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Wedelolactone, a naturally occurring coumestan, enhances IFN-γ signaling through inhibiting STAT1 dephosphorylation.

Zhimin Chen1,*, Xiaoxiao Sun1,*, Shensi Shen1, Haohao Zhang1, Xiuquan Ma2, Jingli Liu1, Shan

Kuang1, Qiang Yu1, ‡. 1 Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

2 Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences. 555 Zuchongzhi Road, Shanghai, China 201203 *Both authors contributed equally to this work.

‡ To whom correspondence should be addressed: E-mail: qyu@sibs.ac.cn

Running title: Wedelolactone enhances IFN-γ signaling.

Keywords: IFN-γ, STAT1, phosphorylation, Wedelolactone

Background: IFN-γ inhibits tumor cell growth through activating STAT1. Results: Wedelolactone, specifically inhibited TCPTP, the major phosphatase of STAT1, prolonged IFN-γ-induced STAT1 phosphorylation and enhanced the anti-tumor effect of IFN-γ. Conclusion: Wedelolactone enhanced the anti-tumor effect of IFN-γ by inhibiting TCPTP-mediated STAT1 dephosphorylation. Significance: We identified a novel anti-tumor drug candidate, a new target and a new mechanism to develop novel anti-cancer therapeutics. Summary STAT1 (signal transducers and activators of transcription 1) transduces signals from cytokines and growth factors, particularly interferon-gamma (IFN-γ), and regulates expression of genes involved in cell survival/death, proliferation, and migration. STAT1 is activated through phosphorylation on its tyrosine 701 by Janus kinases (JAKs) and is inactivated through dephosphorylation by tyrosine phosphatases. We discovered a natural compound Wedelolactone that increased IFN-γ signaling by inhibiting STAT1 dephosphorylation and prolonging STAT1

activation through specific inhibition of TCPTP, an important tyrosine phosphatase for STAT1 dephosphorylation. More interestingly, Wedelolactone inhibited the TCPTP through interacting with the C-terminal auto-inhibition domain of TCPTP. We also found that Wedelolactone synergized with IFN-γ to induce apoptosis of tumor cells. Our data suggest a new target for anticancer or anti-proliferation drugs, a new mechanism to regulate PTPs specifically, and a new drug candidate for treating cancer or other proliferation disorders. Introduction

Signal transducer and activators of transcription 1 (STAT1) is a member of a family of seven proteins who function as both signal transducers and transcription factors. STAT1 mainly transduces signals from interferon-gamma (IFN-γ) and growth hormones. The STAT1-mediated interferon-gamma signaling activates expression of genes critically involved in inflammatory process, such as inducible nitric oxide synthase (iNOS), COX2, vascular cell adhesion molecules (VCAMs), and intercellular cell adhesion molecules (ICAMs)(1).

In addition to its roles in inflammation, STAT1 also functions as a tumor-suppressor

http://www.jbc.org/cgi/doi/10.1074/jbc.M112.442970The latest version is at JBC Papers in Press. Published on April 11, 2013 as Manuscript M112.442970

Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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(2,3). Loss of STAT1 signaling has been found in a large group of diverse tumors. STAT1-knockout mice suffer a higher spontaneous rate of malignancy. Deficiency in STAT1 signaling also allows tumor cells to escape from IFN-γ-dependent immunosurveillance. As a result, STAT1 null mice develop more frequent and rapidly growing tumors when exposed to chemical carcinogens (4) . STAT1 is also required for the growth inhibition effects of IFN-γ (5,6). STAT1 regulates a wide spectrum of genes involved in cell-cycle control, apoptosis, angiogenesis, cell invasion, metastasis, and immune recognition (7). STAT1 can trigger the expression of proapoptotic genes such as the caspases 2, 3, and 7, while suppresses the expression of antiapoptotic genes such as the BCL2L1 and BCL2 (8).

STAT1 is activated through phosphorylation on tyrosine 701, mainly by Janus kinases (JAKs), leading to STAT1 dimerization, nuclear translocation, and activation of target gene expression. Activated STAT1 can be negatively regulated through protein tyrosine phosphatases (PTPs). T-cell protein tyrosine phosphatase (TCPTP) has been reported to be involved in the inactivation of STAT1 in IFN-γ treated cells (9). Because TCPTP exists in two isoforms, a nuclear form and a cytoplasmic form, it was speculated that TCPTP was involved in the dephosphorylation of STAT1 in both nucleus and cytoplasm (9) .

TCPTP is linked to the development of several inflammatory disorders, including type1 diabetes, Crohn’s disease, and rheumatoid arthritis (10,11). Therefore, TCPTP regulators may serve as therapeutic agents. Efforts have been made to develop drugs against the PTPs, but the highly conserved architectures of PTP active sites impede the development of selective PTP inhibitors (12). For example, TCPTP and PTP1B have a sequence identity of about 74% in their catalytic domains (13), while they clearly

fulfill different biological functions (14-16), suggesting that the protein sequences outside of the catalytic domains play important roles in determining the specificity of the PTPs. Indeed, TCPTP itself was reported to be regulated by an auto-inhibition mechanism (17). In vitro studies, using proteolytically cleaved fragments of TCPTP, have demonstrated that the catalytic activity of TCPTP is regulated by an intramolecular inhibition involving a carboxy-terminal segment of the 45 KD form of TCPTP (17), indicating that the carboxy-terminal domain of TCPTP has an important regulatory role.

Wedelolactone is a coumestan isolated from Eclipta prostrata L., a medicinal herb that has been used in the treatment of infective hepatitis in Indian, snake venom poisoning in Brazil (18). A collective work of different groups has demonstrated that Wedelolactone has multiple biological effects, including inhibition of IKK kinase activity in NF-κB signaling (18), Na+,K+-ATPase activities(19), and phospholipase A (2) activity in snake venom(20). It has also been found to inhibit NS5B RNA polymerase activity, which is critical for hepatitis C virus replication (21), suggesting a potential hepatoprotective activity (22). Recently, Wedelolactone has also been found to have anti-tumor effects by both in vitro and in vivo studies in a wide range of tumor types. The growth inhibition effects of Wedelolactone on tumor cells were believed to be accomplished through its inhibition of IKK(23,24), androgen receptor(25,26), or topoisomerase II (27).

