Abstract
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.
Highlights
Prostate cancer is the most common cancer as well as the second leading cause of cancerrelated deaths in men [1]
Their finding indicated that the apoptosis inducing factor (AIF) did not translocate to nuclei as in the case of apoptosis, but rather it remained within mitochondria throughout the course of autophagy.[24]
Our present findings show that DMDP-1 induces autophagy and activates calpain-2, while DMDP-2 inhibits autophagy, activates cathepsin B and calpain-2, both leading to caspase-independent cell death in human prostate cancer cells
Summary
Prostate cancer is the most common cancer as well as the second leading cause of cancerrelated deaths in men [1]. Despite the availability of multiple treatment options, there are currently no effective therapies available for treatment of apoptotic-resistant androgen-independent prostate cancer which often arises after hormonal deprivation or ablation therapy [2]. Yang and colleagues, demonstrated fifteen isoprenylated coumarins isolated from Mammea americana exhibited significant cytotoxic effects and high anti-oxidant activity in human colon cancer cell lines [12]. In a study with both coumarin and 7-hydroxycoumarin, inhibition of cell growth in lung carcinoma cell lines by inducing G1 phase cell cycle arrest and apoptosis was demonstrated [13]. In another report, geranylated coumarins were seen to exert anti-proliferative actions through apoptotic cell death in leukemia cells [14]
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