Crucial Role of C-Jun Phosphorylation at Ser63/73 Mediated by PHLPP Protein Degradation in the Cheliensisin a Inhibition of Cell Transformation

Published OnlineFirst October 3, 2014; DOI: 10.1158/1940-6207.CAPR-14-0233

Cancer Prevention Research Article Research

Crucial Role of c-Jun Phosphorylation at Ser63/73 Mediated by PHLPP Protein Degradation in the Cheliensisin A Inhibition of Cell Transformation

Junlan Zhu1,2, Jingjie Zhang1, Haishan Huang1,2, Jingxia Li1, Yonghui Yu1, Honglei Jin1,2, Yang Li1,2, Xu Deng3, Jimin Gao2, Qinshi Zhao3, and Chuanshu Huang1

Abstract Cheliensisin A (Chel A), as a novel styryl-lactone isolated from Goniothalamus cheliensis Hu, has been demonstrated to have an inhibition of EGF-induced Cl41 cell transformation via stabilizing p53 protein in a Chk1-dependent manner, suggesting its chemopreventive activity in our previous studies. However, its underlying molecular mechanisms have not been fully characterized yet. In the current study, we found that Chel A treatment could increase c-Jun protein phosphorylation and activation, whereas the inhibition of c-Jun phosphorylation, by ectopic expression of a dominant-negative mutant of c-Jun, TAM67, reversed the Chel A inhibition of EGF-induced cell transformation and impaired Chel A induction of p53 protein and apoptosis. Moreover, our results indicated that Chel A treatment led to a PHLPP downregulation by promoting PHLPP protein degradation. We also found that PHLPP could interact with and bind to c-Jun protein, whereas ectopic PHLPP expression blocked c-Jun activation, p53 protein and apoptotic induction by Chel A, and further reversed the Chel A inhibition of EGF-induced cell transformation. With the ﬁndings, we have demonstrated that Chel A treatment promotes a PHLPP protein degradation, which can bind to c-Jun and mediates c-Jun phosphorylation, and further leading to p53 protein induction, apoptotic responses, subsequently resulting in cell transformation inhibition and chemopreventive activity of Chel A. Cancer Prev Res; 7(12); 1270–81. 2014 AACR.

Introduction growth (5). However, the following studies have found that Cheliensisin A (Chel A), as a novel styryl-lactone isolated PHLPP could also act as a tumor suppressor in several types from Goniothalamus cheliensis Hu, has been reported to of cancer due to its ability to block growth factor–induced possess the potent chemoprevention effect (1–3). Our signaling in cancer cells (5, 6). Most recently, the studies published studies have demonstrated that chemopreventive from our group have indicated that PHLPP1 downregula- activity is mediated by its induction of apoptosis via trig- tion serves as cell apoptosis controller by promoting p53 gering p53 protein expression and activation (4). The protein translation via activation of Akt/p70S6K cascade pleckstrin homology domain leucine-rich repeat protein (7). We found here that PHLPP was downregulated in cells phosphatase (PHLPP), including PHLPP1 and PHLPP2, are treated with Chel A, which mediated chemopreventive protein phosphatases, which have been demonstrated to activity of Chel A. speciﬁcally dephosphorylate the hydrophobic motif of Akt, c-Jun, a member of the basic region leucine zipper subsequently triggering apoptosis and suppressing tumor protein family of transcription factors, in combination with itself or other proteins such as c-Fos, forms the transcription factor activator protein 1 (AP-1). c-Jun pro- 1Nelson Institute of Environmental Medicine, New York University School tein consists of a C-terminal DNA-binding domain and of Medicine, Tuxedo, New York. 2Zhejiang Provincial Key Laboratory for an N-terminal transactivation domain. The transcriptional Technology and Application of Model Organisms, School of Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China. 3State Key Lab- activity of c-Jun is increased by phosphorylation of serines oratory of Phytochemistry and Plant Resources in West China and 63 and 73 in the transactivation domain (8, 9). c-Jun Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, phosphorylation at Ser 63 and Ser 73 could be mediated China. by activation of JNKs upon a large variety of external or J. Zhu, J. Zhang, and H. Huang contributed equally to this article. internal stimulations (10–12) or the inhibition of its phos- Corresponding Authors: Chuanshu Huang, Nelson Institute of Environ- phatase. However, to the best of our knowledge, phospha- mental Medicine, New York University School of Medicine, 57 Old Forge Road, Tuxedo, NY 10987. Phone: 845-731-3519; Fax: 845-351-2320; tase that targets phosphorylated c-Jun protein has not been E-mail: [email protected]; Qinshi Zhao, E-mail: identiﬁed yet. Upon activation, c-Jun exerts various biologic [email protected]; and Jimin Gao, E-mail: [email protected] effects on cell proliferation, differentiation, cellular trans- doi: 10.1158/1940-6207.CAPR-14-0233 formation, and apoptosis (10–12). It has been reported 2014 American Association for Cancer Research. that inhibition of c-Jun activation by expressing a c-Jun

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Chel A Inhibits Cell Transformation via Degradation of PHLPP

