Ovarian cancer stem-like cells with induced translineage-differentiation capacity and are suppressed by alkaline phosphatase inhibitor

Oncotarget

Volumn 4, Issue 12, 2013, Pages 2366-2382

  Liu, Kuei Chun ^a   Yo, Yi Te ^a,b   Huang, Rui Lan ^a,c   Wang, Yu Chi ^a,b   Liao, Yu Ping ^a   Huang, Tien Shuo ^a,c   Chao, Tai Kuang ^a,c   Lin, Chi Kang ^a,b   Weng, Shao Ju ^a,b   Ma, Kuo Hsing ^a   Chang, Cheng Chang ^a,b   Yu, Mu Hsien ^a,b   Lai, Hung Cheng ^a,b,c,d  

^a NATIONAL DEFENSE MEDICAL CENTER   (Taiwan)

^b TRI SERVICE GENERAL HOSPITAL   (Taiwan)

^c TAIPEI MEDICAL UNIVERSITY   (Taiwan)

^d TAIPEI MEDICAL UNIVERSITY   (Taiwan)

Author keywords

Alkaline phosphatase; Cancer stem like cells; Epithelial ovarian cancer; Epithelial mesenchymal transition; Levamisole; Trans lineage differentiation

Indexed keywords

ALKALINE PHOSPHATASE; LEVAMISOLE; PLACEBO;

ADOLESCENT; ADULT; ADVANCED CANCER; AGED; ANIMAL EXPERIMENT; ANIMAL MODEL; ANIMAL TISSUE; ARTICLE; CANCER PROGNOSIS; CANCER STAGING; CANCER STEM CELL; CARCINOGENICITY; CELL DIFFERENTIATION; CELL RENEWAL; CELL STRUCTURE; CONTROLLED STUDY; EPITHELIAL MESENCHYMAL TRANSITION; FEMALE; HUMAN; HUMAN CELL; HUMAN TISSUE; MAJOR CLINICAL STUDY; MOUSE; NONHUMAN; OVARY CANCER; OVERALL SURVIVAL; SURVIVAL RATE; TUMOR GROWTH; TUMOR RECURRENCE; TUMOR SPHEROID;

ADOLESCENT; ADULT; AGED; ALKALINE PHOSPHATASE; CELL DIFFERENTIATION; CELL GROWTH PROCESSES; CELL LINE, TUMOR; CELL LINEAGE; EPITHELIAL-MESENCHYMAL TRANSITION; FEMALE; HUMANS; MIDDLE AGED; NEOPLASMS, GLANDULAR AND EPITHELIAL; NEOPLASTIC STEM CELLS; OVARIAN NEOPLASMS; SPHEROIDS, CELLULAR; YOUNG ADULT;

EID: 84891905690     PISSN: None     EISSN: 19492553     Source Type: Journal    
DOI: 10.18632/oncotarget.1424     Document Type: Article

References (78)

