The authors have declared that no competing interests exist.
Conceived and designed the experiments: YY JH SG. Performed the experiments: YY. Analyzed the data: YY. Contributed reagents/materials/analysis tools: SG GQ XF. Wrote the paper: YY JH SG. Technical help: LW HZ HW XZ LZ YZ.
¶ These authors also contributed equally to this work.
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.
Most forms of cancer chemotherapy are unable to eradicate all malignant cells, and they often are highly toxic because of their lack of selectivity to cancer cells. As a result, new efforts have focused on developing interventions that include tumor-specific replicating viruses and siRNA.
A virus-based strategy takes advantage of the fact that the intracellular replication and production of adenoviral progeny requires the cell cycle gatekeeper p53 to be in an inactive status, and in many tumors, p53 is either mutated or epigenetically silenced. The viral early gene
In China, an oncolytic adenovirus called H101 has been clinically approved for the treatment of several malignancies
Notch signaling plays a pivotal role in cellular differentiation, proliferation, and apoptosis
The Notch signaling pathway is disrupted in several malignancies, offering a potential target for therapeutic intervention. There is aberrant activation of Notch signaling in glioblastoma (GBM) cell lines and in human GBM-derived neurospheres. Inhibition of Notch signaling via the expression of a dominant negative form of the Notch co-activator, mastermind-like 1 (DN-MAML1) or the treatment of an γ-secretase inhibitor (GSI) MRK-003 resulted in a significant reduction in GBM cell growth
Notch is also critical in maintaining the ability of cancer stem cells (CSCs to self renew) (see reviews
Cervical cancer cell line Hela-S3 was deficient in p53, and preclinical studies demonstrated that Hela-S3 was very sensitive to H101 oncolytic treatment. We have previously shown that knockdown of the Notch 1 gene could inhibit the proliferation and growth of HeLa cells both
Among the Notch family genes, Notch1 is the best validated target in malignancies, with the highest activating mutations identified in tumors. Our previous
We then tested the suppression of Notch1 by its siRNA in HeLa-S3 tumor cells. Notch1-siRNA and control NC-siRNA were used to transfect HeLa-S3 cells, respectively, and the efficiency of siRNA on Notch1 expression was examined by RT–PCR and Western blot. As expected, no change in the abundance of Notch1 mRNA was detected in the H101 group. As compared with the NC-siRNA control, Notch1-siRNA, used either alone or with H101, suppressed Notch1 expression (
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.
Having established that Notch1-siRNA inhibited Notch1 expression, we used the MTT method to detect the effects of combined treatment of Nocth1-siRNA and H101 on cell growth (
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.
To examine whether the enhanced
As compared with the PBS control group, the Notch1-siRNA or H101 monotherapy showed similar inhibition of tumor growth. The Notch1- siRNA/H101 group, however, had an enhanced anti-tumor effect (
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.
To examine the mechanism underlying the augment of anti-tumor effect by the combined Notch1-siRNA/H101 treatment, cell apoptosis was measured using an Annexin V-FITC apoptosis kit and flow cytometric analysis 48 hours after the cells were transfected with Notch1-siRNA and H101. As seen in
HeLa-S3 cell was harvested 72 h after transfection, and Annexin V staining was used to analyze early-stage cell apoptosis.
We then used Western blot analysis to detect the activity of caspase-3, a critical component in cell apoptosis pathway. We found that the expression of caspase-3 did not change significantly among the treated groups (
Cells were transfected with the Notch1-siRNA and H101 for 72 hours, and total protein was analyzed by Western blot with specific antibodies.
