Silencing of herg gene by shRNA inhibits SH-SY5Y cell growth in vitro and in vivo

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European Journal of Pharmacology 579 (2008) 50 – 57 www.elsevier.com/locate/ejphar

Silencing of herg gene by shRNA inhibits SH-SY5Y cell growth in vitro and in vivo ☆ Jie Zhao 1 , Xiao-Li Wei 1 , Yong-Sheng Jia, Jian-Quan Zheng ⁎ Beijing Institute of Pharmacology and Toxicology, Taiping Road 27, Beijing 100850, China Received 13 July 2007; received in revised form 21 September 2007; accepted 4 October 2007 Available online 13 October 2007

Abstract Overexpression of human ether-à-go-go (eag) related gene (herg) contributes to the progression and metastasis of a variety of tumors of different histogenesis, which implies that the herg gene could provide a promising target on tumor therapy. In the present study, plasmid-mediated expression of shRNA-herg1 and shRNA-herg1/1b was employed to silence the herg gene expression in human neuroblastoma SH-SY5Y cell lines. The inhibition of the target gene expression was confirmed by RT-PCR and Western blot. It was found that shRNA-herg1 or shRNA-herg1/ 1b depressed the cellular growth rate, inhibited cell viability and reduced colony formation of SH-SY5Y cells. The flow cytometry assay revealed that SH-SY5Y cells were retarded in G0–G1 after herg1 or herg1/1b gene was silenced by shRNA-herg1 or shRNA-herg1/1b. In vivo, intratumor injection of shRNA-herg1/1b inhibited the growth of SH-SY5Y tumors inoculated subcutaneously in nude mice. The result suggested that the herg played an important role in regulating the growth and proliferation of SH-SY5Y cells. The block of the HERG channel might be a potential therapeutic strategy for neuroblastoma and some other tumors with overexpression of the herg gene. © 2007 Published by Elsevier B.V. Keywords: RNA interference; herg; SH-SY5Y cell; Tumor

1. Introduction The human ether-à-go-go (eag) related gene (herg) belongs to an evolutionarily conserved multigenic family of voltageactivated K+ channels, the eag family (Warmke and Ganetzky, 1994). All members of the eag family consist of a pore-forming unit with six transmembrane spanning segments, which selectively conduct K+ across the cell membrane in the heart and nervous tissue (Curran et al., 1995; Sanguinetti et al., 1995; De Ponti et al., 2000, Gutman et al., 2005). The human ERG channel (HERG or Kv11.1) mediates the rapidly activated delayed rectifier K current (IKr), which makes a vital contribution to repolarization of the cardiac action potential. Dysfunction of HERG, either because of inherited mutations of the channel or block by any of a variety of medications, causes ☆ This study was supported by the National Natural Science Foundation of China (No. 30472019, 30500620). ⁎ Corresponding author. Tel.: +86 10 66931635; fax: +86 10 68211656. E-mail address: [email protected] (J.-Q. Zheng). 1 These authors contributed equally to this work.

0014-2999/$ - see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.ejphar.2007.10.008

long QT syndrome, which is characterized by abnormalities of repolarization that are predisposed to cardiac arrhythmias and an increased risk of sudden death (Sanguinetti et al., 1995; Trudeau et al., 1995; Keating and Sanguinetti, 2001; Sanguinetti and Mitcheson, 2005; Witchel, 2007). Furthermore, there is accumulating evidence that herg gene and HERG protein are overexpressed in many types of human cancers (Bianchi et al., 1998; Withcel, 2007), including endometrial (Cherubini et al., 2000) and colorectal adenocarcinomas (Lastraioli et al., 2004), as well as acute myeloid (Pillozzi et al., 2002) and lymphoid leukaemias (Smith et al., 2002), whereas it is not expressed in the corresponding normal cells or in benign neoplastic lesions such as endometrial hyperplasias (Cherubini et al., 2000) and most colorectal adenomas (Lastraioli et al., 2004). In mammals, the ERG subfamily comprises three genes erg1, erg2, and erg3 (herg1, herg2, and herg3 in humans), with the latter being specific to the nervous tissues (Shi et al., 1997). The herg gene subtypes preferentially expressed in a variety of tumor cell lines are herg1 gene and herg1b gene (a N-terminal truncated form of herg1 mRNA), which are absent from healthy cells from which the respective tumor cells were derived