We identified Wedelolactone as an enhancer of STAT1 signaling through screening a natural compound library. We found that Wedelolactone prolonged the IFN-γ-induced STAT1 tyrosine phosphorylation by targeting TCPTP and inhibiting STAT1 dephosphorylation. In doing so, Wedelolactone synergistically enhanced IFN-γ-induced apoptosis of tumor cells in a STAT1-dependent manner. Our findings revealed

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a novel drug target, a novel mechanism to regulate PTPs, and a novel mechanism against cell proliferation. Wedelolactone, particularly in combination with IFN-γ may be a new strategy to treat cancer and other proliferation-related diseases. Experimental procedures General reagents:

Wedelolactone was provided by Shanghai Ambrosia Pharmaceuticals Inc. Tyrosine phosphatase inhibitor sodium orthovanadate and sodium fluoride were purchased from Sigma Aldrich. Stock solution of sodium orthovanadate was constituted in H2O at a concentration of 100mM adjusted to pH10, boiled until it became translucent and the pH was readjusted to pH10. Chemicals, if not specified, were dissolved in DMSO. In experiments that cells were treated with the various inhibitors, the same volumes of corresponding solvents were used as controls. Human IFN-γ was purchased from Shanghai Tongren Yaofang, Inc. Interleukin-6 (IL-6) was from BD Biosciences. Human IFN-α was from Peprotech. Recombinant human EGF was from Invitrogen. Cell culture, transfection, and luciferase gene reporter assay.

HepG2, WiDr, A431, A549 cells were purchased from the American Type Culture Collection. A HepG2 cell line stably transfected with a GAS-luciferase reporter gene was obtained from Dr. Xinyuan Fu of Indiana University. The cells, except HepG2, were maintained in DMEM medium (GIBCO) supplemented with 10% fetal calf serum. HepG2 cells were cultured in α-MEM (GIBCO) with 10% fetal calf serum.

For transfection, cells were cultured to near confluence and transfected for 4 hours with various plasmids using Lipofectamine2000 (Invitrogen). Cells were trysinized and cultured for 24 hours after transfection. Equal number of

cells was collected for various assays. The luciferase activity was measured by a luminometer using Luciferase assay system (Promega).

RNA extraction and RT-PCR.

Total cellular RNA was extracted from cells using TRIzol Reagent (Invitrogen). Reverse transcription and PCR were performed as the following. Briefly, 5 μg of total cellular RNA from each sample was reverse-transcribed using Maloney murine leukemia virus reverse transcriptase (MBI). The first strand cDNA was diluted 1:10 as the PCR template. The PCR rTaq polymerase mixture was prepared according to the manufacturer’s instructions (Takara). PCR primers for the different genes were designed as following: SOCS1: sense 5’-CCCCTTCCAGATTTGACCG-3’; antisense 5’-TGAAGAGGTAGGAGGTGCGAGT-3'. IP-10: sense 5’-CCTCCAGTCTCAGCACCAT- 3’; antisense 5’-AGCAGGGTCAGAACATCC- 3’. IL-8: sense 5’-CAGTTTTGCAAGGAGTGC TAA-3’; antisense 5’-AACTTCTCCACAACCC TCTGC-3’. GAPDH: sense 5’-ACCACAGTCC ATGCCATCAC-3’; antisense 5’-TCCACCAC CCTGTTGCTG-3’. IFN-γ-Receptor Binding Assay.

Suspended HepG2 cells were pretreated with vehicle or Wedelolactone for 20 min, and then incubated with (FITC-IFN-γ) (30000 IU/ml) for another 70 min, followed by analysis using FACScan flow cytometric analysis (Becton Dickinson). Antibodies and Immunoblotting Analysis.

The mouse monoclonal antibody to phosphotyrosine (4G10) was obtained from Upstate Biotechnology. Anti -p-STAT1 (Y701) antibody, anti-STAT1 antibody, anti-p-STAT3 (Y705) antibody, anti-STAT3 antibody, anti-p-ERK (T202/Y204) antibody, anti-ERK

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antibody, anti-EGFR antibody, anti-p-JAK1 (Y1022/1023) antibody, anti-p-JAK2 (Y1007/1008) antibody, anti-JAK1 antibody, were purchased from Cell Signaling. Anti-α-tubulin antibody and anti-JAK2 antibody were purchased from Santa Cruz. Anti-p-EGFR (Y1068) was purchased from BioSourse. All antibodies purchased commercially were used as recommended by the manufactures.

For immunoblotting analysis, Cells were lysed in 1X Laemmli sample buffer (Sigma Aldrich) and boiled for 5 min. Proteins were resolved by SDS-PAGE (8%), transferred to a nitrocellulose membrane, and blocked by incubation for 60 min at room temperature with 5% (w/v) nonfat dry milk in TBST. All the membranes were incubated overnight at 4°C with primary antibodies diluted in TBST with 5% BSA. The membranes were then washed and incubated for 1h at room temperature with horseradish peroxidase-conjugated secondary antibodies (Promega). Immune complexes were detected by ECL (Pierce). In vitro Protein Tyrosine Phosphatase Assay.

Cells were washed three times with ice-cold PBS and lysed in 1% Triton-X100 lysis buffer(50mM Hepes pH7.2, 5mM EDTA, 0.25mM Sucrose, 1% Triton-X100) containing protease and serine phosphatase inhibitors including 1mM PMSF, 1mM NaF, protease inhibitor cocktail (Sigma Aldrich). Samples were incubated on ice for 30min and centrifuged at 14,000g for 10min at 4°C to remove insoluble material. The supernatants were transferred into fresh tubes, and protein concentrations were determined using a Bradford protein assay kit (Bio-Rad). Equal amounts of protein lysates (10μg) were used for PTP enzymatic assay: resuspended in 35μl reaction buffer (100mM Hepes pH7.0, 150mM NaCl, 1mM EDTA, 5mM DTT) with 10mM para-nitrophenyl phosphate (pNPP) (Sigma Aldrich) as substrate. The reactions were incubated at 30°C with rocking

for different time periods and 2 volumes of 1N NaOH stop solution were added. The samples were measured extinction (OD) at 405nm.

The LAR Tyrosine Phosphatase Assay Kit (AK-815) was purchased from Enzo Lifescience. Purified human recombinant Leukocyte Antigen Related (LAR) cytoplasmic domain (1275-1613) was used as enzyme and a synthetic 12-mer peptide based on the regulatory auto-phosphorylation region of the IR b subunit cytoplasmic domain (1142-1153) was served as substrate. All the experiments were performed according to the manufacture’s instruction.

The TCPTP Fluorometric Assay Kit was from Abnova. Recombinant human TCPTP (full length, 1-387) was used as the enzyme and the Fluoro-Phospho-Substrate, which comprised a unique PTP substrate containing a phosphor group, was served as a substrate. All experiments were performed according to the manufacture’s instruction.

