Mitochondrial Delivery of Bongkrekic Acid Using a MITO-Porter Prevents the Induction of Apoptosis in Human HeLa Cells

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ABSTRACT

The fact that mitochondrial dysfunction has been implicated in a variety of human diseases suggests that they would be expected as a target organelle for these diseases. Bongkrekic acid (BKA) is a chemical that functions as a ligand of the adenine nucleotide translocator and is known to potently inhibit the mitochondrial permeability transition that is associated with apoptosis. Thus, delivering it to mitochondria would be an innovative therapy for the treatment of mitochondrial diseases that are largely associated with apoptosis. Here, we report on the use of a MITO-Porter, an innovative nanocarrier for mitochondrial delivery via mitochondrial membrane fusion, for delivering BKA to mitochondria. We first constructed a BKA-MITO-Porter, in which BKA is contained in lipid envelopes of a MITO-Porter. We then confirmed that the BKA–MITO-Porter was efficiently internalized into cells and is delivered to mitochondria, similar to a conventional MITO-Porter. Moreover, we evaluated the antiapoptosis effect of the BKA–MITO-Porter in HeLa cells by measuring caspase 3/7 activity. The findings confirmed that the BKA–MITO-Porter showed a strong antiapoptosis effect compared with naked BKA. The results reported here demonstrate its potential for the use in therapies aimed at mitochondrial diseases, as a mitochondrial medicine candidate. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1008–1015, 2013

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INTRODUCTION

Apoptosis is a process of programmed cell death and is a major mechanism involved in the precise regulation of cell numbers and functions as a defense mechanism to remove unwanted and potentially dangerous cells. Mitochondria play a central role in the regulation of apoptosis1., 2. and mitotchondrial dysfunction causes a variety of human disorders.3 Therefore, mitochondria represent an important target organelle for drug therapy,4., 5. and thus therapy promises to be useful and productive for

Materials

1,2-Dioleoyl-sn-glycero-3-phosphatidyl ethanolamine (DOPE) and 7-nitrobenz-2-oxa-1, 3-diazole labeled DOPE (NBD-DOPE) were purchased from Avanti Polar lipids (Alabaster, Alabama). Sphingomyelin (SM)was purchased from Sigma (St. Louis, MO). Stearyl octaarginine (STR-R8)22 was obtained from Kurabo Industries Ltd. (Osaka, Japan). HeLa human cervix carcinoma cells were obtained from RIKEN Cell Bank (Tsukuba, Japan). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and

Construction of BKA-MITO-Porter

We first examined the effect of BKA on the MITO- Porter preparation, where mitochondria-fusogenic lipid composition [DOPE/SM/STR-R8 = 9:2:1 (molar ratio)]17., 19. was used. The BKA–MITO-Porter was prepared using lipid films containing different amounts of BKA (0–40 mol % of total lipid) (Fig 2a). The diameters of the MITO-Porter with different contents of BKA were comparable up to a content ratio of 20 mol% BKA (about 100 nm), while aggregation occurred at higher content of BKA (Fig. 2b, Table S1).

DISCUSSION

Previous studies reported that BKA has a high affinity for the ATP–ADP translocator protein in mitochondria, resulting in the inhibition of mitochondrial oxidative phosphorylation, that is, BKA is clearly a toxic chemical.7., 8. On the other hand, BKA has also been found to be an inhibitor of apoptosis.9., 10., 11. Chen et al.28 recently showed that BKA blocked the loss of mitochondrial membrane potential when apoptosis was induced in cells that had been pretreated with BKA. In this case, it

CONCLUSIONS

In this study, we reported on an approach for the mitochondrial delivery of BKA, an antiapoptosis agent, using a MITO-Porter. We first developed a BKA–MITO-Porter and showed that it is taken up by cells and delivered to mitochondria, similar to that of the conventional MITO-Porter. In addition, the mitochondrial delivery of BKA using the MITO-Porter resulted in an antiapoptosis effect in HeLa cells. These results provide support for the assumption that the mitochondrial delivery of BKA can be

ACKNOWLEDGMENTS

This work was supported, in part by the Advanced research for medical products Mining Programme of the National Institute of Biomedical Innovation (NIBIO), a grant-in-aid for Young Scientists (A) and a grant-in-aid for Scientific Research (S) from the Ministry of Education, Culture, Sports, Science and Technology of Japanese Government (MEXT). We also thank Dr. Milton Feather for his helpful advice in writing the manuscript.

REFERENCES (29)

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