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CYP1B1 prevents proteasome-mediated XIAP degradation by inducing PKCε activation and phosphorylation of XIAP
BBA - Molecular Cell Research 1866 (2019) 118553
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CYP1B1 prevents proteasome-mediated XIAP degradation by inducing PKCε activation and phosphorylation of XIAP Hyoung-Seok Baek, Yeo-Jung Kwon, Dong-Jin Ye, Eunah Cho, Tae-Uk Kwon, Young-Jin Chun
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College of Pharmacy and Center for Metareceptome Research, Chung-Ang University, Seoul 06974, Republic of Korea
ARTICLE INFO
ABSTRACT
Keywords: CYP1B1 4-OHE2 XIAP PKCε Ubiquitination Apoptosis
Cytochrome P450 1B1 (CYP1B1) is a key enzyme that catalyzes the metabolism of 17β-estradiol (E2) into catechol estrogens, such as 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2). CYP1B1 is related to tumor formation and is over-expressed in a variety of cancer cells. In particular, CYP1B1 is highly expressed in hormone-related cancers such as breast cancer, ovarian cancer, or prostate cancer compared to other cancers. However, the detailed mechanisms involving this protein remain unclear. In this study, we demonstrate that CYP1B1 affects X-linked inhibitor of apoptosis protein (XIAP) expression. When CYP1B1 was over-expressed in cells, there was significant increase in the XIAP protein level, whereas the XIAP mRNA level was not affected by CYP1B1 expression. Treatment with 4-OHE2, mainly formed by CYP1B1 activity, also increased XIAP protein levels, whereas treatment with 2-OHE2 did not have a significant effect. Treatment with 4-OHE2 significantly prevented proteasome-mediated XIAP degradation. In addition, phosphorylation of XIAP on serine 87, which is known to stabilize XIAP, was up-regulated by 4-OHE2, indicating that 4-OHE2 affects XIAP stability through XIAP phosphorylation. We also found that phosphorylation of protein kinase C (PKC)ε, which is required for XIAP phosphorylation, increased when cells were treated with 4-OHE2. In summary, our data show that CYP1B1 may play an important role in preventing ubiquitin-proteasome-mediated XIAP degradation through the activation of PKCε signaling in cancer cells.
1. Introduction Cytochrome P450 1B1 (CYP1B1), a member of the CYP family 1, has been implicated in tumor proliferation, metastasis, and survival [1–6]. Previously, we showed that CYP1B1 increases the activation of the Wnt/β-catenin signaling pathway through the induction of Sp1, which leads to cell growth and migration in breast cancer cells [2]. The principal role of CYP1B1 in cells is the hydroxylation of 17βestradiol (E2) to the catechol metabolites such as 2-hydroxyestradiol (2OHE2) and 4-hydroxyestradiol (4-OHE2) [7,8]. Several studies have shown that 4-OHE2 is a potential carcinogen for animals and humans [9–11]. Mueck et al. [12] showed that 4-OHE2 induces Bcl-2 and reduces cytochrome c levels in MCF-7 cells. However, 2-OHE2 was not found to have any significant effects. Furthermore, Gao et al. [13] demonstrated that only 4-OHE2 increases the expression of VEGF-A, which affects endothelial cell growth, angiogenesis, and suppression of apoptosis. It also induces the expression of HIF1-α, which regulates
cancer cell metabolism and migration. These reports provide evidence that 4-OHE2 plays a crucial role in carcinogenesis. X-linked inhibitor of apoptosis protein (XIAP) is a potent antiapoptotic factor, and it contains three amino-terminal BIR domains: BIR-1, BIR-2, and BIR-3. The BIR-2 domain binds to caspase-3 and caspase-7 and suppresses apoptosis, while the BIR-3 domain binds to caspase-9 and inhibits cell death [14,15]. XIAP is upregulated in a variety of cancer cells, and it increases the resistance to radiation- or chemotherapy-induced apoptosis [16–19]. Therefore, it may be important to regulate the expression of XIAP in order to obtain a better effect in cancer therapy. Phosphorylation of XIAP on serine 87 is important for the activity and stability of XIAP. A previous study has shown that the stability of XIAP significantly increases following the mutation of serine 87 to glutamate, possibly because it inhibits BIR1 dimerization and results in XIAP self-ubiquitination [20]. Protein kinase C (PKC) is a member of the family of protein kinases
Abbreviations: CYP1B1, cytochrome P450 1B1; XIAP, X-linked inhibitor of apoptosis protein; PKCε, Protein kinase C ε; TMS, 2, 2′, 4, 6′-tetramethoxystilbene; DMBA, 7, 12–dimethylbenz[a]-anthracene; 2-OHE2, 2-hydroxyestradiol; 4-OHE2, 4-hydroxyestradiol; DR4, death receptor 4 ⁎ Corresponding author at: College of Pharmacy and Center for Metareceptome Research, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 06974, Republic of Korea. E-mail address: [email protected] (Y.-J. Chun). https://doi.org/10.1016/j.bbamcr.2019.118553 Received 22 March 2019; Received in revised form 29 August 2019; Accepted 3 September 2019 Available online 04 September 2019 0167-4889/ © 2019 Published by Elsevier B.V.
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Fig. 1. Expression of CYP1B1 increases the XIAP protein level. (A–B) MCF-7 and HeLa cells were transfected with the pcDNA3.1_CYP1B1 vector for 48 h. (A) XIAP protein levels were measured using western blot analysis. (B) Real-time qPCR was performed to detect the expression of XIAP mRNA. The data represent the mean ± SD (n = 3). (C–D) MCF-7 and HeLa cells were transfected with CYP1B1 siRNA (30 nM) for 48 h. (C) XIAP protein levels were measured using western blot analysis. (D) Real-time qPCR was performed to detect the expression of XIAP mRNA. The data represent the mean ± SD (n = 3). (E) MCF-7 and HeLa cells were cotreated with TMS (5, 10, or 20 μM) and DMBA (10 μM) for 48 h. CYP1B1 and XIAP protein levels were measured using western blot analysis. (F) MCF-7 and HeLa cells were treated with TMS (5 or 10 μM) for 48 h. CYP1B1 and XIAP protein levels were measured using western blot analysis. (G) Clonogenic assay was performed in MCF-7 and HeLa cells after transfecting them with CYP1B1 siRNA (30 nM) for 4 d. The data represent the mean ± SD (n = 3). (H) MCF-7 and HeLa cells were treated with TMS (2.5, 5, 10, 20, or 40 μM) for 48 h. Cell viability was measured using CCK assay (n = 3). (I) MCF-7 and HeLa cells were treated with TMS (20 μM) for 48 h. Apoptosis assay was performed using a Muse annexin V kit. The data represent the mean ± SD (n = 3). ns = not significant; *p < 0.05.
that are involved in the phosphorylation of serine and threonine in target proteins. PKCs play an important role in various intracellular functions such as regulation of the inflammatory response, proliferation, metastasis, cell death, and differentiation [21–29]. Previously, it was reported that PKC stabilizes XIAP by inducing phosphorylation on serine 87 [30]. Pardo et al. [31] showed that PKCε over-expression induces XIAP protein levels, and this increase in the XIAP protein level may be correlated with a complex involving PKCε, B-Raf, and S6K2. In addition, it has been reported that the interaction between Raf-1 and XIAP prevents XIAP degradation through phosphorylation of XIAP [32]. In this study, we demonstrate that the proteasomal degradation of XIAP is prevented by 4-OHE2. We also show that phosphorylation of XIAP on serine 87 by 4-OHE2, which increases XIAP stability and activity, is tightly correlated with the activation of PKCε. Our results suggest that 4-OHE2, catalyzed by CYP1B1 may play a crucial role in preventing the degradation of XIAP and apoptosis by inducing PKCε activation.
mounting medium (sc-24,941) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Actin antibody (A300-491A, polyclonal) and ECS (DDDDK) antibody (A190-102A, polyclonal) were purchased from Bethyl (Montgomery, TX). PARP antibody (46D11, monoclonal) was purchased from Cell Signaling Technology (Beverly, MA). Phospho-XIAP (S87) polyclonal, PKCε polyclonal, and phosphoPKCε (S729) polyclonal antibodies were purchased from Flarebio (College Park, MD). All other chemicals were obtained from commercial sources. 2.3. Transient transfection
2. Material and methods
CYP1B1 siRNA (Qiagen, Venlo, Netherlands), PKCε siRNA (Santa Cruz), the HA-ubiquitin expression vector (a gift from Edward Yeh, Addgene plasmid #18712), the pcDNA3.1/Zeo+ vector containing the CYP1B1-encoding sequence, and the p3xFLAG_CMV10 vector containing the XIAP-encoding sequence were used for transfection. Cells were transfected with 30 nM siRNA or 3 μg plasmid with the Neon Transfection System (Life Technologies).
2.1. Cell culture
2.4. Generation of stable CYP1B1 knock-down MDA-MB-231 cells
The MCF-7 and MDA-MB-231 human breast cancer cell lines and the HEK293T human embryonic kidney cell line were obtained from the American Type Culture Collection (Manassas, VA). The HeLa human cervical cancer cell line and PC-3 human prostate cancer cell line were obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea). MCF-7, MDA-MB-231, HeLa, and PC-3 cell lines were cultured in RPMI 1640 supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. HEK293T cell line was cultured in DMEM supplemented with 10% (v/v) heatinactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/ mL streptomycin. Cells were incubated at 37 °C in a humidified atmosphere of 5% CO2. For treatment of MCF-7, MDA-MB-231, HeLa, and PC-3 cells with 4-OHE2 or 2-OHE2, cells were seeded in growth media as a monolayer in 60-mm dish plates. After 24 h, the medium were changed to phenol red-free RPMI 1640 with 10% (v/v) charcoalstripped FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were incubated for 72 h, and then, the cells were treated with 4-OHE2 or 2-OHE2 for 48 h.