The SHP-2 and TCPTP catalytic domain assays were performed by the National Center for Drug Screening (Shanghai, China).

RNA interference. TCPTP siRNA: sense 5’-GAUUGACAGACAA CUAAUATT-3’; antisense 5’-UAUUAGUUGU CUGUCAAUCTT-3’. siRNAs sequences were derived from each protein’s coding sequence and a random sequence control siRNA were synthesized by Genepharma (Shanghai, China). The synthetic siRNA was transfected into HepG2 cells using DharmaFECT1 (Dharmacon). In order to achieve maximal effect, the siRNA-transfected cells were cultured to confluence and then trypsinized and reseeded into dishes. The luciferase activity was measured by a luminometer using Luciferase assay system (Promega). Cell viability assay.

Cells were seeded in a 96-well plate and

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after treatment with indicated drugs for indicated time, 20 µl of MTT solution (5mg/ml, Sigma Aldrich) was added to each well and incubated for 3 h at 37°C. The supernatant was aspirated, and the MTT-formazan crystals formed by metabolically viable cells were dissolved in 150µl of DMSO. Finally, the absorbance was monitored by a microplate reader (Molecular Devices) at a wavelength of 570 nm. STAT1 in vitro dephosphorylation assay.

HepG2 cells were washed with ice-cold PBS and lysed with HEST buffer (50mM Hepes pH7.2, 5mM EDTA, 0.25M Sucrose, 1% Triton-X100) containing protease and serine phosphatase inhibitors 1mM PMSF, 1mM NaF, and protease inhibitor cocktail (Sigma Aldrich). Samples were incubated on ice for 30 minutes, harvested, and centrifuged for 10 minutes at 4°C (14,000g) to remove insoluble material. The supernatants were transferred into fresh tubes and served as “enzyme lysates”. HepG2 cells treated with 100 IU/ml IFN-γ were lysed the same as above and served as “substrate lysate”. Before mixing with the “enzyme lysates”, the “substrate lysate” was denatured in boiling water bath for 5 minutes then incubated on ice for another 5 minutes. The “enzyme lysates” and “substrate lysate” were then mixed in equal volume and incubated at 35°C with agitation for indicated time duration. The reactions were terminated by adding half-volume Laemmli 2X sample buffer (Sigma Aldrich) following by boiling at 100°C for 5 minutes and then subjected to Western blotting analysis.

Results Wedelolactone increased IFN-γ/STAT1-mediated promoter activity.

We used a cell-based promoter-reporter assay system p-GAS-HepG2, which is a HepG2 cell line stably transfected with a STAT1 responsible promoter-reporter (pGAS-luciferase) plasmid (materials and methods), to identify

compounds that enhance IFN-γ/STAT1 signaling. We screened about 1000 traditional Chinese medicinal herb extracts using the assay and identified a natural compound Wedelolactone from Eclipta prostrata L. as a potent IFN-γ/STAT1 signaling enhancer （ Fig.1 ） . Treatment of the p-GAS-HepG2 cells with Wedelolactone increased the IFN-γ-induced luciferase reporter activity in a dose and time-dependent manner (Figs.1a and 1b). The EC50 of the enhancement was about 20 μM and the maximum enhancement was about 6-7 folds of the control (Fig.1a). The induction time course indicated that both the magnitude and the duration of the IFN-γ-induced luciferase activity were increased (Fig.1b). To ensure that the increased luciferase activity by Wedelolactone was indeed due to the increased STAT1 activity, we analyzed the effects of Wedelolactone on the expressions of three endogenous IFN-γ-induced STAT1 target genes: IP-10, SOCS1, and IL-8. The mRNA levels of all three genes we tested were increased by the Wedelolactone treatment (Fig.1c). These data collectively demonstrate that Wedelolactone enhance the IFN-γ/STAT1 signaling. Wedelolactone did not affect the binding of IFN-γ to its receptor.

To investigate the mechanisms of Wedelolactone in enhancing the IFN-γ/STAT1 signaling, we first tested whether Wedelolactone played a role in the ligand-receptor interaction. We increased the concentration of IFN-γ in the presence or the absence of the same concentration of Wedelolactone (Fig.2a). Wedelolactone alone had very little effects on the luciferase activity, suggesting that Wedelolactone did not act as a ligand (FigS.1). Maximum luciferase activity was achieved when the concentration of IFN-γ reached 1000 IU/ml, suggesting saturation of IFN-γ receptor. But Wedelolactone had similar enhancing effects on

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different concentrations of IFN-γ, suggesting that Wedelolactone do not affect the binding of IFN-γ to its receptor (Fig.2a). To confirm this, we examined the effects of Wedelolactone on the binding of IFN-γ to its receptor directly by using the FITC-labeled IFN-γ which allowed us to detect IFN-γ binding using flow cytometry. Wedelolactone treatment had no effects on the binding of IFN-γ to cells (Fig.2b), although the FITC-labeled IFN-γ seemed to have a relatively low binding affinity. All these results suggest that the enhancing effect of Wedelolactone on the IFN-γ-induced luciferase activity may be not through affecting IFN-γ-receptor interactions. Wedelolactone enhanced IFN-γ/STAT1 signaling by prolonging STAT1 tyrosine phosphorylation.

We next analyzed the effects of Wedelolactone on tyrosine phosphorylation of JAK1, JAK2 and STAT1, which are the upstream signaling events in response to the IFN-γ stimulation. Wedelolactone did not affect the tyrosine phosphorylation of JAK1 nor JAK2 (Fig.2c), nor the initial phosphorylation of STAT1 (Fig.2c), but significantly prolonged the duration of STAT1 tyrosine phosphorylation. The IFN-γ-induced phosphorylation of STAT1 was a transient event, which began to decrease 1-2 hours after stimulation (Fig.2c and 2d). The STAT1 phosphorylation sustained more than 8 hours in the presence of Wedelolactone (Fig.2d). The effect of Wedelolactone on STAT1 phosphorylation was also dose dependent (Fig.2e), consistent with its effect on the STAT1 promoter activity (Fig.1a). It appeared that Wedelolactone did not affect the IFN-γ-induced phosphorylation of STAT1, but prevented or slowed down its decrease after stimulation.

Wedelolactone specifically enhanced IFN-γ signaling.

