A Novel Combinatorial Epigenetic Therapy Using Resveratrol and Pterostilbene for Restoring Estrogen Receptor-α (ERα) Expression in ERα-Negative Breast Cancer Cells.

Breast cancer is the second most common cancer and a leading cause of cancer death in women. Specifically, estrogen receptor-α (ERα)-negative breast cancers are clinically more aggressive and normally do not respond to conventional hormone-directed therapies such as tamoxifen. Although epigenetic-based therapies such as 5-aza-2'-deoxycytidine and/or trichostatin A as DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors, respectively, can regulate the expression of ERα, this can often lead to a number of side effects. Plant-based dietary compounds such as resveratrol and pterostilbene in novel combinatorial therapy provides new avenues to target these side effects and provide similar results with a higher level of safety. Here, we report that combinatorial resveratrol and pterostilbene leads to the reactivation of ERα expression in ERα-negative breast cancer cells in a time-dependent manner. Chromatin immunoprecipitation analysis of the ERα promoter in each cell type revealed an increase in enrichment of acetyl-H3, acetyl-H3lysine9 (H3K9) and acetyl-H4 active chromatin markers in the ERα promoter region after combinatorial treatment. This treatment also resulted in a significant change in HDAC and histone acetyl transferase (HAT) enzyme activity in these cells after 3 days of treatments. The combination resulted in a significant decrease in DNMT enzyme activity and 5-methylcytosine levels in MDA-MB-157 breast cancer cells. Moreover, reactivation of ERα expression by resveratrol combined with pterostilbene was found to sensitize ERα-dependent response to 17β-estradiol (E2)-mediated cellular proliferation and antagonist 4-hydroxytamoxifen (4-OHT)-mediated inhibition of cellular proliferation in ERα-negative breast cancer cells. E2 and 4-OHT further affected the ERα-responsive downstream progesterone receptor (PGR) gene in ERα reactivated MDA-MB-157 cells. Collectively, our findings provide a new and safer way of restoring ERα expression by regulating epigenetic mechanisms with the use of phytochemicals in combinatorial therapy. This combination can further provide effective treatment options for hormonal refractory breast cancer with available anti-hormonal therapy

Combinatorial treatment retrieved responsiveness to E2 and 4-hydroxytamoxifen (4-OHT) treatments in resensitized MDA-MB-157 TNBC cells.

<p><i>PGR</i> expression was detected by quantitative real-time PCR. GAPDH was used as the internal control. MDA-MB-157 cells were treated with or without 10 nM E2 or 5 μM 4-OHT for 48 h after 72 h of treatment with combinatorial treatment. MCF-7 cells served as a positive control. Cells without any E2 or 4-OHT served as the system control within the different treatment sets. Combination treatment along with E2 (ligand activator) treatment resulted in a significant increase in <i>PRG</i> mRNA and, with 4-OHT (antagonist) treatment, resulted in a significant decrease in <i>PGR</i> mRNA expression. MCF-7 cells when treated with E2 (ligand activator) and 4-OHT (antagonist) for 48 h showed a similar <i>PGR</i> mRNA responsiveness. Values are representative of three independent experiments and are relative to control ± SE; *P<0.05, **P<0.01.</p

Combinatorial treatment results in a significant decrease in DNMT enzyme activity and global DNA methylation.

<p>Fig 5a shows a relative decrease in overall DNMT enzyme activity with 5 μM pterostilbene and combination treatments. These decreases in DNMT activity with combination were found to be highly significant when compared with different treatment groups. Fig 5b shows a relative decrease in global DNA methylation pattern with 5 μM pterostilbene and combination treatments. This decrease in 5-mC levels with combination was found to be highly significant and in accordance to our DNMT data, when compared with different treatment groups. Res 15, resveratrol 15 μM; Ptero 5, pterostilbene 5 μM; Combination, 15 μM resveratrol and 5 μM pterostilbene in combination. Values are representative of three independent experiments and represented as relative of DMSO control ± SE; *P<0.05, **P<0.01.</p

Combinatorial resveratrol and pterostilbene resulted in a time-dependent reexpression of ERα in HCC1806 cells.