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(Bianchi et al., 1998; Cherubini et al., 2000; Pillozzi et al., 2002; Crociani et al., 2003). Both full-length HERG1 and HERG1B proteins were co-expressed and could form heterotetrameric channels on the plasma membrane of tumor cells. Moreover, selective pharmacological blockage of the HERG channel dramatically impaired cell growth of HERG-bearing tumor cells (Pillozzi et al., 2002; Crociani et al., 2003). These studies suggested that modulated expression of the HERG channel might be the molecular basis of a novel mechanism regulating neoplastic cell proliferation. It is still unclear, however, how overexpression of this particular voltage-dependent K+ channel contributes to the neoplastic phenotype. One possibility is that the special properties of HERG channels contribute to maintaining a more depolarized membrane potential, thus permitting an easier passage through the cell mitotic cycle (Pardo et al., 2005; Lang et al., 2005). RNA interference (RNAi) was a recently discovered antiviral mechanism in plants and invertebrates induced by small doublestranded RNA (dsRNA), which will lead to sequence-specific gene silencing at the post-transcriptional level (Hannon, 2002). Short hairpin RNAs (shRNAs) driven by polymerase III promoters have been investigated as an alternative strategy to suppress gene expression more stably, and such constructs with well-defined initiation and termination sites have been used to produce various small dsRNA species that inhibit the expression of genes with diverse functions in mammalian cell lines (Lee et al., 2002). In the present study, the regulation of HERG K+ channels on the growth and proliferation of SH-SY5Y cells was studied by silencing the expression of herg1 and herg1b genes with RNAi technique. The therapeutic effect of shRNA-herg1 and shRNAherg1/1b on the inoculated neuroblastoma was investigated in vivo. 2. Materials and methods 2.1. Cell culture and transfection The human neuroblastoma SH-SY5Y cell lines (from the Cell Center at Peking Union Medical College) were used in the study. They were cultured in RPMI 1640 medium (SIGMA) containing 100 U/ml penicillin, 100 μg/ml streptomycin and 15% fetal calf serum (Hyclone) and incubated at 37 °C in a humidified atmosphere with 5% CO2 24 h before transfection. 3 × 105 cells were plated in a 6-well plate to reach a 50–70% confluency. Cells were transfected with Lipofectamine 2000 (Invitrogen, CA) according to the manufacturer's instructions.

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Briefly, for each well, 4 μg of the respective shRNA plasmid was incubated with 250 μl serum-free medium for 5 min. Subsequently, a mixture of 10 μl Lipofectamine 2000 and 250 μl serum-free medium which has been incubated for 5 min was slowly added. After incubation of 20 min at room temperature, 1 ml serum-free medium was added to the dilution, mixed again and 1.5 ml transfection mixture was added to each well. After being incubated at 37 °C for 4–6 h, the medium containing the transfection mix was replaced with the growth medium. Cells were continuously cultured until harvest for analysis. 2.2. Target sequence selection and plasmid vector construction The oligonucleotide sequences of two different hergshRNAs and a control shRNA are as shown in Table 1. The shRNA expression cassette contained 19 nucleotide (nt) of the target sequence followed by a loop sequence (9nt), the reverse complement to the 19nt, stop codon for U6 promoter and XbaI and BbsI sites. The shRNA expression cassette driven by U6 promoter was engineered into the XbaI and BbsI sites of plasmid mU6 pro vector (gift from Dr. Daoid L. Turner, University of Michigan, Mental Health Research Institute & Dept. of Biological chemistry), resulting in the following plasmids: mU6–shRNA-herg1, mU6–shRNA-herg1/1b and mU6–shRNA-control. The mU6–shRNA-control was designed as an unspecific (nonsilencing) shRNA which has a similar base composition to herg-shRNA but has no homology to herg or other known genes. To generate plasmids for cloning shRNAs, mU6 pro vector was linearized with XbaI and BbsI restriction endonuclease (Biolabs) and the annealed oligonucleotides were ligated with the linearized mU6 pro vector at XbaI-BbsI sites using T4 DNA ligase. All the constructed plasmids were confirmed by DNA sequencing. These oligonucleotide sequences encoding shRNAs for targeted gene herg1 and herg1b coding region were taken from GeneBank accession NM_000238 and AJ512214, and they were evaluated for sequence specificity by a BLAST search and did not show homology to other known genes. 2.3. RT-PCR analysis Total RNA was extracted from the cultured SH-SY5Y cells by Trizol Reagent (Invitrogen) according to the manufacturer's instructions. RNA concentration and quality were assessed spectrophotometrically at wavelengths 260 and 280 nm. RTPCR was carried out using a two-step RT-PCR kit (Promega

