Immune Netw.  2009 Dec;9(6):236-242. 10.4110/in.2009.9.6.236.

Selenium Inhibits Metastasis of Murine Melanoma Cells through the Induction of Cell Cycle Arrest and Cell Death

• Song H
• Hur I
• Park HJ
• Nam J
• Park GB
• Kong KH
• Hwang YM
• Kim YS
• Cho DH
• Lee WJ
• Hur DY

• KMID: 2150651
• DOI: http://doi.org/10.4110/in.2009.9.6.236

Abstract

BACKGROUND
Melanoma is the most fatal form of skin cancer due to its rapid metastasis. Recently, several studies reported that selenium can induce apoptosis in melanoma cells. However, the precise mechanism remains to be elucidated. In this study, we investigated the effect of selenium on cell proliferation in murine melanoma and on tumor growth and metastasis in C57BL/6 mice. METHODS: Cell proliferation was measured by MTT assay in selenium-treated melanoma cells. Cell cycle distribution was analysized by staining DNA with propidum iodide (PI). mRNA and protein expression related to cell cycle arrest was measured by reverse transcription PCR and western blot. Tumor growth and metastasis was measured by in vivo model. RESULTS: Selenium was suppressed the proliferation of melanoma cells in a dose dependent manner. The growth inhibition of melanoma by selenium was associated with an arrest of cell cycle distribution at G0/G1 stage. The mRNA and protein level of CDK2/CDK4 was suppressed by treatment with selenium in a time-dependent manner. In vivo, tumor growth was not suppressed by selenium; however tumor metastasis was suppressed by selenium in mouse model. CONCLUSION: These results suggest that selenium might be a potent agent to inhibit proliferative activity of melanoma cells.

Keyword

Selenium; Metastasis; Tumor immunity; Apoptosis

MeSH Terms

Animals
Apoptosis
Blotting, Western
Cell Cycle
Cell Cycle Checkpoints
Cell Death
Cell Proliferation
DNA
Melanoma
Mice
Neoplasm Metastasis
Polymerase Chain Reaction
Reverse Transcription
RNA, Messenger
Selenium
Skin Neoplasms
DNA
RNA, Messenger
Selenium

Figure

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  Figure 1 Effect of selenium on cell viability and morphological changes of B16F10 murine melanoma cells. (A) Selenium inhibits cell viability of B16F10 murine melanoma cells (MTT assay). B16F10 melanoma cells (1×103) were seeded onto a 96-well plate and treated with various doses (0. 1, 5, 10, 25, 50 and 100 uM) of selenium for 72 hr. MTT solution (100 ug/ well) was added to the cells 4 hr later. (B) B16F10 cells (2×105) were seeded onto a 6-well plate overnight to undergo attachment and then treated with various doses (0, 10, 20, and 40 uM) of selenium. After 48 h, photos of cells were taken with an inverted microscope.

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  Figure 2 Selenium-induced cell death and cell cycle arrest of B16F10 murine melanoma cells. (A) B16F10 melanoma cells (2×105) were seeded onto a 6-well plate overnight to undergo attachment. Cells were exposed to selenium (0, 10, 20, and 40 uM) and then incubated for 48 hr. After incubation, cells were harvested with cell dissociation solution and washed once with PBS. The cytotoxic effect of selenium on B16F10 melanoma cells was assessed by Annexin-V-FITC/PI staining. Cell death analysis was performed by flow cytometry. The numbers indicate the percentages of the viable (Annexin-V -; PI -) cell population (lower left quadrant), the apoptotic (Annexin V+) cell population (lower right quadrant), and the necrotic cell populations in the upper right (Annexin-V +; PI +) and the upper left quadrants (Annexin-V -; PI +). (B) B16F10 melanoma cells were treated with selenium (0, 10, 20, and 40 uM) and then incubated for 48 hr. Cells were harvested with cell dissociation solution, washed twice with cold PBS, and centrifuged. The pellet was resuspended in 1 ml of cold PBS and 4 ml of cold ethanol for 30 min at 4℃. Cells were then stained with propidium iodide 30 min and analyzed by flow cytometry. Percentages of G0/G1-, S-, and G2/M-phase cells are shown in this figure.

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  Figure 3 The effect of selenium on the expression of cell cycle arrest related genes in B16F10 melanoma cells. (A) B16F10 melanoma cells were harvested at the indicated times after incubation with selenium (40 uM). Total RNA was extracted from cells at 48 hr and cDNA was prepared using methods described in the Material and Methods section. (B) B16F10 melanoma cells were harvested at the indicated times after incubation with selenium (40 uM). The cells were then lysed, and the supernatants were subjected to western blot analysis and immunoblotted with anti-p21Waf1/Cip1, anti-CDK4, and anti-CDK2 antibodies.

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  Figure 4 The effect of selenium on tumor growth. B16F10 melanoma cells were injected s.c. into mice. Selenium was injected at the tumor site. Selenium was then injected 2 more times in 4-day-period. Tumor size was measured every other day by a vernier calipers.

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  Figure 5 The effect of selenium on metastasis to lung. Affect of intra-peritoneal treatment with 100 uM selenium on B16F10 experimental metastasis to lung (A) Representative photos of the lungs. Cells were injected through intra-optic venous. After 14 days mice were killed and the pulmonary nodules of B16F10 melanoma were counted. (B) Representative hematoxylin and eosin staining sections of the lungs were photographed. The arrows point to the metastatic tumors in lung. The area of the lung occupied by the colonies in the histology sections was measured microscopically. (C) The number of lung metastatic tumor.

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