A novel study of melatonin diffusion in a 3D cell culture model

Melatonin diffusion in 3D cell culture

  • Francisco Artime-Naveda Departamento de Morfología y Biología Celular, School of Medicine, University of Oviedo, Asturias, Spain
  • Lucas Alves-Pérez Departamento de Morfología y Biología Celular, School of Medicine, University of Oviedo, Spain
  • David Hevia Departamento de Morfología y Biología Celular, School of Medicine, University of Oviedo, Spain
  • Sergio Alcón-Rodríguez Departamento de Morfología y Biología Celular, School of Medicine, University of Oviedo, Spain
  • Sheila Fernández-Vega Departamento de Morfología y Biología Celular, School of Medicine, University of Oviedo, Spain
  • Alejandro Alvarez-Artime Departamento de Morfología y Biología Celular, School of Medicine, University of Oviedo, Spain
  • Isabel Quirós-González Departamento de Morfología y Biología Celular, School of Medicine, University of Oviedo, Spain
  • Rafael Cernuda Departamento de Morfología y Biología Celular, School of Medicine, University of Oviedo, Spain
  • Rosa M Sainz Departamento de Morfología y Biología Celular, School of Medicine, University of Oviedo, Spain
  • Juan Carlos Mayo Departamento de Morfología y Biología Celular, Instituto Universitario Oncológico del Principado de Asturias (IUOPA), Universidad de Oviedo http://orcid.org/0000-0002-0882-2047
DOI: https://doi.org/10.32794/mr112500148
Keywords: Melatonin, AFMK, HaCaT, 3D culture, difussion, epidermal equivalent, ROS

Abstract

Melatonin is now considered a major physiological regulator of many different functions including synchronization of circadian rhythms, antioxidant defense at different levels, immunomodulation, cell growth control, neuroprotector and anti-tumor agent. In addition to its membrane receptor-dependent actions, it has been classically assumed that its diffusion through lipid bilayers contribute to its intracellular actions, including direct and indirect free radical scavenging activities. While pineal gland is the major site of nocturnal production of the indolamine, skin is considered an important source of melatonin synthesis. Here, using a 3-D culture model of HaCaT cells in an artificial scaffold (epidermal equivalents), we have quantified diffusion of melatonin in these cells and compared it to 2-D or spheroid cultures. Diffusion in 3-D scaffold cultures was similar to that found in 2-D culture and proportion of intracellular melatonin was low. AFMK, a major oxidative metabolite of melatonin, was also found and quantified. Redox parameters including total ROS, superoxide or mitochondrial mass were also assayed. We also report the effect of melatonin on the cytoskeleton of normal human keratinocyte HaCaT cells. We propose HaCaT epidermal equivalents as an affordable, easy-to-use, 3-D cell culture tool to test diffusion rates of melatonin but also other similar small molecules. This 3-D models can also be studied at cellular and molecular level, including redox parameters, and can provide important information regarding molecules that can be topically added to skin. Similarly, mechanisms of transportation can also be approached with this methodology.


Author Biography

Juan Carlos Mayo, Departamento de Morfología y Biología Celular, Instituto Universitario Oncológico del Principado de Asturias (IUOPA), Universidad de Oviedo