To address whether the enhancing effect of Wedelolactone is specific for IFN-γ signaling, we tested the effects of Wedelolactone on IFN-α,

IL-6 or EGF signaling. HepG2 cells were stimulated with IFN-α, IL-6 or EGF in the presence or absence of Wedelolactone and the phosphorylation status of their downstream proteins were analyzed. There were no obvious effects of Wedelolactone on the IFN-α-induced STAT1/3 tyrosine phosphorylation (Fig.3a), the IL-6-induced STAT3/ERK phosphorylation or the level of GP130 (Fig.3b), nor on the EGF-induced EGFR/ERK phosphorylation (Fig.3c). These data strongly suggest that the enhancing effect of Wedelolactone is specific on IFN-γ signaling. Wedelolactone enhanced IFN-γ signaling through inhibition of STAT1 dephosphorylation.

Since slowdown of the deduction of STAT1 phosphorylation can be achieved either through activating its kinases or inhibiting its phosphatases, we performed a pause-chase experiment to distinguish between these two possibilities. HepG2 cells were stimulated with IFN-γ for 30 min followed by an addition of a general kinase inhibitor Staurosporine to inhibit further phosphorylation of STAT1 by its kinases (28). Wedelolactone, as well as the general PTP inhibitor vanadate, still could prolong the duration of STAT1 phosphorylation in the presence of Staurosporine (Fig.4a), suggesting that Wedelolactone acts through inhibiting STAT1 dephosphorylation rather than enhancing its phosphorylation.

To clarify that Wedelolactone is indeed a STAT1 phosphatase inhibitor, we analyzed the effects of Wedelolactone on STAT1 in vitro dephosphorylation using an in vitro dephosphorylation assay (29). Substrate cell lysate containing the phosphorylated STAT1 was prepared by treating the HepG2 cells with IFN-γ. In parallel, the enzyme cell lysate was prepared from the untreated HepG2 cells. Equal amounts of the substrate cell lysate and the enzyme cell lysate were mixed together to allow the in vitro dephosphorylation. STAT1 dephosphorylation

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could be observed after 30 minutes incubation. Wedelolactone dose-dependently inhibited the STAT1 in vitro dephosphorylation (Fig.4b).

Taken together, these results demonstrate that Wedelolactone serves as a PTP inhibitor in regulation of IFN-γ signaling.

To address whether Wedelolactone is a general PTP inhibitor, we analyzed the effects of Wedelolactone on the general protein tyrosine phosphorylation in the HepG2 cells and compared with that of a general PTP inhibitor vanadate. Vanadate increased the general tyrosine phosphorylation level in the HepG2 cells as expected, whereas Wedelolactone had no obvious effects on the general tyrosine phosphorylation but significantly enhanced the STAT1 phosphorylation, demonstrating that Wedelolactone is not a general tyrosine phosphatase inhibitor (Fig.4c).

We also examined the effects of Wedelolactone on protein dephosphorylation in vitro using whole cell lysates and pNPP as the substrate. Vanadate again inhibited the in vitro phosphatase activities of the whole cell lysate in a dose dependent manner while Wedelolactone had no effects (Fig.4d). These results collectively indicate that Wedelolactone is not a general PTPs inhibitor as vanadate. The Wedelolactone-enhanced STAT1 phosphorylation was TCPTP-dependent and Wedelolactone is a specific TCPTP inhibitor.

TCPTP has been reported as the major phosphatase involved in STAT1 dephosphorylation in IFN-γ signaling (9). We then examined the role of TCPTP in the Wedelolactone-enhanced STAT1 phosphorylation. We overexpressed TCPTP in the p-GAS-HepG2 cells (Fig.5a) and analyzed the effects of TCPTP overexpression on the IFN-γ-induced luciferase activity in the presence or the absence of Wedelolactone. Overexpression of TCPTP reduced the IFN-γ-induced luciferase activity as expected

and decreased the enhancing effects of Wedelolactone (Fig.5a), suggesting that increased level of TCPTP can counterbalance the Wedelolactone-enhanced IFN-γ signaling.

To confirm the role of TCPTP in mediating the Wedelolactone effects, we did the reciprocal experiments by knocking down TCPTP expression in the p-GAS-HepG2 cells using TCPTP-specific siRNA (Fig.5b). Luciferase activity was increased in the TCPTP knocking down p-GAS-HepG2 cells comparing to the mock control. The Wedelolactone’s effects were almost completely abolished in the TCPTP-deficient cells, strongly suggesting that Wedelolactone targets TCPTP to modulate the IFN-γ-induced STAT1 phosphorylation (Fig.5b). The efficiency of overexpression and knocking down TCPTP were evaluated with western blot (Fig.5c)

Similar results in STAT1 dephosphorylation were obtained by knocking down the TCPTP in HepG2 cells (Fig.5d). The dephosphorylation of STAT1 was inhibited by knocking down TCPTP and the enhancing effects of both of Wedelolactone and Vanadate on STAT1 phosphorylation were profoundly reduced (Fig.5c), again suggesting that TCPTP is the target of Wedelolactone in enhancing the IFN-γ-induced STAT1 phosphorylation.

To confirm that Wedelolactone is indeed a TCPTP inhibitor, we examined the effect of Wedelolactone on TCPTP activity in vitro using purified enzymes. Wedelolactone efficiently inhibited the phosphatase activity of the full-length TCPTP (a.a 1-387) in vitro (Fig.5e) while had no obvious effects on that of two control phosphatase: LAR and SHP-2 (Fig.5e).

Interestingly, Wedelolactone had much less effects on inhibiting the phosphatase activity of the TCPTP catalytic fragment (a.a 2-346) (Fig.5e), suggesting that the C-terminal domain of the TCPTP is required for Wedelolactone to inhibit the catalytic activity of the TCPTP. Moreover, the inhibition of Wedelolactone on

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the full length TCPTP was not complete and reached a plateau after 10 μM concentration of Wedelolactone (Fig.5e). These data strongly suggest that Wedelolactone is a specific inhibitor for TCPTP and it specifically interacts with the C-terminal domain of the TCPTP.

Wedelolactone synergized with IFN-γ to induce tumor cell death.

IFN-γ has been known to induce apoptosis and cell-cycle arrest in certain tumor cells (30). We therefore examined whether the prolonged STAT1 tyrosine phosphorylation by Wedelolactone could enhance the ability of IFN-γ to induce cell death or cell cycle arrest in tumor cells. Wedelolactone synergized with IFN-γ, to different extends, to induce cell deaths in hepatocarcinoma cell line HepG2 (Fig.6a), colon cancer cell line WiDr (Fig.6b), and epidermoid carcinoma cell line A431NS (Fig.6c), but had no effects on hepatocarcinoma cell line 97H (Fig.6d) (31), which lacks the STAT1 protein (Fig.6e). These data suggest that Wedelolactone enhances the anti-tumor activity of IFN-γ in a STAT1-dependent manner.