dominant-negative mutant TAM67 inhibits apoptosis due Reagent (SignaGen Laboratories) following the manufac- to survival signal withdrawal (11). In the current study, we turer’s instructions. Their stable transfectants were estab- revealed that Chel A treatment resulted in PHLPP protein lished by G418-resistant selection. PW cells were transfected degradation, which further mediated c-Jun phosphoryla- with TAM67 or its corresponding vector control by using the tion at Ser 63 and 73 through JNK-independent manner. same method as described above, and the stable transfec- Moreover, we found the downregulation of PHLPP and its tants were selected by G418. mediated activation of c-Jun were essential for the induction of apoptosis as well as the inhibition of cell transformation Anchorage-independent growth in soft agar induced by EGF. Soft agar colony formation assay was conducted as described previously (4, 15, 16, 19). Briefly, 2.5 mL of Materials and Methods 0.5% agar in basal modified Eagle medium (BMEM) sup- Reagents and plasmids plemented with 10% FBS and 20 ng/mL EGF, as well as Chel Chel A was isolated from Goniothalamus cheliensis by the A at indicated concentrations, was layered onto each well of Kunming Institute of Botany, Chinese Academy of Sciences 6-well tissue culture plates. A total of 1 104 Cl41 cells, and (Kunming, Yunnan, China) as previously described (1, 3). their stable transfectants, were mixed with 1 mL of 0.33% The chemicals cycloheximide and MG132 were purchased agar BMEM (supplemented with 10% FBS with or without from Calbiochem. Luciferase assay substrate and EGF 20 ng/mL EGF, as well as with or without Chel A), and were from Promega. The antibodies specific against c-Jun, layered on top of the 0.5% agar layer. The plates were c-Jun(D), p-c-Jun Ser63, p-c-Jun Ser73, p-AKT Ser473, p- incubated at 37 Cin5%CO2 for 3 weeks. The colonies AKT Thr308, AKT, p-Erk1/2, Erk1/2, p-p38, p38, p-JNK1/2, were then counted under inverse microscopy. Colonies with JNK1/2, PARP, cleaved PARP, caspase-3, cleaved caspase-3, more than 32 cells were scored. Each experiment was done p53, p-p53 Ser15, GFP, and GAPDH were purchased from at least three independent times. The results were presented Cell Signaling Technology. HA antibody was obtained from as colonies/104 seeded cells. Covance Inc.. Antibodies specific against PHLPP1 and PHLPP2 were purchased from Bethyl Laboratories. Anti- Flow cytometry assay bodies against b-actin and a-tubulin were bought from Flow cytometry assay was conducted as described previ- Sigma. The plasmid, HA-PHLPP1 and HA-PHLPP2 were ously (4, 16, 20). Cl41 cells and their stable transfectants from Addgene. The plasmids, AP-1-luciferase reporter, were cultured in 6-well plates until they reached 70% to dominant-negative c-Jun–mutant plasmid TAM67, and 80% confluence. Cell culture medium was replaced with GFP-c-Jun were used and are described in our previous 0.1% FBS medium for 36 hours. The cells were then treated studies (13–15). with EGF (20 ng/mL) with or without Chel A at indicated concentrations in the medium containing 0.1% FBS. Cells Cell culture and transfection were harvested and fixed in ice-cold 70% ethanol. The cells Normal mouse epidermal Cl41 cells, which have been were stained with propidium iodide (PI) for 15 minutes and previously described (4, 16, 17), and their stable trans- then subjected to flow cytometry (Beckman Coulter) for fectants were maintained in 5% FBS Eagle’s minimum apoptotic analysis. essential medium (MEM), supplemented with 1% peni- cillin/streptomycin and 2 mmol/L L-glutamine(Life Tech- Western blotting nologies) at 37 Cin5%CO2 incubator that have been Cells were cultured using the same method described in described previously (4, 16, 17). PW cells have been flow cytometry assay, followed by pretreated with Chel A for described previously (18), and 293T cells and their stable 30 minutes, and afterwards exposed to EGF as indicated. transfectants were cultured in DMEM with 10% FBS. The The cells were subsequently washed on ice-cold PBS, and human colon cancer cell lines HCT116 cells and their then extracted with lysis buffer (10 mmol/L Tris–HCl, stable transfectants were cultured in McCoy’s 5A medium pH 7.4, 1% SDS, 1 mmol/L Na3VO4, and proteasome (Invitrogen), supplemented with 10% FBS. Cl41 cells inhibitor). The cell extracts were subjected to the Western stably transfected with AP-1 transactivation luciferase blot analysis and the protein bands specifically bound to reporter, TAM67, and their corresponding control vector antibodies were detected using alkaline phosphatase– have been established in our previous studies (15). These linked secondary antibody and ECF Western blotting sys- cells are all authenticated; the ATCC number of Cl41 cell tem as described previously (4, 16). is CRL-2010; of 293T cell is CRL-11268; and of HCT116 cell is CCL-247. Reverse transcription PCR Cl41 cells were transfected with HA-PHLPP1, HA- Total RNAs were extracted after treatment for the indi- PHLPP2, and their vector control (pcDNA3.0), HCT116 cated time periods using TRIzol reagent (Invitrogen). Total cells were transfected with HA-PHLPP1 and its vector con- cDNAs were synthesized by using oligo (dT) 20 primer trol, 293T cells were transfected with HA-PHLPP2 and by Superscript First-Strand Synthesis system (Invitrogen). its vector control, and 293T cells were transfected with PHLPP1, PHLPP2, and b-actin mRNA presented in the GFP-c-Jun together with HA-PHLPP1 or HA-PHLPP2, or cells were determined by semiquantitative reverse GFP-c-Jun, by using PolyJet DNA In Vitro Transfection transcription (RT)-PCR assay. The primers for mouse

