For thousands of years, humans have been using the light emitted by the sun as a therapeutic agent against various skin diseases 37,38. The therapeutic potential of light, in particular light, which alone or in combination with appropriate photosensitizers generates reactive intermediates with therapeutic potential, also including reactive oxygen species (ROS), is of steadily increasing interest in modern medicine 39. In contrast to high-energy types of radiation such as ultraviolet radiation, blue light with a wavelength of 453 nm is not able to directly generate ROS by splitting molecules or applying energy to oxygen molecules due to its relatively low energy content. Nevertheless, the intracellular generation of reactive oxygen species (ROS) appears to be the relevant molecular mechanism for the effects of exposure to blue light 40–42.
Contrary to what is often assumed, the production of reactive oxygen species ensured by cells own enzyme systems represents a normal cell-physiological process for the control or induction of different physiological signaling pathways. Thus, ROS play an important role in immunity, cell growth and cell signaling. However, in excess, ROS are deadly to cells and the overproduction of these molecules leads to a wide variety of serious diseases 43,44. The enzymes involved in ROS production are e.g. flavoenzymes of the mitochondrial respiratory chain, in particular NADH dehydrogenase (complex I) and isoenzymes from the NADPH oxidase family (NOX) but also other like 5-lipoxygenase or xanthine oxidoreductase (XOR) 45. In the functioning of flavoenzymes, the natural substrates NADH or NADPH play the role of reduction equivalents and serve as electron donors for the reduction of the flavin residues. Only the reduced form guarantees a targeted and controlled transfer of electrons to an oxygen molecule and thus the generation of superoxide radical anions (O2−.) and H2O2 46,47. In contrast to the physiological function of the flavoenzymes, the interaction with blue light leads to a photoreduction of the flavin content of the enzyme due to the absorption properties of flavin residues without participating in the natural substrate NADH or NADPH 48. Immediately after the light-induced flavin reduction, a process of flavin reoxidation begins. This process takes place in a light-independent reaction and, as under physiological conditions, leads to electron transport to oxygen molecules and thus also to the formation of ROS in the form of superoxide radical anions or hydrogen peroxide 46. However, since the enzyme has no activity control via a feedback mechanism, e.g. the regulation of substrate consumption, the level of ROS production by the flavoenzyme is solely a function of the light dose. Depending on the light dose, very high amounts of ROS can be generated. So, it is not surprising that we were able to observe such a strong ROS-inducing effect of blue light also in the RT112 bladder cancer cell line used here.
The biological responses observed as a result of blue light increased ROS production include a reduction in migration, proliferation and differentiation of the various cell types exposed to blue light, and above a critical threshold of ROS production it can negatively affect cell viability and become cytotoxic 18,40,48,49. In order to be able to better record additive or synergistic effects of blue light with the chemotherapeutic agent, we were very careful in the study presented here to use a light dose that, when applied alone, could not induce any significant cytotoxicity. It should of course not go unmentioned that blue light can also have a strong cytotoxic effect, depending on the dose used and the frequency of radiation. For example, Zhang et all were able to show that long irradiation of HL-60 myelogenous leukemia cells with blue light (456 nm) alone led to strong cytotoxicity in the light-exposed cell cultures. The predominant mode of induced cell death was apoptosis, accompanied by all the typical features of caspase-3 controlled apoptosis 21.
Nevertheless, using the chosen non-toxic light dose we observed a significant increase in Mitomycin C toxicity in RT112 bladder cancer cell cultures exposed to blue light. We could not clearly determine the type of increased cell death. We originally assumed that the combined use of the chemotherapeutic agent plus blue light would increase cell death via apoptosis. We were able to partially confirm this expectation. We observed a significant increase in apoptosis, accompanied by a significant increase in the expression of the pro-apoptotic protein Bax and a significantly increased rate of the caspase-dependent degradation of PARP. Overall, this scenario indicates a significantly increased rate of caspase-3 mediated apoptosis. We attribute this increased rate of apoptosis to the greatly increased intracellular production of ROS, which are known to be very effective inducers of apoptosis 50,51.
On the other hand, we also observed a strong and significant increase in secondary necrosis. Apoptotic cell death is a finely tuned and programmed cell death which requires energy to be carried out successfully. If a cell carrying out the apoptotic program no longer has the required energy sources in the form of ATP, it stops this cell death program at the corresponding point in the mechanism and becomes necrotic (secondary necrosis) 52. This is primarily the case when a damaged cell no longer has sufficient glycolytic or mitochondrial ATP synthesis or is unable to form ATP due to a lack of substrates. This finding of the increased rate of secondary necrosis prompted us to characterize the influence of blue light on the energy metabolism of the RT112 cancer cells used here. In fact, using SeaHorse technology, we were able to find that irradiation of the RT112 cultures with blue light induced a complete breakdown of the mitochondrial respiratory chain and a significant reduction in ATP synthesis. We consider this blue light-induced breakdown of the mitochondrial respiratory chain to be the causal mechanism for the increased rate of secondary necrosis in irradiated RT112 cultures. Of course, the question arises as to why the irradiation of the cells as a single stimulus led to a greatly increased ROS production and mitochondrial breakdown, but did not lead to increased toxicity of the cells. As we were recently able to show with human skin fibroblasts, the decoupling of the respiratory chain shown here is a reversible process after exposure to blue light. Depending on the light dose used, the mitochondrial respiratory chain recovers quite quickly and showed its original activity potential after just 18 to 24 hours. This temporal course therefore makes sense that in the context of a combination therapy, the addition of the chemotherapeutic agent, as carried out by us, takes place shortly after the radiation, at the time of the greatest ROS exposure and ATP production inhibition.
As a pure in vitro study, the results of our study here are of course only of limited significance with regard to therapeutic clinical use. Nonetheless, our data show that using modern endoscopy techniques, combined simultaneous or sequential local application of a chemotherapeutic agent and blue light can represent an effective treatment option for certain types of bladder cancer. Such a therapy option could be individually adapted to the respective patient by using higher doses of light and further promote the success of the therapy. By using special templates that sharply delimit the area to be treated, one could also protect healthy areas in the treatment area from possible side effects of the therapy.