Brought to you by Portland Press Ltd.
Published on behalf of the International Federation for Cell Biology
Cancer Cell death Cell cycle Cytoskeleton Exo/endocytosis Differentiation Division Organelles Signalling Stem cells Trafficking
Cell Biology International (2008) 32, 210–216 (Printed in Great Britain)
Antisense oligonucleotide Elk-1 suppresses the tumorigenicity of human hepatocellular carcinoma cells
Tsung‑Ho Yingab, Yi‑Hsien Hsiehb, Yih‑Shou Hsiehb and Jer‑Yuh Liub*
aDepartment of Obstetrics and Gynecology, Chung Shan Medical University Hospital, No. 110, Sec. 1, Chien-Kuo N. Road, Taichung 40203, Taiwan
bInstitute of Biochemistry and Biotechnology, School of Medicine, College of Medicine, Chung Shan Medical University, No. 110, Sec. 1, Chien-Kuo N. Road, Taichung 40203, Taichung, Taiwan


Abstract

In previous studies, we showed that reducing Ets-like protein-1 (Elk-1) expression inhibited protein kinase C alpha (PKCα) expression and decreased cell migration and invasion in human hepatocellular carcinoma (HCC). In this study, we have investigated the role of Elk-1 in tumorigenesis. SK-Hep-1 HCC cells were transfected with the ElK-1 antisense oligonucleotide (ODN). In the pretreated cells we detected a reduction of mRNA level using RT–PCR. The inhibitory rate of cell growth was measured by MTT assay. Pretreated-SK-Hep-1 HCC cells were implanted subcutaneously into nude mice to observe the tumor growth and calculate tumor inhibitory rate. The results showed that 5μM of the antisense ODN Elk-1 suppressed both Elk-1 and PKCα production by SK-Hep-1 HCC cells after cationic liposome-mediated transfection, to 8% and 1% of control values, respectively, and the growth of SK-Hep-1 HCC cells was inhibited at 2–5μM doses of the antisense ODN Elk-1. The control reagent, sense ODN Elk-1, showed no effects. In BALB/nude mice, SK-Hep-1 HCC cells transfected with the 5μM antisense ODN Elk-1 formed tumors much smaller than those of sense ODN Elk-1 pretreated cells. The maximum inhibitory rate of tumor growth was 80.8±12.6% and the tumor formation time was prolonged from 13 to 25days. These findings suggested the usefulness of antisense ODN Elk-1 as a new reagent for liver cancer therapy.


Keywords: Elk-1, Hepatocellular carcinoma, Protein kinase C alpha, Tumorigenesis, Antisense oligonucleotide.

*Corresponding author. Tel.: +886 4 2473 0022x11673; fax: +886 4 2324 8195.


1 Introduction

The Ets family (ETS) proteins affect the expression of several oncogenes and tumor suppressor genes by direct regulation of their promoters or protein–protein interactions (Oikawa, 2004). They play important roles in cell proliferation, apoptosis and differentiation in normal cells and deregulated expression of ETS proteins could lead to disruption of these processes. It has also been found that the aberrant of ETS includes those encoded by Ets-1, Fli-1, Erg, E1AF/PEA3, ER81, ESE-1/ESX, PU.1 and TEL gene subfamilies. This has been documented in many types of human tumors and correlates well with the grade of invasiveness and metastasis and therefore can be useful in predicting poor prognosis for cancer patients (Behrens et al., 2001; Katayama et al., 2005; Nakayama et al., 2001). However, the role of the several ETS members in tumor development is not yet clear. Therefore, investigation of additional ETS proteins associated with tumorigenesis may provide insight into the molecular mechanisms underlying tumorigenesis.

