In the same way, the gene, which encodes a pyruvate dehydrogenase kinase that plays a key role in the regulation of glucose and fatty acid metabolism and homeostasis via phosphorylation of the two pyruvate dehydrogenase subunits [57], is upregulated by 5.5- and four-fold at 12 h and 24 h, respectively (Determine 2a). Therefore, MDA-MB-231 cells were treated with 100 M DHA for 12 h or 24 h before RNA-seq analysis. The results show the great impact of DHA-treatment around the transcriptome, especially after 24 h of treatment. The impact of DHA is particularly visible in genes involved in the cholesterol biosynthesis Celecoxib pathway that is strongly downregulated, and the endoplasmic reticulum (ER)-stress response that is, conversely, upregulated. This ER-stress and unfolded protein response could explain the pro-apoptotic effect of DHA. The expression of genes related to migration and invasion (especially or appear as encouraging alternatives for DHA supply [11,12,18]. DHA has shown anticancer effects in several types of cancers and, at the cellular level, has exhibited encouraging antiproliferative and pro-apoptotic effects in several types of cancers and cell lines [9,19,20,21], among which breast malignancy cells [17,22,23]. Celecoxib The mechanisms underlying the anticancer effect of DHA include the regulation of Wnt/-catenin inhibition, oxidative DNA damage, and mitogen-activated protein kinase activation (examined in [18,22,23]). DHA was also shown to induce autophagy whilst suppressing mTOR in human cervical malignancy, prostate malignancy, lung malignancy, and glioblastoma cells [24,25,26,27]. More specifically for breast malignancy, several intracellular targets have been identified as being involved in the DHA effect, among which the Fli1 PKB/akt, and p53 pathways and increased caspase activity (examined in [18,23]). Additional mechanisms involved in the pro-apoptotic effect of DHA in breast cancer cells include, but are not limited to, decreased Erk activity [28,29], increased Bax pro-apoptotic enzyme levels or activity and decreased Bcl-XL, increased death receptors (DR-4, TRAIL, and Fas) expression and mitochondrial release of the caspase activator SMAC/Diablo in the MCF-7 cell collection [30], PPAR- overexpression in breast cancer tissue or cells [31,32], increased expression of the stress-induced growth inhibitor 1 (OSGIN1) and transcription factor NFE2L2 in MCF-7 and Hs578T breast malignancy cells [33]. However, DHA also increased the activity of antioxidant enzymes (SOD, CAT, and GPX) in breast cancer tissues [31], suggesting that this anti-/pro-oxidant effects of DHA may be dependent on the cell type and concentration used. Finally, DHA also functions as an antiproliferative agent by lengthening the cell cycle at the G2/M transition, such as in the MDA-MB-231 breast cancer cell collection [34]. A few studies have shown that diet can affect the metastatic potential of malignancy cells known to have a high metastatic phenotype Celecoxib such as breast malignancy [35,36]. The anti-invasive effect of DHA, at a concentration ranging from 10 to 100 M, was highlighted in breast malignancy cell lines [31,37,38,39,40,41,42,43]. Moreover, this anti-metastatic effect is usually corroborated by in vivo animal studies using transgenic mice capable of generating n-3 FA from your n-6 type, leading to abundant n-3 FA with reduced levels of n-6 FA in their organs and tissues, and this was without the need of a dietary n-3 supply [44]. The use of transgenic mice has shown a decrease in tumor growth and the diminution of lung metastasis of syngeneic breast cancer cells in this DHA-rich environment [42]. Therefore, DHA appears as a safe, natural compound that can greatly improve the anticancer properties of anticancer drugs by additive or synergistic interactions [14,45,46]. In addition, n-3 PUFA reduced the risk of obesity-related breast malignancy [47] and experienced protective effects towards cardiotoxicity of anthracyclines, the most extensively used chemotherapeutics [48,49]. Thus, current results of cohort studies and investigations in cell lines or animal models exhibited that DHA could reduce tumor cell number by acting as soon as the cell begins its neoplastic transformation through a decrease in proliferation and an increase in apoptosis. However, the cellular targets and mechanisms of action of DHA remain to be further comprehended, and genome-wide transcriptomic studies are few. In the present study, MDA-MB-231 cells, which are a generally used model of triple-negative breast malignancy, were submitted to DHA treatment and then analyzed by RNA-sequencing. 2. Materials and Methods 2.1. Cell Culture and Treatment The triple-negative breast cancer cell collection MDA- MB-231 was purchased from ATCC (Manassas, VA, USA) and routinely produced as monolayers at 37 C, in a humidified atmosphere with 5% CO2, in minimum essential medium (MEM) (Sigma-Aldrich, Saint-Quentin Fallavier, France) supplemented with 10% fetal calf serum (FCS) (Gibco; Invitrogen, Cergy Pontoise, France), 20 mM Hepes, and 2 mM L-glutamine, Celecoxib 100 U/ml penicillin/streptomycin (Sigma-Aldrich). For subsequent RNA analysis experiments, cells were plated in four biological replicates in six-wells plates for 24 h, then the medium was.