Colorectal cancer (CRC) is the third most common cancer and the fourth most common cancer cause of death in the world, accounting for roughly 1.23 million new cases and 608,000 cases of deaths every year [
Acyl-CoA synthetase 5 (ACS5) gene encodes an enzyme involved in fatty acid degradation and lipid biosynthesis [
In this study, we investigated the expression of ACS5 in CRC tissues and cell lines using immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR), and western blotting. In addition, we identified the correlations between ACS5 expression levels and clinicopathological features in CRC patients. Furthermore, we explored the functional role of ACS5 in CRC cells proliferation, apoptosis, and invasion by in vitro experiments.
Five CRC cell lines (HCT116, HT29, LOVO, SW620, and SW480), which were obtained from American Type Culture Collection (Manassas, VA, USA), were grown in Dulbecco’s modified Eagle medium (Gibco BRL, Rockville, MD, USA) containing 10% fetal bovine serum (Gibco BRL) and 100 U/ml penicillin/streptomycin at 37°C under 5% CO2.
Immunohistochemistry (IHC) of tissue specimens was treated in routinely processed, formalin-fixed, paraffin-embedded sections using a streptavidin-biotin complex method. The specimens were autoclaved for 10 min and then were incubated with anti-ACS5 antibody overnight. The specimens were washed and incubated with secondary antibodies at 37°C for 2 h. Detection was carried out using 3′,3-diaminobenzidine tetrahydrochloride (DAB). Finally, specimens were counterstained with haematoxylin.
IHC analysis was performed as described elsewhere [
The siRNA targeting human ACS5 (NCBI database NM_016234) was as follows: 5′-GCAAUUACGUGAAGCUGGA-3′. A control siRNA oligonucleotide, which does not match any known human coding cDNA, was used as the negative control. All siRNAs were purchased from Sigma (Deisenhofen, Germany). siRNAs were introduced into the HT29 and SW480 cells with Lipofectamine™ RNAiMAX (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The cells were divided into 3 groups: the blank control group (the untreated cells), the negative control group (cells infected with nonsilencing siRNA), and the ACS5 siRNA group (cells infected with ACS5 siRNA). We employed real-time RT-PCR and western blot to evaluate the inhibitory effects.
The vector pcDNA3.1(+) (Invitrogen), containing a human cytomegalovirus immediate-early (CMV) promoter, was designed for high-level, constitutive expression in a variety of mammalian cell lines. ACS5 cDNAs were amplified by PCR method using the following primers: 5′-cgcggatccgccaccATGGACGCTCTGAAGCCACCC-3′ (BamHI restriction site) and 5′-ccgctcgagCTAATCCTGGATGTGCTCATACAGG-3′ (XhoI restriction site). The ACS5 overexpression vector (pcDNA3.1(+)-ACS5) was created by cloning the ACS5 coding sequence into the BamHI/XhoI site of pcDNA3.1(+). All constructed vectors were confirmed by DNA sequencing. The cells were divided into 3 groups: the blank control group (the untreated cells), the negative control group (transfected with empty vector pcDNA3.1(+)), and the ACS5 overexpression group (transfected with pcDNA3.1(+)-ACS5). LOVO and SW620 cells transfection were performed using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. Overexpression efficiency was measured with real-time RT-PCR and western blot.
Total mRNA was extracted from cultured CRC cells and then reversely transcribed into cDNA using 10 units of Reverse Transcriptase XL (AMV) (TaKaRa, Kyoto, Japan) according to the manufacturer’s recommendations. Real-time quantitative RT-PCR was carried out using SYBR Green qPCR SuperMix (Invitrogen). A cycle threshold (CT) was assigned at the beginning of the logarithmic phase of PCR amplification. The relative gene expression was calculated using the
Cells were lysed with RIPA Lysis Buffer (Thermo Fisher Scientific, Waltham, MA, USA) 48 h after transfection. Supernatants were collected, and protein was measured using the BCA Assay Kit (Thermo Fisher Scientific). Protein samples were subjected to SDS-PAGE followed by transfer of protein to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). We incubated membranes for 1 h with an appropriate dilution of the primary antibodies against ACS5 (Santa Cruz Biotechnology, Santa Cruz, USA) followed by incubation with the horseradish peroxidase-conjugated second-step antibody (Santa Cruz Biotechnology) and visualized them using an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA). A GAPDH (Santa Cruz Biotechnology) was used as an internal control. Each experiment was repeated thrice and all reactions were carried out in triplicate.
The cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) method. In brief, we used an MTS kit (cellTiter 96 AQ, Promega, Madison, WI). At the indicated time points (0 h, 24 h, 48 h, and 72 h), cells (100
Following culture for 48 h, the cells were harvested, washed with PBS, resuspended in the binding buffer, and incubated for 15 min in the dark with propidium iodide (PI) and Annexin V/fluorescein isothiocyanate (FITC). The stained cells were immediately analyzed by flow cytometry (Beckman Coulter, Brea, CA, USA). The experiment was independently repeated three times.
The transwells (Costar, USA) with 8
The statistical software package SPSS 16.0 (SPSS, Inc., Chicago, IL) was applied. Quantitative data were evaluated by independent sample
In order to determine whether the expression of ACS5 was different between CRC cell lines and human normal colonic epithelial cell line, quantitative RT-PCR and western blot analysis were carried out. As shown in Figure
Expression of ACS5 in CRC cell lines. The ACS5 mRNA and protein levels were significantly upregulated in five CRC cells compared with the normal cell line (HCoEpiC).
To investigate the role of ACS5 in CRC, we examined the level of ACS5 in 32 human CRC tissues, 29 adenoma tissues, and 21 normal mucosa tissues (obtained from the Institute of Gastrointestinal Surgery) by immunohistochemistry (IHC) analysis. Typical immunostaining of ACS5 expression in CRC and control tissues is presented in Figure
Relationship between ACS5 expression and clinical pathological features of CRC
Variables | Cases (32) | ACS5 expression |
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High (27) | Low (5) | |||
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<60 | 12 | 8 (29.6) | 4 (80.0) | 0.053 |
≥60 | 20 | 19 (70.4) | 1 (20.0) | |
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Male | 19 | 17 (63.0) | 2 (40.0) | 0.374 |
Female | 13 | 10 (37.0) | 3 (60.0) | |
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Not exceeding muscular layer | 8 | 4 (14.8) | 4 (80.0) |
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Exceeding muscular layer | 24 | 23 (85.2) | 1 (20.0) | |
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Negative | 18 | 13 (48.1) | 5 (100.0) | 0.052 |
Positive | 14 | 14 (51.9) | 0 (0.0) | |
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Well | 8 | 5 (18.5) | 3 (60.0) | |
Moderate | 10 | 8 (29.6) | 2 (40.0) |
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Poor | 14 | 14 (51.9) | 0 (0.0) | |
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A | 7 | 6 (22.2) | 1 (20.0) | |
B | 10 | 7 (25.9) | 3 (60.0) | 0.328 |
C + D | 15 | 14 (51.9) | 1 (20.0) |
ACS5 overexpression in CRC tissues. (a) The ACS5 protein level was significantly upregulated in CRC tissues compared with adenoma tissues and normal mucosa tissues. (b) Representative photographs of ACS5 staining in CRC, adenoma, and normal mucosa tissues (stain, hematoxylin; magnification, ×100).
As shown in Figure
The level of ACS5 expression in CRC cells after transfection. ((a), (c)) ACS5 mRNA and protein levels were significantly lower in HT29 and SW480 cells with ACS5 knockdown than those in the negative control or blank control. ((b), (d)) ACS5 mRNA and protein levels were remarkably upregulated in LOVO and SW620 cells with ACS5 overexpression compared with the negative control or blank control. ((e), (f)) Representative western blot results of ACS5 protein expression.
Effect of ACS5 on the proliferation of CRC cells. Cell proliferation was evaluated by MTS, and the results of cell growth were expressed by absorbance at 490 nm. ((a), (b)) The proliferation inhibition of HT29 and SW480 cells with ACS5 knockdown was observed compared with negative control and blank control at 24 h, 48 h, and 72 h. ((c), (d)) The proliferation promotion of LOVO and SW620 cells with ACS5 overexpression was observed compared with negative control and blank control at 48 h and 72 h.
The effect of ACS5 on CRC cell apoptosis was analyzed using a flow cytometer. Silencing of ACS5 remarkably increased the percentage of early and late apoptotic cells in both HT29 (Figures
Effect of ACS5 on the apoptosis of CRC cells. ((a), (b), (e)) The percentage of early and late apoptotic cells was higher in HT29 and SW480 cells with ACS5 knockdown than that in negative control or blank control cells. ((c), (d), (f)) The percentage of late apoptotic cells was lower in LOVO and SW620 cells with ACS5 overexpression than that in negative control or blank control cells.
To further evaluate the effect of ACS5 on the migration and invasion capability of CRC cells, the transwell assays were performed. Knockdown of ACS5 remarkably decreased the migrated and invaded number of both HT29 (Figures
Effect of ACS5 on the migration and invasion of CRC cells. ((a)–(d), (i)) Analysis of migration capability of CRC cells. ((e)–(h), (j)) Analysis of invasion capability of CRC cells. Both the migrated and invaded number of HT29 and SW480 cells with ACS5 knockdown were significantly decreased compared with negative control or blank control. Inversely, both the migrated and invaded number of LOVO and SW620 cells with ACS5 overexpression were remarkably increased compared with negative control or blank control.
