Selenium is generally known as an antioxidant due to its presence in selenoproteins as selenocysteine, but it is also toxic. The toxic effects of selenium are, however, strictly concentration and chemical species dependent. One class of selenium compounds is a potent inhibitor of cell growth with remarkable tumor specificity. These redox active compounds are pro-oxidative and highly cytotoxic to tumor cells and are promising candidates to be used in chemotherapy against cancer. Herein we elaborate upon the major forms of dietary selenium compounds, their metabolic pathways, and their antioxidant and pro-oxidant potentials with emphasis on cytotoxic mechanisms. Relative cytotoxicity of inorganic selenite and organic selenocystine compounds to different cancer cells are presented as evidence to our perspective. Furthermore, new novel classes of selenium compounds specifically designed to target tumor cells are presented and the potential of selenium in modern oncology is extensively discussed.

Despite progress in the treatment of non-visceral malignancies, the prognosis remains poor for malignancies of visceral organs and novel therapeutic approaches are urgently required. We evaluated a novel therapeutic regimen based on treatment with Se-methylselenocysteine (MSC) and concomitant tumor-specific induction of Kynurenine aminotransferase 1 (KYAT1) in hepatocellular carcinoma (HCC) cell lines, using either vector-based and/or lipid nanoparticle-mediated delivery of mRNA. Supplementation of MSC in KYAT1 overexpressed cells resulted in significantly increased cytotoxicity, due to ROS formation, as compared to MSC alone. Furthermore, microRNA antisense-targeted sites for miR122, known to be widely expressed in normal hepatocytes while downregulated in hepatocellular carcinoma, were added to specifically limit cytotoxicity in HCC cells, thereby limiting the off-target effects. KYAT1 expression was significantly reduced in cells with high levels of miR122 supporting the concept of miR-guided induction of tumor-specific cytotoxicity. The addition of alpha-ketoacid favored the production of methylselenol, enhancing the cytotoxic efficacy of MSC in HCC cells, with no effects on primary human hepatocytes. Altogether, the proposed regimen offers great potential to safely and specifically target hepatic tumors that are currently untreatable.

Kynurenine aminotransferase 1 (KYAT1 or CCBL1) plays a major role in Se-methylselenocysteine (MSC) metabolism. It is a bi-functional enzyme that catalyzes transamination and beta-elimination activity with a single substrate. KYAT1 produces methylselenol (CH₃SeH) via β-elimination activities with MSC as a substrate. This methylated selenium compound is a major cytotoxic selenium metabolite, causing apoptosis in a wide variety of cancer cells. Methylselenol is volatile and possesses extraordinary nucleophilic properties. We herein describe a simple spectrophotometric assay by combining KYAT1 and thioredoxin reductase (TrxR) to detect CH₃SeH in a coupled activity assay. The metabolite methylselenol and its oxidized form from MSC metabolism is utilized as a substrate for TrxR1 and this can be monitored spectroscopically at 340 nm. Our results show the feasibility of monitoring the β-elimination of KYAT1 by our assay and the results were compared to the previously described β-elimination assays measuring pyruvate. By using known inhibitors of KYAT1 and TrxR1, we further validated the respective reaction. Our data provide a simple but accurate method to determine the β-elimination activity of KYAT1, which is of importance for mechanistic studies of a highly interesting selenium compound.