The Ets-like transcription factor 1 (Elk-1) encompasses 6 introns, two of which lie within 5′ untranslated sequences (Rao et al., 1989). It is a member of the ternary complex factor (TCF) family (Seth and Watson, 2005; Wasylyk et al., 1998), and functions as a nuclear transcriptional activator via its association with serum response factor (SRF) in a ternary complex on the serum response element (SRE) present in the promoter of many immediate early genes (c-fos, egr1, egr2, pip92 and nur77) (Price et al., 1995; Treisman, 1992). Elk-1 is expressed in a variety of human tissues, notably in the brain and esophageal squamous cell carcinoma (Cesari et al., 2004; Chen et al., 2006). Elk-1 expression is both necessary for skeletal muscle cell differentiation (Khurana and Dey, 2002) and critical to the regulation of cell proliferation and apoptosis (Hsieh et al., 2006; Shao et al., 1998). In addition, Elk-1 activation is suggested necessary for tumor progression, such as involvement in bombesin-induced prostate cancer cell growth (Xiao et al., 2002) and modulation of ovarian cancer cell migration (Bourguignon et al., 2005). However, no obtained data has yet indicated the role of Elk-1 in hepatocellular carcinoma (HCC) tumorigenesis in vivo.

Recently we found that reducing Elk-1 expression inhibited PKCα expression and decreased cell migration and invasion in HCC (Hsieh et al., 2006). In this study, we established SK-Hep-1 cell lines transfected with antisense oligonucleotide (ODN) of Elk-1. Characterization of these cells with regard to the tumorigenicity in vitro and in vivo was performed. The results showed that Elk-1 inhibition exerts inhibitory effects on SK-Hep-1 cell proliferation. Antisense ODN mediated Elk-1 suppression also inhibits tumor growth in human hepatocellular carcinoma xenografts in nude mice. Thus, Elk-1 may control the proliferative potential of HCC cells, acting as a tumorigenic factor in HCC.

2 Materials and methods

2.1 Cell culture

The SK-Hep-1 cell line was obtained from the American Type Culture Collection (Rockville, MD). The SK-Hep-1 cell line is poorly differentiated (Aden et al., 1979). The cell lines were cultured with DMEM (Gibco BRL) supplemented with 100μM non-essential amino acid, 2mM glutamate, 10% fetal bovine serum (FBS), 100units/ml penicillin G, and 100μg/ml streptomycin (Sigma Chemical Co., St. Louis, MO) in a humidified atmosphere containing 5% CO2 in air at 37°C.

2.2 Antisense knockout assay

The antisense knockout assay was performed as described by Shen et al. (1999) and the following antisense and sense (as a control) sequences were used: Elk-1 (antisense ODN 5′-CAGCGTCACAGATGGGTCCAT-3′, AS-Elk-1; sense ODN 5′-ATGGACCCATCTGTGACGCTG-3′, S-Elk-1) (Hsieh et al., 2006). Cells were plated at 70% density 24h before sense or antisense oligonucleotide treatment. The cells were washed in triplicate with serum-free DMEM and incubated with sense or antisense oligonucleotide (0, 1, 2 or 5μM) in serum-free DMEM containing 10μg/ml lipofectin (Life Technologies, Grand Island, NY) at 37°C. The medium was changed to 10% FBS DMEM medium 6h later before culturing at 37°C for 48h.

2.3 RNA isolation

Total RNA was isolated from cells by the guanidinium thiocyanate-phenol method (Chomczynski and Sacchi, 1987). The HCC cell lines were homogenized (4M guanidine thiocyanate, 25mM sodium citrate, 0.5% (w/v) sodium lauryl sarcosinate, 0.1M β-mercaptoethanol) in a polypropylene tube; total RNA was isolated using a standard method. Phenol/chloroform reagent was added to the samples, and the tube centrifuged at 12,000×g for 30min at 4°C. RNA was precipitated from the aqueous phase with isopropanol. The resultant pellet was then washed twice with 70% ethanol. The RNA content of the resuspended pellet was quantified and checked for purity and condition using spectrophotometry at a wavelength of 260nm. The extract integrity was assessed using 1.5% agarose gel electrophoresis. RNA was visualized by ethidium bromide staining.

2.4 Reverse transcriptase (RT)-PCR

RT–PCR assay was a slightly modified method of De et al. (1998). An aliquot of total RNA (0.5μg) was reverse transcribed using 0.5μM oligo d(T) primers in a reaction solution (50μl) containing 75mM KCl, 50mM Tris–HCl (pH 8.3), 3mM MgCl2, 10mM DTT, 10U RNase inhibitor (Promega, Madison, WI), 0.8mM total dNTPs, and 200units of Moloney murine leukemia virus reverse transcriptase (Promega). The sample was incubated at 42°C for 1h and at 99°C for 5min before chilling on ice for 10min.

