Int J Clin Exp Med 2017;10(2):2850-2857www.ijcem.com /ISSN:1940-5901/IJCEM0042544

Original Article Curcumin supresses the WTX expression via Stat3 sinaling in G401 cells

Shibo Zhu, Wei Jia, Wen Fu, Guochang Liu

Department of Pediatric Surgery, Guangzhou Women and Children’s Medical Center, Affiliated Women and Chil-dren’s Medical center of Guangzhou Medical University, Guangzhou, China

Received October 21, 2016; Accepted November 22, 2016; Epub February 15, 2017; Published February 28, 2017

Abstract: Backgrounds: Wilms’ tumor gene on the X chromosome (WTX) has been considered as a key tumor sup-pressor in Wilms’ tumor. This study aimed to assess the effects of curcumin on WTX expression in G401 cells and explore its underlying mechanism. Methods: The G401 Wilms’ tumor cell line were treated with curcumin for 48 h. The effects of curcumin on G401 cell proliferation was analyzed using CCK8 assay. In addition, the effects of cur-cumin on G401 cell apoptosis was detected by flow cytometry and TUNEL staining. Total RNA and protein was col-lected for qRT-PCR and Western blot analysis, respectively. Results: Curcumin inhibited growth and increased the ex-pression of apoptosis protein in these cells. At the molecular level, curcumin treatment led to a significant increase of WTX expression. Moreover, curcumin also markedly inhibited the phosphorylation level of Signal Transducer and Activator of Transcription-3 (Stat3). In addition, Stat3 siRNA was administered, and it significantly decreased WTX expression in G401 cells. Conclusion: Curcumin exerts anti-cancer effects and increases WTX expression via sup-pressing Stat3 signaling pathway in G401cells.

Keywords: Curcumin, WTX, Stat3, G401 cells, Wilms’ tumor

Introduction

Wilms tumor, the most regular malignant neo-plasm of the kidney in children, is an embryonic tumor that is composed of blastemal, stromal and epithelial cells [1]. Although international trials have been established, which have col-lected a number of Wilms’ tumor samples; the mechanisms underlying its pathogenesis and effective treatment strategies remain to be clearly revealed [2]. Rapidly growing research has identified somatic mutations at several lo- ci in Wilms tumorigenesis, including WT1, CTNNB1, WTX and TP53 [3-5].

WTX has been deemed as a vital tumor sup-pressor in Wilms’ tumor [6, 7]. Inactivation of WTX is the most common genetic episode in sporadic Wilms’ tumor, reported in up to 30% of cases [3]. Protein interaction studies have sug-gested that WTX associates with the APC com-plex and might negatively regulate β-catenin stability [8]. In additional, previous studies have

also uncovered a positive effect on p53 signal-ing through enhancing CBP/P300-mediated acetylation of p53 at Lysine 382 [9]. Accordingly, WTX may involve in Wilms tumor initiation and progression through several mechanisms. How- ever, the molecular determinants of WTX ex- pression remain largely unexplored.

Curcumin, a diferuloylmethane derived from the rhizomes of turmeric, have been extensively proved to be a strong anti-cancer agent against a wide range of cancer cells while being non-toxic normal cells [10-12]. Moreover, curcumin hinders cancer cell proliferation and promotes cancer cell differentiation [13]. These effects contain multiple pathways and intracellular tar-gets, including DNA, mRNA, and proteins [14]. Therefore, the results implying that the mecha-nisms underlying curcumin’s anticancer effects are distinct among tumor cell types suggest a cell-type specific effect of curcumin on the inhi-bition of tumor progression.

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However, it remains unclear whether curcumin directly inhibits proliferation in the G401 human Wilms’ tumor cell line. Thus, the aim of the pre- sent study was to investigate the effect of cur-cumin on G401 cell proliferation and the under-lying molecular mechanisms, to potentially pro-vide results which may aid in the development of effective drugs for the clinical treatment of Wilms’ tumor.

Materials and methods

Cell cultures

The G401 Wilms’ tumor cell line was purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China), and cultured in RPMI-1640 (Dulbecco’s modifed Eagle’s medium) supplemented with 10% fetal calf serum, 100 IU/ml penicillin and 100 mg/ml streptomycin (all obtained from Gibco-BRL, Carlsbad, CA, USA). Cultures were maintained at 37°C in a humidifed 5% CO2 atmosphere.

Cell viability assay

The CCK-8 (Dojindo Laboratories, Japan) assay was used to assess effects of curcumin on pro-liferation and viability of G401 cells. Cells were seeded onto 96-well plates at a concentration of 1 × 104 per well and treated with curcumin or diluent. After treatments, absorbances at 450 nm were registered using a microplate reader (Bio-Rad, USA).

