Researchers discover a method of inducing autophagic cell death

The results, published in Scientific Reports, could pave way to development of new antitumoral compounds.

By Karina Toledo

Agência FAPESP – To guarantee survival in adverse situations such as nutrient deprivation or the presence of pathogens or toxins, cells can activate a defense mechanism known as autophagy, in which they break down and recycle certain intracellular components such as damaged organelles.

However, when autophagy surpasses a given threshold, it ceases to be a survival mechanism and eventually leads to autophagic cell death, or cell death with autophagy. An article published recently in the journal Scientific Reports by Brazilian researchers helps to explain why this transition occurs.

“We show that cell death with autophagy can be induced very efficiently by simultaneously causing damage to the mitochondrial and lysosomal membranes. This discovery could pave the way to the development of new antitumoral drugs,” said Maurício Baptista, a professor at the University of São Paulo’s Chemistry Institute (IQ-USP) in Brazil and a member of the Center for Research on Redox Processes in Biomedicine (Redoxoma), one of FAPESP’s Research, Innovation and Dissemination Centers (RIDCs). Mitochondria and lysosomes are cellular organelles responsible for energy production and the digestion of internal or external waste, respectively.

According to Baptista, the extent of the damage caused to the membranes of key organelles appears to be the trigger that transforms pro-survival autophagy into autophagic death. The research group reached this conclusion after performing in vitro experiments and computer simulations with cultured keratinocytes, the predominant cell type in the outermost layer of the skin (epidermis) and the cells responsible for producing keratin.

The model was chosen because keratinocytes naturally have a highly activated pro-survival autophagic process, similar to that of tumor cells.

“In physiological situations, keratinocytes must differentiate very rapidly, and this transformation depends on autophagy. Tumor cells grow very fast and in a disorderly manner, so they also need robust, highly active autophagy to survive. When we inserted a compound that hindered the process, the cells died,” Baptista said.

Equal but different

In the in vitro experiments, some cells were treated with oleanolic acid, and others were treated with betulinic acid. These two compounds, found in fruit peel and tree bark, protect plants from pests and infections.

Oleanolic acid is widely used as an anti-inflammatory, antiangiogenic and antioxidant agent. Betulinic acid, which has proven antitumor activity, is being tested in clinical trials against melanoma and other hard-to-treat types of cancer.

Although they have the same molecular formula, C30H48O3, the arrangements of the atoms in these compounds are different; they are therefore considered isomers.

A comparison of variables such as cell viability, cell proliferation and cell death mechanisms in the different cultures showed betulinic acid to be more cytotoxic. According to Baptista, this property is associated with greater efficiency in causing damage to organelle membranes. The plasma membrane that forms the external boundary of the cell was not damaged.

“Betulinic acid molecules have a relatively flat structure, so they can more easily penetrate mitochondrial and lysosomal membranes, efficiently inducing cell death with autophagy. In contrast, oleanolic acid molecules have a folded structure, so it’s harder for them to interact with organelle membranes, and they don’t induce death with autophagy,” Baptista said.

Through computer simulations performed in partnership with researchers from the Federal University of the ABC (UFABC), the IQ-USP group altered the spatial arrangement of the oleanolic acid molecules to make their structure flatter and observed that this increased their interactions with organelle membranes.

According to Baptista, other compounds capable of causing the same type of damage to organelle membranes would probably induce death with autophagy efficiently and could be used to combat cancer. “The key point is that this is physical damage, and the development of cellular resistance is harder,” he said.

For Baptista, the discovery may assist screening of betulinic acid derivatives with the aim of identifying the compound that induces death with autophagy most efficiently.

“One possibility would be to use a computer simulation to test the compound’s interaction with organelle membranes,” he said.

The research described in the article published by Scientific Reports was conducted in partnership with Waleska K. Martins during her postdoctoral fellowship at Redoxoma and under the aegis of a Thematic Project, for which Baptista was principal investigator. Researchers at the Ludwig Cancer Research Institute in São Paulo and the University of São Paulo’s Biomedical Science Institute (ICB-USP) also participated, with partial support from the Ministry of Education’s Office for Faculty Development (CAPES) and Farma Service Bioextract.

The article “Parallel damage in mitochondrial and lysosomal compartments promotes efficient cell death with autophagy: The case of the pentacyclic triterpenoids” (DOI:10.1038/srep12425) by Waleska K. Martins, Maurício S. Baptista et al. can be read at www.nature.com/articles/srep12425.