The RT product (2μl) was diluted with the PCR buffer (50mM KCl, 10mM Tris–HCl, and 2mM MgCl2) to a final volume of 50μl, containing 0.5μM dNTPs (final concentration, 0.8mM) and 0.5unit of Super-Therm Taq DNA polymerase (Southern Cross Biotechnology, Cape Town, South Africa). PCR was performed on a GeneAmp PCR system 2400 (Applied Biosystems, Foster City, CA). We tested different amounts of RNA (0.1, 0.5, 1 and 2μg) and different cycle numbers (21, 23, 25, 27, 29, 31, 33 and 35 cycles) in our preliminary trials to avoid obtaining PCR products at the plateau phase. Up to 33 cycles were performed in each experiment to avoid reaching the PCR plateau values. The PCR products were analyzed using 1.5% agarose gel electrophoresis with direct visualization after SYBR Green I (Cambrex Bio Science Rockland Inc., Rockland, ME) staining. The agarose gels were scanned and analyzed using the Kodak Scientific 1D Imaging System (Eastman Kodak Company, New Haven, CT). The accuracy of the amplification reaction for each set of primers was determined by amplifying several dilutions of the same cDNA with the same cycling profiles and amplifying the same cDNA dilution with different cycling profiles. The specificity of the cDNA was also checked using DNA sequence analysis (data not shown).

2.5 Oligonucleotide synthesis

The primers used in RT–PCR were as follows: Elk-1, PKCα, PKCδ, PKCε, PKCι and β2-MG as internal control, described previously by Hsieh et al. (2006).

2.6 Western blotting

Cultured cells were washed twice with PBS and lysed with buffer (50mM Tris–HCl (pH 7.4), 2mM EDTA, 2mM EGTA, 150mM NaCl, 1mM PMSF, 10μg/ml leupeptin, 10μg/ml aprotinin, 10μg/ml trypsin inhibitor, 1mM NaF, 1mM sodium orthovanadate, 1% (v/v) 2-mercaptoethanol, 1% (v/v) Nonidet P40, 0.3% sodium deoxycholate). The lysates were centrifuged at 100,000×g and 4°C for 30min. The supernatant was collected and its protein concentration was determined using the Bradford method. Each sample (40μg) was separated using SDS–PAGE in 10% (w/v) gel and electrophoretically transferred on o a PVDF membrane (Millipore, Belford, MA). The membrane was blocked with 5% (w/v) non-fat dried milk in TBST buffer (20mM Tris–HCl (pH 7.4), 150mM NaCl, 0.1% (v/v) Tween 20) and incubated with the specific anti-Elk-1 (1:500) or α-tubulin (1:3000) antibody at 4°C overnight. After washing with TBST, the blots were incubated with HRP-conjugated anti-mouse antibody (1:3000) at room temperature for 1h and washed with TBST before visualized using chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ). Photographs were used to measure the Elk-1 protein bands by densitometry (Kodak Scientific 1D Imaging System; Eastman Kodak). The relative protein density (Dp) was calculated using a formula: Dp=protein density/α-tubulin protein density. α-Tubulin was considered an internal control.

2.7 Cell viability assay

Cell growth was determined by the yellow tetrazolium MTT assay (Sobottka and Berger, 1992). To evaluate the effects of antisense ODN Elk-1 on the growth of SK-Hep-1 cell lines, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay was carried out. Briefly, 2×104 cells were seeded in 24-well microplates and cultured for 24h. Antisense or sense ODN Elk-1 was added to the wells to the indicated concentrations. The cells were cultured for another 24 and 48h and the medium in each well was replaced with 1ml of fresh medium containing 5mg/ml MTT. After an additional incubation for 4h, the medium was discarded and 1ml isopropanol was added to each well to dissolve the formazan crystals. The optical density was read with an automated microplate reader at 570nm. Experiments were carried out in triplicate, and the results expressed as mean±S.D. of 3 independent experiments.

2.8 Flow cytometric analysis

Cell growth was determined by flow cytometry (Detjen et al., 2000). Cells were treated with sense or antisense ODN (5μM) for 48h and aliquots of 106 cells were fixed in 70% ethanol at -20°C overnight. The fixed cells were washed with PBS and incubated in PBS containing 100mg/ml RNase A, 0.1% Triton X-100, 1mM EDTA and 1.5mg/ml propidium iodide (Sigma) at room temperature for 30min. Cell-cycle analysis was performed on the FACSCalibur flow cytometer and the Cellquest software (Becton Dickinson, San Jose, CA).

