Basic Research

Tanshinone IIA inhibits human prostate cancer cells growth by induction of endoplasmic reticulum stress in vitro and in vivo

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Abstract

BACKGROUND:

Tanshinone IIA (Tan-IIA) is one of the major lipophilic components isolated from the root of Salviae Miltiorrhizae Radix. We explored the mechanisms of cell death induced by Tan-IIA treatment in prostate cancer cells in vitro and in vivo.

METHODS:

Cells were treated with Tan-IIA and growth inhibition was assessed. Cell cycle profiles after Tan-IIA treatment were determined by flow cytometry. Expression levels of cell cycle regulatory proteins and apoptosis-related proteins were determined after Tan-IIA treatment. Expression levels of endoplasmic reticulum (ER) stress-regulated genes were determined to investigate their role in Tan-IIA-induced cell death. GADD153 expression was knocked down by small interfering RNA (siRNA) transfection. Rate of cell death and proliferation was obtained by 3-(4,5-dimethyl thizol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Antitumor activity of Tan-IIA was performed in LNCaP xenograft model.

RESULTS:

Our results showed that Tan-IIA caused prostate cancer cell death in a dose-dependent manner, and cell cycle arrest at G0/G1 phase was noted, in LNCaP cells. The G0/G1 phase arrest correlated with increase levels of CDK inhibitors (p16, p21 and p27) and decrease of the checkpoint proteins. Tan-IIA also induced ER stress in prostate cancer cells: activation and nuclear translocation of GADD153/CCAAT/enhancer-binding protein-homologous protein (CHOP) were identified, and increased expression of the downstream molecules GRP78/BiP, inositol-requiring protein-1α and GADD153/CHOP were evidenced. Blockage of GADD153/CHOP expression by siRNA reduced Tan-IIA-induced cell death in LNCaP cells. Tan-IIA also suppressed LNCaP xenograft tumor growth, causing 86.4% reduction in tumor volume after 13 days of treatment.

CONCLUSIONS:

Our findings suggest that Tan-IIA causes G0/G1 cell cycle arrest in LNCaP cells and its cytotoxicity is mediated at least partly by ER stress induction. These data provide evidence supporting Tan-IIA as a potential anticancer agent by inducing ER stress in prostate cancer.

Introduction

Prostate cancer is the most common malignancy and the second leading cause of deaths in men in the western world.1 The treatment options for prostate cancer patients include radical prostatectomy, radiation, hormonal therapy, chemotherapy and combinations of some of these treatments. The taxane group (for example, paclitaxel and docetaxel) is a classification of chemotherapy drugs used in advanced castrate-resistant prostate cancer.2, 3 However, increased concentrations of cytotoxic drugs may fail to improve the response to chemotherapy and lead to resistance to apoptosis in prostate cancer cells.

Recently, alternative medicines for cancer therapy using herbs and dietary supplements have been considered. Tanshinone IIA (Figure 1a, Tan-IIA; C19H18O3), extracted from Salviae Miltiorrhizae Radix (also called Danshen in Chinese), has been reported to exert diverse biological properties including anti-inflammation,4, 5 antioxidation6, 7 and antiangiogenesis.8 Extensive investigations have also been conducted to explore its potential as an antitumor agent in leukemia,9, 10 breast cancer,11 colon cancer12 and hepatocellular carcinoma cells.13, 14 These studies demonstrated that Tan-IIA could induce cancer cell cycle arrest at G0/G1 phase via activation of p53 signaling and inhibition of androgen receptor (AR) signaling.15 Won et al.16 also reported that Tan-IIA induced mitochondria-dependent apoptosis by inhibiting the phosphoinositide 3-kinase/protein kinase B pathway in LNCaP prostate cancer cells. These findings indicate that Tan-IIA may have the potential to become a new anticancer compound.

Figure 1
figure1

Effects of tanshinone IIA (Tan-IIA) on the viability human prostate cancer cells. (a) Chemical structure of Tan-IIA, C19H18O3, molecular weight: 294.34. (b) LNCaP cells were treated with increasing concentration of Tan-IIA (0.313–5 μg ml−1) for 24 (black) and 48 h (gray), respectively, and analyzed with 3-(4,5-dimethyl thizol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. (c) PC-3 cells were treated with increasing concentration of Tan-IIA (0.313–5 μg ml−1) for 24 (black) and 48 h (gray), respectively, and analyzed with MTT assay. Data are presented as means±s.d. from three different experiments. **P<0.01 versus vehicle.

