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Proteasome inhibitor-I enhances tunicamycin-induced chemosensitization of prostate cancer cells through regulation of NF-κB and CHOP expression
Cellular Signalling 23 (2011) 857–865
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Cellular Signalling j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c e l l s i g
Proteasome inhibitor-I enhances tunicamycin-induced chemosensitization of prostate cancer cells through regulation of NF-κB and CHOP expression Pham Thi Thu Huong a, Dong-Oh Moon a, Sun Ok Kim a, Kyoon Eon Kim b, Sook Jung Jeong a, Ki Won Lee c,f, Kyung Sang Lee d, Jae Hyuk Jang a, Raymond Leo Erikson e, Jong Seog Ahn a,⁎, Bo Yeon Kim a,f,⁎⁎ a
Chemical Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 685-1 Yangcheonri, Ochangeup, Cheongwongun, 363-883, Republic of Korea Department of Biochemistry, College of Natural Sciences, ChungNam National University, 220 Gung-dong, Yuseong-gu, Daejeon, 305-764, Republic of Korea Department of Agricultural Biotechnology, Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921, Republic of Korea d Laboratory of Metabolism, National Cancer Institute, NIH, Rockville Pike, Bethesda, MD 20892-4258, USA e Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA f World Class Institute, Korea Research Institute of Bioscience and Biotechnology, Republic of Korea b c
a r t i c l e
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Article history: Received 8 December 2010 Accepted 13 January 2011 Available online 27 January 2011 Keywords: Proteasome inhibitor-1 Tunicamycin ER-stress NF-κB CHOP
a b s t r a c t Although endoplasmic reticulum (ER) stress induction by some anticancer drugs can lead to apoptotic death of cancer cells, combination therapy with other chemicals would be much more efficient. It has been reported that proteasome inhibitors could induce cancer cell death through ER-stress. Our study, however, showed a differential mechanism of proteasome inhibitor-I (Pro-I)-induced cell death. Pro-I significantly enhanced apoptotic death of PC3 prostate cancer cells pretreated with tunicamycin (TM) while other signaling inhibitors against p38, mitogen activated kinase (MEK) and phosphatidyl-inositol 3-kinase (PI3K) did not, as evidenced by cell proliferation and cell cycle analyses. NF-κB inhibition by Pro-I, without direct effect on ER-stress, was found to be responsible for the TM-induced chemosensitization of PC3 cells. Moreover, TM-induced/enhancer-binding protein (C/EBP) homologous protein (CHOP) expression was enhanced by Pro-I without change in GRP78 expression. CHOP knockdown by siRNA also showed a significant decrease in Pro-I chemosensitization. All these data suggest that although TM could induce both NF-κB activation and CHOP expression through ER-stress, both NF-κB inhibition and increased CHOP level by Pro-I are required for enhanced chemosensitization of PC3 prostate cancer cells. Thus, our study might contribute to the identification of anticancer targets against prostate cancer cells. © 2011 Elsevier Inc. All rights reserved.
1. Introduction NF-κB is activated by a variety of signals through mechanisms that result in phosphorylation and proteasomic degradation of the inhibitory IκBα protein [1]. It has been reported that IκBα mutantmediated inhibition of NF-κB nuclear translocation enhanced apoptotic killing of human fibro-sarcoma and pancreatic carcinoma cells by certain chemotherapeutic drugs [2,3]. Regarding the IκBα regulation, many chemical agents, referred to as biological response or resistance modifiers, have been demonstrated to alter chemosensitivity in refractory tumor cells and are potentially useful in clinical cancer therapeutics [4]. Although ubiquitin–proteasome pathway is an efficient therapeutic target for cancer treatment [5,6],
⁎ Correspondence to: J.S. Ahn, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 685-1 Yangcheonri, Ochangeup, Cheongwongun, 363-883, Republic of Korea. Tel.: + 82 43 240 6160. ⁎⁎ Correspondence to: B.Y. Kim, World Class Institute, Korea Research Institute of Bioscience and Biotechnology, Ochangeup, Republic of Korea. Tel.: + 82 43 240 6163. E-mail addresses: [email protected] (J.S. Ahn), [email protected] (B.Y. Kim). 0898-6568/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2011.01.010
and recently developed proteasome inhibitors have been potent anticancer agents for various cancers, their underlying molecular mechanism remains to be elucidated. Recently, endoplasmic reticulum (ER) has emerged a prominent target for the development of chemotherapeutics against diverse diseases including diabetes and cancer. Tunicamycin (TM), a nucleoside antibiotic, inhibits the first step in the biosynthesis of N-linked oligosaccharides in cells, resulting in the ER stress induction and apoptotic cell death by unfolded protein response (UPR) in certain cancer cells [7–11]. CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), also called GADD153, implicated in the regulation of processes relevant to energy metabolism, cellular proliferation, differentiation, and expression of cell-type-specific genes, is primarily pro-apoptotic and is one of the highly inducible genes during ER stress [12,13]. In many cases of ER stress, induction or over-expression of CHOP sensitizes cells to ER stress-induced apoptosis, whereas CHOP deletion protects the cells from apoptosis. The pro-apoptotic effect of CHOP in ER stress may be mediated by the induction of growth arrest and DNA damage gene 34 (GADD34) that dephosphorylates eukaryotic translation initiation factor 2α (eIF2α) [12]. These results suggested that CHOP regulation could be critical for the treatment of cancer.
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Concerned with the proteasome regulation, there are multiple pathways activated in prostate cancer cells [14]. It has been reported that p38 MAP kinase pathway is required for transcriptional induction by NF-κB [15] and that proteasome inhibitors indeed activated p38 MAPK in prostate cancer models [16,17]. Moreover, proteasome mediated ER tress induction elicited apoptotic proteins such as GRP78 and caspase-12 activations as well as CHOP [18]. Caspase-12 deficient cells are resistant to inducers of ER stress, suggesting that caspase12 is crucial in ER stress-induced apoptosis [18,19]. On the other hand, PI3K-Akt pathway plays a central role in the development and progression of prostate cancer cell [20], activated Akt translocating to the nucleus and mediating various targets for cell survival. Akt also phosphorylates IκBα, inducing the nuclear translocation of NF-κB for transcriptional upregulation of anti-apoptotic genes [20,21]. In this study, we attempted to find out a potential molecular target for overcoming the chemo-resistance in prostate cancer. It was found that chemosensitization of PC3 prostate cancer cells was strongly enhanced by the combined treatment of both tunicamycin and proteasome inhibitor-1 (Pro-I) to the cells. This study indicated that simultaneous targeting of both CHOP and NF-κB downstream of ER stress signaling could be promising for prostate cancer treatment.
and 100 μg/ml streptomycin, and incubated at 37 °C in a humidified atmosphere of 5% CO2. Cells were seeded on 100 mm dishes one day before transfection with 6 μg of IκBα mutant plasmid using Lipofectamin 2000 system. Transfected cells were cultured in complete medium for 24 h, treated with TM (5 μg/ml) for 24 h, and collected for further analyses. 2.2. Antibodies and reagents
2. Materials and methods
Antibody to caspase-12 was from StressGen (Ann Arbor, MI. 48108), caspase-3 was obtained from Imgenex (San Diego, CA 92121). Antibodies to phospho-IκBα, p-Akt1, GAPDH, p38, p-p38, GRP78, and CHOP were purchased from Cell Signaling (Beverley, MA). Antibodies to IκBα, Akt1, p55, p65 and Bcl-2 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Proteasome inhibitor-I (Pro-I) was from Calbiochem (San Diego, CA). Lipofectamine 2000 was obtained from Chemicon (USA and Canada), RPMI1640 and fetal bovine serum were purchased from GIBCO-BRL (Grand Island, NY). Tunicamycin (TM), SB202190, PD98059, wortmannin were purchased from CalBiochem. Polyvinylidene difluoride (PVDF, 0.22 μm) membrane was obtained from Bio-Rad (Hercules, CA, USA). For EMSA, [γ-32P] ATP isotope was from NEN, Dupont (Boston, MA, USA). All the other reagents were obtained from Sigma (St. Louis, MO, USA).
2.1. Cell culture and transfection
2.3. Plasmids
Human prostate cancer PC3 cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin
IκBα mutant was generated by point mutation of the wild-type plasmid and confirmed by DNA sequencing.
