The role of endoplasmic reticulum (ER) stress in quercetin-mediated cell death in human prostate cancer PC-3 cells through mitochondrial pathway
Kuo-Ching Liu1, , Chun-Yi Yen 2, Rick Sai-Chuan Wu3, Jai-Sing Yang4, Hsu-Feng Lu5, Kung-Wen Lu6, Chyi Lo7, Hung-Yi Chen8, Nou-Ying Tang7, Chih-Chung Wu9, Heng- Chien Ho10,*, Jing-Gung Chung2,11,*
Departments of 1Medical Laboratory Science and Biotechnology, 2Biological Science and Technology and 4Pharmacology, Schools of 6Post-Baccalaureate Chinese Medicine,
7Chinese Medicine and 8Pharmacy, China Medical University, Taichung 404, Taiwan,
3Department of Anesthesia, China Medical University Hospital, Taichung 404, Taiwan
5Department of Clinical Pathology, Cheng Hsin Rehabilitation Medical Center, Taipei, Taiwan
9Department of Nutrition and Health Sciences, Chang Jung Christian University, Tainan 711, Taiwan.
Department of Biochemistry
11Department of Biotechnology, Asia University, 500 Liufeg Rd., Wufeng, Taichung County 413, Taiwan, R.O.C.
Running title: Quercetin induced apoptosis in human prostate cancer PC-3 cells
Correspondence to: Jing-Gung Chung, PhD, Department ofBiological Science and Technology, China Medical University, No. 91 Hsueh-Shih Road, Taichung 404, Taiwan Tel: 886-4-22053366-2161, Fax: 886-4-22053764
E-mail: [email protected]
Abstract
Prostate cancer has its highest incidence in the USA. In Asian countries, prostate cancer is becoming a major concern. In Taiwan, prostate cancer is the eight leading cause of cancer- related deaths among men. Quercetin, a natural polyphenolic compound, has been showed to induce apoptosis in many human cancer cell lines including prostate cancer PC-3 cells.
Although numerous evidences show multiple possible signaling pathways of quercetin in apoptosis, however, there is no report to show the role of ER stress in quercetin induced apoptosis in PC-3 cells. The purpose of this study was to investigate the effects of quercetin on the induction of the apoptotic pathway in human prostate cancer pC-3 cells.
When PC-3 cells were treated with quercetin for 24 and 48 h and at various doses (50-200 μM), cell morphology and viability decreased significantly in dose-dependent manners.
Flow cytometric assay indicated that quercetin caused G0/G1 phase arrest ( % to %) at - μM and sub-G1 phase cells ( % to %) for - h treatment and this effect is time-dependent manner. Western blotting indicated that quercetin induced G0/G1 phase arrest via decreased the protein expression of CDK2, CDK4, cyclins E and B proteins. Quercetin increased the protein expression of ATF, Grp78 and GADD153 which is a hall marker of ER stress. Furthermore, PC-3 cells after incubation with quercetin for 48 h showed an apoptotic cell death (which is also confirmed by DAPI and Comet assays) by the decrease the anti-apoptotic Bcl-2 protein and levels of ΔΨm and increase the pro-apoptotic Bax protein and increase the activations of caspase-3, -8 and -9. Moreover, quercetin increased the AIF protein released from mitochondria to nuclei. These data suggested that quercetin may induce apoptosis by direct activation of caspase cascade through mitochondrial pathway in PC-3 cells.
Key Words: Quercetin, Apoptosis, Cell cycle, Caspase-3, Mitochondria, Bladder cancer
PC-3 cells
INTRODUCTION
Prostate cancer, one of the major causing deaths in man, the second leading cause of cancer-related deaths among men in the United States (1, 2), is increasingly the events in men throughout the world. In Taiwan, 2.3 persons per 100 thousand die annually from prostate cancer based on reports 2008 from the People Health Bureau of Taiwan. The major treatments for prostate cancer patients are surgery, radiotherapy and chemotherapy, or combine with radiotherapy and chemotherapy, however, the efficiency of cure rates are not satisfactory. Most chemotherapy and radiation treatments are to kill cancer cells via the induction of apoptosis (programmed cell death) of tumor cells (3, 4). To find new agents for novel targets of prostate cancer are currently under investigation.