Assistant professor of Cell Biology

Departamento de Morfología y Biología Celular

U. de Oviedo

References

1. Arendt J, Lerner A (2007) Who discovered melatonin. J. Pineal Res 43: 106–107?
2. Reiter RJ (1991) Pineal melatonin: Cell biology of its synthesis and of its physiological interactions. Endocr. Rev. 12: 151–180.
3. Huether G (1993) The contribution of extrapineal sites of melatonin synthesis to circulating melatonin levels in higher vertebrates. Experientia 49: 665–670.
4. Vivien-Roels B, Pévet P (1993) Melatonin: presence and formation in invertebrates. Experientia 49: 642–647.
5. Schippers KJ, Nichols SA (2014) (Deep, dark secrets of melatonin in animal evolution. Cell 159, 9–10.
6. Hardeland R, Poeggeler B (2003) Non-vertebrate melatonin. J. Pineal Res. 34: 233–241.
7. Tan DX, Reiter RJ (2020). An evolutionary view of melatonin synthesis and metabolism related to its biological functions in plants. J. Exp. Bot. 71: 4677–4689.
8. Arnao MB, Hernández-Ruiz J (2015) Functions of melatonin in plants: a review. J. Pineal Res. 59: 133–150.
9. Reiter RJ (1993) The melatonin rhythm: both a clock and a calendar. Experientia 49: 654–664.
10. Dardente H (2012) Melatonin-dependent timing of seasonal reproduction by the pars tuberalis: pivotal roles for long daylengths and thyroid hormones. J. Neuroendocrinol. 24: 249–266.
11. Galano A, Tan DX, Reiter RJ (2018) Melatonin: A versatile protector against oxidative DNA damage. Molecules 23.
12. Mayo JC, et al. (2017) IGFBP3 and MAPK/ERK signaling mediates melatonin-induced antitumor activity in prostate cancer. J. Pineal Res. 62: 1–17.
13. Sainz RM, Lombo F, Mayo JC (2012) Radical decisions in cancer: Redox control of cell growth and death. Cancers (Basel) 4: 442–474.
14. Galano A, Tan DX, Reiter RJ (2013) On the free radical scavenging activities of melatonin’s metabolites, AFMK and AMK. J. Pineal Res. 54: 245–257.
15. Costa EJX, Lopes RH, M. Lamy‐Freund T (1995) Permeability of pure lipid bilayers to melatonin. J. Pineal Res. 19: 123–126.
16. Lu H, Marti J (2020) Cellular absorption of small molecules: free energy landscapes of melatonin binding at phospholipid membranes. Sci. Rep. 10: 1–12.
17. Liu L, Labani N, Cecon E, Jockers R (2019) Melatonin target proteins: too many or not enough? Front. Endocrinol. (Lausanne) 10: 791.
18. Fletcher DA, Mullins RD (2010) Cell mechanics and the cytoskeleton. Nature 463: 485–492.
19. Mostowy S, Cossart P (2012) Septins: the fourth component of the cytoskeleton. Nat. Rev. Mol. Cell Biol. 13: 183–194.
20. Benítez-King G (2006) Melatonin as a cytoskeletal modulator: Implications for cell physiology and disease. J. Pineal Res. 40: 1–9.
21. Banerjee S, Margulis L (1973) Mitotic arrest by melatonin. Exp. Cell Res. 78: 314-318.
22. Benítez‐King G, Huerto‐Delgadillo L, Antón‐Tay F (1990) Melatonin effects on the cytoskeletal organization of MDCK and neuroblastoma N1E‐115 cells. J. Pineal Res. 9: 209–220.
23. Matsui DH, Machado-Santelli GM (1997) Alterations in F-actin distribution in cells treated with melatonin. J. Pineal Res. 23: 169–175.
24. Witt-Enderby PA, et al. (2000) Melatonin induction of filamentous structures in non-neuronal cells that is dependent on expression of the human mt1 melatonin receptor. Cell Motil. Cytoskeleton 46: 28–42.
25. Bellon A, Ortíz-López L, Ramírez-Rodríguez G, Antón-Tay F, Benítez-King G (2007) Melatonin induces neuritogenesis at early stages in N1E-115 cells through actin rearrangements via activation of protein kinase C and Rho-associated kinase. J. Pineal Res. 42: 214–221.
26. Ramírez-Rodríguez G, Ortiz-López L, Benítez-King G (2007) Melatonin increases stress fibers and focal adhesions in MDCK cells: participation of Rho-associated kinase and protein kinase C. J. Pineal Res. 42: 180–190.
27. Lee SJ, Jung YH, Oh SY, Yun SP, Han HJ (2014) Melatonin enhances the human mesenchymal stem cells motility via melatonin receptor 2 coupling with Gαq in skin wound healing. J. Pineal Res. 57: 393–407.
28. Kadena M., et al. (2017) Microarray and gene co-expression analysis reveals that melatonin attenuates immune responses and modulates actin rearrangement in macrophages. Biochem. Biophys. Res. Commun. 485: 414–420.
29. Slominski AT, et al. (2017) Melatonin, mitochondria, and the skin. Cell. Mol. Life Sci. 74: 3913–3925.
30. Kim TK, et al. (2013) Metabolism of melatonin and biological activity of intermediates of melatoninergic pathway in human skin cells. FASEB J. 27: 2742–2755.
31. Colombo I, et al. (2017) HaCaT cells as a reliable in vitro differentiation model to dissect the inflammatory/repair response of human keratinocytes. Mediators Inflamm. 2017: 7435621.
32. Jensen C, Teng Y (2020) Is it time to start transitioning from 2D to 3D cell culture? Front. Mol. Biosci. 7: 33.
