SEARCH

SEARCH BY CITATION

Keywords:

  • Notch1;
  • colon cancer;
  • proliferation;
  • cell cycle;
  • apoptosis;
  • cancer stem cells

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONFLICT OF INTEREST DISCLOSURES
  7. REFERENCES

BACKGROUND:

The aberrant activation of the Notch signaling has been associated with the development of colon cancers. However, the role of Notch1 in the pathogenesis of colon cancers is poorly understood.

METHODS:

The expression of Notch1 in colon cancer tissues and nontumor tissues and in colon cancer cell lines was examined by Western blot analysis and immunohistochemistry. The impact of small interfering RNA (siRNA)-mediated Notch1 knockdown or Notch1 intracellular domain (NICD)-based transgene-induced Notch1 overexpression on the proliferation, cell cycling, apoptosis, colony formation, and tumorsphere formation in vitro and the development and growth of implanted tumors in vivo was characterized.

RESULTS:

Notch1 was overexpressed in colon cancer tissues, and the levels of Notch1 expression in different types of colon cancers were associated with the pathologic grade, progression, and metastasis of colon cancers. Furthermore, knockdown of Notch1 significantly inhibited the proliferation, colony formation, and tumorsphere formation of SW480 and HT-29 cells, induced apoptosis and cell cycle arrest at G0/G1 phase, and mitigated the development and growth of implanted colon cancers in vivo. In contrast, Notch1 overexpression promoted the proliferation, colony formation, cell cycling, and tumorsphere formation of colon cancer cells in vitro and the development and growth of implanted colon cancers in vivo, but it inhibited spontaneous apoptosis.

CONCLUSIONS:

The current results indicated that Notch1 signaling positively regulates the growth of colon cancers. Conceivably, the modulation of Notch1-related signaling may be a promising therapy for human colon cancers. Cancer 2010. © 2010 American Cancer Society.

The Notch signaling pathway plays a critical role in the development and homeostasis of tissues by regulating cell-fate decisions, proliferation, differentiation, and apoptosis.1, 2 There are 4 different Notch receptors (Notch1-4) and 5 different Notch ligands (Jagged 1, Jagged 2, Delta-like 1, Delta-like 3, and Delta-like 4) in mammalian cells. Commonly, Notch receptors are engaged and activated by binding of a ligand on neighboring cells. This process usually initiates the γ-secretase mediated proteolytic release of the Notch intracellular domain (NICD), which, in turn, translocates into the nucleus of the cells. The NICD in the nucleus interacts with the C promoter-binding factor-1 (CBF1) transcriptional cofactor and transactivates the target genes, such as those in the hairy and enhancer of split (Hes) and Hes related with YRPW motif (Hey) families.3

It is believed that Notch signaling regulates many aspects of intestinal development and epithelial renewal,4-7 and aberrant activation of the Notch1 signaling pathway has been associated with the development of colon cancers. Notch1 expression has been detected in colon adenocarcinoma but not in normal epithelial cells,8 and Notch1 activation reportedly was essential for the development of adenoma in adenomatous polyposis coli/ multiple intestinal neoplasia (ApcMin) mice.7 Recently, significantly up-regulated expression of the Notch1 and HES1 genes was observed in human colon cancer tissue samples,9 raising the issue of whether Notch1 expression is associated with the pathologic grade of different types of colon cancers. It is noteworthy that the roles of Notch1 signaling in the proliferation, cell cycle, and apoptosis of colon cancer cells and in the development and progression of colon cancers in vivo are poorly understood. Clearly, a better understanding of the role of Notch1 in the development of colon cancers may provide new insights into the tumorigenesis of colon cancers.

