Cytotoxic and Antimetastatic Activity of Hesperetin and Doxorubicin Combination Toward Her2 Expressing Breast Cancer Cells

Figures

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  Figure 1. Cytotoxicity of Hst, Dox, and Their Combination on MCF-7/HER2 Cells. The 1.5x103 MCF-7/HER2 cells were seeded into 96 well plates and treated with Hst, Dox, and the combination on the following days. The cytotoxic assay was performed using the MTT method, as described in methods. (A), Percentage of cell viability after a single treatment of Hst and Dox at various concentrations for 24 h. Profiles of cell viability were expressed as mean ± SD from 3 independent experiments; (B), Change of cells morphology after Hst and Dox treatment for 24 h. Arrows indicate changes in each cell morphology; (C), Effect of the combination of Hst and Dox treatment on cell viability; (D), Index combination value; (E), Combination index plot, and (F), Isobologram for a combination of Hst and Dox measured by CompuSyn® software based on Chou-Talalay method

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  Figure 2. Effect of Hst and Dox on Cell Cycle and HER2 Expression of MCF-7/HER2. MCF-7/HER2 was seeded into a 6-well plate, added with Hst Dox, and the Combination and Incubated for 24 h. Cells were harvested and added with PI reagent and analyzed using a flow cytometer. (A), Cell distribution profile of (i) cell control (ii) Dox 0.2 μM (iii) Hst 95 μM dan (iv) combination Hst and Dox; (B), Percentage of the cell population in each phase after various treatments; (C), HER2 expression was examined using western blot. Band visualization was obtained using Luminograph; (D), Band obtained were quantified using ImageJ software showed an intensity ratio of HER2 and ß-actin after treatment with Hst, Dox, and the combination showed that the treatment decreased HER2 expression

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  Figure 3. Effect of Hst, Dox, and Combination on Cell Apoptosis. The 1.5x105 cells were seeded into a 6-well plate and incubated for 24 hours. The next day, each well was treated with Hst, Dox, and the combination for 24 hours. Furthermore, samples were stained with Annexin V and propidium iodide (PI) and analyzed using flow cytometry. (A), Profile of cell distribution after treatment consists of living cells (bottom-left), cells undergo early apoptosis (bottom-right), late apoptosis (upper-right), and necrosis (upper-left); (B), Percentage of cells undergo early and late apoptosis

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  Figure 4. Effect of Hst, Dox, and Their Combination on Lamellipodia Formation and Cells Migration. An inverted microscope was used to observe lamellipodia formation after treatment with 100x magnification. (A), Morphological change after Hst, Dox, and the combination treatment. White arrow showed lamellipodia of a cell, and observation was done at a certain interval of time; (B), Inhibition of cell migration induced by Dox 10 nM was examined using scratch wound healing assay. Scratch area was observed at a certain time using an inverted microscope at 100x magnification; (C), Percent closure after Hst, Dox, and the combination treatment at 24 and 48 h measured using ImageJ software

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  Figure 5. Effect of Hst and Dox on Rac1 and MMP9 Expression. MMP9 expression was performed using gelatin zymography, meanwhile, Rac1 using western blot. (A), Band of Rac1 and MMP-9 after Hst, Dox, and combination treatment. The band was quantified using ImageJ software; (B), A percentage of MMP9 expression relative to control cells; (C), Percentage of Band Ratio of Rac1 between treatment and control cells