2.9 DAPI stain

Nuclear morphology of apoptosis was assessed by staining with DAPI and cells with condensed was recognized as apoptotic cells by fluorescence microscopy. SK-Hep-1 cells were treated with sense or antisense ODN (5μM) for 48h and fixed with paraformaldehyde (4%) at room temperature for 30min. After fixation, dishes were washed twice with PBS, and incubated at room temperature with 4,6-diamino-2-phenylindole (DAPI) for 15min and washed twice with PBS. Apoptotic cells were determined by the morphological changes after staining DAPI under fluorescence microscopic (DAS Microscope LEICA DAR, 100×).

2.10 Tumorigenicity assay in nude mice

Female BALB/c nude mice, 4–6weeks of age, nude mice were purchased from the National Health Research Institute (Taipei, Taiwan), housed in a dedicated nude mouse facility with microisolator caging. The SK-Hep-1 cells were treated with 5μM antisense or sense ODN Elk-1, and were detached by trypsinization 48h later, washed three times in serum-free DMEM, and 1×107 cells in 100μl volume were injected subcutaneously into the right posterior flank of mice with a 1ml syringe with a 24-gauge needle (Takei et al., 2001). Five mice were randomly assigned to a each treatment group: sense ODN Elk-1 (5μM, 0.1ml/mouse), S-Elk-1; antisense ODN Elk-1 (5μM, 0.1ml/mouse), AS-Elk-1. The experiment was repeated twice. Tumor volume was calculated using the formula: 0.5236×L1(L2)2, where L1 is the long diameter, and L2 the short diameter (Singh et al., 2002). The inhibitory rate of tumor growth was calculated by the formula: (tumor volumeS-Elk-1tumor volumeASElk-1/tumor volumeS-Elk-1)×100%.

2.11 Statistical analysis

The data were expressed as mean±standard deviations and were compared using Student's t-test.

3 Results

3.1 Effect of antisense ODN Elk-1 on the Elk-1 mRNA level and protein level

To verify the effectiveness of antisense ODN in depleting the expression of Elk-1 mRNA, we transfected 0–5μM sense or antisense ODN against Elk-1. Elk-1 mRNA expression was analyzed by semiquantitative RT–PCR. Elk-1 mRNA was downregulated according to the increase in antisense ODN concentration, although sense ODN did not show any effect on the Elk-1 mRNA level (Fig. 1A). The antisense ODN Elk-1 at 5μM suppressed Elk-1 production by SK-Hep-1 cells to 8% of the controls at 0μM dosage. β2-MG did not show any change in SK-Hep-1 cells treated with sense or antisense ODN. These altered patterns of the mRNA expressions were consistent with the protein expression levels determined using Western blotting (Fig. 1B).


Fig. 1

Effect of antisense ODN Elk-1 on the mRNA expression and cell growth. (A) Effect of antisense ODN Elk-1 (AS-Elk-1) or sense ODN Elk-1 (S-Elk-1) on the levels of Elk-1 mRNA detected using semiquantitative RT–PCR in SK-Hep-1 cells on day 2 after treatment as described in Section 2. The quantitative analysis of the semiquantitative RT–PCR was performed. (B) Effect of antisense ODN on the levels of Elk-1 protein detected using western blotting in SK-Hep-1 cells on day 2 after treatment as described in Section 2. The quantitative analysis of the Western blotting was performed. The data represent one of 3 independent experiments with similar results. (C) Effect of antisense ODN Elk-1 on the levels of PKC isoforms mRNA detected using semiquantitative RT–PCR in SK-Hep-1 cells on day 2 after treatment as described in Section 2. The data represent one of 3 independent experiments with similar results. (D) Effect of antisense ODN Elk-1 on cell growth. The SK-Hep-1 cell was transfected with the indicated dose of antisense or sense ODN Elk-1. Cell growth was determined using the MTT assay 48h after treatment as described in Section 2. Absorbance values obtained from untreated cells on day 0 after treatment were taken as 100%. The untreated group was designed as control. Data are presented as means±S.E. of 3 replicates from 2 independent experiments. *P<0.05 versus control; **P<0.01 versus control.


