A novel combinatorial approach using sulforaphane- and withaferin A-rich extracts for prevention of estrogen receptor-negative breast cancer through epigenetic and gut microbial mechanisms

Abstract

Estrogen receptor-negative [ER(-)] mammary cancer is the most aggressive type of breast cancer (BC) with higher rate of metastasis and recurrence. In recent years, dietary prevention of BC with epigenetically active phytochemicals has received increased attention due to its feasibility, effectiveness, and ease of implementation. In this regard, combinatorial phytochemical intervention enables more efficacious BC inhibition by simultaneously targeting multiple tumorigenic pathways. We, therefore, focused on investigation of the effect of sulforaphane (SFN)-rich broccoli sprouts (BSp) and withaferin A (WA)-rich Ashwagandha (Ash) combination on BC prevention in estrogen receptor-negative [ER(-)] mammary cancer using transgenic mice. Our results indicated that combinatorial BSp + Ash treatment significantly reduced tumor incidence and tumor growth (~ 75%) as well as delayed (~ 21%) tumor latency when compared to the control treatment and combinatorial BSp + Ash treatment was statistically more effective in suppressing BC compared to single BSp or Ash intervention. At the molecular level, the BSp and Ash combination upregulated tumor suppressors (p53, p57) along with apoptosis associated proteins (BAX, PUMA) and BAX:BCL-2 ratio. Furthermore, our result indicated an expressional decline of epigenetic machinery HDAC1 and DNMT3A in mammary tumor tissue because of combinatorial treatment. Interestingly, we have reported multiple synergistic interactions between BSp and Ash that have impacted both tumor phenotype and molecular expression due to combinatorial BSp and Ash treatment. Our RNA-seq analysis results also demonstrated a transcriptome-wide expressional reshuffling of genes associated with multiple cell-signaling pathways, transcription factor activity and epigenetic regulations due to combined BSp and Ash administration. In addition, we discovered an alteration of gut microbial composition change because of combinatorial treatment. Overall, combinatorial BSp and Ash supplementation can prevent ER(-) BC through enhanced tumor suppression, apoptosis induction and transcriptome-wide reshuffling of gene expression possibly influencing multiple cell signaling pathways, epigenetic regulation and reshaping gut microbiota.

Figure 1

Early life BSp and/or Ash treatment prevents ER-negative breast tumor development in C3 mice. Effect of different single and combined administration of BSp and Ash on tumor incidence (A), tumor latency (B), tumor growth volume (C,D) and tumor weight measured at the termination point (E). Values are means ± SEMs. The significances of tumor incidence were analyzed using the chi-square test. Means that do not share a common superscript are significantly different at p ≤ 0.05. Significances of tumor volume among treatments were determined using two-way repeated measures ANOVA considering time and treatment as factors. Values are means ± SE, n = 10. Means that do not share a common superscript are significantly different at p ≤ 0.05. Tumor latency and tumor weight comparisons among the dietary groups were performed with One-way ANOVA analysis and Tukey’s HSD. Values are means ± SEM, n = 8–10. Means that do not share a common superscript are significantly different at p ≤ 0.05. Here, BSp: Broccoli sprouts; Ash: Ashwagandha; BSp + Ash: Broccoli sprouts and Ashwagandha combination.

Figure 2

Expressional changes of key tumor suppressors proteins in the mammary tumor of single and combined BSp and Ash treated mice. The top panel shows the cropped images from an imaging system. The bottom panel shows the statistical analysis of band intensity calculated on the images using ImageJ. Graphs represent β-actin normalized protein expression level with each bar representing the mean ± SE (n = 3). Means that do not share a common superscript are significantly different at p ≤ 0.05 upon One-way ANOVA and Tukey’s post-hoc analysis. Here, BSp: Broccoli sprouts; Ash: Ashwagandha; BSp + Ash: Broccoli sprouts and Ashwagandha combination.

Figure 3

Expressional changes of key apoptosis-associated proteins in the mammary tumor of single and combined BSp and Ash treated mice. The top panel shows the cropped images from an imaging system. The bottom panel shows the statistical analysis of band intensity calculated on the images using ImageJ. Graphs represent β-actin normalized protein expression level with each bar representing the mean ± SE (n = 3). Means that do not share a common superscript are significantly different at p ≤ 0.05 upon One-way ANOVA and Tukey’s post-hoc analysis. Here, BSp: Broccoli sprouts; Ash: Ashwagandha; BSp + Ash: Broccoli sprouts and Ashwagandha combination.