RNA isolation and qPCR

Total RNA was isolated from cells by TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), and reverse transcription was con-ducted using a Takara RNA PCR kit (Takara Biotechnology, Dalian, China), according to the manufacturer’s instructions. In order to analyze the transcripts of the genes of interest, qPCR was performed using an SYBR-Green Premix Ex Taq (Takara Biotechnology) on an ABI 7300 machine (Invitrogen Life Technologies).

Western blotting

Western blotting was performed similarly as described previously [18]. In brief, membrane protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electroph- oresis and blotted onto a polyvinylidene difl uoride membrane (Bio-Rad, Hercules, CA). The membranes were blocked with 5% milk and incubated in 4°C overnight with WTX, pStat3, t-Stat3, Bax, Bcl-2, GAPDH (Santa Cruz, CA, USA) (1:500 dilution in TBST). Proteins were visualized with ECL procedure (Bio-Rad, USA). The results were analyzed with Quantity One software (Bio-Rad).

Transient transfections

All the transient transfections were performed by Lipofectamin 2000 (Invitrogen), according to the manufacturer’s instructions. For luciferase reporter assay, G401 cells were seeded in 24-well plates and transfected with the indicat-ed plasmids. Cells were harvested 30 hours after transfection. (Promega, USA).

Flow cytometry analysis

G401 cells were seeded at a concentration of 1×106/ml onto 6-well culture plates. When the cells reached about 70% degree confluency, the medium was changed, and curcumin (10 μM) or diluent was added. Then, G401 cells

Figure 1. Curcumin treatment inhibits cell growth in a dose-dependent manner. G401 cells were incubated with various curcumin concentrations (0-20 μM) for 48 h. A. Cell proliferation was assessed using the CCK8 assay. Cells incubated in a medium without curcumin was defined as control and considered to have a 100% proliferation rate. B. Chemical structure of curcumin. *P < 0.05 vs. Control group.

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were trypsinized to obtain a singlecell suspen-sion. One million cells were double-stained with APC-labeled annexin V and propidium iodide (Becton-Dickinson, USA). Percentage of apop-totic cells was determined by flow cytometry (Becton-Dickinson, USA).

TUNEL staining

TUNEL staining was performed with the use of the In Situ Cell Death Detection kit (Roche) according to the manufacturer’s instructions. Propidium iodide staining was performed to visualize nuclei after TUNEL reaction, and the percentage of TUNEL-positive nuclei was quan-tifed using Image J software. The sections were dehydrated, mounted and observed at 400 × magnification.

Figure 2. Effect of curcumin on apoptosis in G401 cells. A. Apoptosis was quantified by FACS analysis after staining with Annexin V and propidine iodine (PI). B. Representative Western blot analysis of Bax and Bcl-2 in curcumin (10 μM)-treated G401 cells. C. Densitometric analysis was used to quantify the ratio of Bax/Bcl-2. The results represent the mean ± SEM for three experiments. *P < 0.05 vs. Control group.

Statistical analysis

Statistical analysis was performed with a pair- ed Student’s t-test or two-way analysis of vari-ance test. Numerical data are presented as the mean ± SEM. *P < 0.05 was considered to indi-cate a statistically significant difference.

Results

Curcumin treatment inhibits cell growth in a dose-dependent manner

To the best of our knowledge, the effects of cur-cumin on Wilms’ tumor cells has not been previ-ously analyzed. Thus, G401 cells were selected to investigate whether curcumin exhibits po- tential anti-proliferative functions. G401 cells were treated with curcumin at several concen-

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trations. After 48 h of treatment, growth was inhibited in a dose-dependent manner as determined by CCK-8 assays (Figure 1A). Moreover, these results suggested that the concentration of curcumin at 10 µM was opti-mal in G401 lines. Therefore, 10 µM of curcum-in was selected for the further analysis of gene expression in G401 cells.

Effect of curcumin on apoptosis in G401 cells

We further determined the effect of curcum- in on G401 cell apoptosis. As shown in We assayed for annexin V/PI to show that the rela-tive percent of apoptotic cells and the numb- er of annexin V positive cells were significant- ly lower in curcumin treated cells (Figure 2A). Treatment with curcumin at 10 μM pro-foundly attenuated apoptotic morphology and decreased the number of apoptotic cells as evi-

denced by TUNEL staining (Figure 3A, 3B). Furthermore, curcumin treatment increased the expression of several important pro-apop-totic proteins Bax and decreased the expre- ssion of antiapoptotic Bcl-2 (Figure 2B, 2C).

Curcumin increases WTX expression in G401 cells

WTX activation plays a critical role in tumor development and progression. We determined the effect of curcumin treatment on this pro-tein. It was observed that WTX expression was markedly increased following curcumin treat-ment in G401 cells (Figure 3C, 3E).

Curcumin downregulates Stat3 activity in G40- 1 cells

Several studies have indicated that the antipro-liferative effects of cucumin involve the Stat3

Figure 3. Effects of curcumin on apoptosis with TUNEL assay and the WTX expression in G401 cells. A. Fluorescent microscopic images showing TUNEL and nuclear staining. B. Quantification of apoptotic cells. C. The expression of WTX mRNA was determined by real-time PCR using β-actin as the internal reference. D. The protein level of WTX was examined by Western blot analysis. E. Densitometric analysis was used to quantify the expression of WTX. The results represent the mean ± SEM for three experiments. *P < 0.05 vs. Control group.