1. Pastrana E, Silva-Vargas V and Doetsch F. Eyes wide open: a critical review of sphere-formation as an assay for stem cells. Cell Stem Cell. 2011; 8(5):486-498.
2. Frank NY, Schatton T and Frank MH. The therapeutic promise of the cancer stem cell concept. JClinInvest. 2010; 120(1):41-50.
3. Valent P, Bonnet D, De Maria R, Lapidot T, Copland M, Melo JV, Chomienne C, Ishikawa F, Schuringa JJ, Stassi G, Huntly B, Herrmann H, Soulier J, Roesch A, Schuurhuis GJ, Wohrer S, et al. Cancer stem cell definitions and terminology: the devil is in the details. Nature reviews Cancer. 2012; 12(11):767-775.
4. Vermeulen L, Sprick MR, Kemper K, Stassi G and Medema JP. Cancer stem cells--old concepts, new insights. Cell death and differentiation. 2008; 15(6):947-958.
5. Bapat SA, Mali AM, Koppikar CB and Kurrey NK. Stem and progenitor-like cells contribute to the aggressive behavior of human epithelial ovarian cancer. Cancer Res. 2005; 65(8):3025-3029.
6. Kusumbe AP, Mali AM and Bapat SA. CD133-expressing stem cells associated with ovarian metastases establish an endothelial hierarchy and contribute to tumor vasculature. Stem Cells. 2009; 27(3):498-508.
7. Hu L, McArthur C and Jaffe RB. Ovarian cancer stem-like side-population cells are tumourigenic and chemoresistant. BrJCancer. 2010; 102(8):1276-1283.
8. Silva IA, Bai S, McLean K, Yang K, Griffith K, Thomas D, Ginestier C, Johnston C, Kueck A, Reynolds RK, Wicha MS and Buckanovich RJ. Aldehyde dehydrogenase in combination with CD133 defines angiogenic ovarian cancer stem cells that portend poor patient survival. Cancer Res. 2011; 71(11):3991-4001.
9. Zhang S, Balch C, Chan MW, Lai HC, Matei D, Schilder JM, Yan PS, Huang TH and Nephew KP. Identification and characterization of ovarian cancer-initiating cells from primary human tumors. Cancer Res. 2008; 68(11):4311-4320.
10. Alvero AB, Chen R, Fu HH, Montagna M, Schwartz PE, Rutherford T, Silasi DA, Steffensen KD, Waldstrom M, Visintin I and Mor G. Molecular phenotyping of human ovarian cancer stem cells unravels the mechanisms for repair and chemoresistance. Cell Cycle. 2009; 8(1):158-166.
11. Curley MD, Therrien VA, Cummings CL, Sergent PA, Koulouris CR, Friel AM, Roberts DJ, Seiden MV, Scadden DT, Rueda BR and Foster R. CD133 expression defines a tumor initiating cell population in primary human ovarian cancer. Stem Cells. 2009; 27(12):2875-2883.
12. Gao MQ, Choi YP, Kang S, Youn JH and Cho NH. CD24+ cells from hierarchically organized ovarian cancer are enriched in cancer stem cells. Oncogene. 2010; 29(18):2672-2680.
13. Kusumbe AP and Bapat SA. Cancer stem cells and aneuploid populations within developing tumors are the major determinants of tumor dormancy. Cancer Res. 2009; 69(24):9245-9253.
14. Szotek PP, Pieretti-Vanmarcke R, Masiakos PT, Dinulescu DM, Connolly D, Foster R, Dombkowski D, Preffer F, Maclaughlin DT and Donahoe PK. Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance responsiveness. ProcNatlAcadSciUSA. 2006; 103(30):11154-11159.
15. Yo YT, Lin YW, Wang YC, Balch C, Huang RL, Chan MW, Sytwu HK, Chen CK, Chang CC, Nephew KP, Huang T, Yu MH and Lai HC. Growth inhibition of ovarian tumor-initiating cells by niclosamide. Molecular cancer therapeutics. 2012; 11(8):1703-1712.
16. Flesken-Nikitin A, Hwang CI, Cheng CY, Michurina TV, Enikolopov G and Nikitin AY. Ovarian surface epithelium at the junction area contains a cancer-prone stem cell niche. Nature. 2013; 495(7440):241-245.
17. Fang D, Nguyen TK, Leishear K, Finko R, Kulp AN, Hotz S, Van Belle PA, Xu X, Elder DE and Herlyn M. A tumorigenic subpopulation with stem cell properties in melanomas. Cancer research. 2005; 65(20):9328-9337.