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PHLPP1 (forward: 50-ACACCGTGATTG CTCACTCC-30,re- inhibition of EGF-induced cell transformation in mouse verse: 50-TTCCAGTCAGGTCTAGCTCC-30), mouse PHLPP2 epidermal Cl41 cells in a dose-dependent manner (Fig. 1A (forward:50-AGGTTCCTGAGCATCTCTTC-30,reverse:50-GT- and B). AP-1 is a transcription factor, and its inhibition has TCAGGCCCTTCAGTTGAG-30), and mouse b-actin (for- been reported to be involved in chemopreventive effect in ward: 50-ATATCGCTGCGCTGGTCGTC-30 reverse: 50-AGGA- previous studies (15, 24). Therefore, we determined wheth- TGGCGTGAGGGAGAGC-30) were used to determine the er Chel A treatment could inhibit AP-1 activation by utiliz- mRNA amount of PHLPP1, PHLPP2, and b-actin, respective- ing Cl41 cells stably transfected with AP-1-luciferase report- ly. The results were imagined with Alpha Innotech SP er. Unexpectedly, the results indicated that Chel A treatment image system (Alpha Innotech Corporation) as described induced AP-1–dependent transactivation in a dose-depen- previously (15). dent manner (Fig. 1C). c-Jun is the most extensively studied protein of AP-1 components and has also been Luciferase assay reported to be involved in the regulation of numerous cell Cl41 cells stably transfected with AP-l luciferase report- activities, such as proliferation, survival, tumorigenesis, er constructs were seeded into 96-well plates and cultured and apoptosis (24, 25). The transcriptional activation of until 70% to 80% confluent. The cells were treated with c-Jun depends on its phosphorylation at Ser 63 and 73 in various concentrations of Chel A in MEM medium con- the transactivation domain (8, 9, 11). The phosphoryla- taining 0.1% FBS for 12 hours and then lysed for lucif- tion of c-Jun has also been reported to play a role in the erase assay using luciferase substrate as described previ- mediation of apoptosis under withdrawal of survival ously (21, 22). signaling (24). Therefore, the next experiment was carried out to evaluate the effect of Chel A on c-Jun phosphor- Immunoprecipitation ylation at Ser63/73. As shown in Fig. 1D, Chel A treat- Stable transfectants of 293T cells, 293T (GFP-c-Jun, ment alone induced c-Jun phosphorylation at Ser63/73 in pcDNA3.0), 293T (GFP-c-Jun, HA-PHLPP1), and 293T a time-dependent manner with maximum induction at 3 (GFP-c-Jun, HA-PHLPP2), were cultured in 10-cm dishes hours after treatment. Moreover, we found that cotreat- until 70% to 80% confluence. Culture medium was ment of cells with Chel A and EGF increased c-Jun replaced with DMEM containing 0.1% FBS for 12 hours, phosphorylation at Ser63/73 (Fig. 1E and F). These and the cells were then lysed in cell lysis buffer (1% Triton X- results indicated that Chel A treatment led to c-Jun 100, 150 mmol/L NaCl, 10 mmol/L Tris, pH 7.4, 1 mmol/L phosphorylation at Ser63/73 and promoted EGF-induced EDTA, 1 mmol/L EGTA, 0.2 mmol/L Na3VO4,0.5%NP-40, c-Jun phosphorylation in Cl41 cells. and complete protein cocktail inhibitors from Roche) on ice. Lysate was incubated with normal IgG/Protein A/G plus- c-Jun phosphorylation at Ser63/73 was crucial for the agarose or anti-GFP agarose (Santa Cruz Biotechnology, Inc.) Chel A inhibition of EGF-induced transformation in at 4C for 12 hours. The agarose beads were collected by Cl41 cells centrifugation, followed by being washed three times with To evaluate the potential role of c-Jun activation in Chel A cell lysis buffer, and the beads were extracted with Western inhibition of cell transformation, Cl41 cells stably trans- blot sample buffer and subjected to Western blot assay (23). fected with dominant-negative N-terminal–truncated Stable transfectants of Cl41 cells, Cl41 (GFP-c-Jun/HA- mutant of c-Jun, TAM67, and its parental vector control PHLPP1), and Cl41 (GFP-c-Jun/HA-PHLPP2), were cultured plasmid (Cl41 vector) had been established and well char- in 10 cm dishes until 70% to 80% confluence. Culture acterized in our previous publications (15, 26, 27). Verifi- medium was replaced with MEM containing 0.1% FBS for cation of TAM67 expression in Cl41 stable transfectant cells 12 hours, followed by pretreatment with Chel A as indicated. was indicated in Fig. 2D. Our results revealed that ectopic Then, the cells were lysed and extracted for Western blot assay expression of TAM67 dramatically attenuated the inhibitory by using the same method as described above. effect of Chel A on EGF-induced cell transformation (Fig. 2A and B); this strongly indicates that induction of c-Jun Statistical analysis phosphorylation at Ser63/73 is crucial for the chemo- Student t testwasemployedtodeterminethesignifi- preventive activity of Chel A. cance of differences between the different groups in each experiment. The differences were considered significant at c-Jun exerted its chemopreventive effect via P < 0.05. upregulation of p53 protein expression and apoptosis To elucidate the molecular mechanisms underlying c-Jun–mediated chemopreventive activity of Chel A, the Results flow cytometry assay was used to assess the effect of Chel A treatment inhibited EGF-induced cell TAM67 overexpression on cell-cycle progression and apo- transformation with induction of c-Jun ptotic responses, and western blot assay for p53 protein phosphorylation at Ser63/73 in Cl41 cells expression due to Chel A treatment. As shown in Fig. 2C, Our most recent studies have indicated that Chel A could Chel A treatment led to a marked cell death in Cl41 (vector) act as a chemopreventive agent for inhibition of cell trans- cells. Very interestingly, EGF cotreatment slightly increased formation (2, 4). Chel A treatment consistently showed the Chel A–induced cell death, rather than provide a protective

1272 Cancer Prev Res; 7(12) December 2014 Cancer Prevention Research

Chel A Inhibits Cell Transformation via Degradation of PHLPP

A Vehicle EGF 20 ng/mL EGF+ChelA 0.5 μmol/L B 35 C 5 * * 30 * 4 25 cells 4 20 3 EGF+Chel A 1 μmol/L EGF+Chel A 2 μmol/L EGF+Chel A 4 μmol/L * 15 * 2 * 10 * 1 5 * Relative AP-1 activity D 0 1 3 6 12 24 36 48 Chel A (h) Colonies (× 100) /10 p-c-Jun S63 0 0

p-c-Jun S73 0 0.5 1 2 4 0 1 2 4 Chel A (μmol/L) Chel A (μmol/L) c-Jun F GAPDH –––––– + + + + + + Chel A – + + + + + – + + + + + EGF E 0 0 0.5 1.0 2.0 4.0 Chel A (μmol/L) 0 15 30 60 120 180 0 15 30 60 120 180 Time (min) – + + + + + EGF p-c-Jun S63 p-c-Jun S63

