Purpose. To investigate the inhibitory effects of trehalose on malignant melanoma cell growth. Methods. We cultured human malignant melanoma cells in a medium containing trehalose (control/2.5%/5.0%/7.5%/10.0%) and used the MTT assay to evaluate the growth activities. Subsequently, trehalose was topically instilled on subconjunctivally inoculated melanoma cells in F334/NJcl-rmu/rmu rats, followed by a histopathological evaluation of tumor growth. Using flow cytometry, we compared the distribution of the cell cycle, rate of apoptotic cells, and intracellular factors related to the cell cycle in cultured melanoma cells after trehalose treatment. Results. The MTT study showed that proliferation of melanoma cells was significantly inhibited by ≧？5% of trehalose concentrations in the culture media. Subconjunctivally inoculated melanoma cell masses were significantly smaller in eyes administered trehalose as compared to controls. Flow cytometry analyses demonstrated that the trehalose groups had increased rates of G2/M phase cells and apoptotic cells in the cell culture. These cells also exhibited increased expressions of cell-cycle inhibitory factors. Conclusions. The current results show trehalose inhibits malignant melanoma cell growth by inducing G2/M cell cycle arrest and apoptosis, suggesting trehalose as a potential candidate for a topical agent to inhibit proliferation of malignant tumor cells of the ocular surface. 1. Introduction Trehalose, (α-glucopyranosyl-(1→1)-α-D-glucopyranoside), is a disaccharide isomer of sucrose that has a molecular weight of 342.29？Da with numerous interesting properties. This substance is widely present in animals, plants, insects, and microorganisms and plays an important role in preserving cells in situations that could lead to complete dehydration [1, 2]. Trehalose also reportedly has an inhibitory effect on the denaturation of protein and membranes in bacterial [3] and human cells [4], protects cryopreserved cells under freeze-dried conditions [5] and corneal epithelial cells under dry eye conditions [6], and may potentially have a therapeutic effect when treating the progression of Huntington’s disease [7]. The safety and efficacy of topical instillation of trehalose eyedrops have been previously confirmed [8]. In addition to examining the protective effects of trehalose on epithelial cells, we have also previously reported that trehalose exhibited an inhibitory effect on the proliferation of fibroblasts and vascular endothelial cells [9, 10]. Even though trehalose has been shown to have various influences on cell
References
[1]
J. H. Crowe, F. A. Hoekstra, and L. M. Crowe, “Anhydrobiosis,” Annual Review of Physiology, vol. 54, pp. 579–599, 1992.
[2]
R. J. Mayer and D. J. Candy, “Changes in energy reserves during flight of the desert locust, schistocerca gregaria,” Comparative Biochemistry And Physiology, vol. 31, no. 3, pp. 409–418, 1969.
[3]
S. B. Leslie, E. Israeli, B. Lighthart, J. H. Crowe, and L. M. Crowe, “Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying,” Applied and Environmental Microbiology, vol. 61, no. 10, pp. 3592–3597, 1995.
[4]
N. Guo, I. Puhlev, D. R. Brown, J. Mansbridge, and F. Levine, “Trehalose expression confers desiccation tolerance on human cells,” Nature Biotechnology, vol. 18, no. 2, pp. 168–171, 2000.
[5]
T. Nakamura, E. Sekiyama, M. Takaoka et al., “The use of trehalose-treated freeze-dried amniotic membrane for ocular surface reconstruction,” Biomaterials, vol. 29, no. 27, pp. 3729–3737, 2008.
[6]
T. Matsuo, Y. Tsuchida, and N. Morimoto, “Trehalose eye drops in the treatment of dry eye syndrome,” Ophthalmology, vol. 109, no. 11, pp. 2024–2029, 2002.
[7]
M. Tanaka, Y. Machida, S. Niu et al., “Trehalose alleviates polyglutamine-mediated pathology in a mouse model of Huntington disease,” Nature Medicine, vol. 10, no. 2, pp. 148–154, 2004.
[8]
T. Matsuo, “Trehalose versus hyaluronan or cellulose in eyedrops for the treatment of dry eye,” Japanese Journal of Ophthalmology, vol. 48, no. 4, pp. 321–327, 2004.
[9]
K. Takeuchi, M. Nakazawa, Y. Ebina et al., “Inhibitory effects of trehalose on fibroblast proliferation and implications for ocular surgery,” Experimental Eye Research, vol. 91, no. 5, pp. 567–577, 2010.
