Whole Transcriptomic Analysis of Apigenin on TNFα Immuno-activated MDA-MB-231 Breast Cancer Cells.

BACKGROUND

Triple-negative breast cancer is categorized by a lack of hormone receptors, inefficacy of anti-estrogen or aromatase inhibitor chemotherapies and greater mortality rates in African American populations. Advanced-stage breast tumors have a high concentration of tumor necrosis factor-α (TNFα) throughout the tumor/stroma milieu, prompting sustained release of diverse chemokines (i.e. C-C motif chemokine ligand 2 (CCL2)/CCL5). These potent chemokines can subsequently direct mass infiltration of leukocyte sub-populations to lodge within the tumor, triggering a loss of tumor immune surveillance and subsequent rapid tumor growth. Previously, we demonstrated that in the MDA-MB-231 TNBC cell line, TNFα evoked a rise in immune signaling proteins: CCL2, granulocyte macrophage colony-stimulating factor, interleukin (IL)1α, IL6 and inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKBKε) all of which were attenuated by apigenin, a dietary flavonoid found in chamomile and parsley.

MATERIALS AND METHODS

The present work elucidates changes evoked by TNFα in the presence or absence of apigenin by examining the entire transcriptome for mRNA and long intergenic non-coding RNA with Affymetrix Hugene-2.1_ST human microarrays. Differential gene-expression analysis was conducted on 48,226 genes.

RESULTS

TNFα caused up-regulation of 75 genes and down-regulation of 10. Of these, apigenin effectively down-regulated 35 of the 75 genes which were up-regulated by TNFα. These findings confirm our previous work, specifically for the TNFα-evoked spike in IL1A vs. untreated controls [+21-fold change (FC), p<0.0001] being attenuated by apigenin in the presence of TNFa (-15 FC vs. TNFα, p<0.0001). Similar trends were seen for apigenin-mediated down-regulation of TNFα-up-regulated transcripts: IKBKE (TNFα: 4.55 FC vs. control, p<0.001; and TNFα plus apigenin: -4.92 FC, p<0.001), CCL2 (2.19 FC, p<0.002; and -2.12 FC, p<0.003), IL6 (3.25 FC, p<0.020; and -2.85 FC, p<0.043) and CSF2 (TNFα +6.04 FC, p<0.001; and -2.36 FC, p<0.007). In addition, these data further establish more than a 65% reduction by apigenin for the following transcripts which were also up-regulated by TNFα: cathepsin S (CTSS), complement C3 (C3), laminin subunit gamma 2 (LAMC2), (TLR2), toll-like receptor 2 G protein-coupled receptor class C group 5 member B (GPRC5B), contactin-associated protein 1 (CNTNAP1), claudin 1 (CLDN1), nuclear factor of activated T-cells 2 (NFATC2), C-X-C motif chemokine ligand 10 (CXCL10), CXCL11, interleukin 1 receptor-associated kinase 3 (IRAK3), nuclear receptor subfamily 3 group C member 2 (NR3C2), interleukin 32 (IL32), IL24, slit guidance ligand 2 (SLIT2), transmembrane protein 132A (TMEM132A), TMEM171, signal transducing adaptor family member 2 (STAP2), mixed lineage kinase domain-like pseudokinase (MLKL), kinase insert domain receptor (KDR), BMP-binding endothelial regulator (BMPER), and kelch-like family member 36 (KLHL36).

CONCLUSION

There is a possible therapeutic role for apigenin in down-regulating diverse genes associated with tumorigenic leukocyte sub-population infiltration by triple-negative breast cancer. The data have been deposited into the Gene Expression Omnibus for public analysis at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE120550.