS, Aghajanian C, Hensley M, Sabbatini P, Venkatraman E, Spriggs DR. Platinum-retreatment of platinum-resistant ovarian cancer after nonplatinum therapy. Gynecol Oncol 2003, 91:123-129. 10.1016/S0090-8258(03)00464-5, 14529671.
6. Lamberth E, Gregory WM, Nelstrop AE, Rustin GJ. Long-term survival in 463 women treated with platinum analogs for advanced epithelial carcinoma of the ovary: life expectancy compared to women of an age-matched normal population. Int J Gynecol Cancer 2004, 14:772-778. 10.1111/j.1048-891X.2004.014507.x, 15361183.
7. Kontoghiorghes GJ, Efstathiou A, Ioannou Loucaides S, Kolnagou A. Chelators controlling metal metabolism and toxicity pathways: applications in cancer prevention, diagnosis and treatment. Hemoglobin 2008, 32:217-227. 10.1080/03630260701727119, 18274999.
8. Chan A, Wong F, Arumanayagam M. Serum ultrafiltrable copper, total copper and ceruloplasmin concentrations in gynecological carcinoma. Ann Clin Biochem 1993, 30:545-549.
9. Kuo KW, Chen SF, Wu CC, Chen DR, Lee JH. Serum and tissue trace elements in patients with breast cancer in Taiwan. Biol Trace Elem Res 2002, 89:1-11. 10.1385/BTER:89:1:1, 12413046.
10. Yaman M, Kaya G, Simsek M. Comparison of trace element concentrations in cancerous and non cancerous human endometrial and ovary tissues. Int J Gynecol Cancer 2007, 17:220-228. 10.1111/j.1525-1438.2006.00742.x, 17291257.
11. Daniel KG, Chen D, Orlu S, Cui QC, Miller FR, Dou QP. Clioquinol and pyrrolidine dithiocarbamate complex with copper to form proteasome inhibitors and apoptosis inducers in human breast cancer cells. Breast Cancer Res 2005, 7:R897-R908. 10.1186/bcr1322, 1410741, 16280039.
12. Gupte A, Mumper RJ. Copper chelation by D-penicillamine generates reactive oxygen species that are cytotoxic to human leukemia and breast cancer cells. Free Radic Biol Med 2007, 43:1271-1278. 10.1016/j.freeradbiomed.2007.07.003, 17893040.
13. Linder MC, Hazegh-Azam M. Copper biochemistry and molecular biology. Am J Clin Nutrition 1996, 63:797S-811S.
14. Yu Y, Wong J, Lovejoy DB, Kalinowski DS, Richardson DR. Chelators at the cancer coalface: desferrioxamine to Triapine and beyond. Clin Cancer Res 2006, 12:6876-6883. 10.1158/1078-0432.CCR-06-1954, 17145804.
15. Gomes A, Fernandes E, Lima JLFC. Fluorescence probes used for detection of reactive oxygen species. J Biochem Biophys Methods 2005, 65:45-80. 10.1016/j.jbbm.2005.10.003, 16297980.
16. Waris G, Ahsan HJ. Reactive oxygen species: role in the development of cancer and various chronic conditions. Carcinog 2006, 5:1-8.
17. Gupte A, Mumper RJ. Elevated copper and oxidative stress in cancer cells as a target for cancer treatment. Cancer Treat Rev 2009, 35:32-46. 10.1016/j.ctrv.2008.07.004, 18774652.
18. Hileman EO, Liu J, Albitar M, Keating MJ, Huang P. Intrinsic oxidative stress in cancer cells: a biochemical basis for therapeutic selectivity. Cancer Chemother Pharmacol 2004, 53:209-219. 10.1007/s00280-003-0726-5, 14610616.
19. Pelicano H, Carney D, Huang P. ROS stress in cancer cells and therapeutic implications. Drug Resistance Updat 2004, 7:97-110.
20. Brard L, Granai CO, Swamy N. Iron chelators deferoxamine and diethylenetriamine pentaacetic acid induce apoptosis in ovarian carcinoma. Gynecol Oncol 2006, 100:116-127. 10.1016/j.ygyno.2005.07.129, 16203029.
