Colorectal cancer ranks the second among the causes of cancer mortality in the Western world.1 Approximately 14 000 new cases are diagnosed and about 5000 people die from colorectal cancer each year in the United States.2 Epidemiologic studies suggest that men are more likely than women to develop colon cancer at all ages.3–5 Hormone replacement therapy in postmenopausal women reduces the risk of colorectal cancer by 30% to 40% and also confers protection against the incidence of colorectal cancer.6–10 These reports suggest that estrogen plays a role in colorectal cancer prevention.
Previously, it was considered that the main biological function of estrogen is mediated by binding to estrogen receptor α (ERα), but recent studies have shown that estrogen receptor β (ERβ)11 and several splice variants of ERα and ERβ12–18 are involved in estrogen signaling. An exon-1 truncated ERα transcript has been identified in human cell lines including MCF-7 cells,19 osteoblasts,20 and vascular endothelial cells.21 In contrast to its full-length counterpart ERα66, this truncated ERα lacks the transactivation domain AF-1 and encodes a protein with a predicted molecular weight of 46 kDa; which was therefore designated as ERα46. ERα46 transcripts were found to be more abundant in non-reproductive organs than in reproductive tissues,19 and our preliminary work showed that ERα66 is not the only ERα isoforms in the colon mucosa, ERα46 is also expressed at a high level, therefore ERα46 may also play an important physiological role in colorectal tissues.
In this study, we confirmed the expression of ERα46 in both normal and cancerous colorectal tissues by Western blot, and compared the expression levels of ERα46 mRNA in 32 normal colorectal tissues and their matched cancer tissues, and investigated the function of ERα46 on the growth of HT-29 colon cancer cells in the presence of 17β-oestradiol.
This study was approved by the Research Ethics Committee of the First Affiliated Hospital of Zhejiang University. All patients gave written informed consent to donate colorectal tissues. A total of 32 malignant tumor tissues and matched adjacent normal tissues were collected immediately after surgery and stored at -80°C until use. Histological diagnosis was performed by skillful pathologists. Matched normal tissues were collected from a region more than 15 cm away from the edge of tumors. None of patients included had received hormonal therapy, chemo- or radiotherapy before tissue collection.
RNA extraction and TaqMan real-time quantitative polymerase chain reaction
Total RNAs were extracted from frozen colorectal tissues using the Trizol reagent (Invitrogen Life Technologies, USA) according to the manufacturer's instruction. Two micrograms of total RNA of each sample was reverse-transcribed into cDNA using M-MLV reverse transcriptase (Promega Co, USA) in a total volume of 25 μl. Taqman real-time quantitative polymerase chain reaction (PCR) was performed in triplicate for each sample using the Line-Gene K Detection System (Hangzhou Bioer Technology Co., China) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. Sequences for primers and probes used in this study are listed in Table 1. Data analysis was performed using the comparative CT method.22 The amount of ERα46 mRNA relative to GAPDH mRNA is expressed as 2−ΔCt, and the relative levels of ERα46 mRNA in cancer samples versus matched normal tissues is expressed as 2−ΔΔCt.
Construction of expression vector and transfection
The open-reading frame (ORF) of the ERα46 was cloned by PCR from the template of full length of human ERα66 cDNA in the pSG5-HEO plasmid23 with the primer set 5′-GGCATTCTACAGGCCAAA-3′ and 5′-TAGAAG-GCACAGTCGAGG-3′ and verified by DNA sequencing. The cDNA was inserted into the multiple cloning site of the mammalian expression vector pcDNA3.1(+) to generate the ERα46 expression vector pcDNA3.1-ERα46.