3.2 Effect of antisense ODN Elk-1 on the mRNA levels of PKC isoforms

To confirm that Elk-1 could involve in the expression of PKCα mRNA in SK-Hep-1 cells, we transfected with 0–5μM sense or antisense ODN against Elk-1. The mRNA expressions of PKC isoforms were analyzed using semiquantitative RT–PCR. PKCα mRNA downregulated in SK-Hep-1 cells treated with antisense ODN Elk-1, but was not downregulated in SK-Hep-1 cells treated with sense ODN (Fig. 1C). The antisense ODN Elk-1 at 5μM suppressed PKCα production by SK-Hep-1 cells to 1% of the control value. The antisense ODN Elk-1 did not inhibit the other PKC isoforms. β2-MG did not show any change in SK-Hep-1 cells treated with sense or antisense ODN.

3.3 Effect of antisense ODN Elk-1 on cell growth

It has been demonstrated that antisense ODN Elk-1 specifically inhibited Elk-1 mRNA expression, resulting in lower levels of Elk-1 within treated tumor cells. To determine whether such antisense ODN Elk-1 mediated inhibition of Elk-1 resulted in changes in cell growth, the effects of such treatment on in vitro tumor cell growth were examined. Antisense ODN Elk-1 showed a dose-dependent reduction in cell growth compared to cells treated with sense ODN Elk-1 in SK-Hep-1 cells and cell doubling time increased from 105.6% of the control for 1μM antisense ODN to 170.5% for 2μM antisense ODN. Moreover, cell growth was completely inhibited by 5μM antisense ODN (Fig. 1D).

To determine whether antisense ODN Elk-1 affected cell growth by blocking cell proliferation or inducing cell apoptosis, we observed both the morphological change and apoptotic cells in SK-Hep-1 cells in the treated with sense or antisense ODN (5μM) Elk-1 for 48h. No condensed and fragmented nuclei were found in DAPI staining (Fig. 2A). In addition, flow cytometric analysis was also used to quantify apoptosis of cells treated with sense or antisense ODN (5μM) Elk-1 for 48h. In SK-Hep-1 cells, the G0/G1 phase proportion increased from 59% in the control to 73% in the antisense ODN Elk-1-treated group (Fig. 2B). The S-phase proportion decreased from 21% in the control group to 14% in the antisense ODN Elk-1-treated cells (Fig. 2B). These results indicate that antisense ODN Elk-1 suppresses cell growth, at least in part, by inhibiting the G1→S transition in the cell cycle, not induced apoptosis.


Fig. 2

Antisense ODN Elk-1 effect on cell cycle arrest of SK-Hep-1 cells, not induced apoptosis. (A) Effects of antisense ODN Elk-1 on nuclear morphology. The SK-Hep-1 cells were treated with 5μM Elk-1 antisense ODN (AS-Elk-1) or 5μM Elk-1 sense ODN (S-Elk-1) for 48h with ×100 magnification. Nuclear morphological changes were analyzed after DAPI staining and observed by fluorescence microscopy as described in Section 2. (B) Flow cytometry of cell was treated with 5μM Elk-1 antisense ODN (AS-Elk-1) or 5μM Elk-1 sense ODN (S-Elk-1) and assessed for their distribution in the cell cycle as described in Section 2. (C) The untreated cultures were designated as controls. Values are means±S.E. of the percentage of cells in the G1 (open bars), S (closed bars) and G2/M (hatched bars) phases of the cell cycle from three independent experiments. **P<0.01 versus control.


3.4 Effect of antisense ODN Elk-1 on the tumorigenicity of SK-Hep-1 cells

Because antisense ODN Elk-1 specifically inhibited Elk-1 in SK-Hep-1 cells and inhibited their growth, we further examined the effects of sense or antisense ODN Elk-1 on tumor formation using these cells. This was performed by injecting 5×106 sense or antisense ODN Elk-1 pretreated SK-Hep-1 cells into nude mice and monitored the mice for tumor morphological and growth. The sense ODN Elk-1 pretreated cells gave rapid formation and growth of tumors (Fig. 3A–C), like the untreated (control) cells (data not shown). In contrast, mice injected with antisense ODN Elk-1 pretreated cells developed slow-growing tumors. The maximum inhibitory rate of tumor growth was 80.8±12.6% (n=5) and the mean of inhibitory rate of tumor growth from 28days to 56days was 74.4±8.6%. The tumor formation time was prolonged from 13days to 25days, and showed a significant difference (P<0.01) between the antisense-treated group (25.3±4.2days) and the sense-treated group (13.2±2.4days).