Figure 4

Expressional changes of key epigenetics modification-related proteins in the mammary tumor of single and combined BSp and Ash treated mice. The top panel shows the cropped images from an imaging system. The bottom panel shows the statistical analysis of band intensity calculated on the images using ImageJ. Graphs represent β-actin normalized protein expression level with each bar representing the mean ± SE (n = 3). Means that do not share a common superscript are significantly different at p ≤ 0.05 upon One-way ANOVA and Tukey’s post-hoc analysis. Here, BSp: Broccoli sprouts; Ash: Ashwagandha; BSp + Ash: Broccoli sprouts and Ashwagandha combination.

Figure 5

Dietary BSp and/or Ash treatment induce alteration of transcriptome in mammary tumor of C3 mice. (A) Heatmap representing differentially expressed mRNAs in C3 mice due to combinatorial BSp and Ash treatment. Each row represents differentially expressed mRNA, and each column represents biological replicates. Here, Ctl indicates control diet treated control group samples (n = 3) while BA indicated combinatorial BSp + Ash treated test group samples (n = 3). Blue color denotes downregulation and red color denotes upregulation. (B) The volcano plot shows distribution of DEGs in C3 mice as a result combinatorial BSp + Ash treatment. Here, gray dots indicate mRNAs that were not significantly expressed, blue dots represent downregulated mRNAs and red dots indicate upregulated mRNAs [Benjamini–Hochberg FDR < 0.05 and |log2(fold-change) |> 1). (C) The dot plot representing the gene ontology enrichment terms for the differentially expressed mRNAs in C3 mice. The dot size represents the number of enriched genes while the dot color represents activated (blue) and suppressed (red) genes. (D) Validation of differentially expressed mRNA with qRT-PCR. Graphs represent β-actin-normalized expression level of corresponding mRNA with each bar representing the mean ± standard error (SE) of three biological replicates (n = 3). The asterisks indicate statistical significance (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001) upon unpaired T-test. Here, BSp + Ash indicates broccoli sprouts and Ashwagandha combination treatment.

Figure 6

Gut microbial composition changes in dietary treatment groups before and after tumor onset. (A) Alpha-diversity before tumor onset: observed species, PD whole tree, Shannon and Simpson diversity. The asterisk indicates statistical significance (*p ≤ 0.05, **p ≤ 0.01) upon One-way ANOVA and Tukey’s post-hoc analysis. Here, BSp: Broccoli sprouts; Ash: Ashwagandha; BSp + Ash: Broccoli sprouts and Ashwagandha combination. (B) Bray Curtis 3D-PCoA plot before tumor onset. The red dots indicate control group samples, green dots indicate BSp group samples, yellow dots indicate Ash group samples, blue dots indicate combinatorial BSp + Ash group samples. Statistical analysis of Bray–Curtis and weighed Unifrac tests of beta diversity was performed using permutational multivariate analysis of variance (PERMANOVA). Statistical significance: control-BSp (p = 0.007), control-Ash (p = 0.043), control-combination (p = 0.003); and with weighed Unifrac control-BSp (p = 0.462), control-Ash (p = 0.042), control-combination (p = 0.002). (C) Phylum level changes of microbial community in dietary treated groups before tumor onset. The top 10 abundant phyla in respective groups are represented in pie charts. D) Alpha-diversity after tumor onset: observed species, PD whole tree, Shannon and Simpson diversity. The asterisk indicates statistical significance (*p ≤ 0.05) upon One-way ANOVA and Tukey’s post-hoc analysis. Here, BSp: Broccoli sprouts; Ash: Ashwagandha; BSp + Ash: Broccoli sprouts and Ashwagandha combination. (E) Bray Curtis 3D-PCoA plot after tumor onset. The red dots indicate control group samples, green dots indicate BSp group samples, yellow dots indicate Ash group samples, blue dots indicate combinatorial BSp + Ash group samples. Statistical analysis of Bray–Curtis and weighed Unifrac tests of beta diversity was performed using permutational multivariate analysis of variance (PERMANOVA). Statistical significance: control-BSp (p = 0.007), control-Ash (p = 0.043), control-combination (p = 0.003); and with weighed Unifrac control-BSp (p = 0.462), control-Ash (p = 0.042), control-combination (p = 0.002). (F) Phylum level changes of microbial community in dietary treated groups after tumor onset. The top 10 abundant phyla in respective groups are represented in pie charts.