Panepoxydone inhibited NF-kB:

<p>(<b>A</b>) Immunoblotting of IkB and pIkBα showed accumulation of IkBα (inactive) and down-regulation of pIkBα (active) in PP treated breast cancer cells. This indicates the potential of PP in keeping NF-kB in the active state. (<b>B</b>) Bar diagram indicate the increased IkB/pIkB ratio in all breast cancer cells after PP treatment. * indicates statistically significant difference between PP treated and untreated cells at p<0.05 (*), p<0.01(**), and, p<.001(***) levels by student's t-test. (<b>C</b>) Localization of NF-κB was done on MCF-7, MDAMB-231, MDAMB-468 and MDAMB-453 breast cancer cells that were fixed, permeabilized and labeled with anti-p65 subunit of NF-κB then nuclei were stained with DAPI. Controls cells were compared to cells treated with the top dose of PP (D3). Controls cells showed increased expression of NF- κB in the nucleus, whereas following treatment with PP, NF-κB accumulated in the cytoplasm, indicating decreased activity.</p

Effect of Panepoxydone on cell cycle phase distribution in breast cancer cells.

<p>(<b>A</b>) MCF-7, MDAMB-231, MDAMB-468 and MDAMB-453 breast cancer cells (2×10⁵ cells/well) were seeded in 6 well plates, treated with PP or DMSO (0.2%, vehicle control) for 24 hrs and subsequently stained with propidium iodide (PI) followed by flow cytometry. The left triangle crest is the G1 phase, the right crest is the G2/M phase, and the middle area is the S-phase. Data are representative of one of the two independent experiments. (<b>B</b>) Histogram constructed on the mean value of two independent experiments. MCF-7 and MDAMB-468 cells accumulated in G2-phase whereas increase in S-phase was observed for MDAMB-231 and MDAMB-453 cells after PP treatment. (<b>C</b>) Total protein was isolated from control and PP-treated breast cancer cells and subjected to immunoblotting of p27, CDK1, CDK2,cyclin B and cyclin E proteins. Membranes were stripped and re-probed with anti-actin antibody to ensure equal protein loading. This data indicate the possible role of PP in inducing cell cycle arrest at G2 and S phase.</p

Panepoxydone decreases migration and invasion properties of breast cancer cells.

<p>MCF-7, MDA-MB-231, MDA-MB-468 and MDA-MB-453 cells were seeded on noncoated or matrigel-coated membranes for motility (<b>A</b>) and invasion (<b>B</b>) assays, respectively and incubated for 24 hrs. DMEM containing 10% fetal bovine serum in the lower chamber was used as a chemoattractant. After incubation, cells that had migrated or invaded through the membrane/matrigel to the bottom of the insert were washed, fixed, stained and counted in ten random microscopic fields. The histograms show mean of two independent experiments and the error bar represent SEM. * indicates statistically significant difference between PP treated and untreated cells at p<0.05 (*), p<0.01(**), and, p<.001(***) levels by student's t-test. Results indicate reduction in the migrated and invaded cells after PP treatment in a dose dependent manner.</p

Inhibitory effects of panepoxydone on the proliferation of human breast cancer cells.

<p>MCF-7, MDA-MB-231, MDA-MB-468 and MDA-MB-453 breast cancer cells were grown in 96 well microtitre plates (5000 cells/well) and treated with increasing concentrations of PP or with DMSO (0.2% vehicle control) and analyzed by CellTiter Glow assay. (<b>A</b>) Time-dependent anti-proliferative effect of PP on breast cancer cells. Data are presented as the means ± SD (n = 2). (<b>B</b>) Dose-dependent anti-proliferative effect of PP on breast cancer cells after 72 hrs. Data are presented as the means ± SD (n = 4). PP inhibited cell viability in a dose-dependent manner for all the cell types suggesting anti-cancer activity of PP. (<b>C</b>) Morphological changes of breast cancer cells after treatment with PP. Based on the IC₅₀ value, 3 doses were selected for subsequent experiments: D1 (half of IC₅₀, D2 (IC₅₀₎ and D3 (2×IC₅₀). Cells (1.5×10⁴ cells/well) were seeded in 6-well plates and incubated with increasing concentrations of PP or DMSO (0.2% vehicle control) for 24 hrs. Morphological changes were observed under the inverted phase-contrast microscope and photographed. Representative micrographs are from one of the random fields of view (magnification 200X) of cells.</p

Schematic diagram of PP-induced cell death mechanism and EMT reversal.

<p>Schematic diagram of PP-induced cell death mechanism and EMT reversal.</p

Panepoxydone modulates EMT related markers.

<p>Total protein was isolated from control and PP-treated breast cancer cells and subjected to immunoblotting of proteins. Membranes were stripped and re-probed with anti-actin antibody to ensure equal protein loading. (<b>A</b>) Immunoblotting of FOXM1 after PP treatment and silencing of FOXM1 and NF-κB. (<b>B</b>) Immunoblots of cell lysates treated with PP or silenced FOXM1 and NF-kB for E cadherin, vimentin, slug and zeb-1. (<b>C</b>) Bar diagram indicate the fold difference in the EMT markers after PP treatment and above specified silencing. * indicates statistically significant difference between PP treated and untreated cells at p<0.05 (*) and p<0.01(**) levels by student's t-test. Altogether this data indicate the PP induced EMT reversal is through FOXM1 in breast cancer cells.</p

Panepoxydone induces apoptosis in breast cancer cells.

<p>(<b>A</b>) MCF-7, MDA-MB-231, MDA-MB-468 and MDA-MB-453 breast cancer cells were grown in a 6-well plate (1×10⁶ cells/well) and treated with increasing concentrations of PP or DMSO (0.2% vehicle control) for 24 hrs. After treatment, cells were stained with 7-AAD and PE Annexin V followed by flow cytometry. Unstained DMSO-treated cells served as a negative control. Data show a dose-dependent increase in the number of apoptotic cells in all breast cancer cells after treatment with PP as compared to control cells. A representative picture of two independent experiments is shown. (<b>B</b>) Histogram representation of increased number of apoptotic cells in breast cancer cells after treatment with PP. (<b>C</b>) Total protein was isolated from control and PP-treated breast cancer cells and subjected to immunoblotting of apoptosis an dsurvival related proteins. Membranes were stripped and re-probed with anti-actin antibody to ensure equal protein loading. Bax and cleaved PARP was upregulated and Bcl-2, survivin, cyclin D1 and caspase 3 was down-regulated in all breast cancer cells in a dose-dependent manner. (<b>D</b>) Bar diagram indicate the increased Bax/Bcl-2 ratio in all breast cancer cells after PP treatment indicating its role in apoptosis. * indicates statistically significant difference between PP treated and untreated cells at p<0.05 (*), p<0.01(**), and, p<.001(***) levels by student's t-test. Altogether this data indicate the possible role of PP in modulation of apoptosis related genes. Immunoblotting for each protein was performed at least twice.</p