33. Jung MH, Jung SM, Shin HS (2016) Co-stimulation of HaCaT keratinization with mechanical stress and air-exposure using a novel 3D culture device. Sci. Rep. 6: 33889.
34. Hevia D, et al. (2015) Melatonin uptake through glucose transporters: A new target for melatonin inhibition of cancer. J. Pineal Res. 58: 234–250.
35. Alvarez-Artime A, et al. (2020) Melatonin-induced cytoskeleton reorganization leads to inhibition of melanoma cancer cell proliferation. Int. J. Mol. Sci. 21: 548.
36. Bokhari M, Carnachan RJ, Cameron NR, Przyborski SA (2007) Novel cell culture device enabling three-dimensional cell growth and improved cell function. Biochem. Biophys. Res. Commun. 354: 1095-1100.
37. Hevia D, et al. (2008) Melatonin uptake in prostate cancer cells: Intracellular transport versus simple passive diffusion. J. Pineal Res. 45: 247–257.
38. Fernández AS, et al (2022) Evaluation of different internal standardization approaches for the quantification of melatonin in cell culture samples by multiple heart-cutting two dimensional liquid chromatography tandem mass spectrometry. J. Chromatogr. A 1663: 462752.
39. Quiros-Gonzalez I, et al. (2022) Androgen-dependent prostate cancer cells reprogram their metabolic signature upon GLUT1 upregulation by manganese superoxide dismutase. Antioxidants (Basel) 11: 313.
40. Mayo JC, Sainz RM, González-Menéndez P, Hevia D, Cernuda-Cernuda R (2017) Melatonin transport into mitochondria. Cell. Mo. Life Sci.74: 3927–3940.
41. Costa EJX, Shida CS, Biaggi MH, Ito AS, Lamy-Freund MT (1997) How melatonin interacts with lipid bilayers: A study by fluorescence and ESR spectroscopies. FEBS Lett. 416: 103–106.
42. Saija A, et al. (2002) Interaction of melatonin with model membranes and possible implications in its photoprotective activity. Eur. J. Pharm. Biopharm. 53: 209–215.
43. Severcan F, Sahin I, Kazanci N (2005) Melatonin strongly interacts with zwitterionic model membranes-evidence from Fourier transform infrared spectroscopy and differential scanning calorimetry. Biochim. Biophys. Acta Biomembr. 1668: 215–222.
44. García JJ, et al. (2014) Protective effects of melatonin in reducing oxidative stress and in preserving the fluidity of biological membranes: A review. J. Pineal Res. 56: 225–237.
45. Dies H, Cheung B, Tang J, Rheinstädter MC (2015) The organization of melatonin in lipid membranes. Biochim. Biophys. Acta Biomembr. 1848: 1032–1040.
46. Martí J, Lu H, (2021) Microscopic interactions of melatonin, serotonin and tryptophan with zwitterionic phospholipid membranes. Int. J. Mol. Sci. 22: 1–25.
47. Bolmatov D, et al. (2019) Deciphering Melatonin-stabilized phase separation in phospholipid bilayers. Langmuir 35: 12236–12245.
48. Bae S, et al. (2022) Melatonin increases growth properties in human dermal papilla spheroids by activating AKT/GSK3β/β-Catenin signaling pathway. PeerJ 10: e13461.
49. Jacquemet G, Hamidi H, Ivaska J (2015) Filopodia in cell adhesion, 3D migration and cancer cell invasion. Curr. Opin. Cell Biol. 36: 23–31.
50. Sokolov D, et al. (2022) Melatonin and andrographolide synergize to inhibit the colospheroid phenotype by targeting Wnt/beta-catenin signaling. J. Pineal Res. 73: e12808.
51. Phiboonchaiyanan PP, et al. (2021) Melatonin and its derivative disrupt cancer stem-like phenotypes of lung cancer cells via AKT downregulation. Clin Exp. Pharmaco.l Physiol. 48: 1712–1723.
52. Jadid MFS, et al. (2021) Melatonin increases the anticancer potential of doxorubicin in Caco-2 colorectal cancer cells. Environ. Toxicol. 36: 1061–1069.
53. Zhao Y, Wang C, Goel A (2022) A combined treatment with melatonin and andrographis promotes autophagy and anticancer activity in colorectal cancer. Carcinogenesis 43: 217–230.
54. Garcia MA, Nelson WJ, Chavez N (2018) Cell-cell junctions organize structural and signaling networks. Cold Spring Harb Perspect Biol. 10: a029181.
55. Wang L, Ding L, Du Z, Liu J (2020) Effects of hydrophobicity and molecular weight on the transport permeability of oligopeptides across Caco-2 cell monolayers. J. Food Biochem. 44: e13188.
56. Nagarajan SK, Klein S, Fadakar BS, Piontek J (2023) Claudin-10b cation channels in tight junction strands: Octameric-interlocked pore barrels constitute paracellular channels with low water permeability. Comput. Struct. Biotechnol. J. 21: 1711–1727.
Published
2023-06-30
How to Cite
[1]
Artime-Naveda, F., Alves-Pérez, L., Hevia, D., Alcón-Rodríguez, S., Fernández-Vega, S., Alvarez-Artime, A., Quirós-González, I., Cernuda, R., Sainz, R.M. and Mayo, J. 2023. A novel study of melatonin diffusion in a 3D cell culture model. Melatonin Research. 6, 2 (Jun. 2023), 173-188. DOI:https://doi.org/https://doi.org/10.32794/mr112500148.
Issue
Vol 6 No 2 (2023): Melatonin Research
Section
Research Articles
DOI
https://doi.org/10.32794/mr112500148
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