Contributing to cancer treatment by tumor-selective toxicity

Biological impacts of reactive oxygen species (ROS)

Without dioxygen (so-called oxygen, O₂) in the air, most living things including us could not live while the molecule by itself is highly toxic for them, and one call this nature as oxygen toxicity. We can survive owing to the occurrence of an adequate concentration (20%) of oxygen in the air. Imagine that the air was solely composed of oxygen (it means the oxygen concentration in the air was 100%!), we would die at once by severe oxidative damages of virtually all organs especially lung and brain. The toxicity is a result of the excessive activity of oxygen, resulting from its unique biradical structure (•O=O• because the ground state of oxygen is a triplet (³O₂).

Radicals represent chemical species with an unpaired electron. Electrons in molecules can stably exist a priori by making pairs. Accordingly, radicals are chemically very unstable and tend to make an electron pair by robbing electrons from another molecule. So, the biradical oxygen molecule is also chemically active to react with electrons, thereby producing more active, unstable reactive oxygen species (ROS). They include the singlet oxygen (¹O₂), the exciting state of oxygen, superoxide anion radical shortly superoxide (O₂^•-) and hydrogen peroxide (H₂O₂), which are made by one and two electron addition, respectively to biradical oxygen, and more active hydroxy radical (•OH), which is formed by means of the reduction of H₂O₂ by metal ions such as ferrous and cuprous ions (Fe²⁺/Cu⁺). Excess ROS can react with various macromolecules in cells including nucleic acids, proteins, and lipids nonspecifically, thereby damaging them. So, it is generally believed ROS are profoundly harmful to health possibly by causing a variety of chronic diseases such as arteriosclerosis, diabetes, cancer, and aging. However, an increasing body of evidence suggests that an appropriate level of ROS are critical and even essential for maintaining homeostasis by regulating a variety of biological processes including gene expression. Indeed, ROS have a dual action on cell growth and survival depending on the conditions such as cell type and dose, and exposure time. For example, H₂O₂ can lead to cell activation and increased proliferation as well as reduced cell growth and cell death depending on the conditions. The primary site of ROS production in the cell is mitochondria where O₂ is gradually reduced to water in association with the production of energy (ATP) by oxidative phosphorylation in the electron transport complex (ETC). This energy-generating process necessarily produces ROS as a byproduct. Accordingly, the mitochondria are the site where energy and ROS are continuously produced together.

Because ROS act as a “Ninja in Japan” by successively transforming their shapes, it is rather difficult to catch them in the real-time and determine their chemical identity responsible for a given biological response. So, for most researchers, ROS were unfamiliar research theme at that time. However, my policy of research is that avoiding big field and studying based on unique aspects and hypotheses as possible. This policy led me to basic research on the biological impacts of ROS since 1993. In 2008, the achievement of this basic research opened a way to contribute to cancer treatment in the form of “tumor-selective toxicity.”

ROS is a critical factor in tumor-selective toxicity

The term “tumor-selective toxicity” stands for the toxicity selective for neoplasms. I encountered the term of “Magic Bullets” for the first time in 2008 at EHRLICH II, World Congress on Magic Bullets, an International Meeting held in Nürnberg, Germany, to where I was invited to give a talk by German Pharmaceutical Society. This meeting was held to celebrate the 100th Anniversary of Novel Prize Awarded to Paul Ehrich. In 1907, he proposed the concept of chemotherapy. In this idea, he stated “Magic Bullets,” a virtual drug being effective toward microorganisms while being harmless toward the human body. In the case of anticancer drugs, they should be highly toxic toward neoplasms while displaying minimal toxicity toward normal tissues. However, anticancer drugs used in conventional cancer “chemotherapy” have poor tumor-selectivity leading to serious side effects from that time. Strictly speaking, no ideal cancer chemotherapy could be accomplished unless we can hand Magic Bullets against cancers. So, I heartily wished to find the principle of tumor-selectivity and open a way to generate such weapons.

Eventually, we discovered that mitochondrial O₂^•- accumulation triggers mitochondrial and endoplasmic reticulum dysfunctions, thereby leading to non-apoptotic cell death. Our data also strongly suggested that the source of the O₂^•- generation may be the ETC in the mitochondria. H₂O₂ is a relatively stable oxidant species in the absence of destroying enzymes such as glutathione peroxidase, peroxidase, and catalase, and so that can be added exogenously. (The commercially available disinfectant Oxydol usually contains approx. 3% hydrogen peroxide solution). In 2013, to our surprise, as little as 0.00034% (100 μM) of H₂O₂ could kill TRAIL-resistant malignant tumor cells such as lung cancer, melanoma, and osteosarcoma cells. More surprisingly, under the conditions the oxidant had minimal cytotoxicity toward normal cells such as dermal and lung fibroblasts and melanocytes. In conclusion, ROS are emerging as a critical factor in the principle of tumor-selective toxicity through non-apoptotic cell death induction.

