Background
Prostate cancer is the most common malignancy in American men and the second leading cause of deaths from cancer [
1]. In the early stage, prostate cancer usually grows slowly and remains confined to the gland, initially producing few or no symptoms. As the cancer advances, it can, however, spread beyond the prostate into the surrounding tissues and to other areas, such as the bones, lungs, and liver. Therefore, symptoms often appear after the cancer has processed to an advanced stage.
The treatment options for patients with prostate cancer include surgery, radiation therapy, hormonal therapy, chemotherapy, cryotherapy, and combinations of some of these treatments. At the early stage, surgery, radiation therapy, and hormonal therapy are the preferred treatments. As the cancer processes, chemotherapy and cryotherapy become the preferred treatments. One of the most common drug classes for chemotherapy treatments for prostate cancer is the taxanes, which include the first-generation drug paclitaxel (Taxol, a trademark of Bristol-Myers Squibb) [
2,
3]. Because taxanes often cause significant negative side effects, newly developed drugs are valuable.
Recently, non-traditional treatments such as herbs and dietary supplements have been considered as alternative medicines. Nan-Chai-Hu (Chai Hu of the South), the root of
Bupleurum scorzonerifolium, is an important Chinese herb in the treatment of influenza, fever, malaria, cancer, and menstrual disorders in China, Japan, and many other parts of Asia. We previously showed that the crude acetone extract of
B. scorzonerifolium (BS-AE) causes cell cycle arrest at the G2/M phase and apoptosis in the human lung carcinoma cell line A549 [
4‐
6]. After the acetone extract fraction was further purified, a novel lignan, isochaihulactone, which has antitumor activity against A549 cells
in vitro and
in vivo, was identified [
7]. Isochaihulactone induces G2/M arrest and apoptosis in cancer cells. This compound can also be isolated from
Bursera microphylla (Burseraceae) and shows antitumor effects [
8].
Here we describe the anti-tumor activity of isochaihulactone, which causes cell cycle arrest at G2/M phase and cell death in LNCaP cells. We provided evidence that the disruption of the cell cycle at G2/M phase and the activation of phospho-Bcl-2 and caspase-3 are important in isochaihulactone-induced cell death. Recently, we found isochaihulactone induces growth inhibition and apoptosis in A549 cells by activating early growth response gene 1 (EGR-1) and nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1) through an extracellular signal-regulated kinase 1/2 (ERK 1/2)-dependent pathway, but PI3K signaling is not involved [
9]. Here we show that isochaihulactone induced growth inhibition and cell death in prostate cancer cells by activating EGR-1 and NAG-1 through JNK-dependent pathway and that did not involve activation of ERK signaling. Also, isochaihulactone-induced cell death can be restored by siNAG-1 siRNA transfection. Our findings indicate that isochaihulactone is a potential antitumor compound for prostate cancer therapy.
Methods
Cells and cell culture
LNCaP human prostate cells, obtained from ATCC (American Type Culture Collection, Manassas, VA), were cultured in RPMI 1640 medium with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 U/ml streptomycin, 1% sodium pyruvate, 2 mM L-glutamine (all of these reagents are from Invitrogen, Carlsbad, CA) at 37°C in a humidified atmosphere with 5% CO2. Cells were plated in 6-well plates at a seeding density of approximately 2 × 105 cells/well in the presence or absence of isochaihulactone (20 μM).
Chemicals and reagents
Bupleurum scorzonerifolium roots were supplied by Chung-Yuan Co. (Taipei, Taiwan). The plant was identified and deposited at National Defense Medicinal Center (NDMCP No. 900801). Isochaihulactone (4-benzo[
1,
3]dioxol-5-ylmethyl-3-(3,4,5-trimethoxyl-benzylidene)-dihydro-furan-2-one) was prepared as described previously [
7]. RPMI 1640 medium, fetal bovine serum (FBS), penicillin, streptomycin, L-glutamine, sodium pyruvate, trypsin/EDTA were purchased from Invitrogen. The RNA isolation kit was purchased from QIAGEN (Valencia, CA). Dimethyl sulfoxide (DMSO), 3-(4,5-dimethyl thizol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), paclitaxel, and horseradish peroxidase-conjugated secondary antibodies were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The ERK1/2 kinase inhibitor PD98059 and the JNK inhibitor SP600125 were purchased from R&D Systems (Minneapolis, MN). The p38 inhibitor SB203580 and the PI3K/AKT inhibitor LY294002 were purchased from Calbiochem (San Diego, CA). The annexin-V-FLUOS Staining Kit was from Roche Molecular Biochemicals (Mannheim, Germany). Polyvinyldenefluoride (PVDF) membranes, BSA protein assay kit and western blot chemiluminescence reagent were purchased from Amersham Biosciences (Arlington Heights, IL).