Herein, we examined Notch1 expression in different types of colon cancers from Chinese patients and in 4 colon cancer cell lines. We also generated stable Notch1-knockdown and transgenic colon cancer cell lines and characterized the impact of Notch1 knockdown or overexpression on the proliferation, colony formation, cell cycle, apoptosis, and tumorsphere formation of colon cancer cells in vitro and the growth of implanted colon cancer cells in vivo. We observed that Notch1-related signaling was a critical regulator of the proliferation and tumorsphere formation of colon cancer cells in vitro and of the growth of colon cancers in vivo. Our findings suggest that Notch1 signaling positively regulates the growth of colon cancers, and down-regulation of Notch1-related signaling may be a promising therapy for human colon cancers.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONFLICT OF INTEREST DISCLOSURES
  7. REFERENCES

Human Colon Cancer Samples

A tissue array containing 42 human colon samples was obtained from Shaanxi Chaoying Biotechnology (Xi'an, China). It included 5 colon samples from patients without tumors, 22 samples from patients with adenocarcinoma, 7 samples from patients with mucinous adenocarcinoma, and 8 samples from patients with signet ring cell carcinoma. The clinical and pathologic features of these samples are presented in Table 1. Thirteen pairs of matched colon cancer tissues and adjacent nontumor tissues were collected from the Department of General Surgery, Second Affiliated Hospital of Medical School, Xi'an Jiaotong University. All tissue samples were obtained from untreated patients who were undergoing surgery. For the use of these clinical materials for research purposes, we obtained prior consent from all patients and approval from the Institutional Research Ethics Committee. All histopathologic diagnoses and malignant classifications were determined by 2 pathologists in a blinded fashion, according to World Health Organization International Histological Classification of Tumors. This study was approved by the Internal Review Board of Medical School of Xi'an Jiaotong University.

Table 1. Correlation Between Notch1 Expression and Clinicopathologic Parameters in Patients With Colon Carcinoma
VariableNo. of PatientsNotch1 IRSP
++++++
  • IRS indicates immunoreactivity score; −, negative Notch1 expression; +, weak Notch1 expression; ++, moderate Notch1 expression; +++, strong Notch1 expression.

  • a

    P values were determined with the Wilcoxon rank-sum test.

  • b

    P value was determined with the Kruskal-Wallis H test.

Sex     .21a
 Men25 7117 
 Women12 246 
Age, y     .5a
 ≤5523 6107 
 >5514 356 
Grade of differentiation     <.001b
 Well8 8   
 Moderate6 141 
 Poor23  1112 
Lymphatic metastases     <.001a
 Negative22 8122 
 Positive15 1311 

Immunohistochemistry

Immunohistochemistry was performed on 5-μm sections that were prepared from paraffin-embedded tissue arrays. Briefly, tissue sections were successively deparaffinized and rehydrated, followed by pretreated with Tris/ethylene diamine tetracetic acid (EDTA) buffer (10 mM Tris-HCl and 1 mM EDTA, pH 9.0) in a steam pressure cooker. After treating with 3% H2O2 and blocking with 20% normal rabbit serum for 1 hour at room temperature, the expression of Notch1 was analyzed with goat polyclonal antibodies against Notch1 (1:100 dilution; sc-6014; Santa Cruz Biotechnology, Santa Cruz, Calif) overnight at 4°C. The tissue sections were incubated with biotinylated rabbit antigoat immunoglobulin G (IgG) for 30 minutes at room temperature. After washing, the sections were incubated in streptavidin-peroxidase complex for 30 minutes, and immunostaining was developed using 0.05% 3′,3′-diaminobenzidine tetrahydrochloride followed by counterstaining with hematoxylin. Sera from nonimmunized goats were used as negative controls. The immunoreactivity and subcellular localization of Notch1 were evaluated by 3 separate pathologists in a blinded manner, as described previously.10 Briefly, a Notch1 immunoreactivity score (IRS) was determined by multiplying the Notch1 staining intensity (scored as 0, no staining; 1, weak staining; 2, moderate staining; or 3, strong staining) and the percentage of Notch1-positive cells (scored as 1, 0%-25% positive cells; 2, 26%-74% positive cells; 3, 75%-89% positive cells; or 4, 90%-100% positive cells). To determine the Notch1 IRS, 1000 cells were observed in 5 to 10 adjacent high-power fields at ×400 magnification in the strongest staining area of each sample.

In addition, cells were cultured on glass coverslips for 48 hours, fixed with 4% paraformaldehyde for 20 minutes, and permeabilized with 0.3% Triton X-100 for 20 minutes at room temperature. The expression of Notch1 in those cells was determined by immunocytochemistry, as described above.