Table 1 The oligonucleotide sequences of shRNA driven by U6 promotor in mU6 pro vector shRNA

Forward strand

Reverse strand

shRNA-herg1 (1071–1088 nt)

5′-TTTGCCAGTGACCGTGAGATCATTTCAAGAGAATGATCTCACGGTCACTGGTTTTT-3′, 5′-TTTGCCACCTACGTCAATGCCAATTCAAGAGATTGGCATTGACGTAGGTGGTTTTT-3′ 5′-TTTGGAGCTATTGTCGACGCTTCTTCAAGAGAGAAGCGTCGACAATAGCTCTTTTT-3′

5′-CTAGAAAAACCAGTGACCGTGAGATCATTCTCTTGAAATGATCTCACGGTCACTGG-3′; 5′-CTAGAAAAACCACCTACGTCAATGCCAATCTCTTGAATTGGCATTGACGTAGGTGG-3′ 5′-CTAGAAAAAGAGCTATTGTCGACGCTTCTCTCTTGAAGAAGCGTCGACAATAGCTC-3′

shRNA-herg1/1b (1431–1449 nt) shRNA-control

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NO.A3500). cDNA was synthesized at 42 °C for 15 min, 95 °C for 5 min, 0 °C for 5 min, and PCR was performed as following: 95 °C 5 min, 94 °C 15 s, 62 °C 15 s, 72 °C 15 s for 30 cycles, 72 °C 10 min. The primers used for herg1 amplification were 5′-CCGTAAGTTCATCATCGCCAAC-3′ and 5′-CATCCTCAATTTCGAGGTGGTG-3 and the predicted product was 319 bp. The primers used for herg1/1b amplification were 5′CAGCGGCTGTACTCGGGCACAG-3′ and 5′-GAGTTCTCCGACCACTTCTG-3′ and the predicted product was 569 bp. As housekeeping gene, the primers used for 495 bp GAPDH1 amplification were 5′-GTCAACGGATTTGGTCGTATTG-3′ and 5′-AGTGATGGCATGGACTGTGGT-3′ and the primers used for 336 bp GAPDH2 amplification were 5′GATTTGGTCGTATTGGGGCGC-3′ and 5′-CAGAGATGACCCTTTTGGCTCC-3′. The PCR products were separated on 1% agarose gel. 2.4. Western blotting The levels of HERG1 and HERG1B protein expression were determined by western blot analysis. Briefly, the cell monolayer was washed three times with ice-cold phosphate-buffered salt solution (PBS) before collection by scraping and were lysed with RIPA buffer containing 50 mM Tris–HCl (pH 7.4), 1% NP-40, 0.25% sodium-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF and protease inhibitor mixture (phenylmethylsulfonyl fluoride 1 mM, aprotinin, leupeptin, pepstatin 1 mg/ml each) at 4 °C for 20 min. Cell debris was removed by centrifugation at 14,000 g for 30 min at 4 °C, and protein concentration was determined by the Bradford method with BSA as a standard. The protein samples (50 μg) were separated on 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membrane. After being blocked in TBST (25 mM Tris, pH7.5, 135 mM NaCl, 2.7 mM KCl, 0.05% Tween 20) containing 5% nonfat dry milk the membrane was incubated overnight at 4 °C with anti-HERG antibody (1:1000 dilution; Santa Cruz, sc-15968). After that, the membrane was washed

