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. 2024 May 30;17(6):712.
doi: 10.3390/ph17060712.

β-Tocotrienol Decreases PDGF-BB-Induced Proliferation and Migration of Human Airway Smooth Muscle Cells by Inhibiting RhoA and Reducing ROS Production

Aditya Sri Listyoko  1 , Ryota Okazaki  1 , Tomoya Harada  1 , Miki Takata  1 , Masato Morita  1 , Hiroki Ishikawa  1 , Yoshihiro Funaki  1 , Akira Yamasaki  1
Affiliations

β-Tocotrienol Decreases PDGF-BB-Induced Proliferation and Migration of Human Airway Smooth Muscle Cells by Inhibiting RhoA and Reducing ROS Production

Aditya Sri Listyoko et al. Pharmaceuticals (Basel). .

Abstract

Background: Tocotrienols exhibit antioxidant and anti-inflammatory activities. RhoA, a small GTPase protein, plays a crucial role in regulating contractility in airway smooth muscle (ASM). Previous studies have demonstrated that γ-tocotrienols reduce ASM proliferation and migration by inhibiting the activation of RhoA. In this present study, we investigate the effect of another vitamin E isoform, β-tocotrienols, on human ASM cell proliferation and migration stimulated by platelet-derived growth factor-BB (PDGF-BB).

Methods: Human ASM cells were pre-treated with β-tocotrienol prior to being stimulated with PDGF-BB to induce ASM cell proliferation and migration. The proliferation and migration of PDGF-BB-induced human ASM cells were assessed using colorimetric and transwell migration assays. The intracellular ROS assay kit was employed to quantify reactive oxygen species (ROS) in human ASM cells. Additionally, we explored the effect of β-tocotrienols on the signaling pathways involved in PDGF-BB-induced ASM proliferation and migration.

Results: β-tocotrienol inhibited PDGF-BB-induced ASM cell proliferation and migration by reducing RhoA activation and ROS production. However, in this present study, β-tocotrienol did not affect the signaling pathways associated with cyclin D1, phosphorylated Akt1, and ERK1/2.

Conclusions: In conclusion, the inhibition of RhoA activation and ROS production by β-tocotrienol, resulting in the reduction in human ASM proliferation and migration, suggests its potential as a treatment for asthma airway remodeling.

Keywords: ASM; ROS; Rho-A; airway remodeling; asthma; tocotrienol; vitamin E.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Effect of β-tocotrienols on ASM cell proliferation. Human ASM cells were cultured with 10 ng/mL PDGF-BB, 5–25 μM β-tocotrienols, or pre-treated with 5–25 μM β-tocotrienols before 10 ng/mL PDGF-BB stimulation. The proliferation of ASM cells was measured using the Cell Counting Kit (CCK-8). The results are presented as a percentage of the non-stimulated (control) group and expressed as the mean ± standard deviation (SD) of at least three independent experiments (*: p < 0.01 compared to PDGF-BB stimulation alone).
Figure 2
Figure 2
Effect of β-tocotrienols on ASM cell migration. Human ASM cells were cultured with 10 ng/mL PDGF-BB, 5–50 μM β-tocotrienols, or pre-treated with 5–50 μM β-tocotrienols before 10 ng/mL PDGF-BB stimulation. The migration of ASM cells was evaluated using a transwell chamber. The results are presented as a percentage of the non-stimulated (control) group and expressed as the mean ± standard deviation (SD) of at least three independent experiments (*: p < 0.01 compared to PDGF-BB stimulation alone).
Figure 3
Figure 3
Effect of β-tocotrienols on ROS production. Human ASM cells were cultured with 10 ng/mL PDGF-BB, 25 μM β-tocotrienols, or pre-treated with 25 μM β-tocotrienols before 10 ng/mL PDGF-BB stimulation. The level of ROS in ASM cells was quantified after stimulation with 10 ng/mL PDGF-BB using the Oxiselect intracellular ROS assay kit. The results are presented as a percentage of the non-stimulated (control) group and expressed as the mean ± standard deviation (SD) of at least three independent experiments (*: p < 0.01 compared to PDGF-BB stimulation alone).
Figure 4
Figure 4
Effect of β-tocotrienols on cyclin D1 expression: (a) human ASM cells were cultured with 10 ng/mL PDGF-BB or pre-treated with 25 μM β-tocotrienols before 10 ng/mL PDGF-BB stimulation. Western blot was used to evaluate the expression of cyclin D1. (b) The graphic represents densitometry calculations of relative protein expression/β-actin. The results are presented as the mean ± standard deviation (SD) of at least three independent experiments.
Figure 5
Figure 5
Effect of β-tocotrienols on Akt1 and ERK1/2 phosphorylation: (a) Human ASM cells were cultured with 10 ng/mL PDGF-BB or pre-treated with 25 μM β-tocotrienols before 10 ng/mL PDGF-BB stimulation. Western blot was used to evaluate the expression of Akt1 and ERK1/2. (b) The graphic represents densitometry calculations of phosphorylated protein targets to total protein. The results are presented as the mean ± standard deviation (SD) of at least three independent experiments.
Figure 6
Figure 6
Effect of β-tocotrienols on RhoA activity: (a) human ASM cells were cultured with 10 ng/mL PDGF-BB or pre-treated with 25 μM β-tocotrienols before 10 ng/mL PDGF-BB stimulation. Rho activity was evaluated using a Rho pull-down assay, and the expression was determined with Western blot analysis. (b) The graphic represents densitometry calculations of active RhoA to total RhoA. The data are presented as the mean ± standard deviation (SD) of at least three independent experiments. (*: p < 0.05, **: p < 0.01, compared to PDGF-BB stimulation).
Figure 7
Figure 7
Schematic representation of the effects of β-tocotrienol on inhibiting ASM proliferation and migration. β-tocotrienol directly inhibits ROS production and induces RhoA inactivation. However, β-tocotrienol may not inhibit the Akt1, ERK, and cyclin signaling pathways. Platelet-derived growth factor-BB (PDGF-BB), G protein-coupled receptor (GPCR), extracellular signal-regulated kinase (ERK), phosphatidylinositol 3′-kinase (PI3K), reactive oxygen species (ROS), and nicotinamide adenine dinucleotide phosphate (NADPH).
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