Abstract
Purpose: Breast cancer cells with overexpression of HER2 are known to be more aggressive, invasive, and resistant to chemotherapeutic agent. Brazilin, the major compound in the Caesalpinia sappan L. (CS) heartwood, has been studied for it's anticancer activity. The purpose of this study was to investigate the cytotoxic and antimigratory activity of brazilin (Bi) in combination with doxorubicin (Dox) on MCF-7/HER2 cells.Methods: Cytotoxic activities of Bi individually and in combination with Dox were examined by MTT assay. Synergistic effects were analyzed by combination index (CI). Apoptosis and cell cycle profiles were observed by using flow cytometry. Migrating and invading cells were observed by using a Boyden chamber assay. Levels of MMP2 and MMP9 activity were observed by using a gelatin zymography assay. Levels of HER2, Bcl-2, Rac1, and p120 protein expression were observed by using an immunoblotting assay.Results: The results of the MTT assay showed that Bi inhibited MCF-7/HER2 cell growth in a dose-dependent manner with an IC50 of 54 ± 3.7 µM. Furthermore, the combination of Bi and Dox showed a synergistic effect (CI <1). Flow cytometric analysis of Bi and its combination with Dox showed cellular accumulation in the G2/M phase and induction of apoptosis through suppression of Bcl-2 protein expression. In the Boyden chamber assay, gelatin zymography, and subsequent immunoblotting assay, the combination Bi and Dox inhibited migration, possibly through downregulation of MMP9, MMP2, HER2, Rac1, and p120 protein expression.Conclusion: We conclude that Bi enhanced cytotoxic activity of Dox and inhibited migration of MCF-7/HER2 cells. Therefore, we believe that it has strong potential to be developed for the treatment of metastatic breast cancer with HER2 overexpression.
Highlights
Metastasis is the latest stage of cancer progression and is difficult to overcome.[1]
To confirm whether brazilin enhanced the cytotoxic activity of doxorubicin, we analyzed the synergistic combination by using the combination index (CI)
Combinations of 1/10, 1⁄8, 1⁄4, and 1⁄2 IC50 of brazilin/doxorubicin showed a synergistic effect on inhibition of MCF-7/HER2 cell growth (CI
Summary
Metastasis is the latest stage of cancer progression and is difficult to overcome.[1]. HER2 is a member of the epidermal growth factor receptor family that is overexpressed in many human cancers, especially breast cancer, and is related to invasiveness, drug resistance, and poor prognosis.[4] Overexpression of HER2 induces proliferation, migration, and invasion of cancer cells through its downstream signaling pathway. Overexpression of this protein increases Src synthesis and activates Vav[2], followed by activation of Ras homolog-Guanosine Triphosphate-ases (Rho-GTPases) such as Rac[1], cell division cycle 42 (Cdc42), and Ras homolog A (RhoA) and modulation of cell migration.[5] to activate HER2 signaling-induced migration, p120 catenin (p120) is needed as a Vav[2] substrate. Trastuzumab (Herceptin; Genentech, South San Francisco, CA) is an agent that competitively binds to the extracellular domain of HER2 and inhibits the HER2 signaling
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