Plasmid Construction

The DNA fragment that encodes the NICD was amplified by polymerase chain reaction using combinational DNA derived from human glioma U251MG cells as a template along with specific primers. The sequences of primers were 5′-CTCGAGAATATGGTGCTGCTGTCCCGCAAG-3′ and 5′-GGATCCGCACACAGACGCCCGAAGG-3′. The DNA fragment was cloned into pGEM-T Easy Vector (Promega, Madison, Wis) and subjected to sequence analysis. The correct DNA fragment subsequently was cloned into the XhoI and BamHI sites of the internal ribosome entry site (IRES) vector pIRES2-enhanced green fluorescent protein (EGFP) (Clontech, Mountain View, Calif) to generate pIRES2-EGFP-NICD.

To generate plasmids that express Notch1-specific small interfering RNA (siRNA), the oligonucleotide inserts for hairpin siRNA targeting the Notch1 messenger RNA (mRNA) were designed using an online siRNA design tool (Ambion, Austin, Tex). The oligonucleotide sequences were 5′-GATCCTGGCGGGAAGTGTGAAGCGTTCAAGAGACGCTTCACACTTCCCGCCATTA-3′ and 5′-AGCTTAATGGCGGGAAGTGTGAAGCGTCTCTTGAACGCTTCACACTTCCCGCCAG-3′. The synthesized oligonucleotide inserts were annealed and cloned into a linearized pSilencer4.1-cytomegalovirus neo siRNA expression vector (Ambion), according to the manufacturer's instructions, to generate pSilencer4.1-siNotch1. The plasmid pSilencer4.1-siControl, which was provided by the supplier, was used as a negative control and encoded a hairpin siRNA with a sequence that is not found in human genome databases.

Cell Cultures and Generating Stable Transfectants

The human colon adenocarcinoma cell lines SW480, SW620, HCT-116, and HT-29 were maintained in Dulbecco's Modified Eagle's Medium (DMEM) (Sigma-Aldrich, St. Louis, Mo) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, Calif) and 1% penicillin-streptomycin. All cell lines were maintained at 37°C in an atmosphere of 5% carbon dioxide.

To generate stable transfected cell lines, SW480 and HT-29 cells were transfected with pSilencer4.1-siNotch1, pSilencer4.1-siControl, pIRES2-EGFP, or pIRES2-EGFP-NICD, respectively, using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocols. One day later, the cells were treated with 1 mg/mL G418 reagent (Calbiochem, La Jolla, Calif) and exposed to fresh media containing the same concentration of G418 every 3 days for 4 weeks. Individual drug-resistant clones were collected and expanded for further identification.

Western Blot Analysis

All fresh tissues and cells were lysed in a radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 2 mM EDTA; 1% NP-40; and 0.1% sodium dodecyl sulfate [SDS]) that contained a protease inhibitor cocktail (Complete Mini; Roche Diagnostics, Branchburg, NJ). The lysate samples were separated by 8% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. After blocking with 5% fat-free milk in Tris-buffered saline, Notch1 and control β-Actin were probed with goat anti-Notch1 antibody (1:100 dilution; sc-6014; Santa Cruz Biotechnology) or with mouse anti-β-Actin antibody (1:500 dilution; sc-47778; Santa Cruz Biotechnology) overnight at 4°C, respectively. After washing, the bound antibodies were visualized with horseradish peroxidase (HRP)-conjugated antigoat or antimouse IgG (Thermo Fisher Scientific Inc., New York, NY) and the Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, Mass).

Cell Proliferation and Colony Formation Assays

Cells (5 × 104) were cultured in triplicate in 35-mm tissue culture dishes for 7 days. The cells were harvested longitudinally, and the cells were counted using a hemocytometer under light microscopy. To test the ability of colony formation, 1000 cells of each type were cultured in triplicate in 10-cm tissue culture dishes and exposed to fresh media every 3 days for 2 weeks. Then the cell colonies that were formed were stained with 0.01% crystal violet and counted.