p-c-Jun S73 p-c-Jun S73

c-Jun c-Jun α-Tubulin GAPDH

Figure 1. The inhibition on EGF-induced cell transformation and the induction of c-Jun phosphorylation and AP-1 transactivation by Chel A in Cl41 cells. A and B, Cl41 cells were exposed to indicate concentrations of Chel A in combination with EGF for cell transformation assay in soft agar as described previously (4). The colony formation was photographed (A), and the number of colonies was scored and presented as colonies/104 seeded cells (B). , a significant decrease as compared with that of EGF treatment alone (P < 0.05). Each bar indicates the mean and SD of three independent experiments. C, Cl41 cells (1 103) stably expressing AP-1-luciferase reporter were seeded into each well of a 96-well plate. After synchronization, cells were cultured in 0.1% FBS medium for 48 hours and then treated with various concentrations of Chel A for 12 hours, and then extracted for determination of luciferase activity as described previously (19, 41). , a significant increase in AP-1 activity (P < 0.05). Each bar indicates the mean and SD of three independent experiments. D–F, Cl41 cells were seeded into each well of 6-well plates and cultured as described in ref. (4). Then, the cells were treated with EGF and Chel A at different concentrations (E) or for different time periods (D and F). The cell extracts were subjected to Western blotting as described in Materials and Methods. GAPDH and a-tubulin were used as a control for protein loading. The results shown are data represented from three independent experiments. effect on cell death induced by Chel A in Cl41(vector) cells. bition of c-Jun activation by ectopic expression of TAM67 Importantly, Chel A–induced cell death was dramatically blocked p53 protein expression in comparison with that attenuated by ectopic expression of TAM67 in Cl41 cells observed in Cl41 vector cells, demonstrating that c-Jun (Fig. 2C), suggesting that c-Jun activation was crucial for activation mediated p53 protein expression. Chel A–induced cell death. Consistently, the results obtained from flow cytometry also showed that sub-G1 Chel A treatment promoted PHLPP protein DNA content (cell death peak) increased significantly degradation and such PHLPP protein degradation upon Chel A treatment in Cl41 vector cells, and stable mediated c-Jun phosphorylation at Ser63 and Ser73 expression of TAM67 abolished Chel A–induced increase due to Chel A treatment in sub-G1 cells (Fig. 2E). Moreover, our results indicated MAPK, including the ERK1/2, JNK, and p38 have been that TAM67 abrogated Chel A–induced cleaved PARP and reported to be responsible for the regulation of various cell caspase-3 as demonstrated in Western blot analysis (Fig. functions in different experimental systems (28). Activated 2F and G), clearly revealing that Chel A–induced cell MAPK causes phosphorylation and activation of transcrip- death was an apoptotic response. As our recent studies tion factors in the cytoplasm and/or nucleus (29). c-Jun has have demonstrated that p53 protein induction was essen- been reported as one of the most important transcription tial for Chel A–induced apoptotic responses (4), we assess factors that can be phosphorylated at Ser63/73 and activat- the relationship between c-Jun activation and p53 protein ed by MAPKs, especially JNKs. Thus, to elucidate the mech- expression by comparison of p53 protein expression due anism underlying c-Jun phosphorylation upon Chel A to Chel A treatment between Cl41 (vector) and Cl41 treatment, we first examined whether Chel A treatment (TAM67) transfectants. As indicated in Fig. 2D, the inhi- could induce the activation of MAPKs. The results showed

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Chel A 4 μmol/L Vehicle μ μ Chel A 4 mol/L Chel A 2 mol/L Vehicle Cl41 TAM67 Cl41 Vector Ap Ap o/ EGF+ChelA4μmol/L Medium controlEGFEGF+ChelA2μmol/L o/ EGF+ChelA4μmol/L Medium controlEGFEGF+ChelA2μmol/L 2 4 0 24ChelA(μmol/L) 0 24 0 G G Medium Cl41 (vector)(TAM67) 0 0 –G –G cancerpreventionresearch.aacrjournals.org 1 Published OnlineFirstOctober3,2014;DOI:10.1158/1940-6207.CAPR-14-0233 Cl41(vector) 1 S S Medium G G Ap Ap G G G G 2 2 S –M S –M 2 0 2 0 –M –G –M –G 1 1 89.92 62.11 21.12 1.13 7.26 5.50 7.00 0.51 EGF Cl41 (vector) Ap Ap Mdu EGF Medium G G 0 0 –G –G S 1 M G 1 S EGF 2 Cl41(TAM67) Ap G G G Caspase-3 Cleaved Caspase-3 Ap β-Actin PARP Cancer Research. G G Cleaved PARP S 2 2 0 S –M 2 0 –M –G –M –G 1 1 10.91 48.70 26.41 8.10 16.57 71.97 6.86 0.48 on September 23, 2021. © 2014American Association for G Medium Ap Ap G G 0 0 –G –G PW (vector) PW (TAM67)PW (vector) 0 D 1 1 S S 0 24 Cl41 (vector)(TAM67) Ap G Ap G G G G G S S 2 2 0 2 0 2 –M –M –G –M –G –M 4 0 1 1 20.23 61.14 10.56 10.77 82.46 5.82 3.11 0.54 Cl41 (TAM67) Relative colony formation B 0.2 0.4 0.6 0.8 1.0 0 024ChelA(μmol/L) acrPeeto Research Prevention Cancer Ap Ap 0 Cl41 (vector)(TAM67) G G 4 EGF +ChelA2μmol/L EGF +ChelA4μmol/L EGF Medium 1.0 0 0 –G –G 0.35 0.25 1 1 Chel A(μmol/L) S S G EGF G Ap G Ap G G G PARP β-Actin Caspase-3 Cleaved Caspase-3 PARP Cleaved 2 2 S S 2 0 –M 2 0 –M –M –G –M –G 0 1 1.0 1 15.84 66.93 0.70 * 8.32 3.75 11.70 77.57 c-Jun TAM67 p53 α-Tubulin Ser15 p-p53 4.99 0.43 0.63 * Published OnlineFirst October 3, 2014; DOI: 10.1158/1940-6207.CAPR-14-0233

Chel A Inhibits Cell Transformation via Degradation of PHLPP

– + + + + + − + + + + + EGF A 0 1 2 4 Chel A (h) C ––– – – – + + + + + + Chel A p-Erk1/2 0 15 30 60 120 180 0 15 30 60 120 180 Time (min) p-Erk1/2 Erk1/2 p-p38 Erk1/2

p38 p-p38 p-JNK1/2 p38 p-JNK1/2 JNK1/2

PHLPP1 JNK1/2 PHLPP2 D 0 1 2 3 Chel A (h) β-Actin β-Actin phlpp1 B – + + + + + EGF 0 0 0.5 1.0 2.0 4.0 ChelA (μmol/L) β-Actin p-Erk1/2 E phlpp2 – + + + + + + + + + MG132 (10 μmol/L) Erk1/2 –– + + + + + + + + CHX (50 μg/mL) –––––– + + + + Chel A (4 μmol/L) p-p38 0 0 1 2 3 6 1 2 3 6 Time (h) p38 PHLPP1