Apoptosis, which is known as programmed cell death, plays a crucial role in the maintenance of cell homeostasis, have been recognized to be as a cascade of a caspases and endonucleases responsible for the proteolytic cleavage of cellular proteins to form plasma membrane blebbing, cell shrinkage, apoptotic body, chromatin condensation, and DNA fragmentation (5). Apoptosis can be divided into a death receptor-dependent (extrinsic) which involved in caspase-8 or independent (intrinsic) which involved in mitochondria pathways. Thus, apoptosis also can be occurred via caspase-dependent and -indepndent pathways. Caspases (14 family members), are synthesized as inactive zymogens, will be proteolytically cleaved at two or three aspartate residues to generate the active mature enzyme which then interact with specific adapter molecules to turn cleave and activate the downstream ‘‘executioner’’ caspases for induced apoptosis in cells (6).
Thus, it is recognized to be the best strategy to kill cancer cells is to induce apoptotic cell death.
Epidemiological studies in humans and in the animals studies have shown that regular consumption of fruits, vegetables, spices, and tea exert potential anticarcinogenic activities
and reduced risk of cancer development (7). Quercetin (3, 3’, 4’,5,7-pentahydroxy flavones), one of an anti-oxidant flavonoid that is widely presented in the natural plants, has been shown to have anticancer activities in many human cancer cell lines including leukemia (8, 9), colon (10), breast (11), lung (12), hepatoma (13), oral (14) and prostate (15-18) cancer cells. Recently, it was reported that quercetin decreases the survival of androgen independent prostate cancer cells by modulating the expression of insulin-like growth factors (IGF) system components, signaling molecules and induces apoptosis, which could be very useful for the androgen independent prostate cancer treatment (19).
Furthermore, quercetin is mediated by the dissociation of Bax from Bcl-xL and the activation of caspase families (20) and enhances TRAIL-induced apoptosis via increased protein stability of death receptor 5 in human prostate cancer cells (18). Quercetin may have anti-tumor effects on human cervical cancer HeLa cells via AMPK-induced HSP70 and down-regulation of EGFR (21). Quercetin induces apoptosis via AMPK activation and p53-dependent apoptotic cell death in HT-29 colon cancer cells (22).
Our previous studies showed that quercetin enhances immune responses in WEHI-3 cell leukemia mice (23) and quercetin induced apoptosis in human breast cancer cells (24).
Other investigators also reported that quercetin induced apoptosis in human prostate cancer pC-3 cells (15-18). However, knowledge of the molecular mechanisms of quercetin-induced apoptosis in human cancer cells was rudimentary and not exact clearly the signal pathway especially may be cell type dependent. This study was undertaken to evaluate the induction of apoptosis in human prostate cancer PC-3 cells which caused by treatment with quercetin. The possible other signally pathways was associated with ES stress and the probable apoptotic molecular mechanisms.
MATERIALS AND METHODS
Chemicals and reagents
Quercetin, propidium iodide (PI), dimethyl sulfoxide (DMSO), penicillin and streptomycin, Tris-HCl, trypan blue, and triton X-100 were purchased from Sigma Chemical. (St. Louis, MO, USA). The fluorescent probe 2’, 7’-dichlorofluorescin diacetate (DCFH-DA), Indo 1/AM and DiOC6 were obtained from Calbiochem (La Jolla, CA, USA). Anti-caspase-3, -caspase-8 and -caspase-9 and anti-PARP were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-Bcl-2, anti-Bax, anti-BID, anti-pro- ATF6, anti-PERK, anti-GRP78, anti-GADD153, anti-p57, anti-Thymidylate synthase, anti-cyclins A and E, anti-p53, anti-Fas, anti-TRAIL, anti-XIAP,anti-GADD153 and anti- AIF were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Caspase-3, -8 and -9 substrates were purchased from R&D system Inc. (Minneapolis, MN, USA).
Materials and chemicals for electrophoresis were from BioRad (Hercules, CA, USA). Cell culture dishes and cell culture medium were purchased from Falcon (San Diego, CA, USA).