Fig. 1. Pro-I enhanced TM-induced apoptosis of PC3 prostate cancer cells. (A) Pro-I increased TM-induced cell death. PC3 cells (4 × 104 cells/ml) were seeded in 96 well-plate overnight and then pretreated with Pro-I at various concentrations (0, 0.5, 1, and 3 μM) for 30 min before treatment with TM for 48 h. Cell proliferation was measured by CCK-8 Kit. (B) Enhancement of caspase activation by co-treatment of Pro-I and TM. Cells were exposed to Pro-I in the presence or absence of TM for 24 h. Cell lysates were subjected to western blotting analysis and immune-blotted with antibodies specific to caspase 12 and caspase 3. (C) Enhanced DNA fragmentation by Pro-I and TM co-treatment. Cells as in (B) were lysed and subjected to agarose gel analysis. M; 1 kb DNA ladder marker. (D) Flow cytometry analysis of Pro-I enhancement of apoptosis in PC3 cells through arresting sub-G1 phase of the cell cycle. Cells were seeded in 6 well plates and then challenged for 30 min with Pro-I (1 μM) before treatment with TM (5 μg/ml). (D1) After treatment with TM and Pro-I for 24 h and 48 h, cell cycle arrest was analyzed using Cycle Test™ Plus DNA Reagent Kit. (D2) Dead cells are counted and positive for Annexin V-FITC staining (D2). Under the same condition, cell morphology (D3) and the propidium iodide (PI)-stained DNA (D4) were recorded. Cells were stained with fluorescent DNA-binding dye Hoechst 33342 for 10 min at room temperature, visualized and analyzed by fluorescence microscopy. DNA condensation and fragmentation (arrow) were increased in cells exposed to both TM (5 μg/ml) and Pro-I (1 μM).
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Fig. 1 (continued).
2.4. Proliferation assays Cells (4 × 104 cells/ml) in 96 well plate (100 μl/well) were treated with Pro-I for 30 min followed by incubation with TM (5 μg/ml) for 48 h. Ten microliters of CCK solution was added to each well and the plates were further incubated at 37 °C for 2.5 h. Utilizing Dojindo's highly water-soluble tetrazolium salt, the absorbance was measured at 450 nm with a reference wavelength at 650 nm using a microplate reader MR700 (Dynatech Inc., Chantilly, VA, USA).
cytometric analysis was performed using a FACSCalibur (Becton Dickinson, San Jose, CA). 2.6. Hoechst 33342 (HO)/propidium iodide (PI) Cells were seeded in 96-well plate and treated with Pro-I in the presence or absence of TM. Cells were stained with fluorescent DNAbinding dye Hoechst 33342 for 10 min at room temperature and were visualized and analyzed by fluorescence microscopy. 2.7. Western blotting
2.5. Cell cycle analyses To analyze cell cycle arrest, flow cytometry analysis was carried out using a FACScan cell sorter (Becton Dickinson, San Jose, CA), and data were analyzed by the Flowjo program (Tree Star, San Carlos, CA). In order to analyze the percentage of apoptotic cells, all cultural cells were harvested and washed twice with cold PBS. The collected cells were resuspended in annexin-V binding Ca2+ buffer in annexin-V-FITC staining solution (1.0 μg/ml) and incubated for 15 min at room temperature in the dark. Flow
After washing with cold PBS buffer (pH 7.4), cells were lysed with ice-cold lysis buffer [50 mM Tris–HCl, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 20 mM NaF, 100 mM Na3VO4, 0.5% NP40, 1% Triton X100, 1 mM phenylmethylsulfonyl fluoride (pH 7.4)] containing freshly added protease inhibitor mixture (Protease Inhibitor Cocktail Set III; Calbiochem, La Jolla, CA) on ice for 30 min. Whole cell lysates were centrifuged at 14,000 rpm for 15 min, and then the upper part of solution was transferred into a new tube. For western blot analysis, appropriate amount of cell lysate containing 50 μg protein was
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subjected to 10–14% SDS-PAGE, then transfered onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). Membrane was immunoblotted with specific antibodies at 4 °C for the detection with chemiluminescence kit (ECL; Amersham Life Sciences Inc.). 2.8. Small interfering RNAs CHOP small interfering RNA (siRNA) sequences were: sihChop F: 5′-AAGAA CCA GCA GAG GTC ACA A-3′. SihChop R: 5′-AAGA CCA GCA GAG GTC ACA A-3′ which were synthesized by Bioneer Ltd, Korea. The LacZ siRNA was used as a siRNA control. In brief, one day prior to the transfection, PC-3 cells were seeded without antibiotics at a density of 30% to 40%. CHOP siRNAs (25 nmol/L) were transfected into cells using N-TER™ Nanoparticle siRNA Trasfection System (Sigma), in which the concentration of N-TER™ peptide was reduced to one-third of the recommended volume to limit toxic effects. After 24 h of transfection, cells were treated with TM (5 μg/ml) for another 24 h and then harvested. 2.9. Reverse transcription-polymerase chain reaction (RT-PCR) Cells were treated with TM (5 μg/ml) or transfected with IκBα mutant plasmid before TM-treatment. After appropriate time, RNA was extracted from the cells using Trizol reagent (Gibco-BRL). First-strand cDNA synthesis was performed with the SuperScript first-strand synthesis system (Invitrogen, Carlsbad, CA, USA). To amplify GRP78 mRNA, PCR was performed 22 cycles by 95 °C, 30 s; 55 °C, 30 s; and 72 °C, 1 min using primers F: 5′-GTT CTT CAA TGGCAA GGA ACC ATC-3′ and R: 5′-CCA TCC TTT CGA TTT CTT CAG GTG-3′. To amplify CHOP (GADD153) mRNA, PCR was followed 25 cycles for 94 °C — 30 s; 60 °C — 30 s; and 72 °C — 1 min using primers F: 5′-GCA CCT CCC AGA GCC CTC ACT CTC C-3′ and R: 5′-GTC TAC TCC AAG CCT TCC CCC TGC G-3′. And to amplify XBP1 mRNA, PCR was performed 35 cycles by 94 °C, 30 s; 55 °C, 30 s; and 72 °C, 30 s using primers F: 5′-ACA CGC TTG GGG ATG AAT GC3′ and R: 5′-CCA TGG GAA GAT GTT CTG GG-3′. Control primers for GAPDH were 5′-TAG ACG GGA AGC TCA CTG GC-3′ and 5′-AGG TCC ACC ACC CTG TTG CT-3′ for 20 cycles by 94 °C, 30 s; 60 °C, 30 s; and 72 °C, 30 s. Three fragments, representing spliced (XBP-1 s, 215 bp), unspliced (XBP-1u, 241 bp) and a hybrid (XBP-1 h, 267 bp), were produced and detected by running on a 2% agarose gel and staining with ethidium bromide. The other PCR products were loaded on a 1–1.5% agarose gel. 2.10. Electrophoretic mobility Shift Assay (EMSA) EMSA analysis was performed as described by Janssen and Sen [22]. In brief, PC3 cells were grown at 5 × 105 cells/ml in 100 mm dishes, pretreated 30 min with Pro-I in the presence or absence of TM. Cells were lysed on ice for 15 min in a hypotonic solution containing 10 mM HEPES–KOH (pH 7.8), 10 mM KCl, 2 mM MgCl2, 0.1 mM EDTA (sodium salt), 0.2 mM NaF, 0.2 mM Na3VO4, 0.4 mM phenylmethylsulfonyl fluoride (PMSF), leupeptin (10 μg/ml), 1 mM dithiothreitol (DTT) and 0.15% NP-40. The lysate was centrifuged at 16,000 rpm for 1 min at 4 °C and the resulting nuclear pellet was resuspended in ice-cold extraction buffer [50 mM HEPES–KOH (pH 7.8), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 0.2 mM NaF, 0.2 mM Na3VO4, 0.4 mM PMSF, 1 mM DTT and 10% glycerol] and incubated for 30 min at 4 °C with occasional vortex. The nuclear lysate was then centrifuged at 16,000 rpm for 30 min at 4 °C and the resulting supernatant was stored at −80 °C or immediately subjected to EMSA analysis. An oligonucleotide having NF-κB binding site (3.5 pmol) (Santa Cruz) was incubated for 10 min at 37 °C in 10 μl of reaction solution containing 10 μCi of [γ-32P] ATP, 5 U of T4 polynucleotide kinase and 1× kinase buffer (supplied with the kinase). The labeling reaction was terminated by the addition of 100 mM EDTA, followed by centrifugation through a Sephadex G-25 column to remove unincorporated 32P. The 32P-labeled oligonucleotide was then stored at −80 °C until use. For EMSA assay, nuclear protein extract (10 μg) was
