Molecular Biology Reports

, Volume 37, Issue 3, pp 1627–1632

Curcumin decreases the expression of Pokemon by suppressing the binding activity of the Sp1 protein in human lung cancer cells

Authors

  • Jiajun Cui
    • Zhoukou Normal University
    • Beijing Institute of Biotechnology
  • Xianfeng Meng
    • Tanshan Medical University
  • Xudong Gao
    • 307 Hospital of PLA
    • Zhoukou Normal University
Article

DOI: 10.1007/s11033-009-9575-6

Cite this article as:
Cui, J., Meng, X., Gao, X. et al. Mol Biol Rep (2010) 37: 1627. doi:10.1007/s11033-009-9575-6

Abstract

Pokemon, which stands for POK erythroid myeloid ontogenic factor, can regulate expression of many genes and plays an important role in tumorigenesis. Curcumin, a natural and non-toxic yellow compound, has capacity for antioxidant, free radical scavenger, anti-inflammatory properties. Recent studies shows it is a potential inhibitor of cell proliferation in a variety of tumour cells. To investigate whether curcumin can regulate the expression of Pokemon, a series of experiments were carried out. Transient transfection experiments demonstrated that curcumin could decrease the activity of the Pokemon promoter. Western blot analysis suggested that curcumin could significantly decrease the expression of the Pokemon. Overexpression of Sp1 could enhance the activity of the Pokemon promoter, whereas knockdown of Sp1 could decrease its activity. More important, we also found that curcumin could decrease the expression of the Pokemon by suppressing the stimulation of the Sp1 protein. Therefore, curcumin is a potential reagent for tumour therapy which may target Pokemon.

Keywords

PokemonCurcuminPromoterSp1Expression

Abbreviations

Sp1

Specificity protein 1

ChIP

Chromatin immunoprecipitation assay

GAPDH

Glyceraldehydes-3-phsphate dehydrogenase

Introduction

Curcumin is a natural and non-toxic yellow compound isolated from the root of a kind of plant, Curcuma longa Linn. Curcumin is known for its antioxidant, free radical scavenger and anti-inflammatory properties [1, 2]. Recently, anticarcinogenic properties have also been reported for curcumin since that it can inhibit the proliferation and induce the apoptosis of cancer cells, such as gastric, colon, breast and other cancer cells [35].

Pokemon, which stands for POK erythroid myeloid ontogenic factor, was known by several names (LRF, OCZF, and FBI-1) and originally identified as a protein that binds specifically to the inducer of short transcripts (IST) element in the HIV-1 promoter region [6]. It was first named FBI-1 or factor binding to IST-1, which is encoded by the Zbtb7 gene. Many reports revealed that Pokemon not only regulates HIV-1 Tat trans-activation process [7, 8], but also is involved in the human and murine adipogenesis [9].

As a transcription factor, Pokemon can regulate the expression of many proteins, such as extracellular matrix collagen type I, II, IX, X, and XI, fibronectin, elastin, human cartilage oligomeric matrix protein (COMP) [10, 11], c-fos and c-myc oncoproteins [12]. More importantly, Pokemon is a repressor of the ARF tumour suppressor gene. Overexpression of the Pokemon gene gave rise to the decreased expression of ARF gene, which in turn resulted in the p53 degradation and oncogenic transformation. Conversely, depletion of the Pokemon gene could inhibit the oncogene-mediated cellular transformation and induce cell senescence and apoptosis [13, 14]. Therefore, Pokemon plays a critical role in cell tumorigenesis and may be a potential therapeutic target for human cancer therapy.

Sp1 (specificity protein 1) is a sequence-specific transcription factor that can recognize a motif 5′-GGGCGG-3′ or related GC-rich sequences [15]. It belongs to the Krüppel-like C2H2-type zinc finger superfamily and can activate a wide range of viral and cellular genes. Since a large number of genes contain the Sp1-binding GC-box in their promoters, Sp1 often plays a role in the transcription activation of many genes [16].

Although many studies have reported that curcumin efficiently suppressed the growth of the tumour cells and induced tumour cell apoptosis, it is still not known whether curcumin has the capacity to regulate the expression of Pokemon gene. In this study, we found that curcumin decreased the activity of the Pokemon promoter and the expression of the Pokemon gene. More important, curcumin could decrease the expression of Pokemon by suppressing the stimulation of Sp1. These observations help us to better understand the anticancer mechanism of curcumin.

Materials and methods

Cell culture and treatment

Human lung carcinoma A549 cells were grown in F12-K medium with 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Curcumin (Sigma) was dissolved in DMSO and added to the medium to a given final concentration. After cells reached about 80–90% confluence, they were exposed to 10, 20, 30, 40 and 50 μM curcumin for 24 and 48 h, respectively.