To investigate the potential mechanism by which ACS5 affects cellular proliferation and invasion, we explored the change in cell apoptosis and invasion-related molecules in these transfected cells. The downregulation of ACS5 in HT29 and SW480 cells resulted in a significant reduction in survivin and CD44 expression and striking increase in caspase-3 and E-cadherin expression (Figures
ACS5 affects CRC cells growth and metastasis through the modulation of survivin and CD44 expression. ((a), (b), (c), (e), (g)) The downregulation of ACS5 in HT29 and SW480 cells resulted in a significant reduction in survivin and CD44 expression and striking increase in caspase-3 and E-cadherin expression. ((a), (b), (d), (f), (h)) The expression of survivin and CD44 was significantly elevated, while the levels of caspase-3 and E-cadherin expression were markedly decreased in ACS5 overexpressed SW620 and LOVO cells.
Colorectal cancer is notorious for its high incidence and poor prognosis [
ACS5 gene is located on chromosome 10q25.1-q25.2, spans approximately 46 kb, comprises 21 exons and 22 introns, and encodes a 683-amino acid protein [
In the current study, we first used quantitative RT-PCR and western blot analysis to detect the expression of ACS5 mRNA and protein levels in CRC cell lines (HT29, SW480, LOVO, SW620, and HCT116) and human normal colonic epithelial cell line (HCoEpiC). The results showed that the ACS5 mRNA and protein levels were higher in CRC cell lines than in human normal colonic epithelial cell line, suggesting that ACS5 overexpression may be associated with the development of CRC at the cellular level.
On the other hand, we revealed that the IHC scores of ACS5 protein in CRC tissues were significantly higher than those in adenoma tissues and normal mucosa tissues. There was no significant difference in the scores between adenoma tissues and normal mucosa tissues. Additionally, clinicopathological data showed that high ACS5 expression was more frequent in CRC patients with excess muscular layer and with poor tumor differentiation. These findings suggest the possibility that ACS5 overexpression may play an important role in the development of CRC and may be linked with tumor differentiation and invasion. In a previous study, Gassler et al. measured ACS5 expression in 15 CRC patients using RT-PCR and western blot. They revealed that ACS5 expression level was significantly upregulated in adenoma tissues and CRC tissues compared with normal mucosa tissues and that the expression of ACS5 expression level was also higher in adenoma tissues than that in CRC tissues [
Next, we silenced ACS5 in HT29 and SW480 cells with high levels of endogenous ACS5, and we utilised a plasmid to upregulate ACS5 expression in the LOVO and SW620 cells with low endogenous ACS5 levels. The quantitative RT-PCR and western blot data showed that ACS5 gene expression was successfully silenced in HT29 and SW480 cells and overexpressed in LOVO and SW620 cells.
Then, we employed in vitro experiments to further explore the role of ACS5 in the development and progression of CRC. Knockdown of ACS5 expression led to decreased proliferation, apoptosis induction, and decreased migration and invasion capability of HT29 and SW480 cells. In contrast, the ectopic overexpression of ACS5 resulted in increased proliferation, apoptosis inhibition, and increased migration and invasion of LOVO and SW620 cells. Similarly, Mashima et al. demonstrated that the stable expression of ACS5 strongly inhibited brain cancer cell death [
The molecular mechanisms by which ACS5 promotes cancer cell proliferation and metastasis remain unclear. Some data argue for an involvement of LKB1/AMPK/mTOR signal transduction pathway, whereas others indicate ACS5-related changes in cytochrome C release from mitochondria as apoptosis-relevant mechanisms. The serine/threonine kinase LKB1 is a master kinase involved in cellular responses such as energy metabolism, cell polarity, and cell growth. LKB1 regulates these crucial cellular responses mainly via AMPK/mTOR signaling. Various cancers have been associated with impaired AMPK activation and/or mTOR inhibition [
In conclusion, our study has demonstrated that ACS5 expression was increased in CRC cells and CRC tissues and its upregulation closely correlated to poor tumor differentiation and excess muscular layer in patients with CRC. In addition, we verified that ACS5 can promote CRC cells growth and invasion in vitro, possibly through modulating survivin and CD44 expression. Our findings provide useful information for understanding the role of ACS5 in the development and progression of CRC and as a potential target for CRC gene therapy.
The authors declare no conflicts of interest.
Shihua Ding, Shaohui Tang, and Min Wang contributed equally to this work.