20 - World Journal of Gastroenterology

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A 120 P = 0.04 B 120 P = 0.07 C 120 P = 0.24 Cell viability (%) 100 80 60 40 20 0 0 25 50 100 150 200 μmol/L HCT 116-18 h-MTT tential in vivo effects of siRNA on inhibition of the proliferation of colon cancer cells. Viable HCT 116 colon cancer cells were injected subcutaneously into the right flank of the nude mice. In the first series of experiments, the inoculated mice were divided randomly into four groups of ten mice each and were treated with different doses of siRNA (control, 10, 20 and 50 μmol/L) weekly, for 4 wk. Over a 4 wk period after siRNA injection, the tumor volumes were estimated every week. As seen in Figure 6, the rates of tumor growth significantly decreased in higher than 20 μmol/L transfected groups with siRNA as compared with the control group 2 wk following treatment (P = 0.03). How- WJG|www.wjgnet.com 100 80 60 40 20 0 0 25 50 100 150 200 μmol/L HCT 116-24 h-MTT 100 80 60 40 20 0 0 25 50 100 150 200 μmol/L HCT 116-30 h-MTT Figure 4 MTT assay after small interfering RNA transfection in HCT116 cell line. A: The proliferation of cells transfected with siRNA was remarkably inhibited at 18 h posttransfection (P = 0.04); B and C: Cellular proliferation at 24 and 30 h post-transfection. The inhibitory effect shown A disappeared, cellular proliferation was similar with control. A B C PI fluorescence intensity D PI fluorescence intensity 10 4 10 3 10 2 10 1 10 0 10 4 10 3 10 2 10 1 10 0 unstaing.001 annexin/PI.004 50.005 10 0 10 1 10 2 10 3 10 4 Annexin-V FITC intensity Unstaing 100.006 10 0 10 1 10 2 10 3 10 4 Annexin-V FITC intensity Livin-siRNA 100 μmol/L Cell viability (%) PI fluorescence intensity E PI fluorescence intensity 10 4 10 3 10 2 10 1 10 0 10 4 10 3 10 2 10 1 10 0 10 0 10 1 10 2 10 3 10 4 Annexin-V FITC intensity Control 150.007 10 0 10 1 10 2 10 3 10 4 Annexin-V FITC intensity Livin-siRNA 150 μmol/L Oh BY et al . siRNA therapy in colon cancer PI fluorescence intensity F PI fluorescence intensity 10 4 10 3 10 2 10 1 10 0 10 4 10 3 10 2 10 1 10 0 10 0 10 1 10 2 10 3 10 4 Annexin-V FITC intensity Livin-siRNA 50 μmol/L 200.008 10 0 10 1 10 2 10 3 10 4 Annexin-V FITC intensity Livin-siRNA 200 μmol/L Figure 5 Results of Annexin V staining after small interfering RNA transfection in HCT116 cell line. The apoptotic portion (right lower quadrant) was increased continuously after siRNA transfection, but the necrotic portion (right upper quadrant) was also increased. A: The apoptotic rate of staining was 0.26%; B: The apoptotic rate of control was 3.91%; C: The apoptotic rate of cells transfected with siRNA 50 μmol/L was 30.32%; D: The apoptotic rate of cells transfected with siRNA 100 μmol/L was 32.88%; E: The apoptotic rate of cells transfected with siRNA 150 μmol/L was 36.91%; F: The apoptotic rate of cells transfected with siRNA 200 μmol/L was 45.08%. Cell viability (%) ever, there was no difference in antitumor effect between 20 μmol/L and 50 μmol/L in the transfected group. In the first study series, we detected that siRNA 20 μmol/L was the most effective dose for reduction of tumor growth in xenograft models. Thereafter, in the second series of experiments, we divided the inoculated mice randomly into two groups (control group and siRNA 20 μmol/L treated group) of ten mice per group. The mice of the siRNA 20 μmol/L treated group were injected with siRNA daily for 4 wk, and then the tumor volumes were measured every week during that period. The result was similar with the previous experiment; the mean volume of tumors decreased in transfected groups with siRNA 2567 May 28, 2011|Volume 17|Issue 20|
Tumor volume (cm 3 ) Oh BY et al . siRNA therapy in colon cancer 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0 1 2 3 4 t /wk 20 μmol/L as compared with control groups (Figure 7). To evaluate the toxic effect of siRNA in vivo, the body weights of mice were taken during the experimental period. The body weight of mice in all groups slightly increased over the entire experimental period; there were no significant body weight changes after siRNA injection compared to the control group. Histologic examination was performed with tissues taken from sacrificed mice after experiment completion. It revealed no significant toxic reaction in kidney, liver and brain of siRNA treated mice compared with the control group (data not shown). DISCUSSION control siRNA-10 μmol/L siRNA-20 μmol/L siRNA-50 μmol/L Figure 6 Tumor volume after small interfering RNA intratumoral injection in vivo. The inoculated mice were divided into four groups and were treated with different doses of siRNA (10, 20 and 50 μmol/L) weekly for 4 wk; tumor volumes were checked every week. In higher than 20 μmol/L siRNA, the tumor volumes were significantly decreased as compared with control group from 2 wk after transfection (P = 0.03). Eight human IAP molecules have been reported to date; NAIP (BIRC1), c-IAP1 (BIRC2), c-IAP2 (BIRC3), X-linked IAP (XIAP, BIRC4), Survivin (BIRC5), Apollon (BRUCE, BIRC6), Livin/ML-IAP (BIRC7), and IAP-like protein 2 (BIRC8) [12] . They are categorized as IAP by the presence of a baculovirus IAP repeat (BIR) protein domain, which forms a zinc-fold, a critical motif for regulating apoptosis. Many in this family also harbor a COOH-terminal RING finger domain. The BIR domain constitutes the zinc-fold, which is the important motif in delivering the anti-apoptotic signals [9] . It has been proposed that endogenous Livin has a minor direct effect on caspase activity whereas its anti-apoptotic effect could be ascribed to its antagonizing activity on the XIAP-Smac/DIABLO interaction and has been regarded as counteraction molecules against apoptosis through the blocking the terminal effectors such as caspases, thus far [12] . That the IAPs are highly conserved from yeast to mammals suggests the importance of this family in maintain life [11] . There are numerous reports on overexpression of the IAP family in cancer tissues to normal counterparts. The role of Survivin and Livin, in particular, in carcinogenesis has been well confirmed to date. Livin was reported in 2001 as a novel IAP family protein [9] . The overactivity of Livin was regarded as a predic- WJG|www.wjgnet.com P > 0.05 P = 0.03 P = 0.03 A Control B Livin-siRNA 20 μmol/L Figure 7 Tumor xenograft in nude mice. The inoculated mice were divided into two groups (control and siRNA-20 μmol/L treated group), and then tumor volume was measured in the the same manner as described in Figure 6. A: Control group; B: siRNA-20 μmol/L treated group. The tumor volume was dramatically decreased as compared with control. tor of poor prognosis in cancer patients and a resistance factor to chemotherapeutic agents or radioactive drugs in several cancers [13,14] . The overexpression of Livin mRNA has been found in some tumors including melanoma, breast, cervical, colon and prostate cancers, as well in leukemia, in lymphoma and in hepatoma cell lines [12] . Moreover, researchers have shown that only overexpression of Livin-α isoform is correlated with high risk of relapse in bladder cancer [10] . In the previous study, Livin gene was expressed well in the stools of colon cancer patients as opposed to the healthy control group. This finding suggests that the exfoliated cancer cells from the gastrointestinal tumors were contained in the patients’ stool and overexpressed tumor related genes such as Livin were detected from the stool. Based on this result, we chose Livin gene as a treatment target in this experiments. In recent years, numerous genetic engineering techniques were introduced in drug development markets worldwide such as antisense oligonucleotides, aptamer (single stranded nucleic acids), use of ribozymes, dominant negative mutant construction and siRNA. We focused on siRNA technology because of its target sequence-specific degradation ability and relatively simple application procedure. In higher organisms, defense mechanisms are more complex but long dsRNAs should not be used as an experimental tool to trigger RNAi in mammalian cells. Long dsRNAs are processed by the endonuclease Dicer 2568 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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Online Submissions: http://www.wjgn
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Mandrekar P. Epigenetics and alcoho
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Moghe A et al . Altered nutrients,
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James SJ. Intracellular S-adenosylh
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69: 562-573 91 Murphy FR, Issa R, Z
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Aziz-Seible RS et al . Fibronectin
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mechanisms accounted for by autoimm
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to autocrine TNF-a signaling after
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Sondek J, Zuobi-Hasona K, Aukhil I.
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stress in alcohol toxicity. Free Ra
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HIV, VIRAL HEPATITIS AND ALCOHOL US
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Table 1 Common mechanisms of liver/
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perpermeability and endotoxemia pri
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Page 117 and 118: Bardag-Gorce F. Proteasome inhibito
Page 119 and 120: Bardag-Gorce F. Proteasome inhibito
Page 121 and 122: Oh BY et al . siRNA therapy in colo
Page 123: A Livin expression (%) 120 100 80 6
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Page 132 and 133: A B C D E Figure 1 Change in coloni
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Page 145 and 146: Instructions to authors ISSN and EI
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