18. Vermeulen L, Todaro M, de Sousa Mello F, Sprick MR, Kemper K, Perez Alea M, Richel DJ, Stassi G and Medema JP. Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity. Proceedings of the National Academy of Sciences of the United States of America. 2008; 105(36):13427-13432.
19. Ramgolam K, Lauriol J, Lalou C, Lauden L, Michel L, de la Grange P, Khatib AM, Aoudjit F, Charron D, Alcaide-Loridan C and Al-Daccak R. Melanoma spheroids grown under neural crest cell conditions are highly plastic migratory/invasive tumor cells endowed with immunomodulator function. PloS one. 2011; 6(4):e18784.
20. Zhau HE, He H, Wang CY, Zayzafoon M, Morrissey C, Vessella RL, Marshall FF, Chung LW and Wang R. Human prostate cancer harbors the stem cell properties of bone marrow mesenchymal stem cells. Clinical cancer research: an official journal of the American Association for Cancer Research. 2011; 17(8):2159-2169.
21. Wang YC, Yo YT, Lee HY, Liao YP, Chao TK, Su PH and Lai HC. ALDH1-bright epithelial ovarian cancer cells are associated with CD44 expression, drug resistance, and poor clinical outcome. The American journal of pathology. 2012; 180(3):1159-1169.
22. Shekhani MT, Jayanthy AS, Maddodi N and Setaluri V. Cancer stem cells and tumor transdifferentiation: implications for novel therapeutic strategies. American journal of stem cells. 2013; 2(1):52-61.
23. Fok EY and Zandstra PW. Shear-controlled single-step mouse embryonic stem cell expansion and embryoid body-based differentiation. Stem Cells. 2005; 23(9):1333-1342.
24. Yeung TM, Gandhi SC, Wilding JL, Muschel R and Bodmer WF. Cancer stem cells from colorectal cancer-derived cell lines. ProcNatlAcadSciUSA. 2010; 107(8):3722-3727.
25. Meng F, Glaser SS, Francis H, DeMorrow S, Han Y, Passarini JD, Stokes A, Cleary JP, Liu X, Venter J, Kumar P, Priester S, Hubble L, Staloch D, Sharma J, Liu CG, et al. Functional analysis of microRNAs in human hepatocellular cancer stem cells. Journal of cellular and molecular medicine. 2012; 16(1):160-173.
26. Zhang JQ, Yu XB, Ma BF, Yu WH, Zhang AX, Huang G, Mao FF, Zhang XM, Wang ZC, Li SN, Lahn BT and Xiang AP. Neural differentiation of embryonic stem cells induced by conditioned medium from neural stem cell. Neuroreport. 2006; 17(10):981-986.
27. Greco SJ, Liu K and Rameshwar P. Functional similarities among genes regulated by OCT4 in human mesenchymal and embryonic stem cells. Stem Cells. 2007; 25(12):3143-3154.
28. Ma W, Tavakoli T, Derby E, Serebryakova Y, Rao MS and Mattson MP. Cell-extracellular matrix interactions regulate neural differentiation of human embryonic stem cells. BMC developmental biology. 2008; 8:90.
29. Cristiano L, Cimini A, Moreno S, Ragnelli AM and Paola Ceru M. Peroxisome proliferator-activated receptors (PPARs) and related transcription factors in differentiating astrocyte cultures. Neuroscience. 2005; 131(3):577-587.
30. Kaushik S, Rodriguez-Navarro JA, Arias E, Kiffin R, Sahu S, Schwartz GJ, Cuervo AM and Singh R. Autophagy in hypothalamic AgRP neurons regulates food intake and energy balance. Cell metabolism. 2011; 14(2):173-183.
31. Ohkubo S, Kimura J and Matsuoka I. Ecto-alkaline phosphatase in NG108-15 cells: a key enzyme mediating P1 antagonist-sensitive ATP response. British journal of pharmacology. 2000; 131(8):1667-1672.
32. Palmqvist L, Glover CH, Hsu L, Lu M, Bossen B, Piret JM, Humphries RK and Helgason CD. Correlation of murine embryonic stem cell gene expression profiles with functional measures of pluripotency. Stem cells (Dayton, Ohio). 2005; 23(5):663-680.
33. Takahashi K and Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006; 126(4):663-676.
