Geranylated 4-Phenylcoumarins Exhibit Anticancer Effects against Human Prostate Cancer Cells through Caspase-Independent Mechanism.

Geranylated 4-phenylcoumarins, DMDP-1 & -2 isolated from Mesua elegans were investigated for anticancer potential against human prostate cancer cells. Treatment with DMDP-1 & -2 resulted in cell death in a time and dose dependent manner in an MTT assay on all cancer cell lines tested with the exception of lung adenocarcinoma cells. DMDP-1 showed highest cytotoxic efficacy in PC-3 cells while DMDP-2 was most potent in DU 145 cells. Flow cytometry indicated that both coumarins were successful to induce programmed cell death after 24 h treatment. Elucidation on the mode-of-action via protein arrays and western blotting demonstrated death induced without any significant expressions of caspases, Bcl-2 family proteins and cleaved PARP, thus suggesting the involvement of caspase-independent pathways. In identifying autophagy, analysis of GFP-LC3 showed increased punctate in PC-3 cells pre-treated with CQ and treated with DMDP-1. In these cells decreased expression of autophagosome protein, p62 and cathepsin B further confirmed autophagy. In contrary, the DU 145 cells pre-treated with CQ and treated with DMDP-2 has reduced GFP-LC3 punctate although the number of cells with obvious GFP-LC3 puncta was significantly increased in the inhibitor-treated cells. The increase level of p62 suggested leakage of cathepsin B into the cytosol to trigger potential downstream death mediators. This correlated with increased expression of cathepsin B and reduced expression after treatment with its inhibitor, CA074. Also auto-degradation of calpain-2 upon treatment with DMDP-1 &-2 and its inhibitor alone, calpeptin compared with the combination treatment, further confirmed involvement of calpain-2 in PC-3 and DU 145 cells. Treatment with DMDP-1 & -2 also showed up-regulation of total and phosphorylated p53 levels in a time dependent manner. Hence, DMDP-1 & -2 showed ability to activate multiple death pathways involving autophagy, lysosomal and endoplasmic reticulum death proteins which could potentially be manipulated to develop anti-cancer therapy in apoptosis resistant cells

Isolation and characterization of two 4-phenylcoumarins from <i>Mesua elegans</i>.

<p><b>(A)</b> Chemical structure of DMDP-1 & -2. <b>(B)</b> (I) HPLC chromatogram of fraction A2 with the following experimental conditions (analytical): column, ZORBAX Eclipse Plus C18, 4.6 mm i.d. x 150 mm x 3.5 μm; mobile phase, two solvents: A, 0.1% formic acid in H₂0 and B, 0.1% formic acid in MeOH; the elution program at 0.6 mL/min as isocratic with 90% B (0–30 min) to afford isomers DMDP-1 & -2. (II) HPLC chromatogram of fraction A2 with the following experimental conditions (semi-preparative): column, ZORBAX Eclipse Plus C18, 9.4 mm i.d. x 250 mm x 3.5 μm; mobile phase, two solvents: A, 0.1% formic acid in H₂0 and B, 0.1% formic acid in MeOH; the elution program at 3.0 mL/min as isocratic with 90% B (0–50 min) to afford isomers DMDP-1 & -2.</p

Photomicrograph of PC-3 and DU 145 cell lines depicting increased number of vacuoles after treatment with DMDP-1 & -2 for 24 h.

<p>All images are shown at 400x magnification, and formation of vacuoles is indicated by white arrows.</p

DMDP-1 & -2 induces cell death-mediated cytotoxicity in NP69, PC-3 and DU 145 cells.

<p>(A) Live/Dead assay after treatment with DMDP-1 & -2 for 24 h and DMSO as solvent control. Green fluorescence denotes viable cells stained with calcein-AM, while red fluorescence represents dead cells stained with ethidium homodimer. (B)Annexin V-FITC/PI flow cytometry dot plot analysis after treatment over 24 h. Percentage of cell death was calculated based on top right and bottom right quadrants from a total of 1.0 x 10⁴ cells.</p

Activation of proteins involved in caspase-independent pathways.

<p>(A) Cells were treated with DMDP-1 & -2 over 24 h, followed by examination of key proteins involved in multiple mode of cell death by western blotting. (B) Quantification of band intensities were determined by densitometry analysis and normalized to GADPH using the ImageJ v1.43 software. All results were presented as mean normalized intensity ±S.D. of three independent experiments.</p

Western blotting confirmation on the effects of DMDP-1 & -2 treatment over 24 h on PC-3 and DU 145 caspase levels.

<p>(A) Treatment with DMDP-1 & -2 showed full length of protein levels and absence of cleaved PARP and caspases-9, -3 and -8. (B) Analysis of anti-apoptotic Bcl-2 and pro-apoptotic Bax protein levels. Quantification of protein band intensities were determined by densitometry analysis and normalized to GADPH using the ImageJ v1.43 software. All results were presented as mean normalized intensity ±S.D. of three independent experiments.</p

Summary of DMDP-1 & -2 mean IC₅₀ values and mean viability levels obtained from MTT assays for ten human cancers and one normal cell lines after 24 h treatment.

<p>All values were presented as mean ±SD of three technical and three biological replicates.</p