Flow Cytometry Analysis

Cells (6 × 105 per well) were cultured in 6-well plates for 24 hours and labeled with bromodeoxyuridine (BrdU) solution for 20 minutes to determine their rate of proliferation. The cells were harvested and stained with anti-BrdU using the APC BrdU Flow Kit (BD Pharmingen), according to the manufacturer's instructions, and they were subjected to fluorescence-activated cell sorter (FACS) analysis.

The cultured cells were harvested at 105 cells per tube and stained in duplicate with APC annexin V and propidium iodide (PI) (BD Pharmingen) to characterize spontaneous cell apoptosis, according to the manufacturer's instructions. In addition, the cells were harvested, washed twice with phosphate-buffered saline (PBS), and fixed with ice-cold ethanol overnight at 4°C. After washing twice with PBS, the cells at 106 cells per tube were treated with RNase A and stained with PI (Sigma-Aldrich) in the dark, followed by FACS analysis on a FACS Calibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ).

Tumorsphere Formation Assay

To obtain tumorspheres, each cell type at 103 cells per well was cultured in DMEM/F12 with 1 × B-27 serum-free supplement (Invitrogen), 20 ng/mL epidermal growth factor (EGF), and basic fibroblastic growth factor (PeproTech Inc., Rocky Hill, NJ) in 24-well, ultra-low attachment plates for 14 days. Under these culture conditions, only cancer stem cells (CSCs) and early progenitor cells, and not differentiated cancer cells, could survive and proliferate.11-13 The formed tumorspheres were examined and counted under a microscope.

Tumor Xenotransplantation

Groups of Balb/c nude mice at ages 6 to 8 weeks were injected subcutaneously with 2 × 106 SW480 cells or 1 × 106 HT-29 cells and housed in a pathogen-free facility. The development and progression of solid tumors were monitored longitudinally for 6 to 8 weeks. The tumor volume (V) was determined by the length (a) and width (b) as V = ab2/2. At the end of the experiment, the formed tumors were dissected out, and their net weights were measured. The experimental protocols were evaluated and approved by the Animal Care and Use Committee of the Medical School of Xi'an Jiaotong University.

Statistical Analysis

The ordinal data from semiquantitative analysis of immunoreactivity were analyzed with Wilcoxon rank-sum tests and Kruskal-Wallis H tests. Measurement data were analyzed with Student t tests. Statistical analyses were performed with the Statistical Package of Social Science (SPSS) version 16.0 for Windows (SPSS Inc., Chicago, Ill). A P values <.05 were considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONFLICT OF INTEREST DISCLOSURES
  7. REFERENCES

Notch1 Expression in Human Colon Cancer Tissues and Cell Lines

To determine Notch1 expression, Western blot analysis was performed on 13 pairs of colon cancer tissues and corresponding adjacent nontumor tissues. Notch1 expression in all 13 cancer tissues was significantly higher than that in matched nontumor tissues (P < .001) (Fig. 1A,B). To examine further whether Notch1 protein up-regulation was linked to the clinical progression of colon cancer, a tissue array of 42 tissue samples from 37 patients with colon cancer and 5 patients without tumors was characterized for the expression of Notch1 by immunohistochemistry. Although Notch1 was undetectable in 4 of 5 samples of nontumor colon epithelia, broadly weak cytoplasmic staining of Notch1 was observed in 1 nontumor colon tissue sample (Fig. 1C1). In contrast, Notch1 expression was detected in all colon cancer samples (Fig. 1C2-C6). It is noteworthy that moderate to high levels of Notch1 expression were observed predominately in the cytoplasm of grade 2/3 adenocarcinomas and mucinous adenocarcinomas, whereas positive nuclear staining for anti-Notch1 was observed in signet ring cell carcinomas (Fig. 1C6). In addition, the level of Notch1 expression was associated positively with the pathologic grade of colon cancer in these patients (P < .001) (Fig. 1D). Distribution of the Notch1 IRS in different groups of colon cancer samples is illustrated in Figure 1E. Notch1 expression in mucinous adenocarcinomas and signet ring cell carcinomas tended to be higher than that observed in the other samples that were analyzed. Stratification analysis revealed that the Notch1 IRS in patients with lymphatic metastases was statistically higher than that in patients without lymphatic metastases (P < .001) (Table 1), but the IRS was not associated with age or sex. Notch1 expression was observed in all colon cancer samples and appeared to be associated with the pathologic grade and progression of colon cancer. In addition, we characterized the expression of Notch1 in colon cancer cell lines. Notch1 expression was detected in both the nucleus and cytoplasm of these cells, suggesting the activation of Notch1 signaling in colon cancer cells (Fig. 1F). Therefore, we established that the aberrant activation of Notch1 signaling contributes to the development and progression of colon cancer.

