Annatto-Derived Tocotrienol Promotes Mineralization of MC3T3-E1 Cells by Enhancing BMP-2 Protein Expression Via Inhibiting RhoA Activation and HMG-CoA Reductase Gene Expression

Overview

Journal Drug Des Devel Ther

Specialty Pharmacology

Date 2020 Mar 19

PMID 32184566

Citations 9

Authors

Wan Nuraini Wan Hasan

Kok-Yong Chin

Norzana Abd Ghafar

Ima Nirwana Soelaiman

Affiliations

Soon will be listed here.

Abstract

Purpose: Annatto-derived tocotrienol (AnTT) has been shown to improve bone formation in animal models of osteoporosis and promote differentiation of pre-osteoblastic cells. However, the mechanism of action of AnTT in achieving these effects is unclear. This study aims to investigate the mechanism of action of AnTT on MC3T3-E1 pre-osteoblasts via the mevalonate pathway.

Methods: Murine pre-osteoblastic cells, MC3T3-E1, were cultured with the density of 1 × 10 cells/mL and treated with 4 concentrations of AnTT (0.001-1 µg/mL). Expression of HMG-CoA reductase (HMGR) gene was carried out using qPCR after treatment with AnTT for 21 days. RhoA activation and bone morphogenetic protein-2 (BMP-2) were measured using immunoassay after 9 and 15 days of AnTT treatment. Lovastatin was used as the positive control. Mineralized nodules were detected using Von Kossa staining after 21 days of AnTT treatment.

Results: The results showed that HMGR was up-regulated in the lovastatin group on day 9 and 21 compared to the control. Lovastatin also inhibited RhoA activation (day 9 and 15) and increased BMP-2 protein (day 15). On the other hand, AnTT at 0.001 μg/mL (day 3) and 0.1 μg/mL (day 21) significantly down-regulated HMGR gene expression compared to the control. On day 21, HMGR gene expression was significantly reduced in all groups compared to day 15. AnTT at 0.1 μg/mL significantly decreased RhoA activation on day 9 compared to the control. AnTT at 1 μg/mL significantly increased BMP-2 protein on day 15 compared to the control (P<0.05). Mineralized calcium nodules were more abundant in AnTT treated groups compared to the control on day 21.

Conclusion: AnTT suppresses the mevalonate pathway by downregulating HMGR gene expression and inhibiting RhoA activation, leading to increased BMP-2 protein in MC3T3-E1 cells. This explains the stimulating effects of AnTT on osteoblast mineralization.

Citing Articles

Palm Tocotrienol Activates the Wnt3a/β-Catenin Signaling Pathway, Protecting MC3T3-E1 Osteoblasts from Cellular Damage Caused by Dexamethasone and Promoting Bone Formation.

Abdullah Sani N, Kamaruddin N, Soelaiman I, Pang K, Chin K, Ramli E Biomedicines. 2025; 13(1).

PMID: 39857826 PMC: 11762645. DOI: 10.3390/biomedicines13010243.

Prospective and challenges of locally applied repurposed pharmaceuticals for periodontal tissue regeneration.

El-Nablaway M, Rashed F, Taher E, Abdeen A, Taymour N, Soliman M Front Bioeng Biotechnol. 2024; 12:1400472.

PMID: 39605747 PMC: 11600316. DOI: 10.3389/fbioe.2024.1400472.

Updates in the skeletal and joint protective effects of tocotrienol: a mini review.

Chin K Front Endocrinol (Lausanne). 2024; 15:1417191.

PMID: 38974581 PMC: 11224474. DOI: 10.3389/fendo.2024.1417191.

Role of vitamins beyond vitamin D in bone health and osteoporosis (Review).

Skalny A, Aschner M, Tsatsakis A, Rocha J, Santamaria A, Spandidos D Int J Mol Med. 2023; 53(1).

PMID: 38063255 PMC: 10712697. DOI: 10.3892/ijmm.2023.5333.

Molecular Mechanism of Tocotrienol-Mediated Anticancer Properties: A Systematic Review of the Involvement of Endoplasmic Reticulum Stress and Unfolded Protein Response.

Pang K, Mai C, Chin K Nutrients. 2023; 15(8).

PMID: 37111076 PMC: 10145773. DOI: 10.3390/nu15081854.

