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γ-Tocotrienol prevents 5-FU-induced reactive oxygen species production in human oral keratinocytes through the stabilization of 5-FU-induced activation of Nrf2.

Takano H, Momota Y, Kani K, Aota K, Yamamura Y, Yamanoi T, Azuma M - Int. J. Oncol. (2015)

Bottom Line: When cells were treated with 5-FU alone, significant growth inhibition was observed as compared to untreated cells.Simultaneous treatment of cells with these agents resulted in the significant recovery of cell growth, owing to the suppression of ROS generation by γ-tocotrienol.In addition, expression of Nrf2-dependent antioxidant genes, such as heme oxygenase-1 (HO-1) and quinone oxidoreductase-1 (NQO-1), was significantly augmented by treatment of cells with both agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Medicine, Institute of Health Biosciences, The University of Tokushima Graduate Faculty of Dentistry, Tokushima, Japan.

ABSTRACT

Unlabelled: Chemotherapy-induced oral mucositis is a common adverse event in patients with oral squamous cell carcinoma, and is initiated through a variety of mechanisms, including the generation of reactive oxygen species (ROS). In this study, we examined the preventive effect of γ-tocotrienol on the 5-FU-induced ROS production in human oral keratinocytes (RT7). We treated RT7 cells with 5-FU and γ-tocotrienol at concentrations of 10 µg/ml and 10 nM, respectively. When cells were treated with 5-FU alone, significant growth inhibition was observed as compared to untreated cells. This inhibition was, in part, due to the ROS gene-rated by 5-FU treatment, because N-acetyl cysteine (NAC), a ROS scavenger, significantly ameliorated the growth of RT7 cells. γ-tocotrienol showed no cytotoxic effect on the growth of RT7 cells. Simultaneous treatment of cells with these agents resulted in the significant recovery of cell growth, owing to the suppression of ROS generation by γ-tocotrienol. Whereas 5-FU stimulated the expression of NF-E2-related factor 2 (Nrf2) protein in the nucleus up to 12 h after treatment of RT7 cells, γ-tocotrienol had no obvious effect on the expression of nuclear Nrf2 protein. Of note, the combined treatment with both agents stabilized the 5-FU-induced nuclear Nrf2 protein expression until 24 h after treatment. In addition, expression of Nrf2-dependent antioxidant genes, such as heme oxygenase-1 (HO-1) and

Nad(p)h: quinone oxidoreductase-1 (NQO-1), was significantly augmented by treatment of cells with both agents. These findings suggest that γ-tocotrienol could prevent 5-FU-induced ROS generation by stabilizing Nrf2 activation, thereby leading to ROS detoxification and cell survival in human oral keratinocytes.

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Effects of reactive oxygen species (ROS) on the growth of RT7 cells. (A) Generation of ROS in 5-FU-treated RT7 cells. The levels of ROS production were measured at 24 and 48 h after treatment of RT7 cells with 5-FU (10 μg/ml) using the ROS detection dye DCFH-DA. The total amount of ROS produced was significantly augmented at 48 h after treatment. (B) Effect of N-acetyl cysteine (NAC) on the generation of 5-FU-induced ROS. NAC (0.1 mM), a ROS scavenger, significantly suppressed the production of ROS at 48 h after treatment with 5-FU (10 μg/ml). (C) NAC improvement of cell growth in 5-FU-treated RT7 cells. Cells were treated with 5-FU (△), NAC (●), or both (▲). Combined treatment of RT7 cells with 5-FU (10 μg/ml) and NAC (0.1 mM) significantly recovered the impairment of cell growth as compared to the treatment with 5-FU alone. Statistically significant at *P<0.05 (Mann-Whitney U test).
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f2-ijo-46-04-1453: Effects of reactive oxygen species (ROS) on the growth of RT7 cells. (A) Generation of ROS in 5-FU-treated RT7 cells. The levels of ROS production were measured at 24 and 48 h after treatment of RT7 cells with 5-FU (10 μg/ml) using the ROS detection dye DCFH-DA. The total amount of ROS produced was significantly augmented at 48 h after treatment. (B) Effect of N-acetyl cysteine (NAC) on the generation of 5-FU-induced ROS. NAC (0.1 mM), a ROS scavenger, significantly suppressed the production of ROS at 48 h after treatment with 5-FU (10 μg/ml). (C) NAC improvement of cell growth in 5-FU-treated RT7 cells. Cells were treated with 5-FU (△), NAC (●), or both (▲). Combined treatment of RT7 cells with 5-FU (10 μg/ml) and NAC (0.1 mM) significantly recovered the impairment of cell growth as compared to the treatment with 5-FU alone. Statistically significant at *P<0.05 (Mann-Whitney U test).