thumbnail image

Figure 1. The expression of Notch1 is illustrated in colon cancer tissues, nontumor colon tissues, and colon cancer cell lines. (A) These representative Western blot analyses show Notch1 expression in 13 pairs of matched colon cancer tissues (CC) and adjacent nontumor tissues (NT). (B) Notch1 protein expression was compared between matched, adjacent NT and CC tissues in 13 patients. The paired t test was used for statistical analyses. (C) Immunohistochemical staining is shown for Notch1 in human colon tissues, including (C1) NT colon tissue, (C2) grade 1 adenocarcinoma, (C3) grade 2 adenocarcinoma, (C4) grade 3 adenocarcinoma, (C5) mucinous adenocarcinoma, and (C6) signet ring cell carcinoma. Each inset shows a negative control. (D) Semiquantitative analysis of Notch1 expression is illustrated. Data are shown as the mean (±standard error) immunoreactivity score (IRS) for each group. P values were determined with the Kruskal-Wallis H test. (E) The distribution according to individual IRS is shown in different colon cancer tissues. Data shown are the mean IRS of individual colon cancers. (F) Immunocytochemistry staining for Notch1 is illustrated in colon cancer cell lines. Each inset shows a negative control.

Download figure to PowerPoint

Notch1 Regulates the Proliferation of Colon Cancer Cells In Vitro

Next, we examined how Notch1 modulated the proliferation of colon cancer cells. We designed 4 siRNAs that targeted Notch1 mRNA and observed that 1 of the siRNAs effectively inhibited the expression of Notch1 in SW480 cells and HT-29 cells, as evidenced by remarkable reduction of Notch1 expression in stable SW480-siNotch1 and HT-29–siNotch1 cells compared with control cells (Fig. 2A). An analysis of these cell lines revealed that significantly reduced numbers of SW480-siNotch1 and HT-29–siNotch1 cells were present throughout the observation period (Fig. 2B), and there were lower percentages of BrdU-positive SW480-siNotch1 and HT-29–siNotch1 cells (Fig. 2C) compared with the percentages in control cells. Similarly, the numbers of formed colonies in SW480-siNotch1 and HT-29–siNotch1 cells also were significantly smaller than in SW480-siControl and HT-29–siControl cells (P < .001) (Fig. 2D). Apparently, knockdown of Notch1 expression significantly inhibited the proliferation of SW480 and HT-29 cells in vitro.

thumbnail image

Figure 2. Notch1 regulates the proliferation of colon cancer cells in vitro. (A) Western blot assays were used to characterize the expression of small interfering RNA (siRNA)-mediated Notch1 (siNotch1) in SW480-siNotch1 cells, in SW480 plasmid pSilencer4.1-siRNA control (siControl) cells, in HT-29–siNotch1 cells, and in HT-29–siControl cells. (B) The proliferation of SW480-siNotch1 cells (green line), SW480-siControl cells (blue line), HT-29–siNotch1 cells (purple line), and HT-29–siControl cells (yellow line) was determined by counting cells longitudinally. (C) The proliferation of siNotch1 and siControl cells also was evaluated in both cell lines with bromodeoxyuridine (BrdU) incorporation followed by fluorescence-activated cell sorter (FACS) analysis. (D) These photomicrographs show colony-formation assays, and the graph illustrates quantitative analysis. (E) Western blot analysis of Notch1 expression is shown in SW480 enhanced green fluorescent protein (EGFP) cells, in SW480 Notch1 intracellular domain (NICD) cells, in HT-29–EGFP cells, and in HT-29–NICD cells. (F) This chart shows growth curves for SW480-EGFP cells (blue line), SW480-NICD cells (green line), HT-29–EGFP cells (yellow line), and HT-29–NICD cells (purple lines). (G) Cell proliferation after EGFP and NICD treatment also was evaluated in both cell lines with BrdU incorporation followed by FACS analysis. (H) These photomicrographs show colony-formation assays, and the graph illustrates quantitative analysis. Data shown are the means ± standard error for representative images from 3 separate experiments. P values were determined with the Student t test.

