Oxidative stress sensitivity of cancer cells could be altered by plasma membrane status
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.
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
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