The major cellular antioxidant is glutathione (GSH) which has the ability to be produced and regenerated after oxidation. The enzyme responsible for the process is glutathione reductase (GSR1), which depends on the NADPH2 cofactor in order to regenerate this compound. The other major antioxidant is thioredoxin (Trx), a small protein with an active sulfur center, which oscillates between the oxidized state (S-S) and the reduced state (-SH) depending on the intensity of the presence of oxidizing species. The enzyme responsible for its regeneration is thioredoxin reductase (TxR), which in addition to NADPH2 as a cofactor, has a selenium atom in its active center. Humans need the chemical element selenium for good health. This is because it enters as a cofactor of special enzymes, most of which have free radical removal capabilities. While the major function of TrxR is to provide electrons to Trx, having a broad substrate specificity, TrxR is also involved in regenerating the small protein cytochrome C and small cellular antioxidants like ascorbic acid, coenzyme Q and lipoic acid.
Thioredoxin reductase enzyme (TxR), together with thioredoxin and NADPH, composes the thioredoxin system, which is a broad-spectrum thiol reduction system and plays an important role in maintaining the redox balance within the cells. Reactive oxygen species are produced in cancer cells as a byproduct of their high glucose metabolism, especially in tumors bearing mutations in oncogenes like K-Ras which are represented averagely in 30% of human cancers. To counteract this, cancer cells often upregulate their antioxidant defense machinery. This, however, leaves a small margin for these cells to cope with additional stress. Cancer cells with elevated ROS production are thus more vulnerable to a compromised antioxidant defense system, which will give rise to a further increase in the levels of ROS, giving rise to oxidative stress and, ultimately, cell death. Therefore, manipulating ROS levels by redox modulation is an encouraging way to selectively kill cancer cells, without causing significant toxicity to normal cells.
Elevated levels of thioredoxin reductase 1 can be observed in several forms of cancer and are linked to worse prognosis in head and neck cancers, lung, liver and breast cancer. That’s why processing drugs that selectively target this enzyme can be an effective way to kill cancer cells. Indeed, there are a large number of TrxR inhibitors that have been developed and identified over the years, and several of these compounds exhibit cytotoxic properties in tumor cells. The TrxR inhibitors’ mode of action on TrxR inhibition is diverse and includes both competitive, noncompetitive, irreversible and mechanism-based inhibition. Perhaps the first thioredoxin reductase inhibitor has been auranofin (Ridaura). This drug was approved in 1985 as an antirheumatic agent against arthritis, though its mechanism of action has been eluded for many years. Then it was serendipitously found to bind covalently to TxR1, blocking its activity. Though the mechanism was pinpointed, this does not explain its effectiveness as an antinflammatory. It is indeed a contradiction that enhancing oxidative stress would ameliorate arthritis evolution.
Nevertheless, its mechanism of action drew attention in the context of malignancies. Indeed, auranofin and close derivative are effective TxR1 inhibitors and exert rapid killing on cancer cells. Their specificity is however modest but, even so, higher than some chemotherapy drugs currently employed. For example, melphalan, an alkylating agent used against multiple myeloma also blocks TxR1. So do nitrosoureas (carmustine, semustine, etc.), cisplatin and other platinum-based anticancers (oxaliplatin, carboplatin, etc.), antitumor antibiotics like anthracyclines that are hinge aids against aggressive cancers like lung, pancreatic, breast carcinomas and glioblastoma, the most aggressive form of brain cancer. Scientists have been recently attracted by the idea to replace the gold atom in these drugs and replace it with silver. Even though silver has been used as colloidal material for many years, only recent advances in synthesis and characterization techniques have led to this metal being incorporated into many metal complexes for drug development purposes.
Silver complexes, have been mainly studied as antibacterials in the last decade with good results and an acceptable safety threshold. The idea to employ silver as an anticancer arose from the knowledge that exposure to Ag leads to the accumulation of silver selenide (Ag2Se) in mammalian cells. Then, keeping in mind the reactivity of selenocysteine residues inside TxR1 catalytic pocket, researchers recently evaluated some silver complexes as TrxR inhibitors and compared their activity with those elicited by their gold parent compounds. Like their corresponding gold complexes, the silver derivatives were effective in inhibiting 50% of isolated TrxR1 activity at nanomolar concentrations. Remarkably, the cytotoxic effects induced by Ag(I) complexes were slightly higher than those elicited by corresponding Au(I) complexes. Moving on, arsenic is another inorganic element that have been intensely studied as an anticancer. In the form of arsenic trioxide (ATO), has been used for several centuries in traditional medicine for curing cancers, being shown to be very effective in the treatment of both leukemia and solid tumors.
