Oxidative stress is defined as “A status that the balance between the oxidation reaction and the antioxidant reaction is lost in our body, and that the oxidation state is dominant.” Oxidative stress is generally adverse for our bodies because it can impair DNA, protein, and lipids. Oxidative stress is caused by reactive oxygen species (ROS) such as active oxygen (O₂⁻), hydrogen peroxide (H₂O₂), hydroxyl radicals (•OH), and singlet oxygen (¹O₂), which are generated as a result of electron reduction of oxygen.

ROS are key molecules in maintaining cell proliferation, inflammation, and cell death. ROS protect us by killing bacteria that invade our bodies, but these are also involved in aging and may cause diseases such as Parkinson’s disease, Alzheimer’s disease, cancer, diabetes mellitus, and periodontal disease. In addition, when oxidative stress, such as due to radiation or drugs, is externally applied, the amount of intracellular ROS increases or cell membrane lipid peroxidization occurs and eventually results in cell death. The primary site of ROS generation in vivo is mitochondria. It has been revealed that ROS produced in mitochondria exhibit biological defense, as mentioned above.

Fig. 1. The scheme of relationship between H2O2 and changes in plasma membrane peroxidation status in parent and ρ⁰ cells. The red membrane lipids indicate lipoperoxide. Dysfunction of mitochondria in ρ⁰ cells could reduce the plasma membrane potential through the production of ATP or accelerate membrane lipid peroxidation status, which could cause cell death by external H₂O₂ administration. The enhancement of lipid peroxidation in ρ0 cells might be explained by the increase in internal ROS via mitochondrial dysfunction due to depleted mtDNA (complete lack of mtDNA). (Tomita K. et al., J. Tohoku Med. Pharm. Univ.64 49-55 2017.)

Mitochondria have their own DNA (mtDNA) that partly encodes oxidative phosphorylation component proteins, and mitochondrial dysfunction occurs when mtDNA is impaired. Therefore, mtDNA impaired cells can serve as valuable cell models for studying oxidative stress and overcoming ROS-derived diseases. In addition, H₂O₂ is recognized as a sensitizer of anti-cancer therapy. A further understanding of H₂O₂ action mechanisms in cell models is necessary for the development of new anti-cancer therapeutic agents, protocols, or strategies.

To analyze the mechanism of oxidative stress and ROS-derived diseases, mtDNA depleted cells (ρ⁰ cells) were developed by treatment with low-dose ethidium bromide. We established two ρ⁰ cell lines, namely cervical cancer (HeLa) and tongue squamous cell carcinoma (SAS).

We investigated whether established ρ⁰ cells contain mtDNA, extracted RNA from the cells, and performed quantitative PCR (qPCR) for ND1, which is an mtDNA-encoded gene. Complete ND1 expression silencing was observed in ρ⁰ cells. Next, we tested H₂O₂ sensitivity. Higher sensitivity was seen in ρ⁰ cells than in their parental cells. We also investigated the activity of catalase, which is an enzyme that decomposes H₂O₂ in cells. In ρ⁰ cells, higher catalase activity was observed despite the higher H₂O₂ sensitivity. The expression of other antioxidative enzyme genes was investigated, but their expression seemed unrelated to H₂O₂ sensitivity. We further investigated cell surface plasma membrane status using DiBAC₄(3), a potential-dependent fluorescence dye. The fluorescence intensities were statistically lower in ρ⁰ cells with plasma membrane potential. We confirmed the internal amount of H₂O₂ after H₂O₂ administration and found that H₂O₂ membrane permeability was facilitated in ρ⁰ cells. We also found that the internal amount of H₂O₂ was higher in ρ⁰ cells than in the parent cells after 1h of H₂O₂ administration.

Our results suggest that changes in the plasma membrane status induced by increased internal ROS generation provide a hyper-permeability of H₂O₂ in ρ⁰ cells after H₂O₂ administration. The extracellular H₂O₂ administration to ρ⁰ cells may lead to higher internal ROS generation including the external H₂O₂ permeating the cell. The lipid peroxidation of plasma membranes induced by internal ROS (including the external permeated H₂O₂) will eventually result in cell death.

These results suggest a new anti-cancer therapeutic protocol if the membrane peroxidation status can be changed using selective anti-cancer agents or mitochondrial functional inhibitors. In future, we will further investigate how membrane status may be used to enhance the therapeutic effect on cancer.

Yuko Takashi ^1,2, Kazuo Tomita ¹, Yoshikazu Kuwahara ^1,3,4, Akihiro Kurimasa ³,
Manabu Fukumoto ^4,5, Yoshihiro Nishitani ², Tomoaki Sato ^1
1Department of Applied Pharmacology, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima,
Kagoshima, 8908544, Japan
²Department of Restorative Dentistry and Endodontology, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka,
Kagoshima, Kagoshima, 8908544, Japan
³Department of Radiation Biology and Medicine, Faculty of Medicine, Tohoku Medical and Pharmaceutical University, 4-4-1, Komatsushima, Aoba-ku,
Sendai, Miyagi, 9818558, Japan
⁴Department of Pathology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi 4-1, Aoba-ku, Sendai, Miyagi, 9808575, Japan
⁵Department of Molecular Pathology, Tokyo Medical University, 6-1-1, Shinjuku, Shinjuku-ku, Tokyo, 1608402, Japan

Publication

Sensitivity of mitochondrial DNA depleted ρ0 cells to H2O2 depends on the plasma membrane status.
Tomita K, Kuwahara Y, Takashi Y, Tsukahara T, Kurimasa A, Fukumoto M, Nishitani Y, Sato T
Biochem Biophys Res Commun. 2017 Aug 19

Read offline:     

Related Articles:

	Short hydrogen bonds in proteins and their quantum… The assembly of linear polypeptide chains into functional three-dimensional protein architectures involves a unique force called hydrogen bonding. A typical hydrogen bond forms when the donor and acceptor groups approach…
	Defects in mismatch repair increase cancer risk and… Microsatellites are formed by 1-6 nucleotide base pairs that are repeated in direct order 5-50 times. Repetitive DNA including microsatellites are mainly present in non-coding DNA regions that occupy more…
	Intestinal inflammation mediated by silver… The interest in silver nanoparticles (AgNP) has been growing exponentially around the world, due to their unique physical, chemical and biological properties, as evidenced by their wide application in numerous…
	Murburn concept explains why oxygen is acutely… Murburn concept (from “mured burning”, signifying a restricted uncontrolled oxidative process) provides a tangible rationale why oxygen is so quintessential for immediate maintenance of life-order. It postulates that oxygen-centered diffusible…
	The complex regulation and functional significance… Across animals, variation in body size is often influenced by environmental conditions and is considered to have adaptive value. In bees, in which many species show various degrees of sociality,…
	Could mometasone furoate be a viable glucocorticoid… Glucocorticoids are medications widely used to treat inflammation and allergies, and in contexts of immunosuppression such as after transplants. They are well-established and safe. The problem is that, when used…