Western blot analysis
LNCaP cells were lysed on ice with 200 μl of lysis buffer (50 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 5 mM MgCl2, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoridefor, 1 μg/ml pepstatin, and 50 μg/ml leupeptin) and centrifuged at 13,000 × g at 4°C for 5 min. The protein concentrations in the supernatants were quantified using a BSA Protein Assay Kit. Electrophoresis was performed on a NuPAGE Bis-Tris Electrophoresis System using 30 μg of reduced protein extract per lane. Resolved proteins were then transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk for 1 h at room temperature and probed with appropriately dilution of primary antibodies at 4°C overnight: NAG-1/PTGF-b (1:1000, Upstate Biotechnology, Lake Placid, NY), phospho-ERK1/2 (1:2000), ERK1/2 (1:2000), phospho-p38 (1:1000), p38 (1:1000), phospho-JNK1/2 (1:1000), JNK1/2 (1:1000), cyclin B1 (1:1000), cdc2 (1:1000), cleaved Caspase-3 (Asp175) (1:1000), cleaved Caspase-8 (1:1000), cleaved Caspase-9 (Asp330) (1:1000), PARP (46D11) (1:1000), phospho-Bcl-2 (ser70) (1:1000), p53 (1:1000), were purchased from Cell Signaling Technology, Inc. (Danvers, MA). After the PVDF membrane was washed three times with TBS/0.2% Tween 20 at room temperature, it was incubated with appropriate secondary antibody (goat anti-mouse or anti-rabbit, 1:10000, Sigma Chemical, St. Louis, MO) labeled with horseradish peroxidase for 1 h at room temperature. All proteins were detected using Western Lightning™ Chemiluminescence Reagent Plus (Amersham Biosciences, Arlington Heights, IL) and quantified with densitometers.
Growth inhibition assay
The viability of the cells after treatment with various chemicals was evaluated using MTT assay preformed in triplicate. Briefly, the LNCaP cells (2 × 105/well) were incubated in 6-well plates containing 2 ml of serum-containing medium. Cells were allowed to adhere for 18-24 h and then were washed with phosphate-buffered saline (PBS). Solutions were always prepared fresh by dissolving 0.2% DMSO (control) or drugs in culture medium before their addition to LNCaP cells. For inhibitor treatment experiments, cells were pre-incubated for 1 h with 25 μM and 50 μM ERK1/2 kinase inhibitor PD98059, 10 μM and 20 μM p38k inhibitor SB203580, or 10 μM and 20 μM JNK inhibitor SP600125 and then were treated with 20 μM isochaihulactone for 24 h. The drug-containing medium was removed, cells were washed with PBS, and culture medium containing 300 μg/ml MTT was added for 1 h at 37°C. After the medium were removed, 2 ml of DMSO were added to each well. Absorbance at 570 nm of the maximum was detected by a PowerWave × Microplate ELISA Reader (Bio-Tek Instruments, Winooski, VT). The absorbance for DMSO-treated cells was considered as 100%. The results were determined by three independent experiments.
Cell cycle analysis
The cell cycle was determined by flow cytometry following DNA staining to reveal the total amount of DNA. Approximately 5 × 105 of LNCaP cells were incubated with 20 μM isochaihulactone for the indicated time. Cells were harvested with trypsin/EDTA, collected, washed with PBS, fixed with cold 100% ethanol overnight, and then stained with a solution containing 45 mg/ml PI, 10 mg/ml RNase A, and 0.1% Triton X-100 for 1 h in the dark. The cells were then passed through FACScan flow cytometer (equipped with a 488-nm argon laser) to measure the DNA content. The data were obtained and analyzed with CellQuest 3.0.1 (Becton Dickinson, Franklin Lakes, NJ) and ModFitLT V2.0 software.
Transfection with siRNA
NAG-1 siRNA was designed by siGENOME SMARTpool duplex siRNA and purchased from Dharmacon RNAi Technologies (Chicago, IL). LNCaP cells at 50 to 60% confluence were transfected with NAG-1 siRNA (10-50 nM) for 48 h using RNAifect Transfection Reagent (QIAGEN). The medium was removed, and the cells were treated with isochaihulactone or vehicle for up to 48 h. Proteins were then isolated for western blotting, or cells were collected for the MTT assay.