Patient-derived tissue culture models are valuable tools to investigate drug effects and targeted treatment approaches. Resected tumor slices cultured ex vivo have recently gained interest in precision medicine, since they reflect the complex microenvironment of cancer tissue. In this study, we examined the treatment response to an internally developed ex vivo tissue culture model from pancreatic ductal adenocarcinoma (PDAC) and in vitro analysis. Seven PDAC tissues were cultured and subsequently treated with indole-3-pyruvic acid (IPA). IPA, which is known as an agonist of the aryl hydrocarbon receptor (AHR) pathway, has antioxidant properties. Genome-wide transcriptome sequencing analysis revealed activation of AHR pathway genes (CYP1A1 and CYP1B1, p ≤ 0.05). Additionally, significant upregulation of AHR repressor genes AHRR and TiPARP was also observed (p ≤ 0.05), which is indicative of the negative feedback loop activation of AHR pathway signaling. The overall transcriptomic response to IPA indicated that the tissues are biologically active and respond accordingly to exogenous treatment. Cell culture analysis confirmed the significant induction of selected AHR genes by IPA. A morphological examination of the paraffin-embedded formalin-fixed tissue did not show obvious signs of IPA treatment related to tumor cell damage. This study is a proof of concept that ex vivo patient-derived tissue models offer a valuable tool in precision medicine to monitor the effect of personalized treatments.

Se-methylselenocysteine (MSC) is a redox-active selenium-containing amino acid with notable anticancer potential, requiring enzymatic activation for cytotoxicity. Human kynurenine aminotransferase 1 (hKYAT1) catalyzes MSC through transamination and β-elimination pathways, generating β-methylselenopyruvate and methylselenol, both of which induce oxidative stress and epigenetic modulation. To enhance MSC metabolism and its therapeutic efficacy, we performed site-directed mutagenesis targeting three critical hKYAT1 residues: Tyr101, Asp126, and Phe278. These mutants, along with wild-type hKYAT1, were expressed in hepatocellular carcinoma cell lines HepG2 and Huh7, and their impact on enzymatic activity, cytotoxic effects, apoptosis and chromatin remodeling were evaluated. Several mutations significantly enhanced MSC metabolism, with Y101H and F278A increasing both transamination and β-elimination activity, and D126L favoring β-elimination. These modifications led to a five-to 30-fold increase in MSC-induced cytotoxicity compared to wild-type hKYAT1. Additionally, mutant hKYAT1 expression altered histone deacetylase (HDAC) profiles, increased histone H4 acetylation, and activated apoptotic signaling through caspase cleavage and cytochrome c release. Collectively, our findings demonstrate that rational engineering of hKYAT1 can potentiate MSC metabolism and amplify its anticancer effects, offering a promising enzyme-targeting strategy for selenium-based cancer therapies.

We report development and characterization of small non-immunoglobulin affibody affinity proteins directed to the highly glycosylated human carcinoembryonic antigen-related adhesion molecule 5 (CEACAM5, CEA), and their use in immunohistochemical (IHC) analyses of human pancreatic cancer samples and for in vivo tumor imaging. A total of nineteen unique anti-CEA affibodies were identified from large phage display libraries constructed using combinatorial protein engineering of a small 58 amino acid three-helix bundle protein domain. Molecular modeling suggested that all enriched clones share a binding surface with several clustered tryptophan residues interacting with a hydrophobic patch in the N1 domain of CEA centered around a phenylalanine residue. One variant, designated as C9, exhibited the highest affinity in biosensor analyses and was reformatted into a 15 kDa homodimer expressed in Escherichia coli. The biotinylated form, C9-C9-Bio, was evaluated for its IHC performance on matched frozen and formalin-fixed, paraffin-embedded (FFPE) sections of human pancreatic cancer samples (n = 7). Compared to clinical-grade monoclonal antibodies II-7 and CEA31, as well as a polyclonal reagent, C9-C9-Bio demonstrated highly sensitive CEA detection with minimal background staining. Statistical analyses including intraclass correlation and Bland-Altman assessments revealed excellent agreement between C9-C9-Bio and the two monoclonal antibodies in FFPE tissue samples. Further, a ^99mTc[Tc]-labeled C9-C9 construct showed CEA-dependent binding to human cancer cell lines in vitro, and selectively bound to CEA-expressing BxPC3 xenografts in mice when investigated as a tracer for in vivo imaging, allowing for a visualization of tumors after four hours. In summary, these findings highlight the potential use of the easily produced CEA-binding C9 affibody for various clinical applications, including IHC and medical imaging, and as a targeting moiety for directing various therapeutic modalities to CEA-expressing tumors.