Discussion

STATs mediate important signals from cytokines, growth factors, and extracellular matrix to regulate diverse biological activities. STATs phosphorylation is a nodal point for regulating the STATs-mediated signaling. The collective work of multiple research groups has uncovered complex molecules and mechanisms in regulating STATs phosphorylation, including kinases (32), phosphatases (9,33,34), conformational reorientation (35,36), and scaffold proteins (37). We know a great deal about the function and regulation of the kinases and phosphorylation of STATs and have developed various compounds to target the kinases that inhibit STAT signaling for treating STATs signaling-related diseases (38-40). We however know very little about the function and

regulation of the phosphatases and dephosphorylation of STATs. There have been no compounds developed to target the dephosphorylation of STATs. Here we report the identification of Wedelolactone, a natural compound from Eclipta prostrata L. that interfered with the dephosphorylation of STAT1. Wedelolactone specifically prolonged the IFN-γ-induced STAT1 phosphorylation by inhibiting the TCPTP-dependent dephosphorylation of STAT1 and synergized with IFN-γ in inducing cell death among certain STAT1 expressing tumor cells. Our results suggest that Wedelolactone acts as a TCPTP specific inhibitor to inhibit the dephosphorylation of STAT1 after IFN-γ stimulation and enhances the biological effects of IFN-γ.

The transient activation of STAT1 after IFN-γ stimulation has been noticed before (28), but the biological significance of the regulation of the duration of STAT1 phosphorylation has not been revealed. STAT1 regulates hundreds of genes with diverse biological activities. The level and duration of the STAT1 activation may have significant impact on the biological outcome of the STAT1-mediated signaling. In deed inhibition of STAT1 dephosphorylation by Wedelolactone facilitated the anti-tumor function of IFN-γ in several tumor cell lines (Fig. 6), demonstrating the biological significance of the duration of the STAT1 phosphorylation.

IFN-γ is a multifunctional cytokine which is involved in many diseases, including cancer (41-43), tuberculosis (44), hepatitis (45), and Rheumatoid Arthritis (46). IFN-γ1b was approved for the treatments of chronic granulomatous disease (CGD) and adult T cell leukemia (ATL) (47). Much effort has been made to explore its therapeutic potential to treat cancer. It has been shown that certain genotoxic agents could sensitize cells in response to very low doses of either interferon-α or γ through activation of STAT1 (48). Enhanced STAT1

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phosphorylation caused by a mutation in SH2 domain of STAT2 promoted type-I IFN-induced apoptosis (49). Therefore, increased STAT1 phosphorylation appears to be a valid strategy to induce cell death of tumor cells and Wedelolactone is a unique chemical modulator of IFN-γ signaling to enhance the biological functions of the cytokine in clinical use.

TCPTP (gene name PTPN2) was originally cloned from a human peripheral T-cell cDNA library (50). Human PTPN2 is located within chromosomal region 18p11.2-11.3 (51), where are thought to be associated with susceptibility to three autoimmune diseases: Crohn’s disease (CD), rheumatoid arthritis (RA), and type 1 diabetes (T1D) (10). TCPTP is also involved in many signaling pathways including EGF signaling (52), IFN-γ signaling (9), and cell-cycle regulation (53). Efforts have been made to develop specific small molecule inhibitors for TCPTP (12,54). However, the highly conserved catalytic domains of the PTPs remain a challenge for developing specific small molecule inhibitors for the PTPs (13,55). It has been reported that TC-PTP can be regulated through conformational changes mediated by extracellular matrix (56,57) In vitro studies, using proteolytically cleaved fragment of TCPTP, have suggested that the catalytic activity of TCPTP is regulated by an intramolecular inhibition involving a carboxy-terminal segment of the TCPTP (17). In this regard, our finding that Wedelolactone specifically inhibited the phosphatase activity of the full-length TC-PTP but not that of the C-terminus-deleted TC-PTP suggests that Wedelolactone may interact with the amino acid residues within the C-terminal regulatory domain of TCPTP to influence the catalytic activity of TCPTP. Further detailed understanding of the interactions of Wedelolactone with TCPTP will uncover a new strategy to develop specific TCPTP inhibitors. Wedelolactone holds enormous potential to be a novel drug candidate for the treatment of the

various TCPTP- related diseases. Comparing to its effects on IFN-γ signaling,

Wedelolactone only showed a mild effect on the IFN-α-induced phosphorylation of STAT1 (Fig.3a). This result suggests that the inactivation of STAT1 after IFN-α and IFN-γ stimulation are distinct. The IFN-α treatment leads to STAT1 and STAT2 hetero-dimer formation while the IFN-γ treatment results in STAT1 homo-dimer formation. The conformation of phosphorylated STAT1 has been shown to be a crucial factor in facilitating enzyme-substrate interaction during its dephosphorylation (36). Therefore, dephosphorylation of STAT1 in these two specific dimers could be regulated by distinct mechanisms. Wedelolactone could interfere with the dephosphorylation of STAT1 homo-dimer but not with the STAT1/STAT2 hetero-dimer.

Several studies have shown that Wedelolactone alone could induce growth arrest and apoptosis in several cancer cell lines through targeting IKK (23,24) , androgen receptor (25,26), or topoisomerase II (27), which is consistent with our observation (Figs.6a, 6b, and 6c). However, Wedelolactone could achieve higher efficacy in inducing grow inhibition of tumor cells when combined with IFN-γ, confirming that the STAT pathway is an important target of Wedelolactone in inhibiting tumor cell growth.

In conclusion, we identified Wedelolactone as a potent enhancer of IFN-γ/STAT1 signaling. Wedelolactone specifically enhances the IFN-γ-induced STAT1 phosphorylation by inhibiting the STAT1 phosphatase TCPTP, possibly interacting with the C-terminal auto-inhibitory domain of the TCPTP. In doing so, Wedelolactone facilitates IFN-γ to induce tumor cell death. Our results revealed a new strategy to target TCPTP and IFN-γ/STAT1 signaling, and uncovered a new drug candidate to treat cancer and other proliferation-related diseases.