Human colon adenocarcinoma cell line HT-29 cells were maintained in RPMI1640 medium (Gibco Life Technologies Inc, USA) supplemented with 10% fetal calf serum (Hyclone, USA) and 1% penicillin/streptomycin at 37°C in a humidified atmosphere with 5% CO2 in air. The plasmids pcDNA3.1-ERα46 and pcDNA3.1 were transfected into HT-29 cells by Lipofectamine™ 2000 (Invitrogen Life Technologies, USA) and stable transfectants were selected by 800 μg/ml G418 (Invitrogen Life Technologies Inc., USA) for 2 weeks. Single clones were picked, subcultured in 6 well plates and examined for ERα expression by Western blot. Positive clones were maintained in culture medium with 400 μg/ml G418. Cells were cultured in phenol red-free RPMI1640 medium containing 10% dextran-coated, charcoal-treated fetal calf serum (Hyclone, USA) for 48 hours before they were used in the following experiments with 17β-oestradiol treatment.
For clinical samples the normal and cancerous colorectal tissues were homogenized in lysis buffer, 50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 0.1% SDS, and 1% NP-40, and sonicated on ice before insoluble debris was removed by centrifugation at 10 000 r/min for 20 minutes at 4°C. For in vitro cultures, the cells were harvested, washed twice with ice cold PBS and lysed in lysis buffer. Protein concentration of each sample was determined by DC protein assay kit (Bio-Rad Laboratories, USA). Equal amounts of proteins (40 μg/lane) were loaded onto 10% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes. The membrane was probed with rabbit anti-ER polyclonal antibody (Lab Vision Corporation, USA) which recognizes both ERα46 and ERα66. HRP-conjugated secondary antibody (Santa Cruz Biotechnology, USA) and the enhanced ECL system (Millipore Corporation, Billerica) were used before exposure to X-ray film. Monoclonal antibody against GAPDH (Sigma, USA) was used for normalization of protein loading. Protein band densities were analyzed by using Glyko Bandscan (Glyko, USA).
17β-oestradiol treatment and methyl thiazolyl tetrazolium assay
Cells were seeded into 96-well plates at a density of 1×103/well and treated with 10−8 mol/L 17β-oestradiol for 0, 48, 96 and 144 hours. Cell viability was evaluated by adding 0.5 mg/ml methyl thiazolyl tetrazolium (MTT) solution into the 96-well plates and incubating for an additional 4 hours. Two hundred microlitre of dimethyl sulfoxide was then added to dissolve the formazan crystals and the 570 nm absorbance was measured by a microplate reader (Model 680, Bio-Rad, USA).
Flow cytometry analysis
Cells were plated into 6-well plates at density of 5×106/well and treated with 10−8mol/L 17β-oestradiol for 0, 4, 24 and 48 hours. The cells were harvested, fixed in 70% ethanol at 4°C overnight, stained with 40 μg/ml propidium iodide at room temperature for 0.5 hour and analyzed with flow cytometery (Counter EPICS XL, USA).
Cells were plated into 6-well plates at a density of 5×106/well and treated with 10−8 mol/L 17β-oestradiol for 48 hours. Cells were harvested, washed twice with ice-cold phosphate buffered saline, lysed in 10 mmol/L Tris-HCl, 0.1 mol/L EDTA, 0.5% (v/v) SDS, 20 μg/ml RNaseA for 1 hour at 37°C and digested with proteinase K (100 μg/ml) at 37°C overnight. DNA was precipitated by 2.5 volumes of ethanol and analyzed by electrophoresis in a 1.5% agarose gel with 0.5 μg/ml ethidium bromide.
Cells were plated into 6-well plates at density of 1×105/well and treated with 10−8 mol/L 17β-oestradiol for 48 hours, and fixed with freshly prepared 4% paraformaldehyde. TUNEL staining was performed using an in situ cell death detection kit (Roche, USA) according to the manufacturer's instructions. Positive TUNEL staining (brown staining) cells were characterized as apoptotic cells and 10 randomly selected microscopic fields in each group were used to calculate the relative ratio of TUNEL positive cells.