The pLKO.1 puro (a gift from Bob Weinberg, Addgene plasmid # 8453) [33], pMD2.G, and psPAX2 (a gift from Didier Trono, Addgene plasmid # 12259 and #12260) plasmid vectors were obtained from Addgene. HEK293T cells were co-transfected with the CYP1B1-pLKO.1 puro, pMD2.G, and psPAX2 vectors. After 48 h, the medium containing lentiviral CYP1B1 shRNA was collected, and MDA-MB-231 cells were subsequently treated with lentiviral soup containing CYP1B1 shRNA and polybrene (8 μg/mL) for 24 h. The CYP1B1 knock-down MDA-MB231 cells were selected using puromycin (1 μg/mL). 2.5. Quantitative PCR Total RNA was extracted using RiboEx™ solution (GeneALL, Seoul, Korea). Total RNA (1 μg) was reverse-transcribed at 37 °C for 1 h in a 25 μL total volume containing 5× RT buffer, 10 mM dNTPs, 40 U RNase inhibitor, 200 U Moloney murine leukemia virus reverse transcriptase, and 100 pmol oligo-dT primer. qPCR was performed using the RotorGene SYBR1 PCR Kit, as recommended by the manufacturer, and analyzed using QIAGEN Rotor-Gene Q Series software. Each reaction contained 10 μL of 2× SYBR1 Green PCR Master Mix, 1 μM oligonucleotide primers, and 20 ng of cDNA in a final volume of 20 μL. Amplification was conducted as follows: one cycle at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/ extension at 56 °C for 10 s. The following primer sets were used for qPCR: XIAP, 5′-CTTGGGCACAGAGAGCA-3′ and 5′-AACTGCTCATCAC CCCATTC-3′, and 18S rRNA, 5′-GTAACCCGTTGAACCCCATT-3′ and 5′-CCATCCAATCGGTAGTAGCG-3′.
2.2. Reagents 7,12-Dimethylbenz[a]anthracene (DMBA), embelin, docetaxel, 4OHE2, and 2-OHE2 were purchased from Sigma-Aldrich (St. Louis, MO). 2,2′,4,6′-Tetramethoxystilbene (TMS) was kindly provided by Dr. Sanghee Kim (Seoul National University, Seoul, Korea). All chemicals were prepared in dimethyl sulfoxide, stored as small aliquots at −20 °C, and then diluted as needed in cell culture medium. FBS and RPMI 1640 medium were purchased from HyClone (Logan, UT). Charcoal-stripped FBS was purchased from Tissue Culture Biologicals (Long Beach, CA). Phenol red-free RPMI 1640, bicinchoninic acid (BCA) protein assay kits, and the Neon transfection system were purchased from ThermoFisher Scientific (Waltham, MA). D-Plus™ ECL solution was purchased from Dongin LS (Seoul, Korea). Antibodies against XIAP (sc-11,426, monoclonal), goat anti-rabbit IgG-Texas Red (sc-2780), and Ultra Cruz™
2.6. Western blot analysis Cells were solubilized with ice-cold lysis buffer containing 50 mM Tris-HCl (pH 7.4), 1% NP-40, 150 mM NaCl, 0.5% sodium deoxycholate, 2 mM EDTA, 0.1% SDS, and 50 mM NaF. The extracted proteins (20 μg) were separated by sodium dodecyl sulfate-polyacrylamide 3
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Fig. 2. Rescue of XIAP expression in embelin-treated breast and cervical cancer cells by 4-OHE2. (A) MCF-7 cells were treated with 2-OHE2 or 4-OHE2 (0.2 or 2 μM). XIAP protein levels were measured using western blot analysis. The data represent the mean ± SD (n = 3). (B) MCF-7 cells were transfected with the CYP1B1-wildtype, L432V, or N203S vector for 48 h. CYP1B1, XIAP, and phospho-XIAP protein levels were measured using western blot analysis. (C) MCF-7, MDA-MB-231, or HeLa cells were co-treated with embelin (20 μM) and 4-OHE2 (2 μM) for 48 h. XIAP and phospho-XIAP levels were measured using western blot analysis. (D) MCF-7 cells were co-treated with embelin (20 μM) and 4-OHE2 (2 μM) for 48 h. Confocal microscopy analysis was performed to assess XIAP expression. The data represent the mean ± SD (n = 3). Microscopy scale bar = 50 μm. *p < 0.05.
gel electrophoresis (SDS-PAGE) on 10 or 12% polyacrylamide gels and then electrophoretically transferred to 0.45 μm PVDF membranes. Membranes were blocked with 5% (w/v) bovine serum albumin in Trisbuffered saline for 5 h at 4 °C and then incubated overnight with primary antibodies at a 1:1000 dilution in 5% (w/v) Tris-buffered saline containing 0.1% Tween-20. After incubation with secondary antibodies for 2 h, proteins were visualized by D-Plus™ ECL solution (Dongin LS, Seoul, Korea), and the band intensity was analyzed using a ChemiDoc XRS densitometer and quantified using Quantity One software (BioRad, Richmond, CA). Protein concentrations were estimated using the BCA method according to the supplier's recommendations using bovine serum albumin as a standard.
Care and Use Committee (IACUC) of Chung-Ang University and performed according to the guidelines of an approved protocol (201900045). The BALB/c nude mice were purchased from Central Lab. Animal Inc. (Seoul, Korea). The mice (5 weeks old) were divided into two groups, with five mice in each. Control and CYP1B1 shRNA-infected MDA-MB-231 cells (5 × 106 cells/100 μL PBS and Matrigel mixture/animal) were injected subcutaneously into nude mice. The mice were sacrificed after 60 d, and the tumors were harvested. 2.12. Immunohistochemistry Xenograft tissues were fixed in 10% formalin solution, dehydrated through a graded ethanol series, cleared in xylene, and processed for embedding in paraffin wax according to routine protocols. Sections were incubated in a solution of 0.3% H2O2 for 15 min to inhibit endogenous peroxidase activity. Sections were then incubated for 1 h at 21 °C with primary antibodies against phospho-XIAP antibody and phospho-PKCε antibody diluted 1:100. The EnVision+ system (DAKO, Glostrup, Denmark) was applied according to the manufacturers' instructions. Slides were stained with diaminobenzidine tetrahydrochloride. Counterstaining was performed with Meyer's hematoxylin. The images on the slides were visualized with an Olympus CKX53 microscope (Tokyo, Japan).
2.7. Clonogenic assay MCF-7 cells were transfected with CYP1B1 siRNA and then seeded in 6-well plates (1 × 103 cells/well). After 4 days, cells were fixed with 4% formaldehyde for 15 min at 24 °C and stained with 0.2% crystal violet for 15 min. After washing three times using PBS, the number of colonies was counted. 2.8. Cell viability assay MCF-7 cells (1 × 104 cells/well) were seeded in 96-well plates and incubated for 24 h. Cells were treated with TMS (2.5, 5, 10, 20, or 40 μM) for 48 h or co-treated with 4-OHE2 (2 μM), cisplatin (12 μM), docetaxel (80 μM), and PKCε siRNA (30 nM) for 12, 24, or 48 h. EZCyTox solution (DoGenBio, Korea) was added to plate and then incubated for 2 h. Cell viability was measured using spectrophotometry at 450 nm using a Sunrise™ microplate reader (Tecan, Männedorf, Switzerland).
2.13. Statistical analysis Statistical analysis was performed using one-way analysis of variance, followed by Dunnett's pairwise multiple comparison t-test when appropriate, with GraphPad Prism 4 software (GraphPad Software Inc., CA). Differences were considered statistically significant at *p < 0.05. 3. Results
2.9. Apoptosis and caspase 3/7 assay
3.1. CYP1B1 induces cancer cell growth and survival through the upregulation of XIAP
Cells were treated with 4-OHE2 (2 μM) and embelin (20 μM) for 48 h. Cells were harvested with 0.05% trypsin-EDTA and washed twice with PBS. Cells were labeled with Muse® annexin V & dead cell assay kit or Muse® Caspase-3/7 Assay Kit (Merck Millipore, Germany) for 30 min. Fluorescence-labeled cells were detected by a Muse® cell analyzer (Merck Millipore).