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Acknowledgments We thank Dr. Xinyuan Fu for providing the

HepG2 cells stably transfected with the p-GAS-luciferase. We thank Dr. N.K.Tonks for providing the human TC-PTP plasmid. We thank National Center for Drug Screening (Shanghai, China) for helping the PTP activity assays. This work was supported by China Ministry of Science and Technology Key New Drug Creation and Manufacturing Program Grant 2013ZX09102015, China Ministry of Science and Technology Research Grant 2013ZX10002010-009, China National Natural Science Foundation Grants 31129004, 91129701,

and 91213304, and National Science and Technology 973 Grants 2012CB910704 and 2013CB910900. Author contributions

Zhimin Chen, Xiaoxiao Sun and Qiang Yu designed the research; Zhimin Chen, Xiaoxiao Sun, Shensi Shen, Haohao Zhang, Xiuquan Ma, Jingli Liu, and Shan Kuang performed the research; Zhimin Chen, Xiaoxiao Sun and Qiang Yu analyzed the data; Zhimin Chen and Qiang Yu wrote the paper.

References 1. Horvath, C. M. (2000) Trends Biochem Sci 25(10), 496-502 2. Battle, T. E., and Frank, D. A. (2002) Curr Mol Med 2(4), 381-392 3. Stephanou, A., and Latchman, D. S. (2003) Int J Exp Pathol 84(6), 239-244 4. Kaplan, D. H., Shankaran, V., Dighe, A. S., Stockert, E., Aguet, M., Old, L. J., and Schreiber, R. D.

(1998) Proc Natl Acad Sci U S A 95(13), 7556-7561 5. Bromberg, J. F., Horvath, C. M., Wen, Z., Schreiber, R. D., and Darnell, J. E., Jr. (1996) Proc Natl Acad

Sci U S A 93(15), 7673-7678 6. Buard, A., Vivo, C., Monnet, I., Boutin, C., Pilatte, Y., and Jaurand, M. C. (1998) Cancer Res 58(4),

840-847 7. Chin, Y. E., Kitagawa, M., Su, W. C., You, Z. H., Iwamoto, Y., and Fu, X. Y. (1996) Science 272(5262),

719-722 8. Huang, Y. Q., Li, J. J., and Karpatkin, S. (2000) J Biol Chem 275(9), 6462-6468 9. ten Hoeve, J., de Jesus Ibarra-Sanchez, M., Fu, Y., Zhu, W., Tremblay, M., David, M., and Shuai, K.

(2002) Mol Cell Biol 22(16), 5662-5668 10. Consortium, W. T. C. C. (2007) Nature 447(7145), 661-678 11. Todd, J. A., Walker, N. M., Cooper, J. D., Smyth, D. J., Downes, K., Plagnol, V., Bailey, R., Nejentsev,

S., Field, S. F., Payne, F., Lowe, C. E., Szeszko, J. S., Hafler, J. P., Zeitels, L., Yang, J. H., Vella, A., Nutland, S., Stevens, H. E., Schuilenburg, H., Coleman, G., Maisuria, M., Meadows, W., Smink, L. J., Healy, B., Burren, O. S., Lam, A. A., Ovington, N. R., Allen, J., Adlem, E., Leung, H. T., Wallace, C., Howson, J. M., Guja, C., Ionescu-Tirgoviste, C., Simmonds, M. J., Heward, J. M., Gough, S. C., Dunger, D. B., Wicker, L. S., and Clayton, D. G. (2007) Nat Genet 39(7), 857-864

12. Zhang, S., Chen, L., Luo, Y., Gunawan, A., Lawrence, D. S., and Zhang, Z. Y. (2009) J Am Chem Soc 131(36), 13072-13079

13. Iversen, L. F., Moller, K. B., Pedersen, A. K., Peters, G. H., Petersen, A. S., Andersen, H. S., Branner, S., Mortensen, S. B., and Moller, N. P. (2002) J Biol Chem 277(22), 19982-19990

14. Elchebly, M., Payette, P., Michaliszyn, E., Cromlish, W., Collins, S., Loy, A. L., Normandin, D., Cheng, A., Himms-Hagen, J., Chan, C. C., Ramachandran, C., Gresser, M. J., Tremblay, M. L., and Kennedy, B. P. (1999) Science 283(5407), 1544-1548

by guest on Novem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from

11

15. Klaman, L. D., Boss, O., Peroni, O. D., Kim, J. K., Martino, J. L., Zabolotny, J. M., Moghal, N., Lubkin, M., Kim, Y. B., Sharpe, A. H., Stricker-Krongrad, A., Shulman, G. I., Neel, B. G., and Kahn, B. B. (2000) Mol Cell Biol 20(15), 5479-5489

16. You-Ten, K. E., Muise, E. S., Itie, A., Michaliszyn, E., Wagner, J., Jothy, S., Lapp, W. S., and Tremblay, M. L. (1997) J Exp Med 186(5), 683-693

17. Hao, L., Tiganis, T., Tonks, N. K., and Charbonneau, H. (1997) J Biol Chem 272(46), 29322-29329 18. Kobori, M., Yang, Z., Gong, D., Heissmeyer, V., Zhu, H., Jung, Y. K., Gakidis, M. A., Rao, A., Sekine,

T., Ikegami, F., Yuan, C., and Yuan, J. (2004) Cell Death Differ 11(1), 123-130 19. Pocas, E. S., Lopes, D. V., da Silva, A. J., Pimenta, P. H., Leitao, F. B., Netto, C. D., Buarque, C. D.,

Brito, F. V., Costa, P. R., and Noel, F. (2006) Bioorg Med Chem 14(23), 7962-7966 20. Diogo, L. C., Fernandes, R. S., Marcussi, S., Menaldo, D. L., Roberto, P. G., Matrangulo, P. V., Pereira,

P. S., Franca, S. C., Giuliatti, S., Soares, A. M., and Lourenco, M. V. (2009) Basic Clin Pharmacol Toxicol 104(4), 293-299

21. Kaushik-Basu, N., Bopda-Waffo, A., Talele, T. T., Basu, A., Costa, P. R., da Silva, A. J., Sarafianos, S. G., and Noel, F. (2008) Nucleic Acids Res 36(5), 1482-1496

22. Upadhyay, K., Gupta, N. K., and Dixit, V. K. (2011) Drug Dev Ind Pharm 23. Idris, A. I., Libouban, H., Nyangoga, H., Landao-Bassonga, E., Chappard, D., and Ralston, S. H. (2009)

Mol Cancer Ther 8(8), 2339-2347 24. Vender, J. R., Laird, M. D., and Dhandapani, K. M. (2008) Neurosurgery 62(5), 1122-1127; discussion

1027-1128 25. Tsai, C. H., Lin, F. M., Yang, Y. C., Lee, M. T., Cha, T. L., Wu, G. J., Hsieh, S. C., and Hsiao, P. W.