We were also curious whether the combined Notch1-siRNA/H101 therapy would affect the expression of endogenous p53, an important component that affect H101 oncolysis and apoptosis. Three days after transfection with Notch1-siRNA and H101, HeLa-S3 cells were collected and total cellular protein was extracted. We found that the treatment of Notch1-siRNA, whether used alone or combined with H101, did not have significant effect on p53 protein level in treated cells (
We then used Western blot analysis to examine the expression of MDM2, a downstream target gene of p53. Three days after transfection with Notch1-siRNA and H101, both the H101 treatment and the combined H101/Notch-siRNA treatment significantly affected MDM2 protein expression in treated cells. Although the H101/siRNA combined therapy showed a slightly better effect than the H101/siNC control
We then focused on whether the Notch-siRNA alters viral replication in H101-infected tumors. Hexon protein is a component of the adenovirus capsid and is synthesized after cell infection. The synthesis of hexon protein marks the packaging of virus particles in the final replication stage. Thus, the amount of protein synthesized is considered to be a reliable indicator of viral replication.
To test whether Notch1-siRNA would affect H101 DNA replication, the expression of the late gene hexon was determined by real-time RT-PCR. We found that after Notch1-siRNA interference, the H101/Notch1-siRNA group had a significant increase of hexon mRNA expression compared with the H101 group (P<0.05,
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.
The Notch signaling pathway plays an important role in the regulation of cell growth and differentiation, tissue renewal, and cell homeostasis, and the pathway may be disregulated in several carcinomas
There is also a functional link between Notch and p53 activities. Notch1 is a
H101, which lacks E1B55-kDa, can specifically lyse tumor cells. However, H101 has limited potential to eradicate tumors when used as monotherapy. Thus, H101 is often used in combination with traditional modalities, such as chemotherapy. In this communication, we studied the antitumor efficacy of H101 in conjunction with siRNA to Notch1. As demonstrated in
We also tested this combined therapy in other three tumor cell lines that had different status of p53 mutations, including lung cancer cells (A549) and uveal melanoma cells (OCM1 and VUP). Both OCM1 and VUP cell lines contain a common mutation (C. 797G>A, P. Gly133Glu) in the exon 7 of p53
We also examined the cytotoxic effect of this combined therapy on cancer stem cells (CSCs). We first infected HeLa S3 cells with H101 and/or siNotch1. After 24 hours, cancer cells were cultured in CSC sphere culture medium and CSC spear numbers were recorded
The mechanism underlying the additive effect of the combined therapy remains uncharacterized. In tumor cells treated by the combined Notch1-siRNA and H101, we found an enhanced induction of apoptosis in HeLa-S3 cells, but we did not detect significant alteration of caspase-3 or activated caspase-3 expression. Neither Notch1-siRNA nor H101 appears to induce apoptosis by a non-caspase-3 pathway, and neither agent alters p53 expression. We also examined the expression of MDM2 and p21, which are the downstream targets of p53. MDM2 is an E3 ubiquitin ligase that targets p53 for ubiquitination and degradation. Both the MDM2 C-terminal region including the RING finger and the acidic domain are essential for p53 ubiquitination
Numerous studies have shown that the efficiency of adenovirus infection is related to the tumor cell surface receptor CAR. However, the expression of CAR in some tumor cells is relatively low. Extensive studies have attempted to alter viral tropism to increase infection rates and improve the anti-cancer effect
In an attempt to improve H101 anti-tumor efficacy, we previously demonstrated that H101 therapy was potentiated by concomitant use of a
In summary, this study provides support for the combined use of an oncolytic adenovirus and Notch1-siRNA as a promising approach in cancer gene therapy. We demonstrated an anticancer augmentation of the combined therapy of Notch1-siRNA and H101. Future studies will combine two therapies as a single adenoviral agent by integrating Notch1-siRNA into the H101 viral backbone. In addition, it will be interesting to examine whether the strategy used here would be more potent to target cancer stem cells (CSCs), particularly those CSCs cultured from clinical surgery or biopsy tumors.
