Beneficial effects of δ-tocotrienol against oxidative stress in osteoblastic cells: studies on the mechanisms of action

Abstract

Purpose Natural antioxidants are considered as promising compounds in the prevention/treatment of osteoporosis. We studied the ability of purified δ-tocotrienol (δ-TT) isolated from a commercial palm oil (Elaeis guineensis) fraction to protect osteoblast MC3T3-E1 and osteocyte MLO-Y4 cells against tert-butyl hydroperoxide (t-BHP)-induced oxidative damage and the mechanisms involved in its protective action in MC3T3-E1. Methods MC3T3-E1 and MLO-Y4 cells were treated with δ-TT (1.25-20 µg/ml for 2 h) followed by t-BHP at 250 µM or 125 µM for 3 h, respectively. MTT test was used to measure cell viability. Apoptotic cells were stained with Hoechst-33258 dye. Intracellular ROS levels were measured by dichlorofluorescein CM-DCFA. The OPT fluorimetric assay was used to detect the reduced glutathione to oxidized glutathione ratio (GSH/GSSG) contents. Results δ-TT significantly prevented the effects of t-BHP on cell viability and apoptosis reaching a maximum protective activity at 10 and 5 µg/ml in MC3T3-E1 and MLO-Y4 cells, respectively. This protective effect was due to a reduction of intracellular ROS levels and an increase in the defense systems shown by the increase in the GSH/GSSG. GSH loss induced by an inhibitor of GSH synthesis significantly reduced the δ-TT-positive effect on ROS levels. δ-TT prevention of oxidative damage was completely removed by combined treatment with the specific inhibitors of PI3K/AKT (LY294002) and Nrf2 (ML385). Conclusions The δ-TT protective effect against oxidative damage in MC3T3-E1 cells is due to a reduction of intracellular ROS levels and an increase of the GSH/GSSG ratio, and involves an interaction between the PI3K/Akt-Nrf2 signaling pathways. Keywords Tocotrienol • Oxidative stress • MC3T3-E1 cells • MLO-Y4 cells • Signaling pathways Abbreviations δ-TT δ-Tocotrienol γ-TT γ-Tocotrienol ROS Reactive oxygen species t-BHP Tert-butyl hydroperoxide FPP Farnesylpyrophosphate GGPP Geranylgeranylpyrophosphate CM-DCFA 5(6)-Carboxy-2′:7-dichlorofluorescein diacetate GSH Glutathione GSSG Glutathione disulfide Nrf2 Nuclear factor-erythroid-2-related factor 2 PI3K/Akt Phosphatidyl-inositol-3-kinase BSO l-Buthionine-sulfoximine

Food for Bone: Evidence for a Role for Delta-Tocotrienol in the Physiological Control of Osteoblast Migration

“…When we studied the involvement of PI3K/Akt pathway in the wound healing activity of δ-TT, we found that δ-TT increased the phosphorylated (activated) form of AKT and that pretreatment with LY294002, a PI3K-specific inhibitor, reversed the wound healing action of δ-TT on MC3T3-E1 cells. These results are in line with previous data showing that PI3K/Akt pathway is involved in the δ-TT-protective action against oxidative stress on MC3t3-E1 cells [16].…”

“…The percentage of cells in each cell cycle phase (G 0 /G 1 , S, and G 2 /M) was determined by flow cytometry and PI DNA staining. We used δ-TT at the concentration (10 μg/mL) previously shown to increase MC3T3-E1 cell viability [ 16 ]. As shown in Figure 1 , δ-TT significantly increased the percentage of cells in G 0/ G 1 and reduced the percentage of the cell in both S and n G 2/ M phases.…”

“…Furthermore, annatto TTs have been shown to enhance the expression of genes involved in both bone formation and osteoblast activity [14,15]. In our previous studies, we have shown that purified δ-TT exerts a stimulatory effect on MC3T3-E1 cell viability and protects MC3T3-E1 cells against oxidative stress [16] suggesting that δ-TT could represent a valuable approach in the prevention and management of osteoblast dysfunction.…”

Comment on: Food for Bone: Evidence for a Role for Delta-Tocotrienol in the Physiological Control of Osteoblast Migration. Int. J. Mol. Sci. 2020, 21, 4661

“…Previously, Casati et al demonstrated that δ-TT induced MC3T3-E1 cell proliferation upon 2 h of treatment [19]. However, in the current study, a 24 h 10 µg/mL of δ-TT inhibited the MC3T3-E1 cell proliferation by causing G1 arrest (Figure 1).…”

Biological Redox Impact of Tocopherol Isomers Is Mediated by Fast Cytosolic Calcium Increases in Living Caco-2 Cells

“…Certainly, the longer the exposure with tocopherol/tocotrienol, the more chances the compounds have to interact with cell components. In osteocyte-like cells, 2 h of treatment with δ-tocotrienol (25 µM) was effective to protect cells against death induced by 3 h of tert-butyl hydroperoxide (250 µM), a protection that was sensitive to the pharmacological inhibition of Nrf2 as well as PI3K signalling [27], supporting the idea that antioxidant properties of tocopherol/tocotrienol are not limited by their ROS scavenging capacity, but also to functional interaction with cellular components.…”