Indeed the FDA approved this molecule in 2010 for the treatment of acute promyelocytic leukemia (AML). Past evidence indicated protein sulfhydryl groups as the main targets of the drug, since in form of organic derivatives was shown to affect enzymes, structural proteins and even transription factors bearing free cysteine residues, e.g. AP-1 and members of the zinc finger family, one of the best known of which is estrogen receptor (ER-alpha). On the other hand, a mechanism involving a strong and irreversible inhibition of mammalian TrxR, involving both the C-terminal and N-terminal redox active sites of the enzyme, was recognized by Holmgren and co-workers. Notably, the inhibition of TrxR in breast cancer cells subsequently resulted in the inactivation of the Trx system and, ultimately, the induction of cell death. It has not completely demonstrated that ATO treatment of breast cancer cells result in simultaneous inactivation of TxR1, ER-alpha and other proteins, even though this would be the case. However, the strategy to hit as many as possible redox targets inside cancer cells is preferably to maximize the chance for an effective tumor killing. The only deterrent is coping with the onset of general toxicity.
That is why it was thought to resort to alternative formulations of the drug, including nanoformulation to increase bioavailability. Through this strategy, encouraging results have been obtained on an experimental level against colon cancer, but especially in the last decade against breast cancer. In vitro studies on MCF7 (estrogen-positive) cells, ATO induces oxidative stress, blockade of potassium channels to culminate in apoptotic cell death. It is likely that the oxidative stress induced by the molecule includes the inhibition of TxR1, but also of other antioxidant enzymes and estrogen-dependent transduction. Arsenic is also a poison of the mitochondria, because these organelles have their own isoform of thioredoxin (Trx2) and a corresponding regenerating enzyme (TxR2). In addition, the glutathione system is essential to protect them from the intense oxidative activity that derives from the oxidative phosphorylation chain (respiration). This mechanism has been seen to confer resistance during antiestrogen therapy in breast cancer. But the ATO is able to reverse it, indicating that the redox state in the mitochondria is its target.
Hence it is logical to expect that the ATO can only be used in tumors that show positivity to the estrogen receptor. Well, the surprise is that the ATO is also active on ER-negative tumors with a “harm and insult” mode: following exposure to arsenic trioxide, the MDA-MB-231 ER-negative breast cancer cells in previously unresponsive, they become sensitive to 4-hydroxytamoxifen antagonists and ICI 182,780. Furthermore, specific methylation and sequencing tests show that arsenic trioxide induces partial demethylation of the ERα gene promoter. This would happen because arsenic suppresses the expression of the enzymes DNMT1 and DNMT3a necessary to methylate DNA, including some regions of the ERα gene. Through this mechanism of remodeling of epigenetic events, the ATO also manages to confer the lost resistance to breast tumors that have become resistant to conventional drugs such as paclitaxel, doxorubicin and irinotecan. A very recent development of intervention on thioredoxin reductase against breast cancer was published a few months ago, showing the synergy of action between ATO and EGCG, the major antioxidant polyphenol in green tea.
Aside from its direct inflammatory, antioxidant and antitumor action, as proven in various in vitro and in vivo experiments, EGCG is also proven to be a competitive inhibitor of TxR1. Furthermore, as a molecule, it is well tolerated after oral administration and, besides anti-oxidant effects, EGCG can help us to improve the adverse side effects of cancer therapy. Therefore, EGCG can be combined with regular anticancer drugs to reduce their side effects. In summary, there is objectively a feasibility in using TxR inhibitors to selectively treat some of the more common as well as aggressive forms of cancer. The challeng is to find a perfect way of delivery and effectiveness, eventual synergism with other drugs and a minimal patient toxicity. An avenue which nowadays belong to almost none of the anticancers known to man. They say that cancer must be fought with an “iron” fist: perhaps the solution is with another kind of metal.
- Edited by Dr. Gianfrancesco Cormaci, PhD, specialist in Clinical Biochemistry.
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