Figure 4. Curcumin downregulates Stat3 activity in G401 cells. A, B G401 cells were pretreated with curcumin for 48 h. The phosphorylation level of Stat3 was determined by Western blot analysis. C-E. G401 cells strains with stable expression of Stat3 siRNA or negtive control (NC) siR-NA were established. The phosphorylation level of Stat3and protein level of WTX were examined by Western blot analysis. The re-sults represent the mean ± SEM for three experiments. *P < 0.05 vs. Control group.

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pathway [15]. We first determined the effect of curcumin treatment on this pathway. For this, we documented expression of phosphorylated versus total Stat3 in G401 cells exposed to cur-cumin. As expected, curcumin did not affect expression of total Stat3 (Figure 4A). In con-trast, phosphorylation of Stat3 was markedly reduced in G401 cells treated with curcumin (Figure 4A, 4B). Thereby, we demonstrated that curcumin negatively modulates the Stat3 sig-naling pathway.

Stat3 inhibits WTX expression in G401 cells

At the molecular level, Stat3 modulates the transcription of a variety of genes involved in the regulation of critical functions, including cell differentiation, proliferation, apoptosis, angiogenesis, metastasis and immune re- sponses [16, 17]. However, whether Stat3 in- volved in the roles of WTX remain poorly under-stood. To determine whether the reduction of WTX by Stat3 is required for the anti-prolifer- ative effect in G401 cells, Stat3 knockdown experiments using siRNA were conducted (Figure 4C, 4D). As a result, the siRNA increase the expression of WTX in G401 cells (Figure 4C, 4E). Therefore, the results indicate that cur-cumin can inhibit the WTX expression in G401 cells via Stat3 sinaling.

Discussion

In this study, the involvement of curcumin and its molecular mechanism in Wilms’ tumor cells was investigated. Curcumin was shown to inhib-it cell proliferation in G401 cells as demonstrat-ed by CCK8, flow cytometry and TUNEL assays. At the molecular level, the results demonstrat-ed that curcumin activated Stat3 activation. In addition, Stat3 was identifed to be a novel molecular target of curcumin. Stat3 invalida-tion, using siRNA, reduced the WTX expression in G401 tumor cells. Collectively, the data su- ggested that curcumin may be benefcial in the treatment of Wilms’ tumor.

Accumulating evidence indicates that curcumin inhibits tumor progression via multiple cellular signaling pathways [18-20]. It has been docu-mented that curcumin suppresses pancreatic cancer migration and invasion through the inhi-bition of the ROS/ERK/NF-κB signaling path-way [21]. Zhang et al demonstrate that curcum-in exerts anti-cancer effects by negative modu-lation of the PI3K/AKT signaling pathway [22]. In the present study, curcumin treatment could

markedly exacerbate the apoptosis of G401 cells, and boost up the expression of WTX. Co- llectively, our findings indicate that curcum- in treatment may exert the anti-cancer effect in G401 cells through up-regulation WTX ex- pression.

Stat3, a member of the STAT family, is capable of regulating the expression of target genes implicated in cell cycle progression, apoptosis, promotion of cellular transformation, and aber-rant cell proliferation [23, 24]. Previous studi- es have suggested that the antiproliferative effects of curcumin involved the Stat3 pathway [15, 25, 26]. Therefore, Stat3 represents an attractive target for therapeutic intervention. Early in vitro work reported that structurally modified curcumin analogs inhibit Stat3 phos-phorylation and promote apoptosis of human renal cell carcinoma [27]. Moreover, Cao et al uncovered that Stat3 inhibits WTX express- ion through up-regulation of microRNA-370 in Wilms tumor [28]. In the current work, we dis-closed that the phosphorylation level of Stat3 was distinctly supressed by curcumin in G401 cells. Besides, Stat3 siRNA markedly decreased the expression of WTX. Together, we suggested the protective effects of curcumin to WTX acti-vation by regulating the Stat3 signaling in G401 cells.

In conclusion, the results suggest the under- lying mechanisms that may contribute to the antineoplastic effects of curcumin. Further stu- dies are required to investigate the potential of curcumin as a therapy for Wilms’ tumor preven-tion and treatment.

Acknowledgements

This study was supported by the national sci-ence foundation of China (87376482).

Disclosure of conflict of interest

None.

Address correspondence to: Dr. Shibo Zhu, De- partment of Pediatric Surgery, Guangzhou Wom- en and Children’s Medical Center, Affiliated Wom- en and Children’s Medical Center of Guangzhou Medical University, No.9 Jinsui Road, Guangzhou 510623, China. E-mail: zhushibogdgz@163.com

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