Download figure to PowerPoint

Previous studies have indicated that Notch1 regulates the transformation and oncogenesis of nontumor cells in a dose-dependent manner.14-16 To further confirm the role of Notch1, we established stable SW480-NICD, HT-29–NICD, and control cell lines. After confirming several-fold increases in Notch1 expression in SW480-NICD and HT-29–NICD cells (Fig. 2E), we characterized their proliferation and colony formation. Obviously, the induction of Notch1 overexpression promoted the proliferation of colon cancer cells, as evidenced by significantly increased numbers of SW480-NICD and HT-29–NICD cells over a 7-day culture (Fig. 2F), higher percentages of BrdU-positive cells (Fig. 2G), and increased numbers of colonies in SW480-NICD and HT-29–NICD cells (Fig. 2H) compared with their control cells. Collectively, our data indicate that Notch1 is a critical regulator of colon cancer cell proliferation in vitro.

Notch1 Regulates the Cell Cycle and Apoptosis of Colon Cancer Cells In Vitro

Changes in cell proliferation usually are associated with modulation of the cell cycle. To explore the potential mechanism underlying the action of Notch1 expression in regulating colon cancer cell proliferation, cell cycles were characterized by FACS analysis. The percentages of SW480-siNotch1 cells and HT-29–siNotch1 cells in G0/G1-phase increased significantly to 86.8% and 80.67%, respectively, accompanied by decreasing percentages of cells in S-phase to 6.6% and 14.38%, respectively (Fig. 3A,B), suggesting that the knockdown of Notch1 expression induced cell cycle arrest in G0/G1-phase. Conversely, the percentages of HT-29–NICD cells in S-phase and in G2/M-phase increased significantly by 47.38% and 213%, respectively, compared with the percentages of control HT-29–EGFP cells (Fig. 3A,B). Similar cell cycle changes were observed in SW480-NICD cells, indicating that the overexpression of Notch1 promoted cell cycle progression in colon cancer cells.

thumbnail image

Figure 3. Notch1 regulates the cell cycle and apoptosis of colon cancer cells in vitro. The impact of Notch1 knockdown and overexpression on cell cycling and apoptosis was determined by fluorescence-activated cell sorter (FACS) analysis. Cells were stained with propidium iodide (PI), and cell cycling was measured by (A) FACS analysis and (B) quantitative analysis. (C) Similarly, cells were stained with allophycocyanin-annexin V/PI, and spontaneous apoptosis was examined by FACS analysis. Data shown are the means of representative images from 3 independent experiments. siControl indicates plasmid pSilencer4.1-small interfering RNA (siRNA) control; siNotch1, siRNA-mediated Notch1; EGFP, enhanced green fluorescent protein; NICD, Notch1 intracellular domain.

Download figure to PowerPoint

We also analyzed the frequency of apoptotic cells and observed that 21.61% of SW480-siControl cells and 22.57% of SW480-EGFP cells had undergone apoptosis, whereas 35.38% and only 14% of apoptotic cells were detected in SW480-siNotch1 cells and SW480-NICD cells, respectively (Fig. 3C). In addition, parallel trends were observed in HT-29–siNotch1 cells and HT-29–NICD cells, indicating that the knockdown of Notch1 expression promoted colon cancer cell apoptosis and that the overexpression of Notch1 inhibited spontaneous colon cancer cell apoptosis. Clearly, modulation of the cell cycle and apoptosis by Notch1-mediated signaling regulates the proliferation of colon cancer cells in vitro.

