- Research article
- Open Access
- Open Peer Review
Silibinin induces mitochondrial NOX4-mediated endoplasmic reticulum stress response and its subsequent apoptosis
© The Author(s). 2016
- Received: 30 July 2015
- Accepted: 27 June 2016
- Published: 12 July 2016
Silibinin, a biologically active compound of milk thistle, has chemopreventive effects on cancer cell lines. Recently it was reported that silibinin inhibited tumor growth through activation of the apoptotic signaling pathway. Although various evidences showed multiple signaling pathways of silibinin in apoptosis, there were no reports to address the clear mechanism of ROS-mediated pathway in prostate cancer PC-3 cells. Several studies suggested that reactive oxygen species (ROS) play an important role in various signaling cascades, but the primary source of ROS was currently unclear.
The effect of silibinin was investigated on cell growth of prostate cell lines by MTT assay. We examined whether silibinin induced apoptosis through production of ROS using flow cytometry. Expression of apoptosis-, endoplasmic reticulum (ER)-related protein and gene were determined by western blotting and RT-PCR, respectively.
Results showed that silibinin triggered mitochondrial ROS production through NOX4 expression and finally led to induce apoptosis. In addition, mitochondrial ROS caused ER stress through disruption of Ca2+ homeostasis. Co-treatment of ROS inhibitor reduced the silibinin-induced apoptosis through the inhibition of NOX4 expression, resulting in reduction of both Ca2+ level and ER stress response.
Taken together, silibinin induced mitochondrial ROS-dependent apoptosis through NOX4, which is associated with disruption of Ca2+ homeostasis and ER stress response. Therefore, the regulation of NOX4, mitochondrial ROS producer, could be a potential target for the treatment of prostate cancer.
- Reactive oxygen species
- Endoplasmic reticulum stress
Reactive oxygen species (ROS) can act as secondary messengers in cancer cell signaling. It plays an important role in various cellular response, including cell growth, differentiation, survival, death, inflammation and immune response [1, 2]. The sources of ROS are various organelles and enzymes system including mitochondria, endoplasmic reticulum, peroxisomes and NADPH oxidase (NOX) . ROS are generated in forms of superoxide anion (O2–•), hydroxyl radical (OH•) and hydrogen peroxide (H2O2) in living organisms . Most of O2–• are produced by NOXs, xanthine oxidase and the mitochondrial electron-transport chain . Extracellular stresses including toxin, growth factors, ROS, ultraviolet radiation, viral infection and anti-cancer agents are known to trigger apoptosis in many studies [6–9]. In addition, apoptosis occurs from the changes of mitochondrial membrane potential (MMP, ΔΨ m ) and release of pro-apoptotic factor such as cytochrome c . ROS are critically important signals to activate endoplasmic reticulum (ER) stress as well as apoptosis by a variety of stimulating conditions .
Recently, several studies have reported that the apoptosis is related with ER stress responses in cancer cell lines [11, 12]. ER stress is involved in the initiation of apoptosis by at least two different mechanisms, namely the unfolded protein response (UPR) and Ca2+ signaling [13, 14]. The UPR-mediated signals consist of a complex interaction between three signaling, inositol requiring enzyme 1 (IRE1)-X box-bonding protein 1 (XBP1) signaling, pancreatic ER kinase (PKR)-like ER kinase (PERK)-eukaryotic initiation factor 2α (eIF2α) signaling and activating transcription factor 6 (ATF6) signaling . The activation of ER membrane-resident caspase-4/12 (human/mice) and induction of CHOP stimulate ER stress-mediated apoptosis [16, 17]. Subsequently, the typical signaling of apoptosis mainly lead to the activation of intracellular caspases, major activator of the mitochondrial-dependent pathway . ER is the main intracellular storage compartment for Ca2+, which is an important secondary messenger required for cellular functions. Changes of cellular Ca2+ homeostasis including cytosolic Ca2+ overload, ER Ca2+ depletion and mitochondrial Ca2+ increase induce apoptosis [19, 20]. The ER-mitochondria interaction supports communication between the two organelles, including the exchange of Ca2+, which controls ER chaperone protein, mitochondrial ATP production and apoptosis .