21. Neufeld EJ. Oral chelators deferasirox and deferiprone for transfusional iron overload in thalassemia major: new data, new questions. Blood 2006, 107:3436-3441. 10.1182/blood-2006-02-002394, 1895765, 16627763.
22. Turner J, Koumenis C, Kute TE, Planalp RP, Brechbiel MW, Beardsley D, Cody B, Brown KD, Torti FM, Torti S. Tachpyridine, a metal chelator, induces G2 cell cycle arrest, activates checkpoint kinases, and sensitizes cells to ionizing radiation. Blood 2005, 106:3191-3199. 10.1182/blood-2005-03-1263, 1895322, 16014567.
23. Liu MC, Lin TS, Sartorelli AC, Liu MC, Lin TS, Sartorelli AC. Synthesis and anti-tumor activity of amino derivatives of pyridine-2-carboxaldehyde thiosemicarbazone. J Med Chem 1992, 35:3672-3677. 10.1021/jm00098a012, 1433178.
24. Rakba N, Loyer P, Gilot D, Delcros JG, Glaise D, Baret P, Pierre JL, Brissot P, Lescoat G. Antiproliferative and apoptotic effects of O-Trensox, a new synthetic iron-chelator, on differentiated human hepatoma cell lines. Carcinogenesis 2000, 21:943-951. 10.1093/carcin/21.5.943, 10783316.
25. Crichton RR, Ward RJ. Iron metabolism-new perspectives in view. Biochemistry 1992, 31:11255-11264. 10.1021/bi00161a001, 1445865.
26. Huang X. Iron overload and its association with cancer risk in humans: evidence for iron as a carcinogenic metal. Mutation Res 2003, 533:153-171.
27. Toyokuni S. Iron-induced carcinogenesis: the role of redox regulation. Free Radical Biol Med 1996, 20:553-566.
28. Faulk WP, Hsi B, Stevens PJ. Transferrin and transferrin receptors in carcinoma of the breast. Lancet 1980, 23:390-392.
29. Seymour GJ, Walsh MD, Lavin MF, Strutton G, Gardiner RA. Transferrin receptor expression by human bladder transitional cell carcinomas. Urol Res 1987, 15:341-344. 10.1007/BF00265663, 3324443.
30. Fassl S, Leisser C, Huettenbrenner S, Maier S, Rosenberger G, Strasser S, Grusch M, Fuhrmann G, Leuhuber K, Polgar D, Stani J, Tichy B, Nowotny C, Krupitza G. Transferrin ensures survival of ovarian carcinoma cells when apoptosis is induced by TNFalpha, FasL, TRAIL, or Myc. Oncogene 2003, 22:8343-8355. 10.1038/sj.onc.1207047, 14614458.
31. Richardson DR, Kalinowski DS, Lau S, Jansson PJ, Lovejoy DB. Cancer cell iron metabolism and the development of potent iron chelators as anti-tumor agents. Biochim Biophys Acta 2009, 1790:702-717.
32. Melnyk P, Leroux V, Sergheraert C, Grellier P. Design, synthesis and in vitro antimalarial activity of an acylhydrazone library. Bioorg Med Chem Lett 2006, 16:31-35. 10.1016/j.bmcl.2005.09.058, 16263280.
33. Baker E, Richardson DR, Gross S, Ponka P. Evaluation of the iron chelation potential of hydrazones of pyridoxal, salicylaldehyde and 2-hydroxy-1-naphthylaldehyde using the hepatocyte in culture. Hepatology 1992, 15:492-501. 10.1002/hep.1840150323, 1544632.
34. Richardson DR, Tran EH, Ponka P. The potential of iron chelators of the pyridoxal isonicotinoyl hydrazone class as effective antiproliferative agents. Blood 1995, 86:4295-4306.
35. Richardson DR, Milnes K. The potential of iron-chelators of the pyridoxal isonicotinoyl hydrazone class as effective antiproliferative agents II: the mechanism of action of ligands derived from salicylaldehyde benzoyl hydrazone and 2-hydroxy-1-naphthylaldehyde benzoyl hydrazone. Blood 1997, 89:3025-3038.
