Prostate-specific antitumor effects of natural compounds: Isoliquiritigenin and alternol
Yeung, Eddie Dong
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Prostate cancer is the most commonly diagnosed form of cancer among men in the United States. It is second only to lung cancer as the leading cause of cancer-related deaths in males. The lethality of prostate cancer can be attributed to the progression of the tumor in becoming androgen-independent. Conventional chemotherapy is one of the most common and effective approaches used clinically for the treatment of prostate cancer. Most chemotherapeutic agents used currently exert preferential action on cells that rapidly divide. Due to the non-selective natures of these agents between tumorigenic and non-tumorigenic cells, conventional chemotherapy is often associated with numerous adverse side effects. The objective of the present thesis was to first, screen for selective prostate-specific anticancer agent(s) from natural compounds, and then to elucidate possible signaling pathways and mechanism(s) involved in the selective effect exerted by the anticancer agent(s). Results from in vitro experimentation on human prostate cancer cell lines (C4-2, LNCaP, DU-145, and PC-3) in comparison with non-tumorigenic epithelial cell lines (IEC-6, and RWPE-1) reveal two compounds that are selective toward tumorigenic prostate cells: Isoliquiritigenin (ISL) and Alternol. In vitro exposures of these agents were conducted. Cell viability was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Protein expression levels were examined by Western blotting. The fluorescent probe H2DCFDA [5-(6)-chloromethyl-2',7'- dichlorodihydrofluorescein diacetate acetyl ester] was used to measure the levels of intracellular reactive oxygen species (ROS). The mitochondrial membrane potential was measured by using the mitochondrial probe JC-1 [5,5',6,6'- tetrachloro-1,1',3,3' tetra-ethylbenzimidazolo-carbocyanine iodide]. Cell cycle was analyzed by fluorescence activated cell sorting with propidium iodide staining. Part I: Isoliquitigenin . ISL is a simple chalcone-type flavonoid derived from licorice compounds, previously reported to possess antioxidative and anti-tumor activities. The results demonstrated that ISL exposure for 24 hrs markedly inhibited the proliferation of both C4-2 and LNCaP prostate cancer cells in a dose-dependent manner. Conversely, ISL did not affect the viability of IEC-6 normal epithelial cells within a similar dosage range. ISL treatment significantly decreased the ROS level and mitochondrial membrane potential (Δ&PSgr;m) of C4-2 cells, but did not affect the ROS level and Δ&PSgr;m of IEC-6 cells. Furthermore, the level of AMP-activated protein kinase (AMPK) and extracellular-signal regulated kinase (ERK) phosphorylation were noticeably higher in IEC-6 cells than in C4-2 cells. Part II: Alternol . Alternol is an original compound purified from the fermentation products of Alternaria alternata var. monosporus , a microorganism from the bark of the yew tree. It was previously reported to induce apoptosis and inhibit proliferation of mouse leukemia cells and human gastric carcinoma cells. Alternol treatment of 24 hrs resulted in a significant decrease in the viability of prostate cancer cells (C4-2, DU-145, and PC-3) but had lesser effects on RWPE-1 cells. Results from flow cytometry demonstrated that Alternol caused an accumulation in the G 2 M-phase of cell cycle in C4-2 cells. Alternol inhibited AMPK phosphorylation in C4-2 cells but stimulated AMPK phosphorylation in RWPE-1 cells. Inhibition of p27 phosphorylation was observed in C4-2 cells whereas a promotion of p27 phosphorylation was seen in RWPE-1 cells. Alternol treatment resulted in a profound increase in the LC3II/LC3I protein ratio, a marker for autophagy, in RWPE-1 cells but not in C4-2 cells. A dose-dependent down-regulation of Bcl-2 protein was detected in C4-2 cells but not in RWPE-1 cells, suggesting the induction of apoptosis in C4-2 cells. Pretreatment of cells with Compound C (AMPK inhibitor) before Alternol treatment abolished the selective antitumor effect of Alternol. The results from the first set of studies suggest that ISL, a natural antioxidant, selectively inhibits the proliferation of prostate cancer C4-2 cells. The mechanism may be partly attributed to the differential effects of ISL on the AMPK and ERK signaling pathways in C4-2 cells vs . IEC-6 cells. The results from the latter set of studies reveal for the first time that Alternol exerts a selective antitumor effect on prostate cancer cells when compared with non-tumorigenic RWPE-1 prostate epithelial cells. In addition, the data provides the first evidence that the AMPK pathway may be responsible for the selective antitumor effects of Alternol.