RETRACTED: Insulin-like growth factor-binding protein-7 (IGFBP7): a promising gene therapeutic for hepatocellular carcinoma (HCC)

Abstract

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the editor-in-chief. Similarities were found between images within this article. Image analysis performed by the editorial office confirmed findings of image duplication in Figures 1B, 4, and 8A. This reuse (and in part misrepresentation) of data without appropriate attribution represents a severe abuse of the scientific publishing system. No authors responded when contacted about the retraction.

Figure 1

Ad.IGFBP7 generates IGFBP7. (a) Huh7, Hep3B, and HepG3 cells were either uninfected (control) or infected with Ad.vec or Ad.IGFBP7 at an MOI of 100 vp/cell. IGFBP7 mRNA expression was determined by quantitative RT-PCR 24 h post-infection. GAPDH expression was used as control. IGFBP7 expression level in the uninfected cells was considered as one. The data represent mean ± SEM. *P < 0.01. (b) Huh7, Hep3B, and HepG3 cells were either uninfected (control) or infected with Ad.vec or Ad.IGFBP7 at an MOI of 100 vp/cell. Cells in the control and Ad.vec groups were harvested 3 days post-infection while those in Ad.IGFBP7 group were harvested 1, 2, and 3 days post-infection. The lysates were subjected to western blot analyses using anti-HA antibody. EF1-α was used as loading control. (c) Huh7, Hep3B, and HepG3 cells were infected with Ad.vec or Ad.IGFBP7 at an MOI of 100 vp/cell. Conditioned media were collected 24 and 48 hours post-infection and subjected to IGFBP7 detection by ELISA. The data represent mean ± SEM. ^#P < 0.05; *P < 0.01. (d) Huh7 cells were infected with Ad.IGFBP7 at 100 vp/cell. IGFBP7 expression was detected by immunofluorescence analysis 24 hours post-infection. Images were analyzed using a Zeiss confocal laser scanning microscope. DAPI was used to stain the nucleus. A.U., arbitrary unit; DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HA, hemagglutinin; MOI, multiplicity of infection; RT-PCR, reverse transcription-PCR; vp, viral particle.

Figure 2

Ad.IGFBP7 induces apoptosis in human HCC cells. (a) Huh7, Hep3B, and HepG3 cells were either uninfected (control) or infected with Ad.vec (1,000 vp/cell) or Ad.IGFBP7 (100, 500 or 1,000 vp/cell). Cell viability was detected by standard MTT assay 1, 2, and 3 days post-infection. Cell viability of the control uninfected cells at day 1 was considered as 100%. (b) Huh7, Hep3B, and HepG3 cells were either uninfected (control) or infected with Ad.vec (1,000 vp/cell) or Ad.IGFBP7 (100 or 1,000 vp/cell) and subjected to colony formation assay. Colonies were scored after 2 weeks. (c) Huh7, Hep3B, and HepG3 cells were infected with Ad.vec or Ad.IGFBP7 at an MOI of 1,000 vp/cell. Apoptosis was detected by Annexin V staining followed by flow cytometry 1, 2, and 3 days post-infection. For a–c, the data represent mean ± SEM of three independent experiments. ^#P < 0.05; *P < 0.01. (d) Huh7, Hep3B, and HepG3 cells were either uninfected (control) or infected with Ad.vec or Ad.IGFBP7 at an MOI of 1,000 vp/cell. Cells in the control and Ad.vec groups were harvested 3 days post-infection while those in Ad.IGFBP7 group were harvested 1, 2, and 3 days post-infection. The lysates were subjected to western blot analyses using anti-PARP antibody. EF1α was used as loading control. HCC, hepatocellular carcinoma; MOI, multiplicity of infection; vp, viral particle.

Figure 3

Ad.IGFBP7 induces reactive oxygen species (ROS). (a) Huh7, Hep3B, HepG3, and QGY-7703 cells were infected with Ad.vec or Ad.IGFBP7 at an MOI of 1,000 vp/cell. ROS was detected by a commercially available kit as described in Supplementary Materials and Methods. (b) The indicated cells were treated as in a and either untreated or treated with N-acetylcysteine (NAC) at 5 or 10 mmol/l doses. Cell viability was determined by standard MTT assay. The data represent mean ± SEM of three independent experiments. ^#P < 0.05; *P < 0.01. MOI, multiplicity of infection; vp, viral particle.