36. Rose GS, Tocco LM, Grange GA, DiSaia PJ, Hamilton TC, Santin AD, Hiserodt JC. Development and characterization of a clinically useful animal model of epithelial ovarian cancer in the Fischer 344 rat. Am J Obstet Gynecol 1996, 175:593-599. 10.1053/ob.1996.v175.a73595, 8828419.
37. Malich G, Markovic B, Winder C. The sensitivity and specificity of the MTS tetrazolium assay for detecting the in vitro cytotoxicity of 20 chemicals using human cell lines. Toxicol 1997, 124:179-192.
38. Singh RK, Lange TS, Shaw SK, Kim KK, Brard L. A novel Indole Ethyl Isothiocyanate (7Me-IEITC) with anti-proliferative and pro-apoptotic effects on platinum-resistant human ovarian cancer cells. Gynecol Oncol 2008, 109:240-249. 10.1016/j.ygyno.2008.01.042, 2747483, 18329084.
39. Lange TS, Singh RK, Kim KK, Zou Y, Kalkunte SS, Sholler GL, Swamy N, Brard L. Anti-proliferative and pro-apoptotic properties of 3-bromoacetoxy calcidiol (B3CD) in high-risk neuroblastoma. Chem Biol Drug Design 2007, 70:302-310.
40. Gorczyca W, Gong J, Darzynkiewicz Z. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res 1993, 53:1945-1951.
41. Lange TS, Carolyn McCourt C, Singh RK, Kim KK, Singh AP, Luisi BS, Strongin RM, Brard L. Apoptotic and chemotherapeutic properties of iron(III) salophene in an ovarian cancer animal model. Drug Design, Development and Therapy 2008, 3:17-26.
42. Ott I, Gust R. Non platinum metal complexes as anti cancer drugs. Arch Pharm 2007, 340:117-126.
43. Huang R, Wallquist A, Covell DG. Anticancer metal compounds in NCI s tumor screening database: putative mode of action. Biochem Pharmacol 2005, 69:1009-1039. 10.1016/j.bcp.2005.01.001, 15763539.
44. Lange TS, Kim KK, Singh RK, Strongin RM, McCourt CK, Brard L. Iron(III)-salophene: An metallo-organic compound with selective cytotoxic and anti-proliferative properties in platinum-resistant ovarian cancer cells. PLOS One 2008, 3(e2303):1-10.
45. Richardson DR, Bernhardt PV. Crystal and molecular structure of 2 hydroxy 1 naphthaldehyde isonicotinoyl hydrazone (NIH) and its iron (III) complexes: an iron chelator with anti tumour activity. J Biol Inorg Chem 1999, 4:266-273. 10.1007/s007750050312, 10439071.
46. Johnson DK, Murphy TB, Rose NJ. Cytotoxic chelators and chelates 1. Inhibition of DNA synthesis in cultured rodent and human cells by AHCs and by a copper(II) complex of salicyladehyde benzoyl hydrazone. Inorganica Chim Acta 1982, 67:159-165.
47. Koha LL, Konb OL, Loha KW, Longa YC, Ranforda JD, Tanc ALC, Tjana YY. Complexes of salicylaldehyde acylhydrazones: Cytotoxicity, QSAR and crystal structure of the sterically hindered t butyl dimer. J Inorg Biochem 1998, 72:155-162. 10.1016/S0162-0134(98)10075-2, 10094614.
48. Haq RU, Wereley JP, Chitambar CR. Induction of apoptosis by iron deprivation in human leukemic CCRF-CEM cells. Exp Hematol 1995, 23:428-432.
49. Blastt J, Stitely S. Antineuroblastoma activity of desferrioxamine in human cell lines. Cancer Res 1987, 47:1749-1750.
50. Simonart T, Degraef C, Andrei G, Mosselmans R, Hermans P, Van Vooren JP, Noel JC, Boelaert JR, Snoeck R, Heenen M. Iron chelators inhibit the growth and induce the apoptosis of Kaposi's sarcoma cells and of their putative endothelial precursors. J Invest Dermatol 2000, 115:893-900. 10.1046/j.1523-1747.2000.00119.x, 11069629.
51. Hoke EM, Maylock CA, Shacter E. Desferal inhibits breast tumor growth and does not interfere with the tumoricidal activity of doxorubicin. Free Radic Biol Med 2005, 39:403-411. 10.1016/j.freeradbiomed.2005.03.029, 15993339.
