Original Article

Cancer Gene Therapy (2006) 13, 7–12. doi:10.1038/sj.cgt.7700869; published online 5 August 2005

Comparative studies of suppression of malignant cancer cell phenotype by antisense oligo DNA and small interfering RNA

N Hiroi1,2,3,4,6, A Funahashi1,2,3,4,7 and H Kitano1,2,3,4,5

  1. 1ERATO-SORST, Kitano Symbiotic Systems Project, Japan Science and Technology Agency, Shibuya-ku, Tokyo, Japan
  2. 2Faculty of Science and Technology, Keio University, Kohoku-ku, Yokohama, Japan
  3. 3School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan
  4. 4The Systems Biology Institute, Shibuya-ku, Tokyo, Japan
  5. 5Sony Computer Science Laboratories, Inc., Shinagawa, Tokyo, Japan

Correspondence: Dr N Hiroi, JST ERATO-SORST, Kitano Symbiotic Systems Project, 6-31-15 Jingumae, M-31 Suite 6A, Shibuya-ku, Tokyo 150-0001, Japan. E-mail: nhiroi@symbio.jst.go.jp

6Visiting research fellow of Keio University for the period 1 June 2002–31 March 2006.

7Visiting research fellow of Keio University for the period 1 January 2004–31 March 2006.

Received 9 June 2004; Revised 27 March 2005; Accepted 11 April 2005; Published online 5 August 2005.

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Abstract

One of the distinguishing features of malignant tumor cells is the ability to proliferate in an anchorage-independent manner; methods that effectively suppress this phenotype may be applicable to the therapeutic inhibition of the malignancy of cancers. Interfering RNA is a potentially powerful tool for cancer therapy because of its specificity of target selection and remarkably high efficiency in target mRNA suppression. We studied the use of two knockdown strategies, antisense oligo DNA (AS-ODN) and small interfering RNA (siRNA), and showed how the anchorage-independent proliferation of malignant cells could be blocked efficiently. Anchorage-independent proliferation of rat fibroblasts transformed with v-src was suppressed with only a single 1-muM dose of AS-ODN; similar suppression using siRNA required treatment with 1 nM siRNA every 12 h. With our experimental system, the molecular stability of AS-ODN allowed the use of a simple treatment regimen to control the amount of the target molecule, providing that the treatment dose was sufficiently high. In comparison, siRNA treatment was effective at lower doses, but more frequent treatment was necessary to achieve the same suppression of proliferation.

Keywords:

anchorage-independent growth, antisense oligo DNA, small interfering RNA

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Introduction

The antisense oligo DNA (AS-ODN) method is a key procedure for suppressing target molecules. The mechanism of inhibition by AS-ODN seems to involve several complicated steps, including inhibition of translation, splicing, and transport of the target mRNA and degradation of the DNA–RNA hybrid by RNase H.1 One advantage of this method is the simplicity of the knockdown procedure.2 In addition, because the stability of DNA as a chemical reagent further facilitates its use, several attempts have been made to overcome the rapid degradation of AS-ODN in the presence of serum, including chemical modification of the backbone, which results in enhanced resistance to nucleases and prolongs survival times.3 However, before we can use unmodified AS-ODN extensively in vivo, a number of problems must be addressed: (1) poor membrane transport; (2) nonspecific inhibition; and (3) cytotoxicity at high doses.2

RNA interference (RNAi) was discovered recently as a gene knockdown phenomenon in plant, nematode, Drosophila, protozoal, and mammalian cells that is induced by double-stranded RNA (dsRNA).4, 5, 6, 7, 8, 9, 10 The mechanism of RNAi is not fully understood, but recent genetic and biochemical studies have revealed that an RNase III-related enzyme, designated 'Dicer', digests dsRNA inside the cell into fragments of 21–23 nucleotides (nt) with 2-nt 3' overlaps.11 Subsequently, these small fragments, known as small interfering RNAs (siRNAs), are incorporated into the RNA-induced silencing complex.12, 13 The siRNA in the active complex acts as a guide to the cognate target RNA so that the target is recognized and cleaved by the complex.

The activity of siRNAs is high, even at low concentrations, and without apparent toxicity. Further, the siRNA method may suppress in vitro targets, including culture cells, more efficiently than does the AS-ODN method.14, 15 These data suggest that the siRNA method may require only small amounts of nucleotides in vivo for sufficient suppression of the target molecule for reversal of the malignant phenotype that is the cause of the disease. Although in vivo studies of the siRNA method have been performed,16, 17, 18, 19, 20, 21, 22 they have not addressed whether siRNA suppresses its target's phenotype more efficiently than does AS-ODN.