Silibinin, a major biologically active constituent of silymarin from milk thistle extract, has been used clinically for its hepatoprotective action for more than three decades in Europe and recently in Asia and the United States . It has been reported that it induces apoptosis by regulating the cell cycle arrest in human bladder carcinoma cells and human colon carcinoma HT-29 cell [23, 24]. Other reports in murine orthotopic hepatocarcinoma model have indicated that it inhibits tumor growth through the activation of TRAIL/death receptor/apoptotic signaling pathway, both in vitro and in vivo . In addition, it has been shown anti-tumor effect in which induces apoptosis through a p53-dependent pathway involving Bcl-2/Bax, cytochrome c release and caspase activation . So far, it has not been clarified that production of ROS or Ca2+ signaling-mediated ER stress play a critical role in the silibinin-induced apoptosis in human prostate cancer PC-3 cells. Therefore, we investigated whether production of ROS by silibinin causes ER stress through changes of Ca2+ concentration and whether these intracellular signaling pathways lead to cellular apoptosis.
Reagents and antibodies
3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltertrazolium bromide (MTT), propidium iodide (PI), 6-diamidino-2-phenylindole dihydrochloride (DAPI), 2′,7′-dichlorfluorescein-diacetate (DCFH-DA) and 4-(6-Acetoxymethoxy-2,7-dichloro-3-oxo-9-xanthenyl)-4′-methyl-2,2′(ethylenedioxy)dianiline-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl) ester (Fluo-3/AM) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 1,2-bis-(o-Aminophenoxy) ethane-tetraacetic acid tetra-(acetoxymethyl) ester (BAPTA/AM), Diphenyleneiodonium (DPI), Z-DEVD-FMK and Z-YVAD-FMK were purchased from Calbiochem (Merck, Darmstadt, Germany) and R&D Systems (Minneapolis, MN, USA), respectively. FITC Annexin-V apoptosis Detection kit and Caspase-3 Colorimetric Assay Kit were purchased from BD Bioscience (San Jose, CA, USA) and Assay Design Inc. (Ann Arbor, Michigan, USA), respectively. RiboEx_column™ kit was purchased from GeneAll Biotechnology Co. (Seoul, Korea). TOPscript™ RT DryMIX kit (Enzynomics, Deajeon, Korea) and EmeraldAmp PCR masterMIX (TAKARA, Otsu, Japan) were obtained. The ECL Western Kit was purchased from iNtRON Biotechnology (Seongnam, Korea). Antibodies for NOX4, Caspase-3 and β-Actin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies for poly (ADP-ribose) polymerase-1 (PARP-1), Bip, IRE1α, and CHOP were purchased from Cell Signaling (Beverly, MA, USA). MitoSOX was purchased from Invitrogen (Grand Island, NY, USA).
Cell lines and cell culture
Human prostate cell lines, PC-3, LNCaP and RWPE-1 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Prostate cancer PC-3 and LNCaP cells were cultured in Dulbecco’s modified Eagle’s minimal medium (DMEM, WelGENE Inc., Korea) and Roswell Park Memorial Institute (RPMI) 1640 (WelGENE Inc., Korea) supplemented with 10 % FBS and 1 % penicillin-streptomycin solution with 5 % CO2 at 37 °C, respectively. Prostate normal RWPE-1 cells were cultured in keratinocyte serum-free media (K-SFM) containing 2.5 μg of epidermal growth factor (EGF), 25 mg of bovine pituitary extract (BPE, GIBCO) and 1 % penicillin-streptomycin solution with 5 % CO2 at 37 °C.
Cell viability assay
Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The prostate cancer PC-3 cells were seeded at a density of 1 × 104/ml in a 48-well culture dish and treated with silibinin of various concentrations for 24 and 48 h. After incubation, these cells were treated with 0.5 mg/ml of the MTT solution for further 3 h incubation and the precipitates were dissolved in dimethyl sulfoxide to dissolve the MTT-formazan complex. Absorbance was recorded on a microplate reader (Molecular Devices, Sunnyvale, CA, USA) at a wavelength of 540 nm. The cell viability was determined the relatives as their percentage of the treated cells to one of the untreated cells by comparing their optical densities.
