Article pubs.acs.org/JAFC
Antitumor Activity of Garcinol in Human Prostate Cancer Cells and Xenograft Mice Yu Wang,† Mei-Ling Tsai,‡ Li-Yu Chiou,‡ Chi-Tang Ho,§ and Min-Hsiung Pan*,∥,⊥,# †
Department of Food Science and Human Nutrition, Citrus Research and Education Center, University of Florida, 700 Experiment Station Road, Lake Alfred, Florida 33850, United States ‡ Department of Seafood Science, National Kaohsiung Marine University, Kaohsiung 811, Taiwan ∥ Institute of Food Science and Technology, National Taiwan University, Taipei 10617, Taiwan § Department of Food Science, Rutgers University, New Brunswick, New Jersey 08901, United States ⊥ Department of Medical Research, China Medical University Hospital, China Medical University, Taichung 40402, Taiwan # Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan ABSTRACT: Garcinol, which is isolated from fruit rinds of Garcinia indica, is a polyisoprenylated benzophenone. It has been studied for its antitumor activity by inducing apoptosis and inhibiting autophagy in human prostate cancer cells. The Bax/Bcl-2 ratio increased when garcinol was applied to PC-3 cells indicating a presence of apoptosis. Meanwhile, procaspases-9 and -3 were suppressed with attenuating PARP and DFF-45. Autophagy was inhibited through activating p-mTOR and p-PI3 Kinase/AKT by garcinol, which as a result induced the cells to apoptosis directly. In addition, the apoptosis effect of garcinol in a xenograft mouse model was also tested, suggesting a consistent result with PC-3 cell model. The tumor size was reduced more than 80 percent after the mouse accepted the garcinol treatment. Garcinol was demonstrated to have a strong antitumor activity through inhibiting autophagy and inducing apoptosis, which was discovered for the first time. Based on these findings, our data suggests that garcinol deserves further investigation as a potent chemopreventive agent. KEYWORDS: garcinol, apoptosis, autophagy, prostate cancer cells and xenograft mice
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INTRODUCTION In the United States, the most common cancer that men have is prostate cancer. It has become the second leading cause of cancer death in Western countries for men after lung cancer.1 However, distribution of prostate cancer on a worldwide scale shows a geographic distinction, where Asian countries have the lowest occurrence compared with Western countries. Many factors, such as genetics or lifestyle, have been indicated for the induction of prostate cancer. Genetics are always taken as reasons for cancer, but less than 10% of prostate cancer is attributed to heritage.2 On the other hand, some studies demonstrated that once Asian men adapted a Western lifestyle, they had a significantly higher risk of prostate cancer compared to other Asian men.2 More and more evidence supported that lifestyle, particularly diet, plays an important role in the development of prostate cancer,2 and the strategy for controlling prostate cancer is chemoprevention by natural or synthetic compounds. Therefore, identification of new chemotherapeutic agents is needed to induce the death of cancer cells. Programmed cell death mainly occurs through either type I cell death, known as apoptosis, or type II cell death, namely, autophagy. During apoptosis, cell shrinkage, membrane blebbing, and chromatin condensation can be observed. In addition, apoptosis induces internucleosomal DNA cleavage, which leads to the formation of DNA ladders with multiple fragments.3 In the apoptosis cascade, proteolytic cleavage of procaspases is a leading step to caspase activation and its downstream caspases.4,5 For example, releasing cytochrome c into cytoplasm causes the activation of caspase-8 and caspase-9, © 2015 American Chemical Society
which in turn activate executioner caspases, including caspase-3, and then cleave a number of cellular proteins.5 Autophagy is a response of eukaryotic cells to unfavorable conditions such as starvation, pathogen infestation, and chemotherapy. It is characterized by the accumulation of autophagosomes in the cytoplasm that surround a normal nucleus.6 Beclin1 and LC3 (light chain 3) have been indicated as the essential factors for autophagy, and they are associated with the autophagosome membranes. The extent of autophagosome formation is correlated to the distribution of beclin-1 and LC3 to the membrane of autophagosomes.7 In addition, two major signaling pathways that regulate autophagy are MEK/extracellular signal-regulated kinase (ERK1/2) and phosphatidylinositol-3-kinase (PI3K)/active human protein kinase (AKT)/ mammalian TOR (mTOR).8 Garcinol is a polyisoprenylated benzophenone derived from fruit rinds of Garcinia indica. The dried rind has been used in cooking and traditional medicine for centuries. The chemical structure of garcinol is similar to curcumin, which contains a phenolic hydroxyl group and a β-diketone moiety (Figure 1A). The bioactivity of garcinol has been investigated, for example, anti-inflammation, antitumor, or antiulcer.9 Garcinol is able to induce apoptosis in a concentration- and time-dependent manner in human leukemia HL-60 cells. Compared to curcumin, Received: Revised: Accepted: Published: 9047