The endoplasmic reticulum (ER) is an organelle found only in eukaryotic cells that forms an interconnected network of tubules, vesicles and cisternae. ER is responsible for the synthesis of proteins and lipids, metabolism of carbohydrates, regulation of calcium concentration and detoxification of drugs and poisons in eukaryotic cells. However, oxidative stress, calcium dysregulation, glucose deprivation and viral infection can lead to ER stress, a state of accumulation of unfolded protein in the ER lumen.17 ER stress has emerged as a potential cause of cell damage/death in hypoxia/ischemia, insulin resistance and other disorders. Specific apoptotic pathways to eliminate severely damaged cells will be activated when the stress cannot be resolved.18, 19, 20 Upregulation of glucose-regulated protein GRP78/BiP has been widely recognized as a marker for ER stress and the initiation of ER stress. ER stress is initiated by three distinct ER membrane proteins: protein kinase RNA-like ER kinase, inositol-requiring protein-1α (IRE1-α) and activating transcription factor-6 (ATF6).21 The transcription factor CCAAT/enhancer-binding protein-homologous protein (CHOP; also known as DDIT3/GADD153) operates at the convergence of the protein kinase RNA-like ER kinase, IRE1-α and ATF6 pathways.22, 23 Overexpression of GADD153/CHOP has an important role in the ER stress-induced apoptosis.24 Furthermore, oxidation of the ER lumen is induced by ER oxidase 1-like α, a downstream transcriptional target of GADD153/CHOP.25

Our previous studies have demonstrated that ER stress has an important role in the Tan-IIA-mediated cell death.26, 27 Based on these observations, we hypothesize that Tan-IIA may also inhibit prostate cancer cell growth, at least in part, through ER stress-induced apoptosis. Here, we investigated the effects of Tan-IIA on growth inhibition and ER stress-associated apoptosis in prostate cancer cells.

Materials and methods

Cell cultures and chemicals

The human prostate cancer cell lines LNCaP (androgen-sensitive) and PC-3 (androgen-insensitive) were purchased from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U ml−1 penicillin and 100 U ml−1 streptomycin, 1% sodium pyruvate and 2 mM L-glutamine (all from Invitrogen, Carlsbad, CA, USA) at 37 °C in a humidified atmosphere with 5% CO2. Tan-IIA (C19H18O3, >96% HPLC) was purchased from Herbasin (Shenyang, China). Dimethyl sulfoxide (DMSO), 3-(4,5-dimethyl thizol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and horseradish peroxidase-conjugated secondary antibodies were purchased from Sigma Chemical (St Louis, MO, USA). Polyvinyldenefluoride membranes, bovine serum albumin protein assay kit and western blot chemiluminescence reagent were purchased from Amersham Biosciences (Arlington Heights, IL, USA).

Western blot analysis

Five hundred thousand cells per 6-cm plate were lysed on ice with 200 μl 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−1 pepstatin and 50 μg ml−1 leupeptin) and centrifuged at 13 000 g at 4 °C for 5 min. The protein concentration in the supernatants was quantified using a bovine serum albumin Protein Assay Kit. Electrophoresis was performed on a NuPAGE Bis-Tris Electrophoresis System using 20 μg of reduced protein extract per lane. Resolved proteins were transferred to polyvinyldenefluoride membranes, blocked with 5% skim milk for 1 h at room temperature, finally probed with the following primary antibodies at 4 °C overnight: GADD153/CHOP, Bip, calnexin, PDI, IRE1-α, cyclin D1, CDk2, phospho-Rb (Ser807/811), cleaved caspase-3 (Asp175), caspase-9, PARP, Bax, p16, p21, p27 (all from Cell Signaling Technology, Danvers, MA, USA) and ATF6 (Abcam, Cambridge, MA, USA). After the polyvinyldenefluoride membrane was washed three times with TBS (50 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 5 mM MgCl2)/0.2% Tween-20 at room temperature, it was incubated with appropriate secondary antibody labeled with horseradish peroxidase (goat anti-mouse or anti-rabbit, 1:10 000, Sigma Chemical) for 1 h at room temperature. All resolved protein bands were detected using Western Lightning Chemiluminescence Reagent Plus (Amersham Biosciences).

