Trans-resveratrol boronic acid exhibits enhanced anti-proliferative activity on estrogen-dependent MCF-7 breast cancer cells

Figures & data

Figure 1. (A) Structures of known boronic acid compounds and (B) Design of boronic acid analogs of resveratrol: cis-4 and trans-4.

Figure 2. Synthetic scheme of cis-4 and trans-4.

Table 1. Effects of cis-4, trans-4 or RSV on growth inhibition of the ER+, estrogen-dependent human breast cancer MCF-7 cell line

MCF-7 Cell growth inhibition (WST-1 assay, G150, µM)
Compound	48 h	72 h
trans-4	36.6 ± 0.06 mM	31.1 ± 0.05 mM
cis-4	155.8 ± 0.10 mM	173.8 ± 0.10 mM
RSV	97.7 ± 0.10 mM	105.2 ± 0.09 mM

Note: cis-4, trans-4 and RSV were tested at various concentrations for effects on cell growth inhibition of MCF-7 breast cancer cells. Cell growth inhibition was estimated 48 and 72 h after the addition of each compound using the WST-1 reduction assay. The GI50 value (the concentration yielding 50% growth inhibition) was interpolated from the graph of the log of compound concentration vs. the fraction of surviving cells. The GI50 was calculated using Graph Pad Prism. Data are expressed as mean (± SEM) of triplicate experiments.

Table 2. Effects of trans-4 or paclitaxel on the growth inhibition of multi-drug resistant breast cancer cell lines

CL 10.3 Cell growth inhibition (WST-1 assay, GI50, µM 72 h)
Cell line	Origin	trans-4	Paclitaxel
MCF-7	breast	31.10 ± 0.05 mM	0.002 ± 1.12 mM
CL 10.3	breast (MDR)	49.09 ± 0.001 mM	> 50 mM
MDR GI50/non-MDF GI50		1.57 mM	> 25,000 mM

Note:trans-4 or Paclitaxel were tested at various concentrations for effects on cell growth inhibition of breast cancer cells. Cell growth inhibition was estimated 72 h after the addition of each compound using the WST-1 reduction assay. The GI50 value (the concentration yielding 50% growth inhibition) was interpolated from the graph of the log of compound concentration vs. the fraction of surviving cells. The GI50 was calculated using Graph Pad. Data are expressed as mean (± SEM) of triplicate experiments.

Figure 3. A. Effect of compounds on cell cycle distribution in MCF-7 breast cancer cell lines. Asynchronous MCF-7 cells were treated with 30 µM of cis-4, trans-4, or vehicle control for different time intervals (16, 24, and 48 h), fixed in 70% ethanol and DNA content was determined by labeling with PI followed by flow cytometric analysis. B. Fraction of compound treated cells with trans-4 in G₁ relative to control-treated cells. Results are represented as a mean of triplicate samples.

Figure 4. Irreversible of trans-4 on MCF-7 breast cancer cell growth inhibition under different conditions. After 24 h post incubation, cells were treated with increasing concentrations of trans-4 (1–100 µM) under three different conditions. In the first method, cells were exposed for 48 h to trans-4. In the second method cells were exposed to trans-4 for 48 h, washed with serum free media and cultured for additional 48 h without compound. In the third method, cells were treated every 24 h to fresh media with trans-4 for 72 h. Cells were harvested at the indicated times and analyze for cell growth inhibition by the WST-1 method. Values represent the mean ± SD of triplicate wells. The experiment was repeated thrice with identical results.

Figure 5. Effect of trans-4 on the expression of G₁-S transition regulatory proteins. MCF-7 cells were treated with the indicated concentrations of trans-4 for 24 and 48 h. Cell lysates were subjected to immunoblotting to detect cyclin D1, cdk4, cyclin E, cdk2 and pRb proteins by their specific antibodies. Human -actin was used as a loading control. Twenty-micrograms of lysate was used for each experimental condition.

Figure 6. western blot analysis of PARP cleavage. (A) MCF7 cells were treated with trans-4 for 48 and 72 h and equal amounts of cell lysate were resolved using SDS-PAGE and analyzed by immunoblot using anti-PARP antibody. The blots were re-probed with anti -actin antibody to confirm equal protein loading. (B) Comparison of density of cleaved PARP band (normalized with actin) of trans-4 and resveratrol were determined by densitometry NIH image analysis.

Figure 7. Effect of trans-4 or RSV on E2-mediated MCF-7 cell growth. MCF-7 cells were treated with estradiol (E2, 10^-9M) alone or in combination with the indicated concentrations of trans-4 or RSV. After 5 d incubation, the DNA content of the treated cells was measured using a DNA fluorescence kit (BioRad # 170–2480). Results are shown as the mean of triplicate samples ± SD. Repeated the experiment in twice with identical results.