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

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 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 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