Mentions: To determine whether or not the 5-FU treatment would stimulate ROS generation in RT7 cells, we measured the levels of ROS using the ROS detection dye DCFH-DA. No increase in ROS was detected at 24 h after treatment with 10 μg/ml of 5-FU (Fig. 2A). Further incubation with 10 μg/ml of 5-FU for 48 h resulted in a significant increase in ROS production in RT7 cells. To examine whether NAC, a ROS scavenger, actually abolishes ROS, we measured the ROS level using the method described above. When RT7 cells were treated with both 5-FU and NAC (0.1 mM) for 24 h, the generation of ROS was not affected. However, ROS production was significantly suppressed at 48 h after treatment with NAC (Fig. 2B). Since ROS affects the suppression of cell growth, we examined the effect of NAC on the growth of RT7 cells. As shown in Fig. 2C, although NAC alone did not affect the growth of RT7 cells, the cell growth that was suppressed by 5-FU was significantly restored through the elimination of ROS in RT7 cells. Therefore, these results indicate that growth suppression by 5-FU may be at least partly due to the generation of ROS from RT7 cells.


γ-Tocotrienol prevents 5-FU-induced reactive oxygen species production in human oral keratinocytes through the stabilization of 5-FU-induced activation of Nrf2.

Takano H, Momota Y, Kani K, Aota K, Yamamura Y, Yamanoi T, Azuma M - Int. J. Oncol. (2015)

Effects of reactive oxygen species (ROS) on the growth of RT7 cells. (A) Generation of ROS in 5-FU-treated RT7 cells. The levels of ROS production were measured at 24 and 48 h after treatment of RT7 cells with 5-FU (10 μg/ml) using the ROS detection dye DCFH-DA. The total amount of ROS produced was significantly augmented at 48 h after treatment. (B) Effect of N-acetyl cysteine (NAC) on the generation of 5-FU-induced ROS. NAC (0.1 mM), a ROS scavenger, significantly suppressed the production of ROS at 48 h after treatment with 5-FU (10 μg/ml). (C) NAC improvement of cell growth in 5-FU-treated RT7 cells. Cells were treated with 5-FU (△), NAC (●), or both (▲). Combined treatment of RT7 cells with 5-FU (10 μg/ml) and NAC (0.1 mM) significantly recovered the impairment of cell growth as compared to the treatment with 5-FU alone. Statistically significant at *P<0.05 (Mann-Whitney U test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356501&req=5