incubated with the gel shift binding buffer for 10 min at room temperature and then with 32P-labeled probe in each reaction sample according to the manufacture's protocol (Gel shift assay system, Promega Kit., USA). For supershift analysis, the nuclear extract was incubated with specific antibodies (2 μg) for 30 min at room temperature prior to the addition of the labeled oligonucleotide. The binding reaction was terminated by the addition of electrophoresis sample buffer, and the samples were fractionated on 5% non-denaturing polyacrylamide gels in 0.5× Tris-boric acid-EDTA (TBE) buffer. The gels were dried and subjected to autoradiography. 2.11. DNA fragmentation assay After treatment with the indicated drugs, cells were lysed in 20 μ l lysis buffer (0.8% Sodium lauryl sarcarosin, 20 mM EDTA, 100 mM Tris pH8.0). The cell extract was treated with 10 μ l of RNase A (1 mg/ml) for 30 min at 37 °C then incubated for 1.5 h at 50 °C in the presence of 10 μ l of proteinase K (20 mg/ml). After mixing with 10× Loading dye buffer, the lysates were loaded on a 2% agarose gel electrophothesis at 100 V for 1.5 h. Gel was stained and DNA fragment was detected with ethidium bromide. 3. Results 3.1. Pro-I enhanced TM-induced apoptosis of PC3 prostate cancer cells In order to determine the effect of Pro-I on TM-induced ER-stress and apoptosis, PC3 prostate cancer cell proliferation was exploited after treatment with tunicamycin (TM) and Pro-I. Cells were dosedependently treated with TM for 48 h in the presence or absence of Pro-I at various concentrations. It was observed that Pro-I even at 0.5 μM significantly reduced the cell proliferation when treated with TM together while Pro-I alone showed only moderate effect (Fig. 1A). Enhanced DNA fragmentation could also be obtained when the cells were simultaneously treated with both Pro-I and TM (Fig. 1B), which was in accordance with the activation of caspase-3 and ER-stress associated caspase-12 (Fig. 1C), suggesting the involvement of ERstress in this cell death signaling. Enhanced cell death by Pro-I was confirmed by flow cytometry analysis. Pro-I significantly enhanced both the sub-G1 phase and annexin-5-staining of the cells in the presence of TM (Fig. 1D, D1 and D2). Cell morphological change and nuclear staining with Hoechst 33342 (HO) further supported the chemosensitizing effect of Pro-I (Fig. 1D, D3 and D4). Given the report demonstrating that inhibition of Akt and p38 augmented proteasome inhibitor-mediated apoptosis [23], involvement of other signaling pathways in enhancing chemosensitization of PC3 cells was exploited. PC3 cells were treated with SB203580, PD98059 and wortmannin, inhibitors of p38, mitogen activated protein kinase (MAPK) and Akt, respectively, in the presence of TM for 48 h. As shown in Fig. 2A, these inhibitors did not show any significant enhancement of cell chemosensitivity (Fig. 2A), although SB203580 and wortmannin could completely block the TM-induced phosphorylation of p38 and Akt (Fig. 2B); there could not be seen, however, ERK phosphorylation. These data suggested that NF-κB might be the target of enhanced chemosensitization of TM-induced cancer cells. 3.2. TM-induced NF-кB activation through ER-stress signaling is inhibited by Pro-I Apoptosis is usually antagonized by NF-κB activation. In cancer cells, NF-κB is highly activated and some chemotherapeutic agents could block NF-κB signaling pathways [24]. ER-stress was also reported to induce NF-κB [25]. In order to determine whether ER-stress and NF-кB activation could be achieved by TM and could also be affected by Pro-I, PC3 cells were treated with TM for various times. In addition to the IRE-
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Fig. 2. Effect of other signaling regulators on PC3 cell proliferation. Cells (4 × 104 cells/ml) were seeded in 96 well plate overnight and pretreated for 30 min with various concentrations of SB203580, PD98059 or wortmannin before exposure to TM for 48 h. Cell proliferation was measured with CCK-8 kit (A). Bars represent means ± SE from a representative triplicate experiment. Effects of the inhibitors on the phosphorylation of the respective proteins, p38, ERK, and Akt were shown on the right (B) to determine whether the compounds were really active in the cells. Cells were exposed to SB203580 (10 μM), PD98059 (20 μM) and wortamanin (200 nM) for 30 min followed by incubation with TM (5 μg/ml) for 24 h. Cells were lysed and subjected to immuno-blot analysis with specific antibodies to p38, ERK and Akt.
Fig. 3. TM-induced NF-κB activation and IRE-1α phosphorylation are inhibited by Pro-I. (A) TM induced phosphorylation of IRE-1α and IκBα. Cells were treated with TM at 5 μg/ml and lysed at the indicated time points for application to western blotting. (B) Pro-I inhibited TM-induced IκBα phosphorylation and degradation. After incubation with Pro-I (1 μM) for various times (30, 60, and 120 min), cells were treated with TM (5 μg/ml) for 24 h. Cell lysate was subjected to immune-blot analysis with specific antibodies. (C) EMSA analysis for NF-κB activation. TM (5 μg/ml) was treated to the cells for 1–2 h or in the presence or absence of Pro-I (10 μM) pretreated for 30 min. Nuclear fraction (10 μg) was applied to EMSA analysis with or without an antibody specific to p50 (p50 AB) or p65 (p65 AB) (2 μg) for super shift (*).NF-κB fold induction was shown in the lower panel.
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1α phosphorylation, TM also increased the phosphorylation and degradation of IκBα in the cells (Fig. 3A). Pro-I, however, significantly reversed TM-induced IκBα phosphorylation and degradation (Fig. 3B). The effect of Pro-I on NF-κB was also confirmed by EMSA analysis. TMinduced NF-κB DNA binding activity was strongly inhibited by Pro-I, and among NF-κB subunits p65/p50 heterodimer was found to be involved as evidenced by supershift assay (Fig. 3C, upper). The result of NF-κB fold induction was also shown as a graph (Fig. 3C, lower). These results suggest that Pro-I could effect on ER-stress-mediated NF-κB signaling by targeting IκBα degradation. 3.3. NF-κB inhibition enhances TM-induced apoptosis To further confirm that NF-κB inhibition enhances TM-induced apoptosis, PC3 cells were transiently transfected with IκBα mutant (IκBα-mt) plasmid followed by challenging with TM for various times. IκBα-mt significantly reduced the phosphorylation of endogenous IκBα in both TM treated and non-treated conditions (Fig. 4A). Cell death was also increased by IκBα-mt in the presence of TM compared to TM alone (Fig. 4B). Enhanced chemosensitization by IκBα-mt was further confirmed by cell cycle analysis. Cotreatment of both IκBα-mt and TM significantly increased the sub-G1 phase of the cells as well as
the cell death (Fig. 4C, upper and lower, respectively). Similarly, the level of anti-apoptosis factor bcl-2 was decreased by combined treatment of Pro-I and TM and was shown as a fold induction, too (Fig. 4D). These data indicate that inhibition of NF-κB enhances TMinduced chemosensitization of PC3 cancer cells. 3.4. Pro-I enhanced TM-induced CHOP expression independently of ER-stress One of the primary effectors of ER stress-mediated cell death is CCAAT/enhancer-binding protein homologous protein (CHOP), also called GADD153 [26]. In our study, it is shown that CHOP also plays a crucial role in Pro-I induced chemosensitization. As TM induced the phosphorylation of ER-stress protein IRE-1α (Fig. 3A), XBP-1 splicing (Fig. 5A) and expression of CHOP and GRP78 (Fig. 5B) were also increased by TM. Phosphorylation of another ER-stress associated protein, Protein kinase-like endoplasmic reticulum eIF2α kinase (PERK), was also found to be slightly and transiently increased by TM. Interestingly, however, there was no change in the extent of TM-induced phosphorylation of IRE-1α and PERK between the cells treated with or without Pro-I (Fig. 5C). Accordingly, increased XBP-1 splicing and GRP78 expression, the ER-stress
Fig. 4. NF-κB inhibition enhanced TM-induced Apoptosis. Enhancement of TM-induced cell death by IκBα mutant (IκBα-mt) (A,B). (A) PC3 cells were transfected with IκBα-mt plasmid (6 μg) for 24 h and then exposed to TM (5 μg/ml) for another 24 h. After cell lysis, total lysate was subjected to immune-blot analysis with antibodies to endogenous IκBα (enIκBα) and exogenous IκBα (exIκBα). (B) After 24 h of transfection with IκBα-mt, cells were further incubated with TM (0.5, 1, 2, and 5 μg/ml) for 48 h. Cell proliferation was recorded by CCK-8 kit. (C) Cell cycle arrest by NF-κB inhibition and TM. IκBα-mt transfected PC3 cell cycle arrested in the presence or absence of TM (5 μg/ml). Cells were seeded in 6 well plate overnight and then transfected with 2 μg of IκBα-mt plasmid for 24 h followed by TM treatment (5 μg/ml). Cell cycle arrest was analyzed by flow cytometry, showing the DNA content at the sub-G1 phase of the cell cycle. Microscopic cell morphology was also observed. (D) Decrease of anti-apoptosis factor bcl-2 decreased by combined-treatment of Pro-I and TM. Cells were incubated with TM with or without Pro-I for 24 h. After lysis, the lysate was applied to western blotting using bcl-2 antibody. Bcl-2 fold induction was normalized and shown as a graph.