Human prostate cell culture
Human prostate cancer cell line (PC-3) was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). The cells were placed into 75 cm2 tissue culture flasks under a humidified 5% CO2 and 95% air grown at 37°C and one atmosphere in RPMI 1640 medium (Gibco/Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS, 1% penicillin-streptomycin (100 units/ml penicillin and 100 μg/ml streptomycin) and 2 mM L-glutamine. All the chemicals used were extra pure and were of culture grade.
Morphological changes and viable cells determination
PC-3 cells at 2×105 cells/well in 12-well plate were treated with 0, 50, 100, 150 and
200 μM quercetin (dissolved in DMSO) for 24 and 48 h. For examining the morphological changes of PC-3 cells, after exposed to quercetin, cells on the well plates were examined and photographed under phase contrast microscope at 200x. For determining cell viability, the trypan blue exclusion and PI- exclusion method by flow cytometric protocol were used, as previously described (23,24). Trypan blue exclusion protocol was performed. An aliquot of the total cell suspension was mixed with an equal volume of trypan blue in PBS and incubated for 5 min at room temperature. Dead cells were turned to light blue and live cells come out white color. Viable cells were calculated by counting the cells in a Neubauer chamber. For PI exclusion protcol, cells after exposed to quercetin for various time then were collected, resuspended in PBS containing 4 μg/ml PI and then analyzed by a flow cytometer (FACSCalibur, Becton Dickinson, NJ, USA). All experiments were performed in triplicate. The percentage of cell viability was calculated as a ratio between quercetin-treated cells and 0.1% DMSO vehicle-control cells.
Cell cycle and sub-G1 group assays
PC-3 cells at 2×105 cells/well in 12-well plate were treated with 0 and 150 μM quercetin (dissolved in DMSO) for 0, 6, 12, 24, 36 and 48 h. Meidum containing float cells were sucked by pipetman, then trypsin were added to the cells of each well in plate for 3 min then cells were harvested then were centrifuged by centrifugation at 110g for 5 min, pellet were washed twice with cold PBS then fixed by using 70% ethanol (in PBS) in 4°C overnight as described previously (24). Then cells were washed twice with cold PBS then cells were re-suspended in PBS containing 40 μg/mL PI and 0.1 mg/mL RNase and 0.1% triton X-100 in dark room for 30 minutes at 37°C then the cells were analyzed by flow cytometry. Then the cell cycle and sub-G1 (apoptosis) group were determined and analyzed (24)
DAPI staining
PC-3 cells at 2×105 cells/well in 12-well plate were treated with 0, 10, 50, 100, 150 and 175 μM quercetin (dissolved in DMSO) for 24 and 48 h, while only adding DMSO (solvent) for the control regimen, and grown in 5% CO2 and 95% air at 37°C. Cells in each treatment and control were fixed in 4% paraformaldehyde-PBS solution for 15 min then were stained by DAPI (300 nmol/l) according to the manufacturer’s instructions then were examined and photographed under fluorescence microscopy as described elsewhere (24).
Then nuclear morphology was visualized and photographed by fluorescence microscopy.
For Comet assay, cells were harvested by centrifugation, isolated and examined for DNA damage by using the Comet assay as previously described (25).
Determination of Ca2+ concentrations and mitochondrial membrane potential (ΔΨm) PC-3 cells at 2×105 cells/well in 12-well plate were treated with 0, 150 μM quercetin (dissolved in DMSO) for 48 h, while only adding DMSO (solvent) for the control regimen, and grown in 5% CO2 and 95% air at 37°C. At the end of incubation, cells from each treatment were harvested and were washed twice by PBS, then were re-suspended in 500 μl of Indo 1/AM (3 μg/ml) for the levels of Ca2+ concentrations and in 500 ul of DiOC6 (4 μM) for the levels of mitochondrial membrane potential (ΔΨm). Then cells were incubated at 37°C under dark room for 30 min and were analyzed immediately by flow cytometry as described previously (24).
