SELENOT–Knockdown Leads to Reducing the Expression of Key Enzymes of ERAD–System in Cancer Cells Under ER–Stress

SELENOT is one of the seven selenoproteins localized in the ER. The purpose of this work was to study the effect of SELENOT–knockdown on mRNA expression of a large number of genes (ER–stress markers, physiological partners of SELENOT, ER–resident selenoproteins, other selenoproteins) under conditions of ER–stress caused by both selenium–containing compounds (sodium selenite and methylseleninic acid) and non–selenium–containing compounds (dithiothriitol) by the example of two human cancer cell lines A–172 (human glioblastoma) and Caco–2 (human colorectal adenocarcinoma). In the course of this work, it was found that SELENOT–knockdown does not significantly affect viability and proliferative properties, redox homeostasis in A–172 and Caco–2 cells and the acquisition of signs of normal cells by them, does not significantly affect the expression of ER–stress marker genes, ER–resident selenoproteins, with the exception of DIO2 and SELENOM. But it has a significant effect on reducing the levels of mRNA expression of AMFR and RNF5 are important enzymes of the ERAD–system, disrupting its work and leading to the accumulation of proteins with incorrect folding, thereby only aggravating ER stress.

EGFR-IL-6 Signaling Axis Mediated the Inhibitory Effect of Methylseleninic Acid on Esophageal Squamous Cell Carcinoma

Epidemiological and experimental evidence indicate that selenium is associated with a reduced risk of some cancers, including esophageal cancer. However, the exact mechanism is still unclear. In the present study, we used esophageal squamous cell carcinoma (ESCC) cell lines and animal models to explore the anti-cancer mechanism of methylseleninic acid (MSA). Firstly, MSA treatment dramatically attenuated Epidermal Growth Factor Receptor (EGFR) protein expression but did not alter mRNA levels in ESCC cells. On the contrary, EGFR overexpression partly abolished the inhibitory effect of MSA. With a microRNA-array, we found MSA up-regulated miR-146a which directly targeted EGFR, whereas miR-146a inhibitor antagonized MSA-induced decrease of EGFR protein. We further used 4-nitroquinoline-1-oxide (4NQO)-induced esophageal tumor mice model to evaluate the inhibitory effect of MSA in vivo. MSA treatment significantly decreased the tumor burden and EGFR protein expression in tumor specimens. Furthermore, MSA treatment inhibited EGFR pathway and subsequntly reduced Interleukin-6 (IL-6) secretion in the supernatant of cancer cell lines. MSA-induced IL-6 suppression was EGFR-dependent. To further evaluate the association of IL-6 and the anti-tumor effect of MSA on esophageal cancer, we established the 4NQO-induced esophageal tumor model in IL-6 knock-out (IL-6 KO) mice. The results showed that IL-6 deficiency did not affect esophageal tumorigenesis in mice, but the inhibitory effect of MSA was abolished in IL-6 KO mice. In conclusion, our study demonstrated that MSA upregulated miR-146a which directly targeted EGFR, and inhibited EGFR protein expression and pathway activity, subsequently decreased IL-6 secretion. The inhibitory effect of MSA on esophageal cancer was IL-6 dependent. These results suggested that MSA may serve as a potential drug treating esophageal cancer.

THE MAIN CYTOTOXIC EFFECTS OF METHYLSELENINIC ACID ON VARIOUS CANCER CELLS

Studies of recent decades have repeatedly demonstrated the cytotoxic effect of selenium-containing compounds on cancer cells of various origins. Particular attention in these studies is paid to methylseleninic acid, a widespread selenium-containing compound of organic nature, for several reasons: it has a selective cytotoxic effect on cancer cells, it is cytotoxic in small doses, it is able to generate methylselenol, excluding the action of the enzyme β-lyase. All these qualities make methylseleninic acid an attractive substrate for the production of anticancer drugs on its basis with a well-pronounced selective effect. However, the studies available to date indicate that there is no strictly specific molecular mechanism of its cytotoxic effect in relation to different cancer cell lines and cancer models. This review contains generalized information on the dose- and time-dependent regulation of the toxic effect of methylseleninic acid on the proliferative properties of a number of cancer cell lines. In addition, special attention in this review is paid to the influence of this selenium-containing compound on the regulation of endoplasmic reticulum stress and on the expression of seven selenoproteins, which are localized in the endoplasmic reticulum.