(2009) Clin Cancer Res 15(17), 5435-5444 26. Lin, F. M., Chen, L. R., Lin, E. H., Ke, F. C., Chen, H. Y., Tsai, M. J., and Hsiao, P. W. (2007)

Carcinogenesis 28(12), 2521-2529 27. Benes, P., Knopfova, L., Trcka, F., Nemajerova, A., Pinheiro, D., Soucek, K., Fojta, M., and Smarda, J.

(2011) Cancer Lett 303(1), 29-38 28. Haspel, R. L., Salditt-Georgieff, M., and Darnell, J. E., Jr. (1996) Embo J 15(22), 6262-6268 29. Chen, Z., Ma, X., Zhang, H., Sun, X., Shen, S., Li, Y., Gu, Y., Wang, Y., Yan, S., and Yu, Q. (2011) Cell

Signal 23(8), 1404-1412 30. Ning, Y., Riggins, R. B., Mulla, J. E., Chung, H., Zwart, A., and Clarke, R. (2010) Mol Cancer Ther

9(5), 1274-1285 31. Li, Y., Tang, Z. Y., Ye, S. L., Liu, Y. K., Chen, J., Xue, Q., Chen, J., Gao, D. M., and Bao, W. H. (2001)

World J Gastroenterol 7(5), 630-636 32. Leonard, W. J. (2001) Int J Hematol 73(3), 271-277 33. Wu, T. R., Hong, Y. K., Wang, X. D., Ling, M. Y., Dragoi, A. M., Chung, A. S., Campbell, A. G., Han,

Z. Y., Feng, G. S., and Chin, Y. E. (2002) J Biol Chem 277(49), 47572-47580 34. Liu, D., Scafidi, J., Prada, A. E., Zahedi, K., and Davis, A. E., 3rd. (2002) Biochem Biophys Res

Commun 299(4), 574-580 35. Mertens, C., Zhong, M., Krishnaraj, R., Zou, W., Chen, X., and Darnell, J. E., Jr. (2006) Genes Dev

20(24), 3372-3381 36. Zhong, M., Henriksen, M. A., Takeuchi, K., Schaefer, O., Liu, B., ten Hoeve, J., Ren, Z., Mao, X.,

Chen, X., Shuai, K., and Darnell, J. E., Jr. (2005) Proc Natl Acad Sci U S A 102(11), 3966-3971 37. Mo, W., Zhang, L., Yang, G., Zhai, J., Hu, Z., Chen, Y., Chen, X., Hui, L., Huang, R., and Hu, G. (2008)

Mol Cell 31(5), 695-707

by guest on Novem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from

12

38. Vijayakrishnan, L., Venkataramanan, R., and Gulati, P. (2011) Trends Pharmacol Sci 32(1), 25-34 39. Pardanani, A., Vannucchi, A. M., Passamonti, F., Cervantes, F., Barbui, T., and Tefferi, A. (2011)

Leukemia 25(2), 218-225 40. Wang, Y., Ma, X., Yan, S., Shen, S., Zhu, H., Gu, Y., Wang, H., Qin, G., and Yu, Q. (2009) Cancer Res

69(18), 7302-7310 41. Detjen, K. M., Farwig, K., Welzel, M., Wiedenmann, B., and Rosewicz, S. (2001) Gut 49(2), 251-262 42. Gooch, J. L., Herrera, R. E., and Yee, D. (2000) Cell Growth Differ 11(6), 335-342 43. Ruiz-Ruiz, C., Munoz-Pinedo, C., and Lopez-Rivas, A. (2000) Cancer Res 60(20), 5673-5680 44. Condos, R., Raju, B., Canova, A., Zhao, B. Y., Weiden, M., Rom, W. N., and Pine, R. (2003) Infect

Immun 71(4), 2058-2064 45. Gattoni, A., Parlato, A., Vangieri, B., Bresciani, M., and Derna, R. (2006) Clin Ter 157(4), 377-386 46. Cannon, G. W., Pincus, S. H., Emkey, R. D., Denes, A., Cohen, S. A., Wolfe, F., Saway, P. A., Jaffer, A.

M., Weaver, A. L., Cogen, L., and et al. (1989) Arthritis Rheum 32(8), 964-973 47. Miller, C. H., Maher, S. G., and Young, H. A. (2009) Ann N Y Acad Sci 1182, 69-79 48. Youlyouz-Marfak, I., Gachard, N., Le Clorennec, C., Najjar, I., Baran-Marszak, F., Reminieras, L., May,

E., Bornkamm, G. W., Fagard, R., and Feuillard, J. (2008) Cell Death Differ 15(2), 376-385 49. Scarzello, A. J., Romero-Weaver, A. L., Maher, S. G., Veenstra, T. D., Zhou, M., Qin, A., Donnelly, R.

P., Sheikh, F., and Gamero, A. M. (2007) Mol Biol Cell 18(7), 2455-2462 50. Cool, D. E., Tonks, N. K., Charbonneau, H., Walsh, K. A., Fischer, E. H., and Krebs, E. G. (1989) Proc

Natl Acad Sci U S A 86(14), 5257-5261 51. Sakaguchi, A. Y., Sylvia, V. L., Martinez, L., Smith, E. A., Han, E. S., Lalley, P. A., Shows, T. B., and

Choudhury, G. G. (1992) Genomics 12(1), 151-154 52. Tiganis, T., Bennett, A. M., Ravichandran, K. S., and Tonks, N. K. (1998) Mol Cell Biol 18(3),

1622-1634 53. Ibarra-Sanchez, M. J., Wagner, J., Ong, M. T., Lampron, C., and Tremblay, M. L. (2001) Oncogene

20(34), 4728-4739 54. Blaskovich, M. A. (2009) Curr Med Chem 16(17), 2095-2176 55. Romsicki, Y., Kennedy, B. P., and Asante-Appiah, E. (2003) Arch Biochem Biophys 414(1), 40-50 56. Mattila, E., Auvinen, K., Salmi, M., and Ivaska, J. (2008) J Cell Sci 121(Pt 21), 3570-3580 57. Mattila, E., Pellinen, T., Nevo, J., Vuoriluoto, K., Arjonen, A., and Ivaska, J. (2005) Nat Cell Biol 7(1),

78-85 Figure legends Figure 1: Wedelolactone increased IFN-γ/STAT1-mediated promoter activity. (a) p-LUC-HepG2 cells were stimulated with 1,000 IU/ml IFN-γ plus vehicle or Wedelolactone at indicated concentrations, and luciferase activity was measured following stimulation of IFN-γ for 6 hours. Indicated treatment was compared to IFN-γ treatment alone by Student’s t-test. **: p<0.01 and ***: p<0.0001. Data were mean ± s.e.m. of a representative experiment done in triplicate. (b) p-LUC-HepG2 cells were stimulated with 1,000 IU/ml IFN-γ plus vehicle or 50 μM Wedelolactone, and luciferase activity was measured after stimulation of IFN-γ for indicated time. Data were mean ± s.e.m. of a representative experiment done in triplicate. (c) HepG2 cells were stimulated with 1,000 IU/ml IFN-γ plus vehicle or 50 μM Wedelolactone. After indicated time, cells were harvested for total RNA followed by RTPCR analysis of three indicated IFN-γ response gene: IP-10, SOCS1, and IL-8. Densitometric quantification of indicated gene normalized to GAPDH was graphed.