p-JNK1/2 PHLPP2 β-Actin JNK1/2

Figure 3. Chel A decreased PHLPP1 and PHLPP2 protein expression by inducing PHLPP1 and PHLPP2 protein degradation. A and B, Cl41 cells were exposed to Chel A for indicated time periods or concentrations, and the cell extracts were applied to Western blotting for determination of the protein expressions using speciﬁc antibodies. b-Actin was used as a protein loading control. The result represents one of three independent experiments. C, Cl41 cells were treated with EGF and/or Chel A (4 mmol/L) for indicated time periods, and the cell extracts were applied to Western blotting for determination of the protein expressions using speciﬁc antibodies as indicated. D, Cl41 cells were exposed to Chel A (4 mmol/L) for indicated time periods and PHLPP1 and PHLPP2 mRNA was determined by RT-PCR. E, Cl41 cells were treated with MG132 for 4 hours, followed by exposure with cycloheximide (CHX) combined with Chel A or cycloheximide alone as indicated. Then cell extracts were subjected to Western Blotting and b-actin protein expression was used as a protein loading control. The result represents one of three independent experiments. that Chel A treatment alone only induced a slight increase in c-Jun phosphorylation at Ser63/73 (Fig. 1D–F), suggesting the activation of ERKs, p38, and JNKs at high dose (4 mmol/ that MAPK activations were not major mediators responsi- L) and did not promote kinase activation in dose–response ble for c-Jun activation by Chel A treatment. Thus, we studies (Fig. 3A and B). In contrast, to increase cell death in anticipated that phosphatases might play a major role in cotreatment of Cl41 cells with Chel A and EGF, as compared c-Jun phosphorylation following Chel A treatment. with treatment of cells with either one alone, Chel A The recent discovery of the PHLPP Ser/Thr phosphatases cotreatment of cells with EGF slightly inhibited EGF- added a new player to the cast of phosphate-controlling induced activation of ERKs and JNKs 15 minutes after enzymes in cell signaling responses (30). PHLPPs, consist- treatment, as compared with EGF treatment alone (Fig. ing of PHLPP1 and PHLPP2, catalyzes the dephosphoryla- 3C). Thus, the results obtained from determination of ERKs, tion of a conserved regulatory motif, (the hydrophobic p38, and JNKs activation were completely inconsistent with motif) on the AGC kinases Akt, PKC, and S6 kinase, as well

Figure 2. Expression of TAM67 reversed the biologic effect of Chel A in Cl41 cells. A–C, effect of Chel A on EGF-induced cell transformation in Cl41 TAM67 and Cl41 vector cells was determined in soft agar assays. The colony formation was observed under inverted microscope and photographed (A). The numbers of colonies were scored and presented as colonies per 10,000 seeded cells (B). , a significant increase in Cl41 TAM67 cells compared with Cl41 Vector cells (P < 0.05). Each bar indicates the mean and SD from three independent experiments. C, the Cl41 (TAM67) transfectant and its scramble vector transfectant, Cl41 (vector), were treated with Chel A and EGF as indicated. Images were taken under microscopy at 48 hours after Chel A treatment. D, the stable transfectant of Cl41 cells transfected with TAM67 and its scramble vector were treated with Chel A as indicated, and the cell extracts were applied to Western blotting for the determination of the protein expressions using specific antibodies. a-Tubulin was used as protein loading control. E, Cl41 cells and Cl41 TAM67 stable transfected cells (2 105) were seeded into each well of 6-well plates and cultured the same as those for Western blot analysis, whereas cells were treated with various concentrations of EGF and Chel A as indicated, for 48 hours, and then were fixed and stained with PI as described in Materials and Methods. Cell apoptosis was determined by flow cytometry. The result shown represents one of three independent experiments. F and G, Cl41 TAM67 stably transfected cells and its vector control cells, PW TAM67 stable transfected cells and its vector control cells were seeded into 6-well plates. The cells were treated with Chel A for indicated concentrations for 48 hours and the cell extracts were applied to Western blotting to determine the expression of cleavages of PARP and caspase-3. b-Actin was used as a protein loading control. The result represents one of three independent experiments.

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as an inhibitory site on the kinase Mst1, to inhibit cellular To evaluate the role of PHLPPs protein degradation in proliferation and induce apoptosis (30). Our most recent c-Jun activation upon Chel A treatment, we tested whether studies demonstrated that PHLPP1 inhibited cell apoptosis the overexpression of HA-PHLPP proteins could regulate c- via downregulation of p53 translation (7). Hence, we first Jun phosphorylation at Ser63/73 in Cl41 cells. The results determined the effect of Chel A on PHLPP protein expres- revealed the c-Jun protein phosphorylation at Ser63/73 was sion in Cl41 cells. The results showed that Chel A treatment abolished upon ectopic expression of PHLPP1 in either markedly attenuated the expression of PHLPPs in a time- transfectants of Cl41 HA-PHLPP1, Cl41 HA-PHLPP2, or dependent manner (Fig. 3A). To elucidate the mechanisms HCT116 HA-PHLPP2 (Fig. 4A). To further buttress this underlying Chel A downregulation of PHLPPs protein notion, Chel A–induced c-Jun phosphorylation was com- expression, we first examined the effect of Chel A on phlpp1 pared among the transfectants of Cl41 Vector, Cl41 HA- and phlpp2 mRNA levels by RT-PCR assay. The results PHLPP1, and Cl41 HA-PHLPP2. As anticipated, either over- showed that Chel A treatment had no observable effect on expression of HA-PHLPP1 or HA-PHLPP2 blocked c-Jun mRNA levels of either phlpp1 or phlpp2 (Fig. 3D), exclud- phosphorylation at Ser63/73 due to Chel A treatment as ing the possibility of Chel A affecting phlpp gene transcrip- comparison with Cl41 Vector cells (Fig. 4B). It was noted tion or mRNA stability. Thus, we further assessed the that basal level of c-Jun phosphorylation at Ser63/73 potential effect of Chel A on PHLPPs protein degradation. was not inhibited in Cl41 HA-PHLPP1 and HA-PHLPP2 To test whether Chel A treatment was able to promote transfectants as compared with Cl41 (vector) transfectant PHLPPs protein degradation, Cl41 cells were first pretreated (Fig. 4B), which is different with the results observed in Fig. with proteasome inhibitor MG132 to accumulate PHLPPs 4A. This could be caused by various cell culture conditions protein. The MG132 was then removed from cell culture in two experiments. The cell culture medium used in Fig. 4A medium and the protein synthesis inhibitor cycloheximide is 5% FBS MEM, whereas the cell culture medium used for was added to the cells alone or in combination with Chel A. experiment shown in Fig. 5B is 1% FBS MEM. It is well The effect of Chel A on the dynamics of PHLPP protein known that low serum of cell culture causes cell stress degradation was determined during the indicated time responses. We therefore anticipated that low concentration periods. As shown in Fig. 3E, the PHLPP1 and PHLPP2 of serum for experiment shown in Fig. 4B might cause cell protein degradation rates were markedly increased when stress, which could increase the basal level of c-Jun phos- cells were coincubated with Chel A plus cycloheximide in phorylation through activation of stress kinases, such as comparison with the cells incubated with cycloheximide JNKs. These results demonstrated that PHLPP1/2 could alone. Those results strongly indicated that Chel A treat- repress c-Jun phosphorylation at Ser63/73, which might be ment decreased PHLPP expression via promoting PHLPP responsible for the effect of Chel A on apoptosis and cell protein degradation. transformation in Cl41 cells.