Growth inhibition assay

The viability of the cells after treatment with Tan-IIA was evaluated using MTT assay preformed in triplicate. Briefly, cells (2 × 105 per well) were incubated in 6-well plates containing 2 ml of serum-containing medium. Cells were allowed to adhere for 18–24 h and were washed with phosphate-buffered saline (PBS). Solutions were always prepared fresh by dissolving 0.1% DMSO (control) or Tan-IIA in culture medium before their addition to cells. The Tan-IIA-containing medium was removed after treatment for 48 h, cells were washed with PBS, and culture medium containing 300 μg ml−1 MTT was added for 1 h at 37 °C. After the MTT medium was removed, 2 ml of DMSO were added to each well. Absorbance at 570 nm was detected by a PowerWave X Microplate ELISA Reader (Bio-Tek Instruments, Winooski, VT, USA). The absorbance for DMSO-treated cells was considered as 100%.

Cell cycle analysis

The cell cycle was determined by flow cytometry using DNA staining dye to reveal the total amount of DNA. Approximately 5 × 105 of LNCaP cells were incubated with 2.5 μg ml−1 Tan-IIA for indicated times. Cells were harvested with trypsin/EDTA, collected, washed with PBS, fixed with cold 100% ethanol overnight and then stained with a solution containing 20 μg ml−1 propidium iodide, 0.2 mg ml−1 RNase A and 0.1% Triton X-100 for 30 min in the dark. The cells were then analyzed with 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, USA) and ModFit LT V2.0 software (Verity Software House, Topsham, ME, USA).

Small interfering RNA (siRNA) transfection

GADD153/CHOP and IRE1-α siRNA were designed by siGENOME ON-TARGET plus SMARTpool siRNA purchased from Dhamarcon RNAi Technologies. GADD153/CHOP (DDIT3) target sequences are: 5′-IndexTermGGUAUGAGGACCUGCAAGA-3′, 5′-IndexTermCACCAAGCAUGAACAAUUG-3′, 5′-IndexTermGGAAACAGAGUGGUCAUUC-3′ and 5′-IndexTermCAGCUGAGUCAUUGCCUUU-3′. IRE1-α(ERN1) target sequences are: 5′-IndexTermCUACCCAAACAUCGGGAAA-3′, 5′-IndexTermCUCCAGAGAUGCUGAGCGA-3′, 5′-IndexTermAUAAUGAAGGCCUGACGAA-3′ and 5′-IndexTermGUCCAGCUGUUGCGAGAAU-3′. Individual siRNA for GADD153/CHOP (DDIT3) sequences: #6: 5′-IndexTermGGUAUGAGGACCUGCAAGA-3′ and #7: 5′-IndexTermCACCAAGCAUGAACAAUUG-3′. Non-targeting control sequences were not provided. LNCaP cells at 50–60% confluence were transfected with siRNA (20 or 50 nM) using the RNAifect Transfection Reagent (QIAGEN, Taiwan) according to the manufacturer’s protocol. Cells were cultured for 48 h, and then treated with Tan-IIA or vehicle for an additional 24 or 48 h. Protein were then isolated for western blotting, or cells were collected for the MTT assay.

TdT-mediated dUTP nick end-labeling (TUNEL) assay

Cells were cultured in the presence or absence of Tan-IIA (2.5 μg ml−1 for LNCaP, 5 μg ml−1 Tan-IIA for PC-3) for 48 h and then examined for apoptosis with TUNEL assay (In Situ Cell Death Detection kit, Roche Diagnostics, Taiwan).

