HomeENGLISH MAGAZINEWhen the fight against diseases gets colorful: originally tissue dyes as bioactive...

When the fight against diseases gets colorful: originally tissue dyes as bioactive molecules as well

Histological staining is a series of technique processes undertaken in the preparation of sample tissues by staining using histological stains to aid in the microscope study. Histological studies are used in forensic investigations, autopsy, diagnosis and in education. In addition, histology is used extensively in medicine especially in the study of diseased tissues to aid treatment. In comparison with the end of the XIX century used for histological staining through chemical, molecular biology assays and immunological techniques, collectively and have facilitated greatly in the study of organs and tissues. Staining is a commonly used medical process in the medical diagnosis of tumors in which a dye color is applied on the posterior and anterior border of the sample tissues, to locate the diseased or tumorous cells or other pathological cells. In biological studies staining is used to mark cells and to flag nucleic acids, proteins or the gel electrophoresis to aid in the microscopic examination.

The first histological staining experiments were carried out using Haematoxylin, an aromatic substance extracted from a small tree in Central America. It colors the nuclear structures black and once its polycyclic nature was known, the scientists probed its possible link with macromolecular structures. And interactions with known and unknown substrates were found. It inhibits the replication of Herpesvirus 8 and of the mononucleosis virus (EBV) with an unknown mechanism. In addition, due to the urge to find antiviral molecules, under the pressure of the current pandemic, a drug screening has found that it can also interfere with the replication of the SARS-CoV2 coronavirus. On the contrary, independent screenings have shown that it is a good inhibitor of protein kinases such as c-Kit, c-Met, c-Fms, HER-2 and FGFR1. These receptor proteins are often overexpressed in human tumors such as lung, breast and thyroid cancer.

Today, the prevalence of synthetic dyes has prevailed and all the analyses on biopsies, cellular staining, study of cell organelles and whatever else happens with synthetic dyes. Basically, dyes with basic reaction are used to color acidic substrates such as polysaccharides and nucleic acids, while those with acid reaction dye basic structures generally based on proteins. Precisely for this reason, scientists became curious, since nucleic acids and all other cellular organelles are complex structures with accessory molecules. The binding of dyes to proteins or other macromolecules can reveal potential pharmacological effects to be exploited in the medical-clinical field. For example, the well-known dye Methylene Blue is used as a venous contrast in endoscopic examinations to reveal the presence of cells in fluids, such as metastases in the lymphatic tract, or to stain endometrial polyps as it stains pre-cancerous cells (dysplasia).

This dye is also used as an antidote for methemoglobinemia, a situation where external toxicants or certain types of drugs cause oxidation of hemoglobin, preventing red blood cells from carrying oxygen. One of its mechanisms of action is to help the regeneration of glutathione in red blood cells and other cells, but since it has side effects and has caused the so-called “serotonin toxicity”, investigations with molecular screening have shown that it is a competitive inhibitor of monoamine oxidase A (MAO-A). That is why investigations are underway on the possibility of obtaining derivatives with greater specificity for use as potential antidepressant drugs. In addition, it was found to be an inhibitor of the function of the nuclear proteins YDP-1 and RecQ1: these are enzymes involved in certain aspects of DNA repair. Nile Blue A, a benzophenazine dye, was also found to be a competitive inhibitor of MAO-A, but of the cyclic AMP-dependent protein kinase as well, a ubiquitous enzyme that mediates cellular actions for many hormones.

Given these properties, it could represent a structural lead to simultaneously condition the biological actions of hormones or neurotransmitters that use the AMPc-PKA pathway (e.g. subtypes of receptors for dopamine, serotonin, noradrenaline, etc.) and that use the MAO- A to terminate their actions. Another dye called Sulfan Blue, from the triphenyl-methane dye family, and used as a ribosome dye was found to interact with the HP-1 beta protein, which serves to shrink inactive chromatin (heterochromatin). One of its precursors or Aurinic acid (ATA), in addition to being a histological dye, is a multiple enzyme inhibitor. It has been used in cell death studies (apoptosis) since it blocks endonuclease III, an enzyme responsible for terminal fragmentation of DNA. It block also DNA menthyl-transferase 1 (DNMT-1), meaning it is also able to affect cellular differentiation. It was also, in addition, found to be an inhibitor of the insulin-like growth factor receptor (IGF1R) and of the PTP-1B protein phosphatase which deactivates the insulin receptor.