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Figure 2: Wedelolactone enhanced IFN-γ/STAT1 signaling by prolonging STAT1 tyrosine phosphorylation. (a) p-LUC-HepG2 cells were stimulated with IFN-γ at indicated concentrations plus vehicle or 50 μM Wedelolactone, and luciferase activity was measured following stimulation of IFN-γ for 6 hours. Data were mean ± s.e.m. of a representative experiment done in triplicate. (b) Effects of Wedelolactone on binding of IFN-γ to its receptors. Suspended HepG2 cells were incubated with FITC-labeled IFN-γ plus vehicle or 50 μM Wedelolactone for 60 min, followed by analysis using flow cytometry. (c,d) HepG2 cells were stimulated with IFN-γ plus vehicle or 50 μM Wedelolactone for various length of time (c: 0-120 min; d: 0-8 h) as indicated. Whole cell lysates were processed for Western blot analysis and probed with the indicated antibodies. (e) HepG2 cells were stimulated with IFN-γ plus vehicle or Wedelolactone at various concentrations as indicated. After 2-hour treatment whole cell lysates were subjected to Western blot analysis. Densitometric quantification of tyrosine phosphorylated STAT1 normalized to total STAT1 comparing to IFN-γ alone was graphed at the right side of the corresponding blot. Figure 3: Wedelolactone specifically enhanced IFN-γ signaling. (a-c) HepG2 cells were stimulated with (a) IFN-α or (b) IL-6 or (c) EGF plus vehicle or 50 μM Wedelolactone for various length of time (a: 0-180 min; b: 0-240 min; c: 0-240 min) as indicated. Whole cell lysates were processed for Western blot analysis and probed with the indicated antibodies. Figure 4: Wedelolactone regulated IFN-γ signaling through inhibiting STAT1 dephosphorylation. (a) HepG2 cells were stimulated with 100 IU/ml IFN-γ plus vehicle or Wedelolactone or Vanadate at various concentrations as indicated for 30 min followed by an addition of 150nM Staurosporin. After 30 min chasing with Staurosporin, the whole cell lysates were processed for Western blot analysis and probed with the indicated antibodies. Tyrosine phosphorylated STAT1 normalized to total STAT1 comparing to IFN-γ alone was quantitated densitometrically and graphed at the right side of the corresponding blots. (b) In vitro dephosphorylation of p-STAT1 in the cell lysates mixed with Wedelolactone at various concentrations as indicated. (c) HepG2 cells were stimulated with 100 IU/ml IFN-γ plus vehicle or 50 μM Wedelolactone for various length of time as indicated. Levels of general tyrosine phosphorylation in whole cell lysates were analyzed by Western blot using 4G10 antibody. 500 μM Vanadate treatment was served as positive control. (d) Whole cell lysates from HepG2 cells were mixed with Vanadate or Wedelolactone at various concentrations as indicated and proceed to pNPP in vitro dephosphorylation assay. Figure 5: Wedelolactone prolonged IFN-γ induced STAT1 phosphorylation through TCPTP. (a) p-LUC-HepG2 cells were transfected with Mock or TCPTP pMT2 plasmid 24 hours before being stimulated with IFN-γ plus vehicle or 50 μM Wedelolactone, and luciferase activity was measured following stimulation of IFN-γ for 6 hours. IFN-γ plus 500 μM Vanadate treatment was served as positive control. Data were mean ± s.e.m. of a representative experiment done in triplicate and analyzed by Student’s t-test. (b) p-LUC-HepG2 cells were transfected with control siRNA or TCPTP siRNA 72 hours before being stimulated with IFN-γ plus vehicle or 50 μM Wedelolactone, and luciferase activity was measured following stimulation of IFN-γ for 6 hours. IFN-γ plus 500 μM Vanadate treatment was served as positive control. Data were mean ± s.e.m. of a representative experiment done in triplicate and analyzed by Student’s t-test. (c) Transfection efficiency in (a) and (b)

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were tested by Western blot using TCPTP antibody. (d) HepG2 cells were transfected with control siRNA or TCPTP siRNA 72 hours before being stimulated with IFN-γ plus vehicle or 50 μM Wedelolactone for various length of time as indicated. Whole cell lysates were processed for Western blot analysis and probed with the indicated antibodies. IFN-γ plus 250 μM Vanadate treatment was served as positive control. (e) Wedelolactone was tested for its inhibitory effect on in vitro PTP activity of various PTPs as described in Material and Method. Each value was mean ± s.e.m of a representative experiment done in triplicate. Figure 6: Synergistic effect of IFN-γ and Wedelolactone in tumor cell viability reduction. (a-d) Different tumor cell lines were treated with Wedelolactone alone or together with 1000 IU/ml IFN-γ at various concentrations. After (a) HepG2 cells: 24 hours; (b) WiDr cells: 72 hours; (c) A431NS cells: 36 hours; (d) 97H cells: 72 hours treatment, cells were proceeded for MTT assay. Each value was mean ± s.e.m of a representative experiment done in triplicate. Data were analyzed by Student’s t-test. The first two columns in each histogram are the control cells (the hollow column) and the cells treated with IFN-γ only (the solid column). (e) 97H cells were stimulated with 100IU/ml IFN-γ or 20 ng/ml IL-6 as indicated. 30 minutes after stimulation whole cell lysate of 97H cells was harvested analyzed by Western blot with indicated probes.

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Kuang and Qiang YuZhimin Chen, Xiaoxiao Sun, Shensi Shen, Haohao Zhang, Xiuquan Ma, Jingli Liu, Shan

inhibiting STAT1 dephosphorylation signaling throughγWedelolactone, a naturally occurring coumestan, enhances IFN-

published online April 11, 2013J. Biol. Chem. 

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