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

ABSTRACT: 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-? (TNF?) 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, TNF? evoked a rise in immune signaling proteins: CCL2, granulocyte macrophage colony-stimulating factor, interleukin (IL)1?, IL6 and inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKBK?) 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 TNF? 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:TNF? 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 TNF?. These findings confirm our previous work, specifically for the TNF?-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. TNF?, p<0.0001). Similar trends were seen for apigenin-mediated down-regulation of TNF?-up-regulated transcripts: IKBKE (TNF?: 4.55 FC vs. control, p<0.001; and TNF? 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 (TNF? +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 TNF?: 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.

SUBMITTER: Bauer D

PROVIDER: S-EPMC6885362 | biostudies-literature | 2019 Nov-Dec

REPOSITORIES: biostudies-literature

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

Bauer David D Mazzio Elizabeth E Soliman Karam F A KFA

Cancer genomics & proteomics 20191101 6

<h4>Background</h4>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 ...[more]

PMID: 31659097

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Project description:The lack of hormone receptors in triple negative breast cancer (TNBC) is associated with the inefficacy of anti-estrogen chemotherapies, leaving fewer options for patient treatment and higher mortality rates. Additionally, as with numerous types of inflammatory breast cancer, infiltration of tumor associated macrophages and other leukocyte sub-populations within the tumor inevitably lead to aggressive, chemo-resistant, metastatic and invasive types of cancer which escape immune surveillance. These processes are orchestrated by the release of potent cytokines, including TNF?, IL-6 and CCL2 from the stroma, tumor and immune cells within the tumor microenvironment. The present study evaluated apigenin modulating effects on the pro-inflammatory activating action of TNF? in TNBC MDA-MB-468 cells, derived from an African American woman. Initially, cell viability was determined to establish an optimal sub-lethal dose of TNF? and apigenin in MDA-MB-468 cells. Subsequently, various treatments effects were evaluated using whole transcriptomic analysis of mRNA and long intergenic non-coding RNA with Affymetrix HuGene-2.1-st human microarrays. Gene level differential expression analysis was conducted on 48,226 genes where TNF? caused significant upregulation of 53 transcripts and downregulation of 11 transcripts. The largest upward differential shift was for CCL2 [+61.86 fold change (FC); false discovery rate (FDR), P<0.0001]; which was down regulated by apigenin (to +10.71 FC vs. Control; FDR P-value <0.001), equivalent to an 83% reduction. Several TNF? deferentially upregulated transcripts were reduced by apigenin, including CXCL10, C3, PGLYRP4, IL22RA2, KMO, IL7R, ROS1, CFB, IKBKe, SLITRK6 (a checkpoint target) and MMP13. Confirmation of CCL2 experimentally induced transcript alterations was corroborated at the protein level by ELISA assays. The high level of CCL2 transcript in the cell line was comparable to that in our previous studies in MDA-MB-231 cells. The differential effects of TNF? were corroborated by ELISA, where the data revealed a >10-fold higher releasing rate of CCL2 in MDA-MB-468 cells compared with in MDA-MB-231 cells, both of which were attenuated by apigenin. The data obtained in the present study demonstrated a high level of CCL2 in MDA-MB-468 cells and a possible therapeutic role for apigenin in downregulating TNF?-mediated processes in these TNBC cells.

Project description:Since bone metastatic breast cancer is an incurable disease, causing significant morbidity and mortality, understanding of the underlying molecular mechanisms would be highly valuable. Here, we describe in vitro and in vivo evidence for the importance of serine biosynthesis in the metastasis of breast cancer to bone. We first characterized the bone metastatic propensity of the MDA-MB-231(SA) cell line variant as compared to the parental MDA-MB-231 cells by radiographic and histological observations in the inoculated mice. Genome-wide gene expression profiling of this isogenic cell line pair revealed that all the three genes involved in the L-serine biosynthesis pathway, phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH) were upregulated in the highly metastatic variant. This pathway is the primary endogenous source for L-serine in mammalian tissues. Consistently, we observed that the proliferation of MDA-MB-231(SA) cells in serine-free conditions was dependent on PSAT1 expression. In addition, we observed that L-serine is essential for the formation of bone resorbing human osteoclasts and may thus contribute to the vicious cycle of osteolytic bone metastasis. High expression of PHGDH and PSAT1 in primary breast cancer was significantly associated with decreased relapse-free and overall survival of patients and malignant phenotypic features of breast cancer. In conclusion, high expression of serine biosynthesis genes in metastatic breast cancer cells and the stimulating effect of L-serine on osteoclastogenesis and cancer cell proliferation indicate a functionally critical role for serine biosynthesis in bone metastatic breast cancer and thereby an opportunity for targeted therapeutic interventions. Parental MDA-MB-231 cells and MDA-MB-231(SA) cells were cultured in cell culture flasks. RNA was isolated in order to compare the gene expression profiles of these cell variants. Total of two samples. No replicates.

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

Publications

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

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