We studied the effect of suppression (to approximately 10%) of SELENOT mRNA expression (approximately 10 times) and protein content of SELENOT is quite high in them compared in two human cancer cell lines. Moreover, these two cell lines A-172 and Caco-2, were found most suitable for efficient transduction with lentiviral particles. The main objectives of this study were (i) to explore explanation of the role of SELENOT in the regulation of molecular mechanisms of ER–stress caused by selenium containing as well as non-selenium sources of ER-stress, (ii) to study the effect of SELENOT–knockdown on the expression of its previously identified physiological partners, other ER–resident selenoproteins, selenoproteins, enzymes that are key regulators of redox homeostasis, such as glutathione peroxidases and thioredoxin reductases. (iii) In addition, a series of experiments was carried out, the purpose of which was to find out whether suppression of SELENOT mRNA expression affects conversion to normal cells as detected by enhanced features of normal cells in them. The main goal of this study was to explore the role of SOLENOT in the ER with and without ER-stress in the processes of carcinogenesis, using as model cell lines of two widespread types of cancer, glioblastoma and colorectal adenocarcinoma. The choice of these cell lines was made because the mRNA expression and protein content in them is quite high compared to other available cell lines. In addition, these cells of both lines were found to be most suitable for efficient transduction, since the percentage of transduced cells was over 90%.
We selected MSA and SS as selenium-containing ER-stress sources for A-172 and Caco-2 [13,14]. The choice of these ER–stress inducers was not made by chance. We previously carried out studies on cytotoxic effects of MSA and SS in these cell lines and analysed the expression of SELENOT at various concentrations. It was found that when both cell lines were treated with 0.1 μM MSA or SS, a slight increase in SELENOT expression was observed. In addition, when these cell lines were treated with 0.1 μM MSA or SS, a greater percentage of cells were in a state of apoptosis compared to 0.01 μM concentrations of these compounds. In addition, earlier, a similar series of experiments was carried out using the well–known non–selenium ER–stress inducer DTT; it was shown that DTT in Caco–2 cells promoted an increase in the expression of SELENOT.
It is known that the cytotoxic effect of selected ER–stress inducers is mediated by various molecular mechanisms, primarily due to the production of various metabolites. The final metabolite of SS is hydrogen selenide, which, after being converted into selenophosphate, actively participates in the synthesis of selenoproteins through the interaction with selenocysteine tRNA. The active metabolite of MSA is methylselenol. The manifold antitumor mechanisms of MSA include: glutathione-dependent induction of lipid peroxidation should be distinguished , inhibition of the PI3K/AKT/mTOR pathway and activation of FOXO proteins , inhibition of the activity of deacetylase and DNA methyltransferase , inhibition of angiogenesis by suppressing β3–integrin and interrupting its clustering . DTT is a strong reducing agent of disulfide bonds in proteins due to two consecutive exchanges of thiol disulfides between cysteine residues in proteins. In addition, it is known that DTT is a pharmaceutical nonspecific inducers of ER–stress, since it blocks the formation of disulfide bonds in all proteins, including those newly synthesized in the cytosol. We selected this agent as a non–selenium source of ER stress, first of all, to establish the presence/ absence of a specific effect of MSA and SS on the expression of the studied genes, especially selenoproteins, under SELENOT–knockdown.
In the course of this work, it was established that SELENOT–knockdown does not affect either the viability and proliferative properties of A-172 and Caco-2 cells, and does not contribute to their acquisition of the properties of normal cells.
SELENOT–knockdown had practically no effect on the mRNA expression of key ER–stress genes; only when the cells were treated with 1 mM DTT, a slight decrease in the expression of the following genes was observed: GADD34, PUMA, BAX, CHOP and BAK. However, these small fluctuations in the mRNA expression of these genes did not have a conspicuous effect on the pathological state of cells under conditions of prolonged ER–stress as compared to cells in which SELENOT–expression was not disturbed.
According to the results of of their study, the greatest SELENOT–knockdown effect was found in the mRNA gene expression and protein content of the selenoprotein SELENOM as well as its two physiological partners AMFR and RNF5 under ER-stress conditions.
According to the results of our studies, it became obvious that among all seven selenium–containing proteins localized in the ER, only DIO2 and SELENOM most vividly reacted to a decrease in mRNA expression of SELENOT in the studied cancer cells. Moreover, DIO2 mRNA expression decreased in the absence of ER–stress and did not change when cells were treated with all three sources of ER–stress. Conversely, the expression of SELENOM mRNA decreased only under ER–stress, especially when cells were treated with 1 mM DTT and 5 mM DTT.
It is difficult to interpret the direct dependence of a decrease in the SELENOT mRNA expression on the expression of DIO2 mRNA; these data require confirmation by other independent approaches. As for SELENOM, this relationship is understandable. First, both proteins have a thioredoxin–like folding, which may explain the similarity of their functional role . Previously, we have shown that, when DU 145, HT–1080 and MCF–7 cancer cells were treated with 0.1 µM MSA, the expression of SELENOT mRNA changed synchronously with SELENOF, which also, along with SELENOT and SELENOM, have a thioredoxin–like folding, and always asynchronously with the expression of SELENOM mRNA . Therefore, in this case, it would be more logical to have an inverse correlation in the expression of mRNA of both selenoproteins. In general, out of all seven proteins, the function of SELENOM in the regulation of ER–stress and its role for ER in general remains less clear. In many cancer cells, the expression of its mRNA is high. We have repeatedly shown that its expression reacts to a large extent to the effects of selenium–containing sources of ER–stress [12–14, 20-25]. It is interesting that, like SELENOM protein, mRNA expression also decreased in two proteins that are components of the ERAD system: AMFR and RNF5. Earlier, the same tendency of these proteins upon SELENOT–knockout was observed for endocrine cells . AMFR–E3 ubiquitin–protein ligase mediates polyubiquitinylation of lysine and cysteine residues of target proteins for subsequent proteasome degradation. It was initially identified as a tumor autocrine motility factor receptor that promotes the invasion and metastasis of tumors. RNF5, Ring finger protein 5, is an E3 ubiquitin–protein ligase, knockdown of which significantly reduced AMFR–mediated ubiquitinylation of CFTR, the cystic fibrosis transmembrane conductance regulator . AMFR and RNF5 can be linked to each other via members of the Derlins family proteins (Derlin 1, 2, 3) and function as a Derlin–containing complex for the polyubiquitinylation of a number proteins. It has been shown that SELENOT is involved in the N–glycosylation of endogenous glycoproteins and is a subunit of the A–type oligosaccharyltransferase complex (OST) . The results of this work are consistent with the previously revealed dependence of a decrease in the mRNA expression of AMFR and RNF5 on a decrease in the mRNA expression of SELENOT . Since a similar pattern of changes in mRNA expression in A–172 and Caco–2 cells under ER–stress was also established for SELENOM, it is possible that this selenoprotein is also involved in the processes associated with cellular protein quality control (PQC) system and proteolytic machinery of cells. However, this remains to be elucidated by further research.
It has been shown that SELENOT interacts with KRTCAP2 (KCP2), STT3A, DDOST (OST 48), but not with STT3B . However, according to our results, the mRNA expression of these proteins remained practically unchanged upon SELENOT–knockdown both under ER–stress and in intact cells.
It has been reported that a decrease in the expression of two important enzymes of the ERAD–system upon SELENOT–knockdown leads to the accumulation of proteins with incorrect folding in the ER lumen, which, apparently, only aggravates ER–stress in cancer A–172 and Caco–2 cells, therefore, no significant changes in cells morphology, redox homeostasis were observed in our experiments.