Finally, it interferes with the initial stages of general protein synthesis and with the APE-1 enzyme involved in DNA repair, which is why basic research in oncology has used it to elucidate various aspects of signal transduction. On the contrary, Basic Violet 5 (Safranine family), that stains cartilages to check the presence of osteoarthritis, is a potential anticancer agent and antinflammatory as well for some reasons. Indeed, it has been found to suppresses IKK-alpha kinase (IKK-a) upstream to NF-kB, to disactivate the protective transcription factor Nrf-2 and the NRLP-3 component of inflammasome. Different function has the Diacetyl-O-Fluorescein derived from the fluoresceins used in the fluorescence immunohistochemical analyses. Apart from coloring the target structures in yellow-green, it can interfere with the cellular activity of the immune tolerance receptor TLR4, which is notoriously involved in the recognition of bacterial lipopolysaccharide and in the onset of sepsis symptoms.

Completely divergent action has Gallocyanin or Fast Violet dye, which has been found to be an inhibitor of the Wnt-CAT-DKK1 cellular pathway. The Wnt pathway is essential for certain stages of embryogenesis and in adults it is aberrantly activated in many tumors. In the brain it is studied for neurogenesis: the fact that gallocyanin interferes with the interaction of its components DKK1 and LRP-6, represents the discovery of a molecule that interferes only with some portions of this general transduction pathway. In fact, molecules or drugs that affect the Wnt-catenin beta pathway have been known for at least twenty years. Most of them affect the interaction between the Wnt receptor and the imediate downstream beta-catenin, preventing it to translocate in the cell nucleus. Some of them have passed even phase I and II clinical trials for human carcinomas and sarcomas. However, gallocyanin seems to have a moderate effects on this protein interaction and researchers have developed modified derivatives with a better affinity for DKK-1.

This is an example of how often the lead molecule cannot undergo drug repurposing. In fact, not all tissutal or cellular dyes can be used as such to devolve them to possible pharmacological uses. This is the case of the histological dye Acridine Orange, which is notoriously toxic and also carcinogenic. But from its core structure, chemists have developed derivatives that have medicinal activity but not the original toxic one. Mention should be made of tacrine, an acetylcholinesterase (AChE) inhibitor that has found use in delaying cognitive decline from Alzheimer’s-type senile dementia. Or amsacrine (AMSA), an anticancer that does not show action on solid tumors but for acute lymphoblastic leukemia (ALL) only. As can be seen, both Nature and the keeness of the human brain can be put at the service of healing needs. Tens of millions of natural and synthetic compounds are known to man: as they say, imagination is the only limit.

  • Edited to Dr. Gianfrancesco Cormaci, PhD, specialist in Clinical Biochemistry.

Scientific references

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Dott. Gianfrancesco Cormaci

Medico Chirurgo, Specialista; PhD. a CoFood s.r.l.
- Laurea in Medicina e Chirurgia nel 1998 (MD Degree in 1998) - Specialista in Biochimica Clinica nel 2002 (Clinical Biochemistry residency in 2002) - Dottorato in Neurobiologia nel 2006 (Neurobiology PhD in 2006) - Ha soggiornato negli Stati Uniti, Baltimora (MD) come ricercatore alle dipendenze del National Institute on Drug Abuse (NIDA/NIH) e poi alla Johns Hopkins University, dal 2004 al 2008. - Dal 2009 si occupa di Medicina personalizzata. - Guardia medica presso strutture private dal 2010 - Detentore di due brevetti sulla preparazione di prodotti gluten-free a partire da regolare farina di frumento enzimaticamente neutralizzata (owner of patents concerning the production of bakery gluten-free products, starting from regular wheat flour). - Responsabile del reparto Ricerca e Sviluppo per la società CoFood s.r.l. (Leader of the R&D for the partnership CoFood s.r.l.) - Autore di articoli su informazione medica e salute sul sito www.medicomunicare.it (Medical/health information on website) - Autore di corsi ECM FAD pubblicizzati sul sito www.salutesicilia.it
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