Curcumin decreases the expression of Pokemon by suppressing the binding activity of the Sp1 protein in human lung cancer cells
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- Cui, J., Meng, X., Gao, X. et al. Mol Biol Rep (2010) 37: 1627. doi:10.1007/s11033-009-9575-6
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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.
Specificity protein 1
Chromatin immunoprecipitation assay
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 [3–5].
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 . 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 .
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 . 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 . 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 .
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.
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
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 . 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 . 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 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 .
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 [21–24].
Curcumin can not decrease the expression of the Sp1 gene
Curcumin can decrease the binding activity between Sp1 protein and the Sp1 element in the Pokemon promoter
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.