three times with TBS for 10 min each and incubated with antigoat IgG-horseradish peroxidase conjugate secondary antibody (1:500 dilution; Zhongshan Company, China) for 1 h at room temperature and developed with the enhanced chemiluminescence method (ECL; Santa cruz). Membranes probed for HERG were reprobed for β-actin (1:5000 dilution; Sigma) to normalize for loading. 2.5. Cell viability assay The effects of HERG1 and HERG1/1B RNA interference on the viability of SH-SY5Y cells were determined by 3-(4,5dimethylthia-zol,2-yl)-2,5-diphenyltetrazolium bromide (MTT) (AMRESCO) based colorimetric assay in three separate experiments performed in triplicates. 12 h after transfection, cells were seeded into the 96-well plate at a density of 1000 cells/well and cultured at 37 °C in a humidified atmosphere containing 5% CO2. 96 h later, the cells were incubated with 100 μg/well MTT solution. 4 h later, the medium was replaced with 150 μl dimethyl sulfoxide (DMSO) (Sigma) and vortexed for 10 min. Absorbance (A) was recorded at 490 nm using an automatic microwell plate reader (Bio-Rad 3350). Cell viability (%) was calculated as percentage of the untreated cells. 2.6. Cell growth curve 12 h after transfection, SH-SY5Y cells at a concentration of 1 × 104 per well were seeded into 24-well plate and further cultured at 37 °C in a humidified atmosphere containing 5% CO2 for 7 days. The total cell numbers were counted every 24 h with a hemocytometer. Experiments were performed in triplicate. Cell viability was assessed by using trypan blue. 2.7. Colony formation assay 12 h after transfection, a total of 100 cells were seeded into 60 mm Petri dishes separately and allowed to grow undisturbed for 10 days in complete culture medium at 37 °C incubator.

Fig. 1. RNAi reduces expression of herg1 and herg1b gene in SH-SY5Y cells. (A) RT-PCR analysis of herg1 mRNA levels in SH-SY5Y cells treated with shRNAherg1. (B) RT-PCR analysis of herg1 and herg1b gene mRNA levels in SH-SY5Y cells treated with shRNA-herg1/1b. (C) Western blot analysis of HERG1 and HERG1B protein levels in SH-SY5Y cells treated with shRNA-herg1 and shRNA1/1b respectively. GAPDH and β-actin were respectively employed as a loading control. RT-PCR cycle number was optimized in several experiments with determination of linear phase PCR reaction. The results shown are representative of three independent experiments. 1: untreated-control; 2: shRNA-control; 3: shRNA-herg1; 4: shRNA-herg1/1b.

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After that, cells were washed three times with phosphatebuffered saline (PBS), fixed in methanol for 15 min at room temperature and stained with Giemsa for 15 min, then washed with water and air-dried. Cell colonies were visualized. Experiments were performed in triplicate. 2.8. Flow cytometry analysis At 24, 36, 48 h post-transfection respectively, adherent cells were collected by trypsinization, washed in PBS and centrifugated at 500 g. Cells were resuspended at 1 × 106 cells/ml in PBS containing 5% fetal bovine serum and fixed in ice-cold ethanol overnight at 4 °C. Fixed cells were centrifugated and washed once with PBS. Each sample was resuspended in propidium iodide (PI) stain buffer (0.1% Triton X-100, 200 μg of DNase–free RNase A, 20 μg of PI) in PBS for 15–30 min at room temperature. After staining, 1 × 104 cells per sample were analyzed using a FACScan flow cytometer (Becton Dickinson; San José, CA, USA).

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cells and their effects on HERG mRNA levels were determined by comparison with the untreated cells by RT-PCR analysis. As shown in Fig. 1A and B, HERG mRNA expression was effectively suppressed by shRNA-herg1 and shRNA-herg1/1b, compared with nontransfected (untreated-control) cells, whereas shRNA-control transfection could not affect the HERG mRNA expression. In parallel, further western blot analysis showed that as compared to the scrambled shRNA-control transfected cells, shRNA-herg1 transfected cells indeed reduced HERG1 protein (135 kDa–155 kDa) expression, which was a full-length herg1 mRNA encoded protein, and shRNA-herg1/