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Fig. 5. Pro-I enhanced TM-induced CHOP expression independently of ER-stress. (A) Effect of Pro-I and TM on XBP-1 splicing. Cells were pretreated with Pro-I (1 μM) for 30 min before incubation with TM (5 μg/ml) for the indicated times. Total mRNA was extracted using Trizol reagent and XBP-1 splicing was determined by RT-PCR as described in Materials and methods. (B) TM induces expression of both CHOP and GRP78 ER-stress markers. Cells treated with TM (5 μg/ml) for various times as indicated were lysed for application to western blotting. (C) Specific enhancement of CHOP but not GRP78 expression by Pro-I in PC3 cells treated with TM. Cells incubated with Pro-I (1 μM) for 30 min were further exposed to TM (5 μg/ml) for 12 h or 24 h. Cell lysate was subjected to western blot analysis with specific antibodies to CHOP, GRP78, IRE-1α, PERK and their phospho-proteins. (D–F) Blocking CHOP expression reduced TM- and Pro-I-induced apoptosis. (D) Cells were transfected with control siRNA or CHOP siRNA at 100 nM for 24 h and then further exposed to Pro-I (1 μM) and TM (5 μg/ml) for 24 h. Cell lysate was prepared and subjected to western blot using specific antibodies to CHOP. (E) Cell cycle analysis. Cells were grown in 6 well plate overnight and then transiently transfected with control siRNA or CHOP siRNA at 5 μM for 24 h, followed by further incubation with Pro-I (1 μM) and TM (5 μg/ml). Whole cultural medium and cells were collected for application to flow cytometry analysis as described in Materials and methods. (F) Reduction in DNA fragmentation by CHOP knockdown in cells treated with both Pro-I and TM. Cells as in (D) were harvested and lysed for application to agarose gel electrophoresis as described in Materials and methods. M means 1 kb DNA ladder Marker.
markers, could not be seen even when the cells were co-treated with both Pro-I and TM (Fig. 5A and C, respectively). On the other hand, CHOP expression was enhanced by combined treatment of Pro-I and TM (Fig. 5C). These results suggest that Pro-I enhance CHOP expression downstream of ER-stress, leading to the increased cell apoptosis. 3.5. Blocking CHOP expression reduces TM/Pro-I-induced apoptosis In order to confirm that CHOP is required for Pro-I-induced chemosensitization, cells were transfected with siRNA of CHOP
followed by Pro-I and TM treatment (Fig. 5D). It was shown that bcl-2 anti-apoptotic protein was decreased by the co-treatment of Pro-I and TM (Fig. 4D), however, there was no difference in its expression between control and CHOP siRNA treatment (data not shown). This suggests that CHOP-mediated apoptosis might not be associated with bcl-2 even though bcl-2 was decreased by simultaneous treatment of the two compounds to the cells. Subsequent analysis of cell cycle showed that CHOP knockdown by siRNA significantly reduced the amount of the cells in sub-G1 phase when the cells were treated with both Pro-I and TM (Fig. 5E). In support of this result, DNA fragmentation was also reduced by CHOP siRNA in
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Fig. 6. Simplified model for Pro-I chemo-sensitization. Pro-I enhances TM-induced apoptosis through both inactivation of NF-κB and induction (or inhibition of degradation) of CHOP expression.
cells treated with both compounds (Fig. 5F). These results suggest that CHOP act as a mediator of enhanced chemosensitization of PC3 prostate cancer cells treated with Pro-I and TM.
apoptotic processes involve the ER-stress-induced expression of transcriptional factor CHOP/GAD153 or activation of caspase 12 [29,30], both NF-κB and CHOP might be regulated through IRE-1α signaling pathway. Pro-I inhibited the TM-induced NF-κB activation but enhanced the CHOP expression, consequently leading to the increased apoptotic cell death. Given that IRE-1α phosphorylation by TM could not be changed even after Pro-I treatment (Figs. 3A and 5C), it is speculated that Pro-I directly affected proteasome-mediated IκBα degradation (Fig. 3B). This possibility could be supported by the observation demonstrating that Pro-I did not affect the XBP-1 splicing closely associated with IRE-1α activation (Fig. 3A). PERK phosphorylation also did not change even when Pro-I was treated to the cells in the presence of TM (Fig. 5C). Because CHOP expression could be regulated both through IRE-1α and PERK, Pro-I was at first not expected to affect CHOP. Unexpectedly, however, Pro-I enhanced the expression of CHOP while there could not be seen any effect on GRP78, an ER resident chaperone protein (Fig. 5C). Based on the observation that CHOP mRNA level did not change regardless of Pro-I in TM-pretreated PC3 cells (data not shown), it could be suggested Pro-I affect CHOP expression at the translational or protein degradation level. Since Pro-I is an inhibitor of proteasome, it is expected that Pro-I regulates both NF-κB and CHOP by proteomic degradation, consequently leading to the enhanced chemosensitization of TMtreated PC3 prostate cancer cells. Taken together, our results suggest that NF-κB, but not other signaling pathways including Akt, MAPK and p38, could be a strong target for the enhancement of chemosensitization and development of anticancer chemotherapeutics, particularly in androgen-independent PC3 prostate cancer cells.
4. Discussion Inducible activation of NF-κB inhibits the apoptotic response to chemotherapy [27]. Activation of NF-κB via phosphorylation of an inhibitor protein IκBα leads to the degradation of IκBα through the ubiquitin–proteasome pathway [28]. The effect of proteasome inhibition on chemotherapy response in human cancer has been investigated in many studies involving cell cycle control, tumor growth, and induction of apoptosis [5]. In this study, we provided more evidence demonstrating that NF-κB, but not MAPK, Akt or p38, could be a strong target for the treatment of PC3 prostate cancer cells. NF-κB inhibition by Pro-I was found to significantly enhance TMinduced apoptosis of prostate cancer cells, as shown by cell proliferation and cell cycle analysis (Fig. 1A and D). Proteasome inhibition by Pro-I increased TM-elicited apoptotic signaling such as CHOP expression (Fig. 5) and caspase 12 activation (Fig. 1C), leading to caspase-3 activation and DNA fragmentation (Fig. 1B and C). Since it has been reported that caspase-12 is specifically activated after induction of ER-stress [18,19], these results indicate that ER-stress is involved in Pro-I chemosensitization of PC3 cells. Moreover, based on the observation showing that inhibitors of other signaling pathways, such as p38, MAPK and Akt [20,21], did not affect cell proliferation although the inhibitors were functional in the cells (Fig. 2), all these results strongly suggest that NF-κB could be an efficient target for PC3 prostate cancer cell death. The effect of Pro-I on enhancing the TMinduced apoptosis could be further supported by using IκBα-mt. Transient transfection of PC3 cells with IκBα-mt followed by TM treatment further increased the cell death compared to TM alone (Fig. 4B and C). Bcl-2 expression was also dramatically decreased by the combined treatment of Pro-I and TM (Fig. 4D), suggesting that mitochondrial apoptosis could also be partially involved in this Pro-I enhanced chemosensitization, however, more investigation would be required for the detailed interaction of ER-stress and mitochondrial function for this increased cell death. The effect of Pro-I on TM-induced PC3 cell death could be simplified as in Fig. 6. TM induces ER-stress, leading to the activation of NF-κB and increased expression of pro-apoptotic protein CHOP. Based on the reports demonstrating that many apoptotic and/or anti-
Acknowledgments This work was supported by the World Class Institute (WCI) Program, Nuclear Research Program (2009–0073109) of the National Research Foundation of Korea (NRF), Global Partnership Program of Korea Foundation for International Cooperation of Science and Technology (M60602000001-06E0200-00100), KRIBB Research Initiative Program, all grants from the Ministry of Education, Science and Technology (MEST), National R&D Program for Cancer Control (0820260) from the Ministry of Health & Welfare, Korea. References [1] I. Alkalay, A. Yaron, A. Hatzubai, A. Orian, A. Ciechanover, Y. Ben-Neriah, Proc. Natl Acad. Sci. USA 92 (23) (1995) 10599. [2] C.Y. Wang, M.W. Mayo, A.S. Baldwin Jr., Science 274 (5288) (1996) 784. [3] A. Arlt, J. Vorndamm, M. Breitenbroich, U.R. Folsch, H. Kalthoff, W.E. Schmidt, H. Schafer, Oncogene 20 (7) (2001) 859. [4] A.H. Dantzig, D.P. de Alwis, M. Burgess, Adv. Drug Deliv. Rev. 55 (1) (2003) 133. [5] H. Ostrowska, Cell. Mol. Biol. Lett. 13 (3) (2008) 353. [6] R.Z. Orlowski, D.J. Kuhn, Clin. Cancer Res. 14 (6) (2008) 1649. [7] A.D. Elbein, Annu. Rev. Biochem. 56 (1987) 497. [8] R. Kramer, T.K. Weber, R. Arceci, N. Ramchurren, W.V. Kastrinakis, G. Steele Jr., I.C. Summerhayes, Br. J. Cancer 71 (4) (1995) 670. [9] G. Wang, Z.Q. Yang, K. Zhang, Am. J. Transl. Res. 2 (1) (2010) 65. [10] K. Bedard, N. MacDonald, J. Collins, A. Cribb, Basic Clin. Pharmacol. Toxicol. 94 (3) (2004) 124. [11] J. Hacki, L. Egger, L. Monney, S. Conus, T. Rosse, I. Fellay, C. Borner, Oncogene 19 (19) (2000) 2286. [12] S.J. Marciniak, C.Y. Yun, S. Oyadomari, I. Novoa, Y. Zhang, R. Jungreis, K. Nagata, H.P. Harding, D. Ron, Genes Dev. 18 (24) (2004) 3066. [13] V.I. Rasheva, P.M. Domingos, Apoptosis 14 (8) (2009) 996. [14] W. Yang, J. Monroe, Y. Zhang, D. George, E. Bremer, H. Li, Cancer Lett. 243 (2) (2006) 217. [15] W. Liu, M. Kato, M. Itoigawa, H. Murakami, M. Yajima, J. Wu, N. Ishikawa, I. Nakashima, J. Cell. Biochem. 83 (2) (2001) 271. [16] Y. Li, R.F. Schwabe, T. DeVries-Seimon, P.M. Yao, M.C. Gerbod-Giannone, A.R. Tall, R.J. Davis, R. Flavell, D.A. Brenner, I. Tabas, J. Biol. Chem. 280 (23) (2005) 21763. [17] K. Pan, H. Wang, W.L. Liu, H.K. Zhang, J. Zhou, J.J. Li, D.S. Weng, W. Huang, J.C. Sun, X.T. Liang, J.C. Xia, Immunobiology 214 (5) (2009) 350. [18] T. Nakagawa, H. Zhu, N. Morishima, E. Li, J. Xu, B.A. Yankner, J. Yuan, Nature 403 (6765) (2000) 98. [19] N. Morishima, K. Nakanishi, H. Takenouchi, T. Shibata, Y. Yasuhiko, J. Biol. Chem. 277 (37) (2002) 34287.