[10]
K. Takeuchi, M. Nakazawa, and Y. Ebina, “Effects of trehalose on VEGF-stimulated angiogenesis and myofibroblast proliferation: implications for glaucoma filtration surgery,” Investigative Ophthalmology and Visual Science, vol. 52, no. 9, pp. 6987–6993, 2011.
[11]
Y. Kitazawa, K. Kawase, H. Matsushita, and M. Minobe, “Trabeculectomy with mitomycin: a comparative study with fluorouracil,” Archives of Ophthalmology, vol. 109, no. 12, pp. 1693–1698, 1991.
[12]
Y. Kitazawa, H. Suemori-Matsushita, T. Yamamoto, and K. Kawase, “Low-dose and high-dose mitomycin trabeculectomy as an initial surgery in primary open-angle glaucoma,” Ophthalmology, vol. 100, no. 11, pp. 1624–1628, 1993.
[13]
R. Sihota, T. Dada, S. D. Gupta, S. Sharma, R. Arora, and H. C. Agarwal, “Conjunctival dysfunction and mitomycin C-induced hypotony,” Journal of Glaucoma, vol. 9, no. 5, pp. 392–397, 2000.
[14]
A. Solomon, U. Ticho, and J. Frucht-Pery, “Late-onset, bleb-associated endophthalmitis following glaucoma filtering surgery with or without antifibrotic agents,” Journal of Ocular Pharmacology and Therapeutics, vol. 15, no. 4, pp. 283–293, 1999.
[15]
E. J. Poulsen and R. Rand Allingham, “Characteristics and risk factors of infections after glaucoma filtering surgery,” Journal of Glaucoma, vol. 9, no. 6, pp. 438–443, 2000.
[16]
D. S. Greenfield and R. K. Parrish, “Bleb rupture following filtering surgery with mitomycin-C: clinicopathologic correlations,” Ophthalmic Surgery and Lasers, vol. 27, no. 10, pp. 876–877, 1996.
[17]
D. A. Belyea, J. A. Dan, R. L. Stamper, M. F. Lieberman, and W. H. Spencer, “Late onset of sequential multifocal bleb leaks after glaucoma filtration surgery with 5-fluorouracil and mitomycin C,” American Journal of Ophthalmology, vol. 124, no. 1, pp. 40–45, 1997.
[18]
P. W. DeBry, T. W. Perkins, G. Heatley, P. Kaufman, and L. C. Brumback, “Incidence of late-onset bleb-related complications following trabeculectomy with mitomycin,” Archives of Ophthalmology, vol. 120, no. 3, pp. 297–300, 2002.
[19]
B. P. Bouchet, C. Caron de Fronmentel, A. Puisleux, and C. M. Galmarini, “P53 as a target of anti-cancer drug development,” Critical Reviews in Oncology/Hematology, vol. 53, pp. 190–207, 2006.
[20]
T. H. Stracker, T. Usui, and J. H. J. Petrini, “Taking the time to make important decisions: the checkpoint effector kinases Chk1 and Chk2 and the DNA damage response,” DNA Repair, vol. 8, no. 9, pp. 1047–1054, 2009.
[21]
S. Shangary, D. Qin, D. McEachern et al., “Temporal activation of p53 by a specific MDM2 inhibitor is selectively toxic to tumors and leads to complete tumor growth inhibition,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 10, pp. 3933–3938, 2008.
[22]
J. S. Fridman and S. W. Lowe, “Control of apoptosis by p53,” Oncogene, vol. 22, no. 56, pp. 9030–9040, 2003.
[23]
H. C. Reinhardt and M. B. Yaffe, “Kinases that control the cell cycle in response to DNA damage: Chk1, Chk2, and MK2,” Current Opinion in Cell Biology, vol. 21, no. 2, pp. 245–255, 2009.
[24]
F. Uhlmann, C. Bouchoux, and S. López-Avilés, “A quantitative model for cyclin-dependent kinase control of the cell cycle: revisited,” Philosophical Transactions of the Royal Society B, vol. 366, no. 1584, pp. 3572–3583, 2011.
[25]
A. Baldi, A. DeLuca, V. Esposito, M. Camprioni, E. P. Spugnini, and G. Citro, “Tumor suppressors and cell-cycle proteins in lung cancer,” Pathology Research International, vol. 2011, Article ID 605042, 12 pages, 2011.
[26]
U. Strausfeld, A. Fernandez, J. P. Capony et al., “Activation of p34(cdc2) protein kinase by microinjection of human cdc25C into mammalian cells. Requirement for prior phosphorylation of cdc25C by p34(cdc2) on sites phosphorylated at mitosis,” The Journal of Biological Chemistry, vol. 269, no. 8, pp. 5989–6000, 1994.