2.9. Tumor xenografts in nude mice Male Balb/c nude mice of 6–8 weeks old were purchased from Beijing Animal Center, China. All animals were kept under specific pathogen-free conditions and tended to in accordance with institutional guidelines. Tumor was raised by subcutaneous injection of 1 × 106 SH-SY5Y cells/mouse in 200 μl culture medium without FBS into the flanks of the nude mice. The tumors were measured every 2 days with a caliper, and the diameters were recorded. When the tumor nodules reached 4 × 4 mm, the tumor-bearing mice were randomly divided into three groups (n = 6). Group 1 mice were used as normal saline controls. Group 2 received intra-tumor injections (50 μg/mouse) of shRNA-control weekly. Group 3 received intratumor injections with 50 μg shRNA-herg1/1b weekly as described for group 2. Tumor size was measured as described above. Tumor volume (cm3) was calculated by the formula: ab2 / 2, where a was the length and b was the width of the tumor. 2.10. Statistical analysis Data are expressed as means ± S.E.M. Statistical significance of differences was determined by analysis of variance (ANOVA), followed by Dunnett's t test for individual group comparison. A value of P b 0.05 was considered significant. 3. Results 3.1. Vector-mediated RNAi inhibits HERG expression Three shRNA-expressing plasmids (shRNA-herg1, shRNAherg1/1b, shRNA-control) were constructed using the mU6 pro vector. shRNA-herg1 was targeted to inhibit herg1 expression, shRNA-herg1/1b was to inhibit herg1 and herg1b expression, and shRNA-control, the scrambled version of siRNA, was not to affect herg1 or herg1b expression. These plasmids were respectively transfected into human neuroblastoma SH-SY5Y

Fig. 2. RNAi directed against herg gene significantly decreased the cellular growth rate (A) and inhibited the cell viability assayed by MTT (B) and reduces the colony formation (C) in the SH-SY5Y cells as compared with the untreatedcontrol or the shRNA-control. All data were expressed as means ± SEM from three independent experiments. ⁎P b 0.05, ⁎⁎P b 0.01, as compared with the untreated-control.

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Fig. 3. Cell cycle arrest induced by shRNA-herg1 or shRNA-herg1/1b in SH-SY5Y cells. Cells were treated with shRNA-herg1, shRNA-herg1/1b and shRNA-control for different durations (24, 36 and 48 h, respectively shown in A, B and C). Then the cells were harvested and analyzed by the flow cytometry. Cell cycle distributions displayed here were representative of three experiments.

1b transfected cells reduced both expression of HERG1 and HERG1B protein ranging from 85 kDa–100 kDa, which was encoded by a truncated form of full-length herg1 mRNA, lacking part or the entire N terminus (Fig. 1C). 3.2. Herg shRNA inhibits cell growth and proliferation in SH-SY5Y cells To further evaluate whether silencing herg gene in SHSY5Y cells may inhibit cell growth and proliferation, the growth curves of cells nontransfected and transfected with shRNA-herg1, shRNA-herg1/1b or shRNA-control were determined and MTT assay and colony formation assay were performed. As shown in Fig. 2A, RNAi directed against HERG1 and HERG1/1B both significantly decreased the growth rate of SH-SY5Y cells. Compared with the untreated group, cell counts in shRNA-herg1 and shRNA-herg1/1b group respectively decreased 40% and 50–60% at different time points in three separate experiments (P b 0.01), while shRNA-control transfection had no effect on cell growth (P N 0.05). MTT analysis revealed a cell growth inhibition consistent with the results of growth curve analysis. As shown in Fig. 2B, 96 h after shRNA-herg1 or shRNA-herg1/1b transfection, the percentages of viable cells respectively decreased 70% and 80%, as compared to the untreated group (P b 0.01). Similarly, shRNA-

control transfection had no distinct effect on cell viability (P N 0.05). Furthermore, the results of colony formation assay also showed that RNAi directed against HERG1 and HERG1/1B both resulted in a significant decrease (about 20% and 40%, respectively) in the number of colonies in SH-SY5Y cells, as compared to the untreated group (P b 0.01, Fig. 2C). In contrast, the number of colonies in the shRNA-control group was not affected. 3.3. Herg shRNA inhibits SH-SY5Y cells mitotic cycle To determine the effect of herg RNAi on SH-SY5Y cells' mitotic cycle, we analyzed the DNA contents of cells at interval times (24, 36 and 48 h after transfection) by flow cytometric method (Fig. 3A–C). The results suggested that cell numbers in Table 2 Cell percentages in G0–G1 phase determined by flow cytometry (%, mean ± S.E.M.) Time after transfection (h) shRNA-control shRNA-herg1 shRNA-herg1/1b 24 36 48

57.45 ± 0.42 58.23 ± 0.56 57.86 ± 0.33

56.46 ± 0.68 66.49 ± 0.31a 87.72 ± 0.43b

58.14 ± 0.29 65.70 ± 0.37a 86.91 ± 0.42b

P b 0.01, compared with the shRNA-control transfection group. P b 0.01, compared with each cell percentages of 36 h after transfection.

a

b

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Fig. 4. Intra-tumor injection of shRNA-herg1/1b with Lipofectamine 2000 significantly reduced the size of SH-SY5Y tumors implanted subcutaneously in nude mice during the 14-day follow-up period as compared with the saline control and the shRNA-control. ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001, compared with the saline control. ##P b 0.01, compared with the shRNA-control. n = 6.

