Effects of Wild Yam Root (Dioscorea villosa) Extract on the Gene Expression Profile of Triple-negative Breast Cancer Cells

Abstract

Background/aim: Wild yam extract [Dioscorea villosa, (WYE)] is consistently lethal at low IC₅₀s across diverse cancer-lines in vitro. Unlike traditional anti-cancer botanicals, WYE contains detergent saponins which reduce oil-water interfacial tensions causing disintegration of lipid membranes and causing cell lysis, creating an interfering variable. Here, we evaluate WYE at sub-lethal concentrations in MDA-MB-231 triple-negative breast cancer (TNBC) cells.

Materials and methods: Quantification of saponins, membrane potential, lytic death and sub-lethal WYE changes in whole transcriptomic (WT) mRNA, miRNAs and biological parameters were evaluated.

Results: WYE caused 346 differentially expressed genes (DEGs) out of 48,226 transcripts tested; where up-regulated DEGS reflect immune stimulation, TNF signaling, COX2, cytokine release and cholesterol/steroid biosynthesis. Down-regulated DEGs reflect losses in cell division cycle (CDC), cyclins (CCN), cyclin-dependent kinases (CDKs), centromere proteins (CENP), kinesin family members (KIFs) and polo-like kinases (PLKs), which were in alignment with biological studies.

Conclusion: Sub-lethal concentrations of WYE appear to evoke pro-inflammatory, steroid biosynthetic and cytostatic effects in TNBC cells.

Keywords: Dioscorea; Immune stimulation; breast cancer; cell cycle; wild yam.

Figure 1. Cytotoxic and cytostatic effects related to saponin content of WYE in MDA-MB-231 cells. The data represent the mean±S.E.M, n=4 for cell viability (at 24 h), cell proliferation (at 6 days) plotted as % (untreated) controls and WYE saponin foam (where 592 μg/ml was set at 100% control), n=3. Significant differences between the controls and treatment groups were determined by a one way ANOVA, followed by a Tukey post hoc test ,*p<0.05. The gene chip icons, represent the 2 concentrations at which microarray analysis work was conducted representing WYE sublethal Low (15 μg/ml) and WYE sub-lethal High (30 μg/ml).

Figure 2. Effect of WYE on cell membrane potentials (plasma/viability) obtained with FDA (left panel) and mitochondrial TMRE (right panel). (A) Control, WYE Treatment (B) 18.5 μg/ml, (C)37 μg/ml, (D) 74 μg/ml, (E) 148 μg/ml, (F) 296 μg/ml).

Figure 3. Effect of WYE on 3D tumor spheroids. Cell viability and morphological changes in MDA-MB-231 tumor spheroids with increasing concentration of WYE are shown. The data represent basic changes in spheroid structure (main image; black and white), with fluorescent FDA viable cell staining (green) in the lower left section of each main image. (A) Control, WYE Treatment (B) 4.62 μg/ml, (C) 9.25 μg/ml, (D) 18.5 μg/ml, (E) 37 μg/ml, (F) 74 μg/ml, (G) 148 μg/ml, (H) 296 μg/ml.

Figure 4. Microarray gene summary report. Overview of DEGs expressed when comparing controls (untreated) n=3 vs. WYE (Low, sub-lethal at 15 ug/ml) n=3 or WYE (High, sub-lethal cusp of cell death at 30 ug/ml). The data selection criteria were set at -2+2 fold change, p-Value <0.05 and FDR p-Value <0.05.

Figure 5. Stringdb relational network analysis of down-regulated DEGS caused by WYE (Low) 15 μg/ml vs. untreated controls after meeting selection criteria; -2+2 fold change, p-Value <0.05 and FDR p-Value <0.05. The data in the corresponding table: represent database used, ID #s within a database, description of system altered, count in the network for system, strength of relationship and the false discovery rate (FDR) p-Value. Color codes indicate genes (by symbol) in the string diagram corresponding to the table class description.

Figure 6. Cell cycle Wikipathway analysis report for DEGS caused by WYE (Low) 15 μg/ml vs. untreated controls after meeting selection criteria; -2+2, p-Value <0.05 and FDR p-Value <0.05. DEGS are color coded (red=down), (green=up) with color intensity darker with fold higher fold change. Gene symbol denoted by cross bars /// were not altered.

Figure 7. Stringdb relational network analysis of up-regulated DEGS caused by WYE (Low) 15 μg/mL vs. untreated controls after meeting selection criteria; -2+2, p-Value <0.05 and FDR p-Value <0.05. The data represent database used, ID #s within a database, description of system altered, count in the network for system, strength of relationship and the false discovery rate (FDR) p-Value. Color codes indicate genes (by symbol) in the string diagram corresponding to the table class description.

Figure 8. TNF signaling Kegg Pathway report for up-regulated DEGS caused by WYE (Low) 15 μg/ml vs. untreated controls after meeting selection criteria; -2+2 fold change, p-Value <0.05 and FDR p-Value <0.05. Up-regulated DEGS are demarcated by a red star.

Figure 9. Supernatant cytokine detection vs. mRNA transcript. Cytokine released in untreated controls vs. (WYE-Low, 15 μg/ml) in supernatants obtained from the same pellets used for microarray analysis and analyzed by a human cytokine array. The data represents matched changes occurring in both the protein antibody with *p-Values <0.05 in both sets, where array values were set at selection criteria; any fold change, p-Value <0.05 and no filter on FDR p-Values (B). There was a high degree of matching values between proteins released in the supernatant and the mRNA produced for those proteins.

Figure 10. Supernatant cytokine detection. Supernatants collected from pellets used for microarray analysis (WYE-Low, 15ug/ml) show semiquantitative changes in IL-8 by anti-body array (densitometry spot duplicates), demarcated alongside transcriptomic microarray chip spot probe arrays for IL-8 in MDA-MB-231 cells.

Figure 11. Supernatants collected from pellets used for microarray analysis (WYE-Low, 15 μg/ml) show quantitative changes in IL-8 as determined by ELISA. The data represent the mean±S.E.M for optical densitometry values, n=3 and significant differences between the control and treatment was determined by a students t-test; *p<0.05.