To confirm that CYP1B1 expression induces XIAP levels in cancer cells, a CYP1B1 expression vector and siRNA were transfected into MCF-7 and HeLa cells for 48 h. Western blot analysis revealed that CYP1B1 up-regulation induced XIAP levels and that CYP1B1 siRNA was able to inhibit XIAP expression (Fig. 1A and C). However, the XIAP mRNA level was not significantly changed by CYP1B1 expression levels (Fig. 1B and D). Additionally, to determine whether changes in CYP1B1 expression caused by an inducer or inhibitor regulate XIAP expression, MCF-7 and HeLa cells were co-treated with TMS (5, 10, or 20 μM) and DMBA (20 μM) for 48 h or treated with TMS only (5 or 10 μM) for 48 h. TMS is a CYP1B1-specific inhibitor, and DMBA has been reported to induce CYP1B1 expression. As shown in Fig. 1E, XIAP levels induced by DMBA were suppressed by TMS. Treatment with TMS only also reduced XIAP expression in a concentration-dependent manner (Fig. 1F). To determine whether CYP1B1 expression plays a crucial role in cancer cell growth and survival, MCF-7 and HeLa cells were transfected with CYP1B1 siRNA for 4 d, and were then analyzed using a clonogenic assay (Fig. 1G). Down-regulation of CYP1B1 by siRNA caused a decrease in tumor colony formation. Furthermore, when MCF-7 and HeLa cells were treated with TMS (5, 10, 20, or 40 μM) for 48 h, cell viability was considerably decreased (Fig. 1H). Apoptosis assay data also showed that apoptosis is induced by TMS (20 μM) in MCF-7 and HeLa cells
2.10. Immunofluorescence Cells grown on poly D-lysine-coated coverslips were co-treated with 4-OHE2, embelin, or PKCε siRNA in RPMI 1640 or phenol red-free RPMI 1640 medium. The cells were fixed with 4% (w/v) paraformaldehyde in PBS for 30 min at 24 °C. After washing with PBS (3 times), the cells were blocked for 30 min in PBS containing 5% goat serum and 0.2% Triton X-100, then incubated with primary antibody (1:250) for 6 h at 24 °C, washed extensively, and stained for 6 h with goat anti-rabbit IgG-Texas Red (1:250). After further washes, the coverslips were mounted on glass slides using Ultra Cruz™ mounting medium. Fluorescence signals were analyzed using an LSM 800 confocal laser scanning microscope (Carl Zeiss, Germany). 2.11. Mouse xenograft model All animal experiments were approved by the Institutional Animal 5
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Fig. 3. Inhibition of embelin-mediated apoptosis by 4-OHE2 in breast and cervical cancer cells. MCF-7, MDA-MB-231, or HeLa cells were co-treated with embelin (20 μM) and 4-OHE2 (2 μM) for 48 h. (A) Apoptosis assay was performed using a Muse annexin V kit. The data represent the mean ± SD (n = 3). (B) Caspase-3/7 activity was measured using a Muse caspase-3/7 assay kit. The data represent the mean ± SD (n = 3). (C) Confocal microscopy analysis was performed to assess apoptosis and necrosis (yellow fluorescence: apoptosis; red fluorescence: necrosis). (D) PARP cleavage was measured using western blot analysis. Microscopy scale bar = 50 μm, *p < 0.05.
Fig. 4. Prevention of XIAP proteasomal degradation by 4-OHE2. (A) MCF-7 cells were pre-treated with 4-OHE2 (2 μM) for 24 h and subsequently treated with cycloheximide (CHX; 40 μg/mL) for 0, 6, 12, or 24 h. XIAP protein levels were measured using western blot analysis. The data represent the mean ± SD (n = 3). (B) MCF-7 cells were pre-treated with 4-OHE2 (2 μM) for 24 h and subsequently co-treated with CHX (40 μg/mL) and MG132 (20 μg/mL) for 24 h. XIAP protein levels were measured using western blot analysis. (C) MCF-7 cells were transfected with the FLAG-XIAP (wild type), FLAG-XIAP (S87A), or FLAG-XIAP (S87E) mutant expression vector for 24 h and subsequently treated with CHX (40 μg/mL) for 12 or 24 h. XIAP protein levels were measured using western blot analysis. The data represent the mean ± SD (n = 3). (D) MCF-7 cells were transfected with HA-ubiquitin and the FLAG-XIAP vector for 24 h. After 24 h, cells were co-treated with TMS (10 μM) and DMBA (10 μM) for 24 h and MG132 (20 μg/mL) for 6 h before harvesting. Immunoprecipitation (IP) was performed using anti-FLAG antibody. *p < 0.05.
(Fig. 1I). These data suggest that increasing CYP1B1 expression causes an increase in XIAP protein levels and subsequently affects cell growth and survival in cancer cells.
allelic variant with higher enzymatic activity induced cell growth in the MCF-10A human breast epithelial cell line [2]. To determine whether XIAP expression is regulated by CYP1B1 activity, MCF-7 cells were transfected with CYP1B1 L432V or CYP1B1 N203S allelic variant expression vectors, which resulted in higher or lower CYP1B1 activity, respectively. CYP1B1 L432V induced XIAP expression more than the wild-type or N203S (Fig. 2B). Additionally, to determine whether 4OHE2 prevents the suppression of XIAP by embelin, which inhibits XIAP activity and expression, MCF-7, MDA-MB-231, or HeLa cells were co-treated with embelin (20 μM) and 4-OHE2 (2 μM) for 48 h. The suppression of XIAP expression by embelin was recovered by 4-OHE2 treatment in these three cancer cell lines (Fig. 2C). The data from the
3.2. The estrogen metabolite 4-OHE2 formed by CYP1B1 activity increases XIAP expression To determine whether CYP1B1 metabolites affect XIAP expression, MCF-7 cells were treated with 4-OHE2 or 2-OHE2 (0.2 or 2 μM) for 48 h. XIAP protein levels were increased by 4-OHE2 in a concentrationdependent manner. However, 2-OHE2 had no significant effects on XIAP expression (Fig. 2A). Our previous report showed that a CYP1B1 7
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Fig. 5. Induction of XIAP phosphorylation by 4-OHE2 through PKCε signaling. (A) MCF-7 cells were treated with 4-OHE2 (0.2 or 2 μM) for 48 h. PKCε protein levels were measured using western blot analysis. (B) MCF-7 cells were treated with 4-OHE2 (2 μM) for 48 h. Immunoprecipitation was performed using anti-IgG and antiPKCε antibody. PKCε and XIAP protein levels were measured using western blot analysis. (C) MCF-7, MDA-MB-231, or HeLa cells were co-treated with PKCε siRNA (30 nM) and 4-OHE2 (2 μM) for 48 h. PCKε, phospho-PKCε (S729), XIAP, and phospho-XIAP (S87) protein levels were measured using western blot analysis. (D) MCF7 cells were co-treated with PKCε siRNA (30 nM) and 4-OHE2 (2 μM) for 48 h. Confocal analysis was performed to assess phospho-XIAP (S87) and phospho-PKCε (S729) expression. The data represent the mean ± SD (n = 3). (E) MCF-7 cells were co-treated with 4-OHE2 (2 μM), PKCε siRNA (30 nM), docetaxel (80 nM), or cisplatin (12 μM) for 12, 24, and 48 h. Cell viability was measured using CCK assay. The data represent the mean ± SD (n = 3). Microscopy scale bar = 50 μm. *p < 0.05.
confocal microscopy analysis also showed that the inhibition of XIAP expression by embelin was recovered by treatment with 4-OHE2 in MCF-7 cells (Fig. 2D). Taken together, our data suggest that the formation of 4-OHE2 by CYP1B1 may play an important role in XIAP expression in cancer cells.
phosphorylation of XIAP on serine 87. 3.5. 4-OHE2-mediated PKCε phosphorylation is a key determinant of XIAP phosphorylation To determine whether 4-OHE2 induces phosphorylation of PKCε on serine 729, MCF-7 cells were treated with 4-OHE2 (0.2 or 2 μM) for 48 h. The phospho-PKCε level was increased by 4-OHE2 in a concentration-dependent manner, whereas the total PKCε level did not significantly change (Fig. 5A). Additionally, to determine whether phosphorylated PKCε directly interacts with XIAP, an immunoprecipitation (IP) assay was performed after 4-OHE2 treatment. The results indicated that the binding of XIAP to PKCε in the 4-OHE2treated cells was higher than that in untreated cells (Fig. 5B). After transfecting PKCε siRNA into MCF-7, MDA-MB-231, or HeLa cells to decrease PKCε levels, PKCε siRNA-transfected cells were treated with 4OHE2 for 48 h. As expected, the decrease in phospho-PKCε and phospho-XIAP levels were prevented by treatment with 4-OHE2 in cancer cell lines (Figs. 5C and S1A). Confocal microscopy analysis also revealed similar results in MCF-7 and PC-3 cells (Figs. 5D and S1B). To determine whether PKCε-XIAP signaling can reduce cell death induced by anti-cancer drugs, such as docetaxel or cisplatin, MCF-7 cells were treated with docetaxel or cisplatin for 12, 24, or 48 h. Cell death induced by docetaxel or cisplatin was decreased by simultaneous treatment with 4-OHE2 for 24 h. However, this protection by 4-OHE2 against cell death was suppressed by transfection with PKCε siRNA (Fig. 5E). In order to confirm the effect of CYP1B1 on phosphorylation of PKCε and XIAP in vivo, mouse tumor xenografts were established using CYP1B1 shRNA-infected MDA-MB-231 cells. The increase in tumor volume was more prominent in the control shRNA group after 36–42 days of cell injection, and the body weight remained stable during the experiment (Fig. 6A and B). When tumors were finally harvested, the mean volume of tumors between the two groups was about 10 fold higher in the control shRNA group (Fig. 6C). Immunohistochemistry data showed that suppression of CYP1B1 expression by shRNA reduced phosphorylation of XIAP and PKCε (Fig. 6D). Collectively, these data demonstrate that the induction of PKCε phosphorylation by 4-OHE2 may promote XIAP phosphorylation on serine 87.