Immuocytochemistry

Cells cultured on glass slides were treated with Tan-IIA (2.5 μg ml−1 for LNCaP, 5 μg ml−1 for PC-3) for 12 h before 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-GADD153 at 4 °C overnight. The cells were washed with PBS three times and then incubated with FITC-conjugated secondary antibody for 1 h at room temperature. The cells were then washed with PBS three times and stained with 300 nM 4′-6-diamidino-2-phenylindole for 10 min. Images were obtained with the confocal microscope (Carl Zeiss, Oberkochen, Germany).

Animal studies

Ethics statement: The animal use protocol listed below has been reviewed and approved by Institutional Animal Care and Use Committee, Buddhist Tzu Chi General Hospital, approval No: 101–06.

To examine the antitumor effects of Tan-IIA in vivo, the LNCaP human prostate cancer cells were used in male NOD-SCID mice experiments (8–10 weeks). The mice were purchased from the National Laboratory Animal Center (Taipei, Taiwan). All procedures were performed in compliance with the standard operating procedures of the Tzu Chi University Laboratory Animal Center (Hualien, Taiwan). All experiments were carried out using 6- to 8-week-old mice weighing 18–22 g. The animals were subcutaneously implanted with 5 × 105 LNCaP cells into the back of mice. When the tumor reached 80–150 mm3 in volume, animals were divided randomly into control and test groups consisting of six mice per group (day 0). Subcutaneous injection of corn oil (control group), 60 or 90 mg kg−1 of Tan-IIA (treatment groups) was given for every 2 days till the end of experiments. Tan-IIA was dissolved in corn oil. The injection sites were >1.5 cm from the tumors. Mice were weighed three times a week up to day 13. The tumor volume was also determined by measurement of the length (L) and width (W) of the tumor. The tumor volume at day n (TVn) was calculated as TV (mm3)=(L × W2)/2. The relative tumor volume at day n (RTVn) versus day 0 was expressed according to the following formula: RTVn=TVn/TV0. Tumor regression (T/C (%)) in treated versus control mice was calculated using: T/C (%)=(mean RTV of treated group)/(mean RTV of control group) × 100. Xenograft tumors as well as other vital organs of treated and control mice were harvested and fixed in 4% formalin, embedded in paraffin and cut into 4-μm sections for histological analysis.

Statistical analysis

All 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. Significant differences between groups were evaluated using analysis of variance with Games-Howell test as post-hoc test. P<0.05 was considered to be significant.

Results

Tan-IIA inhibited proliferation in prostate cancer cells

To determine the cytotoxicity effect and the optimize dosage of Tan-IIA in prostate cancer cell lines, cells were treated with increasing concentration of Tan-IIA for 24 and 48 h, and subsequently evaluated by the MTT assay. As shown in Figures 1b and c, Tan-IIA significantly decreased the viability of LNCaP and PC-3 cells in dose- and time-dependent manners. Treatment of LNCaP cells with 2.5 μg ml−1 Tan-IIA for 24 and 48 h resulted in 48.4% and 10.8% cell survival, respectively (Figure 1b). Treatment of PC-3 cells with 5 μg ml−1 Tan-IIA for 24 and 48 h resulted in 61.8% and 38.9% cell survival, respectively (Figure 1c). The IC50 at 24 h of Tan-IIA treatment in LNCaP and PC-3 cells were 2.54 and 5.77 μg ml−1, respectively. Based on these data, we used 2.5 μg ml−1 (LNCaP) and 5 μg ml−1 (PC-3) Tan-IIA for subsequent studies.

Tan-IIA induced cell cycle arrest at G0/G1 phase and changed the expression levels of G0/G1 regulatory proteins

We next examined the effects of Tan-IIA on cell cycle progression. Flow cytometric analysis showed that Tan-IIA treatment resulted in a time-dependent accumulation of cells in G0/G1 phase (Figure 2a). Quantification of proliferating vehicle-treated LNCaP cells showed 67.4% of cells were in the G0/G1 phase, 24% in the S phase and 8.9% in the G2/M phase of cell cycle 12 h after plating. Treatment of LNCaP cells with 2.5 μg ml−1 Tan-IIA for 12 h increased the percentage of cells in the G0/G1 phase to 75.5% and reduced the percentage of the cells in the S and G2/M phases to 15.1% and 9.3%, respectively. The percentages of cells in G0/G1 phase increased to 83.9% (24 h) and 88.8% (48 h), respectively, after 2.5 μg ml−1 Tan-IIA treatment.