Apoptotic cells were quantified and analyzed using an annexin V-FITC detection kit and flow cytometry. Briefly, the PC-3 cells were treated with 150 μM of silibinin for 48 h. Then the cells were washed with phosphate buffered saline (PBS) and were collected. Harvested cells were mixed in 1X binding buffer and incubated with an annexin V/PI double staining solution at room temperature for 15 min. Then, the staining cells were analyzed by flow cytometry (FACS Calibur, Becton Dickinson, San Jose, CA, USA) and the CellQuest software (Becton Dickinson Co.).
Measurement of MMP, Ca2+ flux and ROS concentration
MMP, Ca2+ and ROS levels were determined by the DiOC6, Fluo-3/AM and DCFH-DA, respectively. Briefly, silibinin-treated PC-3 cells in the presence or absence of each inhibitor were trypsinized, washed and incubated with each dye, a fluorescent marker, at 37 °C for 30 min. Fluorescence positive cells were measured by flow cytometry with CellQuest analysis software.
Immunofluorescence confocal microscopy
The mitochondrial ROS production was measured by confocal microscopy after staining with MitoSOX (Invitrogen, Waltham, MA, USA). Briefly, PC-3 cells were seeded on coverglass bottom dish and treated with 150 μM of silibinin for 24 h. Then the cells were incubated with 5 μM MitoSOX and fixed with 4 % paraformaldehyde for 10 min at room temperature. After fixation, the cells were washed twice with PBS and then incubated with 1 μg/ml DAPI solution at 4 °C for 15 min. Images were acquired using a confocal microscope (Olympus, Tokyo, Japan). For NOX4 localization assay, PC-3 cells were stained with 5 μM MitoSOX and fixed. Cells were subsequently permeabilized with 0.1 % triton X-100 for 10 min and blocked with 5 % BSA, and incubated with NOX4 primary antibody. Cells were washed with PBS and incubated in FITC-conjugated secondary antibody for 1 h at room temperature and then stained with DAPI solution. Stained cells were visualized by an Olympus confocal microscope.
RNA extraction and reverse transcription PCR
Total RNA was isolated using RiboEx_column™ kit according to the manufacturer’s instructions. Reverse transcription (RT) was carried out with 2 μg RNA and Oligo dT using TOPscript™ RT DryMIX kit, and the resulting cDNA was subjected to PCR. The sequence of the primers used in the PCR was the following: NOX1 (F: 5′ GTA CAA ATT CCA GTG TGC AGA CCA C 3′; R: 5′ CAG ACT GGA ATA TCG GTG ACA GCA 3′), NOX2 (F: 5′ GCT GTT CAA TGC TTG TGG CT 3′; R: 5′ TCT CCT CAT CAT GGT GCA CA 3′), NOX3 (F: 5′ GGA TCG GAG TCA CTC CCT TCG CTG 3′; R: 5′ ATG AAC ACC TCT GGG GTC AGC TGA 3′), NOX4 (F: 5′ CTC AGC GGA ATC AAT CAG CTG TG 3′; R: 5′ AGA GGA ACA CGA CAA TCA GCC TTA G 3′), NOX5 (F: 5′ TTA TGG GCT ACG TGG TAG TGG G 3′; R: 5′ GAA CCG TGT ACC CAG CCA AT 3′), XBP1 (F: 5′ CCT TGT AGT TGA GAA CCA GG 3′; R: 5′ GGG GCT TGG TAT ATA TGT GG 3′), Bip (F: 5′ TGC AGC AGG ACA TCA AGT 3′; R: 5′ CGC TGG TCA AAG TCT TCT CC 3′), CHOP (F 5′ GCG TCT AGA ATG GCA GCT GAG TCA TTG CC 3′; R: 5′ GCG TCT AGA TCA TGC TTG GTG GAG ATT C 3′), and GAPDH (F: 5′ CCA CCC ATG GCA AAT TCC ATG GCA 3′; R: 5′ GCG TCT AGA TCA TGC TTG GTG GAG ATT C 3′). The amplification was performed with EmeraldAmp PCR master MIX in Mycycler Thermal Cycler (Bio-Rad Laboratories Inc., Hercules, CA).
