Ann Surg Treat Res.  2025 Feb;108(2):108-123. 10.4174/astr.2025.108.2.108.

Synergistic anticancer effects of mitochondria-targeting peptide combined with paclitaxel in breast cancer cells: a preclinical study

• Ahn J
• Kim OH
• Jin S
• Ryu JH
• Lee D
• Park WC
• Kim SJ

• KMID: 2564250
• DOI: http://doi.org/10.4174/astr.2025.108.2.108

Abstract

Purpose
Mitochondria-accumulating amphiphilic peptide (Mito-FF) was designed to selectively target mitochondria in cancer cells and enhance anticancer effects through its unique structure. Mito-FF consists of (1) diphenylalanine, a β-sheet-forming building block critical for self-assembly; (2) triphenylphosphonium, a mitochondrial targeting moiety guiding the peptide to mitochondria; and (3) pyrene, a fluorescent probe enabling visualization of its accumulation and selfassembly. This study evaluates the anticancer efficacy of Mito-FF in breast cancer cells and explores its combination with paclitaxel, a standard therapy for breast cancer, focusing on its modulation of the epithelial-mesenchymal transition (EMT) pathway.
Methods
In vitro and in vivo experiments were performed using MCF-7 and MDA-MB-231 breast cancer cell lines and their respective xenograft models. Cell viability, migration, EMT marker expression, and apoptosis-related proteins were analyzed.
Results
Mito-FF demonstrated superior inhibition of cell viability and migration compared to paclitaxel alone in both cell lines. Combination therapy with Mito-FF and paclitaxel resulted in enhanced reduction of cell viability and migration. EMT markers were significantly modulated, with decreased mesenchymal markers (Snail and vimentin) and increased epithelial marker (E-cadherin) following combination treatment. Furthermore, the combination therapy synergistically elevated proapoptotic markers such as poly (adenosine diphosphate-ribose) polymerase and reduced anti-apoptotic markers such as myeloid cell leukemia 1. In vivo experiments revealed a marked reduction in tumor volume with combination therapy, accompanied by the highest expression levels of E-cadherin and pro-apoptotic marker Bim.
Conclusion
Mito-FF, designed for mitochondrial targeting and visualization, exhibited potent anticancer effects when combined with paclitaxel, in the breast cancer cells.

Keyword

Breast neoplasms; Epithelial-mesenchymal transition; Mito-FF; Paclitaxel; Triple negative breast neoplasms

Figure

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  Fig. 1 Mito-FF mechanism and synergistic activity of Mito-FF and paclitaxel on cell viability in breast cancer models. (A) The structure of Mito-FF and its mode of action. (B, C) Cell viability assay in MCF-7 cells (B) and MDA-MB-231 cells (C) following treatment of Mito-FF, paclitaxel (PTX), or a combination of Mito-FF + PTX each treatment. The assay found that Mito-FF and PTX reduced the viability of both breast cancer cell lines in a concentration-dependent manner, with a greater reduction observed when the drugs were used in combination, indicating a synergistic effect. Values are presented as mean ± standard deviation of 3 independent experiments. FF, fiber-forming peptides; TPP, triphenylphosphonium; Mito-FF, mitochondria-accumulating amphiphilic peptide; Ct, control. *P < 0.05.

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  Fig. 2 In vitro measurement of cell migration with a wound healing assay. The present study employed a wound healing assay to evaluate the impact of mitochondria-accumulating amphiphilic peptide (Mito-FF), paclitaxel (PTX), and their combination on the migratory capacity of MCF-7 (A) and MDA-MB-231 (B) breast cancer cells. The combination of Mito-FF and paclitaxel significantly reduced cell migration compared to each drug administered individually. Values are presented as mean ± standard deviation of 3 independent experiments. Ct, control. *P < 0.05.

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  Fig. 3 Western blot analysis showing the impact of Mito-FF, paclitaxel (PTX), and their combination on epithelial-mesenchymal transition (EMT) marker expression. The combination of Mito-FF and PTX exhibited a more pronounced effect on EMT inhibition in both MCF-7 (A) and MDA-MB-231 (B) cells, demonstrating the synergistic impact of these drugs. Relative densities of individual markers had been quantified using National Institutes of Health ImageJ software and then were normalized to that of β-actin in each group. Mito-FF, mitochondria-accumulating amphiphilic peptide; Ct, control; E-cadherin, epithelial cadherin. *P < 0.05.

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  Fig. 4 Immunofluorescence of the synergistic impact of Mito-FF and paclitaxel (PTX) on epithelial-mesenchymal transition (EMT) markers in vitro. Immunofluorescence of EMT markers for determining the effects of Mito-FF, PTX, and their combination on EMT in MCF-7 (A) and MDA-MB-231 (B) cells. The combination therapy of Mito-FF and PTX most effectively inhibited EMT, as demonstrated by the highest increase in E-cadherin expression and the lowest decrease in Snail and vimentin expression among all treatment groups. Percentages of immunoreactive areas were measured using National Institutes of Health ImageJ (and expressed as relative values to those in control cells. Values are presented as mean ± standard deviation of 3 independent experiments. Ct, control; Mito-FF, mitochondria-accumulating amphiphilic peptide; E-cadherin, epithelial cadherin. *P < 0.05.