3.3. 4-OHE2 prevents apoptosis through the regulation of caspase 3/7 activity in cancer cells To determine whether apoptosis can be inhibited by 4-OHE2mediated XIAP induction in cancer cells, MCF-7, MDA-MB-231, and HeLa cells were co-treated with embelin (20 μM) and 4-OHE2 (2 μM) for 48 h, and the rate of apoptotic cell death was then measured using flow cytometry analysis. Embelin alone increased cell death by approximately 25%, whereas, co-treatment increased cell death rate by only approximately 8% (Fig. 3A). The caspase 3/7 activity was increased by approximately 30% by embelin. However, an increase in caspase 3/7 activity by approximately 22% was found in cells treated with embelin and 4-OHE2 (Fig. 3B). Confocal microscopy analysis also revealed that 4-OHE2 inhibited apoptosis and necrosis, which are induced by embelin, in the three cancer cell lines (Fig. 3C). In addition, embelin-mediated PARP cleavage was prevented by co-treatment with 4-OHE2 (Fig. 3D). 3.4. Phosphorylation of XIAP by 4-OHE2 inhibits ubiquitin-proteasomemediated XIAP degradation Previous studies have shown that phosphorylation of XIAP on serine 87 plays a crucial role in XIAP protein stability [20]. The level of phospho-XIAP (S87) was measured after co-treatment with embelin and 4-OHE2 to determine whether the increase in XIAP levels induced by 4OHE2 in cancer cells is caused by phosphorylation of XIAP. The phospho-XIAP levels, which were suppressed by embelin, were recovered by 4-OHE2 (Fig. 2C). To determine whether the increase in phospho-XIAP levels induced by 4-OHE2 affects XIAP protein stability, cells were treated with cycloheximide (CHX; 40 μg/mL) for 6, 12, and 24 h in the presence or absence of 2 μM 4-OHE2. The degradation of XIAP protein in 4-OHE2-treated cells was markedly lower than that in CHX-only-treated cells (Fig. 4A). Additionally, to confirm that XIAP degradation was prevented by proteasome inhibition, cells were treated with MG132, a proteasome inhibitor, in the presence of CHX. The degradation of XIAP by CHX was recovered by MG132 (Fig. 4B). As a previous report suggested that phosphorylation of XIAP on serine 87 actually plays an important role in the stability of XIAP in cancer cells, cells were transfected with XIAP S87A and S87E mutant expression vectors (S87A; loss of function, S87E; gain of function) and then treated with CHX for 12 and 24 h. The inhibition of XIAP degradation in S87Etransfected cells was stronger than that in wild-type or S87A cells (Fig. 4C). Finally, to demonstrate that these effects were caused by ubiquitin-proteasome-mediated degradation, the ubiquitination level of XIAP was measured after simultaneous treatment of MCF-7 cells with TMS and DMBA. The data showed that ubiquitination increased in TMStreated cells. However, the increase in XIAP ubiquitination following CYP1B1 inhibition was strongly reduced in the presence of DMBA (Fig. 4D). Taken together, our data demonstrate that 4-OHE2 prevents ubiquitin-proteasome-mediated XIAP degradation through the
4. Discussion There is growing evidence that CYP1B1 and 4-OHE2, catalyzed by CYP1B1 are able to induce cancer progression, metastasis, and survival in several cancers, including breast, prostate, lung, and ovarian cancers [34–39]. In a previous study, we demonstrated that CYP1B1 induces the inhibition of death receptor 4 (DR4), which transduces a cell death signal and induces apoptosis through DNA-methylation, and it may cause the suppression of apoptosis in cancer cells [40]. Here, we found that the DMBA-mediated induction of XIAP is prevented by TMS in MCF-7 cells. The expression pattern of XIAP protein was changed in the same manner as that of CYP1B1 expression, but the level of mRNA was not changed. Some factors that modulate XIAP protein level have been identified in previous studies. Liu et al. [41] have shown that Notch interacts with RING domain of XIAP and prevents ubiquitination of XIAP. In addition, Fu et al. [42] have shown that Smac3, a novel 9
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Fig. 6. Reduction of PKCε and XIAP phosphorylation by CYP1B1 knock-down decrease tumor growth in vivo. CYP1B1 shRNA infected MDA-MB-231 cells (5 × 106) were injected subcutaneously into the flank of BALB/c nude mice. Xenografts were harvested 60 days after injection. (A) Tumor volume was measured every 3 days with vernier caliper. (B) Mouse weight was measured every 3 days. (C) Photographic images of representative tumor size. (D) Immunohistochemistry images of antiphospho-PKCε (S729) and anti-phospho-XIAP (S87). The data represent the mean ± SD (n = 3). Microscopy scale bar: 100 μm. *p < 0.05.
isoform of Smac/DIABLO accelerates auto-ubiquitination of XIAP and it may be related to the RING domain of XIAP. Therefore, CYP1B1 may induce XIAP expression at the protein level. CYP1B1 catalyzes the hydroxylation of E2 to 2-OHE2 and 4-OHE2 [43]. 4-OHE2 has been shown to increase HIF1-α and VEGF-A, thus promoting carcinogenesis [13], and Fernandez et al. [9] showed that 4-
OHE2 and a higher concentration of 2-OHE2 promoted a 5 bp deletion in exon 4 of p53. Because p53 is an important factor in the inhibition of cell growth and induction of apoptosis by acting on cell-cycle checkpoint in the G1 phase, changes in 4-OHE2 levels in cancer cells may play a significant role in cancer cell survival. In our study, we showed that 4-OHE2 induces XIAP expression in a concentration-dependent 10
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Fig. 7. Scheme showing how CYP1B1 inhibits XIAP degradation. CYP1B1 inhibits the ubiquitin-proteasome-mediated degradation of XIAP via the activation of PKCε signaling in cancer cells.
manner. However, 2-OHE2 did not have a significant effect. Moreover, the embelin-mediated inhibition of XIAP expression and concomitant apoptosis was rescued by 4-OHE2 in three cancer cell lines. As shown in Fig. 2B, as expected, the CYP1B1 L432V polymorphic gene induced a significant increase in XIAP expression compared to that with the wildtype or N203S. These data indicate that CYP1B1 activity is important in inducing XIAP expression. In previous study, Li et al. [44] showed that CYP1B1 activity of L432V variant was at least three times higher than wildtype and Chavarria-Soley et al. [45] showed that N203S variant has low enzymatic activity. In addition, we showed that the increase of tumor progression factors such as β-catenin, c-myc, or Sp1 by L432V is higher than wildtype or N203S [2]. Thus, we concluded that CYP1B1 activity is crucial to modulate XIAP expression and apoptosis. In a previous study, Lin et al. [20] showed that phosphorylation of XIAP on serine 87 is able to prevent XIAP self-ubiquitination and degradation. In the present study, we found that 4-OHE2 reduced the degradation of XIAP, and as reported previously, the mutation of serine 87 to glutamate prevented XIAP degradation by cycloheximide in MCF7 cells. In addition, as shown in Fig. 4D, the inhibition of CYP1B1 by TMS caused XIAP ubiquitination. These data demonstrate that CYP1B1 plays a crucial role in the prevention of XIAP ubiquitination. Thus, we believe that 4-OHE2 formation by CYP1B1 may play protective roles in XIAP proteasomal degradation by increasing XIAP phosphorylation on serine 87. Recently, Kato et al. [30] showed that the PMA-dependent activation of PKC leads to the induction of XIAP stability through phosphorylation on serine 87 and that it prevents etoposide-mediated apoptosis in SH-SY5Y cells. Tian et al. [32] demonstrated that Raf-1 interacts with XIAP and induces phosphorylation of XIAP on serine 87. A previous study showed that PKC-α and PKC-ε induce the Raf-1/MEK/ ERK signaling pathway [46]. Furthermore, treatment with a PKC inhibitor (calphostin C) prevents the cyclic strain-induced Raf-1 activation and sequential activation of PKC-α and that PKC-ε binds to Raf-1 directly and induces activation [47]. Accordingly, we hypothesized that 4-OHE2 induces the activation of PKCε and subsequently interacts with XIAP directly or induces an interaction between PKCε and Raf-1 to
phosphorylate XIAP on serine 87. We found that 4-OHE2 induced PKCε activation and PKCε siRNA-mediated suppression of XIAP expression and that phosphorylation was rescued by 4-OHE2. Whether activated PKCε increases phosphorylation of XIAP through interacting with Raf-1 needs to be clarified, our immunoprecipitation data showed that the binding of PKCε to XIAP increased when PKCε was activated by 4OHE2. Moreover, we found that the ability of 4-OHE2 to inhibit docetaxel- or cisplatin-mediated apoptosis was decreased by the suppression of PKCε expression. Indeed, according to the Kaplan-Meier curves, ovarian cancer patients with high expression of both CYP1B1 and XIAP or PKCε have poor prognosis on progression-free survival, overall survival, and post-progression survival (Fig. S2A–C). Thus, we suggest that the activation of PKCε by 4-OHE2 may play an important role in phosphorylation of XIAP and the resistance of cancer cells to anticancer agents. In summary, we demonstrated that 4-OHE2, catalyzed by CYP1B1 prevents the proteasome-mediated degradation of XIAP through phosphorylation on serine 87 by activating PKCε. The scheme shown in Fig. 7 summarizes the results of this study. Although, 4-OHE2 has been shown to be important for the activity of PKCε, it remains unclear how PKCε phosphorylation is induced by CYP1B1. Thus, further studies are needed to elucidate the detailed mechanism of the activation of PKCε by CYP1B1. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bbamcr.2019.118553. Transparency document The Transparency document associated with this article can be found, in online version. Declaration of competing interest The authors declare that they have no conflicts of interest. 11
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Acknowledgements
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CYP1B1 prevents proteasome-mediated XIAP degradation by inducing PKCε activation and phosphorylation of XIAP Hyoung-Seok Baek, Yeo-Jung Kwon, Dong-Jin Ye, Eunah Cho, Tae-Uk Kwon, Young-Jin Chun
T
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College of Pharmacy and Center for Metareceptome Research, Chung-Ang University, Seoul 06974, Republic of Korea
ARTICLE INFO
ABSTRACT
Keywords: CYP1B1 4-OHE2 XIAP PKCε Ubiquitination Apoptosis
Cytochrome P450 1B1 (CYP1B1) is a key enzyme that catalyzes the metabolism of 17β-estradiol (E2) into catechol estrogens, such as 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2). CYP1B1 is related to tumor formation and is over-expressed in a variety of cancer cells. In particular, CYP1B1 is highly expressed in hormone-related cancers such as breast cancer, ovarian cancer, or prostate cancer compared to other cancers. However, the detailed mechanisms involving this protein remain unclear. In this study, we demonstrate that CYP1B1 affects X-linked inhibitor of apoptosis protein (XIAP) expression. When CYP1B1 was over-expressed in cells, there was significant increase in the XIAP protein level, whereas the XIAP mRNA level was not affected by CYP1B1 expression. Treatment with 4-OHE2, mainly formed by CYP1B1 activity, also increased XIAP protein levels, whereas treatment with 2-OHE2 did not have a significant effect. Treatment with 4-OHE2 significantly prevented proteasome-mediated XIAP degradation. In addition, phosphorylation of XIAP on serine 87, which is known to stabilize XIAP, was up-regulated by 4-OHE2, indicating that 4-OHE2 affects XIAP stability through XIAP phosphorylation. We also found that phosphorylation of protein kinase C (PKC)ε, which is required for XIAP phosphorylation, increased when cells were treated with 4-OHE2. In summary, our data show that CYP1B1 may play an important role in preventing ubiquitin-proteasome-mediated XIAP degradation through the activation of PKCε signaling in cancer cells.