August 8, 2015 October 6, 2015 October 7, 2015 October 7, 2015 DOI: 10.1021/acs.jafc.5b03851 J. Agric. Food Chem. 2015, 63, 9047−9052
Article
Journal of Agricultural and Food Chemistry
DNA Extraction and Electrophoretic Analysis. The PC-3 cells were harvested and washed with phosphate-buffered saline (PBS). The extraction method followed the previous procedure.10 The DNA was extracted using phenol/chloroform/isoamyl alcohol (25:24:1) and was analyzed by 2% agarose gel electrophoresis. DNA was stained with ethidium bromide before being visualized under UV light and photographed. Flow Cytometry. PC-3 cells (2 × 105) were cultured in 60 mm Petri dishes in RPMI-1640 medium and incubated for 24 h. The details of the flow cytometry methodology followed the previous experiment design.10 The propidium iodide−DNA complex was quantitated using FACScan cytometry (Becton Dickinson, San Jose, CA). ModFit LT for Mac 3.0 software (Becton Dickinson) was used to quantify the fraction of each cell cycle stage. Analysis of the Mitochondrial Trans-Membrane Potential. Flow cytometry was used to measure the potential change of the mitochondrial trans-membrane. PC-3 cells were treated with garcinol (30 μM) for different time periods, and the potential of mitochondrial trans-membrane was measured with the direct application of 40 nM 3,3′-dihexyloxacarbocyanine [DiOC6(3)] (Molecular Probes, Eugene, OR). The fluorescence intensity was quantified by flow cytometry after the cells were stained for 30 min at 37 °C. Histograms were analyzed using Cell Quest software. Histograms from treated cells were compared with those from untreated, control cells. Western Blot Analysis. The basic procedure of Western blot analysis was the same as the previous study.10 The primary antibodies used included anticaspase-3, -9 (PharMingen, San Diego, CA), anti-Bcl2, anti-Bcl-XL, anti-Bax, anti-Bad (Santa Cruz Biotechnology, Santa Cruz, CA), antipoly(ADP-ribose) polymerase (PARP), LC3 I/II, Beclin-1, Atg 12, Atg 5, ERK1/2, phosphoERK1/2, JNK1/2, phospho-1/2, p38, phosphor-p38, PI3K, phospho-PI3K, PDK1, phosphor-PDK1, Akt, phospho-Akt, GSK3β, phosphor-GSK3β, mTOR, phosphor-mTOR (UBI Inc., Lake Placid, NY, USA), antiBag-1, anti-Mcl-1 (R and D System Inc., Minneapolis, MN, USA), antiβ-actin (Transduction Laboratory, Lexington, KY, USA), and antiDFF45/inhibitor of caspase activated DNase (ICAD) (MBL, Naka-Ku, Nagoya, Japan) antibodies. Human Prostate Tumor Xenograft Mouse Model. Each nude mouse was injected with prostate cancer PC-3 cells (3 × 106) in 200 μL of a PBS solution between the scapulae. The tumor size was measured using a caliper, and the tumor volume was estimated via the following formula: tumor volume (mm3) = L × W2/2, where L was the length and W was the width. When the tumor size reached the average of 50−100 mm3, the mice were randomly divided into six groups (6−8 animals/ group). In the groups with intraperitoneal treatment, the mice were injected with garcinol (50 mg/kg/d) in 200 μL of coil oil for 5 days per week, while the control group received just corn oil injections. In the groups with oral treatment, the animals were orally administered with either 200 μL of corn oil or garcinol in corn oil (50 mg/kg/d) for 5 days per week. The dietary intake and the body weight of each animal (average weight = 17 g) were monitored every day. The tumor volume was calculated twice per week. After 40−50 days, the mice were sacrificed by CO2 asphyxiation. The livers, kidneys, spleens, and solid tumors were excised immediately and weighed. The average tumor volumes and weights of each group were represented as the means ± standard deviation (SD). The tumor tissue was cut into small portions for Western blot analysis. Statistical Analysis. All results were obtained through three independent experiments and presented as means ± standard error of the mean (SE). The differences between the control group and the different concentrations of garcinol were analyzed by Student’s t test. The data was considered statistically significant at a P-value of