Figure 2
figure2

Tanshinone IIA (Tan-IIA) induced G0/G1 phase arrest and changed the expression profiles of G0/G1 regulatory proteins. (a) Tan-IIA induced cell cycle G0/G1 arrest in LNCaP cells. LNCaP cells were seeded at 5 × 105 per 6-cm plate and treated with 2.5 μg ml−1 Tan-IIA for 12–48 h. Data are presented as means±s.d. from three different experiments. **P<0.01. (b) Western blot analysis of cyclin D1, CDK2, p16, p21, p27 and phospho-Rb (ser807/811) was performed in LNCaP cells treated with 2.5 μg ml−1 of Tan-IIA for 0–48 h. β-Actin was used as an internal control.

To determine the molecular mechanisms underlying the G0/G1 cell cycle arrest in LNCaP cells induced by Tan-IIA, we examined the expression of certain G0/G1 regulatory proteins in LNCaP cells treated with 2.5 μg ml−1 Tan-IIA. We first examined the level of cyclin D1, the main cyclin controlling the G0/G1 checkpoint. Tan-IIA treatment led to rapid reduction of cyclin D1 protein level (Figure 2b). Upregulation of G0/G1 cell cycle regulatory proteins, such as p16, p21 and p27, and downregulation of CDK2 were also observed in a time-dependent manner (Figure 2b). We also observed a rapid reduction of Rb phosphorylation level (Figure 2b) indicating the compromised activation of the CDK4/6.

Tan-IIA induced mitochondrial-mediated apoptosis in prostate cancer cells

To evaluate the role of apoptosis in Tan-IIA-induced LNCaP cell death, sub-G1 fraction, western blotting and TUNEL staining were performed. In flow cytometry analyses (Figures 3a–c), Tan-IIA-induced sub-G1 population significantly increased to 10.8% and 35.8%, respectively, at 24 and 48 h (Figure 3d).

Figure 3
figure3

Tanshinone IIA (Tan-IIA) induced apoptotic cell death in prostate cancer cells. Cell cycle profiles of LNCaP cells treated with 0.1% dimethyl sulfoxide (a), 2.5 μg ml−1 of Tan-IIA for 24 h (b) or 48 h (c), respectively. Cell cycle analysis was done as described in Materials and methods. (d) Tan-IIA-induced sub-G1 population significantly increased at 48 h (Bar C, 35.8%) and 24 h (Bar B, 10.8%) as compared with the control (0.17%). The data represent the means±s.d. from three independent experiments. **P<0.01 versus vehicle.

TUNEL staining at 48 h after Tan-IIA treatment (2.5 μg ml−1 for LNCaP, 5 μg ml−1 for PC-3) also revealed increased number of apoptotic cells in both LNCaP and PC-3 cells (FITC-positive cells in Figures 4b and d, respectively). Activation of caspase family proteins and cleavage of PARP are crucial events for apoptosis induction. Among them, caspase-9 and -3 are key cysteine-protease associated with mitochondria-dependent apoptosis. Their involvement in Tan-IIA-induced apoptosis was investigated in LNCaP cells. Cleavages of caspase-9, -3 and PARP increased dose dependently in LNCaP cells treated with Tan-IIA (Figure 4e). Bax expression also increased slightly after Tan-IIA treatment.

Figure 4
figure4

Tanshinone IIA (Tan-IIA) induced mitochondrial-mediated apoptosis in prostate cancer cells. LNCaP and PC-3 cells were incubated in the absence (a and c, respectively) or presence (b and d, respectively) of Tan-IIA (2.5 μg ml−1 for LNCaP, 5 μg ml−1 for PC-3) for 48 h and then subjected to TdT-mediated dUTP nick end-labeling (TUNEL) assay. (e) Western blot analysis of cleavaged caspase 9, cleavaged caspase 3, PARP and Bax was performed in LNCaP cells. β-Actin was used as an internal control.