Fig. 3

Suppression of tumorigenesis in antisense ODN-transfected cells. (A) Tumor growth curve in S-Elk-1 (5μM; ■) or AS-Elk-1 (5μM; ●) transfected human SK-Hep-1 xenografts. A total of 5×106 sense or antisense ODN Elk-1 pretreated cells were injected into the flank region of nude mice. Tumor development was followed for up to 2months, when the mice were euthanized. Cross-sectional tumor diameters were measured externally, and the approximate tumor volume was calculated as described in Section 2. The results represent the mean±S.D. of 5 developed tumors in the experimental mice. **P<0.01 versus S-Elk-1 pretreated group. (B) The visible tumor formed by the indicated pretreated cells on day 56. (C) Photographs of the representative tumors removed from mice on day 56.


4 Discussion

We have used the antisense ODN technique to specifically knock-out Elk-1 expression in HCC cells. The antisense ODN Elk-1 produced efficient and specific downregulation of Elk-1 mRNA. Hsieh et al. (2006) showed a significant reduction in PKCα mRNA expression occurred. Significantly, antisense ODN Elk-1-mediated knock-out expression of Elk-1, inhibited cellular proliferation and tumor formation in nude mice. These studies suggested a novel role of Elk-1 in tumorigenesis regulation in HCC cells.

Elk-1 is known as a multifunctional protein. Recent data obtained from mice experiments have begun to provide in vivo evidence of the role of Elk-1, including the role of Elk-1 in brain development (Cesari et al., 2004). It has also been reported that nerve growth factor (NGF) induces neuronal differentiation of rat pheochromocytoma PC12 cells through induction of the brain-special short isoform of Elk-1. This isoform of Elk (sElk-1) plays an opposite role to the wild-type of Elk-1 in neuronal cell differentiation and proliferation (Vanhoutte et al., 2001). In contrast, Shao et al. (1998) reported that, when cotreated with calcium ionophore, constitutive expression of Elk-1 triggered apoptosis in Rat-1 fibroblasts and MCF-7 human breast cancer cells. It may be either activating death-inducing genes such as fos (Preston et al., 1996), Bad, Bax, Bak, etc. or repressing death-inhibiting genes, such as Bcl-2 (Oltvai and Korsmeyer, 1994), Mcl-1, Al, Bag-1, etc. leading to apoptosis. Recently, the SRF gene has also been identified as a target for Elk-1, thereby providing a positive-feedback loop where Elk-1 activation leads to enhanced expression of its partner protein, SRF (Kasza et al., 2005). Although Elk-1 is considered as a transcriptional regulator and to be involved in tumor progression (Chai et al., 2001; Cesari et al., 2004; Chen et al., 2006; Xiao et al., 2002), little information is available concerning its involvement in HCC tumorigenesis. Our data is the first to suggest that Elk-1 may be involved in the tumorigenesis of human HCC.

The antisense strategies were employed in a variety of eukaryotic systems both to understand normal gene function and to block gene expression therapeutically in vitro (Agrawal, 1992). For example, the antisense oligonucleotides for the c-myc, myb and mdm2 oncogenes or bcl-2 and bcl-xL anti-apoptotic genes have been used experimentally in a variety of tumors (Agarwal and Gewirtz, 1999). So far, however, no satisfactory results in cancer treatment have been obtained by the in vivo use of antisense oligonucleotides due to their fragility and toxicity, although many successful examples in vitro have been reported. We have demonstrated that human HCC cells can be successfully treated using liposome-mediated antisense ODN Elk-1. In addition, the incomplete delivery of the antisense ODN Elk-1 might be responsible for the lack of a complete anticancer effect. Although we show tumor inhibition, we did not observe tumor regression. One explanation for this fact is that non-transfected cells can still release Elk-1 and induce tumorigenesis. In the future, transfecting liposome/vector complexes and multiple injection of the complex into the tumor may be necessary to improve the delivery of the antisense molecules and either induce tumor regression or inhibit tumor growth for a long period of time. However, our results demonstrate for the first time a role for Elk-1 anti-tumorigenesis effect of antisense ODN Elk-1 in human HCC, and further study on Elk-1 may provide insight into the mechanisms of tumorigenic of HCC.