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Cellular Signalling j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c e l l s i g
Proteasome inhibitor-I enhances tunicamycin-induced chemosensitization of prostate cancer cells through regulation of NF-κB and CHOP expression Pham Thi Thu Huong a, Dong-Oh Moon a, Sun Ok Kim a, Kyoon Eon Kim b, Sook Jung Jeong a, Ki Won Lee c,f, Kyung Sang Lee d, Jae Hyuk Jang a, Raymond Leo Erikson e, Jong Seog Ahn a,⁎, Bo Yeon Kim a,f,⁎⁎ a
Chemical Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 685-1 Yangcheonri, Ochangeup, Cheongwongun, 363-883, Republic of Korea Department of Biochemistry, College of Natural Sciences, ChungNam National University, 220 Gung-dong, Yuseong-gu, Daejeon, 305-764, Republic of Korea Department of Agricultural Biotechnology, Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921, Republic of Korea d Laboratory of Metabolism, National Cancer Institute, NIH, Rockville Pike, Bethesda, MD 20892-4258, USA e Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA f World Class Institute, Korea Research Institute of Bioscience and Biotechnology, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 8 December 2010 Accepted 13 January 2011 Available online 27 January 2011 Keywords: Proteasome inhibitor-1 Tunicamycin ER-stress NF-κB CHOP
a b s t r a c t Although endoplasmic reticulum (ER) stress induction by some anticancer drugs can lead to apoptotic death of cancer cells, combination therapy with other chemicals would be much more efficient. It has been reported that proteasome inhibitors could induce cancer cell death through ER-stress. Our study, however, showed a differential mechanism of proteasome inhibitor-I (Pro-I)-induced cell death. Pro-I significantly enhanced apoptotic death of PC3 prostate cancer cells pretreated with tunicamycin (TM) while other signaling inhibitors against p38, mitogen activated kinase (MEK) and phosphatidyl-inositol 3-kinase (PI3K) did not, as evidenced by cell proliferation and cell cycle analyses. NF-κB inhibition by Pro-I, without direct effect on ER-stress, was found to be responsible for the TM-induced chemosensitization of PC3 cells. Moreover, TM-induced/enhancer-binding protein (C/EBP) homologous protein (CHOP) expression was enhanced by Pro-I without change in GRP78 expression. CHOP knockdown by siRNA also showed a significant decrease in Pro-I chemosensitization. All these data suggest that although TM could induce both NF-κB activation and CHOP expression through ER-stress, both NF-κB inhibition and increased CHOP level by Pro-I are required for enhanced chemosensitization of PC3 prostate cancer cells. Thus, our study might contribute to the identification of anticancer targets against prostate cancer cells. © 2011 Elsevier Inc. All rights reserved.
1. Introduction NF-κB is activated by a variety of signals through mechanisms that result in phosphorylation and proteasomic degradation of the inhibitory IκBα protein [1]. It has been reported that IκBα mutantmediated inhibition of NF-κB nuclear translocation enhanced apoptotic killing of human fibro-sarcoma and pancreatic carcinoma cells by certain chemotherapeutic drugs [2,3]. Regarding the IκBα regulation, many chemical agents, referred to as biological response or resistance modifiers, have been demonstrated to alter chemosensitivity in refractory tumor cells and are potentially useful in clinical cancer therapeutics [4]. Although ubiquitin–proteasome pathway is an efficient therapeutic target for cancer treatment [5,6],
⁎ Correspondence to: J.S. Ahn, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 685-1 Yangcheonri, Ochangeup, Cheongwongun, 363-883, Republic of Korea. Tel.: + 82 43 240 6160. ⁎⁎ Correspondence to: B.Y. Kim, World Class Institute, Korea Research Institute of Bioscience and Biotechnology, Ochangeup, Republic of Korea. Tel.: + 82 43 240 6163. E-mail addresses: [email protected] (J.S. Ahn), [email protected] (B.Y. Kim). 0898-6568/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2011.01.010
and recently developed proteasome inhibitors have been potent anticancer agents for various cancers, their underlying molecular mechanism remains to be elucidated. Recently, endoplasmic reticulum (ER) has emerged a prominent target for the development of chemotherapeutics against diverse diseases including diabetes and cancer. Tunicamycin (TM), a nucleoside antibiotic, inhibits the first step in the biosynthesis of N-linked oligosaccharides in cells, resulting in the ER stress induction and apoptotic cell death by unfolded protein response (UPR) in certain cancer cells [7–11]. CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), also called GADD153, implicated in the regulation of processes relevant to energy metabolism, cellular proliferation, differentiation, and expression of cell-type-specific genes, is primarily pro-apoptotic and is one of the highly inducible genes during ER stress [12,13]. In many cases of ER stress, induction or over-expression of CHOP sensitizes cells to ER stress-induced apoptosis, whereas CHOP deletion protects the cells from apoptosis. The pro-apoptotic effect of CHOP in ER stress may be mediated by the induction of growth arrest and DNA damage gene 34 (GADD34) that dephosphorylates eukaryotic translation initiation factor 2α (eIF2α) [12]. These results suggested that CHOP regulation could be critical for the treatment of cancer.
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Concerned with the proteasome regulation, there are multiple pathways activated in prostate cancer cells [14]. It has been reported that p38 MAP kinase pathway is required for transcriptional induction by NF-κB [15] and that proteasome inhibitors indeed activated p38 MAPK in prostate cancer models [16,17]. Moreover, proteasome mediated ER tress induction elicited apoptotic proteins such as GRP78 and caspase-12 activations as well as CHOP [18]. Caspase-12 deficient cells are resistant to inducers of ER stress, suggesting that caspase12 is crucial in ER stress-induced apoptosis [18,19]. On the other hand, PI3K-Akt pathway plays a central role in the development and progression of prostate cancer cell [20], activated Akt translocating to the nucleus and mediating various targets for cell survival. Akt also phosphorylates IκBα, inducing the nuclear translocation of NF-κB for transcriptional upregulation of anti-apoptotic genes [20,21]. In this study, we attempted to find out a potential molecular target for overcoming the chemo-resistance in prostate cancer. It was found that chemosensitization of PC3 prostate cancer cells was strongly enhanced by the combined treatment of both tunicamycin and proteasome inhibitor-1 (Pro-I) to the cells. This study indicated that simultaneous targeting of both CHOP and NF-κB downstream of ER stress signaling could be promising for prostate cancer treatment.
and 100 μg/ml streptomycin, and incubated at 37 °C in a humidified atmosphere of 5% CO2. Cells were seeded on 100 mm dishes one day before transfection with 6 μg of IκBα mutant plasmid using Lipofectamin 2000 system. Transfected cells were cultured in complete medium for 24 h, treated with TM (5 μg/ml) for 24 h, and collected for further analyses. 2.2. Antibodies and reagents
2. Materials and methods
Antibody to caspase-12 was from StressGen (Ann Arbor, MI. 48108), caspase-3 was obtained from Imgenex (San Diego, CA 92121). Antibodies to phospho-IκBα, p-Akt1, GAPDH, p38, p-p38, GRP78, and CHOP were purchased from Cell Signaling (Beverley, MA). Antibodies to IκBα, Akt1, p55, p65 and Bcl-2 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Proteasome inhibitor-I (Pro-I) was from Calbiochem (San Diego, CA). Lipofectamine 2000 was obtained from Chemicon (USA and Canada), RPMI1640 and fetal bovine serum were purchased from GIBCO-BRL (Grand Island, NY). Tunicamycin (TM), SB202190, PD98059, wortmannin were purchased from CalBiochem. Polyvinylidene difluoride (PVDF, 0.22 μm) membrane was obtained from Bio-Rad (Hercules, CA, USA). For EMSA, [γ-32P] ATP isotope was from NEN, Dupont (Boston, MA, USA). All the other reagents were obtained from Sigma (St. Louis, MO, USA).