Caspase-3, caspase-8 and caspase-9 activity assay
Approximately 5x105 cells/well of PC-3 cells were plated on 12-well plates for 24 h then 0 and 10 μM of quercetin individually added to the well then incubation for 0, 12, 24 and 48 h. At the end of incubation, all cells were trypsinized and centrifuged from each treatment then were centrifuged, collected and washed twice with PBS. All samples were
re-suspended in 50 μl of 10 μM substrate solution (PhiPhiLux-G1D1 for caspase-3, CaspaLux 8-L1D2 for caspase-8 and CaspaLux9-M1D2 for caspase-9) (OncoImmunin, Inc.
Gaithersburg, MD, USA) before being incubated at 37°C for 60 min. All samples were washed again by PBS and were analyzed by flow cytometry as described previously (21, 24).
Determination of apoptotic associated proteins by Western blotting
Approximately 1×106 cells/well of PC-3 cells in 6-well plate were treated with 150 μM of quercetin were incubated for 0, 6, 12, 24 and 48 h for examining the various proteins correlated with ER stress, cell cycle arrest and apoptosis. At the end of incubation, cell from each treatment were harvested and washed with cold PBS. Cells were lysed with RIPA buffer containing protease inhibitor. The total proteins from each treatments were collected and quantiated by Biorad method. About 50 μg protein from each sample was resolved over 12% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS- PAGE) and transferred to nitrocellulose membrane. The blot was soaked in blocking buffer (5% non-fat dry mik/0.05% Tween 20 in 20 mM TBS at pH 7.6) at room temperature for 1 h then incubated with individual primary monoclonal antibodies in blocking buffer at 4°C for overnight. Then followed by secondary antibody horseradish peroxidase conjugate and detected by chemiluminescence and autoradiography using X- ray film (20, 21). To ensure equal protein loading, each membrane was stripped and reprobed with anti-β-actin antibody (20, 21, 24).
Confocal laser microscopy
Approximately 5×104 cells/well of PC-3 cells were plated on 4-well chamber slides, then were treated with or without (control) 150 μM of quercetin for 24 h, cells were
washed then were fixed with 4% formaldehyde in PBS for 15 min, permeabilized with 0.3% Triton-X 100 in PBS for 1 h containing with blocking of non-specific binding sites using 2% BSA. The fixed cells were then incubated with anti-human AIF antibody (1:100 dilution, respectively) overnight, washed twice with PBS then exposed to the secondary antibody (FITC-conjugated goat anti-mouse IgG at 1:100 dilution) for 40 min, followed by DNA staining with PI then the photomicrographs were undertaken by using a Leica TCS SP2 Confocal Spectral Microscope as described previously (24).
Statistical analyses
The results were presented as means ± S.D. of three replicate assays. The difference between the quercetin-treated and control groups were analyzed by Student’s t-test, a probability of p<0.05 being considered significant.
Results
The effects of quercetin on the morphological changes and percentage of viable PC-3 cells
To verify the effect of quercetin on cell morphology and viability, PC-3 cells were treated with increasing concentrations of quercetin for 24 and 48 h, and cell morphology was examined and photographed under phase contrast microscope. As shown in Figure 1A, cell morphological changes was increased, the concentrations of quercetin was increased. And this effect is dose-dependent manner. Cell viability was measured by flow cytometric assay and also was confirmed by trypan blue exclusion. As shown in Figure 1B, cell viability was inversely correlated with quercetin concentration. Significant loss of viability was detected at 50, 100, 150 and 200 μM of quercetin in a dose- and time- dependent manners. Cell viability of PC-3 cells after exposed to quercetin was further confirmed by trypan blue dye exclusion method (data not shown).
The effects of quercetin on the cell cycle distribution and apoptotic associated proteins from PC-3 cells
To determine if decrease in cell viability involved cell cycle distribution, we investigated cell cycle phase distribution of PC-3 cells by flow cytometry and result are shown in Figure 2A. When cells were treated with over 150 μM of quercetin for 48 h, the G2/M phase arrest was observed in a time-dependent patterns (Fig. 2B), for example, 150 μM of quercetin treatment for 24 and 48 h resulted in an increase in the percentage of cells in the G2/M phase from 12.4% to 42.1%, and 38.6% (Fig. 2B) when compared to control, respectively. Concomitant with this increase in the percentage of cells in the G2/M phase was a significant decrease in the percentage of cells in the G0/G1 phase from 47.7% to 24.3% at 48 h. Sub-G1 phase was increased after PC-3 cells was exposed to 150 μM of quercetin and this effects is time-dependent manner (Fig. 2B).