A Cl41 HCT116 Cl41 B Cl41 Vector HA-PHLPP1 HA-PHLPP2 Vector HA-PHLPP1Vector HA-PHLPP1 Vector HA-PHLPP2 0 6 0 6 0 6 Chel A (h) HA HA HA HA-PHLPP1 HA-PHLPP2 HA-PHLPP1 p-c-Jun S73 p-c-Jun S73 HA-PHLPP2 p-c-Jun S63 p-c-Jun S63 c-Jun c-Jun p-c-Jun S73

p-AKT S473 p-AKT S473 p-c-Jun S63

p-AKT T308 p-AKT T308 c-Jun AKT AKT β-Actin β β-Actin -Actin

Figure 4. PHLPP1 and PHLPP2 are crucial for inhibition of c-Jun phosphorylation by Chel A treatment. A, Cl41 cells were stably transfected with HA-PHLPP1 and HA-PHLPP2; HCT116 cells were stably transfected with HA-PHLPP1; the cell extracts were applied to Western blotting to determine the expression of c-Jun and its phosphorylation. B, the stable transfectant of Cl41 cells transfected with HA-PHLPP1, HA-PHLPP2, and its scramble vector were treated with Chel A for 6 hours, and the cell extracts were applied to Western blotting for determination of the protein expressions using speciﬁc antibodies. b-Actin was used as protein loading control.

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Chel A Inhibits Cell Transformation via Degradation of PHLPP

A Vehicle Chel A (2 μmol/L) Chel A (4 μmol/L) B Vector HA-PHLPP1 HA-PHLPP2 0 2 4 0 2 4 0 2 4 Chel A (μmol/L) Caspase-3

Cleaved Caspase-3 PARP

Cleaved PARP Cl41 p-p53 S15

p53

GAPDH HA-PHLPP2 HA-PHLPP1 Vector HA-PHLPP1 HA-PHLPP2 C D Medium control EGF EGF+Chel A 4 μmol/L 35 Medium EGF μ 30 EGF+ChelA 4 mol/L 25.2

Medium control EGF EGF+Chel A 4 μmol/L 20.1 19.1 cells

4 20

15 11.8 11.9 **

10 Medium control EGF EGF+Chel A 4 μmol/L 5.1 Colonies (×100)/10 5 0 0 0 0 HA-PHLPP2 HA-PHLPP1 Vector Control HA-PHLPP1 HA-PHLPP2

Figure 5. HA-PHLPP1 and HA-PHLPP2 overexpression blocked the biologic effect of Chel A in Cl41 cells. A, Cl41 cells were stably transfected with HA-PHLPP1 and HA-PHLPP2, Cl41 vector were treated with indicated Chel A. The cultured cells showed a declined tendency to apoptosis by Chel A. B, Cl41 HA-PHLPP1 cells, Cl41 HA-PHLPP2 cells, and Cl41 vector cells were treated with Chel A for indicated concentrations and the cell extracts were applied to Western blotting to determine the expression and cleavages of PARP and caspase-3, p53, and phosphorylation at Ser15. b-Actin was used as a protein loading control. The result represents one of three independent experiments. C and D, effect of Chel A on EGF-induced cell transformation in Cl41 HA- PHLPP1 cells, Cl41 HA-PHLPP2 cells, and Cl41 vector cells was determined by soft agar assay. The colony formation was observed under inverted microscope and photographed (C). The numbers of colonies were scored and presented as colonies per 10,000 seeded cells (D). , a signiﬁcant increase in Cl41 HA-PHLPP1 cells and Cl41 HA-PHLPP2 cells as compared with those in Cl41 vector cells in response to Chel A (P < 0.05). Each bar indicates the mean and SD from three independent experiments.

PHLPP protein degradation contributed to apoptotic in either cell morphology alteration or western blot results response, p53 induction, and the inhibition of EGF- (caspase-3 cleavage, as well as PARP cleavage). These results induced cell transformation by Chel A treatment demonstrated that PHLPPs degradation by Chel A played a To evaluate whether PHLPP downregulation contributed crucial role in its apoptotic induction. Consistent with the to Chel A–induced apoptotic induction, Cl41 HA-PHLPP1 alteration of cell apoptosis, PHLPP overexpression also and Cl41 HA-PHLPP2 were employed to compare their attenuated p53 protein expression and phosphorylation at apoptotic responses due to Chel A treatment. As shown Ser15, due to Chel A treatment as compared with those in in Fig. 5A and B, cell apoptotic response was observed in Cl41 vector cells (Fig. 5B). Very importantly, the inhibition Cl41 (vector) cells treated with Chel A at 4 mmol/L, whereas of cell transformation by Chel A was partially reversed by such apoptotic induction was impaired by overexpression ectopic expression of HA-PHLPP1 and HA-PHLPP2 as of either PHLPP1 or PHLPP2 in Cl41 cells, as demonstrated shown in Fig. 5C and D. Those results demonstrated that

