A research group at Okayama University is working on a way to prevent healthy cells from incurring damage during cancer treatment. The group is developing a light-induced method for triggering cell apoptosis in targeted cells only, using a light-activated protein rather than chemicals. While drug therapy remains the primary means of cancer treatment, many of the medicines share a common problem: They act not only on cancer cells, but also on the surrounding healthy cells, causing unwanted adverse reactions. Several therapies already exist that use light to kill cancer. Photoimmunotherapy, for example, is a molecular-targeted form of phototherapy that combines photodynamic therapy of the tumor with immunotherapy to kill cancer cells. “These methods use chemical substances and cause necrosis physically by relying on active enzymes or heat,” professor Yuki Sudo said. “Thus, we cannot eliminate adverse reactions no matter how much we improve them.” Rather than using necrosis to eliminate cancerous cells, Sudo and his team developed an optical method to regulate apoptotic cell death — a process by which unwanted cells are actively killed to allow the organism to survive. The researchers achieved apoptosis by controlling the intracellular pH with light-absorbing proton pump proteins from the rhodopsin family. “We thought that if we could stealthily induce apoptosis in the target cancer cells using proteins rather than chemicals, we could help realize a breakthrough in cancer treatment without the accompanying side effects,” Sudo said. As part of an optical approach to cancer therapy developed by a team at Okayama University, AR3, a light-activated protein from the rhodopsin family, is synthesized inside cancer cells and then light is applied, inducing cell apoptosis. Courtesy of Okayama University and JST. The researchers focused on archaerhodopsin-3 (AR3), a light-absorbing protein from the rhodopsin category that has demonstrated the ability to pump hydrogen ions out of a cell. As a cell’s hydrogen ion concentration decreases, the cell becomes more alkaline; and cell alkalinization can trigger apoptosis. Based on this knowledge, the research group started testing cells to see whether they could be made alkaline enough using AR3 to induce apoptosis. The researchers synthesized AR3 in human cancer-derived cells and then exposed the cells to green light with an approximate wavelength of 550 nm. The alkalization-induced shrinking of human HeLa cells cultured at pH 9.0 was significantly accelerated by light-activated AR3. The researchers observed that most of the cells underwent apoptosis within three hours. Researcher Shin Nakao, a student of Sudo’s, conducted a parallel experiment in which the pH conditions applied to the cells were at a physiologically neutral pH 7.4. A biochemical analysis revealed that the intracellular alkalization caused by AR3 triggered the mitochondrial apoptotic signaling pathway, which resulted in cell death accompanied by morphological changes. PH is a measure of the hydrogen ion concentration in an environment, and it is used as an index of acidity, neutrality, and alkalinity. “I thought that a test of our approach could definitely not be performed in neutral conditions,” Sudo said. “Cells die when soaked in an alkaline solution and I wondered if this process could be accelerated using AR3. Thus, I experimented only at pH 9 (alkaline). However, the student also ran an experiment with pH 7 (neutral), that is to say, the conditions that normally occur within the human body.” At neutral pH, it was necessary to observe the results over a longer time period, but the experiment ultimately succeeded. “If our strategy works at neutral pH, it can be used to develop treatments,” Sudo said. “I wanted to make the conditions applicable to patients, which cannot be done at pH 9,” Nakao added. “Thus, I tried our approach at pH 7. This happened to work, so I’m glad I tried it.” The researchers then tested their approach in in vivo experiments. They synthesized AR3 on the sensory neurons of C. elegans, targeting the neuronal cells only. When the bodies of the C. elegans were exposed to green light, only the neurons synthesizing AR3 showed a reduced sensory response to chemicals. The hydrogen ions appeared to have been pumped out of these neurons by AR3, causing the cells to alkalize and die. Based on these results, the researchers concluded that AR3 can trigger apoptosis in targeted cells when it is exposed to light. The researchers compare their approach to cell alkalinization to the way that the messenger RNA (mRNA) vaccine for COVID-19 works. Just as mRNA is injected into cells to synthesize the necessary proteins, the AR3 genes must be introduced into the cancer cells for the cells to synthesize AR3. Genetic markers can be used to synthesize AR3 in targeted cells only. “By using the light-induced cell apoptosis method we developed, in which AR3 is synthesized only in human cancer cells, it is possible to kill the diseased cells without causing adverse reactions in the surrounding healthy cells,” Sudo said. The group said it plans to experiment in mammalian tissue. The researchers’ approach to photo-triggered apoptosis has potential as an optogenetic tool to selectively eliminate target cells with a high spatiotemporal resolution. “Some people may think that because our approach kills cancer with light, it is the same as previous methods. However, our strategy is centered around apoptosis rather than necrosis and therefore is fundamentally different. Our approach could lead to radically new treatment methods,” Sudo said. The research was published in the Journal of the American Chemical Society (www.doi.org/10.1021/jacs.1c12608).