<p><b>a)</b> Relative real-time <i>ER</i>α mRNA expression after 24, 48 and 72 h of treatments in HCC1806 breast cancer cell lines. There was no significant effect of compounds alone or in combination at 24 h and 48 h of treatment; however, with combination treatment and 15 μM resveratrol after 72 h there was a significant increase in ERα mRNA expression. The increase with combination treatment was found to be more significant in comparison to other treatments at 72 h. GAPDH was used as the internal control. <b>b and c)</b> Effects of compounds alone as well as in combination on ERα protein expression after 24, 48 and 72 h of treatments. Fig b represents western blot analysis at three different time intervals and Fig c represents a densitometry analysis of ERα protein expression after 72 h. Treatment with pterostilbene as well as with combination of resveratrol and pterostilbene at 15 μM and 5 μM respectively, resulted in a significant increase in protein expression in HCC1806 cells after 72 h. This increase in expression with combination of compounds was found to be highly-significant when compared with other treatments. MCF-7 ERα protein extracts were used as the positive control and βactin was used as an internal control. Res 15, resveratrol 15 μM; Ptero 5, pterostilbene 5 μM; Combination, 15 μM resveratrol and 5 μM pterostilbene in combination. Values are representative of three independent experiments and are shown as percent of control ± SE; *P<0.05, **P<0.01.</p

Combinatorial treatments result in sensitizing ERα-negative cells towards E2 and 4-hydroxytamoxifen (4-OHT) treatments.

<p>MDA-MB-157 cells were treated with or without 10 nM E2 or 5 μM 4-OHT for 48 h after 72 h of treatment with the dietary compounds. MCF-7 cells served as a positive control. Cells without any E2 or 4-OHT served as the system control within the different treatment sets. Interestingly, with combination treatment and E2 (ligand activator) there was a significant increase in cellular viability and, with 4-OHT (antagonist) there was a significant decrease in cellular viability, as analyzed by MTT assay. MCF-7 cells when treated with E2 (ligand activator) and 4-OHT (antagonist) for 48 h showed a similar responsiveness. Res 15, resveratrol 15 μM; Ptero 5, pterostilbene 5 μM; Combination, 15 μM resveratrol and 5 μM pterostilbene in combination. Values are representative of three independent experiments and represented as relative to control ± SE; *P<0.05, **P<0.01.</p

Combinatorial resveratrol and pterostilbene resulted in a time-dependent reexpression of ERαin MDA-MB-157 cells.

<p><b>a)</b> Relative real-time <i>ERα</i>mRNA expression after 24, 48 and 72 h of treatments in MDA-MB-157 breast cancer cell lines. After 72 h of combinational treatment, there was a significant increase in <i>ERα</i> mRNA expression. GAPDH was used as the internal control. This increase in mRNA was found to be significant with all the treatments. <b>b and c)</b> Effects of compounds alone as well in combination on ERα protein expression after 24, 48 and 72 h of treatments. Fig b represents western blot at different time intervals and Fig c represents 72 h densitometry analysis of ERα protein expression at different treatments. Treatment for 72 h with compounds in combination in MDA-MB-157 cells resulted in a highly-significant increase in ERα protein levels. MCF-7 ERα protein extracts were used as the positive control and βactin was used as an internal control. Res 15, resveratrol 15 μM; Ptero 5, pterostilbene 5 μM; Combination, 15 μM resveratrol and 5 μM pterostilbene in combination. Values are representative of three independent experiments and are shown as percent of control ± SE; *P<0.05, **P<0.01.</p

Resveratrol and pterostilbene alters epigenetic enzymes activity with no significant effects on MCF10A control cells.

