Korean J Physiol Pharmacol.  2022 Jan;26(1):15-24. 10.4196/kjpp.2022.26.1.15.

Gallic acid-mitochondria targeting sequence-H₃R₉ induces mitochondria-targeted cytoprotection

• Bae Y
• Kim GY
• Jessa F
• Ko KS
• Han J

• KMID: 2524507
• DOI: http://doi.org/10.4196/kjpp.2022.26.1.15

Abstract

The development of selective targeting of drug molecules towards the mitochondria is an important issue related to therapy efficacy. In this study, we report that gallic acid (GA)-mitochondria targeting sequence (MTS)-H₃R₉ exhibits a dual role as a mitochondria-targeting vehicle with antioxidant activity for disease therapy. In viability assays, GA-MTS-H₃R₉ showed a better rescue action compared to that of MTS-H₃R₉ . GA-MTS-H₃R₉ dramatically exhibited cell penetration and intercellular uptake compared to MTS and fit escape from lysosome release to the cytosol. We demonstrated the useful targeting of GA-MTS-H₃R₉ towards mitochondria in AC16 cells. Also, we observed that the antioxidant properties of mitochondrial-accrued GA-MTSH₃R₉ alleviated cell damage by reactive oxygen species production and disrupted mitochondrial membrane potential. GA-MTS-H₃R₉ showed a very increased cytoprotective effect against anticancer activity compared to that of MTS-H₃R₉ . We showed that GA-MTS-H₃R₉ can act as a vehicle for mitochondria-targeting and as a reagent for therapeutic applications intended for cardiovascular disease treatment.

Keyword

Antioxidant activity; Gallic acid; Mitochondria

Figure

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  Fig. 1 Schematic description for the localization of the GA-MTS-H3R9 inside cells and its cytoprotective effect.

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  Fig. 2 Cell viability assay with GA-MTS-H3R9. Cell viability was determined using WST-1 and lactate dehydrogenase (LDH) assay. AC16 cells were exposed to different concentrations of GA, MTS, MTS-H3R6, MTS-H3R9, GA-MTS-H3R6, and GA-MTS-H3R9 up to 50 μM for 24 h. Panel (A) presents the results of the WST-1 assay and Panel (B) presents the results of LDH assay. GA, gallic acid; MTS, mitochondria targeting sequence. Data are indicted as the mean ± SD (n = 3).

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  Fig. 3 Membrane internalization by GA-MTS-H3R9. AC16 cells were treated with 3.125 and 6.25 μM of GA, MTS, MTS-H3R6, MTS-H3R9, GA-MTS-H3R6, and GA-MTS-H3R9 for 16 h and were subsequently incubated with 5 μl of PI for 20 min. (A) PI internalization using flow cytometry analysis. (B) Quantitative graph showing each peptide from the PI fluorescence data shown in panel (A). GA, gallic acid; MTS, mitochondria targeting sequence; PI, propidium iodide.

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  Fig. 4 Intracellular uptake of GA-MTS-H3R9. AC16 cells were treated with 3.125 and 6.25 μM of FITC-labeled GA, MTS, MTS-H3R6, MTS-H3R9, GA-MTS-H3R6, and GA-MTS-H3R9 for 16 h. (A) Cellular uptake of each FITC-labeled peptide using flow cytometry analysis. (B) Quantitative graph showing each peptide from uptake data depicted in panel (A). GA, gallic acid; MTS, mitochondria targeting sequence.

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  Fig. 5 Intracellular distribution of GA-MTS-H3R9. AC16 cells were treated with 1 μM of FITC-labeled GA, MTS, MTS-H3R6, MTS-H3R9, GA-MTS-H3R6, and GA-MTS-H3R9 for 16 h. The cells were stained with LysoTracker (red) for 20 min and counterstained with Hoechst stain (blue) for 10 min and imaged by confocal microscopy. Scale bar is 10 µm. GA, gallic acid; MTS, mitochondria targeting sequence.

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  Fig. 6 Intracellular mitochondria localization of GA-MTS-H3R9. AC16 cells were exposed to 1 μM of FITC-labeled GA, MTS, MTS-H3R6, MTS-H3R9, GA-MTS-H3R6, and GA-MTS-H3R9 for 24 h. The cells were stained with MitoTracker (red) for 20 min and counterstained with Hoechst stain (blue) and then imaged by confocal laser scanning microscopy. Scale bar is 20 µm. GA, gallic acid; MTS, mitochondria targeting sequence.

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  Fig. 7 ROS levels and mitochondrial membrane potential (MMP) with GA-MTS-H3R9. (A) AC16 cells were exposed to 6.25 μM of GA, MTS, MTS-H3R6, MTS-H3R9, GA-MTS-H3R6, and GA-MTS-H3R9 for 16 h. Intracellular ROS levels were assessed by GSH assay. (B) AC16 cells were treated using the same conditions as those panel (A). The cells were treated with 20 μM MitoSox for 16 h. Cells were imaged by confocal microscopy. Scale bar is 10 µm. (C) AC16 cells were treated using the same conditions as those in panel (A). The cells were added to 2 μM JC-1 and then incubated for 20 min. MMP was determined by flow cytometry analysis. Data are indicted as the mean ± SD (n = 3). ROS, reactive oxygen species; GA, gallic acid; GSH, glutathione; MTS, mitochondria targeting sequence; CCCP, carbonyl cyanide 3-chlorophenylhydrazone. **p < 0.01.

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  Fig. 8 Induction of cytoprotection by GA-MTS-H3R9. (A) Anticancer activity of each peptide. AC16 cells were exposed to 6.25 μM of GA, MTS, MTS-H3R6, MTS-H3R9, GA-MTS-H3R6, and GA-MTS-H3R9 for 24 h. Apoptosis levels for each group were measured by flow cytometry analysis after annexin V staining. Q1 indicates necrotic cells. Q2 indicates late apoptotic cells, Q3 indicates intact cells, and Q4 indicates early apoptotic cells. (B) Caspase-3 activity of each peptide. AC16 cells were treated using the same conditions as those in panel (A). Caspase-3 activity was assessed as described in Methods. Data are indicted as the mean ± SD (n = 3). GA, gallic acid; MTS, mitochondria targeting sequence. **p < 0.01.

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