Plasmids

The 2,220-bp upstream region of the Pokemon gene was amplified by PCR from human blood genomic DNA and the PCR products were cloned into pGL-3 basic vector (Promega). Sp1 were generated by PCR amplification using the normal human blood cDNA as a template. Then the products were cloned downstream of CMV promoter into the pcDNA 3.1(+) vector (Invitrogen). To construct Sp1 siRNA expression vector, a DNA fragment containing an inverted repeat of the target sequence: CTTGCAGCAGAATTGAGTC, corresponding to the coding region 200–218 relative to the first nucleotide of the start codon, spaced by the 9-nt sequence TTCAAGAGA and a poly (T) stretch as a terminator for RNA polymerase III, was synthesized and cloned under control of the U6 promoter into the BamHI/HindIII sites of pSilencer2.1-U6neo (Ambion). Plasmid pSilencer2.1-U6neo negative control (Ambion) was used as a control vector. This control vector produces universal scramble siRNA that has no significant homology to mouse, rat, or human gene sequences.

Transient transfection and luciferase assay

To analyze the effect of different amounts of curcumin on the activity of the Pokemon promoter, plasmid pRL-TK vector (Promega) was cotransfected as an internal control. Cells were plated in 24-well plate with 1 × 10 P5P cells/well, and triple wells were set for each group. After reaching about 90% confluent, cells were transfected with 30 ng of pRL-TK vector and 300 ng of F-2220 for each well using Lipofectamine PTMP2000 (Invitrogen). After 24 h transfection, cells were treated with different amounts of curcumin for another 24 h. Then cells were harvested and lysed in 200 μl of the reporter lysis buffer (Promega). To study the effect of Sp1 on the activity of the Pokemon promoter, 0–2 μg of the pcDNA-Sp1, 300 ng of the F-2220 plasmid and 30 ng pRL-TK plasmids were cotransfected into cells, respectively. After 48 h transfection, cells was harvested and lysed in 200 μl of the reporter lysis buffer (Promega). The luciferase assay was carried out using the dual luciferase assay kit (Promega), and the enzymatic activity of luciferase was measured with a luminometer (Promega).

Western blot analysis

A549 cells were transfected with indicated plasmids. The total proteins were separated on 10% SDS-polyacrylamide gels and transferred to PVDF membrane. The membrane was blocked with 5% nonfat milk in phosphate-buffered saline containing 0.05% Tween-20 for 1 h, followed by incubating with a 1:200 dilution of rabbit polyclonal antibody against Pokemon or Sp1(Sigma). The membrane was then incubated with a monoclonal anti-rabbit IgG conjugated with peroxidase. Finally, the blots were developed with the ECL plus western blotting detection system (Amersham).

Chromatin immunoprecipitation assay (ChIP)

A549 cells were used in ChIP assays to examine Sp1 binding of Pokemon promoters in vivo. We used a ChIP Assay kit (Upstate) and followed the manufacturer’s protocol. The following primers were used to detect Pokemon promoter sequences: sense primer, 5′-ACCATTCTCATGCACAGCT-3′, antisense primer, 5′-AGCCTGGGCAACAGAGCAAG-3′.

Results and discussion

Curcumin could decrease the expression of Pokemon

Since Pokemon plays a crucial role in cell oncogenesis, we first investigated whether curcumin could decrease the activity of the Pokemon promoter. The 2,220-bp of the Pokemon promoter construct was co-transfected with the pRL-TK plasmid into A549 cells. 24 h later, A549 cells were treated with various content of curcumin for another 24 h and then the luciferase activities were detected. As shown in Fig. 1a, luciferase assays showed that curcumin could decrease the activity of the Pokemon promoter in a dose-dependent manner, suggesting that curcumin may regulate the Pokemon gene expression.
https://static-content.springer.com/image/art%3A10.1007%2Fs11033-009-9575-6/MediaObjects/11033_2009_9575_Fig1_HTML.gif
Fig. 1

a Curcumin can decrease the activity of the Pokemom promoter. A549 cells were cotransfected with F-2220 and pRL-TK for 24 h. Thereafter, cells were treated with various concentrations of curcumin (10–50 μM) for another 24 h. The cells were harvested for the luciferase assay. The values are the mean ± SE of three independent experiments performed in triplicate, and are normalized to Renilla luciferase activity. * P < 0.05. b Effect of various concentration curcumin on the expression of Pokemon. A549 cells were treated with 0, 30 and 50 μM curcumin for 24 h. Whole-cell extracts were prepared and probed with anti-Pokemon or GAPDH antibody. All the data were confirmed in at three least independent experiments

In order to further confirm whether curcumin could reduce the expression of Pokemon, we carried out Western blot analysis. As shown in Fig. 1b, 30 μM curcumin reduced the expression of Pokemon, and 50 μM curcumin yielded more significant reduction than 30 μM curcumin. This result indicated that curcumin is a potential reagent for anticancer therapy which designates Pokemon as a target.