52. Richardson DR, Ponka P, Baker E. The effect of the iron(III) chelator, desferrioxamine, on iron and transferrin uptake by the human malignant melanoma cell. Cancer Res 1994, 54:685-689.
53. Hann HW, Stahlhut MW, Hann CL. Effect of iron and desferoxamine on cell growth and in vitro ferritin synthesis in human hepatoma cell lines. Hepatology 1990, 11:566-569. 10.1002/hep.1840110407, 2158479.
54. Hoffbrand AV. Prospects for oral iron chelation therapy. J Lab Clin Med 1994, 123:492-494.
55. Whitnall M, Howard J, Ponka P, Richardson DR. A class of iron chelators with a wide spectrum of potent antitumor activity that overcomes resistance to chemotherapeutics. PNAS 2006, 103:14901-14906. 10.1073/pnas.0604979103, 1595448, 17003122.
56. Renschler MF. The emerging role of reactive oxygen species in cancer therapy. Eur J Cancer 2004, 40:1934-1940. 10.1016/j.ejca.2004.02.031, 15315800.
57. Mookerjee A, Basu JM, Majumder S, Chatterjee S, Panda GS, Dutta P, Pal S, Mukherjee P, Efferth T, Roy S, Choudhuri SK. A novel copper complex induces ROS generation in doxorubicin resistant Ehrlich ascitis carcinoma cells and increases activity of antioxidant enzymes in vital organs in vivo. BMC Cancer 2006, 6. article#267, 10.1186/1471-2407-6-267, 1660546, 17107616.
58. Gao J, Richardson DR. Multiple effects of iron chelators on molecules controlling cell cycle progression. J Transfusion Sci 2000, 23:247-248.
59. Gao J, Richardson DR. The potential of iron chelators of the pyridoxal isonicotinoyl hydrazone class as effective antiproliferative agents, IV: The mechanisms involved in inhibiting cell-cycle progression. Blood 2001, 98:842-850. 10.1182/blood.V98.3.842, 11468187.
60. Hoyes KP, Hider RC, Porter JB. Cell cycle synchronization and growth inhibition by 3 hydroxypyridin 4 one iron chelators in leukemia cell lines. Cancer Res 1992, 52:4591-4599.
61. Hartwell LH, Kastan MB. Cell-cycle control and cancer. Science 1994, 266:1821-1828. 10.1126/science.7997877, 7997877.
62. Gladden AB, Diehl JA. Cell cycle progression without cyclin E/CDK2: breaking down the walls of dogma. Cancer Cell 2003, 4:160-162. 10.1016/S1535-6108(03)00217-4, 14522248.
63. Shapiro GI, Harper JW. Anticancer drug targets: cell cycle and checkpoint control. J Clin Invest 1999, 104:1645-1653. 10.1172/JCI9054, 409893, 10606615.
64. Mazumder S, DuPree EL, Almasan A. A dual role of cyclin E in cell proliferation and apoptosis may provide a target for cancer therapy. Curr Cancer Drug Targets 2004, 4:65-75. 10.2174/1568009043481669, 1307511, 14965268.
65. Pawlik TM, Keyomarsi K. Role of cell cycle in mediating sensitivity to radiotherapy. Int J Radiat Oncol Biol Phys 2004, 59:928-942. 10.1016/j.ijrobp.2004.03.005, 15234026.
66. Salvesen GS, Abrams JM. Caspase activation stepping on the gas or releasing the brakes? Lessons from humans and flies. Oncogene 2004, 23:2774-27784. 10.1038/sj.onc.1207522, 15077141.
67. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science 1998, 281:1312-1316. 10.1126/science.281.5381.1312, 9721091.
68. Oliver FJ, de la Rubia G, Rolli V, Ruiz Ruiz MC, de Murcia G, Murcia JM. Importance of poly(ADP ribose) polymerase and its cleavage in apoptosis. Lesson from an uncleavable mutant. J Biol Chem 1998, 73:33533-33539.