Tan-IIA induced ER stress in prostate cancer cells

As the elevation of Bax has been implicated in ER stress-induced apoptosis, the induction of ER stress-related genes by Tan-IIA in prostate cancer cells were investigated (Figures 5a and b). Expression of BiP/GRP78 increased, but not calnexin and PDI, after Tan-IIA treatment in LNCaP cells. Upregulation of ER stress transducer IRE1-α was observed but not ATF6 and p-eIF-2α in both prostate cancer cell lines. Tan-IIA-induced IRE1-α expression was evident as early as 3 h after treatment (1.48-folds) and increased till 48 h (2.13-folds) in LNCaP cells (Figure 5a). Increased expression of IRE1-α in PC-3 cells started to occur after 12 h (1.3-folds; Figure 5b). Tan-IIA-induced GADD153/CHOP expression increased 8.9- and 12.1-folds after 48 h treatment in LNCaP and PC-3 cells, respectively (Figures 5a and b). In addition, Tan-IIA dose dependently increased the expressions of BiP, IRE1-α and GADD153/CHOP in LNCaP cells (Figure 5c).

Figure 5
figure5

Tanshinone IIA (Tan-IIA) induced endoplasmic reticulum (ER) stress-related genes expression in prostate cancer cells. (a) Western blot analysis of BiP, calnexin, PDI, ATF6, phospho-eIF2α (Ser51), IRE1-α and GADD153/CHOP was performed in LNCaP cells (a) and PC-3 cells (b). β-Actin was used as an internal control. (c) Western blot analysis of BiP, IRE1-α and GADD153/CHOP was performed in LNCaP cells. β-Actin was used as an internal control.

We further investigated the nuclear translocation of GADD153/CHOP in Tan-IIA-treated prostate cancer cells. Nuclear translocation of GADD153/CHOP indicated the transduction of stress signal into the nucleus and therefore represented a functional activation. At 12 h after Tan-IIA treatment (2.5 μg ml−1 for LNCaP, 5 μg ml−1 for PC-3), GADD153/CHOP was apparently more abundant in the nuclei of Tan-IIA-treated LNCaP and PC-3 cells than that of the controls (Figures 6a and b).

Figure 6
figure6

Nuclear translocation of GADD153/CHOP after tanshinone IIA (Tan-IIA) treatment in prostate cancer cells. (a) LNCaP cells and (b) PC-3 cells were treated with 0.1% dimethyl sulfoxide (DMSO; control) or Tan-IIA (2.5 μg ml−1 for LNCaP, 5 μg ml−1 for PC-3) for 12 h and then fixed and stained with GADD153/CHOP. Fluorescein isothiocyanate (FITC)-labeled secondary antibody was used (green fluorescence), nuclei were stained with 4′-6-diamidino-2-phenylindole (DAPI; blue fluorescence). Images were captured by a confocal laser microscope.

These data revealed Tan-IIA treatment can cause increasing and sustaining expressions of IRE1-α and its downstream target GADD153/CHOP in time- and dose-dependent manners.

The role of ER stress in Tan-IIA-induced cell death

We further used a siRNA approach to determine the role of GADD153/CHOP in Tan-IIA-induced cell death. LNCaP cells were transfected with siRNAs for GADD153/CHOP, with or without post treatment of 2.5 μg ml−1 Tan-IIA for 48 h. Western blot analysis demonstrated that transfection of si-GADD153/CHOP resulted in a suppression of GADD153/CHOP expression induced by Tan-IIA in LNCaP cells, as compared with cells transfected with control scrambled siRNA (Figure 7a). MTT assay showed 12.4 and 15.8% of cell death, which was inhibited by 20 and 50 nM siRNA transfection, respectively, after exposure of cells to 2.5 μg ml−1 Tan-IIA (Figure 7b). These results indicated that Tan-IIA-induced cell death may be involved, at least in part, through the induction of ER stress.