Methylselenol Produced In Vivo from Methylseleninic Acid or Dimethyl Diselenide Induces Toxic Protein Aggregation in Saccharomyces cerevisiae

Methylselenol (MeSeH) has been suggested to be a critical metabolite for anticancer activity of selenium, although the mechanisms underlying its activity remain to be fully established. The aim of this study was to identify metabolic pathways of MeSeH in Saccharomyces cerevisiae to decipher the mechanism of its toxicity. We first investigated in vitro the formation of MeSeH from methylseleninic acid (MSeA) or dimethyldiselenide. Determination of the equilibrium and rate constants of the reactions between glutathione (GSH) and these MeSeH precursors indicates that in the conditions that prevail in vivo, GSH can reduce the major part of MSeA or dimethyldiselenide into MeSeH. MeSeH can also be enzymatically produced by glutathione reductase or thioredoxin/thioredoxin reductase. Studies on the toxicity of MeSeH precursors (MSeA, dimethyldiselenide or a mixture of MSeA and GSH) in S.cerevisiae revealed that cytotoxicity and selenomethionine content were severely reduced in a met17 mutant devoid of O-acetylhomoserine sulfhydrylase. This suggests conversion of MeSeH into selenomethionine by this enzyme. Protein aggregation was observed in wild-type but not in met17 cells. Altogether, our findings support the view that MeSeH is toxic in S. cerevisiae because it is metabolized into selenomethionine which, in turn, induces toxic protein aggregation.

Selenium Compounds as Novel Potential Anticancer Agents

The high number of new cancer incidences and the associated mortality continue to be alarming, leading to the search for new therapies that would be more effective and less burdensome for patients. As there is evidence that Se compounds can have chemopreventive activity, studies have begun to establish whether these compounds can also affect already existing cancers. This review aims to discuss the different classes of Se-containing compounds, both organic and inorganic, natural and synthetic, and the mechanisms and molecular targets of their anticancer activity. The chemical classes discussed in this paper include inorganic (selenite, selenate) and organic compounds, such as diselenides, selenides, selenoesters, methylseleninic acid, 1,2-benzisoselenazole-3[2H]-one and selenophene-based derivatives, as well as selenoamino acids and Selol.

Optimizing Mixtures of Se Compounds as Adjuvants to Therapeutic Agents

Objectives Different chemical forms of selenium (Se) regulate distinct sets of genes and have different metabolic effects. We previously showed that mixtures of different Se compounds can have greater inhibitory effects on prostate cancer cell viability than individual chemical forms. Further, we showed that blends of different Se forms can be optimized using response surface methodology. Previous investigators showed that single forms of Se can potentiate the effects of chemotherapeutic and other agents. We hypothesized that optimized mixtures of different forms of Se would be more effective than individual Se compounds in enhancing the efficacy of Se-drug combinations. Our objective was to provide proof of principle for this hypothesis. Methods Response surface methodology was used to optimize a blend of methylseleninic acid (MSA), selenite (SEL) and Nano Se to achieve maximum reduction of cell viability in LNCaP prostate cancer cells. Apalutamide (APA), Enzalutamide (ENZ), Docetaxel (DOC), and Mitoxantrone (MIT) were each combined with a single Se compound, or with the optimized blend. Cell viability assays were done using Alamar Blue. Results The optimized mix of Se forms, determined using response surface methodology, was a 50:50 blend of MSA and SEL, that surprisingly did not include Nano Se. All medium supplemented with Se compounds contained 10 μM Se. MIT treatment alone reduced cell viability >90% and showed no further reductions due to added Se. Addition of either form of Se to APA, DOC, or ENZ significantly decreased cell viability compared to treatment with the drug alone (P values: 0.008 - 0.041). Similarly, in 5 out of 6 cases, combination of the optimized blend of Se compounds with the drug resulted in further decreases in cell viability, compared to the mix of drug plus one chemical form of Se (P values: 0.013 – < 0.001). Conclusions These results provide proof of principle that the substitution of an optimized blend of Se compounds for a single Se form, may increase the efficacy of previously reported drug-Se combinations and treatments. Optimization of blends of different Se compounds can be done under conditions customized to their intended use to achieve a desired outcome. Funding Sources This work was funded by the BYU Simmons Center for Cancer Research.