1. Introduction Cytochrome P450 1B1 (CYP1B1), a member of the CYP family 1, has been implicated in tumor proliferation, metastasis, and survival [1–6]. Previously, we showed that CYP1B1 increases the activation of the Wnt/β-catenin signaling pathway through the induction of Sp1, which leads to cell growth and migration in breast cancer cells [2]. The principal role of CYP1B1 in cells is the hydroxylation of 17βestradiol (E2) to the catechol metabolites such as 2-hydroxyestradiol (2OHE2) and 4-hydroxyestradiol (4-OHE2) [7,8]. Several studies have shown that 4-OHE2 is a potential carcinogen for animals and humans [9–11]. Mueck et al. [12] showed that 4-OHE2 induces Bcl-2 and reduces cytochrome c levels in MCF-7 cells. However, 2-OHE2 was not found to have any significant effects. Furthermore, Gao et al. [13] demonstrated that only 4-OHE2 increases the expression of VEGF-A, which affects endothelial cell growth, angiogenesis, and suppression of apoptosis. It also induces the expression of HIF1-α, which regulates
cancer cell metabolism and migration. These reports provide evidence that 4-OHE2 plays a crucial role in carcinogenesis. X-linked inhibitor of apoptosis protein (XIAP) is a potent antiapoptotic factor, and it contains three amino-terminal BIR domains: BIR-1, BIR-2, and BIR-3. The BIR-2 domain binds to caspase-3 and caspase-7 and suppresses apoptosis, while the BIR-3 domain binds to caspase-9 and inhibits cell death [14,15]. XIAP is upregulated in a variety of cancer cells, and it increases the resistance to radiation- or chemotherapy-induced apoptosis [16–19]. Therefore, it may be important to regulate the expression of XIAP in order to obtain a better effect in cancer therapy. Phosphorylation of XIAP on serine 87 is important for the activity and stability of XIAP. A previous study has shown that the stability of XIAP significantly increases following the mutation of serine 87 to glutamate, possibly because it inhibits BIR1 dimerization and results in XIAP self-ubiquitination [20]. Protein kinase C (PKC) is a member of the family of protein kinases
Abbreviations: CYP1B1, cytochrome P450 1B1; XIAP, X-linked inhibitor of apoptosis protein; PKCε, Protein kinase C ε; TMS, 2, 2′, 4, 6′-tetramethoxystilbene; DMBA, 7, 12–dimethylbenz[a]-anthracene; 2-OHE2, 2-hydroxyestradiol; 4-OHE2, 4-hydroxyestradiol; DR4, death receptor 4 ⁎ Corresponding author at: College of Pharmacy and Center for Metareceptome Research, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 06974, Republic of Korea. E-mail address: [email protected] (Y.-J. Chun). https://doi.org/10.1016/j.bbamcr.2019.118553 Received 22 March 2019; Received in revised form 29 August 2019; Accepted 3 September 2019 Available online 04 September 2019 0167-4889/ © 2019 Published by Elsevier B.V.
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Fig. 1. Expression of CYP1B1 increases the XIAP protein level. (A–B) MCF-7 and HeLa cells were transfected with the pcDNA3.1_CYP1B1 vector for 48 h. (A) XIAP protein levels were measured using western blot analysis. (B) Real-time qPCR was performed to detect the expression of XIAP mRNA. The data represent the mean ± SD (n = 3). (C–D) MCF-7 and HeLa cells were transfected with CYP1B1 siRNA (30 nM) for 48 h. (C) XIAP protein levels were measured using western blot analysis. (D) Real-time qPCR was performed to detect the expression of XIAP mRNA. The data represent the mean ± SD (n = 3). (E) MCF-7 and HeLa cells were cotreated with TMS (5, 10, or 20 μM) and DMBA (10 μM) for 48 h. CYP1B1 and XIAP protein levels were measured using western blot analysis. (F) MCF-7 and HeLa cells were treated with TMS (5 or 10 μM) for 48 h. CYP1B1 and XIAP protein levels were measured using western blot analysis. (G) Clonogenic assay was performed in MCF-7 and HeLa cells after transfecting them with CYP1B1 siRNA (30 nM) for 4 d. The data represent the mean ± SD (n = 3). (H) MCF-7 and HeLa cells were treated with TMS (2.5, 5, 10, 20, or 40 μM) for 48 h. Cell viability was measured using CCK assay (n = 3). (I) MCF-7 and HeLa cells were treated with TMS (20 μM) for 48 h. Apoptosis assay was performed using a Muse annexin V kit. The data represent the mean ± SD (n = 3). ns = not significant; *p < 0.05.
that are involved in the phosphorylation of serine and threonine in target proteins. PKCs play an important role in various intracellular functions such as regulation of the inflammatory response, proliferation, metastasis, cell death, and differentiation [21–29]. Previously, it was reported that PKC stabilizes XIAP by inducing phosphorylation on serine 87 [30]. Pardo et al. [31] showed that PKCε over-expression induces XIAP protein levels, and this increase in the XIAP protein level may be correlated with a complex involving PKCε, B-Raf, and S6K2. In addition, it has been reported that the interaction between Raf-1 and XIAP prevents XIAP degradation through phosphorylation of XIAP [32]. In this study, we demonstrate that the proteasomal degradation of XIAP is prevented by 4-OHE2. We also show that phosphorylation of XIAP on serine 87 by 4-OHE2, which increases XIAP stability and activity, is tightly correlated with the activation of PKCε. Our results suggest that 4-OHE2, catalyzed by CYP1B1 may play a crucial role in preventing the degradation of XIAP and apoptosis by inducing PKCε activation.
mounting medium (sc-24,941) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Actin antibody (A300-491A, polyclonal) and ECS (DDDDK) antibody (A190-102A, polyclonal) were purchased from Bethyl (Montgomery, TX). PARP antibody (46D11, monoclonal) was purchased from Cell Signaling Technology (Beverly, MA). Phospho-XIAP (S87) polyclonal, PKCε polyclonal, and phosphoPKCε (S729) polyclonal antibodies were purchased from Flarebio (College Park, MD). All other chemicals were obtained from commercial sources. 2.3. Transient transfection
2. Material and methods
CYP1B1 siRNA (Qiagen, Venlo, Netherlands), PKCε siRNA (Santa Cruz), the HA-ubiquitin expression vector (a gift from Edward Yeh, Addgene plasmid #18712), the pcDNA3.1/Zeo+ vector containing the CYP1B1-encoding sequence, and the p3xFLAG_CMV10 vector containing the XIAP-encoding sequence were used for transfection. Cells were transfected with 30 nM siRNA or 3 μg plasmid with the Neon Transfection System (Life Technologies).
2.1. Cell culture
2.4. Generation of stable CYP1B1 knock-down MDA-MB-231 cells
The MCF-7 and MDA-MB-231 human breast cancer cell lines and the HEK293T human embryonic kidney cell line were obtained from the American Type Culture Collection (Manassas, VA). The HeLa human cervical cancer cell line and PC-3 human prostate cancer cell line were obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea). MCF-7, MDA-MB-231, HeLa, and PC-3 cell lines were cultured in RPMI 1640 supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. HEK293T cell line was cultured in DMEM supplemented with 10% (v/v) heatinactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/ mL streptomycin. Cells were incubated at 37 °C in a humidified atmosphere of 5% CO2. For treatment of MCF-7, MDA-MB-231, HeLa, and PC-3 cells with 4-OHE2 or 2-OHE2, cells were seeded in growth media as a monolayer in 60-mm dish plates. After 24 h, the medium were changed to phenol red-free RPMI 1640 with 10% (v/v) charcoalstripped FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were incubated for 72 h, and then, the cells were treated with 4-OHE2 or 2-OHE2 for 48 h.
