Ellagic acid (EA) inhibits cell growth and induces apoptosis in cultured cells; however, the precise molecular mechanism involved in EA-induced apoptosis in prostate cancer cells is unknown. The aim of the present study was to delineate possible apoptotic pathway(s) involved in the EA-mediated chemotherapeutic effects in the LNCaP human prostatic cancer cell line. EA produced anti-proliferative effects through inhibition of rapamycin (mTOR) activation and a reduction in intracellular levels of β-catenin. Moreover, we demonstrated that EA induced apoptosis via downregulation of the anti-apoptotic proteins, silent information regulator 1 (SIRT1), human antigen R (HuR) and heme oxygenase-1 (HO-1). EA modulated the expression of apoptosis-inducing factor (AIF) resulting in a significant increase in reactive oxygen species (ROS) levels and the activation of caspase-3. Finally, we demonstrated that EA reduced both transforming growth factor-β (TGF-β) and interleukin-6 (IL-6) levels. EA treatment resulted in the increased expression of the tumor suppressor protein p21 and increased the percentage of apoptotic cells. In conclusion, the results suggest that EA treatment represents a new and highly effective strategy in reducing prostate cancer carcinogenesis.
Prostate cancer (PC) is the most common cancer in men over the age of 50 years. PC represents one of the leading causes of cancer-related mortality in Western countries (
Our previous research demonstrated a dose-dependent cytotoxic effect of EA, resulting in a reduction in the proliferation rate and a marked increase in DNA damage in prostatic cancer cell lines (
Tumorigenesis is a multistep process activated by various environmental carcinogens, inflammatory agents and tumor promoters. These carcinogens modulate transcription factors, anti-apoptotic proteins, pro-apoptotic proteins, protein kinases, cell cycle proteins, cell adhesion molecules and growth factor signaling pathways. EA was found to inhibit cell growth and induce apoptosis in a variety of cell cultures (
The present study examined the involvement of apoptotic markers in the cytotoxic effects exerted by EA on the LNCaP human prostatic cancer cell line. In particular, we investigated the anti-carcinogenic properties of EA by evaluating its ability to induce cell cycle arrest and apoptosis. We evaluated mTOR, SIRT1, β-catenin, HUR, AIF, caspase-3, p21, IL-6 and TGF-β. In addition we examined the effects of EA on the cell cycle and showed that EA regulates apoptosis in the LNCaP prostatic cancer cell line.
Frozen LNCaP cells were purchased from the American Type Culture Collection (Rockville, MD, USA). After thawing, LNCaP cells were re-suspended in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% heat inactivated fetal bovine serum (FBS) and 1% antibiotic/antimycotic solution (both from Invitrogen Life Technologies, Carlsbad, CA, USA). The cells were plated at a density of 1–5×106 cells/T75 flask. Cell cultures were maintained at 37°C in a 5% CO2 incubator, and the medium was changed after 3–4 days. Subconfluent cells were treated for 48 h with 2 different concentrations (25 and 50 μM) of freshly prepared EA dissolved in dimethyl sulfoxide (DMSO). Control groups received DMSO alone.
Cells were cultured in T75 flasks for 48 h. They were then washed with PBS and trypsinized (0.05% trypsin w/v with 0.02% EDTA). The pellets were lysed in buffer (50 mM Tris-HCl, 10 mM EDTA, 1% v/v Triton X-100, 1% PMSF, 0.05 mM pepstatin A and 0.2 mM leupeptin) and after mixing with sample loading buffer (50 mM Tris-HCl, 10% w/v SDS, 10% v/v glycerol, 10% v/v 2-mercaptoethanol and 0.04% bromophenol blue) at a ratio of 4:1, were boiled for 5 min. Samples (20 μg proteins) were loaded onto 8 or 12% SDS-polyacrylamide (SDS-PAGE) gels and subjected to electrophoresis (120 V, 90 min). The separated proteins were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). After transfer, the blots were incubated with Li-Cor blocking buffer for 1 h, followed by overnight incubation with a 1:1,000 dilution of the primary antibody. Primary polyclonal antibodies directed against AIF, β-catenin, p-mTOR, SIRT-1, caspase-3 and p21 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA) while HuR and TGF-β were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). After washing with TBS, the blots were incubated for 1 h with the secondary antibody (1:1,000). Protein detection was carried out using a secondary infrared fluorescent dye-conjugated antibody absorbing at 800 and 700 nm as described below. The blots were visualized using an Odyssey Infrared imaging scanner (LI-COR Biosciences) and quantified by densitometric analysis performed after normalization with β-actin (Santa Cruz). Results are expressed as arbitrary units (A.U.).
Cells were cultured as previously described, fixed in 70% ethanol overnight at −20°C and washed with phosphate-buffered saline (PBS). Aliquots of 1×106 cells were re-suspended in 1 ml of PBS containing 1 mg/ml of RNase A and 0.5 mg/ml propidium iodide (PI). After a 30-min incubation, the cells were analyzed by flow cytometry using a FACScan flow cytometer (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA) and evaluated by fluorescence-activated cell sorting (FACS) analysis to identify the cells at different stages of the cell cycle.
IL-6 levels were determined in the culture supernatant using an ELISA kit (AssayGate Inc., Ijamsville, MD, USA). The assays were performed according to the manufacturer's guidelines. Results are expressed as pg/ml.