Cervical cancer cell line HeLa-S3 is deficient in p53, and preclinical studies demonstrated that HeLa-S3 was very sensitive to H101 oncolytic treatment
Tumor cell lines HeLa-S3 (cervical cancer) and A549 (lung cancer cells) were obtained from American Type Culture Collection (Manassas, VA, USA). OCM1 and VUP (uveal melanoma were kindly provided by Professor John F. Marshall (Tumor Biology Laboratory, Cancer Research UK Clinical Center, John Vane Science Centre, London, UK)
The Notch1 siRNA duplexes were produced by Shanghai Genepharma Co. Inc. (Genepharma, Shanghai, China) against human Notch1 (5′-AAG GUG UCU UCC AGA UCC UGA-3′). Scrambled fluorescent-labeled siRNA (5′-AAA UGU GUG UAC GUC UCC UCC-3′) (siNC)
HeLa-S3 cells at 30–50% confluence in 24-well plates were transfected with 80 nmol/l Notch1 siRNA using Lipofectamine 2000 following the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). After overnight incubation, cells were infected with H101 at a multiplicity of infection of 100 MOI. Control groups included cells that were transfected with siNC or PBS.
Total RNA was isolated from treated cells using Trizol reagent (Invitrogen) following the protocol provided by the manufacturer. Total RNA (1µg) was reverse-transcribed into cDNA using M-MLV reverse transcriptase (Invitrogen)
Quantitative real-time RT-PCR amplification was carried out using Real-Time MIX (SYBR Premix Ex TaqTM, TaKaRa, Tokyo, Japan). Specifically, total RNA was extracted by Trizol reagent (Invitrogen), and cDNA was synthesized with RNA reverse transcriptase. The CT (threshold cycle) value of
Cells were harvested at the indicated time, and proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis in 10% SDS–polyacrylamide Tris–glycine gels for protein expression. The immunoblotting was performed with the Notch1 (Epitomics, CA, USA), p53 (Cell Signaling Technology, Inc., Danvers, MA, USA), Caspase 3 (Thermo Fisher Scientific, Loughborough, UK), Hexon (Abcam, Cambriadge, UK) and mouse β-actin (Sigma-Aldrich, St. Louis, MO, USA), followed by detection with a horseradish peroxidase–conjugated secondary antibody
Cells were seeded at 2×105 cells per well in flat-bottomed 6-well plates. At the end of the incubation time, the cells were harvested and the Caspase-3 Colorimetric Activity Assay Kit was used to detect the activity of caspase-3
The 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was performed to assess the effect of H101 in combination with RNA interference on cell proliferation
Apoptosis was determined by dual staining with annexin-V-fluorescein isothiocyanate and propidium iodide and analyzed by flow cytometry
Animal experiments were performed in accordance with institutional guidelines for animal care by Jiao Tong University. Specifically, HeLa-S3 cell tumor xenografts were established by s.c. injection of 1×106 cells into the right flank of 4–6-week-old male athymic nude mice. Based on the data from a pilot study, we initiated an early treatment when the tumor volume reached about 100 mm3 (volume = length×width2×0.5). Animals were randomly assigned into four groups. The Notch1-siRNA plus H101 group received intratumoral injections of 10 µg Notch1-siRNA on day 1, 4, 8, 11, 15, and H101 adenovirus at 1×108 plague-forming units on day 2, 5, 9, 12, and 16. The Notch1-siRNA group received five intratumoral injections of 10 µg Notch1-siRNA. The H101 adenovirus group received five intratumoral injections of H101. The control group mice received five injections of PBS. The tumor size was measured by vernier calipers every 4 days. Mice from each group were selected randomly and killed on day 7 after treatment for hematoxylin-eosin staining.
Specimens were dehydrated in ethanol series (80, 85, 80, 90, 95 and 100%), embedded in paraffin, and cut into 5 µm-thick sections. Sections were then deparaffinized, stained with hematoxylin/eosin (H–E) following standard protocol, and observed using a light microscope.
All experiments were performed in triplicate, and the data were expressed as mean ± SD. The data were analyzed with one-way analysis of variance (ANOVA), and results were considered statistically significant at P≤0.05.
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