The pLKO.1 puro (a gift from Bob Weinberg, Addgene plasmid # 8453) [33], pMD2.G, and psPAX2 (a gift from Didier Trono, Addgene plasmid # 12259 and #12260) plasmid vectors were obtained from Addgene. HEK293T cells were co-transfected with the CYP1B1-pLKO.1 puro, pMD2.G, and psPAX2 vectors. After 48 h, the medium containing lentiviral CYP1B1 shRNA was collected, and MDA-MB-231 cells were subsequently treated with lentiviral soup containing CYP1B1 shRNA and polybrene (8 μg/mL) for 24 h. The CYP1B1 knock-down MDA-MB231 cells were selected using puromycin (1 μg/mL). 2.5. Quantitative PCR Total RNA was extracted using RiboEx™ solution (GeneALL, Seoul, Korea). Total RNA (1 μg) was reverse-transcribed at 37 °C for 1 h in a 25 μL total volume containing 5× RT buffer, 10 mM dNTPs, 40 U RNase inhibitor, 200 U Moloney murine leukemia virus reverse transcriptase, and 100 pmol oligo-dT primer. qPCR was performed using the RotorGene SYBR1 PCR Kit, as recommended by the manufacturer, and analyzed using QIAGEN Rotor-Gene Q Series software. Each reaction contained 10 μL of 2× SYBR1 Green PCR Master Mix, 1 μM oligonucleotide primers, and 20 ng of cDNA in a final volume of 20 μL. Amplification was conducted as follows: one cycle at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/ extension at 56 °C for 10 s. The following primer sets were used for qPCR: XIAP, 5′-CTTGGGCACAGAGAGCA-3′ and 5′-AACTGCTCATCAC CCCATTC-3′, and 18S rRNA, 5′-GTAACCCGTTGAACCCCATT-3′ and 5′-CCATCCAATCGGTAGTAGCG-3′.
2.2. Reagents 7,12-Dimethylbenz[a]anthracene (DMBA), embelin, docetaxel, 4OHE2, and 2-OHE2 were purchased from Sigma-Aldrich (St. Louis, MO). 2,2′,4,6′-Tetramethoxystilbene (TMS) was kindly provided by Dr. Sanghee Kim (Seoul National University, Seoul, Korea). All chemicals were prepared in dimethyl sulfoxide, stored as small aliquots at −20 °C, and then diluted as needed in cell culture medium. FBS and RPMI 1640 medium were purchased from HyClone (Logan, UT). Charcoal-stripped FBS was purchased from Tissue Culture Biologicals (Long Beach, CA). Phenol red-free RPMI 1640, bicinchoninic acid (BCA) protein assay kits, and the Neon transfection system were purchased from ThermoFisher Scientific (Waltham, MA). D-Plus™ ECL solution was purchased from Dongin LS (Seoul, Korea). Antibodies against XIAP (sc-11,426, monoclonal), goat anti-rabbit IgG-Texas Red (sc-2780), and Ultra Cruz™
2.6. Western blot analysis Cells were solubilized with ice-cold lysis buffer containing 50 mM Tris-HCl (pH 7.4), 1% NP-40, 150 mM NaCl, 0.5% sodium deoxycholate, 2 mM EDTA, 0.1% SDS, and 50 mM NaF. The extracted proteins (20 μg) were separated by sodium dodecyl sulfate-polyacrylamide 3
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Fig. 2. Rescue of XIAP expression in embelin-treated breast and cervical cancer cells by 4-OHE2. (A) MCF-7 cells were treated with 2-OHE2 or 4-OHE2 (0.2 or 2 μM). XIAP protein levels were measured using western blot analysis. The data represent the mean ± SD (n = 3). (B) MCF-7 cells were transfected with the CYP1B1-wildtype, L432V, or N203S vector for 48 h. CYP1B1, XIAP, and phospho-XIAP protein levels were measured using western blot analysis. (C) MCF-7, MDA-MB-231, or HeLa cells were co-treated with embelin (20 μM) and 4-OHE2 (2 μM) for 48 h. XIAP and phospho-XIAP levels were measured using western blot analysis. (D) MCF-7 cells were co-treated with embelin (20 μM) and 4-OHE2 (2 μM) for 48 h. Confocal microscopy analysis was performed to assess XIAP expression. The data represent the mean ± SD (n = 3). Microscopy scale bar = 50 μm. *p < 0.05.
gel electrophoresis (SDS-PAGE) on 10 or 12% polyacrylamide gels and then electrophoretically transferred to 0.45 μm PVDF membranes. Membranes were blocked with 5% (w/v) bovine serum albumin in Trisbuffered saline for 5 h at 4 °C and then incubated overnight with primary antibodies at a 1:1000 dilution in 5% (w/v) Tris-buffered saline containing 0.1% Tween-20. After incubation with secondary antibodies for 2 h, proteins were visualized by D-Plus™ ECL solution (Dongin LS, Seoul, Korea), and the band intensity was analyzed using a ChemiDoc XRS densitometer and quantified using Quantity One software (BioRad, Richmond, CA). Protein concentrations were estimated using the BCA method according to the supplier's recommendations using bovine serum albumin as a standard.
Care and Use Committee (IACUC) of Chung-Ang University and performed according to the guidelines of an approved protocol (201900045). The BALB/c nude mice were purchased from Central Lab. Animal Inc. (Seoul, Korea). The mice (5 weeks old) were divided into two groups, with five mice in each. Control and CYP1B1 shRNA-infected MDA-MB-231 cells (5 × 106 cells/100 μL PBS and Matrigel mixture/animal) were injected subcutaneously into nude mice. The mice were sacrificed after 60 d, and the tumors were harvested. 2.12. Immunohistochemistry Xenograft tissues were fixed in 10% formalin solution, dehydrated through a graded ethanol series, cleared in xylene, and processed for embedding in paraffin wax according to routine protocols. Sections were incubated in a solution of 0.3% H2O2 for 15 min to inhibit endogenous peroxidase activity. Sections were then incubated for 1 h at 21 °C with primary antibodies against phospho-XIAP antibody and phospho-PKCε antibody diluted 1:100. The EnVision+ system (DAKO, Glostrup, Denmark) was applied according to the manufacturers' instructions. Slides were stained with diaminobenzidine tetrahydrochloride. Counterstaining was performed with Meyer's hematoxylin. The images on the slides were visualized with an Olympus CKX53 microscope (Tokyo, Japan).
2.7. Clonogenic assay MCF-7 cells were transfected with CYP1B1 siRNA and then seeded in 6-well plates (1 × 103 cells/well). After 4 days, cells were fixed with 4% formaldehyde for 15 min at 24 °C and stained with 0.2% crystal violet for 15 min. After washing three times using PBS, the number of colonies was counted. 2.8. Cell viability assay MCF-7 cells (1 × 104 cells/well) were seeded in 96-well plates and incubated for 24 h. Cells were treated with TMS (2.5, 5, 10, 20, or 40 μM) for 48 h or co-treated with 4-OHE2 (2 μM), cisplatin (12 μM), docetaxel (80 μM), and PKCε siRNA (30 nM) for 12, 24, or 48 h. EZCyTox solution (DoGenBio, Korea) was added to plate and then incubated for 2 h. Cell viability was measured using spectrophotometry at 450 nm using a Sunrise™ microplate reader (Tecan, Männedorf, Switzerland).
2.13. Statistical analysis Statistical analysis was performed using one-way analysis of variance, followed by Dunnett's pairwise multiple comparison t-test when appropriate, with GraphPad Prism 4 software (GraphPad Software Inc., CA). Differences were considered statistically significant at *p < 0.05. 3. Results
2.9. Apoptosis and caspase 3/7 assay
3.1. CYP1B1 induces cancer cell growth and survival through the upregulation of XIAP
Cells were treated with 4-OHE2 (2 μM) and embelin (20 μM) for 48 h. Cells were harvested with 0.05% trypsin-EDTA and washed twice with PBS. Cells were labeled with Muse® annexin V & dead cell assay kit or Muse® Caspase-3/7 Assay Kit (Merck Millipore, Germany) for 30 min. Fluorescence-labeled cells were detected by a Muse® cell analyzer (Merck Millipore).