As anchorage-independent growth is critical for the malignancy of cancer cells,23 we sought to use an AS-ODN and various siRNAs to suppress the anchorage-independent proliferation ability of v-src-transfected 3Y1 rat fibroblasts. Comparison of the two methods revealed that a high concentration of AS-ODN was required to suppress the target molecule sufficiently to negate v-src-associated anchorage-independent proliferation of 3Y1 cells. Although siRNAs blocked cell growth at concentrations lower than the effective doses of AS-ODN, frequent retreatment with siRNA was necessary to successfully suppress anchorage-independent proliferation, whereas a single treatment with AS-ODN was sufficient.

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Materials and methods

Oligo DNA design

The AS-ODN used was the reverse sequence of 20 bases of Rous sarcoma virus phosphoprotein p60 (src) gene (GenBank accession no. M84475), beginning from the first Met of v-src: 5'-TTG CTC TTG TTG CTG CCC AT-3'. Sense (5'-ATG GGC AGC AAC AAG AGC AA-3') and randomized (5'-TGT CTG TGC TCT CTG ATC TC-3') oligo DNAs were used as controls. All oligo DNAs were synthesized as s-oligos by Qiagen K.K. (Tokyo, Japan).

siRNA synthesis and transfection

Templates for siRNA synthesis were designed on the basis of the v-src sequence such that the total length was 21 bases, which comprised the 19 nucleotides after an AA doublet, and the relative %GC of the template was 35–55%. The sequences used were: antisense325 (5'-AAC AAC ACG GAA GGT GAC TGG CCT GTC TC-3'), sense325 (5'-AA CCA GTC ACC TTC CGT GTT GTT CCT GTC TC-3'), antisense531 (5'-AAA AGG TGC CTA TTG CCT CTC CCT GTC TC-3'), sense531 (5'-AA GAG AGG CAA TAG GCA CCT TTT CCT GTC TC-3'), antisense805 (5'-AAG CTG GGG CAG GGC TAC TTT CCT GTC TC-3'), and sense805 (5'-AA AAA GTA GCC CTG CCC CAG CTT CCT GTC TC-3'). These target sequences were selected by following the Tuschl method.24 The specificity of each siRNA sequence was checked by BLAST search: one sequence of Yamazaki sarcoma virus was homologous with 325, and the others matched Rous sarcoma virus sequences only. Synthesis was performed using the Ambion Silencer siRNA Construction Kit (Ambion Inc., Austin, TX). After synthesis, sense and antisense oligos were annealed to generate siRNAs. For lipofection of the siRNAs, LipofectAMINE reagent (Invitrogen Corp., Carlsbad, CA) was used according to the manufacturer's instructions, with slight modification: we resuspended 1.14 nM siRNA in 88 mul of serum-free Dulbecco's modified essential medium (DMEM), then mixed in 12 mul of LipofectAMINE reagent. After incubation of the mixture at room temperature for 30 min, the cells were treated with the mixture of siRNA and LipofectAMINE reagent according to the manufacturer's instructions. After 48 h of incubation with the siRNAs, we checked the expression levels of the mRNA and protein of the target molecule.

Cell culture

3Y1 rat embryonal fibroblasts and v-src-transfected 3Y1 (SR3Y1) cells were cultured in DMEM supplemented with 10% fetal calf serum (FCS) and maintained in a 37°C incubator with 5% CO2 in air. For anchorage-independent growth, 4 times 104 cells were resuspended in 2 ml of 1.3% methylcellulose semisolid medium overlaid on a 0.5% agar base in a 6-cm dish. When the anchorage-independent cultures were established, 1 muM of antisense, sense, or random oligo DNA was added to the dish or 1, 10, 100, or 1000 nM siRNA was added every 12 h from culture initiation. To induce malignant morphology, 50 nM of epidermal growth factor (EGF) and 10% FCS were added. Cell morphology was observed 48 h after initiation of semisolid medium culture.

