Antitumor metallodrugs have deserved increasing interest in recent years, but because of their adverse effects, they still pose a challenge to scientists. One example is platinum compounds used as chemotherapeutic drugs against various cancers, which cause intense side effects as well as cellular resistance to treatment. Therefore the importance of the development of alternative drugs, based on different metallic compounds, such as copper(II) complexes with iminic ligands, synthesized, characterized, and studied as antitumor agents against melanoma by the group of Professor Ana Maria da Costa Ferreira, from Instituto de Química at Universidade de São Paulo (USP) and a member of RIDC Redoxoma.

In a new study, recently published in the journal Chemico-Biological Interactions, the researchers show that the dinuclear copper(II) complex is more cytotoxic to SKMEL-05 and SKMEL-147 melanoma cells than the analogous mononuclear complex and that this effect is stimulated by ultraviolet B (UVB) radiation, which induces melanin formation. The results of the work extend data obtained by the group previously, indicating different mechanisms of action of such complexes, depending on their nuclearity. According to the authors of the article, dinuclear copper complexes may constitute a new therapeutic approach against melanoma.

“We have studied the mono and dinuclear copper complexes since Cléia Justino Nunes master’s degree and the new results were obtained from her doctorate – she is the first author of the article. Both complexes are reactive against melanoma cells, but the dinuclear complex, which has two copper centers in its structure, has a much higher reactivity, not just double the reactivity of mononuclear. The difference between them is the mechanism of action and this led us to investigate the relation between structure and reactivity. The dinuclear complex acts by dielectronic reactions, that is, by two electrons transfer, similar to the tyrosine enzymes, which catalyze the formation of melanin,” the researcher said.

Melanoma is the most dangerous type of skin cancer. It is aggressive, metastatic, highly fatal if diagnosed late and often resistant to usual therapies. It arises from the malignant transformation of melanocytes, which are cells located in the barrier between the epidermis and dermis responsible for the synthesis of melanin, the pigment that gives color to the skin, eyes, and hair. Melanin has a dual anti and pro-oxidant role. Besides giving protection to the skin against UVB rays, it can also have a harmful effect, as it can coordinate metal ions, mainly iron and copper, participating in reactions that generate reactive species.

Mononuclear copper compounds usually act as redox agents against tumors, damaging biomolecules and organelles via the formation of reactive oxygen species. Dinuclear compounds have other modes of action, although some of them also act by reactive species-mediated pathways targeting mitochondria. For example, some species described in the literature act as metal-containing nucleases, breaking phosphate bonds into nucleic acids (DNA and RNA); others with different ligands act as inhibitors of human telomerase, HIV reverse transcriptase, and other polymerases; other complexes may block DNA synthesis.

Extended Data

In previous work, the group had already detected the higher activity of copper(II) dinuclear complexes against melanoma compared to mononuclear complexes, but the compounds studied were targeting DNA by an oxidative mechanism. The study also indicated a correlation between melanin content and the sensitization of the cells, especially towards the dinuclear complexes.

Now the researchers have confirmed and expanded the data. In collaboration with the group of oncologist Roger Chammas of the Instituto do Câncer at Faculdade de Medicina at USP (ICESP), they evaluated the cytotoxicity of the mono and dinuclear copper(II) complexes in the human melanoma cell lines SKMEL-05 and SKMEL-147 after 24 and 48h incubation, and found that the dinuclear complex causes more damage to tumors. Clonogenic tests, used to evaluate cell proliferation, also indicated higher activity of this compound, with a significant decrease in the number of surviving cells after treatment. Both mono- and dinuclear complexes are able to form radical species.

In the study, the researchers also tested the influence of UVB radiation and they found that in the presence of the dinuclear complex, the exposure of cells to radiation increased the damage. They also observed that melanoma cells with higher melanin concentration are more sensitive to the complex. “Irradiation causes cells to produce more melanin. The dinuclear complex is more efficiently reduced by melanin than the mononuclear, triggering the oxidative catalytic cycle with the formation of reactive oxygen species, thus sensitizing cells with more melanin” explains Costa Ferreira.

To find out how the compounds cause cell death, the researchers performed autophagy assays, which showed increased levels of cytoplasmic vacuoles following treatment with the dinuclear complex, and measured autophagy marker proteins LC3, which were also increased. “We have seen that tumor cells incubated with the copper dinuclear complex die by two processes: by apoptosis, which is programmed death, and also by autophagy, which is a death in which the cell recycles,” said the researcher.

Research results also showed that the toxic effect of the compounds on non-tumor cells was negligible, indicating that the complexes show selectivity for pigmented tumor cells. They evaluated this effect on keratinocytes, which account for 80% of epidermal cells.

Copper Metallodrugs

Copper is an essential metal, which is present in proteins of various organisms and participates in important biological reactions. Humans have a copper concentration of 1.7 mg/kg corporeal, incorporated in many proteins and enzymes. It is obtained through diet and its homeostasis is well controlled, as both excess and lack of copper cause health problems.

According to Costa Ferreira, because they are already present in the body, these essential metals are the preferred candidates for the development of metallodrugs, which are medicines with at least one metallic center. Because copper is very reactive, however, metallodrugs based on this metal have always been considered toxic. For the researcher, the higher reactivity of metals is a positive feature, as it opens up more possibilities for chemical bonds, and toxicity can be regulated by the ligands. “Due to the reactivity, the concentration of free copper in cells is very low (<10^-10 mol/L). It is always coordinated precisely to modulate its activity. In metal complexes, ligands modulate the biological activity of the metal. Our ligands are designed to provide specific characteristics to metal ions, and this is how we can modulate copper activity. And our compounds are very stable, the metal is always bonded to the ligands, and that's how it gets into the cells”, she explains.

Another advantage of metallic complexes, according to the researcher, is that they are multifunctional, acting on several molecules. “Copper compounds, for example, also inhibit kinases, which are very abundant proteins that control cell cycles, including tumors.”

Costa Ferreira’s group is testing the complexes on functionalized silica matrices. “Having a material that releases the compound would be a possibility for tumors treatment.” The next step will be to test the effects of copper(II) complexes on different melanomas in artificial skin.

The article Unlike reactivity of mono- and binuclear imine-copper(II) complexes toward melanoma cells via tyrosinase-dependent mechanism, by Cléia Justino Nunes, Andréia Hanada Otake, Silvina Odete Bustos, Rodrigo Boni Fazzi, Roger Chammas, and Ana Maria da Costa Ferreira, can be accessed here