G0–G1 phase had significantly been increased 36 h after transfection with shRNA-herg1 and shRNA-herg1/1b plasmids, compared with the shRNA-control transfection group (P b 0.01). Moreover, 48 h post-transfection, the cell numbers in G0–G1 phase in shRNA-herg1 and shRNA1/1b group were distinctly higher than the values of the corresponding groups 36 h post-transfection (P b 0.01). That indicated the SH-SY5Y cells were retarded in G0–G1 after herg1 or herg1/1b gene silencing (data are shown in Table 2). 3.4. Inhibition of tumor growth in nude mice To address the potential effect of herg RNAi on tumor growth in vivo, we performed this experiment using a mouse model of human neuroblastoma xenograft. Intra-tumor injection with shRNA-expressing plasmids was initiated when tumor size reached 4 × 4 mm. As shown in Fig. 4, the tumor size was smaller in shRNA-herg1/1b treated animals as compared with the saline and shRNA-control injected animals at each evaluating time point. At the last two time points of the observation (days 12 and 14), the tumor volume difference between saline control group and shRNA-herg1/1b treated group was statistically significant (n = 6), as the tumor volume was respectively 2.21 ± 0.48 cm3 and 2.80 ± 0.51 cm3 in saline control mice, 1.06 ± 0.32 cm3 (P b 0.01) and 1.21 ± 0.37 cm3 (P b 0.001) in shRNA-herg1/1b injected ones. Moreover, at the last time point (day 14), the tumor from the shRNA-herg1/1b transfected group was also significantly smaller in size than the tumor from the shRNA-control group (2.47 ± 0.68 cm 3 , P b 0.01, n = 6). And the mock shRNA-control injection had no distinct effect on tumor growth, as tumor size at the endpoint of the study was no different from that of saline control animals (P N 0.05). 4. Discussion Potassium channels have long been known to be involved in the regulation of a variety of biological functions ranging from the control of cell excitability to the regulation of cell volume

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and proliferation (Lang et al., 2000; Wonderlin and Strobl, 1996; Pardo et al., 2005; Lang et al., 2005). HERG K+ channel overexpression was a common phenomenon in the development and progression of many human cancers (Bianchi et al., 1998; Cherubini et al., 2000; Pillozzi et al., 2002; Smith et al., 2002). Both the herg gene and the HERG protein were indeed expressed in tumor cell lines of different histogenesis, as well as in primary human cancers. The HERG channel therefore provides a potential target for cancer gene therapy. Many studies have shown that the resting potential (Vrest) in tumor cells could be modulated by the activity of K+ channels, encoded by herg gene. The corresponding current, IHERG, contributed to driving the Vrest of tumor cells to more depolarized values, due to the peculiar biophysical properties of IHERG, permitting an easier passage through the cell mitotic cycle (Arcangeli et al., 1995; Pardo et al., 2005; Lang et al., 2005). Therefore, specific down-regulation of herg might be a potential therapeutic strategy against human cancers. In fact, selective pharmacological blockage of the HERG channel in several primary leukemic cells significantly reduced cell proliferation (Pillozzi et al., 2002; Crociani et al., 2003; Smith et al., 2002), and other studies showed that inhibition of eag1 expression with antisense oligonucleotides was sufficient to decrease the proliferation of some cancer cell lines (Pardo et al., 1999). However, it was successful only in some cases rather than applicable universally. Recently, the advent of RNAidirected ‘knock-down’ has sparked a revolution in somatic cell genetics, allowing the inexpensive, rapid analysis of gene function in mammals. Thus RNAi technique might be exploited for gene therapy in the near future. By means of the RNAi method, in the present study, several cellular growth and proliferation assays were used to determine the functional consequences of shRNA-mediated herg silencing in established SH-SY5Y cells. Our results demonstrated that herg RNAi could effectively down-regulate herg overexpression with great specificity. The shRNA-expressing plasmids could successfully suppress the expression of herg gene and HERG protein. Furthermore, a strong anti-tumor effect of herg RNAi in vivo was observed, as tumor growth in nude mice with xenograft was significantly suppressed when herg gene was silenced by intra-tumor injection of herg shRNA. Our results revealed that the growth and proliferation of SHSY5Y cells were inhibited remarkably, after treatment with shRNA-herg1 or shRNA-herg1/1b. Moreover, the inhibition treated by shRNA-herg1/1b was more obvious than that of shRNA-herg1. As above mentioned, shRNA-herg1 only targeted at herg1 gene and shRNA-herg1/1b targeted at both herg1 and herg1b gene. It was reported previously that the corresponding protein, HERG1 and HERG1B, are differentially expressed during cell cycle phases and that HERG currents are capable of modulating cell proliferation in tumor cells (Crociani et al., 2003). In HERG-bearing tumor cells, an increase in the HERG1B/HERG1 ratio on the plasma membrane occurs as cells proceed through the S phase. Moreover, the truncated HERG1B isoform is not expressed in adult hearts (Pond et al., 2000) but in tumors. The expression of herg1b in neoplastic cells could