34. O'Connor MD, Kardel MD, Iosfina I, Youssef D, Lu M, Li MM, Vercauteren S, Nagy A and Eaves CJ. Alkaline phosphatase-positive colony formation is a sensitive, specific, and quantitative indicator of undifferentiated human embryonic stem cells. Stem cells (Dayton, Ohio). 2008; 26(5):1109-1116.
35. Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, Brooks M, Reinhard F, Zhang CC, Shipitsin M, Campbell LL, Polyak K, Brisken C, Yang J and Weinberg RA. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. 2008; 133(4):704-715.
36. Kabashima A, Higuchi H, Takaishi H, Matsuzaki Y, Suzuki S, Izumiya M, Iizuka H, Sakai G, Hozawa S, Azuma T and Hibi T. Side population of pancreatic cancer cells predominates in TGF-beta-mediated epithelial to mesenchymal transition and invasion. International journal of cancer Journal international du cancer. 2009; 124(12):2771-2779.
37. Cao L, Shao M, Schilder J, Guise T, Mohammad KS and Matei D. Tissue transglutaminase links TGF-beta, epithelial to mesenchymal transition and a stem cell phenotype in ovarian cancer. Oncogene. 2012; 31(20):2521-2534.
38. Kang Y and Massague J. Epithelial-mesenchymal transitions: twist in development and metastasis. Cell. 2004; 118(3):277-279.
39. Jordan NV, Johnson GL and Abell AN. Tracking the intermediate stages of epithelial-mesenchymal transition in epithelial stem cells and cancer. Cell Cycle. 2011; 10(17):2865-2873.
40. Xu Y, Zhu X, Hahm HS, Wei W, Hao E, Hayek A and Ding S. Revealing a core signaling regulatory mechanism for pluripotent stem cell survival and self-renewal by small molecules. Proceedings of the National Academy of Sciences of the United States of America. 2010; 107(18):8129-8134.
41. Li L, Wang S, Jezierski A, Moalim-Nour L, Mohib K, Parks RJ, Retta SF and Wang L. A unique interplay between Rap1 and E-cadherin in the endocytic pathway regulates self-renewal of human embryonic stem cells. Stem Cells. 2010; 28(2):247-257.
42. Karpowicz P, Willaime-Morawek S, Balenci L, DeVeale B, Inoue T and van der Kooy D. E-Cadherin regulates neural stem cell self-renewal. The Journal of neuroscience: the official journal of the Society for Neuroscience. 2009; 29(12):3885-3896.
43. Chen T, Yuan D, Wei B, Jiang J, Kang J, Ling K, Gu Y, Li J, Xiao L and Pei G. E-cadherin-mediated cell-cell contact is critical for induced pluripotent stem cell generation. Stem Cells. 2010; 28(8):1315-1325.
44. Redmer T, Diecke S, Grigoryan T, Quiroga-Negreira A, Birchmeier W and Besser D. E-cadherin is crucial for embryonic stem cell pluripotency and can replace OCT4 during somatic cell reprogramming. EMBO reports. 2011; 12(7):720-726.
45. Eastham AM, Spencer H, Soncin F, Ritson S, Merry CL, Stern PL and Ward CM. Epithelial-mesenchymal transition events during human embryonic stem cell differentiation. Cancer research. 2007; 67(23):11254-11262.
46. Chao TK, Yo YT, Liao YP, Wang YC, Su PH, Huang TS and Lai HC. LIM-homeobox transcription factor 1, alpha (LMX1A) inhibits tumourigenesis, epithelial-mesenchymal transition and stem-like properties of epithelial ovarian cancer. Gynecologic oncology. 2013; 128(3):475-482.
47. Kurman RJ and Shih Ie M. Pathogenesis of ovarian cancer: lessons from morphology and molecular biology and their clinical implications. International journal of gynecological pathology: official journal of the International Society of Gynecological Pathologists. 2008; 27(2):151-160.
48. Michor F and Polyak K. The origins and implications of intratumor heterogeneity. Cancer Prev Res (Phila). 2010; 3(11):1361-1364.
49. Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, Henkelman RM, Cusimano MD and Dirks PB. Identification of human brain tumour initiating cells. Nature. 2004; 432(7015):396-401.