Acknowledgments

We thank Dr. Jaw-Ji Yang for his valuable comments and suggestions.

References

Aden DP, Fogel, A, Plotkin, S. Controlled synthesis of HBsAg in a differentiated human liver carcinoma-derived cell line. Nature 1979:282:615-6
Crossref   Medline   1st Citation  

Agrawal S. Antisense oligonucleotides as antiviral agents. Trends Biotechnol 1992:10:152-8
Crossref   Medline   1st Citation  

Agarwal N, Gewirtz, AM. Oligonucleotide therapeutics for hematologic disorders. Biochim Biophys Acta 1999:1489:85-96
Medline   1st Citation  

Behrens P, Rothe, M, Florin, A, Wellmann, A, Wernert, N. Invasive properties of serous human epithelial ovarian tumors are related to Ets-1, MMP-1 and MMP-9 expression. Int J Mol Med 2001:8:149-54
Medline   1st Citation  

Bourguignon LY, Gilad, E, Rothman, K, Peyrollier, K. Hyaluronan-CD44 interaction with IQGAP1 promotes Cdc42 and ERK signaling, leading to actin binding, Elk-1/estrogen receptor transcriptional activation, and ovarian cancer progression. J Biol Chem 2005:280:11961-72
Crossref   Medline   1st Citation  

Cesari F, Brecht, S, Vintersten, K, Vuong, LG, Hofmann, M, Klingel, K. Mice deficient for the ets transcription factor elk-1 show normal immune responses and mildly impaired neuronal gene activation. Mol Cell Biol 2004:24:294-305
Crossref   Medline   1st Citation   2nd   3rd  

Chai Y, Chipitsyna, G, Cui, J, Liao, B, Liu, S, Aysola, K. c-Fos oncogene regulator Elk-1 interacts with BRCA1 splice variants BRCA1a/1b and enhances BRCA1a/1b-mediated growth suppression in breast cancer cells. Oncogene 2001:20:1357-67
Crossref   Medline   1st Citation  

Chen AG, Yu, ZC, Yu, XF, Cao, WF, Ding, F, Liu, ZH. Overexpression of Ets-like protein 1 in human esophageal squamous cell carcinoma. World J Gastroenterol 2006:12:7859-63
Medline   1st Citation   2nd  

Chomczynski P, Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987:162:156-9
Medline   1st Citation  

De Petro G, Tavian, D, Copeta, A, Portolani, N, Giulini, SM, Barlati, S. Expression of urokinase-type plasminogen activator (u-PA), u-PA receptor, and tissue-type PA messenger RNAs in human hepatocellular carcinoma. Cancer Res 1998:58:2234-9
Medline   1st Citation  

Detjen KM, Brembeck, FH, Welzel, M, Kaiser, A, Haller, H, Wiedenmann, B. Activation of protein kinase Calpha inhibits growth of pancreatic cancer cells via p21(cip)-mediated G(1) arrest. J Cell Sci 2000:113:3025-35
Medline   1st Citation  

Hsieh YH, Wu, TT, Tsai, JH, Huang, CY, Hsieh, YS, Liu, JY. PKC alpha expression regulated by MZF and Elk-1 in human HCC cells. Biochem Biophys Res Commun 2006:339:217-25
Crossref   Medline   1st Citation   2nd   3rd   4th   5th  

Kasza A, O'Donnell, A, Gascoigne, K, Zeef, LA, Hayes, A, Sharrocks, AD. The ETS domain transcription factor Elk-1 regulates the expression of its partner protein, SRF. J Biol Chem 2005:280:1149-55
Crossref   Medline   1st Citation  

Katayama S, Nakayama, T, Ito, M, Naito, S, Sekine, I. Expression of the ets-1 proto-oncogene in human breast carcinoma: differential expression with histological grading and growth pattern. Histol Histopathol 2005:20:119-26
Medline   1st Citation  