Sp1 can increase the activity of the Pokemon promoter and the expression of Pokemon

Sp1 belongs to the Krüpple-like C2H2-type zinc finger superfamily, and binds GC boxes to activate a wide range of viral and cellular genes [16]. To evaluate whether Sp1 protein regulates the expression of the Pokemon gene, 2,220-bp of the 5′-upstream region of the Pokemon gene was delivered in the TRANSFAC 7.0 database [17]. After scanning with the TRANSFAC 7.0 database, we found nine putative Sp1 sites in the upstream of the Pokemon gene (data not shown), suggesting that Sp1 protein may play a role in the regulation of the Pokemon gene expression.

To assess whether Sp1 protein can increase the activity of the Pokemon promoter, we constructed an expression vector harbouring the Sp1 gene. Then, 0–2 μg of pcDNA-Sp1 plasmid, 300 ng of the F-2220 plasmid and 30 ng of the pRL-TK plasmids were transfected into the A549 cells, respectively. As shown in Fig. 2a, Sp1 could enhance the activity of the Pokemon promoter and 2 μg of the expression vector plasmid yielded the largest increase compared with the control, suggesting that overexpression of Sp1 can increase the transcription of the Pokemon gene. Western blot analysis was performed and the result showed Sp1 could enhance the expression of Pokemon (Fig. 2b).
https://static-content.springer.com/image/art%3A10.1007%2Fs11033-009-9575-6/MediaObjects/11033_2009_9575_Fig2_HTML.gif
Fig. 2

a Sp1 can increase the activity of the Pokemon promoter. A549 cells were cotransfected with F-2220, pRL-TK and different amount of pcDNA-SP1. 48 h later, the cells were harvested for the luciferase assay. * P < 0.05. b Western blotting analysis of overexpression of Sp1 on the expression of Pokemon. A549 cells were transfected with 1.0 μg of pcDNA-SP1. 48 h after transfection, whole-cell extracts were prepared and probed with anti-Sp1, anti-Pokemon or GAPDH antibody. c Luciferase reporter assays in the control and Sp1 knockdown cells. Cells were cotransfected with F-2220, pRL-TK and different amount of Sp1 siRNA as indicated. The values are the mean ± SE of three independent experiments performed in triplicate, and are normalized to Renilla luciferase activity. * P < 0.05. d Western blotting with various antibodies showing the specific knockdown effect of the Sp1 siRNA on the endogenous Sp1 protein level and the expression of Pokemon. Cells were transfected with Sp1 siRNA or scramble siRNA (control) plasmid. 48 h after transfection, whole-cell extracts were prepared and probed with anti-Sp1, Pokemon, or GAPDH antibody. At least three independent experiments were performed

To investigate the role of endogenous Sp1 in the activity of the Pokemon promoter, we constructed Sp1 siRNA expression vector and transfected the vector into A549 cells. As shown in Fig. 2d, the Sp1 siRNA effectively inhibited the expression of Sp1 protein 48 h after transfection. Western blot analysis showed knockdown of the endogenous Sp1 could decrease the expression of Pokemon. Moreover, suppression of the normal expression of Sp1 in A549 cells by the specific Sp1 siRNA significantly decreased the activity of the Pokemon promoter (Fig. 2c). These results further suggested that Sp1 can enhance the expression of Pokemon. Interestingly, recent studie showed that Pokemon can decrease the transcription of the P21gene by inhibition of transcription activation by Sp1, suggesting that Pokemon and Sp1 could regulate each other [18].

Curcumin can decrease the activity of the Pokemon promoter by suppressing the stimulation of the Sp1 protein

Curcumin has been studied for its wide range effects on tumorigenesis, angiogenesis, apoptosis and signal transduction pathways [3, 19, 20]. Up to date, many mechanisms have been proposed to explain the anti-carcinogenic effect of curcumin, including its anti-inflammatory and antioxidant activity, induction of phase-II detoxification enzymes, inhibition of cyclooxygenase 2 (COX-2), effect on AP-1 and NF-κB transcription factors, inhibition of matrix metalloproteinase (MMP), effect on protein kinases and more [2124].