Wilcoxon signed-ranks, Mann-Whitney and Kruskal-Wallis tests were applied to determine the expression of ERα46 mRNA in normal colorectal tissues and colorectal cancer tissues and their association with single clinical factors (gender, age, Dukes' stage, histological differentiation). A Student's t test was used to determine the statistical difference between ERα46-transfected and control groups. Differences were considered statistically significant at a level of P <0.05.
Expression of ERα46 in colorectal cancer and matched normal tissues
The expression of ERα46 in colorectal tissues was confirmed by Western blot, as shown in Figure 1A, which also shows a tendency of decreased expression in cancer tissues compared to normal tissues. To quantitatively analyze the expression level changes of ERα46 in normal and cancerous colorectal tissues, we detected the mRNA of ERα46 by real-time RT-PCR. Thirty-two paired normal and tumor samples were used for real-time quantitative PCR analysis of ERα46 expression. The mean values of ERα46 mRNA expression were significantly reduced in the tumor tissues compared with their matched normal tissues (Table 2), which may be related to their neoplastic development. The difference of ERα46 expression was not significantly different between colorectal cancer tissues of different genders, histological differentiation and Dukes' stage (Table 3).
ERα46 can mediate the growth arrest of HT-29 cells in the presence 17β-oestradiol
Human colon cancer cell line HT-29 expresses low levels of ERα46 as determined by real-time quantitative PCR (data not shown). To investigate the role of ERα46 in human colon cancer cells in the presence of 17β-oestradiol, stable transfectants of ERα46 were established. Western blotting confirmed the expression of exogenous ERα46 (Figure 1B).
Two ERα46 positive clones were used to determine the influence of 17β-oestradiol on the growth of ERα-transfectanted HT-29 cells. The MTT assay was performed after cells were exposed to 10−8 mol/L 17β-oestradiol for 0, 48, 96, and 144 hours. As shown in Figure 2, the growth rate of ERα46 transfected cells was significantly decreased compared to the control cells in the presence of 10−8 mol/L 17β-oestradiol. However, no difference of viability between ERα46-transfected cells and the control cells was observed in the absence of 17β-oestradiol. These results suggest that ERα46 expressed in HT-29 cells inhibit cell proliferation in the presence of 17β-oestradiol.
ERα46 can mediate G0/1 arrest of HT-29 cells in the presence of 17β-oestradiol
To better understand ERα46 mediated growth inhibition of HT-29 cells, we performed flow cytometric assays on cells treated with 17β-oestradiol for different time periods. As shown in Figure 3, an accumulation of cells in the G0/1 phase and a reduced proportion of cells in G2/M phase were observed in the ERα46 expressing cells, and the proportion of ERα46-transfected cells in the G0/1 phase showed significant difference compared to that of control cells after exposed to 17β-oestradiol for 48 hours (t=11.307, P=0.008). But the cell cycle pattern was unchanged for control cells. These results demonstrated that ERα46 expressed in HT-29 cells could block the progress of cell cycle in the presence of 17β-oestradiol.
ERα46 can mediate apoptosis of HT-29 cells in the presence of 17β-oestradiol
To test whether ERα46 can mediate colon cancer apoptosis, DNA fragmentation assay and TUNEL assay were performed. As shown in Figure 4, the yields of apoptotic DNA fragments in ERα46 transfectants were higher than the control group treated with 17β-oestradiol or the groups in the absence of 17β-oestradiol. In the TUNEL assay, there were more apoptotic cells among the ERα46 transfected cells than among the control cells after treatment with 17β-oestradiol (t=9.526, P=0.017), as shown in Figure 5. These results showed that ERα46 expressed in HT-29 cells can induce apoptosis in the presence of 17β-oestradiol.
This study demonstrated that the expression of ERα46 mRNA was decreased in human colorectal tumor tissue compared with normal adjacent tissue. It also shows that transfected ERα46 can mediate growth inhibition and apoptosis of HT-29 colon adenocarcinoma cells in the presence of 17β-oestradiol, which gives us some clues for the role that ERα46 may play in the development of colorectal cancer.