50. Stewart JM, Shaw PA, Gedye C, Bernardini MQ, Neel BG and Ailles LE. Phenotypic heterogeneity and instability of human ovarian tumor-initiating cells. Proceedings of the National Academy of Sciences of the United States of America. 2011; 108(16):6468-6473.
51. Tang DG. Understanding cancer stem cell heterogeneity and plasticity. Cell research. 2012; 22(3):457-472.
52. Shackleton M, Quintana E, Fearon ER and Morrison SJ. Heterogeneity in cancer: cancer stem cells versus clonal evolution. Cell. 2009; 138(5):822-829.
53. Mao P, Joshi K, Li J, Kim SH, Li P, Santana-Santos L, Luthra S, Chandran UR, Benos PV, Smith L, Wang M, Hu B, Cheng SY, Sobol RW and Nakano I. Mesenchymal glioma stem cells are maintained by activated glycolytic metabolism involving aldehyde dehydrogenase 1A3. Proceedings of the National Academy of Sciences of the United States of America. 2013; 110(21):8644-8649.
54. Dombrowski GJ, Jr., Swiatek KR and Chao KL. Alkaline phosphatase activity can interfere with the enzymatic determination of triglycerides. Journal of lipid research. 1985; 26(4):513-514.
55. Einarsson K, Benthin L, Ewerth S, Hellers G, Stahlberg D and Angelin B. Studies on acyl-coenzyme A: cholesterol acyltransferase activity in human liver microsomes. Journal of lipid research. 1989; 30(5):739-746.
56. Scher BM, Fuksina I, Hellinger N, Waxman S and Scher W. The phosphatase inhibitors, orthovanadate and levamisole, inhibit induction of erythroid differentiation and abrogate the associated inhibition of glycolysis. International journal of oncology. 1998; 12(5):987-996.
57. Quintana E, Shackleton M, Foster HR, Fullen DR, Sabel MS, Johnson TM and Morrison SJ. Phenotypic heterogeneity among tumorigenic melanoma cells from patients that is reversible and not hierarchically organized. Cancer Cell. 2010; 18(5):510-523.
58. Rawadi G, Vayssiere B, Dunn F, Baron R and Roman-Roman S. BMP-2 controls alkaline phosphatase expression and osteoblast mineralization by a Wnt autocrine loop. Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research. 2003; 18(10):1842-1853.
59. Delgado-Calle J, Sanudo C, Sanchez-Verde L, Garcia-Renedo RJ, Arozamena J and Riancho JA. Epigenetic regulation of alkaline phosphatase in human cells of the osteoblastic lineage. Bone. 2011; 49(4):830-838.
60. Hu X, Zhang X, Dai L, Zhu J, Jia Z, Wang W, Zhou C and Ao Y. Histone deacetylase inhibitor trichostatin A promotes the osteogenic differentiation of rat adipose-derived stem cells by altering the epigenetic modifications on Runx2 promoter in a BMP signaling-dependent manner. Stem cells and development. 2013; 22(2):248-255.
61. Kozlenkov A, Le Du MH, Cuniasse P, Ny T, Hoylaerts MF and Millan JL. Residues determining the binding specificity of uncompetitive inhibitors to tissue-nonspecific alkaline phosphatase. Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research. 2004; 19(11):1862-1872.
62. Gusdon JP, Jr., Homesley HD, Muss HB, Heise ER, Herbst GA, Richards F, 2nd, Spurr CL, Lovelace JV and Di Saia PJ. Chemotherapy of advanced ovarian epithelial carcinoma with melphalan and levamisole: a pilot study of the Gynecologic Oncology Group. American journal of obstetrics and gynecology. 1981; 141(1):65-70.
63. Khoo SK, Whitaker SV, Jones IS and Thomas DA. Levamisole as adjuvant to chemotherapy of ovarian cancer. Results of a randomized trial and 4-year follow-up. Cancer. 1984; 54(6):986-990.
64. Kovach JS, Svingen PA and Schaid DJ. Levamisole potentiation of fluorouracil antiproliferative activity mimicked by orthovanadate, an inhibitor of tyrosine phosphatase. Journal of the National Cancer Institute. 1992; 84(7):515-519.
