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. 2012;7(10):e46627.
doi: 10.1371/journal.pone.0046627. Epub 2012 Oct 10.

A novel anticancer therapy that simultaneously targets aberrant p53 and Notch activities in tumors

Yuting Yao  1 , Li Wang, He Zhang, Haibo Wang, Xiaoping Zhao, Yidan Zhang, Leilei Zhang, Xianqun Fan, Guanxiang Qian, Ji-Fan Hu, Shengfang Ge
Affiliations

A novel anticancer therapy that simultaneously targets aberrant p53 and Notch activities in tumors

Yuting Yao et al. PLoS One. 2012.

Abstract

Notch signaling pathway plays an important role in tumorigenesis by maintaining the activity of self-renewal of cancer stem cells, and therefore, it is hypothesized that interference of Notch signaling may inhibit tumor formation and progression. H101 is a recombinant oncolytic adenovirus that is cytolytic in cells lacking intact p53, but it is unable to eradicate caner stem cells. In this study, we tested a new strategy of tumor gene therapy by combining a Notch1-siRNA with H101 oncolytic adenovirus. In HeLa-S3 tumor cells, the combined therapy blocked the Notch pathway and induced apoptosis in tumors that are p53-inactive. In nude mice bearing xenograft tumors derived from HeLa-S3 cells, the combination of H101/Notch1-siRNA therapies inhibited tumor growth. Moreover, Notch1-siRNA increased Hexon gene expression at both the transcriptional and the translational levels, and promoted H101 replication in tumors, thereby enhancing the oncolytic activity of H101. These data demonstrate the feasibility to combine H101 p53-targted oncolysis and anti-Notch siRNA activities as a novel anti-cancer therapy.

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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 gene knockdown by siRNA.
A. Semi-quantitative RT-PCR analysis of Notch1gene transcripts in HeLa-S3 cells. The experiment was performed 48 hours after siNotch1 (100 nmol/l) transfection with or without H101 infection (multiplicity of infection (MOI)  = 100). β-actin was used as the internal control for normalization. B. Western Blot analysis of Notch1 protein in HeLa-S3 cells. The experiment was performed 72 hours after siNotch1 (100 nmol/l) transfection with or without H101 infection (multiplicity of infection (MOI)  = 100). Western bands were scanned and normalized over the internal control β-actin.
Figure 2
Figure 2. Cell proliferation of HeLa-S3 cells following the combined treatment of Notch1-siRNA and H101.
Cell proliferation was measured by MTT assays 24, 48, 72, 96 hours after co-treatment of Notch1-siRNA (100 nmol/l) and 24, 48, 72 hours after H101 infection (multiplicity of infection (MOI)  = 100). All data are presented as means ± SD of three independent experiments. *p<0.05, #p<0.01 as compared with negative control.
Figure 3
Figure 3. Antitumor activity of the cocktail treatment of Notch-siRNA and H101.
A. HeLa-S3 xenograft tumors in nude mice. Average volume of subcutaneous tumors after treatment with H101(triangle), H101 plus Notch1-siRNA (cross), Notch1-siRNA (square), or PBS (rhombus). Values represent the means ± SD for five animals per group. (*p<0.05). B. Histological analysis of HeLa-S3 derived tumors. Original magnification: ×200.
Figure 4
Figure 4. Apoptosis analysis of HeLa-S3 tumor cell induced by the H101- Notch1-siRNA treatment.
HeLa-S3 cell was harvested 72 h after transfection, and Annexin V staining was used to analyze early-stage cell apoptosis.
Figure 5
Figure 5. Expression of caspase-3 activation, p53 (A) and MDM2 (B) in HeLa-S3 tumor cells.
Cells were transfected with the Notch1-siRNA and H101 for 72 hours, and total protein was analyzed by Western blot with specific antibodies.
Figure 6
Figure 6. The effect of Notch1-siRNA on viral DNA replication in HeLa-S3 cells.
A. Viral DNA replication was determined by real-time RT-PCR quantification of adenoviral late Hexon gene expression at 24, 48, and 72 hours after co-treatment, respectively. For comparison, the mRNA expression of Hexon at 24 hours in H101 group was arbitrarily set as 1, and β-actin was used as the internal control in the calculation. P<0.05: compared to Hexon mRNA expression of the H101 group. B. Western blot analysis of Hexon protein at 72 hours. Bar: average band density of quantified Hexon protein after normalization by the internal control β-actin. Protein expression of Hexon in H101 group was arbitrarily set as 1. *P<0.05: relative to Hexon protein expression in the H101-treated group.
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