Data_Sheet_1_Carnosol, a Natural Polyphenol, Inhibits Migration, Metastasis, and Tumor Growth of Breast Cancer via a ROS-Dependent Proteasome Degradation of STAT3.PDF

We have previously demonstrated that carnosol, a naturally occurring diterpene, inhibited in vitro cell viability and colony growth, as well as induced cell cycle arrest, autophagy and apoptosis in human triple negative breast cancer (TNBC) cells. In the present study, we evaluated the ability of carnosol to inhibit tumor growth and metastasis in vivo. We found that non-cytotoxic concentrations of carnosol inhibited the migration and invasion of MDA-MB-231 cells in wound healing and matrigel invasion assays. Furthermore, gelatin zymography, ELISA, and RT-PCR assays revealed that carnosol inhibited the activity and downregulation the expression of MMP-9. Mechanistically, we demonstrated that carnosol suppressed the activation of STAT3 signaling pathway through a ROS-dependent targeting of STAT3 to proteasome-degradation in breast cancer cells (MDA-MB-231, Hs578T, MCF-7, and T47D). We show that blockade of proteasome activity, by MG-132 and bortezomib, or ROS accumulation, by N-acetylcysteine (NAC), restored the level of STAT3 protein. In addition, using chick embryo tumor growth assay, we showed that carnosol significantly and markedly suppressed tumor growth and metastasis of breast cancer xenografts. To the best of our knowledge, this is the first report which shows that carnosol specifically targets signal transducer and activator of transcription 3 (STAT3) for proteasome degradation in breast cancer. Our study further provide evidence that carnosol may represent a promising therapeutic candidate that canmodulate breast cancer growth and metastasis.</p

Induction of NAG-1 expression by salinomycin.

<p>Cells were exposed to different concentrations of salinomycin, and total RNA was extracted after 2 and 24 hours and analyzed for NAG-1 mRNA expression in A549 cells (<b>A</b>) and after 24 h for protein expression in both A549 and LNM35 cells (<b>B</b>).</p

Inhibition of cell viability by salinomycin.

<p>Exponentially growing LNM35 (<b>A, C</b>) and A549 (<b>B, D</b>) cells were treated with vehicle (0.1% DMSO) or the indicated concentrations of salinomycin. The left panel represents 24 h exposure, while the right panel represents 48 h exposure. Viable cells were assayed as described in Materials and Methods. Induction of caspase-3/7 activity was analyzed in LNM35 (<b>E</b>) and A549 (<b>F</b>) cells treated for 24 h with salinomycin (10 and 50 µM), normalized to the number of viable cells per well and expressed as fold induction compared with the control group. All experiments were repeated at least three times. *Significantly different at P<0.05, **Significantly different at P<0.01, ***Significantly different at P<0.001.</p

The induction of NAG-1 expression by salinomycin mediate its anti-invasive potential.

<p>Silencing of NAG-1 suppressed the increased expression of NAG-1 induced by salinomycin (<b>A</b>) without impact on the inhibition of the A549 cell viability induced by salinomycin (<b>B</b>) leading to complete suppression on anti-invasive potential of salinomycin (<b>C</b>).</p

Salinomycin impairs lung cancer cell migration and invasion.

<p>Wounds were introduced in LNM35 (<b>A</b>) and A549 (<b>B</b>) confluent mono-layers cultured in the presence or absence (control) of salinomycin (0.05–0.5 µM). The mean distance that cells travelled from the edge of the scraped area for 6, 24, and 30 h at 37°C was measured in a blinded fashion, using an inverted microscope. <b>C</b>) LNM35 cells were incubated for 24 h in the presence or absence of salinomycin (0.05–0.1 µM). <b>D</b>) A549 cells were incubated for 24 h in the presence or absence of salinomycin (0.1–0.5 µM). Cells that invaded into Matrigel were scored as described in Materials and Methods.</p

Salinomycin impairs colony growth in soft agar.

<p>LNM35 (<b>A</b>) and A549 (<b>B</b>) cells were grown at a density of 5,000 cells/well in soft agar medium into 6-well plates. After 14 days, formed colonies were treated for 7 days with different concentrations of salinomycin (2.5 and 5 µM). At the end colonies were stained with Giemsa and scored. Representative pictures of the colonies formed in soft agar from LNM35 (<b>C</b>) and A549 (<b>D</b>) were photographed.</p

Inhibition of colony growth by <i>O. majorana</i> extract.

<p>Inhibition of colony growth was assessed by measuring the size of the colonies obtained in vehicle (ethanol)- and OME-treated plates. Data were compared with those obtained for the 2 weeks colonies. Two types of colonies were counted and depending on their diameter were categorized as large (≥200 µm) and small (<200−≥50 µm).</p

Induction of G2/M cell cycle arrest and apoptosis by <i>O. majorana</i> extract in MDA-MB-231 cells.

<p>(A) MDA-MB-231 cells (1.8×10⁶) seeded on 100 mm culture dish were exposed various concentrations of <i>O. majorana</i> extract or equal volume of vehicle (ethanol) as control for 24 h. Following treatment, cells were harvested, fixed, stained with propidium iodide, and analyzed for cell cycle distribution by flow cytometry. Data represent the mean of three independent experiments. The percentage of cells in sub-G1 (apoptosis), G1, S and G2/M appears at the upper right of each graph. (B) Expression of cell cycle regulator in OME-treated MDA-MB-231. Western blot analysis of phospho(ser10)-H3, and cyclin B1 in MDA-MB231 cells exposed for 24 h to ethanol or indicated concentrations of OME. (C) Stimulation of caspase 3/7 activity in MDA-MB-231 cells after exposure to OME (0–600 µg/mL) for 24 h and 48 h, relative to a similar amount of viable ethanol-treated cells. The relative caspase 3/7 activity was normalized to the number of viable cells per well and is expressed as fold of induction compared to the control. (D) Concentration-dependent induction of PARP cleavage in OME-treated MDA-MB231 cells. Cells were treated with or without increasing concentrations of the extract and proteins were extracted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056649#s2" target="_blank">Materials and Methods</a>. Western blot analysis was carried out using anti-PARP antibodies. (*<i>p</i><0.05, **<i>p</i><0.005 and ***<i>p</i><0.0005).</p

Differential regulation of survivin expression by <i>OME</i> in MDA-MB-231 cells.

<p>Western blot analysis showing a differential effect on survivin expression by different concentrations of OME in MDA-MB-231 cells. Whole cell protein were extracted from OME or vehicle (ethanol)-treated cells and subjected to Western blot analysis, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056649#s2" target="_blank">Materials and Methods</a>, for survivin and β-actin (loading control) proteins.</p

The proposed signal pathways on <i>O. majorana-</i>induced cell cycle arrest, at low concentrations, and apoptosis, at high concentrations, in the triple negative mutant p53 human breast cancer MDA-MB-231 cells.

<p>The proposed signal pathways on <i>O. majorana-</i>induced cell cycle arrest, at low concentrations, and apoptosis, at high concentrations, in the triple negative mutant p53 human breast cancer MDA-MB-231 cells.</p