Northern blotting

Cells were harvested in GTC solution (4.0 M guanidine tricyanate, 25 mM sodium citrate, 0.5% N-lauroylsarcosine (Sarkosyl)) after removal of the growth medium. RNA was separated on 1% agarose and subsequently transferred to a nylon filter (Millipore Corporation, Billerica, MA) by using 20 times SSC (3 M NaCl, 0.3 M sodium citrate (pH 7.0)). After transfer, the RNA was UV crosslinked, and the membrane was baked for 2 h at 80°C. For a v-src RNA (target molecule) probe, a cDNA fragment was generated by PCR using the forward primer 5'-GCC CTG CTA CCT ACC AGC CA-3' and reverse primer 5'-ACG AGG AAG GTC CCT CTG GG-3'. For a 28S ribosomal RNA (control) probe, a cDNA fragment was generated using the forward primer 5'-TGG TTC CCT CCG AAG TTT C-3' (nt 1635–1653 of GenBank accession no. M11167) and the reverse primer 5'-CGG ATT CCG ACT TCC ATG-3' (nt 1973–1956). Probes were labeled with biotin by using an Atlas SpotLight Labeling Kit (BD Biosciences Clontech, Franklin Lakes, NJ).

Western blotting

In preparation for Western blotting, 5 times 105 cells were lysed in 100 mul of radioimmunoprecipitation (RIPA) buffer (150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl (pH 7.5), 0.1 mM Na-orthovanadate, 0.1 mM NaF, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride, 1 mug/ml pepstatin, 1 mug/ml leupeptin, and 1 mug/ml aprotinin). After a 10-min incubation on ice, lysed cells were centrifuged at 20 000 g for 10 min at 4°C; after adjustment of the protein concentration, the supernatants were used for Western blotting. The proteins in SDS loading buffer (2% SDS, 10% glycerol, 60 mM Tris-HCl, 100 mM DTT, and 0.001% bromophenol blue) were boiled for 5 min, separated by SDS–polyacrylamide gel electrophoresis (10% polyacrylamide gels), and blotted onto Immobilon-P membrane (Millipore Corporation). The filters were blocked with 5% skim milk in TBS-T (150 mM NaCl, 20 mM Tris-HCl (pH 7.6), 0.1% Tween-20) for 100 min and incubated with primary antibodies (diluted 1:1000 to 1:2000 with 5% skim milk in TBS-T) for 1 h at room temperature. The filters were then washed, incubated for 1 h at room temperature with the secondary antibody (sheep anti-mouse or donkey anti-rabbit) conjugated with horseradish peroxidase (Amersham Biosciences, Piscataway, NJ), and washed with TBS-T. Immunoblotted bands were detected by using the ECL system (Amersham Biosciences) with the same exposure time for all uses of a particular antibody.

Oligonucleotide degradation assay

AS-ODNs and siRNAs were incubated in culture medium for 48 h at 37°C. After ethanol precipitation, they were resuspended in loading buffer (0.21% bromophenol blue, 0.21% xylene cyanol FF, 0.2 M EDTA (pH 8.0), and 50% glycerol) and subjected to electrophoresis in a 3% agarose gel for 30 min at 50 mV.

Colony formation assay

Cells were treated with 1 nM siRNA for 4, 8, 12, 24, 36, 48, or 72 h. The colonies in approx9.8 cm2, which is 1/8 the area of a 10-cm dish, were then counted; in the absence of siRNA, >400 colonies (diameter, >50 mum) formed in an area of this size. The reciprocal of the colony count was plotted against the reciprocal of the interval time between successive siRNA treatments (i.e., 1/4, 1/8, 1/12, 1/24, 1/36, 1/48, and 1/72). This assay was repeated three times.

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Results

AS-ODN halted anchorage-independent cell growth

We treated control 3Y1 cells and v-src-transfected SR3Y1 cells with antisense and control oligonucleotides. None of these DNA oligonucleotides impaired the proliferation of normal and transfected cells growing as monolayers (data not shown). Although 3Y1 cells could proliferate in semisolid medium (Figure 1, panel 3Y1), SR3Y1 cells could make foci in this medium (Figure 1, panel N). However, treatment with 1 muM AS-ODN abolished this anchorage-independent growth (Figure 1, panel AS). The control oligonucleotides lacked this suppressive ability (Figure 1, panels S and R). These results suggest that an AS-ODN can act as a silencer of v-src mRNA and its malignant products. Further, Northern and Western blot analyses indicated that only AS-ODN reduced the src mRNA level and amount of active Src protein (Figure 2a and b). These results suggest that AS-ODN suppressed the anchorage-independent growth of SR3Y1 by downregulating src mRNA and active Src protein levels.

Figure 1.
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Morphologic change after treatment with 1-muM antisense oligo DNA (AS-ODN) in semisolid culture medium. 3Y1: untransformed 3Y1 cells, serum and epidermal growth factor added to medium (S+E); N: v-src-transformed SR3Y1 cells+S+E; AS: v-src-transformed SR3Y1 cells+S+E+AS-ODN; S: v-src-transformed SR3Y1 cells+S+E+sense ODN; and R: v-src-transformed SR3Y1 cells+S+E+random ODN. Magnification, times 1 (left panels); times 100 (right panels).