References

Dhanwal D, Dennison E, Harvey N, Cooper C . Epidemiology of hip fracture: Worldwide geographic variation. Indian J Orthop. 2011; 45(1):15-22. PMC: 3004072. DOI: 10.4103/0019-5413.73656. View

. Assessment of fracture risk and its application to screening for postmenopausal osteoporosis. Report of a WHO Study Group. World Health Organ Tech Rep Ser. 1994; 843:1-129. View

Matozaki T, Nakanishi H, Takai Y . Small G-protein networks: their crosstalk and signal cascades. Cell Signal. 2000; 12(8):515-24. DOI: 10.1016/s0898-6568(00)00102-9. View

Yang Z, Xiao H, Jin H, Koo P, Tsang D, Yang C . Synergistic actions of atorvastatin with gamma-tocotrienol and celecoxib against human colon cancer HT29 and HCT116 cells. Int J Cancer. 2009; 126(4):852-63. PMC: 2981082. DOI: 10.1002/ijc.24766. View

Siegel P, Massague J . Cytostatic and apoptotic actions of TGF-beta in homeostasis and cancer. Nat Rev Cancer. 2003; 3(11):807-21. DOI: 10.1038/nrc1208. View

Sakakura Y, Shimano H, Sone H, Takahashi A, Inoue N, Toyoshima H . Sterol regulatory element-binding proteins induce an entire pathway of cholesterol synthesis. Biochem Biophys Res Commun. 2001; 286(1):176-83. DOI: 10.1006/bbrc.2001.5375. View

Abdul-Majeed S, Mohamed N, Soelaiman I . The use of delta-tocotrienol and lovastatin for anti-osteoporotic therapy. Life Sci. 2014; 125:42-8. DOI: 10.1016/j.lfs.2014.12.012. View

Rachner T, Khosla S, Hofbauer L . Osteoporosis: now and the future. Lancet. 2011; 377(9773):1276-87. PMC: 3555696. DOI: 10.1016/S0140-6736(10)62349-5. View

Cejka D, Benesch T, Krestan C, Roschger P, Klaushofer K, Pietschmann P . Effect of teriparatide on early bone loss after kidney transplantation. Am J Transplant. 2008; 8(9):1864-70. DOI: 10.1111/j.1600-6143.2008.02327.x. View

10.

Kempf T, Sinning J, Quint A, Bickel C, Sinning C, Wild P . Growth-differentiation factor-15 for risk stratification in patients with stable and unstable coronary heart disease: results from the AtheroGene study. Circ Cardiovasc Genet. 2009; 2(3):286-92. DOI: 10.1161/CIRCGENETICS.108.824870. View

11.

Tsuji K, Cox K, Gamer L, Graf D, Economides A, Rosen V . Conditional deletion of BMP7 from the limb skeleton does not affect bone formation or fracture repair. J Orthop Res. 2009; 28(3):384-9. PMC: 2862367. DOI: 10.1002/jor.20996. View

12.

Zhang F, Casey P . Protein prenylation: molecular mechanisms and functional consequences. Annu Rev Biochem. 1996; 65:241-69. DOI: 10.1146/annurev.bi.65.070196.001325. View

13.

Ohnaka K, Shimoda S, Nawata H, Shimokawa H, Kaibuchi K, Iwamoto Y . Pitavastatin enhanced BMP-2 and osteocalcin expression by inhibition of Rho-associated kinase in human osteoblasts. Biochem Biophys Res Commun. 2001; 287(2):337-42. DOI: 10.1006/bbrc.2001.5597. View

14.

Chin K, Ima-Nirwana S . The biological effects of tocotrienol on bone: a review on evidence from rodent models. Drug Des Devel Ther. 2015; 9:2049-61. PMC: 4396581. DOI: 10.2147/DDDT.S79660. View

15.

Kempf T, Wollert K . Growth differentiation factor-15: a new biomarker in cardiovascular disease. Herz. 2009; 34(8):594-9. DOI: 10.1007/s00059-009-3317-3. View

16.

Park Y, Kim J, Kim D, Kim E, Park S, Park J . The Bone Morphogenesis Protein-2 (BMP-2) is associated with progression to metastatic disease in gastric cancer. Cancer Res Treat. 2009; 40(3):127-32. PMC: 2697466. DOI: 10.4143/crt.2008.40.3.127. View

17.

Bandyopadhyay A, Tsuji K, Cox K, Harfe B, Rosen V, Tabin C . Genetic analysis of the roles of BMP2, BMP4, and BMP7 in limb patterning and skeletogenesis. PLoS Genet. 2006; 2(12):e216. PMC: 1713256. DOI: 10.1371/journal.pgen.0020216. View

18.

Oxlund H, Andreassen T . Simvastatin treatment partially prevents ovariectomy-induced bone loss while increasing cortical bone formation. Bone. 2004; 34(4):609-18. DOI: 10.1016/j.bone.2003.12.014. View

19.

Wong S, Chin K, Suhaimi F, Ahmad F, Ima-Nirwana S . Exploring the potential of tocotrienol from Bixa orellana as a single agent targeting metabolic syndrome and bone loss. Bone. 2018; 116:8-21. DOI: 10.1016/j.bone.2018.07.003. View

20.

Liberman U, Weiss S, Broll J, Minne H, Quan H, Bell N . Effect of oral alendronate on bone mineral density and the incidence of fractures in postmenopausal osteoporosis. The Alendronate Phase III Osteoporosis Treatment Study Group. N Engl J Med. 1995; 333(22):1437-43. DOI: 10.1056/NEJM199511303332201. View