Previous studies have shown conflicting results of the ERα expression in normal colorectal mucosa with biochemical assays.24,25 When using the immunohistochemical method to define the localization of the ERα protein in the colorectal epithelium, colorectal tissues were frequently considered as ERα negative tissues.26 This suggested that a more reliable and sensitive method was needed to evaluate the ERα expression in the colorectal tissues, especially those with very low expression levels. In the current study, real-time quantitative PCR was used to compare the expression of ERα46 mRNA in 32 colorectal cancers and their matched normal colorectal tissues, which allows us to detect ERα expression in colorectal tissues with very low ERα levels.27,28
The expression of ERα is important for estrogen-mediated suppression of colorectal cancer. DNA hyper-methylation of the ERα gene at the CpG islands, which results in gene silencing and therefore decreased expression of ERα, has been reported in colorectal cancer in two independent studies.29,30 Loss of ERα expression led to colonic adenomas and invasive adenocarcinomas in a mouse model.31 However, only a few studies have been performed on the expression and function of the ERα in the colorectal tissues until now. Although the expression of ERα was detected in colon cancer cell lines as well as in normal and cancerous tissues, the 66 kDa full length ERα66 expression appeared to be unchanged between normal mucosa and colorectal cancer,1 which implies that ERα66 may not be the isoform responsible for ERα's role in colorectal cancer development. Other alternative ERα splice variants with differential expression levels between normal and cancerous tissues may play more important roles in this process. In this study, we first examined the expression level of ERα46 mRNA in 32 normal colorectal tissues and corresponding cancer tissues. We found a decreased expression of ERα46 mRNA in neoplastic tissue compared to the matched normal colorectal tissues, which suggested that the expression of ERα46 is negatively associated with the colorectal malignancy development.
However, whether this ERα46 isoform can mediate 17β-oestradiol-induced cellular response in colorectal cancer cells and play a role in colorectal cancer prevention has not been investigated. ERα, which belongs to the nuclear receptor superfamily, can be divided into six distinct regions (A-F). The N-terminal domain A/B is responsible for the ligand-independent transactivation function (AF-1). Domain C is responsible for receptor dimerization and the binding to specific DNA sequences. The C-terminal domain E/F is responsible for the ligand-dependent transactivation (AF-2).32 Most ERα splicing variants lack one or more exons compared with the full length ERα mRNA, therefore encode proteins lacking some functional domains of the receptor. ERα46 mRNA is devoid of exon-1 compared with full-length ERα mRNA and encodes a protein lacking AF-1 function. However, it retains the DNA-binding, ligand-binding and AF-2 transactivation domains. This isoform could translocate to the nucleus, interact with estrogen receptor responsive elements (EREs) of target genes and bind to co-repressors or co-activators of ERα to modulate the function of full-length ERα66 as a competitor of ERα66. It has been reported that ERα46 plays a role in the proliferation of breast cancer.19
In human endothelial cells ERα46 is also found to be similar to ERα66 in its subcellular localization and ability to mediate estrogen binding at the surface of the membrane in a ligand-specific fashion, leading to estrogen-induced eNOS activation.21 In the current study, human colon cancer cell line HT-29 lacks endogenous ERα46 expression which allows this cell to be a model for studying the impact of exogenous ERα46 after stable transfection by ERα46 expression vectors. The results strongly suggest that ERα46 can mediate the growth inhibition of ERα46-transfected cells in the presence of 17β-oestradiol, probably via arresting cells at G0/1 phase and inducing apoptosis.
The expression profile of ERα46 in more clinical samples is desired for a sounder molecular epidemiological conclusion. More in vitro and in vivo models are to be established for elucidating the detailed mechanisms that may be involved, and for evaluating the importance of ERα46 in chemoprevention of colorectal cancer as well.
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