Synergistic Cytotoxic and Antimigratory Effect of Brazilein and Doxorubicin on HER2-Overexpressing Cells

Abstract

Objective: The present research aims to report cytotoxic and antimigratory activities of the oxidized form of brazilin, i.e., brazilein, and the effects of the combination of brazilein-doxorubicin on MCF-7/HER2 cells.

Methods: The MTT assay was conducted to test the cytotoxic activity, while flow cytometry with PI and PI-annexin V staining were respectively performed for cell cycle and apoptosis analyses. Migration and invasion analyses were assessed via Boyden chamber assay, while HER2, Rac1, p120, MMP2, and MMP9 protein levels were determined by immunoblotting and gelatin zymography. Molecular docking of ligands with HER2, Src, PI3Kα, PI3KΔ, and PI3Kγ proteins was evaluated using MOE 2010.

Results: The MTT assay showed that the IC50 value of brazilein against MCF-7/HER2 cells was 51 ± 2.1 µM. Moreover, brazilein and its combination with doxorubicin-induced G2/M accumulation and apoptosis. Combination of brazilein-doxorubicin inhibited cell migration and tended to decrease HER2, Rac1, p120, MMP2, and MMP9 protein expression levels. Based on our molecular docking study, the docking score of brazilein with PI3Kγ is comparable to that of the native ligand.

Conclusion: Taken together, a combination of brazilein-doxorubicin exhibited synergistic cytotoxic and antimigratory effects on MCF-7/HER2 cells.<br />.

Keywords: Caesalpinia sappan L; Combination therapy; Molecular docking; PI3K; matrix metalloproteinase.

Figure 1

Cytotoxic Activity of Brazilein (Be) Alone and in Combination with Doxorubicin (Dox) in MCF-7/HER2 Cells. Structure of brazilin (1A, left) and brazilein (1A, right). Cytotoxic activity was measured by MTT assay after 24 h of treatment. (B) Effects of Be on the viability of MCF-7/HER2 cells. (C) The combination of Be and Dox at sub IC₅₀ as indicated in the graph. (D) Combination indices (CIs) between Be and Dox in MCF-7/HER2 cells. Data are expressed as the mean ± SD of three independent experiments. *P < 0.05

Figure 2

Alterations in MCF-7/HER2 Cell Cycle Profiles after Single and Combination Treatment with Brazilein (Be) and Doxorubicin (Dox). (A) Control cells (treated with vehicle) or cells treated with ½ IC₅₀ Be (B), ½ IC₅₀ Dox (C), or the combination of ½ IC₅₀ Be or ½ IC₅₀ Dox (D) for 24 h were subjected to cell cycle analysis by flow cytometry with PI/RNase staining as described in the Methods section

Figure 3

Effects of Co-Treatment of Brazilein (Be) and Doxorubicin (Dox) on MCF-7/HER2 Cell Migration and Invasion. Cells were treated with ¼ IC₅₀ Be alone or in combination with Dox for 24 h in serum-starved medium. Cell (A) migration and (B) invasion below the kit chamber were observed using the protocol described in the Methods section. (C) Detection of MMP protein bands by using gelatin zymography. (D) Quantification of the gelatinase activity of the MMPs. (E) Expression levels of migration- and invasion-regulatory proteins detected by using Western blot. Data are expressed as the mean ± SD of three independent experiments. *P < 0.05

Figure 4

Docking Visualization of the Ligand–Protein Interactions of Brazilein and Brazilin with Src, PI3Kα, PI3KΔ, and PI3Kγ Proteins