To confirm that CYP1B1 expression induces XIAP levels in cancer cells, a CYP1B1 expression vector and siRNA were transfected into MCF-7 and HeLa cells for 48 h. Western blot analysis revealed that CYP1B1 up-regulation induced XIAP levels and that CYP1B1 siRNA was able to inhibit XIAP expression (Fig. 1A and C). However, the XIAP mRNA level was not significantly changed by CYP1B1 expression levels (Fig. 1B and D). Additionally, to determine whether changes in CYP1B1 expression caused by an inducer or inhibitor regulate XIAP expression, MCF-7 and HeLa cells were co-treated with TMS (5, 10, or 20 μM) and DMBA (20 μM) for 48 h or treated with TMS only (5 or 10 μM) for 48 h. TMS is a CYP1B1-specific inhibitor, and DMBA has been reported to induce CYP1B1 expression. As shown in Fig. 1E, XIAP levels induced by DMBA were suppressed by TMS. Treatment with TMS only also reduced XIAP expression in a concentration-dependent manner (Fig. 1F). To determine whether CYP1B1 expression plays a crucial role in cancer cell growth and survival, MCF-7 and HeLa cells were transfected with CYP1B1 siRNA for 4 d, and were then analyzed using a clonogenic assay (Fig. 1G). Down-regulation of CYP1B1 by siRNA caused a decrease in tumor colony formation. Furthermore, when MCF-7 and HeLa cells were treated with TMS (5, 10, 20, or 40 μM) for 48 h, cell viability was considerably decreased (Fig. 1H). Apoptosis assay data also showed that apoptosis is induced by TMS (20 μM) in MCF-7 and HeLa cells
2.10. Immunofluorescence Cells grown on poly D-lysine-coated coverslips were co-treated with 4-OHE2, embelin, or PKCε siRNA in RPMI 1640 or phenol red-free RPMI 1640 medium. The cells were fixed with 4% (w/v) paraformaldehyde in PBS for 30 min at 24 °C. After washing with PBS (3 times), the cells were blocked for 30 min in PBS containing 5% goat serum and 0.2% Triton X-100, then incubated with primary antibody (1:250) for 6 h at 24 °C, washed extensively, and stained for 6 h with goat anti-rabbit IgG-Texas Red (1:250). After further washes, the coverslips were mounted on glass slides using Ultra Cruz™ mounting medium. Fluorescence signals were analyzed using an LSM 800 confocal laser scanning microscope (Carl Zeiss, Germany). 2.11. Mouse xenograft model All animal experiments were approved by the Institutional Animal 5
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Fig. 3. Inhibition of embelin-mediated apoptosis by 4-OHE2 in breast and cervical cancer cells. MCF-7, MDA-MB-231, or HeLa cells were co-treated with embelin (20 μM) and 4-OHE2 (2 μM) for 48 h. (A) Apoptosis assay was performed using a Muse annexin V kit. The data represent the mean ± SD (n = 3). (B) Caspase-3/7 activity was measured using a Muse caspase-3/7 assay kit. The data represent the mean ± SD (n = 3). (C) Confocal microscopy analysis was performed to assess apoptosis and necrosis (yellow fluorescence: apoptosis; red fluorescence: necrosis). (D) PARP cleavage was measured using western blot analysis. Microscopy scale bar = 50 μm, *p < 0.05.
Fig. 4. Prevention of XIAP proteasomal degradation by 4-OHE2. (A) MCF-7 cells were pre-treated with 4-OHE2 (2 μM) for 24 h and subsequently treated with cycloheximide (CHX; 40 μg/mL) for 0, 6, 12, or 24 h. XIAP protein levels were measured using western blot analysis. The data represent the mean ± SD (n = 3). (B) MCF-7 cells were pre-treated with 4-OHE2 (2 μM) for 24 h and subsequently co-treated with CHX (40 μg/mL) and MG132 (20 μg/mL) for 24 h. XIAP protein levels were measured using western blot analysis. (C) MCF-7 cells were transfected with the FLAG-XIAP (wild type), FLAG-XIAP (S87A), or FLAG-XIAP (S87E) mutant expression vector for 24 h and subsequently treated with CHX (40 μg/mL) for 12 or 24 h. XIAP protein levels were measured using western blot analysis. The data represent the mean ± SD (n = 3). (D) MCF-7 cells were transfected with HA-ubiquitin and the FLAG-XIAP vector for 24 h. After 24 h, cells were co-treated with TMS (10 μM) and DMBA (10 μM) for 24 h and MG132 (20 μg/mL) for 6 h before harvesting. Immunoprecipitation (IP) was performed using anti-FLAG antibody. *p < 0.05.
(Fig. 1I). These data suggest that increasing CYP1B1 expression causes an increase in XIAP protein levels and subsequently affects cell growth and survival in cancer cells.
allelic variant with higher enzymatic activity induced cell growth in the MCF-10A human breast epithelial cell line [2]. To determine whether XIAP expression is regulated by CYP1B1 activity, MCF-7 cells were transfected with CYP1B1 L432V or CYP1B1 N203S allelic variant expression vectors, which resulted in higher or lower CYP1B1 activity, respectively. CYP1B1 L432V induced XIAP expression more than the wild-type or N203S (Fig. 2B). Additionally, to determine whether 4OHE2 prevents the suppression of XIAP by embelin, which inhibits XIAP activity and expression, MCF-7, MDA-MB-231, or HeLa cells were co-treated with embelin (20 μM) and 4-OHE2 (2 μM) for 48 h. The suppression of XIAP expression by embelin was recovered by 4-OHE2 treatment in these three cancer cell lines (Fig. 2C). The data from the
3.2. The estrogen metabolite 4-OHE2 formed by CYP1B1 activity increases XIAP expression To determine whether CYP1B1 metabolites affect XIAP expression, MCF-7 cells were treated with 4-OHE2 or 2-OHE2 (0.2 or 2 μM) for 48 h. XIAP protein levels were increased by 4-OHE2 in a concentrationdependent manner. However, 2-OHE2 had no significant effects on XIAP expression (Fig. 2A). Our previous report showed that a CYP1B1 7
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Fig. 5. Induction of XIAP phosphorylation by 4-OHE2 through PKCε signaling. (A) MCF-7 cells were treated with 4-OHE2 (0.2 or 2 μM) for 48 h. PKCε protein levels were measured using western blot analysis. (B) MCF-7 cells were treated with 4-OHE2 (2 μM) for 48 h. Immunoprecipitation was performed using anti-IgG and antiPKCε antibody. PKCε and XIAP protein levels were measured using western blot analysis. (C) MCF-7, MDA-MB-231, or HeLa cells were co-treated with PKCε siRNA (30 nM) and 4-OHE2 (2 μM) for 48 h. PCKε, phospho-PKCε (S729), XIAP, and phospho-XIAP (S87) protein levels were measured using western blot analysis. (D) MCF7 cells were co-treated with PKCε siRNA (30 nM) and 4-OHE2 (2 μM) for 48 h. Confocal analysis was performed to assess phospho-XIAP (S87) and phospho-PKCε (S729) expression. The data represent the mean ± SD (n = 3). (E) MCF-7 cells were co-treated with 4-OHE2 (2 μM), PKCε siRNA (30 nM), docetaxel (80 nM), or cisplatin (12 μM) for 12, 24, and 48 h. Cell viability was measured using CCK assay. The data represent the mean ± SD (n = 3). Microscopy scale bar = 50 μm. *p < 0.05.
confocal microscopy analysis also showed that the inhibition of XIAP expression by embelin was recovered by treatment with 4-OHE2 in MCF-7 cells (Fig. 2D). Taken together, our data suggest that the formation of 4-OHE2 by CYP1B1 may play an important role in XIAP expression in cancer cells.
phosphorylation of XIAP on serine 87. 3.5. 4-OHE2-mediated PKCε phosphorylation is a key determinant of XIAP phosphorylation To determine whether 4-OHE2 induces phosphorylation of PKCε on serine 729, MCF-7 cells were treated with 4-OHE2 (0.2 or 2 μM) for 48 h. The phospho-PKCε level was increased by 4-OHE2 in a concentration-dependent manner, whereas the total PKCε level did not significantly change (Fig. 5A). Additionally, to determine whether phosphorylated PKCε directly interacts with XIAP, an immunoprecipitation (IP) assay was performed after 4-OHE2 treatment. The results indicated that the binding of XIAP to PKCε in the 4-OHE2treated cells was higher than that in untreated cells (Fig. 5B). After transfecting PKCε siRNA into MCF-7, MDA-MB-231, or HeLa cells to decrease PKCε levels, PKCε siRNA-transfected cells were treated with 4OHE2 for 48 h. As expected, the decrease in phospho-PKCε and phospho-XIAP levels were prevented by treatment with 4-OHE2 in cancer cell lines (Figs. 5C and S1A). Confocal microscopy analysis also revealed similar results in MCF-7 and PC-3 cells (Figs. 5D and S1B). To determine whether PKCε-XIAP signaling can reduce cell death induced by anti-cancer drugs, such as docetaxel or cisplatin, MCF-7 cells were treated with docetaxel or cisplatin for 12, 24, or 48 h. Cell death induced by docetaxel or cisplatin was decreased by simultaneous treatment with 4-OHE2 for 24 h. However, this protection by 4-OHE2 against cell death was suppressed by transfection with PKCε siRNA (Fig. 5E). In order to confirm the effect of CYP1B1 on phosphorylation of PKCε and XIAP in vivo, mouse tumor xenografts were established using CYP1B1 shRNA-infected MDA-MB-231 cells. The increase in tumor volume was more prominent in the control shRNA group after 36–42 days of cell injection, and the body weight remained stable during the experiment (Fig. 6A and B). When tumors were finally harvested, the mean volume of tumors between the two groups was about 10 fold higher in the control shRNA group (Fig. 6C). Immunohistochemistry data showed that suppression of CYP1B1 expression by shRNA reduced phosphorylation of XIAP and PKCε (Fig. 6D). Collectively, these data demonstrate that the induction of PKCε phosphorylation by 4-OHE2 may promote XIAP phosphorylation on serine 87.