Cell extracts were prepared by incubating the cells in lysis buffer [150 mM NaCl, 10 mM Tris (pH 7.4), 5 mM EDTA (pH 8.0), 1 % Triton X-100, 1 mM PMSF, 20 mg/ml aprotinin, 50 μg/ml leupetin, 1 mM benzamdine, 1 mg/ml pepstatin]. Forty micrograms of proteins determined by the BSA method were electrophoretically separated on 8–15 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5 % skim milk in TBS-T buffer [20 mM Tris (pH 7.4), 150 mM NaCl, 0.1 % Tween 20] at room temperature for 1 h. The membranes were incubated with primary and secondary antibodies and then washed 3 times with TBS-T buffer for 10 min. Finally, the proteins were detected with an ECL western blotting detection reagent. The densities of each band were determined with a fluorescence scanner (LAS 3000, Fuji Film, Tokyo, Japan) and analyzed with Multi Gauge V3.0 software.
Experiments were repeated at least 3 times with consistent results. Unless otherwise stated, data are expressed as the mean ± SD. ANOVA was used to compare the experimental groups to the control values, whereas comparisons between multiple groups were performed using a Tukey’s multiple comparison test. The results were statistically significant at P <0.05.
Silibinin stimulated ROS production from mitochondria
Silibinin triggered mitochondrial ROS derived from NOX4 expression
Silibinin induced apoptosis through mitochondrial ROS
Silibinin induced ER stress through disruption of intracellular Ca2+ homeostasis
Silibinin induced ROS-mediated Ca2+ signaling and ER stress response
Currently, more effective strategies must be necessary to develop novel therapeutic targets and molecular regulatory agents for cancer diseases. Recently, the role of phytochemicals has been suggested in various cancer managements, especially polyphenolic compounds . Silibinin isolated from milk thistle is a polyphenolic flavonoid and it has been studied for applications as anticancer agents . In addition, some report proposed that silibinin suppresses the tumor growth and exhibits anti-proliferative, pro-apoptotic and anti-angiogenic effects of dietary feeding of silibinin on PC-3 xenograft in vivo . However, complete mechanism on anticancer effect of silibinin has not been clearly identified until now. To further elucidate the silibinin-induced intracellular mechanism, we investigated the various phenomena involved in cell death such as ROS and ER stress response.
First, to investigate silibinin induce cancer selectively death mechanism, apoptosis and ROS were measured in prostate cell lines including androgen-independent PC-3, androgen-dependent LNCaP and normal prostate epithelial RWPE-1 cells. The results indicated that silibinin possesses selective effects on apoptotic cell death and ROS in PC-3 and LNCaP cells, while not affecting the RWPE-1 cells (Additional file 1: Figure S2A and B). ROS are major molecules of intracellular signaling cascades and trigger mitochondria-associated events including apoptosis , which is generated from various cellular sources such as NOXs, mitochondrial respiration and extracellular stress. Our results showed that silibinin significantly stimulated the intracellular ROS production in a time-dependent manner. Among various cellular sources of ROS production, NOX4 produces ROS of superoxide type in the mitochondria. In our system, whereas early ROS production by silibinin was inhibited by all ROS inhibitors, persistent and excessive ROS production were attenuated by only DPI treatment, suggesting that silibinin mainly stimulated NOX4-dependent ROS of superoxide type. Also, we observed the mitochondrial ROS production and mRNA expression of the variety of NOX isoforms in silibinin-treated PC-3 cells. NOX plays an important role in regulation of mitochondrial ROS. Also it was reported that NOX4 is localized at various subcellular compartments such as mitochondria, ER, plasma membrane and nucleus [40–42]. Our results showed that silibinin-stimulated ROS were colocalized with NOX4 in mitochondrial portion confirmed by MitoSOX. Expression of NOX4 increased by silibinin was suppressed by DPI, a specific NOX inhibitor. Although some studies have reported that DPI inhibits NOX activity, like our results, another reported that DPI inhibited NOX4 expression in PMA-stimulated U87MG cells . In addition, DPI inhibited silibinin-induced apoptosis and the expression of apoptosis proteins. These results suggested that NOX4 are the primary source of silibinin-stimulated mitochondrial ROS production and these ROS are involved in apoptosis process by modulating NOX4-driven ROS generation from mitochondria.
