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Monitoring intracellular melatonin levels in human prostate normal and cancer cells by HPLC

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Abstract

Melatonin (N-acetyl-5-methoxytryptamine) is a potent endogenous antioxidant and free radical scavenger that has attracted much attention as a consequence of its multiple biological functions. In addition to other physiological properties, it has clear antiproliferative activity in several types of cancer cell. The concentration of melatonin necessary to inhibit cell growth is much higher than its blood physiological concentrations in some tumor types. For years its indolic nature has impeded proper monitoring, by molecular or immunological techniques, of its uptake by cancer cells. In this work we developed a simple, rapid, and validated analytical method for detection and quantification of MEL inside normal and cancer cells. For this purpose we performed high-performance liquid chromatographic analysis after liquid–liquid extraction of the indole from biological samples. The method was validated, and the correlation coefficient for amounts from 0.125 to 1.25 μg was higher than 0.999, with a range of recovery near 100%. Precision was evaluated as repeatibility, and for intermediate precision, the relative standard deviation was less than 5%. The method was used to study the stability of the indole in solution and to determine intracellular melatonin concentrations in normal (PNT1A) and several cancer (LNCaP, DU-145, PC-3) prostate cell lines. Intracellular LOQ/LOD were 7.23/2.83, 23.17/9.07, 4.03/1.83, and 6.51/2.53 nmol L−1, or 1.82/4.66, 0.56/1.45, 3.26/8.34, and 2.02/5.17 attogram in each cell in PNT1A, LNCaP, DU145, and PC-3 cells, respectively. Because there was no information about intracellular levels of melatonin inside normal or tumor prostate cells after treatment with the indole, nor a relationship between its antiproliferative activity and its intracellular concentration, this is the first time that, by using an analytical method combined with measurement of cellular volume by flow cytometry, the intracellular concentration of MEL has been estimated. Also, data obtained here explain why the antiproliferative properties of MEL vary in different cell types. This is, moreover, the first time that by increasing the intracellular concentration of melatonin, its antitumor properties have been promoted in prostate cancer cells. This process can be monitored by the method developed here.

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Acknowledgements

D.H. acknowledges sponsorship from “Fundación para el Fomento en Asturias de la Investigación Científica Aplicada y la Tecnología” FYCIT, Asturias, Spain (COF07-12) and “Programa JAE-Doc” from CSIC. R.M.S. acknowledges support from “Programa Ramón y Cajal” and the financial sponsorship of the “Fondo Social Europeo” from the EC. J.C.M. acknowledges finantial support from IUOPA “Obra Social y Cultural Cajastur”. I.Q. acknowledges finantial support from FICYT. This work was supported by grants from “Fondo de Investigación Sanitaria” (FISS), Instituto de Salud Carlos III (FISS-07-PI061715), Gobierno Regional de Asturias (COF08-37). C. G.-C. acknowledges financial support from the “Plan Nacional: I+D+I (CICYT)” (AGL2007-66772). We truly thank Maria Navarro for her helpful technical assistance (financial support from RTICC, FISS-08-RD06/0020/1042)

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Authors and Affiliations

  1. Departamento de Morfologia y Biologia Celular, Instituto Universitario de Oncologia del Principado de Asturias (IUOPA), Universidad de Oviedo, C/Julian Claveria N°6, 33006, Oviedo, Principado de Asturias, Spain

    D. Hevia, J. C. Mayo, I. Quiros & R. M. Sainz

  2. Instituto de Fermentaciones Industriales (IFI), CSIC, Tecnologías sectoriales C/Juan de la Cierva N°3, 28006, Madrid, Comunidad de Madrid, Spain

    D. Hevia & C. Gomez-Cordoves

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  1. D. Hevia
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  2. J. C. Mayo
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  3. I. Quiros
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  4. C. Gomez-Cordoves
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  5. R. M. Sainz
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Correspondence to R. M. Sainz.

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Awarded an ABC Poster Prize on the occasion of XXXII Reunion of the Royal Spanish Society of Chemistry, held from 13–18 September 2009 in Oviedo, Spain

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Hevia, D., Mayo, J.C., Quiros, I. et al. Monitoring intracellular melatonin levels in human prostate normal and cancer cells by HPLC. Anal Bioanal Chem 397, 1235–1244 (2010). https://doi.org/10.1007/s00216-010-3653-4

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  • DOI: https://doi.org/10.1007/s00216-010-3653-4

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