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. 2012;7(8):e44301.
doi: 10.1371/journal.pone.0044301. Epub 2012 Aug 28.

Therapeutic efficacy by targeting correction of Notch1-induced aberrants in uveal tumors

Xiaolin Huang  1 , Li Wang, He Zhang, Haibo Wang, Xiaoping Zhao, Guanxiang Qian, Jifan Hu, Shengfang Ge, Xianqun Fan
Affiliations

Therapeutic efficacy by targeting correction of Notch1-induced aberrants in uveal tumors

Xiaolin Huang et al. PLoS One. 2012.

Abstract

There is a need for more effective treatments for uveal melanoma. The recombinant oncolytic adenovirus H101 replicates specifically in p53-depleted tumor cells, and has been approved for use by the Chinese State Food and Drug Administration. However, this treatment is associated with subsequent remission. Transfection of uveal melanoma cells with a small interfering RNA against Notch1 (siNotch1) effectively suppressed Notch1 expression, resulting in significant cell growth inhibition when combined with H101 treatment. Combined treatment with siNotch1 and H101 (H101-Notch1-siRNA) greatly enhanced apoptosis and cell cycle arrest in vitro as compared to treatment with H101 or siNotch1 alone. For in vivo treatments, the combined treatment of siNotch1 and H101 showed remarkable tumor growth inhibition and prolonged mouse survival in the OCM1 xenograft model. We predict that Notch pathway deregulation could be a feature of uveal melanoma, and could be a therapeutic target, especially if p53 is concurrently targeted.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Notch1 and p53 status in UM cells.
(A) Western blot analysis of Notch1 expression in UM cells. HEK293, ARPE-19, VUP and OCM1 cells were assessed for Notch1 protein levels. The HEK293 cells were used as positive controls. The normal cell lines ARPE-19 were used as non-malignant controls. Notch1 protein: 120 kDa, β-actin protein: 42 kDa (B) Protein expression were normalized using the internal control β-actin and the positive control band value was set as 1(100%) according to HEK293 cell lines. (C) Sequence analysis of ARPE-19 cell line showing the sequence of wild-type p53 exon 7. (D) and (E) A heterozygous missense mutation of p53 (C. 797G>A, P. Gly133Glu, arrows indicated) was identified in OCM1 and VUP cell lines. Data represent three independent experiments. (*: p<0.05, **: p<0.01).
Figure 2
Figure 2. Sensitivity of UM cells to H101 oncolytic adenovirus.
(A) RT-PCR analysis of CAR gene in UM cells. The HEK293 cells were used as positive controls. The ARPE-19 cells were used as non-malignant controls. CAR gene: 325 bp, GAPDH gene: 496 bp (B) Densitometric measurement for mRNA expression. The HEK293 band value was set as 100% normalized with the internal control GAPDH. (C) FACS analysis for cell membrane protein CAR. (D) and (E) Immunofluorescence detection of CAR in OCM1 and VUP cells. Nuclei were stained with PI (red), and CAR was visualized with IgG goat anti-mouse secondary antibody (green; white arrows). Infectivity of (F) HEK293, (G) ARPE-19, (H) OCM1 and (I) VUP cells with H101. Cells were incubated in non-FBS culture media and infected with H101 at an MOI of 1, 10, 100, and 500 pfu/cell. The MTT assay was performed at 24, 48, 72 and 96 hours following H101 infection. All data are presented as mean ± SD. of three independent experiments. (*: p<0.05, **: p<0.01, compared with untreated tumor cells).
Figure 3
Figure 3. Notch1 gene knockdown by siRNA.
(A) RT-PCR results of Notch1 in UM cells. OCM1 and VUP cells were analyzed using specific primers for Notch1 mRNA. A 100 bp DNA ladder molecular marker served as the reference. PCR bands were normalized using the internal control β-actin. (B) Western blot analysis of Notch1 protein in UM cells. All experiments were performed 72 hours following siNotch1(50nmol/L) and control siRNA(50nmol/L) transfection with or without H101 infection (MOI = 100). (C) Western bandScan was used to analyze the gray scale values for different electrophoretic bands, and the relative ratio of the gray scale values between the target Notch1 band and the β-actin internal reference was determined. Notch1 protein: 120 kDa, β-actin protein: 42 kDa.
Figure 4
Figure 4. Growth inhibition of combined H101-Notch1-siRNA on UM cells.
Survival index of (A) HEK293, (B) ARPE-19, (C) OCM1 and (D) VUP cells by the combined treatment of H101 and siNotch1. Cell survival index was measured by the MTT assay at 24, 48, 72 and 96 hours. Untreated tumor cells were used as controls. SiNotch1 or siNC was used at a concentration of 50nmol/L. H101 infection was performed at an MOI of 100. All data are presented as mean ± SD. of three independent experiments. (*: p<0.05, **: p<0.01, compared with untreated tumor cells).
Figure 5
Figure 5. Cell cycle distribution and apoptotic activity of combined H101-Notch1-siRNA on UM cells.
(A) and (B) Cell cycle distribution of OCM1 and VUP cells following treatment with siNotch1 and/or H101. OCM1 and VUP cells were harvested 72 hours after co-treatment with siNotch1 (50nmol/L) and H101 (MOI = 100), and propidium iodide staining and FACS analysis were used to analyze the cell cycle distribution. S-phase arrest was detected in the H101 and H101-Notch1-siRNA groups. (C) and (D) Apoptotic activity of OCM1 and VUP cells. Cells were measured by flow cytometry analysis 72 hours after co-treatment with siNotch1 (50nmol/L) and/or H101 (MOI = 100). Upper left: cells affected by necrosis only; upper right: cells affected with both apoptosis and necrosis; lower left: normal cells; lower right: cells affected by apoptosis only. Data are expressed as mean ± SD. of three independent experiments. (*: p<0.05, **: p<0.01, compared with untreated tumor cells). (E) and (F) Relative ratio percentage of apoptosis (cells in lower right group) and necrosis (cells in upper left group) in OCM1 and VUP cells. The percentages of apoptosis and necrosis cells were analyzed according to (C) and (D).
Figure 6
Figure 6. Antitumor effect of combined H101-Notch1-siRNA treatment in an OCM1 tumor xenograft mouse model.
(A) Tumor volume following treatments. Subcutaneous tumors were established by implanting OCM1 cells in nude mice (n = 10). (B) Tumor weight on day 24 after first injection (n = 5). (C) Representative pictures of tumor specimens of each treatment group 24 days after first injection. (D) Percentage of mouse survival over 120 days (n = 5). Percent survival was analyzed by Kaplan-Meier survival analysis. Data represent mean ± SD. (**: p<0.01, ***: p<0.001, compared with control group).
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Grants and funding

This work was supported by a National Key Program for Basic Research of China grant (2010CB529902) to GXQ, a National Natural Science Foundation of China grant (10979034) to SFG, and a Shanghai Leading Academic Discipline Project grant (S30205) to XQF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.