<p><b>a)</b> Overall HDAC percent enzyme activity relative to DMSO control was analyzed after 72 h of treatments in MDA-MB-157 and HCC1806 breast cancer cells using 20 μg of nuclear extract. After 72 h, 5 μM pterostilbene and combination treated group results in a significant down-regulation of enzyme activity in comparison to control in MDA-MB-157 cells. However, in HCC1806 cells there was a significant increase in overall enzyme activity with 15 μM resveratrol and combination treatments, but no significant change with 5 μM pterostilbene treatment. <b>b)</b> Overall HAT percent enzyme activity relative to DMSO control was analyzed after 72 h of treatments in MDA-MB-157 and HCC1806 breast cancer cells using 20 μg of nuclear extract. After 72 h, 5 μM pterostilbene results in a significant increase in overall enzyme activity with no significant change in any of the other treatment groups in MDA-MB-157 cells. Whereas, in HCC1806 cells there was a significant increase in overall enzyme activity in all the treatment groups, but no significant change within the treatment groups itself was found. <b>c)</b> Overall HDAC and HAT enzyme activities in MCF10A control cells. After 72 h, there was no significant change in the enzyme activity in comparison to DMSO control in MCF10A control cells further highlighting the effectiveness of this combination regimen. Res 15, resveratrol 15 μM; Ptero 5, pterostilbene 5 μM; Combination, 15 μM resveratrol and 5 μM pterostilbene in combination. Values are representative of three independent experiments and represented as percent of control ± SE; *P<0.05, **P<0.01.</p

Combinational treatment results in an open chromatin structure in the <i>ER</i>α promoter region of TNBC cells.

<p>Histone modification enrichment in the <i>ERα</i> promoter was calculated from the corresponding DNA fragments amplified by ChIP-(RT) PCR. TNBC cells were treated as described previously and analyzed by ChIP assays using chromatin markers such as acetyl-H3, acetyl-H3K9 and acetyl-H4 for 48 h and 72 h. Percent input method for ChIP analysis-relative to control was used for analysis. <b>a and d)</b> Significant increase in acetyl-H3 enrichment in the <i>ERα</i> promoter region in both the tested cell lines after 48 to 72 h. MDA-MB-157 cells shown an increase enrichment of acetyl-H3 after 72 h of combination treatment which was highly significant when compared with DMSO, 48 h of treatment and 72 h of individual treatments. HCC1806 breast cancer cells (Fig 3d) shown an increase in acetyl-H3 enrichment with single doses at 72 h and pterostilbene 5 μM at 48 h, but the enrichment with combination of 15 μM resveratrol and 5 μM pterostilbene at 72 h was found to be more significant when compared within the treatment groups at both 48 h and 72 h in both the TNBC cell lines. <b>b and e)</b> Significant increase in acetyl-H3K9 enrichment at 72 h in the <i>ERα</i> promoter region in both of the tested cell types when compared with different groups at 48 h. However, in MDA-MB-157 cells the increase in H3K9 acetylation was found to be highly significant with combination of resveratrol and pterostilbene with no significant change within the 72 h treated group. Whereas, in HCC1806 breast cancer cells there was a significant increase with all the treatments groups at 72 h and 5 μM pterostilbene at 48 h when compared with DMSO control. <b>c and f)</b> A significant increase in acetyl-H4 enrichment in the <i>ERα</i> promoter region in both of the tested cell types with all the treatments when compared with DMSO group after 72 h. However, in MDA-MB-157 cells there was no significant difference found within the treatment group itself. In HCC1806 breast cancer cells there was a significant increase in acetyl-H4 enrichment within all the treatment groups after 72 h, but 5 μM pterostilbene at 48 h and 72 h when compared within the tested groups was found to be highly significant. <b>g)</b> Represents an open chromatin structure in the <i>ERα</i> promoter of ERα-positive MCF-7 breast cancer cells. Acetyl-H3 enrichment was found to be more predominant when compared with acetyl-H4 enrichment in MCF-7 cells. Res 15, resveratrol 15 μM; Ptero 5, pterostilbene 5 μM; Combination, 15 μM resveratrol and 5 μM pterostilbene in combination. Values are representative of three independent experiments and represented as percent of control ± SE; *P<0.05, **P<0.01.</p

Additional file 2: of Epigenetic-based combinatorial resveratrol and pterostilbene alters DNA damage response by affecting SIRT1 and DNMT enzyme expression, including SIRT1-dependent Îł-H2AX and telomerase regulation in triple-negative breast cancer

Is the report generated after using CompuSyn software to calculate the synergism in MDA-MB-157 breast cancer cells. It reflects the 72 h MTT values and compares single dose treatment with combination doses and determines the most effective interaction and can be interpreted by combination index (CI) values. CI values range from 0 to 1. The lower the CI values, the more effective is the interaction and the stronger the synergism and vice versa. (PDF 110 kb