Cellular effects of weitaichun, a natural compound, in human prostate cancer
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In the United States, prostate cancer is the most common cancer among men and is the second leading cause of cancer death. For patients who have recurrent or advanced prostate cancer that has not responded to hormone treatment, chemotherapy can improve survival rate and assist with pain control. Because chemotherapeutic drugs travel throughout the body to target and kill fast dividing cells, they can kill quickly dividing cells including those in the hair follicles, skin, gastrointestinal tract, and bone marrow in addition to cancerous cells, resulting in significant side effects. While a major challenge is to make chemotherapy more selective at targeting cancer cells (thus have less side effect), a number of natural compounds have been shown to possess selective cytotoxicity and usage of such compounds have demonstrated targeted killing of cancer cells while leaving normal cells intact. Weitaichun (WTC) is a compound isolated during the fermentation process of micro organisms found within the bark of yew trees. Due to its similar structure as Alternol which has been recently shown to successfully inhibit cancer cell growth, WTC may possess anticancer properties as well. The aim of this study was to determine the effect of WTC on inhibition of cell growth and induction of apoptosis in human prostate cancer cells (compared to normal prostate cells) and investigate signaling pathways and mechanisms underlying the selective effect exerted by WTC. Results from in vitro experimentation on human prostate cancer cell line (C4-2) in comparison with non-tumorigenic prostate epithelial cell line (RWPE-1) revealed that the effects of WTC were selective toward prostate cancer cells at specific dose range. In vitro exposures of WTC 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. Cell cycle was analyzed by fluorescence activated cell sorting with propidium iodide staining. 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]. The fluorescent probe H 2 DCFDA [5-(6)-chloromethyl-2',7'- dichlorodihydrofluorescein diacetate acetyl ester] was used to measure the levels of intracellular reactive oxygen species (ROS). Reduced glutathione and oxidized glutathione levels were measured using the DTNB-GSSG reductase recycling assay method. Weitaichun (WTC) treatment for 24 hours resulted in a significant decrease in the viability of C4-2 cells in a dose-dependent manner. Conversely, WTC did not affect the viability of RWPE-1 cells within a similar dosage range. WTC treatment activated Mitogen-activated protein kinase (MAPK) pathways including P38 MAP kinases and SAPK/JNK in C4-2 cells. While SAPK/JNK was also activated in RWPE-1 cells after WTC treatment (p38 was not activated in RWPE-1 cells), C-Jun, a downstream of SAPK/JNK, was activated only in C4-2 cells; this demonstrates that c-Jun is regulated by not only JNK but also other upstream kinases. Results from flow cytometry demonstrated that WTC caused cell cycle arrest at G 2 /M phase whereas there was no change in cell cycle distribution of RWPE-1 cells after WTC treatment. Interestingly, pretreatment with p38 inhibitor, SB 203580, prevented WTC-induced G 2 /M arrest in C4-2 cells; this suggests that the activation of p38 is responsible for the selective effect of WTC on induction of cell cycle arrest and apoptosis in C4-2 cells. In addition, WTC treatment significantly decreased mitochondrial membrane potential (ΔΨm) and the expression of anti-apoptotic Bcl-2 protein only in C4-2 cells; both changes are early signals of apoptosis. Because the depletion of ROS scavenger glutathione (GSH) is considered to cause cell growth inhibition and enhanced apoptosis in cancer cells, the levels of GSH after WTC treatment were measured for both cell lines. WTC treatment of 24 hrs also resulted in a significant decrease in the GSH/GSSG ratio in C4-2 cells, but not in RWPE-1 cells. Basal GSH levels were significantly higher in C4-2 cells compared to RWPE-1 basal levels. WTC treatment (1.2 μM, 24hrs) also reduced the intracellular ROS levels in C4-2 cells while basal ROS levels in C4-2 cells were significantly higher than that in RWPE-1 cells. The high basal levels of ROS in C4-2 cells is materially correlated with growing evidence that suggest ROS within cancer cells can increase cell proliferation and survival, as well as induce DNA damage leading to genetic lesions that initiate cancer.