The effects of quercetin on apoptosis and DNA damage were examined by DAPI staining and Comet assay in PC-3 cells
To examine whether quercetin inhibits the cell viability of PC-3 cells via inducing apoptosis. We investigated the apoptotic characterizations of PC-3 cells exposed to quercetin then cells were harvested then were stained by DAPI and results as shown in Figure 3A which indicated that condensation of chromatin of the nucleus and cell shrinkage, were seen in quercetin-treated cells (Fig. 3A), moreover, the DNA cleavage was checked by Comet assay. From the results it was clear that with increasing concentration of quercetin, the number of viable cells decreased tremendously. DNA damages were detected in a dose-response manner in quercetin-treated cells (Fig. 3B). Taken together, we conclude that quercetin decrease the percentage of viability of PC-3 cells through the induction of apoptosis and caused DNA damage.
The effects of quercetin on Ca2+ concentrations and ΔΨm levels in PC-3 cells
To examine whether Ca2+ and ΔΨm are involved in the apoptosis of PC-3 cells after exposed to quercetin. The results of Ca2+ concentrations and levels of ΔΨm from flow cytometric analysis are shown in Figure 4A and B. The results indicated that quercetin treated PC-3 cells led to the significant changes in Ca2+ concentrations of PC-3 cells from 3 h and up to 24 h (Fig. 4A). Numerous evidences have been shown that apoptosis is associated with the loss of ΔΨm, of mitochondria. To investigate the change in ΔΨm of PC-3 after quercetin treatment, DiOC6, a mitochondria-specific and voltage-dependent dye, was employed. The results indicated that quercetin significantly decreased the levels of ΔΨm in PC-3 cells in a time-dependent course (Fig. 4B).
Quercetin promoted the activations of caspase-3, caspase-8 and caspase-9 in PC-3 cells
To further characterize the apoptotic pathway activated by quercetin and to investigate whether caspase-3, -8 and -9 are involved in quercetin-induced apoptosis, the enzymatic activity of caspases were detected by using three fluorogenic peptide substrates for caspases-3, -8 and -9, respectively. The results are present in Figure 5, indicating the kinetic activity of various caspases, quercetin induced a rapid rise in caspase-3, -8 and -9 activities.
Quercetin affect the apoptotic associated proteins levels in PC-3 cells
In order to further understand the molecular mechanism involved in cell cycle arrest and apoptosis of PC-3 cells caused by quercetin, the expression of the cell cycle associated and apoptotic associated proteins were assessed in PC-3 cells. The results are as shown in Figure 6A, B, C and D. As shown in Figure 6, the protein level of cyclin E and D (Fig. 6A)
were decreased but the levels of Cdc25, p21, p53, p18 and p25 were increased (Fig. 6A) that were associated with cell cycle arrest. Morover, the levels of Bid, Bcl-2 (Fig. 6B), pro- caspase-3, PARP (Fig. 6C) were decreased but the levels of Bax, cytochrome c (Fig. 6B), caspase-9, AIF and Endo G (Fig. 6C) were increase that were associated with apoptosis.
The levels of pro-caspase-12 and Pro-ATF-6αwere decreased and Grp 78 ATF-4α, and IRE-1α(Fig. 6D) were increased.
Effects of quercetin on AIF nuclear translocation in PC-3 cells
In order to further confirm the release of AIF from mitochondria of PC-3 cells after exposed to quercetin. Cells were treated with or without quercetin then were harvested for examining the tranlocation of AIF which were measured by confocal laser microscope and results are shown in Figure 7. As illustrated in Figure 7, quercetin-treated PC-3 cells indicated that AIF were released from mitochondria then translocated to nuclei.