65. Poplin EA, Benedetti JK, Estes NC, Haller DG, Mayer RJ, Goldberg RM, Weiss GR, Rivkin SE and Macdonald JS. Phase III Southwest Oncology Group 9415/Intergroup 0153 randomized trial of fluorouracil, leucovorin, and levamisole versus fluorouracil continuous infusion and levamisole for adjuvant treatment of stage III and high-risk stage II colon cancer. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2005; 23(9):1819-1825.
66. Dahl O, Fluge O, Carlsen E, Wiig JN, Myrvold HE, Vonen B, Podhorny N, Bjerkeset O, Eide TJ, Halvorsen TB and Tveit KM. Final results of a randomised phase III study on adjuvant chemotherapy with 5 FU and levamisol in colon and rectum cancer stage II and III by the Norwegian Gastrointestinal Cancer Group. Acta Oncol. 2009; 48(3):368-376.
67. Sargent DJ, Marsoni S, Monges G, Thibodeau SN, Labianca R, Hamilton SR, French AJ, Kabat B, Foster NR, Torri V, Ribic C, Grothey A, Moore M, Zaniboni A, Seitz JF, Sinicrope F, et al. Defective mismatch repair as a predictive marker for lack of efficacy of fluorouracil-based adjuvant therapy in colon cancer. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2010; 28(20):3219-3226.
68. Tsai WS, Hsieh PS, Yeh CY, Chiang JM, Tang R, Chen JS, Changchien CR and Wang JY. Long-term survival benefits of adjuvant chemotherapy by decreasing incidence of tumor recurrence without delaying relapse in stage III colorectal cancer. International journal of colorectal disease. 2011; 26(10):1329-1338.
69. Myklebust MP, Li Z, Tran TH, Rui H, Knudsen ES, Elsaleh H, Fluge O, Vonen B, Myrvold HE, Leh S, Tveit KM, Pestell RG and Dahl O. Expression of cyclin D1a and D1b as predictive factors for treatment response in colorectal cancer. British journal of cancer. 2012; 107(10):1684-1691.
70. Kermer V, Ritter M, Albuquerque B, Leib C, Stanke M and Zimmermann H. Knockdown of tissue nonspecific alkaline phosphatase impairs neural stem cell proliferation and differentiation. Neuroscience letters. 2010; 485(3):208-211.
71. Yoon BS, Yoo SJ, Lee JE, You S, Lee HT and Yoon HS. Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment. Differentiation; research in biological diversity. 2006; 74(4):149-159.
72. Li R, Liang J, Ni S, Zhou T, Qing X, Li H, He W, Chen J, Li F, Zhuang Q, Qin B, Xu J, Li W, Yang J, Gan Y, Qin D, et al. A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts. Cell Stem Cell. 2010; 7(1):51-63.
73. Roesch A, Fukunaga-Kalabis M, Schmidt EC, Zabierowski SE, Brafford PA, Vultur A, Basu D, Gimotty P, Vogt T and Herlyn M. A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth. Cell. 2010; 141(4):583-594.
74. Gavert N, Vivanti A, Hazin J, Brabletz T and Ben-Ze'ev A. L1-mediated colon cancer cell metastasis does not require changes in EMT and cancer stem cell markers. Molecular cancer research: MCR. 2011; 9(1):14-24.
75. Liu X, Sun H, Qi J, Wang L, He S, Liu J, Feng C, Chen C, Li W, Guo Y, Qin D, Pan G, Chen J, Pei D and Zheng H. Sequential introduction of reprogramming factors reveals a time-sensitive requirement for individual factors and a sequential EMT-MET mechanism for optimal reprogramming. Nature cell biology. 2013; 15(7):829.
76. Floor S, van Staveren WC, Larsimont D, Dumont JE and Maenhaut C. Cancer cells in epithelial-to-mesenchymal transition and tumor-propagating-cancer stem cells: distinct, overlapping or same populations. Oncogene. 2011; 30(46):4609-4621.
77. Kelly KA, Bardeesy N, Anbazhagan R, Gurumurthy S, Berger J, Alencar H, Depinho RA, Mahmood U and Weissleder R. Targeted nanoparticles for imaging incipient pancreatic ductal adenocarcinoma. PLoS medicine. 2008; 5(4):e85.
78. Tsurumi C, Esser N, Firat E, Gaedicke S, Follo M, Behe M, Elsasser-Beile U, Grosu AL, Graeser R and Niedermann G. Non-invasive in vivo imaging of tumor-associated CD133/prominin. PloS one. 2010; 5(12):e15605.