Investigating an enzyme that stops cells from dividing could be a fruitful avenue for research into how to slow aging and treat aging-related diseases. This was the conclusion that researchers at Kobe University in Japan came to after studying the enzyme D-amino acid oxidase (DAO) and its role in cells. DAO metabolizes D-amino acids, which, unlike their mirror-image cousins the L-amino acids, only have a small presence in mammal biology. For this reason, until the recent study, scientists knew little about the impact of DAO in the body. The new finding reveals that, in the process of metabolizing D-amino acids, DAO produces reactive oxygen species (ROS). Stressors such as DNA damage and ROS prompt cells into senescence, an irreversible state in which they can no longer replicate. The finding uncovers a molecular mechanism that has been missing in previous studies that have linked ROS to cell senescence and aging. ROS are important players in the biology of aging and many diseases that tend to increase with advancing age, such as Parkinson’s, Alzheimer’s, diabetes and many cancers.
This study adds to a growing understanding of the role of senescence in this relationship. Entering an irreversible state in which it can no longer divide and proliferate, does not necessarily diminish a cell’s capacity for change and influence. Early research suggested that the main impact of cell senescence on human biology involved protecting against cancer. Confined to a senescent state, cells with damaged DNA cannot multiply and give rise to tumors. Since then, however, studies have revealed that senescent cells are active in tissue repair, wound healing, embryonic development, and aging. A major focus of continuing research is on the various stressors that can trigger cells to enter the irreversible state. In addition, there is a growing body of knowledge about how aging-related biological changes and diseases involve ROS and senescence. In previous work, the Kobe University researchers had discovered that senescence triggers the tumor suppressor protein p53; that, in turn, activates the gene for DAO. However, the study did not fully explore the direct relationship between DAO and senescence.
P53 is an tumor suppressor gene that is involved in DNA damage sensing, cell cycle arrest, aging and cellular death. Although the p53 transcriptional activity is essential for senescence induction, the downstream genes that are crucial for senescence remain unsolved. One of such genes is proline dehydrogenase (PRODH), which is a mitochondrial enzyme which starts catabolism of proline, an aminoacid. Its alternative name is PIG-6 standing for P53-Indiced Gene 6 and it causes production of hydrogen peroxide, the main cellular trigger for oxidative stress. In their more recent investigation, the researchers coaxed cancer cells into senescence by exposing them to low levels of an anticancer drug that induces DNA double-strand breaks. They found, however, that reducing DAO activity, either with drugs or by silencing its gene, reduced senescence and ROS production. In another experiment, they used a mutant of DAO that stopped it behaving like an enzyme. This version (dominant negative) neither produced ROS nor promoted cell aging.
The team suggests that this proves that it is DAO’s ability as an enzyme to make ROS that allows it to promote senescence in cells. However, even though D-aminoacids are not that much available compared to L-forms, the researchers speculated that substrate availability could be a limiting factor for DAO to execute its action. Among a mixture of D-aminoacids, D-serine and D-arginine were found to be preferential substrates of the enzyme. The researchers also observed that the addition of D-arginine and D-serine by itself did not affect senescence induction and the treatment with L-arginine and L-serine did not enhance the senescence promotion by DAO. This indicates the specific effect of D-forms of arginine and serine in inducing cell senescence. DAO induced generation of hydrogen peroxide, a general trigger for cellular oxidative stress and DNA damage. All the toxic effects were abolished by acetyl-cysteine (NAC), a sulfur aminoacid commonly used as a mucolytic and antioxidant supplement.
In further experiments, the scientists discovered other pathways that help DAO to promote senescence triggered by DNA damage. A key factor in this process would be the transporter protein SLC52A1, which transfers inside cells vitamin B2, a FAD precursor. Flavin adenine dinucleotide (FAD) is a coenzyme; DAO needs FAD to work and SLC52A1 ensures this supply by increasing the availability of vitamin B2. However, it is to be specified that ROS are not always deleterious: they are essential for some functions in the immune system, bone marrow, blood vessels, liver and even nervous system. Perhaps it is the overproduction of ROS that causes problems and tips the balance toward cell stress, disease, and aging. In this respect, the study identifies a previously unknown role for DAO, as a promoter of DNA damage-induced cell aging, which may provide new insights into the roles of D-amino acids in various physiological and pathological processes including neurodegenerative disease and cancer.