Discussion
Numerous evidences have been shown that some of the anti-cancer agents and DNA- damaging agents can induce cell cycle arrest at the G0/G1, S, or G2/M phase and then may also induced apoptotic cell death (26-27). Moreover, many studies also demonstrated that the cell cycle checkpoints can led to cells for DNA repair but apoptotic cell death may led to eliminate irreparable or unrepaired damaged cells. Herein, our results showed that quercetin induced cytotoxic effect on PC-3 cells and these effects are dose- and time- dependent manners (Fig. 1). Figure 2 also showed that quercetin induced G2/M phase arrest and Sub-G1 phase occur in PC-3 cells (Fig. 2A). It is well known that cell cycle events were regulated by a number of Cdks such as Cdk1 and Cdk2 kinases are activated primarily in association with cyclins A and B1, Wee1 and Cdc25c in the G2/M (28-29). It was reported that the cyclinB1/Cdk1 complex is the primary regulator of transition from
G2 to M phase (30). Our result showed that quercetin decreased the levels of cyclin B1 and Wee1, in contrast, the proteins level of Cdk1, cyclin B1 and cyclin E were not changed (Fig. 4) which may led to the G2/M phase arrest (Fig. 2B). It is well known that the G2/M transition is triggered by regulation of cyclin A, B1, E, Cdk1, Cdk2, and Cdc25C which promote the breakdown of the nuclear membrane, chromatin condensation, and microtubule spindle formation (31).
We also used DAPI staining to confirm quercetin induced apoptosis and also used Comet assay for measuring quercetin induced DNA damage in PC-3 cells. Flow cytometric assay also showed that quercetin promoted Ca2+ production and decreased the levels of ΔΨm of PC-3 cells. In our experiment, quercetin exposure can cause cell apoptosis of PC-3, Dum disruption, Ca2+ generation and cytochrome c release that due to mitochondrial dynamics affects mitochondrial function and vice versa. It was reported that mitochondrial dysfunction including as ROS overproduction, intracellular [Ca2+] elevation, and ATP reduction could damage mitochondrial dynamics (32-34).
Apoptosis is induced by a series activation of caspases for causing cell death and, caspase is present as an inactive procaspase, however, after triggered the procaspase will be formed as a cleaved form. Here, our results also showed that quercetin induced the formation of a cleaved form of caspase-8, -9 and -3 (Fig. 4). Caspase-3 is an executioner caspase, which upon activation can systematically dismantle cells through cleaving PARP.
It is well documented that apoptosis can be divided into the extrinsic and intrinsic pathways, the extrinsic pathway are involves the activation of caspase-8, which can cause downstream caspases activation such as caspase-3, -6, and -7 (35). Thus, quercetin induced apoptosis in PC-3 cells through extrinsic pathway. However, the intrinsic pathway are involved the mitochondria-dependent apoptosis, which can cause activation of caspase-9 followed by apoptotic protease activating factor-1 (APAF-1) and cytochrome c release
from mitochondria (36). Thus, quercetin induced apoptosis in PC-3 cells through intrinsic pathway. The present data showed that quercetin can induce apoptosis of PC-3 cells through the activations of caspase-3, -8, -9, bid, and PARP (Fig. 4).
Our results also showed that quercetin promoted the cleavage of Bid to form tBid in PC-3 cells (Fig. 6B). Several reports have been shown that the cleavage of Bid to tBid which is triggered by caspase-8 through the death receptor pathway of apoptosis and mediates mitochondrial apoptosis (37-39). In the present results showed that quercetin induced apoptosis of PC-3 cells through the activation of caspase-3, -8, -9, bid, and PARP (Fig. 3), thus, we suggesting that quercetin induces apoptosis through cross-talk between the extrinsic and the intrinsic pathway mediated by the activation of bid which is also reported by other investigators (40, 41).
In conclusion, quercetin arrested the cell cycle at the G0/G1 phase and induced apoptosis of PC-3 cells. Quercetin induced cell cycle arrest was associated with reduction of cyclins E and B, Cdk2 and Cdk4. Quercetin caused a marked increase in apoptosis, which was accompanied by activated caspase-3, -8, and -9. Overall, the whole signalling pathways of quercetin, which induced cell cycle arrest and apoptosis in PC-3 cells are summarized in Figure 8. Taken together, these findings provide the role of ER stress into the possible molecular mechanisms of the anti-cancer activity of quercetin.
