20 - World Journal of Gastroenterology

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Online Submissions: http://www.wjgnet.com/1007-9327office wjg@wjgnet.com doi:10.3748/wjg.v17.i20.2563 ORIGINAL ARTICLE siRNA targeting Livin decreases tumor in a xenograft model for colon cancer Bo-Young Oh, Ryung-Ah Lee, Kwang Ho Kim Bo-Young Oh, Ryung-Ah Lee, Kwang Ho Kim, Department of Surgery, Ewha Womans University School of Medicine, Seoul 158-710, South Korea Author contributions: Oh BY performed statistical analysis and drafted the manuscript; Lee RA planned the investigation, supervised the laboratory work, helped with data analysis, helped to draft the manuscript, and revised the manuscript; Kim KH participated in the study design and coordination. Supported by the Korea Research Foundation Grant #2007- E00037 funded by Korea government (MOEHRD, Basic Research Promotion Fund) Correspondence to: Ryung-Ah Lee, MD, PhD, Department of Surgery, Ewha Womans University School of Medicine, Seoul 158-710, South Korea. ralee@ewha.ac.kr Telephone: +82-2-26502659 Fax: +82-2-26447984 Received: October 6, 2010 Revised: February 15, 2011 Accepted: February 22, 2011 Published online: May 28, 2011 Abstract AIM: To evaluate the effect of silencing Livin gene expression with siRNA to apoptosis and proliferation in a colon cancer cell line. METHODS: To investigate the anticancer effect of silencing Livin gene expression, we established an siRNA transfected cell line using the HCT116 colon cancer cell line. After confirming the successful transfection, MTT assay, flow cytometry and annexin V staining were employed to evaluate the antiapoptotic effect. To confirm the in vivo effect of Livin-siRNA, different doses of LivinsiRNA were injected into xenografted tumors in BALB/c nude mice model. RESULTS: Livin expression was dramatically decreased after siRNA transfection, especially at 25 μmol/L of siRNA, but this suppression was not dose-dependent. The cell count at 18 h after transfection was significantly reduced as compared with controls (P < 0.01), but tended not to decrease proportionally depending on transfected dose WJG|www.wjgnet.com 2563 World J Gastroenterol 2011 May 28; 17(20): 2563-2571 ISSN 1007-9327 (print) ISSN 2219-2840 (online) © 2011 Baishideng. All rights reserved. or time. MTT assay revealed that silencing the Livin gene suppressed cellular proliferation at 18 h after transfection (P = 0.04); however, the inhibitory effect disappeared thereafter. Also, there was no significant difference in cellular proliferation depending on siRNA dose. The rate of apoptosis also increased with silencing of the Livin gene. In vivo, the tumor size significantly decreased after LivinsiRNA injection at 20 μmol/L concentration (P = 0.03). There were no significant body weight changes of mice after siRNA injection. Histologic examination revealed no significant toxic reaction in kidney, liver and brain of mice. CONCLUSION: siRNA-mediated downregulation of Livin expression can induce apoptosis in colon cancer in vitro and in vivo, which suggests the possibility of new cancer therapeutics using siRNA. © 2011 Baishideng. All rights reserved. Key words: siRNA; Livin; Inhibitor of apoptosis; Colon cancer Peer reviewer: Jian Wu, Associate Professor of Medicine, Internal Medicine/Transplant Research Program, University of California, Davis Medical Center, 4635 2nd Ave. Suite 1001, Sacramento CA 95817, United States Oh BY, Lee RA, Kim KH. siRNA targeting Livin decreases tumor in a xenograft model for colon cancer. World J Gastroenterol 2011; 17(20): 2563-2571 Available from: URL: http://www. wjgnet.com/1007-9327/full/v17/i20/2563.htm DOI: http://dx.doi. org/10.3748/wjg.v17.i20.2563 INTRODUCTION Colon cancer is one of the most common malignancies worldwide and has shown increased incidence, especially in developing countries [1] . Until recently, a major treatment strategy has been surgical resection that shows good outcome over other treatments. With the activation of a screen- May 28, 2011|Volume 17|Issue 20|
Oh BY et al . siRNA therapy in colon cancer ing program, early cancer, less than stage I, is curable with only surgical treatment. But the treatment results for metastatic disease are still unsatisfactory with surgery alone. In these cases, clinicians consider other treatment options such as chemotherapeutic agents and targeted agents. Recently, development of techniques in manipulation of nucleic acids makes gene therapy possible to adopt in cancer therapy. RNA interference (RNAi) is a fundamental protective process in eukaryotic cells including invertebrates and vertebrates, which is able to block harmful signal by targeting complementary mRNA and cleaving thereof [2] . Small interfering RNA (siRNA) is fragments from double-stranded RNA by the enzyme Dicer [3-5] . The natural role of RNAi is supposed to be a defense mechanism against some viral infection or deleterious genomic instability. This special mechanism has been of great interest recently as siRNA targeting small genes are easily manufactured and applied to major clinical problems such as cancer, asthma, inflammatory disease and infection [6] . This methodology is becoming a powerful tool for new drug development in such areas and is replacing the techniques of using antisense oligonucleotide and ribozymes [7,8] . Livin, a 280 amino acid protein and a