2.1. Cell culture and transfection
2.3. Plasmids
Human prostate cancer PC3 cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin
IκBα mutant was generated by point mutation of the wild-type plasmid and confirmed by DNA sequencing.
Fig. 1. Pro-I enhanced TM-induced apoptosis of PC3 prostate cancer cells. (A) Pro-I increased TM-induced cell death. PC3 cells (4 × 104 cells/ml) were seeded in 96 well-plate overnight and then pretreated with Pro-I at various concentrations (0, 0.5, 1, and 3 μM) for 30 min before treatment with TM for 48 h. Cell proliferation was measured by CCK-8 Kit. (B) Enhancement of caspase activation by co-treatment of Pro-I and TM. Cells were exposed to Pro-I in the presence or absence of TM for 24 h. Cell lysates were subjected to western blotting analysis and immune-blotted with antibodies specific to caspase 12 and caspase 3. (C) Enhanced DNA fragmentation by Pro-I and TM co-treatment. Cells as in (B) were lysed and subjected to agarose gel analysis. M; 1 kb DNA ladder marker. (D) Flow cytometry analysis of Pro-I enhancement of apoptosis in PC3 cells through arresting sub-G1 phase of the cell cycle. Cells were seeded in 6 well plates and then challenged for 30 min with Pro-I (1 μM) before treatment with TM (5 μg/ml). (D1) After treatment with TM and Pro-I for 24 h and 48 h, cell cycle arrest was analyzed using Cycle Test™ Plus DNA Reagent Kit. (D2) Dead cells are counted and positive for Annexin V-FITC staining (D2). Under the same condition, cell morphology (D3) and the propidium iodide (PI)-stained DNA (D4) were recorded. Cells were stained with fluorescent DNA-binding dye Hoechst 33342 for 10 min at room temperature, visualized and analyzed by fluorescence microscopy. DNA condensation and fragmentation (arrow) were increased in cells exposed to both TM (5 μg/ml) and Pro-I (1 μM).
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Fig. 1 (continued).
2.4. Proliferation assays Cells (4 × 104 cells/ml) in 96 well plate (100 μl/well) were treated with Pro-I for 30 min followed by incubation with TM (5 μg/ml) for 48 h. Ten microliters of CCK solution was added to each well and the plates were further incubated at 37 °C for 2.5 h. Utilizing Dojindo's highly water-soluble tetrazolium salt, the absorbance was measured at 450 nm with a reference wavelength at 650 nm using a microplate reader MR700 (Dynatech Inc., Chantilly, VA, USA).
cytometric analysis was performed using a FACSCalibur (Becton Dickinson, San Jose, CA). 2.6. Hoechst 33342 (HO)/propidium iodide (PI) Cells were seeded in 96-well plate and treated with Pro-I in the presence or absence of TM. Cells were stained with fluorescent DNAbinding dye Hoechst 33342 for 10 min at room temperature and were visualized and analyzed by fluorescence microscopy. 2.7. Western blotting
2.5. Cell cycle analyses To analyze cell cycle arrest, flow cytometry analysis was carried out using a FACScan cell sorter (Becton Dickinson, San Jose, CA), and data were analyzed by the Flowjo program (Tree Star, San Carlos, CA). In order to analyze the percentage of apoptotic cells, all cultural cells were harvested and washed twice with cold PBS. The collected cells were resuspended in annexin-V binding Ca2+ buffer in annexin-V-FITC staining solution (1.0 μg/ml) and incubated for 15 min at room temperature in the dark. Flow
After washing with cold PBS buffer (pH 7.4), cells were lysed with ice-cold lysis buffer [50 mM Tris–HCl, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 20 mM NaF, 100 mM Na3VO4, 0.5% NP40, 1% Triton X100, 1 mM phenylmethylsulfonyl fluoride (pH 7.4)] containing freshly added protease inhibitor mixture (Protease Inhibitor Cocktail Set III; Calbiochem, La Jolla, CA) on ice for 30 min. Whole cell lysates were centrifuged at 14,000 rpm for 15 min, and then the upper part of solution was transferred into a new tube. For western blot analysis, appropriate amount of cell lysate containing 50 μg protein was
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subjected to 10–14% SDS-PAGE, then transfered onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). Membrane was immunoblotted with specific antibodies at 4 °C for the detection with chemiluminescence kit (ECL; Amersham Life Sciences Inc.). 2.8. Small interfering RNAs CHOP small interfering RNA (siRNA) sequences were: sihChop F: 5′-AAGAA CCA GCA GAG GTC ACA A-3′. SihChop R: 5′-AAGA CCA GCA GAG GTC ACA A-3′ which were synthesized by Bioneer Ltd, Korea. The LacZ siRNA was used as a siRNA control. In brief, one day prior to the transfection, PC-3 cells were seeded without antibiotics at a density of 30% to 40%. CHOP siRNAs (25 nmol/L) were transfected into cells using N-TER™ Nanoparticle siRNA Trasfection System (Sigma), in which the concentration of N-TER™ peptide was reduced to one-third of the recommended volume to limit toxic effects. After 24 h of transfection, cells were treated with TM (5 μg/ml) for another 24 h and then harvested. 2.9. Reverse transcription-polymerase chain reaction (RT-PCR) Cells were treated with TM (5 μg/ml) or transfected with IκBα mutant plasmid before TM-treatment. After appropriate time, RNA was extracted from the cells using Trizol reagent (Gibco-BRL). First-strand cDNA synthesis was performed with the SuperScript first-strand synthesis system (Invitrogen, Carlsbad, CA, USA). To amplify GRP78 mRNA, PCR was performed 22 cycles by 95 °C, 30 s; 55 °C, 30 s; and 72 °C, 1 min using primers F: 5′-GTT CTT CAA TGGCAA GGA ACC ATC-3′ and R: 5′-CCA TCC TTT CGA TTT CTT CAG GTG-3′. To amplify CHOP (GADD153) mRNA, PCR was followed 25 cycles for 94 °C — 30 s; 60 °C — 30 s; and 72 °C — 1 min using primers F: 5′-GCA CCT CCC AGA GCC CTC ACT CTC C-3′ and R: 5′-GTC TAC TCC AAG CCT TCC CCC TGC G-3′. And to amplify XBP1 mRNA, PCR was performed 35 cycles by 94 °C, 30 s; 55 °C, 30 s; and 72 °C, 30 s using primers F: 5′-ACA CGC TTG GGG ATG AAT GC3′ and R: 5′-CCA TGG GAA GAT GTT CTG GG-3′. Control primers for GAPDH were 5′-TAG ACG GGA AGC TCA CTG GC-3′ and 5′-AGG TCC ACC ACC CTG TTG CT-3′ for 20 cycles by 94 °C, 30 s; 60 °C, 30 s; and 72 °C, 30 s. Three fragments, representing spliced (XBP-1 s, 215 bp), unspliced (XBP-1u, 241 bp) and a hybrid (XBP-1 h, 267 bp), were produced and detected by running on a 2% agarose gel and staining with ethidium bromide. The other PCR products were loaded on a 1–1.5% agarose gel. 2.10. Electrophoretic mobility Shift Assay (EMSA) EMSA analysis was performed as described by Janssen and Sen [22]. In brief, PC3 cells were grown at 5 × 105 cells/ml in 100 mm dishes, pretreated 30 min with Pro-I in the presence or absence of TM. Cells were lysed on ice for 15 min in a hypotonic solution containing 10 mM HEPES–KOH (pH 7.8), 10 mM KCl, 2 mM MgCl2, 0.1 mM EDTA (sodium salt), 0.2 mM NaF, 0.2 mM Na3VO4, 0.4 mM phenylmethylsulfonyl fluoride (PMSF), leupeptin (10 μg/ml), 1 mM dithiothreitol (DTT) and 0.15% NP-40. The lysate was centrifuged at 16,000 rpm for 1 min at 4 °C and the resulting nuclear pellet was resuspended in ice-cold extraction buffer [50 mM HEPES–KOH (pH 7.8), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 0.2 mM NaF, 0.2 mM Na3VO4, 0.4 mM PMSF, 1 mM DTT and 10% glycerol] and incubated for 30 min at 4 °C with occasional vortex. The nuclear lysate was then centrifuged at 16,000 rpm for 30 min at 4 °C and the resulting supernatant was stored at −80 °C or immediately subjected to EMSA analysis. An oligonucleotide having NF-κB binding site (3.5 pmol) (Santa Cruz) was incubated for 10 min at 37 °C in 10 μl of reaction solution containing 10 μCi of [γ-32P] ATP, 5 U of T4 polynucleotide kinase and 1× kinase buffer (supplied with the kinase). The labeling reaction was terminated by the addition of 100 mM EDTA, followed by centrifugation through a Sephadex G-25 column to remove unincorporated 32P. The 32P-labeled oligonucleotide was then stored at −80 °C until use. For EMSA assay, nuclear protein extract (10 μg) was
