Identifying Genes Associated with the Anticancer Activity of a Fluorinated Chalcone in Triple-Negative Breast Cancer Cells Using Bioinformatics Tools

Abstract

Fluorinated chalcones are molecules reported to possess potent anticancer properties against triple-negative breast cancer (TNBC) cells. However, their molecular mechanisms have not yet been fully explored. Using bioinformatics tools, we analyzed the transcriptomes of MDA-MB-231 cells treated with either a novel fluorinated chalcone (compound 3) or a control in order to identify differentially expressed (DE) genes associated with its anticancer activity and determine the biological processes in which these genes are involved. A fluorinated chalcone was synthesized using the Claisen-Schmidt method. The transcriptome of MDA-MB-231 cells was then analyzed on an Illumina NextSeq500, and DE genes with significant changes in expression were identified using the DESeq2 v1.38.0 bioinformatics tool under the strict detection criteria of |log2FC| ≥ 2 and adjusted p < 0.05. We identified 504 DE genes, which were enriched in terms related to "regulation of cell death", "cation transport", "response to topologically incorrect proteins", and "response to unfolded proteins". Surprisingly, these genes were involved in "the HSF1-dependent transactivation pathway" and "the attenuation phase pathway". This bioinformatics-based study suggests that the tested fluorinated chalcone could influence HSF-1 silencing in addition to promoting the up-regulation of several genes involved in stress-induced apoptosis. Therefore, the tested compound could have enormous potential as a novel approach for TNBC treatment.

Keywords: DE genes; TNBC; bioinformatic tools; fluorinated chalcone.

Figure 1

Effects of fluorinated chalcone (compound 3) on MDA-MB-231 cell viability and distribution of the DE genes. (a) Cell viability of MDA-MB-231 cells treated with the indicated concentrations of 3 (0–120 μM) for 48 h. Data are presented as mean ± SD from triplicate data. (b) The anticancer effects of compound 3 at 24, 48, and 72 h. (c) Morphological changes in MDA-MB-231 cells treated with compound 3 suggestive of apoptosis, such as contraction and rounding of the cells (red arrows), as well as the formation of blisters on the cell membrane (blue arrows). Scale bars represent 100 μm. (d) Volcano plot showing the genes that were obtained in the differential expression analysis. The y-axis represents the –log10 adjusted p-values, indicating the level of significance of each gene, while the x-axis represents the log2 fold change, indicating the difference between the expression levels of each gene between both conditions. Red dots represent up-regulated genes, green dots show down-regulated genes, and gray dots indicate those genes that showed no significant changes.

Figure 2

Heatmap of DE genes. The color scale represents log10 (i.e., normalized) expression values of DE genes. The horizontal axis represents the samples and the vertical axis represents DE genes, whose names are shown above. Red indicates up-regulated genes and blue represents down-regulated genes.

Figure 3

GO enrichment analysis representing the biological processes in which the DE genes are involved. The most significant processes are highlighted in red and the least significant in blue, based on -log10 (FDR) values. Larger dots on the graph indicate a greater number of genes involved. Abbreviation: FDR: False discovery rate.

Figure 4

Representation of Reactome pathway analysis. The orange color denotes those pathways in which the DE genes were involved, while those that were found to be significantly represented by these genes are indicated in blue letters.

Figure 5

Protein–protein interaction network visualized using STRING. The nodes indicate proteins and the gray lines indicate the number of interactions. The saturation of lines represents the confidence scores of the functional associations. Disconnected nodes are hidden, and only those interactions with a confidence score of 0.7 are shown. Abbreviations: STRING: Search Tool for the Retrieval of Interacting Genes/Proteins.

Figure 6

Application of the k-means algorithm for the identification of clusters in the STRING interaction network. Nodes represent proteins and edges indicate known interactions. The dotted lines indicate the connections between clusters. Colors differentiate the three obtained groups, highlighting functional modules within the network.

Figure 7

A possible apoptotic mechanism of the tested fluorinated chalcone in MDA-MB-231 cells. (1) Due to the physicochemical characteristics of the fluorinated chalcone, it could enter cells by passive diffusion or via specific cellular transporters such as the basolateral organic cation transporter (OCT-1). (2) Accumulation of the fluorinated chalcone at the intracellular level leads to alterations in nDNA and mtDNA and perturbations in the electron transport system, as well as alterations in the mitochondrial membrane potential (∆Ψm), which leads to an increase in the production of reactive oxygen species (ROS) and oxidative stress. (3) ROS trigger endoplasmic reticulum (ER) stress which, in turn, further enhances ROS production. (4) In response to cellular damage, several cytoprotective mechanisms are activated, including the expression of ATF3 heat shock proteins (HSPs70), and HSF1, as well as the activation of other stress-inducible genes. (5) The overproduction of HSPs70, in turn, triggers a counter-feedback mechanism involving the binding of HSPs70 to the HSF1 trimers, which leads to dissociation from the promoter and its conversion to the inactive monomeric form. (6) As a result, the dissociation of HSF1 increases the susceptibility of MDA-MB-231 cells to stress-induced apoptosis. (7) Finally, several apoptosis-inducing genes are activated, including potassium channels, calcium channels, SLC co-transporters, and KLHL, among others. Abbreviations: ∆ψm: membrane potential; Apaf-1: apoptosis protease-activating factor-1; Cytc: cytochrome C; nDNA: nuclear deoxyribonucleic acid; DISC: death-inducing signaling complex; ER: endoplasmic reticulum; HSF1: Heat Shock Factor 1; HSP: heat shock protein; KLHL: Kelch-like proteins; mtDNA: mitochondrial deoxyribonucleic acid; ROS: reactive oxygen species; Ub: ubiquitin.

Scheme 1

Reaction procedure for the synthesis of the fluorinated chalcone (compound 3). The name of this compound is given according to the IUPAC. Abbreviations: EtOH: ethanol; IUPAC: International Union of Pure and Applied Chemistry; NaOH: sodium hydroxide.