3.3. 4-OHE2 prevents apoptosis through the regulation of caspase 3/7 activity in cancer cells To determine whether apoptosis can be inhibited by 4-OHE2mediated XIAP induction in cancer cells, MCF-7, MDA-MB-231, and HeLa cells were co-treated with embelin (20 μM) and 4-OHE2 (2 μM) for 48 h, and the rate of apoptotic cell death was then measured using flow cytometry analysis. Embelin alone increased cell death by approximately 25%, whereas, co-treatment increased cell death rate by only approximately 8% (Fig. 3A). The caspase 3/7 activity was increased by approximately 30% by embelin. However, an increase in caspase 3/7 activity by approximately 22% was found in cells treated with embelin and 4-OHE2 (Fig. 3B). Confocal microscopy analysis also revealed that 4-OHE2 inhibited apoptosis and necrosis, which are induced by embelin, in the three cancer cell lines (Fig. 3C). In addition, embelin-mediated PARP cleavage was prevented by co-treatment with 4-OHE2 (Fig. 3D). 3.4. Phosphorylation of XIAP by 4-OHE2 inhibits ubiquitin-proteasomemediated XIAP degradation Previous studies have shown that phosphorylation of XIAP on serine 87 plays a crucial role in XIAP protein stability [20]. The level of phospho-XIAP (S87) was measured after co-treatment with embelin and 4-OHE2 to determine whether the increase in XIAP levels induced by 4OHE2 in cancer cells is caused by phosphorylation of XIAP. The phospho-XIAP levels, which were suppressed by embelin, were recovered by 4-OHE2 (Fig. 2C). To determine whether the increase in phospho-XIAP levels induced by 4-OHE2 affects XIAP protein stability, cells were treated with cycloheximide (CHX; 40 μg/mL) for 6, 12, and 24 h in the presence or absence of 2 μM 4-OHE2. The degradation of XIAP protein in 4-OHE2-treated cells was markedly lower than that in CHX-only-treated cells (Fig. 4A). Additionally, to confirm that XIAP degradation was prevented by proteasome inhibition, cells were treated with MG132, a proteasome inhibitor, in the presence of CHX. The degradation of XIAP by CHX was recovered by MG132 (Fig. 4B). As a previous report suggested that phosphorylation of XIAP on serine 87 actually plays an important role in the stability of XIAP in cancer cells, cells were transfected with XIAP S87A and S87E mutant expression vectors (S87A; loss of function, S87E; gain of function) and then treated with CHX for 12 and 24 h. The inhibition of XIAP degradation in S87Etransfected cells was stronger than that in wild-type or S87A cells (Fig. 4C). Finally, to demonstrate that these effects were caused by ubiquitin-proteasome-mediated degradation, the ubiquitination level of XIAP was measured after simultaneous treatment of MCF-7 cells with TMS and DMBA. The data showed that ubiquitination increased in TMStreated cells. However, the increase in XIAP ubiquitination following CYP1B1 inhibition was strongly reduced in the presence of DMBA (Fig. 4D). Taken together, our data demonstrate that 4-OHE2 prevents ubiquitin-proteasome-mediated XIAP degradation through the
4. Discussion There is growing evidence that CYP1B1 and 4-OHE2, catalyzed by CYP1B1 are able to induce cancer progression, metastasis, and survival in several cancers, including breast, prostate, lung, and ovarian cancers [34–39]. In a previous study, we demonstrated that CYP1B1 induces the inhibition of death receptor 4 (DR4), which transduces a cell death signal and induces apoptosis through DNA-methylation, and it may cause the suppression of apoptosis in cancer cells [40]. Here, we found that the DMBA-mediated induction of XIAP is prevented by TMS in MCF-7 cells. The expression pattern of XIAP protein was changed in the same manner as that of CYP1B1 expression, but the level of mRNA was not changed. Some factors that modulate XIAP protein level have been identified in previous studies. Liu et al. [41] have shown that Notch interacts with RING domain of XIAP and prevents ubiquitination of XIAP. In addition, Fu et al. [42] have shown that Smac3, a novel 9
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Fig. 6. Reduction of PKCε and XIAP phosphorylation by CYP1B1 knock-down decrease tumor growth in vivo. CYP1B1 shRNA infected MDA-MB-231 cells (5 × 106) were injected subcutaneously into the flank of BALB/c nude mice. Xenografts were harvested 60 days after injection. (A) Tumor volume was measured every 3 days with vernier caliper. (B) Mouse weight was measured every 3 days. (C) Photographic images of representative tumor size. (D) Immunohistochemistry images of antiphospho-PKCε (S729) and anti-phospho-XIAP (S87). The data represent the mean ± SD (n = 3). Microscopy scale bar: 100 μm. *p < 0.05.
isoform of Smac/DIABLO accelerates auto-ubiquitination of XIAP and it may be related to the RING domain of XIAP. Therefore, CYP1B1 may induce XIAP expression at the protein level. CYP1B1 catalyzes the hydroxylation of E2 to 2-OHE2 and 4-OHE2 [43]. 4-OHE2 has been shown to increase HIF1-α and VEGF-A, thus promoting carcinogenesis [13], and Fernandez et al. [9] showed that 4-
OHE2 and a higher concentration of 2-OHE2 promoted a 5 bp deletion in exon 4 of p53. Because p53 is an important factor in the inhibition of cell growth and induction of apoptosis by acting on cell-cycle checkpoint in the G1 phase, changes in 4-OHE2 levels in cancer cells may play a significant role in cancer cell survival. In our study, we showed that 4-OHE2 induces XIAP expression in a concentration-dependent 10
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Fig. 7. Scheme showing how CYP1B1 inhibits XIAP degradation. CYP1B1 inhibits the ubiquitin-proteasome-mediated degradation of XIAP via the activation of PKCε signaling in cancer cells.
manner. However, 2-OHE2 did not have a significant effect. Moreover, the embelin-mediated inhibition of XIAP expression and concomitant apoptosis was rescued by 4-OHE2 in three cancer cell lines. As shown in Fig. 2B, as expected, the CYP1B1 L432V polymorphic gene induced a significant increase in XIAP expression compared to that with the wildtype or N203S. These data indicate that CYP1B1 activity is important in inducing XIAP expression. In previous study, Li et al. [44] showed that CYP1B1 activity of L432V variant was at least three times higher than wildtype and Chavarria-Soley et al. [45] showed that N203S variant has low enzymatic activity. In addition, we showed that the increase of tumor progression factors such as β-catenin, c-myc, or Sp1 by L432V is higher than wildtype or N203S [2]. Thus, we concluded that CYP1B1 activity is crucial to modulate XIAP expression and apoptosis. In a previous study, Lin et al. [20] showed that phosphorylation of XIAP on serine 87 is able to prevent XIAP self-ubiquitination and degradation. In the present study, we found that 4-OHE2 reduced the degradation of XIAP, and as reported previously, the mutation of serine 87 to glutamate prevented XIAP degradation by cycloheximide in MCF7 cells. In addition, as shown in Fig. 4D, the inhibition of CYP1B1 by TMS caused XIAP ubiquitination. These data demonstrate that CYP1B1 plays a crucial role in the prevention of XIAP ubiquitination. Thus, we believe that 4-OHE2 formation by CYP1B1 may play protective roles in XIAP proteasomal degradation by increasing XIAP phosphorylation on serine 87. Recently, Kato et al. [30] showed that the PMA-dependent activation of PKC leads to the induction of XIAP stability through phosphorylation on serine 87 and that it prevents etoposide-mediated apoptosis in SH-SY5Y cells. Tian et al. [32] demonstrated that Raf-1 interacts with XIAP and induces phosphorylation of XIAP on serine 87. A previous study showed that PKC-α and PKC-ε induce the Raf-1/MEK/ ERK signaling pathway [46]. Furthermore, treatment with a PKC inhibitor (calphostin C) prevents the cyclic strain-induced Raf-1 activation and sequential activation of PKC-α and that PKC-ε binds to Raf-1 directly and induces activation [47]. Accordingly, we hypothesized that 4-OHE2 induces the activation of PKCε and subsequently interacts with XIAP directly or induces an interaction between PKCε and Raf-1 to
phosphorylate XIAP on serine 87. We found that 4-OHE2 induced PKCε activation and PKCε siRNA-mediated suppression of XIAP expression and that phosphorylation was rescued by 4-OHE2. Whether activated PKCε increases phosphorylation of XIAP through interacting with Raf-1 needs to be clarified, our immunoprecipitation data showed that the binding of PKCε to XIAP increased when PKCε was activated by 4OHE2. Moreover, we found that the ability of 4-OHE2 to inhibit docetaxel- or cisplatin-mediated apoptosis was decreased by the suppression of PKCε expression. Indeed, according to the Kaplan-Meier curves, ovarian cancer patients with high expression of both CYP1B1 and XIAP or PKCε have poor prognosis on progression-free survival, overall survival, and post-progression survival (Fig. S2A–C). Thus, we suggest that the activation of PKCε by 4-OHE2 may play an important role in phosphorylation of XIAP and the resistance of cancer cells to anticancer agents. In summary, we demonstrated that 4-OHE2, catalyzed by CYP1B1 prevents the proteasome-mediated degradation of XIAP through phosphorylation on serine 87 by activating PKCε. The scheme shown in Fig. 7 summarizes the results of this study. Although, 4-OHE2 has been shown to be important for the activity of PKCε, it remains unclear how PKCε phosphorylation is induced by CYP1B1. Thus, further studies are needed to elucidate the detailed mechanism of the activation of PKCε by CYP1B1. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bbamcr.2019.118553. Transparency document The Transparency document associated with this article can be found, in online version. Declaration of competing interest The authors declare that they have no conflicts of interest. 11
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Acknowledgements
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