A Novel Class of Mitochondria-Targeted Soft Electrophiles Modifies Mitochondrial Proteins and Inhibits Mitochondrial Metabolism in Breast Cancer Cells through Redox Mechanisms

<div><p>Despite advances in screening and treatment over the past several years, breast cancer remains a leading cause of cancer-related death among women in the United States. A major goal in breast cancer treatment is to develop safe and clinically useful therapeutic agents that will prevent the recurrence of breast cancers after front-line therapeutics have failed. Ideally, these agents would have relatively low toxicity against normal cells, and will specifically inhibit the growth and proliferation of cancer cells. Our group and others have previously demonstrated that breast cancer cells exhibit increased mitochondrial oxygen consumption compared with non-tumorigenic breast epithelial cells. This suggests that it may be possible to deliver redox active compounds to the mitochondria to selectively inhibit cancer cell metabolism. To demonstrate proof-of-principle, a series of mitochondria-targeted soft electrophiles (MTSEs) has been designed which selectively accumulate within the mitochondria of highly energetic breast cancer cells and modify mitochondrial proteins. A prototype MTSE, IBTP, significantly inhibits mitochondrial oxidative phosphorylation, resulting in decreased breast cancer cell proliferation, cell attachment, and migration <i>in vitro</i>. These results suggest MTSEs may represent a novel class of anti-cancer agents that prevent cancer cell growth by modification of specific mitochondrial proteins.</p></div

Time-dependent modification of proteins by MTSEs of different chain lengths in MB231 cells.

<p>MB231 cells were treated with 5μM of indicated MTSEs for the indicated times. At the end of treatment, cell lysates were prepared and protein adducts were visualized by Western blot analysis using an antibody directed against TPP (upper panel). Lane densities were quantified and plotted in the lower panel. Values are mean ± SE of 3–5 samples obtained from two separate experiments; *<i>p</i><0.05 compared to no treatment.</p

Effects of IBTP on proliferation of human breast cancer cells or non-tumorigenic breast epithelial cells.

<p>MB231 or MCF10A cells were treated with the indicated concentrations of IBTP (black diamonds) or BTPP (gray squares) for 24h. The number of cells in each group at the end of the experiment was measured. The values are mean ± SE of duplicates from two independent experiments. (*<i>p</i><0.05 compared to BTPP).</p

Effect of IBTP treatment on mitochondrial respiration of MB231 cells.

<p><b>Panel A:</b> Cells plated on XF24 plates were treated with the indicated concentrations of IBTP or BTPP for 4h in 0.5% FBS-containing medium. After treatment, the medium was removed and replaced with XF assay medium (DMEM, containing 5mM glucose, 0.5% FBS, 5mM HEPES without bicarbonate) and equilibrated 1h before OCR measurement. <b>Panel B:</b> Cells plated on 6-well plates were treated with the indicated concentrations of IBTP or BTPP for 4h. After the incubation, the cells were harvested immediately by trypsinization. The harvested cells were replated in XF24 plates and allowed adhere for an additional 20h in complete medium containing 10% FBS (total 24h). The medium was removed and replaced with assay medium and equilibrated 1h before OCR measurement. <b>Panel C</b>: After 4h of IBTP or BTPP treatment, the medium was replaced with complete medium containing 10% FBS, and incubated for 48h. The cells were harvested after 48h, replated in XF24 plates and allowed adhere for an additional 20h in complete medium. The medium was replaced with assay media and incubated 1h before measurement of OCR (total duration 72h).</p

Effects of IBTP on cell attachment and migration in human breast cancer cells.

<p>MB231 cells were treated with the indicated concentrations of IBTP. At the end of the treatment, the cells were either assayed for the cells ability to attach the substratum or migration by scratch assay. <b>Panel A:</b> To determine the effect of IBTP on cell attachment, viable cells were counted (<b>“Total Viable Cells”</b>) and replated onto a 100mm tissue culture dish for 24h. At the end of the incubation, the media was collected and number of viable cells that failed to attach to the substratum was counted (<b>“Viable non-adherent Cells”</b>). The values represent the mean ± SE of triplicates from two independent experiments. (*<i>p</i><0.05 compared to BTPP). <b>Panel B:</b> To determine the effect of IBTP or BTPP on migration, cells were treated with the indicated concentration of compounds for 4h. A scratch assay was performed as described in the Materials and Methods, where cells were allowed to migrate into the cell-free zone for 5h. The values represent the mean ± SE of three separate images obtained from triplicate wells. (*<i>p</i><0.05 compared to BTPP).</p

Dose-dependent modification of proteins by MTSEs of different chain length in MB231 cells.

<p>MB231 cells were treated with the indicated doses for 4h. At the end of treatment, cell lysates were prepared and protein adducts were visualized by Western blot analysis using an antibody directed against TPP. <b>a</b> = Vehicle; <b>b</b> = 0.5 μM; <b>c</b> = 1μM; <b>d</b> = 2μM; <b>e</b> = 5μM. IPTP = 3 carbons, IBTP = 4 carbons, IHTP = 6 carbons, IOTP = 8 carbons, IDTP = 10 carbons. The values are mean ± SE of 3–5 samples obtained from two separate experiments; *<i>p</i><0.05 compared to no treatment.</p

Structures of mitochondria-targeted electrophiles.

<p>The compounds were synthesized as described under “Materials and Methods.”</p

Bioenergetic parameters in MB231 cells.

<p><b>Panels A-F:</b> Bioenergetic parameters were calculated from the OCR traces in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120460#pone.0120460.g005" target="_blank">Fig. 5</a>, panels A-C. Values are mean ± SE obtained from 10–15 wells in two separate experiments; *<i>p</i><0.05 compared to BTPP; #<i>p</i><0.05 compared to vehicle.</p

Mitochondrial protein modification by IBTP in breast cancer cells.

<p>MB231 cells were treated with 10μM IBTP or EtOH vehicle for 4h, and cell fractions were obtained as described in the Materials and Methods. At the end of treatment, cell lysates were prepared and protein adducts were visualized by Western blot analysis using an antibody directed against TPP. The values are mean ± SE of 3–5 samples obtained from two separate experiments; *<i>p</i><0.05 compared to vehicle.</p

Effect of IBTP treatment on glycolysis of MB231 cells.

<p>Cells were plated, cultured, and treated as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120460#pone.0120460.g005" target="_blank">Fig. 5</a> and a glycolysis stress test was performed as described in the Materials and Methods. <b>Panel A:</b> ECAR is represented as a function of time, and the third untreated time point was used to define 100% ECAR for each group. Arrows indicate times of injection for glucose (G), oligomycin (O), and 2-deoxyglucose (2DG). <b>Panel B:</b> Rate of glycolytic flux in MB231 cells with and without BTPP or IBTP treatment. Glycolysis was defined as the %ECAR after addition of glucose (10mM). <b>Panel C:</b> Glycolytic reserve of MB231 cells with and without BTPP or IBTP treatment. Glycolytic reserve represents the difference in ECAR between glycolysis and the maximum glycolytic capacity observed after addition of oligomycin (1μM). Values are mean ± SE obtained from 10–15 wells in two separate experiments; *<i>p</i><0.05 compared to BTPP; # <i>p</i><0.05 compared to vehicle.</p