Inhibitory effects of magnolol and honokiol on human calcitonin aggregation

Abstract

Amyloid formation is associated with multiple amyloidosis diseases. Human calcitonin (hCT) is a typical amyloidogenic peptide, its aggregation is associated with medullary carcinoma of the thyroid (MTC), and also limits its clinical application. Magnolia officinalis is a traditional Chinese herbal medicine; its two major polyphenol components, magnolol (Mag) and honokiol (Hon), have displayed multiple functions. Polyphenols like flavonoids and their derivatives have been extensively studied as amyloid inhibitors. However, the anti-amyloidogenic property of a biphenyl backbone containing polyphenols such as Mag and Hon has not been reported. In this study, these two compounds were tested for their effects on hCT aggregation. We found that Mag and Hon both inhibited the amyloid formation of hCT, whereas Mag showed a stronger inhibitory effect; moreover, they both dose-dependently disassembled preformed hCT aggregates. Further immuno-dot blot and dynamic light scattering studies suggested Mag and Hon suppressed the aggregation of hCT both at the oligomerization and the fibrillation stages, while MTT-based and dye-leakage assays demonstrated that Mag and Hon effectively reduced cytotoxicity caused by hCT aggregates. Furthermore, isothermal titration calorimetry indicated Mag and Hon both interact with hCT. Together, our study suggested a potential anti-amyloidogenic property of these two compounds and their structure related derivatives.

Figure 1. Structures of hCT and compounds.

(A) Primary sequence of hCT with a disulfide bridge between Cys-1 and Cys-7 and C terminus amidated; (B–D) Chemical structures of EGCG (B), magnolol (C) and honokiol (D).

Figure 2. Inhibitory effects of compounds on hCT amyloid formation.

(A-B) ThT fluorescence of hCT aggregation in the absence or presence of different ratios of magnolol (A) and honokiol (B). *P < 0.05; (C–J) TEM images of hCT aggregates in the absence or presence of different compounds. (C) hCT alone; (D) 1:1 mixture of hCT and EGCG; (E–G) Mixtures of hCT and magnolol with molar ratios of 1:1 (E), 1:3 (F) and 1:5 (G); (H–J) Mixtures of hCT and honokiol with molar ratios of 1:1 (H), 1:3 (I) and 1:5 (J). Scale bar represents 200 nm.

Figure 3. Inhibitory effects of magnolol and honokiol on hCT oligomerization and fibrillation detected by dot blot.

Figure 4. Size distribution of hCT aggregates detected by dynamic light scattering in the absence or presence of different compounds.

Figure 5. Effects of magnolol and honokiol on cytotoxicity and membrane disruption caused by hCT aggregates.

(A) Cell viability of SH-SY5Y cells in the absence or presence of different compounds determined by MTT-based assay; (B) Dye leakage levels induced by hCT in the presence of compounds. 0.2% Triton X-100 was used as the positive control. ***P < 0.05.

Figure 6. Disaggregation effects of magnolol and honokiol on hCT aggregates.

(A,B) ThT fluorescence of disaggregation effects of magnolol (A) and honokiol (B). (C–F) TEM images of disaggregation effects of different compounds. (C) hCT incubated for 96 h; (D) hCT aggregates incubated with equimolar of EGCG for 48 h; (E–F) hCT aggregates incubated with 5-fold amounts of magnolol (E) and honokiol (F) for 48 h; Scale bar represents 500 nm. (G–I) Quantitative analysis of hCT concentration in the supernatant after disaggregation for 0 h (G), 24 h (H) and 48 h (I) in the presence of different compounds determined by bicinchoninic acid (BCA) assay.

Figure 7. A schematic representation of how magnolol and honokiol affected hCT aggregation.