Figure 7
figure7

Tanshinone IIA (Tan-IIA) induced apoptosis via GADD153-dependent endoplasmic reticulum (ER) stress. (a) LNCaP cells were transfected with scramble small interfering RNA (siRNA (#)) or 20, 50 nM GADD153 siRNA, respectively, for 48 h using the RNAifect transfection reagent. Western blot analysis was performed for GADD153 after additional 24 h treatment with 2.5 μg ml−1 Tan-IIA. (b) Tan-IIA-induced anti-proliferative activity was measured with the 3-(4,5-dimethyl thizol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay in LNCaP cells transfected with scramble siRNA (#) or 20, 50 nM GADD153 siRNA for 48 h and then treated with 2.5 μg ml−1 Tan-IIA for 24 h. Data are expressed as means±s.d. from three independent experiments. **P<0.01 versus vehicle.

Tan-IIA inhibits growth of prostate cancer cells in NOD-SCID xenograft model

To evaluate the antitumor activity of Tan-IIA in vivo, LNCaP cell xenograft was established by subcutaneous injection of 5 × 105 cells into the dorsal subcutaneous tissue of NOD-SCID mice. As shown in Figure 8a, the relative tumor volume in mice treated with 60 or 90 mg kg−1 Tan-IIA was smaller than the vehicle-treated control mice on day 13 (52.7%, P=0.101, analysis of variance, and 82.1%, P=0.011, analysis of variance, respectively). Tumors of control and therapeutic groups were removed and recorded (Figure 8b). Tumor weight was significantly decreased 56.7% (60 mg kg−1) and 86.4% (90 mg kg−1) in the Tan-IIA-treated groups as compared with the control group on day 13 (Figure 8c). Besides, upregulation of GADD153/CHOP in the Tan-IIA-treatment tumor was observed by western blot analysis (Figure 8d). Caspase-3 activation was also observed in Tan-IIA-treated tumors (Figure 8d). There were no significant differences of body weight between control and Tan-IIA-treated groups. These results indicated that Tan-IIA-induced prostate cancer cell death is correlated with ER stress in vivo.

Figure 8
figure8

Tanshinone IIA (Tan-IIA) inhibits xenographic growth of prostate cancer cells in NOD-SCID mice. (a) NOD-SCID mice were injected with approximately 5 × 105 LNCaP cells into the dorsal subcutaneous tissue. When the tumor reached 80–150 mm3, LNCaP tumor-bearing mice were administrated s.c. with vehicle control (♦) 60 (▪) or 90 mg kg−1 () Tan-IIA every 2 days. The relative tumor volumes of control and the Tan-IIA-treated groups were evaluated using analysis of variance (ANOVA) with Games-Howell test as post hoc test. P<0.05 was considered to be significant. (b, c) Tumors of control and therapeutic groups were removed and weighted on day 13. A tumor was disappeared from the dorsal subcutaneous part of NOD-SCID mice after Tan-IIA treatment (90 mg kg−1 group). Average tumor weight from the Tan-IIA-treated group was 56.7% and 86.4% (60 and 90 mg kg−1, respectively) smaller than control group. Data were expressed as means±s.d. of tumor weight of the control and the Tan-IIA-treated groups. **P<0.01. (d) Expression of GADD153 and active form caspase-3 in the LNCaP xenograft tumor tissue was upregulated after Tan-IIA administration as compared with control group by western blotting analysis.

Discussion

Several naturally occurring dietary therapeutic compounds including resveratrol, curcumin, genistein, diallyl sulfide and many others have been investigated to induce apoptosis in various cancer cells.28 Tan-IIA possessed strong anti-proliferative effect, apoptosis induction ability and ER stress induction ability in various cancer cell lines in our previous studies.26, 27, 29, 30, 31, 32, 33 However, the role of ER stress in Tan-IIA-induced prostate cancer cell death has never been addressed.

One of the important features of cancer is the loss of cell cycle regulation. Tan-IIA-induced cell cycle arrest was evidenced by downregulation of cyclin D1 and CDK2, and upregulation of p16, p21 and p27. The p16 binds to cdk4/6 to block kinase activity at the mid-G1 phase.34 The p21 (cip1) and p27 (kip1) binds to the cyclin/cdk complex, inhibiting G1 to S phase transition via the reduction of Rb phosphorylation by the cyclin/cdk complex.35 Similar results that Tan-IIA-induced apoptosis and G1 arrest in LNCaP cells also reported by Won et al.15, 16