Cells were seeded in a 96-well cell culture plate. After a 48-h treatment with 25 or 50 μM EA, cells were washed in PBS and directly fixed with 4% of paraformaldehyde (PFA) in PBS for 20 min. Cells were permeabilized with 0.2% Triton X-100, blocked with Li-Cor blocking buffer for 60 min at room temperature, followed by overnight incubation with rabbit HO-1 (1:500) and mouse β-actin primary antibody (1:1,000). β-actin was used as a housekeeping gene to normalize the HO-1 signal for the cell number. After 3 washes, protein detection was carried out using a secondary infrared fluorescent dye-conjugated antibody absorbing at 800 or 700 nm. The whole plate was visualized using an Odyssey Infrared imaging scanner with a 700-nm fluorophore (red dye) and 800-nm fluorophore (green dye). Relative fluorescence units from the scanning allowed a quantitative analysis of the proteins.
Determination of ROS was performed using a fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA), as previously described (
Statistical significance between experimental groups was determined by the Fisher's method of analysis of multiple comparisons (p<0.05). For comparisons among treatment groups, the null hypothesis was tested by a 2-factor ANOVA for multiple groups or unpaired t-test for 2 groups. Data are presented as means ± SD.
We assessed the levels of β-catenin, p-mTOR, and SIRT1 after 48 h of culture in the presence of EA. As shown in
HO-1 protein levels were examined by in-cell western blot analysis, to quantify total endogenous cellular protein (
As shown in
The effect of EA on IL-6 levels is shown in
Flow cytometric analysis of the cell cycle distribution is shown in
Prostrate cancer is a chronic disease that develops from a small lesion to clinical manifestation over an extended period of time. However, once the disease is metastatic, patient prognosis is poor. Thus, the development of new strategies to fight PC has become an important therapeutic mission. The administration of both synthetic and naturally occurring agents to suppress, reverse and delay carcinogenesis, is increasingly being touted as an effective approach for the management of prostatic neoplasia (
β-catenin is a subunit of a protein complex acting as a signal transducer, and aberrant accumulation of intracellular β-catenin is a well-recognized characteristic of several types of cancers, including prostate, colon and liver (
We showed that EA treatment exerts anti-proliferative effects by reducing intracellular levels of β-catenin. We previously demonstrated that EA reduced Akt activation/phosphorylation in prostate cancer cell lines. The capacity of p-Akt to phosphorylate/activate mTOR has been described in several cancer cell lines (
SIRT1 functions as an oncogenic protein and plays a role in tumorigenesis (
AIF is a flavoprotein anchored to the mitochondrial inner membrane. Under physiological conditions, AIF exhibits NADH oxidase activity, important for mitochondrial respiration (
IL-6 is a multifunctional cytokine and a major activator of different signaling pathways. It regulates growth of prostate cancer (
(A) Representative western blotting of β-catenin, p-mTOR, Sirt1 and HuR protein expression in cultured LNCaP cells. (B–E) Effect of EA (25 and 50 μM) on β-catenin, p-mTOR, Sirt1 and HuR expression in cultured LNCaP cells. Results are expressed as arbitrary units (A.U.), and represent the means ± SD of 4 experiments performed in triplicate. *p<0.05, significant result of 25 and 50 μM EA vs. control. #p<0.005, significant result of 50 μM EA vs. 25 μM EA. EA, ellagic acid; Sirt1, silent information regulator 1; HuR, human antigen R; Cntrl, control.
(A) Representative image of HO-1 protein expression in cultured LNCaP cells by in-cell western blotting. (B) Effect of 25 and 50 μM EA on HO-1 expression in cultured LNCaP cells. Results are expressed as relative fluorescence intensity (R.F.I.) and represent the means ± SD of 4 experiments performed in triplicate. *p<0.005, significant result of 25 and 50 μM EA vs. control. EA, ellagic acid; HO-1, heme oxygenase-1; Cntrl, control.
(A) Representative western blotting of AIF, caspase-3, p21, TGF-β protein expression in cultured LNCaP cells. (B–E) Effect of EA (25 and 50 μM) on AIF, caspase-3, p21, TGF-β expression in cultured LNCaP cells. Results are expressed as arbitrary units (A.U.), and represent the means ± SD of 4 experiments performed in triplicate. *p<0.05, significant result of 25 and 50 μM EA vs. (black) control; #p<0.05, significant result of 25 and 50 μM EA vs. (grey) control. EA, ellagic acid; AIF, apoptosis-inducing factor; TGF-β, transforming growth factor-β; Cntrl, control.
Intracellular oxidants in LNCaP cells untreated and treated for 48 h with EA at different concentrations (25 and 50 μM). Values represent the means ± SD of 4 experiments performed in triplicate. *p<0.005, significant result vs. untreated control cells. EA, ellagic acid; Cntrl, control.
IL-6 levels in LNCaP cells untreated and treated for 48 h with EA at different concentrations (25 and 50 μM). Values represent the means ± SD of 4 experiments performed in triplicate. *p<0.05, significant result vs. untreated control cells. IL-6, interleukin-6; EA, ellagic acid; Cntrl, control.
Effect of EA (50 μM) on cell cycle distribution as determined by FACS analysis. (A) Percentage of viable cells. (B) Percentage of apoptotic cells. Values are representative from triplicate experiments ± SD. *p<0.005, significant result vs. untreated control cells. EA, ellagic acid; FACS, fluorescence-activated cell sorting; Cntrl, control.