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  Fig. 5 Western blot analysis showing the impact of Mito-FF, paclitaxel (PTX), and their combination on apoptosis-related marker expression. Combination therapy demonstrated a greater increase in poly (adenosine diphosphate-ribose) polymerase (PARP) and a greater decrease in myeloid cell leukemia 1 (MCL-1) and B-cell lymphoma 2 (BCL-2) than either monotherapy in both MCF-7 (A) and MDA-MB-231 (B) cells, indicating a synergistic effect. Relative densities of individual markers had been quantified using National Institutes of Health ImageJ software and then were normalized to that of β-actin in each group. Values are presented as mean ± standard deviation of 3 independent experiments. Mito-FF, mitochondria-accumulating amphiphilic peptide; Ct, control. *P < 0.05.

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  Fig. 6 Effects of monotherapy and combined Mito-FF and paclitaxel (PTX) therapy on mitochondrial reactive oxygen species (ROS) levels in MCF-7 and MDA-MB-231 breast cancer cells. Quantification of superoxide levels in MCF-7 (A) and MDA-MB-231 (B) cells as determined by MitoSOX immunostaining (red signal, Thermo Fisher Scientific). Combination therapy elicited a greater escalation in mitochondrial ROS compared to Mito-FF monotherapy. Mito-FF, mitochondria-accumulating amphiphilic peptide; Ct, control; DAPI, 4’,6-diamidino-2-phenylindole; RFP, red fluorescent protein. *P < 0.05.

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  Fig. 7 Effects of monotherapy and combined Mito-FF and paclitaxel (PTX) therapy on antioxidant enzymes in MCF-7 and MDA-MB-231 breast cancer cells. (A) Western blot analysis showing the expression of antioxidant enzymes superoxide dismutase (SOD) and catalase in MCF-7 cells following treatment with Mito-FF, PTX, and their combination therapy was evaluated using Western blotting. (B) The expression of antioxidant enzymes in MDA-MB-231 cells showing a synergic decrease of the expression in the combination group. Values are presented as mean ± standard deviation of 3 independent experiments. Mito-FF, mitochondria-accumulating amphiphilic peptide; Ct, control. *P < 0.05.

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  Fig. 8 The impact of combining Mito-FF and paclitaxel (PTX) on the tumor volume in the mouse xenograft model. (A) Animal experimental scheme. Representative tumor volume comparison in MCF-7 (B) and MDA-MB-231 (C) mouse xenograft models. Comparison of tumor volumes (top) and body weights (bottom) in MCF-7 (D) and MDA-MB-231 (E) mouse xenograft models. Values are presented as mean ± standard deviation of 3 independent experiments. Mito-FF, mitochondria-accumulating amphiphilic peptide; Ct, control. *P < 0.05.

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  Fig. 9 Immunohistochemistry analysis of the synergistic impact of Mito-FF and paclitaxel (PTX) on epithelial-mesenchymal transition (EMT) markers and apoptosis-related markers in vivo. In both the MCF-7 (A) and MDA-MB-231 (B) mouse xenograft models, the combination therapy resulted in the highest expression of epithelial cadherin (E-cadherin) and the lowest expression of Snail, indicating a synergistic inhibitory effect on EMT. In addition, in both the MCF-7 (C) and MDA-MB-231 (D) mouse xenograft models, the combination therapy exhibited the highest expression of the pro-apoptotic marker Bim and the lowest expression of the anti-apoptotic marker B-cell lymphoma-extra large (Bcl-xL), indicating a synergistic inhibitory effect on apoptosis. Percentages of immunoreactive areas were measured using National Institutes of Health ImageJ and expressed as relative values to those in control tissues. Values are presented as mean ± standard deviation of 3 independent experiments. Mito-FF, mitochondria-accumulating amphiphilic peptide; Ct, control. *P < 0.05.

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  Fig. 10 Proposed mechanism of action for Mito-FF in regulating epithelial-mesenchymal transition (EMT) in cancer cells. Tumor cells exhibit hypermetabolism characterized by increased electron flow in the electron transport chain (ETC), leading to elevated reactive oxygen species (ROS) levels and reduced antioxidant enzyme activity. This metabolic shift promotes tumor cell survival by impeding apoptosis and facilitating EMT. Mito-FF disrupts tumor cell hypermetabolism by inducing mitochondrial impairment, resulting in enhancing tumor cell apoptosis and inhibiting EMT. Mito-FF, mitochondria-accumulating amphiphilic peptide.

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