Regarding the caspase-activation apoptosis pathways, our results demonstrated that mitochondria intrinsic pathway is involved in Tan-IIA-induced apoptosis in LNCaP cells. Tan-IIA-induced caspase-9, -3 and PARP cleavages occurred in a dose-dependent manner in LNCaP cells. The late-stage apoptosis was also revealed by TUNEL staining. These data are consistent to previous finding by Won et al.16 Similarly, Tan-IIA has been reported to induce apoptosis via mitochondria pathway in A549 lung cancer and Colo-205 colon cancer cells in our previous studies.32, 36

As ER stress has been reported to responsible for the death of various cancer cells, its role is investigated in prostate cancer cells. Although previous reported data showed Tan-IIA induced mitochondria-dependent apoptosis in LNCaP cells,16 we further demonstrated that ER stress may also involve. To clarify the role of ER stress in Tan-IIA-induced prostate cancer cells death, the ER stress-related pathways were examined by western blot. The ER transducer proteins IRE1-α, ATF6 and p-eIF2α constitute the core regulator of unfolded protein response and only the activation of IRE1-α was observed. Increasing and sustaining expression of IRE1-α and its downstream target GADD153/CHOP after Tan-IIA treatment occurred in time- and dose-dependent manners. Our previous studies showed that activation of GADD153/CHOP also involved in Tan-IIA-induced cell death in Hep-J5 cell lines.27

On the other hand, the activation of Bax may implicate the possibility of GADD153/CHOP-mediated apoptosis: GADD153/CHOP-mediated Bax expression in ER stress was observed in cardiomyocyte apoptosis.37 Bax also modulates unfolded protein response by a direct interaction with IRE1-α, and is an important link between IRE1-α-GADD153/CHOP-mediated apoptosis. The GADD153/CHOP siRNA transfection suppressed Tan-IIA-induced cell death in LNCaP cells. The IRE1-α-GADD153/CHOP pathway activation has also been reported in other anti-prostate cancer compound in our previous study.38 Taken together, these findings suggest that Tan-IIA may induce apoptosis through the IRE1-α-GADD153/CHOP pathway in LNCaP cells.

Androgen is involved in cell cycle progression and androgen depletion of LNCaP cells resulted in cell cycle G1 phase arrest.39 Inhibition of AR was involved in Tan-IIA-induced G1 arrest in LNCaP cells.15 The treatment with Tan-IIA showed significant cytotoxic effects in LNCaP cells (androgen sensitive, IC50=2.5 μg ml−1) as compared with PC-3 cells (androgen insensitive, IC50=5 μg ml−1). Won et al.15 also supported the idea that AR signaling partly mediated Tan-IIA-induced cell death in prostate cancer cells. Recently, a novel role for AR signaling in prostate cancer was reported.40 AR signaling promotes eIF2α phosphorylation and as a translational regulator of TMEFF2. Tumor suppressor ability of TMEFF2 correlates with its ability to modulate sarcosine levels.41 The involvement of AR signaling pathway in Tan-IIA-induced ER stress and cell death are interesting and worth for further investigation.

In conclusion, our study demonstrated that Tan-IIA causes cell cycle G0/G1 arrest and induces pro-apoptotic activity more prominently in androgen-sensitive LNCaP prostate cancer cells. We provided evidence that the disruption of the cell cycle regulator at G0/G1 phase and the activation of p16, p21 and p27 are important in Tan-IIA-induced cell cycle arrest. The underlying mechanisms for cell death were found to be due to the activations of caspases-9, -3 and PARP, and the induction of ER stress by the IRE1-α-GADD153/CHOP pathway. Taken together, our findings indicate that Tan-IIA triggers apoptosis of LNCaP cells through multiple apoptotic pathways in vitro and in vivo, and is a potential early-stage antitumor compound for prostate cancer therapy.

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Acknowledgements

We thank Hsin-Rong Wu and Wei-Ping Huang for their assistance in animal studies. This work was supported by grants from the Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien, Taiwan (TCSP-01-02, TCRD-I9801-03). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Correspondence to T L Chiu or C Y Pang.

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Keywords

  • tanshinone IIA
  • LNCaP cells
  • ER stress
  • apoptosis
  • GADD153/CHOP

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