Effect of combined RQC or individual quercetin on breast cancer cell apoptosis.

<p>Quiescent MDA-MB-231 (<b>A</b>) or MDA-MB-435 (<b>B</b>) cells in 5% serum and phenol red-free media were treated with vehicle, combined RQC at 5μM each, or Quercetin 15μM for 48h. Trypsinized cells were incubated with Annexin V conjugate and propidium iodide, and analyzed by flow cytometry, using a four-color flow cytometer (FACSCalibur, BD Biosciences, San Jose, CA). Representative dot plots of 20,000 events/ treatment (N = 3), collected using Cell Quest software 3.3 (BD Biosciences, San Jose, CA) and analyzed using Flow Jo software vX.0.7 (BD Biosciences, San Jose, CA). Cell size and granularity were determined on forward versus side scatter (FSC vs. SSC). Annexin-V fluorescence was measured at 530/30 nm and Propidium Iodide at 585/42nm. The average percentage of early and late apoptotic cells obtained from Annexin V vs. Propidium Iodide dot plots are shown below. <b>(C)</b> Effect of combined RQC or individual quercetin on caspase 3/7 activity. Quiescent MDA-MB-231 cells in 96 well plate at 5% serum and phenol red-free media were treated with vehicle, combined RQC at 5μM each or quercetin 15μM for 48hr or 96hr. Then Caspase-Glo®3/7 reagent were added to each treatment, and after 1hr of incubation the luminescence was measured in a plate-reader luminometer. Relative luminescence to vehicle is shown, N = 3±SEM, asterisk indicates statistical significance (p≤0.05) when compared to vehicle.</p

Effect of individual or combined RQC on Akt activity in breast cancer cells.

<p>Quiescent MDA-MB-231 cells were treated with vehicle, or 1, 3, 5, 9, or 15μM of resveratrol (Res), quercetin (Quer), catechin (Cat), or combined Res, Quer, and Cat (RQC) at 3μM total (1μM each), 9μM total (3μM each), or 15μM total (5μM each). MDA-MB-435 cells were treated only with vehicle or 15μM quercetin. Cells were lysed immediately following treatment for 15min, and western blotted for total or active (phospho-Akt^Ser473) Akt. <b>(A)</b> Relative Akt activity (phospho-Akt/Akt) of MDA-MB-231 cells following RQC or individual Res, Quer, or Cat at the indicated concentrations. <b>(B)</b> Relative Akt activity of MDA-MB-231 cells following vehicle or 15μM quercetin. <b>(C)</b> Relative Akt activity of MDA-MB-435 cells following vehicle or 15μM quercetin. For <b>(B)</b> and <b>(C)</b>, representative western blots from 3 separate experiments are shown. Graphs show the analyses of the integrated densities of positive bands relative to vehicle, as quantified from image J analysis. An asterisk indicates statistical significance (<i>p≤</i>0.05) when compared to vehicle.</p

Effect of quercetin on the growth of MDA-MB-231 mammary fat pad tumors.

<p>GFP-MDA-MB-231 cells (0.5x10⁶) were inoculated at the mammary fat pad of SCID mice. One week after inoculation, mice were treated with vehicle, or quercetin at 15 or 45mg/kg BW three times a week by intraperitoneal injection. Fluorescence images of the mammary fatpad tumors were acquired once a week. <b>(A)</b> Average relative tumor area (N = 12) as a function of days following injection. Relative tumor area was calculated as the area of fluorescence, measured by fluorescence intensity on each day of imaging as a function of the fluorescence intensity of the same tumor on day 1. <b>(B)</b> Representative digital images of MDA-MB-231 tumors following vehicle, or quercetin at 15 or 45mg/kg BW at 13 weeks, followed by the quantification of tumor growth. N = 12±SEM, asterisk indicates statistical significance (<i>p≤</i>0.05) when compared to vehicle. <b>(C)</b> Relative SCID mice weight following vehicle, or quercetin at 15 or 45mg/kg BW treatments.</p

Effect of individual or combined RQC on AMPK activity in breast cancer cells.

<p>Quiescent MDA-MB-231 cells were treated with vehicle, or 1, 3, 5, 9, or 15μM of resveratrol (Res), quercetin (Quer), catechin (Cat), or combined RQC at 3μM total (1μM each), 9μM total (3μM each), or 15μM total (5μM each). MDA-MB-435 cells were treated only with vehicle and 15μM quercetin. Cells were treated for 15 min, lysed immediately, and western blotted for total or active (phospho-AMPK^Thr172) AMPK. <b>(A)</b> Relative AMPK activity (phospho-AMPK/AMPK) of MDA-MB-231 cells following RQC or individual Res, Quer, or Cat at the indicated concentrations. Relative AMPK activity of <b>(B)</b> MDA-MB-231 cells following vehicle or 15μM quercetin, or <b>(C)</b> MDA-MB-435 cells following vehicle or 15μM quercetin. For <b>(B)</b> and <b>(C)</b>, representative western blots from 3 separate experiments are shown. Graphs show the analyses of the integrated densities of positive bands relative to vehicle as quantified from image J analysis. An asterisk indicates statistical significance (<i>p≤</i>0.05) when compared to vehicle.</p

Effect of combined RQC or individual quercetin on breast cancer cell proliferation.

<p>MDA-MB-231 and MDA-MB-435 cells in 5% serum and phenol red-free media were treated with vehicle, combined resveratrol, quercetin, and catechin (RQC) at 5μM each or 15μM quercetin for 48h. Cells were fixed, nuclei stained with Propidium Iodide (PI), and intact (non-apoptotic) nuclei quantified. Percentage of viable cells ± SEM for 30 microscopic fields/triplicate treatments (N = 3) is presented. <b>A)</b> Average cell viability of MDA-MB-231 cells treated with RQC or quercetin relative to vehicle. <b>(B)</b> Average cell viability of MDA-MB-435 cells treated with RQC or quercetin relative to vehicle.</p

Effect of combined RQC or individual quercetin on breast cancer cell migration.