incubated with the gel shift binding buffer for 10 min at room temperature and then with 32P-labeled probe in each reaction sample according to the manufacture's protocol (Gel shift assay system, Promega Kit., USA). For supershift analysis, the nuclear extract was incubated with specific antibodies (2 μg) for 30 min at room temperature prior to the addition of the labeled oligonucleotide. The binding reaction was terminated by the addition of electrophoresis sample buffer, and the samples were fractionated on 5% non-denaturing polyacrylamide gels in 0.5× Tris-boric acid-EDTA (TBE) buffer. The gels were dried and subjected to autoradiography. 2.11. DNA fragmentation assay After treatment with the indicated drugs, cells were lysed in 20 μ l lysis buffer (0.8% Sodium lauryl sarcarosin, 20 mM EDTA, 100 mM Tris pH8.0). The cell extract was treated with 10 μ l of RNase A (1 mg/ml) for 30 min at 37 °C then incubated for 1.5 h at 50 °C in the presence of 10 μ l of proteinase K (20 mg/ml). After mixing with 10× Loading dye buffer, the lysates were loaded on a 2% agarose gel electrophothesis at 100 V for 1.5 h. Gel was stained and DNA fragment was detected with ethidium bromide. 3. Results 3.1. Pro-I enhanced TM-induced apoptosis of PC3 prostate cancer cells In order to determine the effect of Pro-I on TM-induced ER-stress and apoptosis, PC3 prostate cancer cell proliferation was exploited after treatment with tunicamycin (TM) and Pro-I. Cells were dosedependently treated with TM for 48 h in the presence or absence of Pro-I at various concentrations. It was observed that Pro-I even at 0.5 μM significantly reduced the cell proliferation when treated with TM together while Pro-I alone showed only moderate effect (Fig. 1A). Enhanced DNA fragmentation could also be obtained when the cells were simultaneously treated with both Pro-I and TM (Fig. 1B), which was in accordance with the activation of caspase-3 and ER-stress associated caspase-12 (Fig. 1C), suggesting the involvement of ERstress in this cell death signaling. Enhanced cell death by Pro-I was confirmed by flow cytometry analysis. Pro-I significantly enhanced both the sub-G1 phase and annexin-5-staining of the cells in the presence of TM (Fig. 1D, D1 and D2). Cell morphological change and nuclear staining with Hoechst 33342 (HO) further supported the chemosensitizing effect of Pro-I (Fig. 1D, D3 and D4). Given the report demonstrating that inhibition of Akt and p38 augmented proteasome inhibitor-mediated apoptosis [23], involvement of other signaling pathways in enhancing chemosensitization of PC3 cells was exploited. PC3 cells were treated with SB203580, PD98059 and wortmannin, inhibitors of p38, mitogen activated protein kinase (MAPK) and Akt, respectively, in the presence of TM for 48 h. As shown in Fig. 2A, these inhibitors did not show any significant enhancement of cell chemosensitivity (Fig. 2A), although SB203580 and wortmannin could completely block the TM-induced phosphorylation of p38 and Akt (Fig. 2B); there could not be seen, however, ERK phosphorylation. These data suggested that NF-κB might be the target of enhanced chemosensitization of TM-induced cancer cells. 3.2. TM-induced NF-кB activation through ER-stress signaling is inhibited by Pro-I Apoptosis is usually antagonized by NF-κB activation. In cancer cells, NF-κB is highly activated and some chemotherapeutic agents could block NF-κB signaling pathways [24]. ER-stress was also reported to induce NF-κB [25]. In order to determine whether ER-stress and NF-кB activation could be achieved by TM and could also be affected by Pro-I, PC3 cells were treated with TM for various times. In addition to the IRE-
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Fig. 2. Effect of other signaling regulators on PC3 cell proliferation. Cells (4 × 104 cells/ml) were seeded in 96 well plate overnight and pretreated for 30 min with various concentrations of SB203580, PD98059 or wortmannin before exposure to TM for 48 h. Cell proliferation was measured with CCK-8 kit (A). Bars represent means ± SE from a representative triplicate experiment. Effects of the inhibitors on the phosphorylation of the respective proteins, p38, ERK, and Akt were shown on the right (B) to determine whether the compounds were really active in the cells. Cells were exposed to SB203580 (10 μM), PD98059 (20 μM) and wortamanin (200 nM) for 30 min followed by incubation with TM (5 μg/ml) for 24 h. Cells were lysed and subjected to immuno-blot analysis with specific antibodies to p38, ERK and Akt.
Fig. 3. TM-induced NF-κB activation and IRE-1α phosphorylation are inhibited by Pro-I. (A) TM induced phosphorylation of IRE-1α and IκBα. Cells were treated with TM at 5 μg/ml and lysed at the indicated time points for application to western blotting. (B) Pro-I inhibited TM-induced IκBα phosphorylation and degradation. After incubation with Pro-I (1 μM) for various times (30, 60, and 120 min), cells were treated with TM (5 μg/ml) for 24 h. Cell lysate was subjected to immune-blot analysis with specific antibodies. (C) EMSA analysis for NF-κB activation. TM (5 μg/ml) was treated to the cells for 1–2 h or in the presence or absence of Pro-I (10 μM) pretreated for 30 min. Nuclear fraction (10 μg) was applied to EMSA analysis with or without an antibody specific to p50 (p50 AB) or p65 (p65 AB) (2 μg) for super shift (*).NF-κB fold induction was shown in the lower panel.
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1α phosphorylation, TM also increased the phosphorylation and degradation of IκBα in the cells (Fig. 3A). Pro-I, however, significantly reversed TM-induced IκBα phosphorylation and degradation (Fig. 3B). The effect of Pro-I on NF-κB was also confirmed by EMSA analysis. TMinduced NF-κB DNA binding activity was strongly inhibited by Pro-I, and among NF-κB subunits p65/p50 heterodimer was found to be involved as evidenced by supershift assay (Fig. 3C, upper). The result of NF-κB fold induction was also shown as a graph (Fig. 3C, lower). These results suggest that Pro-I could effect on ER-stress-mediated NF-κB signaling by targeting IκBα degradation. 3.3. NF-κB inhibition enhances TM-induced apoptosis To further confirm that NF-κB inhibition enhances TM-induced apoptosis, PC3 cells were transiently transfected with IκBα mutant (IκBα-mt) plasmid followed by challenging with TM for various times. IκBα-mt significantly reduced the phosphorylation of endogenous IκBα in both TM treated and non-treated conditions (Fig. 4A). Cell death was also increased by IκBα-mt in the presence of TM compared to TM alone (Fig. 4B). Enhanced chemosensitization by IκBα-mt was further confirmed by cell cycle analysis. Cotreatment of both IκBα-mt and TM significantly increased the sub-G1 phase of the cells as well as
the cell death (Fig. 4C, upper and lower, respectively). Similarly, the level of anti-apoptosis factor bcl-2 was decreased by combined treatment of Pro-I and TM and was shown as a fold induction, too (Fig. 4D). These data indicate that inhibition of NF-κB enhances TMinduced chemosensitization of PC3 cancer cells. 3.4. Pro-I enhanced TM-induced CHOP expression independently of ER-stress One of the primary effectors of ER stress-mediated cell death is CCAAT/enhancer-binding protein homologous protein (CHOP), also called GADD153 [26]. In our study, it is shown that CHOP also plays a crucial role in Pro-I induced chemosensitization. As TM induced the phosphorylation of ER-stress protein IRE-1α (Fig. 3A), XBP-1 splicing (Fig. 5A) and expression of CHOP and GRP78 (Fig. 5B) were also increased by TM. Phosphorylation of another ER-stress associated protein, Protein kinase-like endoplasmic reticulum eIF2α kinase (PERK), was also found to be slightly and transiently increased by TM. Interestingly, however, there was no change in the extent of TM-induced phosphorylation of IRE-1α and PERK between the cells treated with or without Pro-I (Fig. 5C). Accordingly, increased XBP-1 splicing and GRP78 expression, the ER-stress
Fig. 4. NF-κB inhibition enhanced TM-induced Apoptosis. Enhancement of TM-induced cell death by IκBα mutant (IκBα-mt) (A,B). (A) PC3 cells were transfected with IκBα-mt plasmid (6 μg) for 24 h and then exposed to TM (5 μg/ml) for another 24 h. After cell lysis, total lysate was subjected to immune-blot analysis with antibodies to endogenous IκBα (enIκBα) and exogenous IκBα (exIκBα). (B) After 24 h of transfection with IκBα-mt, cells were further incubated with TM (0.5, 1, 2, and 5 μg/ml) for 48 h. Cell proliferation was recorded by CCK-8 kit. (C) Cell cycle arrest by NF-κB inhibition and TM. IκBα-mt transfected PC3 cell cycle arrested in the presence or absence of TM (5 μg/ml). Cells were seeded in 6 well plate overnight and then transfected with 2 μg of IκBα-mt plasmid for 24 h followed by TM treatment (5 μg/ml). Cell cycle arrest was analyzed by flow cytometry, showing the DNA content at the sub-G1 phase of the cell cycle. Microscopic cell morphology was also observed. (D) Decrease of anti-apoptosis factor bcl-2 decreased by combined-treatment of Pro-I and TM. Cells were incubated with TM with or without Pro-I for 24 h. After lysis, the lysate was applied to western blotting using bcl-2 antibody. Bcl-2 fold induction was normalized and shown as a graph.
