In an article published in the journal Autophagy, scientists from the Center for Research of Redox Processes in Biomedicine (RIDC Redoxoma) led by Mauricio S. Baptista, a professor at the Instituto de Química of Universidade de São Paulo (USP), uncovered a paradigm relating parallel damages in mitochondria and lysosomes, autophagy modulation and the efficiency of photoinduced cell death, by investigating molecular responses of cells challenged by photosensitized oxidation.

"Modulating autophagy is one of the most innovative strategies in the development of antitumor drugs. Our study is quantitative and mechanistic and the results can guide new therapies," said Baptista.

The study revealed details of the mechanism by which parallel damage in the membranes of mitochondria and lysosomes activates and inhibits autophagy concomitantly, leading to a very efficient way to cause cell death. To induce specific photoinduced damage in these organelles, the researchers irradiated normal human keratinocytes (HaCaT) and tumor cells treated with two phenothiazine compounds, methylene blue (MB) and 1,9-dimethyl-methylene blue (DMMB).

The study was initially a master's project conducted by Nayra Fernandes Santos, and it was developed in partnership with Dr. Waleska K. Martins, also a researcher affiliated with the RIDC Redoxoma, previously a postdoctoral fellow in the lab and currently a professor at the Universidade Anhanguera of São Paulo.

Modulating autophagy

Cells challenged by photosensitized oxidations face strong redox stresses and rely on autophagy mechanisms to survive or die. Through a clinical procedure called photodynamic therapy (PDT), medicine uses photosensitized oxidation reactions to induce cell death in diseased tissues and, consequently, to treat various diseases with light.

Photodynamic therapy is based on the generation of cytotoxic oxidant species, such as singlet oxygen and hydroxyl radical, by light activation of a photosensitizer. Autophagy is one of the main causes of the cell resistance to the oxidative effects of the therapy.

Autophagy is a process of degradation and recycling of cytosolic components and damaged cellular organelles, which reestablishes cellular homeostasis against various types of stress, constituting a mechanism of cell survival. However, it can also lead to cell death and, in many pathologies, play either a protective or a destructive role.

In several disorders, such as metabolic diseases, neurodegenerative disorders, infectious diseases and cancer, the autophagy process may be dysregulated. Autophagic activity or its inhibition may, under certain circumstances, combat a disease or promote pathogenesis.

Recent progress in the understanding of the molecular basis of autophagy has revealed promising strategies to treat diseases and therefore, pharmacological approaches to upregulate or inhibit autophagy are receiving considerable attention. For example, autophagy upregulation may be of therapeutic benefit in certain neurodegenerative diseases, whereas its inhibition may be a strategy to treat some types of cancer.

According to Baptista, photodynamic therapy may provide a tool to modulate autophagy.

In 2015, in a study published in the journal Scientific Reports, the team showed that autophagy becomes a destructive process when there is parallel damage to mitochondrial and lysosome membranes. In that work, the researchers assessed the damage caused in HaCaT keratinocytes by the oleanolic and betulinic triterpenoid acids.

"We note that when we cause damage to the two organelles, there is a cellular response that involves the activation of a process called mitofagia, which is the selective degradation of mitochondria by autophagy. In mitofagia, dysfunctional mitochondria are recognized and prepared to be digested in the lysosome. If at the same time we cause damage to the lysosome, the process is not finalized, leading to a type of cell death that is connected with autophagy," Baptista explained.

To study the mechanisms involved in autophagy modulation, the researchers, this time, used photosensitizers and light. They compared the photodamage induced by the dyes methylene blue (MB) and 1,9-dimethyl methylene blue (DMMB) in normal human keratinocytes (HaCaT) and in tumor cells.

The two dyes are efficient photosensitizers; each molecule of MB and DMMB generates, respectively, 1.71 and 2.25 molecules of singlet oxygen per second. Using molecular dynamics simulation, however, the researchers found that DMMB binds more to membranes and causes damage more specifically in its constituents, while MB causes a generalized redox imbalance.

The results showed that, at small concentrations (10 nM), only DMMB induced mitochondrial damage, leading to the activation of mitofagia, which did not progress to completion because of parallel lysosomal damage, triggering cell death. MB-induced photodamage was detected almost instantaneously after irradiation, in response to a massive and non-specific oxidative stress at a higher concentration range (2 μM). In this case, the cells die by a common mechanism of cell death by photodynamic therapy, which is a combination of necrosis and apoptosis.

To prove the hypothesis, the researchers created an experimental condition in which MB behaved similarly to DMMB and the same paradigm was observed.

The conclusion, according to Baptista, is that parallel damage in mitochondria and lysosomes activates and inhibits mitophagy, leading to a more efficient late cell death and offering a significant advantage over photosensitizers that cause non-specific oxidative stress. Cell death by photodamage therefore depends on the extent of photossensitizer interaction with the membranes and, in turn, on the level of photoinduced membrane damage.

By also testing tumor cells, the researchers showed that this process is more comprehensive and not just a specificity of the HaCaT keratinocytes, which are differentiated cells of the epithelial tissue with a very active mechanism of autophagy. Most tumor cells are more disorganized internally and rely on autophagy to survive and proliferate.

This is the first example of the use of a photosensitizer to modulate the mitochondrial-lysosomal axis of cellular stress. Researchers believe that learning to control autophagy can impact the efficiency of photodynamic therapy protocols and, in a broader perspective, the development of more effective antitumor treatments.

The article “Parallel damage in mitochondria and lysosomes is an efficient way to photoinduce cell death”, by Waleska K. Martins, Nayra Fernandes Santos, Cleidiane de Sousa Rocha, Isabel OL Bacellar, Tayana Mazin Tsubone, Ana Cláudia Viotto, Adriana Yamaguti Matsukuma, Aline B of P. Abrantes, Paulo Siani, Luís Gustavo Dias and Mauricio S. Baptista can be read here.