Regarding the nervous system and its aging process, this study has particular implications. D-arginine and D-serine have been reported to be present in the brain and to act as a protector of the brain against neurotoxicity induced by high levels of glucocorticoids or glutamate toxicity. Indeed, d-serine acts as an agonist of NMDA receptors that are ligand-gated ion channels mediating excitatory neurotransmission in the brain. Hence, alteration of D-serine metabolism is relevant for neurological diseases, such as brain ischemia, epilepsy, ALS and other neurodegenerative disorders. A just published review on the link between Alzheimer disease and brain/serum D-serine levels has strenghtened the connection between this aminoacid and this neurodegeneration. There is the surprise: patients with cognitive decline/Alzheimer disease had usually higher D-serine levels, both in liquor and in blood plasma. From these data one may argue that D-serine could result protective against beta-amyloid metabolism in this disease. However, the trick is subtle.
As mentioned earlier, D-serine is a regulator of NMDA ion channel, a type of glutamate receptors connected to neurotoxic and degenerative phenomena beyond doubt. In animal models engineered to produce more beta-amyloid, this peptide may upregulate serine racemase (SeR). This enzyme changes L-serine into D-serine. In beta amyloid-injected mice, D-serine improves motor and cognitive impairments by interfering wit the JNK pathway, well known to induce neuronal cell death in many neurodegenerative conditions. This phenomenon, however, could not reflect the human situation, or it is possible that the system is producing more D-serine for two connected reasons. On one hand, D-serine would maintain neuronal toxicity through the NMDA receptor; on the other, oxidative stress would induce p53 gene in neurons, which in turn would act through DAO and PRODH, to generate more reactive species. Like a dog biting his tail, this mechanism would perpetuate itself in the final aim to induce senescence and loss of brain cells, which is manifested as atrophy and shinking seen in NMR brain scans.
Finally, like for other aminoacids, serine, proline and their catabolic enzymes are becoming intersting targets to fight human cancer. Neoplastic cells find more and more proline along their path while progressing in metastatization: metalloproteases they release, indeed, to break down proteins (i.e. mostly collagen) in the extracellular matrix, are continuous providers for proline. PRODH’s functional importance in tumor mitochondria grew out of studies in cancer cell stress responses to nutrient deprivation and hypoxia. The breakdown of extracellular collagen in sustaining ATP production (energy) is deemed to come from this mechanism. These concepts may lead to think that PRODH in cancer cells would not maintain his job to promote growth arrest and induce their death. The answer is that PRODH is moslty an enzyme residing in mitochondria and his ability to produce ROS is not direct. Instead, it would derive from the respiratory chain by continuous oxidation of glutamate derived by proline breakdown.
This, in turn, fueled the search for specific PRODH inhibitors as possible mean to fight aggressive neoplasms like breast or lung cancer. A classical PRODH inhibitor used in lab research is N-propargyl-glycine (N-PPG), which seem already safe enough to be administered in vivo. However, looking for a higher selectivity, scientist developed the first-generation PRODH inhibitor, L-THFA, and then a second-generation improved analog, S-5-OXO. These molecules work differently from N-propargyl-glycine: this is indeed a suicide irreversible inhibitor that lead to subsequent degradation of PRODH without disturbing mitochondria. Given the stability of L-THFA compared to N-PPG, in vivo attempts to treat animals with xenografted cancers has been reported. Giving daily intraperitoneal injections of up to 60 mg/kg of L-THFA into mice bearing small orthotopic implants of murine breast cancer cells, researchers observed excellent host tolerance to this drug and, after 16–18 days of sequential treatment, it reduceed lung metastasis formation by 50%, without any significant impact on primary tumor growth.
As with other anticancer strategies, inhibiting PRODH is not to be envisioned as a single therapy, especially given that cancer metabolism is generally capable to undergo reprograming, to adapt itself to a wide variety of anticancer agents. One proven approach to identifying more effective anticancer strategies now being considered in the context of cancer metabolism is the identification of synthetic lethality-based treatment combinations. As published earler in this website, other aminoacids are now being targeted by scientists to selectively hit tumor cells. Among these, cysteine, glutamine and phenylalanine. Like treating antibiotic-multiresistent bacteria or multiresistant malarial parasites, drug combinations is nowadays a strategy to reduce the onset of resistant strains to the least. Perhaps, this strategy is also appliable to cancer cells and the magical “silver bullet” of Paul Ehrlich is going to become a “magic cocktail”.
- Edited by Dr. Gianfrancesco Cormaci, PhD, specialist in Clinical Biochemistry.
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