Mitochondrial network aberration and non-apoptotic cell death

In 2014~2016 Our mechanistic studies revealed that mitochondrial network aberration contributed to tumor-selective toxicity of H₂O₂. Mitochondria are highly dynamic organelles with a reticular network organization. Mitochondrial morphology is known to be critical for cell function and apoptosis.

The mitochondrial network depends on the delicate balance between two opposing types of machinery responsible for fission and fusion of the mitochondrial membranes. As a result, the mitochondrion exists separately (fission) or exist an organized form (fusion). An imbalance of this dynamics results in a malfunction in energy production, leading to non-apoptotic cell death. We found that H₂O₂ led to altered status of genes controlling the balance through oxidative stress, thereby causing mitochondrial network disruption. We also discovered that mitochondrial O2•- accumulation facilitated increased plasma membrane depolarization, a promoter of mitochondrial network aberration and that the oxidative and membrane potential dysregulation occurred in tumor cells, but not in normal cells.

A chain of our findings leads to a beautiful hypothesis that tumor cells are more prone than normal cells to mitochondrial oxidative stress, thereby being more vulnerable to mitochondrial network aberration and non-apoptotic cell death induction. It is the decisive moment that the past achievements of the basic research on ROS and mitochondria opened the way to cancer treatment in the viewpoint of tumor-selective toxicity.

How about using plasma as a tool for fighting cancers?

Just when we accumulated the findings regarding the role of ROS in tumor-selective toxicity, I got an e-mail from a researcher working for CSIRO (Commonwealth Scientific and Industrial Research Organization) in Australia. The mail described that non-thermal (cold) atmospheric plasma (CAP) has tumor-selective cytotoxicity by poorly understood mechanism unless ROS mediate it. It was an exciting invitation to join the research group as an expert of ROS. At a glance, I felt that CAP might utilize mechanisms similar to TRAIL and H₂O₂ so that the past achievements could be available. Unfortunately, depending on various reasons I could not accept this challenging invitation at that time, but in the later, I encountered another chance to treat CAP.

Plasma stands for the fourth state of materials that follows solid, liquid, and gas, in which the atoms in a gas are separated into the atomic nuclei and electrons by massive energy, and thereby moving together. Recent technical innovation of the plasma field enabled to make a great plasma under cold (nearly at room temperature) and atmospheric pressure conditions and called as CAP. Because CAP readily and effectively modulates physical properties, it has been widely applied in the field of engineering.

Since CAP has been shown to have tumor-selective cytotoxicity about ten years ago, it has emerged as a promising tool for fighting cancers. However, there were several problems to be solved to serve a practical use. The critical one is that the primary application of CAP may be limited to surface cancerous tissues because of a little outreach except in situ irradiation during laparoscopic or abdominal surgery. Another one is that the precise mechanisms of the action remained largely unclear except the involvement of ROS.

Basic research for practical realization of cancer treatment

To expand CAP application to internal cancers, we used plasma-irradiated liquid (PLAIL) in place of direct CAP irradiation because one can readily administer the solutions systemically or locally by intravenous drip or endoscope. In 2016~2018, we discovered that PLAIL contained H₂O₂, which in turn evoke mitochondrial O₂^•- accumulation, leading to mitochondrial network aberration and non-apoptotic cell death. As observed with H₂O₂, tumor cells were much more vulnerable than normal cells to the cell death induction, these pro-death events including mitochondrial ROS accumulation induced by PLAIL. The emerging view is that altered metabolism and genetic instability under the control of oncogenic transformation cause increased ROS generation and decreased antioxidant system in cancer cells. As a result, they may burden excess oxidative stress over normal cells. Together, it is likely that the susceptibility to mitochondrial ROS accumulation leads to the tumor-selective toxicity of PLAIL.

As PLAIL also contained another unknown component that strengthens the anticancer activity, it exerts greater anticancer effect and mitochondrial modulating ability compared with H₂O₂. Also, our recent data suggest that depending on the plasma species and target solutions, PLAIL can kill cancer cells by different cell death modalities including autophagy and, necroptosis. Many of conventional anticancer drugs target apoptosis while cancers are characteristics to possess various types of machinery preventing apoptosis. By contrast, PLAIL can attack cancer cells by targeting different cell death modalities thereby exerting a potent anticancer activity.

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