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Cell Biology International (2008) 32, 210216 (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
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 5
Keywords: Elk-1, Hepatocellular carcinoma, Protein kinase C alpha, Tumorigenesis, Antisense oligonucleotide.
*Corresponding author. Tel.: +886 4 2473 0022x11673; fax: +886 4 2324 8195.
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 100
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 24
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 (4
2.4 Reverse transcriptase (RT)-PCR
RT–PCR assay was a slightly modified method of De et al. (1998). An aliquot of total RNA (0.5
The RT product (2
2.5 Oligonucleotide synthesis
The primers used in RT–PCR were as follows: Elk-1, PKCα, PKCδ, PKCε, PKCι and β
2.6 Western blotting
Cultured cells were washed twice with PBS and lysed with buffer (50
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
2.8 Flow cytometric analysis
Cell growth was determined by flow cytometry (Detjen et al., 2000). Cells were treated with sense or antisense ODN (5
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 (5
2.10 Tumorigenicity assay in nude mice
Female BALB/c nude mice, 4–6
2.11 Statistical analysis
The data were expressed as mean
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 0–5
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
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 0–5
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 1
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 (5
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 5
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
Suppression of tumorigenesis in antisense ODN-transfected cells. (A) Tumor growth curve in S-Elk-1 (5
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.
We thank Dr. Jaw-Ji Yang for his valuable comments and suggestions.
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
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
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
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
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
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
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
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
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
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
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
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
Sobottka SB, Berger, MR. Assessment of antineoplastic agents by MTT assay: partial underestimation of antiproliferative properties. Cancer Chemother Pharmacol 1992:30:385-93
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
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
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
Received 23 April 2007/3 August 2007; accepted 29 August 2007doi:10.1016/j.cellbi.2007.08.027