Full figure and legend (54K)

Figure 2.
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The amounts of v-src mRNA and Src protein were reduced by treatment with antisense oligo DNA (AS-ODN). (a) Northern blot analysis for v-src mRNA. (b) Western blot analysis for v-Src. Control 3Y1 cells (untransfected by v-src) were loaded into the leftmost lane of each gel. SR3Y1 cells (v-src transfected) were loaded into the other lanes. N: no treatment; A: treated with AS-ODN; S: treated with sense ODN; R: treated with random ODN.

Full figure and legend (29K)

Single doses of siRNAs failed to halt anchorage-independent growth

To test the effect of siRNAs on anchorage-independent growth of SR3Y1, three different siRNAs (Figure 3a) were selected according to the Tuschl method.24 When added to the culture medium as single doses of 1 muM (as done with AS-ODN), none of these siRNAs could suppress anchorage-independent growth of the SR3Y1 cells (Figure 3a–c).

Figure 3.
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A single 1-muM dose of small interfering RNA (siRNA) failed to decrease v-src mRNA and protein levels. (a, b) Control 3Y1 cells (untransfected by v-src) were loaded into the leftmost lane. SR3Y1 cells (v-src transfected) were loaded into the other lanes. N: no treatment; 325, 531, and 805: treated with each respective siRNA. (a) Northern blot analysis. (b) Western blot analysis for v-Src. (c) Morphology of SR3Y1 cells treated with 1 muM of each siRNA. Magnification, times 1 (left panels); times 100 (right panels).

Full figure and legend (86K)

Lipofection reagent did not improve the efficiency of siRNAs

A possible reason for the preceding results is that the siRNAs could not cross the cell membrane by themselves. To test this possibility, a lipofection reagent was added with each siRNA. However, addition of the lipofection reagent failed to adequately enhance the ability of the siRNAs to suppress anchorage-independent growth of SR3Y1 cells although adding lipofection reagent together with siRNA 325 or 531 seemed to affect the level of expression of v-Src protein slightly (Figure 4a–c).

Figure 4.
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Lipofection reagent did not increase the efficiency of small interfering RNAs (siRNAs). (a, b). Control 3Y1 cells (untransfected by v-src) were loaded into the leftmost lane. SR3Y1 cells (v-src transfected) were loaded into the other lanes. N: no treatment; 325, 531, and 805: treated with each respective siRNA and lipofection reagent. (a) Northern blot analysis. (b) Western blot analysis for v-Src. (c) Morphologic change in SR3Y1 cells treated with 1 muM of each siRNA. Magnification, times 1 (left panels); times 100 (right panels).

Full figure and legend (72K)

Repeated dosing of siRNAs inhibited anchorage-independent growth

Another possible explanation for the failure of siRNAs to halt anchorage-independent growth of SR3Y1 cells is that the siRNAs were degraded so quickly that they failed to reach effective levels in the cells. To compare the degradation rates of the siRNAs and AS-ODN, they were incubated at 37°C for 48 h and electrophoresed (Figure 5a). The results showed that the siRNAs were degraded more quickly than was the AS-ODN.

Figure 5.
Figure 5 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Repeated treatment with small interfering RNAs (siRNAs) decreased v-src mRNA and Src protein levels. (a) Comparison of degradation of antisense oligo DNA (AS-ODN) and siRNAs in culture medium. Lanes: A, AS-ODN; S, sense ODN; R, random ODN; 325, 531, or 805: loaded with each respective siRNA. Changes that occurred within 48 h after initiation of incubation at 37°C are shown. (b, c) Control 3Y1 cells (untransfected by v-src) were loaded into the leftmost lane. SR3Y1 cells (v-src transfected) were loaded into the other lanes. N: no treatment; 325, 531, and 805: treated with each respective siRNA. (b) Northern blot analysis for mRNA of v-src. (c) Western blot analysis for v-Src. (d) Morphological changes in SR3Y1 cells given repeated treatment with 1 nM of each siRNA every 12 h for 48 h. Magnification, times 1 (left panels); times 100 (right panels).