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PHLPP degradation caused by Chel A treatment was essen- prompted us to test whether PHLPP proteins could interact tial for c-Jun phosphorylation, p53 protein expression, and with c-Jun protein. To test this possibility, 293T cells were apoptosis, which in turn led to the inhibition of EGF- transfected with GFP-c-Jun, GFP-c-Jun plus HA-PHLPP1, or induced cell transformation in Cl41 cells. GFP-c-Jun plus HA-PHLPP2, respectively, and coimmunoprecipitation was performed to pull down GFP-c-Jun using PHLPPs interacted with c-Jun and mediated its specific anti-GFP antibodies. The stable 293T GFP-c-Jun/ phosphorylation PHLPP1 transfectants were selected and identified as shown PHLPP1 and PHLPP2 act as the phosphatases, and have in Fig. 6A. The HA-PHLPP1 was observed in the immuno- been reported to exert the function through binding with complex pull down with specific anti-GFP antibodies, sug- their substrates (such as AKT and PKC), and subsequently gesting that PHLPP1 protein did interact with c-Jun protein dephosphorylating those targeted proteins. Our abovemen- in the intact cells. Similarly, the binding of GFP-c-Jun with tioned results revealed that the PHLPPs and c-Jun protein PHLPP2 protein was also observed in transfectants of GFP- phosphorylation at Ser63/73 was inversely corelated in c-Jun and HA-PHLPP2 (Fig. 6C). To determine whether Cl41 cells after Chel A treatment. Moreover, ectopic expres- Chel A could regulate the interaction of c-Jun with PHLPP, sion of either PHLPP1 or PHLPP2 could abolish the c-Jun the stable Cl41 GFP-c-Jun/PHLPP1 transfectants and Cl41 protein phosphorylation at Ser63/73. Those results GFP-c-Jun/PHLPP2 were treated with Chel A and the cell

Cl41 A BC D 293T 293T 293T HA-PHLPP1 HA-PHLPP2 GFP-c-Jun GFP-c-Jun – + – + – Chel A pcDNA3.0 HA-PHLPP2 pcDNA3.0HA-PHLPP1 GFP-c-Jun GFP-c-JunGFP-c-Jun pcDNA3.0 GFP-c-Jun + IgG–––– GFP-c-JunHA-PHLPP1GFP-c-Jun –– + IgG + + + + – anti-GFP HA + + – anti-GFP –– + IgG GFP HA- + –+ anti-GFP GFP PHLPP1 GFP- GFP GFP- c-Jun GFP c-Jun GFP-c-Jun GFP- p-GFP- HA

c-Jun S73 IP c-Jun HA- IP c-Jun IP p-GFP- HA PHLPP1 p-c-Jun c-Jun S63 HA- S73 HA- PHLPP2 p-c-Jun PHLPP1 HA p-c-Jun S63 HA- S73 p-AKT GFP PHLPP2 S473 p-c-Jun p-AKT GFP- GFP S63 T308 c-Jun GFP- c-Jun c-Jun GFP

AKT Input GFP- GAPDH HA c-Jun c-Jun HA- p-GFP- c-Jun E Chel A PHLPP1 c-Jun S73 p-c-Jun HA Input S73 HA- PHLPP p-Chk1 p-GFP- PHLPP1

Input c-Jun S63 p-c-Jun HA- p-c-Jun p53 S63 PHLPP2 HA p-c-Jun S73 EGF-induced cell HA- Apoptosis p-c-Jun transformation PHLPP2 S63

Figure 6. PHLPPs were responsible for c-Jun phosphorylation via interaction with c-Jun. A, 293T cells were cotransfected with GFP-c-Jun, along with HA-PHLPP1, and the cell extracts were applied to Western blotting for determination of the protein expressions using speciﬁc antibodies. B and C, coimmunoprecipitation was performed with anti-GFP antibody–conjugated agarose beads. Immunoprecipitates were then subjected to immunoblotting for detection of PHLPP1 and PHLPP2 using HA antibody. D, Cl41 cells cotransfected with GFP-c-Jun, along with HA-PHLPP1 or HA-PHLPP2, were treated with or without Chel A (4 mmol/L) for 6 hours, the cell extracts were used for coimmunoprecipitation by using anti-GFP antibody–conjugated agarose beads. Immunoprecipitates were then subjected to immunoblotting for detection of various protein expressions as indicated. E, a model for Chel A–inhibited EGF-induced cell transformation by inducing apoptosis in JB6 Cl41 cells: Chel A treatment reduced the protein level of PHLPPs which results in upregulating the activation of c-Jun and promoting p53 activation and accumulation, apoptosis, and inhibiting EGF-induced cell transformation.

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Chel A Inhibits Cell Transformation via Degradation of PHLPP