Some studies reported that intracellular Ca2+ flux also mediates multiple cellular signaling cascades of survival and apoptosis. Release of Ca2+ from ER and high Ca2+ concentration in cytosol trigger cell death in human cancer cell lines . Our results indicated that silibinin markedly increased the level of intracellular Ca2+ up to 24 h after treatment. And these effects were prevented by pretreatment with Ca2+ chelator BAPTA/AM. Also BAPTA/AM inhibited silibinin-induced apoptosis and the expression of apoptosis proteins. From these results, silibinin provoked the disruption of Ca2+ homeostasis, leading to cellular apoptosis. Also Ca2+ signaling is considered as one of the second messengers strongly involved in ER stress response . ER stress occurs due to the accumulation of unfolded proteins and disruption of Ca2+ homeostasis, and triggers specific signaling pathway with UPR through three key proteins, including IRE1α, ATF4 and PERK. Silibinin induced the expression of ER stress proteins and spliced XBP1 mRNA. In addition, pretreatment with BAPTA/AM markedly decreased the expression of Bip and CHOP. So far, 14 caspase family members have been identified  and play important roles in ER stress and apoptosis. Silibinin-induced apoptosis was reduced by Z-YVAD-FMK, an inhibitor of caspase-4, which is localized in the ER membrane (Additional file 1: Figure S3). These results indicated that ER stress plays major role in silibinin-induced apoptosis through Ca2+ signaling. On the observation to further clarify the role of ROS in Ca2+ flux and ER stress, blocking of ROS production with DPI caused the reduction of Ca2+ level and inhibition of ER stress proteins. It was suggested that ROS generation by silibinin plays a key role in induction of ER stress triggered from disruption of Ca2+ homeostasis in PC-3 cells.
In conclusion, our studies demonstrated that silibinin induced apoptosis mediated by activation of ER stress that requires the disruption of Ca2+ homeostasis through ROS generation and those ROS were produced, in part, from mitochondrial NOX system, upstream pathway of Ca2+ signaling. Therefore, based on these results, regulation of NOX4-driven mitochondrial ROS production could be a potential target for development of the cancer therapy and management.
ATF6, activating transcription factor 6; BAPTA, 1,2-bis-(o-Aminophenoxy) ethane-tetraacetic acid tetra-(acetoxymethyl) ester; DAPI, 6-Diamidino-2-phenylindole dihydrochloride; DCFH-DA, 2′,7′-dichlorfluorescein-diacetate; DPI, diphenyleneiodonium (DPI); eIF2α, Eukaryotic initiation factor 2; ER, endoplasmic reticulum; Fluo-3/AM, 4-(6-Acetoxymethoxy-2,7-dichloro-3-oxo-9-xanthenyl)-4′-methyl-2,2′(ethylenedioxy)dianiline-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl) ester; IRE1, inositol requiring enzyme 1; MMP, mitochondrial membrane potential; MTT, 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltertrazolium bromide; NOX, NADPH oxidase; PERK, pancreatic ER kinase (PKR)-like ER kinase; PI, propidium iodide; ROS, reactive oxygen species; UPR, Unfolded protein response; XBP1, X box-bonding protein 1
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2012R1A1A2022587). This work was supported by the Brain Busan 21 Project in 2016.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article (and its Additional file 1). Any request of data and material may be sent to the corresponding author.
SHK performed the experiments and wrote the main manuscript. SNY and SSC analyzed the data and prepared figures. KYK and YKS contributed materials tools. HSY revised the manuscript. SCA conceived and designed the experiments. All authors reviewed the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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“Not applicable” in this section.
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