The specific degree-of-polymerization of A-type proanthocyanidin oligomers impacts <i>Streptococcus mutans</i> glucan-mediated adhesion and transcriptome responses within biofilms

<div><p>Cranberry A-type proanthocyanidins (PACs) have been recognized for their inhibitory activity against bacterial adhesion and biofilm-derived infections. However, the precise identification of the specific classes of degree-of-polymerization (DP) conferring PACs bioactivity remains a major challenge owing to the complex chemistry of these flavonoids. In this study, chemically characterized cranberries were used in a multistep separation and structure-determination technique to isolate A-type PAC oligomers of defined DP. The influences of PACs on the 3D architecture of biofilms and <i>Streptococcus mutans</i>-transcriptome responses within biofilms were investigated. Treatment regimens that simulated topical exposures experienced clinically (twice-daily, 60 s each) were used over a saliva-coated hydroxyapatite biofilm model. Biofilm accumulation was impaired, while specific genes involved in the adhesion of bacteria, acid stress tolerance, and glycolysis were affected by the topical treatments (<i>vs</i> the vehicle-control). Genes (<i>rmpC</i>, <i>mepA</i>, <i>sdcBB</i>, and <i>gbpC</i>) associated with sucrose-dependent binding of bacteria were repressed by PACs. PACs of DP 4 and particularly DP 8 to 13 were the most effective in disrupting bacterial adhesion to glucan-coated apatitic surface (>85% inhibition <i>vs</i> vehicle control), and gene expression (eg <i>rmpC</i>). This study identified putative molecular targets of A-type cranberry PACs in <i>S. mutans</i> while demonstrating that PAC oligomers with a specific DP may be effective in disrupting the assembly of cariogenic biofilms.</p> </div

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 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

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

Cranberry Flavonoids Modulate Cariogenic Properties of Mixed-Species Biofilm through Exopolysaccharides-Matrix Disruption

<div><p>The exopolysaccharides (EPS) produced by <i>Streptococcus mutans-</i>derived glucosyltransferases (Gtfs) are essential virulence factors associated with the initiation of cariogenic biofilms. EPS forms the core of the biofilm matrix-scaffold, providing mechanical stability while facilitating the creation of localized acidic microenvironments. Cranberry flavonoids, such as A-type proanthocyanidins (PACs) and myricetin, have been shown to inhibit the activity of Gtfs and EPS-mediated bacterial adhesion without killing the organisms. Here, we investigated whether a combination of cranberry flavonoids disrupts EPS accumulation and <i>S</i>. <i>mutans</i> survival using a mixed-species biofilm model under cariogenic conditions. We also assessed the impact of cranberry flavonoids on mechanical stability and the <i>in situ</i> pH at the biofilm-apatite interface. Topical application of an optimized combination of PACs oligomers (100–300 μM) with myricetin (2 mM) twice daily was used to simulate treatment regimen experienced clinically. Treatments with cranberry flavonoids effectively reduced the insoluble EPS content (>80% reduction vs. vehicle-control; p<0.001), while hindering <i>S</i>. <i>mutans</i> outgrowth within mixed-species biofilms. As a result, the 3D architecture of cranberry-treated biofilms was severely compromised, showing a defective EPS-matrix and failure to develop microcolonies on the saliva-coated hydroxyapatite (sHA) surface. Furthermore, topical applications of cranberry flavonoids significantly weaken the mechanical stability of the biofilms; nearly 90% of the biofilm was removed from sHA surface after exposure to a shear stress of 0.449 N/m² (vs. 36% removal in vehicle-treated biofilms). Importantly, <i>in situ</i> pH measurements in cranberry-treated biofilms showed significantly higher pH values (5.2 ± 0.1) at the biofilm-apatite interface vs. vehicle-treated biofilms (4.6 ± 0.1). Altogether, the data provide important insights on how cranberry flavonoids treatments modulate virulence properties by disrupting the biochemical and ecological changes associated with cariogenic biofilm development, which could lead to new alternative or adjunctive antibiofilm/anticaries chemotherapeutic formulations.</p></div

Mechanical stability and <i>in situ</i> pH within intact mixed-species biofilms treated with CranFlav.

<p>(A) The amount of remained biofilm dry-weight before and after application of shear stress was measured to determine the mechanical stability and attachment strength of CranFlav (or vehicle)-treated biofilms on sHA surface. We also determined biofilm removal at 0.184 and 0.449 N/m² (vs. no shear, 0 N/m²) (n = 8). (B) <i>In situ</i> biofilm pH values were determined every 2 μm from the sHA surface, and averaged based on pH measurements across the biofilm interface (n = 3). The molar concentration of hydrogen ion (H⁺) at the biofilm/sHA interface (10–15 μm from the sHA surface) was estimated via the equation: [H⁺] = 10^{-in situ pH}. Values are significantly different from each other at *p<i><</i>0.01 or **p<0.001.</p

Structure of cranberry-derived flavonoids for combination therapy.

<p>Myricetin, one of the most active cranberry flavonols, is characterized by the presence of an unsaturated double bond between C2 and C3 and three hydroxyl-groups in the B ring. Proanthocyanidins (PACs) in cranberry are predominantly found in oligomeric forms (up to 13 monomeric units) with at least one A-type double interflavan linkage [epicatechin-(4β→8, 2β→<i>O</i>→7)-epicatechin] between the lower or lowest two units of the oligomer. The degree-of-polymerization (DP) is variable depending on the number of monomeric units; DP4 (tetramer) and DP9 (nonamer) are among the most abundant and bioactive cranberry PAC.</p

Experimental design for topical treatment regimen.

<p>Mixed-species cariogenic biofilm model and treatment regimen of selected combination of cranberry flavonoids (CranFlav). CranFlav or vehicle was topically applied twice daily with 5 min exposure.</p