Since curcumin and Sp1 have opposite effect on the expression of Pokemon, we postulated that curcumin may decrease the stimulation of Sp1 on the Pokemon promoter. To confirm our hypothesis, A549 cells were transfected with different amounts of pcDNA-Sp1 or Sp1 siRNA, 300 ng of the F-2220 plasmid and 30 ng of the pRL-TK plasmid. 24 h later, cells were treated with 50 μM curcumin for another 24 h and luciferase activities were measured. As shown in Fig. 3a, curcumin could significantly decrease the stimulatory function of Sp1 on the Pokemon promoter, whereas Sp1 siRNA and curcumin could function synergistically on inhibiting the Pokemon promoter activity (Fig. 3b), suggesting that curcumin may reduce the expression of the Pokemon by suppressing the stimulation of Sp1.
https://static-content.springer.com/image/art%3A10.1007%2Fs11033-009-9575-6/MediaObjects/11033_2009_9575_Fig3_HTML.gif
Fig. 3

Curcumin can decrease the activity of the Pokemon promoter by suppressing the stimulation of the Sp1 protein. A549 cells were cotransfected with F-2220, pRL-TK and 0–2 μg of pcDNA-SP1 (a) or Sp1 siRNA (b) for 24 h, and then cells were treated with various concentrations of curcumin (0–50 μM) for another 24 h. Then, the cells were harvested for the luciferase assay. * P < 0.05. The values are the mean ± SE of three independent experiments performed in triplicate, and are normalized to Renilla luciferase activity

Curcumin can not decrease the expression of the Sp1 gene

Since Sp1 siRNA and curcumin could function synergistically on inhibiting the Pokemon promoter activity, we postulated that curcumin could decrease the expression of Pokemon by suppressing the expression of the Sp1. To test our hypothesis, A549 cells were treated with 30, 50 μM curcumin for 24 h. Thereafter, Western blot analysis was performed to detect the expression of Sp1. As shown in Fig. 4, compared with the control, 30 and 50 μM curcumin could not decrease the expression of Sp1. This result suggested that curcumin decreased the activity of the Pokemon promoter not via inhibiting the expression of Sp1. However, recent report showed curcumin could decrease Sp1 expression in bladder cancer cells. This is possibly due to the different cell-type context [25].
https://static-content.springer.com/image/art%3A10.1007%2Fs11033-009-9575-6/MediaObjects/11033_2009_9575_Fig4_HTML.gif
Fig. 4

Effect of various concentration curcumin on Sp1 expression. A549 cells were treated with 0, 30 and 50 μM curcumin for 24 h. Whole-cell extracts were prepared and probed with anti-Sp1 or GAPDH antibody. All the data were confirmed in at least three independent experiments

Curcumin can decrease the binding activity between Sp1 protein and the Sp1 element in the Pokemon promoter

Recent studies showed that curcumin could result in the generation of rapid reactive oxygen species (ROS) [26]. Therefore, the cysteines residues of Sp1 could be oxidized by ROS generated when cells are incubated with curcumin, Once the cysteines residues were oxidized, it can decrease the DNA-binding activity of Sp1 [27]. Given the evidence that curcumin could decrease the DNA-binding activity of Sp1, we speculate that the curcumin might decrease the binding activity between Sp1 protein and Sp1 element within the Pokemon promoter. In order to test our hypothesis, A549 cells were treated with 0, 30 and 50 μM curcumin for 24 h, and then promoter occupancy at the Sp1 element within the endogenous Pokemon promoters by Sp1 was detected by ChIP using antibodies against Sp1 and semiquantitative PCR with primers flanking the Sp1 element of the Pokemon promoter. As expected, Sp1 displayed a clear recruitment to the Pokemon promoters. Importantly, compared with the control 30 and 50 μM curcumin significantly inhibit the recruitment of Sp1 to the Pokemon promoter. Moreover, 50 μM curcumin produced more significant effect than that of 30 μM curcumin. The specificity of Sp1 association within the Pokemon promoter was confirmed by ChIP analysis using IgG which failed to immunoprecipitate the Pokemon promoter sequences (Fig. 5). This result suggested that curcumin may decrease the stimulation of Sp1 on the Pokemon promoter by suppressing its recruitment to the Pokemon promoter.
https://static-content.springer.com/image/art%3A10.1007%2Fs11033-009-9575-6/MediaObjects/11033_2009_9575_Fig5_HTML.gif
Fig. 5

Curcumin can decrease the binding activity between Sp1 element in the pokemon promoter. A549 cells were treated with 0, 30, 50 μM curcumin for 24 h, and then soluble chromatin was prepared and subjected to immunoprecipitation by using IgG (negative control) or antibodies for Sp1. Immunoprecipitated DNA was PCR-amplified with primers that annealed to the corresponding region of the Pokemon promoter. The results were obtained from at least three independent experiments

In conclusion, our study demonstrated that curcumin could inhibit the expression of the Pokemon by suppressing the stimulation of Sp1 protein, which is helpful to further elucidate how curcumin displays anticarcinogenic properties.

Copyright information

© Springer Science+Business Media B.V. 2009