extracts were used to carry out pull-down GFP-c-Jun assay igenesis, and apoptosis (24, 25). c-Jun appears to be both a using specific anti-GFP antibodies. The results showed that positive and a negative regulator of apoptosis (36). The although co-immunoprecipitated HA-PHLPP1 or HA- exact function of c-Jun is likely to be cell type- and stimulus- PHLPP2 protein by pull-down assay with anti-GFP anti- specific. Eferl and colleagues analyzed the antiapoptotic bodies was decreased in Chel A–treated cells as compared function of c-Jun using a cell culture system and found that with vehicle-treated cells, the decreased levels were consis- c-Jun–deficient hepatocytes are more sensitive to TNFa- tent with the downregulated protein levels of HA-PHLPP1 induced apoptosis and that this sensitization was rescued and HA-PHLPP2 upon Chel A treatment (Fig. 6D). This upon p53 deficiency, showing that c-Jun–deficient liver result revealed that Chel A only inhibited HA-PHLPP pro- tumors accumulate high levels of p53 protein (37). How- tein expression, while it did not affect the binding of GFP-c- ever, elevated levels of p53 could not be detected in c-Jun– Jun with either HA-PHLPP1 or HA-PHLPP2. Thus, our deficient hepatocytes. Our current studies found that Chel studies demonstrated that Chel A treatment resulted in A–initiated c-Jun phosphorylation was crucial for this p53 PHLPP protein degradation, which reduced PHLPP protein protein accumulation and activation. It has been reported interaction with c-Jun, and in turn led to the upregulation of that phosphorylated c-Jun has the ability to bind to p53 c-Jun protein phosphorylation, p53 protein induction, promoter for the induction of p53 transcriptional activity apoptotic responses, as well as the inhibition of EGF- (38). Our recent studies, however, have shown that Chel A induced cell transformation. treatment upregulates p53 expression by decreasing protein degradation level rather than transcription or mRNA stability (4). Although the detailed mechanism involved in c- Discussion Jun regulation of p53 protein expression is undergoing Defects in apoptosis underpin both tumorigenesis and investigation in our group, the phosphorylation of c-Jun is drug resistance, and most anticancer drugs exert their che- a crucial factor for activation of p53 by Chel A. motherapeutic effect by inducing tumor cell apoptosis (31). PHLPP1 and PHLPP2 are the phosphatases that directly Chel A exhibits a potent cytotoxicity in HL-60 cells, and is dephosphorylate Akt, promote apoptosis, and suppress capable of inducing apoptosis of leukemia cells by down- tumor growth (5). PHLPP acts as a tumor suppressor in regulation of Bcl-2 expression (2). In addition, our recent several types of cancer due to its ability to block growth studies have found that Chel A exerts an inhibitory effect on factor–induced signaling in cancer cells (5, 6). However, our EGF-induced cell transformation with the induction of most recent studies have indicated that PHLPP1 down- apoptosis in Cl41 cells through stabilization and activation regulation by miR-190 in arsenite responses could mediate of p53 (4). In the current study, we showed that Chel A apoptosis via promotion of p53 translation via upregula- significantly inhibited EGF-induced cell transformation tion of Akt/p70S6K pathway (7). Our current results accompanied with the induction of c-Jun protein phos- showed that Chel A treatment promoted PHLPP protein phorylation at Ser63/Ser73 and PHLPP protein downregu- degradation, leading to an increase in c-Jun protein phos- lation. Further studies showed that ectopic expression of phorylation at Ser63/73, and in turn resulting in p53 dominant-negative c-Jun–mutant TAM67 attenuated Chel protein induction and further leading to apoptosis and A–induced p53 protein expression, apoptotic induction, inhibition of cell transformation. Given only a limited and the inhibition of cell transformation; these indicated number of Ser/Thr phosphatases to balance the actions of that c-Jun phosphorylation and activation was the upstream more than 400 Ser/Thr kinases in human cells (39), mediator for p53 protein expression. Moreover, we found PHLPPs, a family of Ser/Thr phosphatases with multiple that PHLPP protein could bind to and interact with c-Jun regulatory domains, are predicted to have multiple targets protein and attenuated c-Jun protein phosphorylation. In in addition to Akt and PKC that have been identified. On the addition, we revealed that Chel A treatment could induce basis of previous studies, PHLPPs are critical for tumor PHLPP1 and PHLPP2 protein degradation, which further suppression, whereas their loss can activate p53, leading reduced the PHLPP protein interaction with c-Jun protein, to cellular senescence and apoptosis (40). Nevertheless, our resulting in the upregulation of c-Jun protein phosphory- studies showed that PHLPPs interacted with c-Jun, which lation. Therefore, we identify a novel molecular mechanism inhibited c-Jun phosphorylation at Ser63/73, p53 protein underlying Chel A as a chemopreventive agent by activating expression and activation, as well as apoptosis. Very impor- PHLPPs/c-Jun/p53 apoptotic axis as shown in Fig. 6E. tantly, we found that ectopic expression of HA-PHLPP1 or p53 is a tumor suppressor that has a crucial role in the HA-PHLPP2 could almost completely impair c-Jun phos- inhibition of cancer development (32). One of biologic phorylation at Ser63/73, p53 protein expression, and apo- functions of p53 is to trigger cell apoptosis (33–35). Our ptosis, as well as reverse EGF-induced cell transformation in previous studies have strongly indicated that Chel A treat- Cl41 cells, which strongly demonstrated that Chel A- ment leads to p53 protein accumulation and activation by induced apoptosis via PHLPPs/c-Jun/p53 was crucial for preventing p53 proteins from degradation (4). c-Jun is a key the chemopreventive activity of Chel A. In addition, our member of the AP-1 family of transcription factors which results revealed that PHLPPs might act as phosphatases of c- bind to AP-1 elements in their target genes (36), and c-Jun/ Jun, which is currently under investigation in our group. AP-1 activation is involved in the regulation of numerous In summary, our current studies elucidated a novel effect cell biologic activities, such as proliferation, survival, tumor- of Chel A in regulation of PHLPP protein degradation. We

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also found that PHLPP protein degradation could reduce Analysis and interpretation of data (e.g., statistical analysis, biosta- tistics, computational analysis): J. Zhang, H. Huang, Y. Yu, Y. Li, X. Deng, the interaction of PHLPP protein with c-Jun protein and Q. Zhao, C. Huang resulting in an increase in c-Jun phosphorylation at Ser-63/ Writing, review, and/or revision of the manuscript: J. Zhang, H. Huang, Ser-73, further promoting p53 protein expression, apopto- Y. Yu, Y. Li, X. Deng, J. Gao, C. Huang Administrative, technical, or material support (i.e., reporting or orga- sis, which inhibited EGF-induced cell transformation. nizing data, constructing databases): H. Huang, C. Huang Those results provide a new mechanistic insight into the Study supervision: J. Gao, C. Huang understanding chemopreventive effect of Chel A as a cancer chemopreventive agent. Grant Support This study was partially supported by grants from NIH/NCI RO1 Disclosure of Potential Conﬂicts of Interest R01CA177665, CA112557, CA165980, and NIH/NIEHS ES000260 (to No potential conﬂicts of interest were disclosed. C.S. Huang). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Authors' Contributions advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Conception and design: J. Zhang, H. Huang, J. Gao, Q. Zhao this fact. Development of methodology: J. Zhang, X. Deng Acquisition of data (provided animals, acquired and managed patients, Received July 24, 2014; revised September 16, 2014; accepted September provided facilities, etc.): J. Zhu, J. Li, H. Jin, Y. Li, X. Deng, C. Huang 29, 2014; published OnlineFirst October 3, 2014.

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Crucial Role of c-Jun Phosphorylation at Ser63/73 Mediated by PHLPP Protein Degradation in the Cheliensisin A Inhibition of Cell Transformation

Junlan Zhu, Jingjie Zhang, Haishan Huang, et al.

Cancer Prev Res 2014;7:1270-1281. Published OnlineFirst October 3, 2014.

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