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explain both the peculiar pattern of mRNA expression and the biophysical features of HERG currents observed in tumor cells (Bianchi et al., 1998; Arcangeli et al., 1995; Schönnher et al., 1999). Finally, HERG1B lacks the PAS (Per-Arnt-Sim) domain, an oxygen-sensing domain of basic helix–loop–helix proteins, like hypoxia induced factor-1. The latter is a transcriptional activator that is up-regulated by hypoxia and is responsible for gene activation under hypoxic conditions (Semenza, 1998). It is worth noting here that hypoxia is a main determinant of tumor progression and is currently regarded as a major hindrance to cancer therapy (Guillemin and Krasnov, 1997). The ability of tumor cells to express two types of HERG proteins, one endowed with and the other lacking the PAS domain, could be an advantage for cancer growth and progression. In hypoxia, cells could sense the decreased oxygen tension by PAS and lower the HERG1B/HERG1 ratio, thus leading to a shift of the activation curve of HERG currents and hyperpolarizing the membrane potential (Vm), limiting K+ loss (Fontana et al., 2001). This could permit the cell to survive in G1 without entering into the apoptotic pathway (Yu et al., 2001). When the oxygen supply was restored, the HERG1B/HERG1ratio could be increased by a remodeling of HERG channels on the plasma membrane, thus leading to depolarize Vm and sustain cell growth (Crociani et al., 2003). These might explain that shRNA-herg1/1b was more effective than shRNA-herg1 when used in herg gene silence. As mentioned in the Introduction, dysfunction of HERG might cause long QT syndrome and induce the episode of cardiac arrhythmias and an increasing risk of sudden death (Sanguinetti and Mitcheson, 2005; Witchel, 2007). With regard to the future clinical application, it is important to further evaluate the side effect of shRNA–herg on heart in vivo. However, it is more requisite to find a tissue-targeting technology to deliver the RNAi plasmid into tumor sites. In all, the key challenges for the development of siRNA as human therapeutics are largely dependent on the development of safe and efficacious delivery systems. In the near future the systemic delivery of siRNA will be required, possibly using a tissue-specific or cell-specific gene promoter vector or specific antibody-conjugated carriers, thus reducing applied dose of siRNA and resulting in decreased side effects (Takeshita and Ochiya). In summary, our study demonstrated that inhibition of herg1 or herg1/1b expression effectively depressed tumor cell growth both in vitro and in vivo, which further proved the important role of the HERG channel in tumorgenesis. Therefore, plasmid vector-mediated herg RNA interference holds great promise as a novel approach to human cancer with HERG overexpression and RNAi might have potential therapeutic utility in a variety of diseases, including cancer. Further studies should focus on the delivery strategies that can direct vectors carrying herg RNAi specifically into HERG overexpression caner cells with low toxicity and high efficiency. Acknowledgements This project was supported by the National Natural Science Foundation of China (No. 30472019, 30500620). We thank Dr

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