<p>Quiescent MDA-MB-231 and MDA-MB-435 cells were placed in the top of a transwell chamber with the bottom well containing: vehicle, combined resveratrol, quercetin, and catechin (RQC) at 5μM each, or 15μM quercetin in serum- and phenol red- free media. After 8h incubation, cells that migrated through the 8μM pore membrane were fixed, nuclei stained with Propidium Iodide and quantified. Percentage of migrated cells ± SEM for 15 microscopic fields/duplicate treatments for 3 independent experiments is presented. (<b>A)</b> Average cell migration of MDA-MB-231 cells treated with RQC or quercetin relative to vehicle. <b>(B)</b> Average cell migration of MDA-MB-435 cells treated with RQC or quercetin relative to vehicle.</p

Effect of quercetin on mTOR signaling in breast cancer cells.

<p>Quiescent MDA-MB-231 and MDA-MB-435 cells were treated with vehicle or quercetin at 15μM for 15min, lysed immediately, and western blotted for total or active (phospho-p70S6K^Thr389) p70S6K or (phospho-4EBP-1^Thr37/46) 4EBP-1. <b>(A)</b> Relative p70S6K activity (phospho-p70S6K/p70S6K) from MDA-MB-231 cells. <b>(B)</b> Relative p70S6K activity from MDA-MB-435 cells. <b>(C)</b> Relative 4EBP-1 activity (phospho-4EBP-1/4EBP-1) from MDA-MB-231 cells. <b>(D)</b> Relative 4EBP-1 activity from MDA-MB-435 cells. Representative western blots from 3 separate experiments are shown. Graphs show the analyses of the integrated densities of positive bands relative to vehicle as quantified from image J analysis. An asterisk indicates statistical significance (<i>p≤</i>0.05) when compared to vehicle.</p

Targeting Cdc42 with the anticancer compound MBQ-167 inhibits cell polarity and growth in the budding yeast S. cerevisiae

<p>The Rho GTPase Cdc42 is highly conserved in structure and function. Mechanical or chemical cues in the microenvironment stimulate the localized activation of Cdc42 to rearrange the actin cytoskeleton and establish cell polarity. A role for Cdc42 in cell polarization was first discovered in the budding yeast <i>Saccharomyces cerevisiae</i>, and subsequently shown to also regulate directional motility in animal cells. Accordingly, in cancer Cdc42 promotes migration, invasion, and spread of tumor cells. Therefore, we targeted Cdc42 as a therapeutic strategy to treat metastatic breast cancer and designed the small molecule MBQ-167 as a potent inhibitor against Cdc42 and the homolog Rac. MBQ-167 inhibited cancer cell proliferation and migration <i>in-vitro</i>, and tumor growth and spread <i>in-vivo</i> in a mouse xenograft model of metastatic breast cancer. Since haploid budding yeast express a single Cdc42 gene, and do not express Rac, we used this well characterized model of polarization to define the contribution of Cdc42 inhibition to the effects of MBQ-167 in eukaryotic cells. Growth, budding pattern, and Cdc42 activity was determined in wildtype yeast or cells expressing a conditional knockdown of Cdc42 in response to vehicle or MBQ-167 treatment. As expected, growth and budding polarity were reduced by knocking-down Cdc42, with a parallel effect observed with MBQ-167. Cdc42 activity assays confirmed that MBQ-167 inhibits Cdc42 activation in yeast, and thus, bud polarity. Hence, we have validated MBQ-167 as a Cdc42 inhibitor in another biological context and present a method to screen Cdc42 inhibitors with potential as anti-metastatic cancer drugs.</p

Synthesis of Novel Heterocyclic Ferrocenyl Chalcones and Their Biological Evaluation

Breast cancer is currently the most commonly diagnosed cancer, with 287,850 new cases estimated for 2022 as reported by the American Cancer Society. Therefore, finding an effective treatment for this disease is imperative. Chalcones are α,β-unsaturated systems found in nature. These compounds have shown a wide array of biological activities, making them popular synthetic targets. Chalcones consist of two aromatic substituents connected by an enone bridge; this arrangement allows for a large number of derivatives. Given the biological relevance of these compounds, novel ferrocene-heterocycle-containing chalcones were synthesized and characterized based on a hybrid drug design approach. These heterocycles included thiophene, pyrimidine, thiazolyl, and indole groups. Fourteen novel heterocyclic ferrocenyl chalcones were synthesized and characterized. Herein, we also report their cytotoxicity against triple-negative breast cancer cell lines MDA-MB-231 and 4T1 and the noncancer lung cell line MRC-5. System 3 ferrocenyl chalcones displayed superior anticancer properties compared to their system 1 analogues. System 3 chalcones bearing five-membered heterocyclic substituents (thiophene, pyrazole, pyrrole, and pyrimidine) were the most active toward the MDA-MB-231 cancer cell line with IC50 values from 6.59 to 12.51 μM. Cytotoxicity of the evaluated compounds in the 4T1 cell line exhibited IC50 values from 13.23 to 213.7 μM. System 3 pyrazole chalcone had consistent toxicity toward both cell lines (IC50 ∼ 13 μM) as well as promising selectivity relative to the noncancer MRC-5 control. Antioxidant activity was also evaluated, where, contrary to anticancer capabilities, system 1 ferrocenyl chalcones were superior to their system 3 analogues. Antioxidant activity comparable to that of ascorbic acid was observed for thiophene-bearing ferrocenyl chalcone with EC50 = 31 μM