Honokiol modulates Bax/Bcl-2 and Bax/Bcl-xL ratio in human pancreatic cancer cells.

<p>(A) MiaPaCa and Panc1 cells were treated with either honokiol (20, 40 or 60 µM) or DMSO (control) for 24 h. Immunoblotting was performed for Bcl-xl, Bcl-2 and Bax proteins followed by densitometry of immunoreactive bands. Normalized densitometric values are indicated at the top of the bands. (B) Bar diagram summarizing the effects of honokiol treatment on Bax/Bcl-2 ratio (upper panel) and Bax/Bcl-xL ratio (lower panel). Data suggest that honokiol induces apoptosis by upregulating pro-apoptotic Bax and downregulating anti-apoptotic Bcl-2 and Bcl-xL proteins.</p

Honokiol induces apoptosis in human pancreatic cancer cells.

<p>MiaPaCa and Panc1 cells were grown in 6-well plates (1×10⁶ cells /well) and allowed to attain 70–80% confluence. Cells were treated with either honokiol (20, 40 or 60 µM) or DMSO (control) for 24 h and subsequently stained with 7-AAD and PE Annexin V followed by flow cytometry. The lower left quadrants of each panels show the viable cells (negative for both, PE Annexin V and 7-AAD). The upper right quadrants contain necrotic or late apoptotic cells (positive for both, PE Annexin V and 7-AAD). The lower right quadrants represent the early apoptotic cells (PE Annexin V positive and 7-AAD negative). Data show a dose-dependent increase in the number of apoptotic cells in both MiaPaCa and Panc1 cells after treatment with honokiol as compared to control cells, indicating apoptotis inducing potential of honokiol.</p

Induced pluripotent stem cells derived from human amnion in chemically defined conditions

<p>Fetal stem cells are a unique type of adult stem cells that have been suggested to be broadly multipotent with some features of pluripotency. Their clinical potential has been documented but their upgrade to full pluripotency could open up a wide range of cell-based therapies particularly suited for pediatric tissue engineering, longitudinal studies or disease modeling. Here we describe episomal reprogramming of mesenchymal stem cells from the human amnion to pluripotency (AM-iPSC) in chemically defined conditions. The AM-iPSC expressed markers of embryonic stem cells, readily formed teratomas with tissues of all three germ layers present and had a normal karyotype after around 40 passages in culture. We employed novel computational methods to determine the degree of pluripotency from microarray and RNA sequencing data in these novel lines alongside an iPSC and ESC control and found that all lines were deemed pluripotent, however, with variable scores. Differential expression analysis then identified several groups of genes that potentially regulate this variability in lines within the boundaries of pluripotency, including metallothionein proteins. By further studying this variability, characteristics relevant to cell-based therapies, like differentiation propensity, could be uncovered and predicted in the pluripotent stage.</p

Honokiol causes G₁ phase cell cycle arrest in human pancreatic cancer cells.

<p>MiaPaCa and Panc1cells (1×10⁶ cells/well) were synchronized by culturing in serum free media for 72 h, followed by incubation in serum-containing media for 24 h and subsequent treatment with either honokiol (20, 40 or 60 µM) or DMSO (control) for 24 h. Distribution of cells in different phases of cell cycle was analyzed by propidium iodide (PI) staining followed by flow cytometry. Enhanced accumulation of MiaPaCa and Panc1 cells in the G₁ phase of the cell cycle was observed after treatment with honokiol in a dose-dependent manner (as indicated by flow histograms) with a concomitant decrease in S-phase cells.</p

Honokiol suppresses growth of human pancreatic cancer cells.

<p>(<b>A</b>) MiaPaCa and Panc1 cells were seeded in 6 well plate (1×10⁵ cells/well) and allowed to attain 70–80% confluence prior to honokiol (10–60 µM) treatment for 48 h. Following treatment, significant change in cell morphology was observed of both the cell types as examined under phase-contrast microscope. Cells became round, shrunken and detached from cell surface in a dose-dependent manner. Representative micrographs are from one of the random fields of view (magnification 200X) of cells treated with 20, 40 or 60 µM honokiol. (<b>B</b>) MiaPaCa and Panc1 cells were grown in 96 well microtitre plates (1×10⁴ cells /well) and treated with honokiol (10–60 µM) at 70–80% confluence. Percent viability of cells was measured by WST-1 assay after 24, 48 and 72 h. An OD value of control cells (treated with an equal volume of DMSO, final concentration, <0.1%) was taken as 100% viability. Honokiol inhibited cell viability in a dose- and time- dependent manner for both the cell types suggesting anti-tumor effect of honokiol. Data are expressed as mean± SD; (n = 3).</p

Honokiol attenuates constitutive NF-κB activation by inhibiting nuclear translocation of NF-κB/p65 in human pancreatic cancer cells.

<p>(A) MiaPaCa and Panc1cells (0.5×10⁶ cells/well) were seeded in 12-well plate. Next day at 60% confluence, cells were co-transfected with NF-κB luciferase reporter and TK-Renilla luciferase (control) plasmids. Twenty-four hours post-transfection, cells were treated with honokiol (20, 40, or 60 µM) for next 24 h. Protein lysates were made and luciferase (Fire-fly; test and Renilla, transfection efficiency control) activity assessed using a dual-luciferase assay system. Data is presented as normalized fold-change in luciferase activity (mean± SD; n = 3, * p<0.05). (B) Total, nuclear and cytoplasmic extracts were prepared from cells treated with honokiol (20, 40, or 60 µM) for 6 h and expression of NF-κB/p65, p-IκB-α (S32/36) and IκB-α was determined by Western blot analysis. β-actin was used as a loading control. Intensities of the immunoreactive bands were quantified by densitometry. Normalized densitometry values are indicated at the top of the bands indicating a decreased localization of NF-κB/p65 in nucleus with a concomitant increase in cytoplasm. In contrast, expression of p-IκB-α was decreased leading to increased levels of IκB-α. Altogether, these data clearly suggest that honokiol inhibits NF-κB activity through stabilization of IκB-α.</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