Figure 4

Ad.IGFBP7 activates a DNA damage response pathway. Huh7, Hep3B, and HepG3 cells were either uninfected or infected with Ad.vec or Ad.IGFBP7 at an MOI of 1,000 vp/cell. For control and Ad.vec groups, the samples were collected at day 2. Cell lysates were subjected to western blot analysis using the indicated antibodies. A representative blot for EF1α was presented as loading control. MOI, multiplicity of infection; vp, viral particle.

Figure 5

SB203580 protects from Ad.IGFBP7-induced inhibition of cell viability. Huh7, Hep3B, HepG3, and QGY-7703 cells were infected with Ad.vec or Ad.IGFBP7 at an MOI of 1,000 vp/cell and either untreated or treated with SB203580 (SB) at 1 or 2 µmol/l doses. Cell viability was determined by standard MTT assay. The data represent mean ± SEM of three independent experiments. *P < 0.01. MOI, multiplicity of infection; vp, viral particle.

Figure 6

Ad.IGFBP7 inhibits orthotopic human HCC xenografts and intrahepatic metastasis. QGY-luc cells were orthotopically implanted by intrahepatic injection in athymic nude mice and treated with intravenous injections of Ad.vec or Ad.IGFBP7 (1 × 10⁹ vp/injection; a total of five injections over a 2-week period). (a) Bioluminescence imaging of the mice before and after treatment. (b) Quantification of photon counts from the animals. The data represent mean ± SEM; *P < 0.01. Sixteen animals per group were used. (c) Photomicrograph of the livers at the end of the study. (d) Analysis of intrahepatic metastasis score at the end of the study. (e) Analysis of serum human α-fetoprotein (α-FP) levels at the end of the study. For d and e, the data represent mean ± SEM; *P < 0.01. (f) Ad.IGFBP7 does not induce toxicity to normal mouse liver. Hematoxylin and eosin staining of sections containing QGY-luc orthotopic tumor (T) and the adjacent normal liver (N) from mouse treated with Ad.IGFBP7. Please note the preservation of normal architecture in the adjacent liver while the tumor shows hemorrhagic necrosis. The arrows indicate the border between the orthotopic tumor and normal liver. HCC, hepatocellular carcinoma; ROI, region of interest; vp, viral particle.

Figure 7

Ad.IGFBP7 inhibits primary and distant tumors. QGY-luc cells (1 × 10⁶) were subcutaneously implanted in both flanks of athymic nude mice and after the establishment of the tumors (~100 mm³) only the left-sided tumors were injected with either Ad.vec or Ad.IGFBP7 (1 × 10⁹ vp/injection; a total of five injections over a 2-week period). (a) Photograph of the mice at the end of the experiment. (b) Measurement of tumor volume during the course of the experiment. (c) Measurement of tumor weight at the end of the experiment. Fifteen animals per group were used. (d) IGFBP7 protein is detected in the sera of mice receiving intratumoral injection of Ad.IGFBP7. Blood was collected from the tip of the tail at 2 weeks (i.e., at the end of the last Ad injection) and at 4 weeks (i.e., at the end of the experiment). IGFBP7 was measured by ELISA as described in the Materials and Methods. Five animals were used per group. (e) Apoptotic cells were detected in tumor sections by TUNEL assay. For b–e, the data represent mean ± SEM; *P < 0.01. Lt, left; Rt, right; vp, viral particle.

Figure 8

IGFBP7 inhibits proliferation and angiogenesis in primary and distant tumors. (a) Tumor sections were stained with hematoxylin and eosin (H&E) or stained for IGFBP7, Ki-67 and CD31. Asterisk (*) represents necrotic areas. Arrows indicate blood vessels. Original magnification: ×400. (b) IGFBP7 protein is detected in the right-sided uninjected tumor upon longer exposure. Sections from right-sided uninjected tumors from Ad.IGFBP7-treated group were stained for IGFBP7. (c) Conditioned media from Ad.IGFBP7-infected cells induce apoptosis. QGY-luc cells were either uninfected (control) or infected with Ad.vec or Ad.IGFBP7 at an MOI of 100 vp/cell for 48 hours. The conditioned media was collected, filtered, and added to freshly plated QGY-luc cells for 48 hours. The lysates were subjected to western blot analyses using anti-PARP antibody. EF1α was used as loading control. Lt, left; MOI, multiplicity of infection; Rt, right; vp, viral particle.