69. Shaw TJ, Lacasse EC, Durkin JP, Vanderhyden BC. Downregulation of XIAP expression in ovarian cancer cells induces cell death in vitro and in vivo. Int J Cancer 2008, 122:1430-1434. 10.1002/ijc.23278, 18027851.
70. Li J, Feng Q, Kim JM, Schneiderman D, Liston P, Li M, Vanderhyden B, Faught W, Fung MF, Senterman M, Korneluk RG, Tsang BK. Human ovarian cancer and cisplatin resistance: possible role of inhibitor of apoptosis proteins. Endocrinology 2001, 142:370-380. 10.1210/en.142.1.370, 11145600.
71. Greene BT, Thorburn J, Willingham MC, Thorburn A, Planalp RP, Brechbiel MW, Jennings Gee J, Wilkinson J, Torti FM, Torti SV. Activation of caspase pathways during iron chelator mediated apoptosis. J Biol Chem 2002, 277:25568-25575. 10.1074/jbc.M110345200, 11980894.
72. Putcha GV, Harris CA, Moulder KL, Easton RM, Thompson CB, Johnson EM. Intrinsic and extrinsic pathway signaling during neuronal apoptosis: lessons from the analysis of mutant mice. J Cell Biol 2002, 157:441-453. 10.1083/jcb.200110108, 2173286, 11980919.
73. Bussa JL, Neuzilb J, Gellertc N, Weber C, Ponka P. Pyridoxal isonicotinoyl hydrazone analogs induce apoptosis in hematopoietic cells due to their iron-chelating properties. Biochem Pharmacol 2003, 65:161-172. 10.1016/S0006-2952(02)01512-5, 12504792.
74. Huang Z. Bcl 2 family proteins as targets for anticancer drug design. Oncogene 2000, 19:6627-6631. 10.1038/sj.onc.1204087, 11426648.
75. Paull KD, Shoemaker RH, Hodes L, Monks A, Scudiero DA, Rubinstein L, Plowman J, Boyd MR. Display and analysis of pattern of differential activity of drugs against human tumor cell lines: development of mean graph and COMPARE algorithm. J Natl Cancer Inst 1989, 81:1088-1092. 10.1093/jnci/81.14.1088, 2738938.
76. Weinstein JN, Kohn KW, Grever MR, Viswanadhan VN, Rubinstein LV, Monks AP, Scudiero DA, Welch L, Koutsoukos AD, Chiausa AJ. Neural computing in cancer drug development: predicting mechanism of action. Science 1992, 258:447-451. 10.1126/science.1411538, 1411538.
77. Boyd MR, Paull KD. Some practical considerations and applications of the National Cancer Institute in vivo anticancer drug discovery screen. Drug Dev Res 1995, 34:91-109.
78. Bates SE, Fojo AT, Weinstein JN, Myers TG, Alvarez M, Pauli KD, Chabner BA. Molecular targets in the National Cancer Institute drug screen. J Cancer Res Clin Oncol 1995, 121:495-500. 10.1007/BF01197759, 7559726.
79. Weinstein JN, Myers TG, O'Connor PM, Friend SH, Fornace A, Kohn KW, Fojo T, Bates SE, Rubinstein LV, Anderson NL, Buolamwini JK, van Osdol WW, Monks AP, Scudiero DA, Sausville EA, Zaharevitz DW, Bunow B, Viswanadhan VN, Johnson GS, Wittes RE, Paull KD. An information intensive approach to the molecular pharmacology of cancer. Science 1997, 275:343-349. 10.1126/science.275.5298.343, 8994024.
80. Viola Rhenals M, Rieber MS, Rieber M. Role of peroxidases, thiols and Bak/Bax in tumor cell susceptibility to Cu[DEDTC]2. Biochem Pharmacol 2007, 74:841-850. 10.1016/j.bcp.2007.06.048, 17679146.
81. Ma Y, Cao L, Kawabata T, Yoshino T, Yang BB, Okada S. Cupric nitrilotriacetate induces oxidative DNA damage and apoptosis in human leukemia HL-60 cells. Free Radic Biol Med 1998, 25:568-575. 10.1016/S0891-5849(98)00088-4, 9741594.
82. Harris ZH, Gitlin JD. Genetic and molecular basis of copper toxicity. Am J Clin Nutr 1996, 63:836S-841S.