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Fig. 5. Pro-I enhanced TM-induced CHOP expression independently of ER-stress. (A) Effect of Pro-I and TM on XBP-1 splicing. Cells were pretreated with Pro-I (1 μM) for 30 min before incubation with TM (5 μg/ml) for the indicated times. Total mRNA was extracted using Trizol reagent and XBP-1 splicing was determined by RT-PCR as described in Materials and methods. (B) TM induces expression of both CHOP and GRP78 ER-stress markers. Cells treated with TM (5 μg/ml) for various times as indicated were lysed for application to western blotting. (C) Specific enhancement of CHOP but not GRP78 expression by Pro-I in PC3 cells treated with TM. Cells incubated with Pro-I (1 μM) for 30 min were further exposed to TM (5 μg/ml) for 12 h or 24 h. Cell lysate was subjected to western blot analysis with specific antibodies to CHOP, GRP78, IRE-1α, PERK and their phospho-proteins. (D–F) Blocking CHOP expression reduced TM- and Pro-I-induced apoptosis. (D) Cells were transfected with control siRNA or CHOP siRNA at 100 nM for 24 h and then further exposed to Pro-I (1 μM) and TM (5 μg/ml) for 24 h. Cell lysate was prepared and subjected to western blot using specific antibodies to CHOP. (E) Cell cycle analysis. Cells were grown in 6 well plate overnight and then transiently transfected with control siRNA or CHOP siRNA at 5 μM for 24 h, followed by further incubation with Pro-I (1 μM) and TM (5 μg/ml). Whole cultural medium and cells were collected for application to flow cytometry analysis as described in Materials and methods. (F) Reduction in DNA fragmentation by CHOP knockdown in cells treated with both Pro-I and TM. Cells as in (D) were harvested and lysed for application to agarose gel electrophoresis as described in Materials and methods. M means 1 kb DNA ladder Marker.
markers, could not be seen even when the cells were co-treated with both Pro-I and TM (Fig. 5A and C, respectively). On the other hand, CHOP expression was enhanced by combined treatment of Pro-I and TM (Fig. 5C). These results suggest that Pro-I enhance CHOP expression downstream of ER-stress, leading to the increased cell apoptosis. 3.5. Blocking CHOP expression reduces TM/Pro-I-induced apoptosis In order to confirm that CHOP is required for Pro-I-induced chemosensitization, cells were transfected with siRNA of CHOP
followed by Pro-I and TM treatment (Fig. 5D). It was shown that bcl-2 anti-apoptotic protein was decreased by the co-treatment of Pro-I and TM (Fig. 4D), however, there was no difference in its expression between control and CHOP siRNA treatment (data not shown). This suggests that CHOP-mediated apoptosis might not be associated with bcl-2 even though bcl-2 was decreased by simultaneous treatment of the two compounds to the cells. Subsequent analysis of cell cycle showed that CHOP knockdown by siRNA significantly reduced the amount of the cells in sub-G1 phase when the cells were treated with both Pro-I and TM (Fig. 5E). In support of this result, DNA fragmentation was also reduced by CHOP siRNA in
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Fig. 6. Simplified model for Pro-I chemo-sensitization. Pro-I enhances TM-induced apoptosis through both inactivation of NF-κB and induction (or inhibition of degradation) of CHOP expression.
cells treated with both compounds (Fig. 5F). These results suggest that CHOP act as a mediator of enhanced chemosensitization of PC3 prostate cancer cells treated with Pro-I and TM.
apoptotic processes involve the ER-stress-induced expression of transcriptional factor CHOP/GAD153 or activation of caspase 12 [29,30], both NF-κB and CHOP might be regulated through IRE-1α signaling pathway. Pro-I inhibited the TM-induced NF-κB activation but enhanced the CHOP expression, consequently leading to the increased apoptotic cell death. Given that IRE-1α phosphorylation by TM could not be changed even after Pro-I treatment (Figs. 3A and 5C), it is speculated that Pro-I directly affected proteasome-mediated IκBα degradation (Fig. 3B). This possibility could be supported by the observation demonstrating that Pro-I did not affect the XBP-1 splicing closely associated with IRE-1α activation (Fig. 3A). PERK phosphorylation also did not change even when Pro-I was treated to the cells in the presence of TM (Fig. 5C). Because CHOP expression could be regulated both through IRE-1α and PERK, Pro-I was at first not expected to affect CHOP. Unexpectedly, however, Pro-I enhanced the expression of CHOP while there could not be seen any effect on GRP78, an ER resident chaperone protein (Fig. 5C). Based on the observation that CHOP mRNA level did not change regardless of Pro-I in TM-pretreated PC3 cells (data not shown), it could be suggested Pro-I affect CHOP expression at the translational or protein degradation level. Since Pro-I is an inhibitor of proteasome, it is expected that Pro-I regulates both NF-κB and CHOP by proteomic degradation, consequently leading to the enhanced chemosensitization of TMtreated PC3 prostate cancer cells. Taken together, our results suggest that NF-κB, but not other signaling pathways including Akt, MAPK and p38, could be a strong target for the enhancement of chemosensitization and development of anticancer chemotherapeutics, particularly in androgen-independent PC3 prostate cancer cells.
4. Discussion Inducible activation of NF-κB inhibits the apoptotic response to chemotherapy [27]. Activation of NF-κB via phosphorylation of an inhibitor protein IκBα leads to the degradation of IκBα through the ubiquitin–proteasome pathway [28]. The effect of proteasome inhibition on chemotherapy response in human cancer has been investigated in many studies involving cell cycle control, tumor growth, and induction of apoptosis [5]. In this study, we provided more evidence demonstrating that NF-κB, but not MAPK, Akt or p38, could be a strong target for the treatment of PC3 prostate cancer cells. NF-κB inhibition by Pro-I was found to significantly enhance TMinduced apoptosis of prostate cancer cells, as shown by cell proliferation and cell cycle analysis (Fig. 1A and D). Proteasome inhibition by Pro-I increased TM-elicited apoptotic signaling such as CHOP expression (Fig. 5) and caspase 12 activation (Fig. 1C), leading to caspase-3 activation and DNA fragmentation (Fig. 1B and C). Since it has been reported that caspase-12 is specifically activated after induction of ER-stress [18,19], these results indicate that ER-stress is involved in Pro-I chemosensitization of PC3 cells. Moreover, based on the observation showing that inhibitors of other signaling pathways, such as p38, MAPK and Akt [20,21], did not affect cell proliferation although the inhibitors were functional in the cells (Fig. 2), all these results strongly suggest that NF-κB could be an efficient target for PC3 prostate cancer cell death. The effect of Pro-I on enhancing the TMinduced apoptosis could be further supported by using IκBα-mt. Transient transfection of PC3 cells with IκBα-mt followed by TM treatment further increased the cell death compared to TM alone (Fig. 4B and C). Bcl-2 expression was also dramatically decreased by the combined treatment of Pro-I and TM (Fig. 4D), suggesting that mitochondrial apoptosis could also be partially involved in this Pro-I enhanced chemosensitization, however, more investigation would be required for the detailed interaction of ER-stress and mitochondrial function for this increased cell death. The effect of Pro-I on TM-induced PC3 cell death could be simplified as in Fig. 6. TM induces ER-stress, leading to the activation of NF-κB and increased expression of pro-apoptotic protein CHOP. Based on the reports demonstrating that many apoptotic and/or anti-
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