Full figure and legend (76K)

These results suggest that if cells can be retreated with an siRNA before it is degraded completely, its concentration may exceed the threshold necessary for suppression of its target. Treatment every 12 h with 100, 10, or 1 nM of each siRNA (i.e., 1/10, 1/100, or 1/1000 of the AS-ODN effective concentration) effectively and equivalently suppressed anchorage-independent growth of SR3Y1 cells (data not shown and Figure 5b–d). These results suggest that maintenance of the siRNA concentration is critical to the mRNA suppression mechanism; in the model system used, retreatment every 12 h with 1 nM siRNA was sufficient in light of the effect on the malignant morphology (Figure 6). Figure 6 shows the relationship between the reciprocal of the colony count against the reciprocal of the time interval between sequential treatments. These results show that the efficiency of the sequential treatments increased remarkably when the time interval between treatments decreased from 24 to 12 h. However, decreasing the interval to <12 h did not appreciably further suppress colony formation.

Figure 6.
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Correlation between treatment times and efficiency of siRNAs. Vertical axis shows 1/number of colonies', and horizontal axis shows 1/treatment interval (h) (i.e., 1/4, 1/8, 1/12, 1/24, 1/36, 1/48, and 1/72). This assay was repeated three times.

Full figure and legend (9K)

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Discussion

To determine potentially effective sequences of siRNA, we followed a slightly modified version of the Tuschl method.24 Unfortunately, only three of the 10 siRNAs synthesized had an appreciable effect on the target phenotype. The development of an improved algorithm for predicting effective siRNA sequences is essential to overcome this problem.

Lipofection of siRNAs given as single doses seemed to be at least marginally effective, particularly in that it slightly reduced v-Src protein levels after transduction of siRNA 325 or 531, compared with those in the absence of lipofection reagent. Regardless, this reduction in the amount of Src did not abolish the malignant phenotype of SR3Y1. In comparison, repeated treatment with each siRNA led to dramatic downregulation in the expression of v-src mRNA, protein, and the malignant phenotype. The fact that this strategy did not require transfection-enhancing products may offer two benefits in particular: (1) the single-step strategy extends the range of possible applications and (2) we can discard the need to consider the unknown side effects of another material (in this case, a transfection-enhancing product).

Compared with AS-ODNs, siRNAs may be therapeutically effective at much lower concentrations. However, if siRNAs are metabolized too rapidly, their therapeutic use becomes unrealistic. For example, in a previous study,20 siRNAs remained effective for 20 days, suggesting that their use is practicable when the disease can be overcome in less than or equal to20 days. The flexibility of sequential dosage with siRNA will probably enable treatment to be tailored to specific disease situations. In addition, the SR3Y1 cells used in the present experiments were derived by transfection of a constitutively expressed ectopic gene (v-src). This constant overexpression may mandate simple sequential retreatment with siRNAs for efficient results. In comparison, a physiological gene whose expression is controlled closely may require more strict regulation of the siRNA concentration and timing of treatment.

We first studied an overexpressed ectopic gene (v-src). However, this condition could skew our results regarding the need for frequent treatment with siRNA to knock down a target gene. To address a system more similar to the situation likely to be encountered during cancer therapy, we then attempted to knock down a constitutively expressed physiological gene (beta-actin). Unfortunately, the oligonucleotide reagents were toxic to the cells (data not shown). How the procedure should be changed if the target gene is a physiological one requires further investigation.

We cultured the cells for 48 or 72 h to observe their malignant morphology. The length of time required for observing the results of treatment with oligonucleotide reagents may be critical if frequent treatment is needed for knockdown of the target gene. When the target features could be suppressed in only a few hours, sequential dosage will probably not be necessary. We conclude that one of the causes of the difference in effectiveness of AS-ODN and siRNA is the difference in their stability in the culture medium, instead of within the cell. Our results show that when we select DNA oligomers, we can expose the cells to the oligomers for the time necessary to achieve downregulation of the target phenotype. In contrast, RNA oligomers are degraded before repression of the target phenotype occurs. When considering alternative treatment regimens, note in particular two of our findings: (1) addition of lipofection reagent ultimately had little effect on the target phenotype and (2) changing the siRNA concentration from 1 nM to 1 muM negligibly affected the end result. Together, these results suggest that only a small amount of oligonucleotide needs to cross the cell membrane at any particular time. Therefore, it seems that, like the knockdown mechanism of the siRNA in the cells, the target phenotype and source of the target are key factors in determining an efficient treatment strategy.

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Acknowledgements

We are grateful to Sayuko Kaminosono, Haruna Yamanami, and Kotaro Oka (Faculty of Science and Technology, Keio University, Kohoku-ku, Yokohama, Japan) for their kind help with the experiments and for providing the laboratory environment for this study. This study was supported by Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and from Keio University's 21st Century COE Program for Understanding and Control of Life Function via Systems Biology.