Immunocytochemistry
LNCaP cells cultured on glass slides were treated with 20 μM isochaihulactone for 48 h prior to fixation with cold 4% paraformaldehyde. The fixed cells were washed twice in PBS, and incubated in cold permeabilization solution (0.3% Triton X-100 + 0.1% sodium citrate). After endogenous peroxidase activity was inactivated with 3% H2O2, the cells were washed with PBS and incubated with an anti-cleaved caspase-3 at 4°C overnight. The cells were washed with PBS three times and then incubated with FITC-conjugated secondary antibody 1 h at room temperature. The cells were then washed with PBS three times and stained with 300 nM DAPI for 10 min. Images were obtained with a confocal microscope (Carl Zeiss, Oberkochen, Germany).
TUNEL assay
LNCaP cells were cultured in the presence or absence of isochaihulactone (20 μM) for 60 h and then examined for apoptosis with TUNEL assay (In Situ Cell Death Detection Kit, Roche).
Statistical analysis
The data are shown as mean ± S.D. Statistical differences were analyzed using the Student's t-test for normally distributed values and by nonparametric Mann-Whitney U-test for values with a non-normal distribution. Values of P < 0.05 were considered significant.
Discussion
In our previous study, we demonstrated that isochaihulactone was efficacious against various models of human solid tumors but not prostate cancer [
7]. We also have shown recently that isochaihulactone triggers an apoptotic pathway in human A549 lung cancer cells that occurs via the ERK1/2 and NAG-1 pathway [
9]. To clarify the mechanisms of isochaihulactone-induced tumor apoptosis between different types of cancer cells, we further investigated the antitumor potential and mechanisms of isochaihulactone action in human prostate cancer cells. Three human prostate cell lines were used to test the cytotoxicity of isochaihulactone, only the LNCaP prostate cancer cells showed sensitivity to isochaihulactone treatment. This phenomenon might be important to the antitumor potential of isochaihulactone and is discussed later.
In this study, we demonstrated that isochaihulactone apparently induced G2/M cell cycle arrest and cell death in LNCaP cells. The tumor suppressor protein p53 plays a role in the molecular response to DNA damage and cell cycle arrest. The cyclin-dependent kinase inhibitor p21 also helps to maintain G2/M cell cycle arrest by inactivating the cyclin B1/cdc2 complex, disrupting the interaction between proliferating cell nuclear antigen and cdc25c [
11]. Our result showed that increased levels of p53 and p21 proteins were expressed in LNCaP cells in response to treatment with isochaihulactone (Figure
2B). The transition from G2 phase to mitosis is triggered by the cdc25c-mediated activation of the cyclin B1/cdc2 complex. Cyclin B1/cdc2 activation is triggered when cdc25c dephosphorylates Thr15 [
12,
13]. In our study, isochaihulactone-mediated LNCaP cell cycle arrest at G2/M phase (Figure
2B) was accompanied by decreased expression of cyclin B1 and cdc2 kinase. The decrease in the levels of cdc2 may be due to the decrease in cdc25 activation by phosphorylation, leading to subsequent G2 arrest (Figure
2B).
Activation of aspartate-specific cysteine protease (caspase) represents a crucial step in the induction of drug-induced apoptosis, and cleavage of PARP by caspase-3 is considered to be one of the hallmarks of apoptosis [
14]. Isochaihulactone-induced caspase 3 cleavage was observed by immunocytochemistry (Figure
3B), and late-stage apoptosis was revealed by TUNEL staining (Figure
3D). Furthermore, isochaihulactone inhibited Bcl-2 expression, induced caspase-9 and caspase-3 cleavage, and induced PARP activation were also observed (Figure
3E). It is interesting to note that isochaihulactone-induced Bcl-2 phosphorylation, caspase-9 cleavage, and PARP cleavage were observed at nearly the same time point, suggesting that the isochaihulactone-induced Bcl-2 phosphorylation is related apoptosis (Figure
3E). Recent reports have revealed the involvement of JNK-mediated Bcl-2 phosphorylation and degradation, and also the activation of caspase-9 in the apoptosis of both the androgen-dependent and -independent human prostate cancer cells [
15]. Bcl-2 and Bcl-XL inhibit apoptosis by regulating the mitochondrial membrane potential, whereas cytochrome
c release is required for activation of caspase-9 and subsequent activation of caspase-3 [
16]. Thus, increased levels of Bcl-2 phosphorylation, caspase-9 and -3 activation appeared to correlate with mitochondrial apoptosis in isochaihulactone-induced LNCaP cell death.