f2-ijo-46-04-1453: Effects of reactive oxygen species (ROS) on the growth of RT7 cells. (A) Generation of ROS in 5-FU-treated RT7 cells. The levels of ROS production were measured at 24 and 48 h after treatment of RT7 cells with 5-FU (10 μg/ml) using the ROS detection dye DCFH-DA. The total amount of ROS produced was significantly augmented at 48 h after treatment. (B) Effect of N-acetyl cysteine (NAC) on the generation of 5-FU-induced ROS. NAC (0.1 mM), a ROS scavenger, significantly suppressed the production of ROS at 48 h after treatment with 5-FU (10 μg/ml). (C) NAC improvement of cell growth in 5-FU-treated RT7 cells. Cells were treated with 5-FU (△), NAC (●), or both (▲). Combined treatment of RT7 cells with 5-FU (10 μg/ml) and NAC (0.1 mM) significantly recovered the impairment of cell growth as compared to the treatment with 5-FU alone. Statistically significant at *P<0.05 (Mann-Whitney U test).
Mentions: To determine whether or not the 5-FU treatment would stimulate ROS generation in RT7 cells, we measured the levels of ROS using the ROS detection dye DCFH-DA. No increase in ROS was detected at 24 h after treatment with 10 μg/ml of 5-FU (Fig. 2A). Further incubation with 10 μg/ml of 5-FU for 48 h resulted in a significant increase in ROS production in RT7 cells. To examine whether NAC, a ROS scavenger, actually abolishes ROS, we measured the ROS level using the method described above. When RT7 cells were treated with both 5-FU and NAC (0.1 mM) for 24 h, the generation of ROS was not affected. However, ROS production was significantly suppressed at 48 h after treatment with NAC (Fig. 2B). Since ROS affects the suppression of cell growth, we examined the effect of NAC on the growth of RT7 cells. As shown in Fig. 2C, although NAC alone did not affect the growth of RT7 cells, the cell growth that was suppressed by 5-FU was significantly restored through the elimination of ROS in RT7 cells. Therefore, these results indicate that growth suppression by 5-FU may be at least partly due to the generation of ROS from RT7 cells.

Bottom Line: When cells were treated with 5-FU alone, significant growth inhibition was observed as compared to untreated cells.Simultaneous treatment of cells with these agents resulted in the significant recovery of cell growth, owing to the suppression of ROS generation by γ-tocotrienol.In addition, expression of Nrf2-dependent antioxidant genes, such as heme oxygenase-1 (HO-1) and quinone oxidoreductase-1 (NQO-1), was significantly augmented by treatment of cells with both agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Medicine, Institute of Health Biosciences, The University of Tokushima Graduate Faculty of Dentistry, Tokushima, Japan.

ABSTRACT

Unlabelled: Chemotherapy-induced oral mucositis is a common adverse event in patients with oral squamous cell carcinoma, and is initiated through a variety of mechanisms, including the generation of reactive oxygen species (ROS). In this study, we examined the preventive effect of γ-tocotrienol on the 5-FU-induced ROS production in human oral keratinocytes (RT7). We treated RT7 cells with 5-FU and γ-tocotrienol at concentrations of 10 µg/ml and 10 nM, respectively. When cells were treated with 5-FU alone, significant growth inhibition was observed as compared to untreated cells. This inhibition was, in part, due to the ROS gene-rated by 5-FU treatment, because N-acetyl cysteine (NAC), a ROS scavenger, significantly ameliorated the growth of RT7 cells. γ-tocotrienol showed no cytotoxic effect on the growth of RT7 cells. Simultaneous treatment of cells with these agents resulted in the significant recovery of cell growth, owing to the suppression of ROS generation by γ-tocotrienol. Whereas 5-FU stimulated the expression of NF-E2-related factor 2 (Nrf2) protein in the nucleus up to 12 h after treatment of RT7 cells, γ-tocotrienol had no obvious effect on the expression of nuclear Nrf2 protein. Of note, the combined treatment with both agents stabilized the 5-FU-induced nuclear Nrf2 protein expression until 24 h after treatment. In addition, expression of Nrf2-dependent antioxidant genes, such as heme oxygenase-1 (HO-1) and

Nad(p)h: quinone oxidoreductase-1 (NQO-1), was significantly augmented by treatment of cells with both agents. These findings suggest that γ-tocotrienol could prevent 5-FU-induced ROS generation by stabilizing Nrf2 activation, thereby leading to ROS detoxification and cell survival in human oral keratinocytes.

Show MeSH
Related in: MedlinePlus