ACKNOWLEDGMENTS
This research was supported by Grant NSC 96-2815-C-039-047-B from the National Science Council, Taiwan
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Figure legends
Fig. 1. The effects of quercetin on the cell morphological changes and the percentages of viable PC-3 cells. Cells (2×105 cells/well; 12-well plates) in RPMI 1640 medium with 10% FBS were treated with different doses of quercetin for 24 and 48 h. Cells collected by centrifugation and the viable cells were counted by flow cytometric assay (A), or cells were examined and photographed undertaken by phase contrast microscope (B) as described in Materials and Methods. Each point is mean ± S.D. of three experiments.
***Significantly different from the control at p<0.001.
Fig. 2. The effects of quercetin on the cell cycle distribution from PC-3 cells. Cells (2×105 cells/well; 12-well plates) in RPMI 1640 medium with 10% FBS were treated with 150 μM quercetin for 0, 6, 12, 24, 36 and 48 h. The cells were collected by centrifugation and the cell cycle and sub-G1 phase (A) were examined by flow cytometric assay or the percentage of cells in the cell cycle distribution wer calculated (B) as described in Materials and Methods. Each point is mean ± S.D. of three experiments.
Fig. 3. The effects of quercetin on apoptosis and DNA damage were examined by DAPI staining and Comet assay in PC-3 cells. Cells were incubated with various dose of quercetin for 24 h. Cells were examined by DAPI staining (A) and also were examined by Comet assay (B) as described in Materials and Methods.
Fig. 4. The effects of quercetin on the productions of Ca2+ and ΔΨm levels in PC-3 cells.
Cells were incubated with 150 μM quercetin for various time periods. Isolated cells were stained individually by Indo 1/AM dye for Ca2+ (A) and DiOC6 for ΔΨm (B) then the stained cells were quantitated by flow cytometry as described in the Materials and Methods.
***Differs between quercetin and control. p<0.001.
Fig. 5. The effects of quercetin on the activities of caspase-3, -8 and -9 in PC-3 cells.
About 1x105 cells/well of PC-3 cells in 12-well plate were treated with 150 μM quercetin were incubated for 24 or 48 h to detect the activities of caspase-3, -8 and -9 as described in Materials and Methods. ***Significantly different from the control at p<0.001.
Fig. 6. The effects of quercetin on the levels of cell cycle arrest and apoptosis associated proteins levels in PC-3 cells were examined by Western blotting. Cells (1×106/ml) were treated with 150 μM quercetin for 0, 6, 12, 24 and 48 h then the total protein were prepared and determined. The evaluation of the associated protein levels was carried out by Western blotting as described in Materials and Methods. (A) cyclin E, -D, cdc25, p21, p53, p18 and p25; Bid, Bax, Bcl-2 and cytochrome 2 (B); caspase-9, pro-caspase-3, PARP, AIF and Endo G (C) and Grp 78, pro-caspase-12, ATF-4α, Pro-ATF-6αand IRE-1α(D).
Fig. 7. Effects of quercetin on AIF nuclear translocations in PC-3 cells. PC-3 cells (5x104 cells/well; 4-well chamber slides) were incubated with 150 μM quercetin for 24 h. Cells were fixed and stained with primary antibodies to AIF-labeled secondary antibodies were used (green fluorescence) and the proteins were detected by a confocal laser microscopic system. The nuclei were stained by PI (red fluorescence). Areas of colocalization between AIF expressions and nuclei in the merged panels are yellow. Scale bar, 40 μm.
Fig. 8. Proposed model for the molecular mechanisms of quercetin induced apoptosis in human bladder cancer PC-3 cells. Quercetin induced ER stress (up levels of Grp78 and GADD153) through the productions of Ca2+ and reduced ΔΨm levels leading to caspase-9 and caspase-3 activation before causing apoptosis in PC-3 cells.
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