Many microtubule-destabilizing agents are activators of caspase-9, a major key player in mitochondrial apoptotic pathway [
17,
18]. Microtubule depolymerization agents arrest the cell cycle in G2/M phase by acting through several types of kinases, which lead to phosphorylation cascades, activation of the cyclin B1/cdc2 complex, and the phosphorylation of Bcl-2 [
19]. The MAPK inhibitor PD98059 has been shown to partially inhibit isochaihulactone-induced cdc2 phosphorylation, causing G2/M arrest in A549 cells. The activation of NAG-1 expression via ERK1/2 pathway is involved in isochaihulactone-induced G2/M arrest in A549 cells [
7,
9]. To determine which MAPK family member is involved in the major signaling pathway for isochaihulactone-mediated cell growth inhibition, MAPK inhibitors were used to study the growth inhibition induced by isochaihulactone in LNCaP cells. Only JNK1/2 inhibitor SP600125 significantly decreased the growth inhibition induced by isochaihulactone (Figure
4A), and neither the p38 inhibitor SB203580 nor the ERK1/2 inhibitor PD98059 reversed isochaihulactone-induced growth inhibition. Phosphorylation of JNK kinase was also observed with western blot analysis after isochaihulactone treatment (Figure
4B). In cell cycle analysis, pre-treatment of JNK1/2 inhibitor SP600125 significantly reduces sub-G1 population (Figure
4C). These data suggest that JNK1/2 signaling pathway is involved in isochaihulactone-induce cell death.
Increased NAG-1 expression results in the induction of apoptosis in several cancer cell lines [
20,
21]. NAG-1 is induced not only by NSAIDs but also by several anti-tumorigenic compounds including dietary compounds, peroxisome proliferator-activated receptor-γ ligands, phytochemicals [
16‐
18], as well as resveratrol, genistein, diallyldisulfide, 5F203, and retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN) [
22‐
24]. NAG-1 appears to be a key downstream target of EGR-1[
9].
In our previously studies, we confirmed the antitumor effect of isochaihulactone [
7], and the inhibition of tumor growth that was attributable to NAG-1 protein expression in a nude mice xenograft model [
9]. Thus, NAG-1 is an essential factor in the antitumor activity of isochaihulactone. Our current results show that isochaihulactone induced EGR-1 and NAG-1 protein expression in LNCaP cells in a time-dependent manner (Figure
5A). Furthermore, only the JNK1/2 inhibitor SP600125 reduced isochaihulactone-induced NAG-1 protein expression (Figure
5B). These data support that isochaihulactone-induced JNK1/2 activity is critical in regulating NAG-1 expression. In addition, we further confirmed by using siRNA approach that NAG-1 expression has an apoptosis-promoting effect (Figure
5D).
In summary, we found that isochaihulactone increased NAG-1 expression, suggesting that the antitumor effect of isochaihulactone is mediated via this tumor suppressor protein. NAG-1 mRNA is highly expressed in the human prostate epithelium [
25], suggesting its role in prostate homeostasis. Despite this, NAG-1 negatively affects LNCaP cell survival [
26], and is overexpressed in many tumors including prostate cancer [
27,
28]. NAG-1 may be like other members of the TGF-β superfamily, acting as a tumor suppressor in the early stages but becoming pro-tumorigenic during the later stages of tumor progression. The effects of NAG-1 appear to be ambiguous, and under different conditions, NAG-1 exhibits either tumorigenic or anti-tumorigenic activity [
24]. Epidemiological studies have shown that patients who use NSAIDs for 10-15 years have a reduced risk of developing cancer [
29]. NSAIDs inhibit cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). Several studies have suggested that the tumorigenic or anti-tumorigenic activity of NAG-1 may be due to the interaction of NAG-1 and cyclooxygenase [
21,
30,
31].
Recent study has revealed a new pathway that Retinoblastoma (RB; encoded by
RB1) depletion induced unchecked androgen receptor (AR) activity that underpinned therapeutic bypass and tumor progression [
32]. The hypo-phosphorylation form of RB suppresses E2F1-mediated transcriptional activation and induces cell cycle arrest. Loss of
RB1 was observed in most of the castrate-resistant prostate cancer (CRPC), and
AR as a gene under the control of E2F1, which in turn is stringently regulated by RB. Since hypo-phosphorylation of RB was observed after isochaihulactone treatment in LNCaP cells (data not shown), this might explain why LNCaP is more sensitive to isochaihulactone than the other two androgen-independent prostate cancer cell lines. However, the exact mechanism of these differences needs to be extensively investigated.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SCC carried out the most of the experiments and drafted the manuscript. MJW, HHY, YLC, SZL participated in the design and coordination of the study. SPC carried out the statistical analysis. SYH carried out the immunostaining. HJH and CYP conceived of the study, participated in its design and coordination, and drafted the manuscript. All authors read and approved the final manuscript.