Khurana A, Dey, CS. Involvement of Elk-1 in L6E9 skeletal muscle differentiation. FEBS Lett 2002:527:119-24
Crossref   Medline   1st Citation  

Nakayama T, Ito, M, Ohtsuru, A, Naito, S, Sekine, I. Expression of the ets-1 proto-oncogene in human colorectal carcinoma. Mod Pathol 2001:14:415-22
Crossref   Medline   1st Citation  

Oikawa T. ETS transcription factors: possible targets for cancer therapy. Cancer Sci 2004:95:626-33
Crossref   Medline   1st Citation  

Oltvai AN, Korsmeyer, SJ. Checkpoints of dueling dimers foil death wishes. Cell 1994:79:189-92
Crossref   Medline   1st Citation  

Preston GA, Lyon, TT, Yin, Y, Lang, JE, Solomon, G, Annab, L. Induction of apoptosis by c-Fos protein. Mol Cell Biol 1996:16:211-8
Medline   1st Citation  

Price MA, Rogers, AE, Treisman, R. Comparative analysis of the ternary complex factors Elk-1, SAP-1a and SAP-2 (ERP/NET). EMBO J 1995:14:2589-601
Medline   1st Citation  

Rao VN, Huebner, K, Isobe, M, ar-Rushdi, A, Croce, CM, Reddy, ES. elk, tissue-specific ets-related genes on chromosomes X and 14 near translocation breakpoints. Science 1989:244:66-70
Crossref   Medline   1st Citation  

Seth A, Watson, DK. ETS transcription factors and their emerging roles in human cancer. Eur J Cancer 2005:41:2462-78
Medline   1st Citation  

Shao N, Chai, Y, Cui, JQ, Wang, N, Aysola, K, Reddy, ES. Induction of apoptosis by Elk-1 and deltaElk-1 proteins. Oncogene 1998:17:527-32
Crossref   Medline   1st Citation   2nd  

Shen L, Dean, NM, Glazer, RI. Induction of p53-dependent, insulin-like growth factor-binding protein-3-mediated apoptosis in glioblastoma multiforme cells by a protein kinase Calpha antisense oligonucleotide. Mol Pharmacol 1999:55:396-402
Medline   1st Citation  

Singh RP, Dhanalakshmi, S, Tyagi, AK, Chan, DC, Agarwal, C, Agarwal, R. Dietary feeding of silibinin inhibits advance human prostate carcinoma growth in athymic nude mice and increases plasma insulin-like growth factor-binding protein-3 levels. Cancer Res 2002:62:3063-9
Medline   1st Citation  

Sobottka SB, Berger, MR. Assessment of antineoplastic agents by MTT assay: partial underestimation of antiproliferative properties. Cancer Chemother Pharmacol 1992:30:385-93
Crossref   Medline   1st Citation  

Takei Y, Kadomatsu, K, Matsuo, S, Itoh, H, Nakazawa, K, Kubota, S. Antisense oligodeoxynucleotide targeted to midkine, a heparin-binding growth factor, suppresses tumorigenicity of mouse rectal carcinoma cells. Cancer Res 2001:61:8486-91
Medline   1st Citation  

Treisman R. The serum response element. Trends Biochem Sci 1992:17:423-6
Crossref   Medline   1st Citation  

Vanhoutte P, Nissen, JL, Brugg, B, Gaspera, BD, Besson, MJ, Hipskind, RA. Opposing roles of Elk-1 and its brain-specific isoform, short Elk-1, in nerve growth factor-induced PC12 differentiation. J Biol Chem 2001:276:5189-96
Crossref   Medline   1st Citation  

Wasylyk B, Hagman, J, Gutierrez-Hartmann, A. Ets transcription factors: nuclear effectors of the Ras-MAP-kinase signaling pathway. Trends Biochem Sci 1998:23:213-6
Crossref   Medline   1st Citation  

Xiao D, Qu, X, Weber, HC. GRP receptor-mediated immediate early gene expression and transcription factor Elk-1 activation in prostate cancer cells. Regul Pept 2002:109:141-8
Crossref   Medline   1st Citation   2nd  


Received 23 April 2007/3 August 2007; accepted 29 August 2007

doi:10.1016/j.cellbi.2007.08.027


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)