member of the mammalian type of inhibitor of apoptosis (IAP), are well-conserved proteins across the species. It has a single baculovirus IAP repeat (BIR) domain and a COOHterminal RING domain and is localized predominantly in the nucleus and a filamentous pattern of cytoplasm [9] . The Livin gene spans 46 kb and is located on chromosome 20 at band q13 [10] . The major function of Livin is an inhibition of apoptosis by binding to caspase 3, caspase 7 and caspase 9 and also in inhibition of proteolytic processes of caspase 9 [9,11] . Its overexpression protects cells from various proapoptotic stimuli. The expression of Livin, similar to expression of Survivin, is rarely detected in normal adult tissues but exists abundantly in cancerous tissues and transformed cells [9] . The overexpression of Livin protein has been reported in colorectal cancer, leukemia, hepatocellular carcinoma, and melanoma [12] . Recently, major attention has been focused on the IAP family, especially Survivin and Livin, as they are easy to handle in the genetic field due to their small size. Moreover, the lack of expression in normal adult tissues of Livin is a very attractive characteristic in cancer therapy [9] . In this study, we used siRNA targeting to Livin transfection into the HCT116 colon cancer cell line to confirm the antitumor effect and blockade of Livin gene and performed an additional in vivo study using BALB/c nude mice xenografts to determine the direct tumor regression effect of Livin silencing with siRNA. These results suggested that Livin is an effective target for colorectal cancer treatment using siRNA technique. MATERIALS AND METHODS Materials Cell culture-The HCT116 colon cancer cell line, purchased from Korean cell line bank, was grown in RPMI1640 medium (Life Technologies, Inc., Grand Island, NY) sup- WJG|www.wjgnet.com plemented with 10% fetal bovine serum (FBS, Life Technologies, Inc.), penicillin (100 U/mL) and streptomycin (100 g/mL). Cells were maintained at 37℃ in a humidified atmosphere of 50 mL/L CO2. RT-PCR procedure Total RNA was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA). The method for extracting total RNA was according to the manufacturer’s protocol. RNA samples were quantified at 260 nm using spectrophotometry. One μg of total RNA was reacted for 15 min at 42℃ using the Promega’s Reverse Transcription System (Promega Corp., Madison, WI), reacted for 5 min at 95℃, and cDNA was obtained. For polymerase chain reaction (PCR), cDNA (3 μL) was obtained by reverse transcription, 0.1 μmol primers, 1.25 μL GoTaq DNA polymerase, 0.2 mmol/L deoxynucleotide triphosphate (dNTP), 1 Go Taq reaction buffer and each primer was added, and DNA was amplified by performing PCR with primer of Livin; forward, 5-GTCAGTTCCT- GCTCCGGTCAA-3; reverse, 5-GGGCACTTTCAGACT- GGACCTC-3. Electrophoresis of PCR products was performed on 1.5% agarose gel and examined by staining with ethidium bromide. By measuring the brightness of bands using a densitometer, their amount was quantified using the value of β-actin as the standard value. siRNA design and preparation Synthetic 21-nt RNAs were purchased from Dharmacon Research (Lafayette, CO; in deprotected, desalted, and annealed form.). We used 4 primers for silencing the Livin gene to improve the blocking efficiency. The target sequences of Livin for production of siRNA were 5'-GGAGAGA- GGTCCAGTCTGA-3', 5'-GGAAGAACCGGAAGAC- GCA-3', 5'-GCTCTGAGGAGTTGCGTCT-3' and 5'-GCTCTGAGGAGTTGCGTCTTT-3'. The nonspecific control siRNA duplex was also purchased from Dharmacon Research. Briefly, centrifuge tubes containing siRNA to ensure that the siRNA pellet was collected at the bottom of the tube. Resuspended siRNA to a convenient stock concentration with 1X siRNA buffer (Dharmacon, Inc., Lafayette, CO). After placing the solution on a shaker for 30 min at room temperature, the concentration of siRNA was verified using UV spectrophotometry at 260 nm. HCT116 cells were diluted in fresh media without antibiotics and transferred to six-well plates (1 × 10 5 cells/well) 24 h before transfection and maintained at 37℃ in a humidified atmosphere of 50 mL/L. HCT116 cells grown to a confluence of 40%-50% were transfected with siRNA using Lipofectamine 2000 (Life Technologies, Inc., Grand Island, NY) according to the manufacturer’s recommendations. To detect the transfection efficiency, the cells were analyzed by FACSan (Becton Dickson, San Jose, CA) 6 h after transfection with FITC-labeled siRNA. After transfection, the cells were incubated and then used to various analyses. MTT assay Cell viability was examined by routine 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. 2564 May 28, 2011|Volume 17|Issue 20|
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World Journal of Gastroenterology W
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Robert JL Fraser, Daw Park Jacob Ge
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Italy Donato F Altomare, Bari Piero
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Fernando Azpiroz, Barcelona Ramon B
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S Contents EDITORIAL TOPIC HIGHLIGH
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Contents APPENDIX FLYLEAF EDITORS F
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HIV, VIRAL HEPATITIS AND ALCOHOL US
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