Notch1 Regulates the Growth of Colon Cancer Cells In Vivo

Next, we explored the hypothesis that the modulation of colon cancer cell proliferation, cell cycle, and spontaneous apoptosis by Notch1 should alter the growth of colon cancer cells in vivo. To test this hypothesis, we created nude mice xenograft assays, and the development and growth of solid colon cancers were monitored longitudinally (Fig. 4A,B). The growth of solid tumors in the SW480-siNotch1 group was delayed significantly, and 2 of 5 mice notably failed to develop solid tumors. The tumor weights generated from SW480-siNotch1 and HT-29–siNotch1 cells were reduced significantly (P < .001) (Fig. 4C). In contrast, the growth of tumors in the SW480-NICD and HT-29–NICD group was accelerated, and the tumor weights greatly increased (P = .004 and P = .002, respectively) (Fig. 4C). It is noteworthy that 3 of 6 mice implanted with SW480-NICD cells died from their tumors, and had distant metastasis confirmed. Together, our data indicated that knockdown of Notch1 expression inhibited the growth of tumors, whereas inducing Notch1 overexpression promoted the growth of tumors. Therefore, our results indicate that Notch1 regulates the growth of colon cancers in vivo.

thumbnail image

Figure 4. Notch1 regulates the growth of colon cancers in vivo. (A,B) Groups of Balb/c nude mice were implanted subcutaneously, and the development and growth of solid tumors were monitored longitudinally for 6 to 8 weeks. (C) At the end of the experiments or when the 3 mice in the SW480 Notch1 intracellular domain (NICD) group died because of their tumors at 5 or 6 weeks postimplantation, the mice were killed, the tumors were dissected out, and the tumor net weights were measured. Data shown are the means ± standard error for each group (n=5 for the SW480 plasmid pSilencer4.1-small interfering RNA [siRNA] control [siControl] group and the SW480 siRNA-mediated Notch1 [siNotch1] group; n=6 for the SW480 enhanced green fluorescent protein [EGFP] and SW480-NICD groups and for the HT-29–siControl, siNotch1, EGFP, and NICD cell groups). P values were determined with the Student t test.

Download figure to PowerPoint

Notch1 Regulates the Formation of Tumorspheres In Vitro

Recently, several studies have indicated the existence of CSCs in colorectal cancers,17-20 and CSCs are critical for the maintenance of tumor growth, progression, and metastasis.21, 22 It has been demonstrated that Notch signaling regulates the proliferation and differentiation of CSCs in other types of cancers.21, 23, 24 Conceivably, Notch1 may regulate CSCs in colon cancer. Given the lack of widely recognized surface markers for the identification of colon CSCs, we used a tumorsphere culture system to explore the potential role of Notch1 on tumorsphere-forming cells. Figure 5A illustrates how the knockdown of Notch1 expression inhibited the tumorsphere formation and growth of SW480-siNotch1 and HT-29–siNotch1 cells, as evidenced by the significantly reduced numbers of tumorspheres compared with the numbers in control cells (P < .001). Conversely, the induction of Notch1 overexpression promoted the formation and growth of tumorspheres, and the numbers of tumorspheres formed by SW480-NICD and HT-29–NICD cells were significantly greater than the numbers in control cells (P < .001 and P = .001, respectively) (Fig. 5B). Similar data were obtained in a 96-well–based, single-cell tumorsphere culture (data not shown). Our findings indicate that Notch1-related signaling is a critical regulator of the proliferation of tumorsphere-forming colon CSCs.

thumbnail image

Figure 5. Notch1 regulates the formation of tumorspheres in vitro. The impact of Notch1 knockdown and overexpression on the formation of tumorspheres was determined by tumorsphere formation assays. The formed tumorspheres were imaged and counted (A) under a light microscope or (B) under a fluorescent microscope. Charts on the right illustrate quantitative analysis of the tumorspheres. Data are expressed as representative images or as the means ± standard error for each group from 3 separate experiments. P values were determined with the Student t test. siControl indicates plasmid pSilencer4.1-small interfering RNA (siRNA) control; siNotch1, siRNA-mediated Notch1; EGFP, enhanced green fluorescent protein; NICD, Notch1 intracellular domain.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONFLICT OF INTEREST DISCLOSURES
  7. REFERENCES

In the current study, we examined the expression of Notch1 in colon cancers and in nontumor colon tissues and observed that Notch1 was overexpressed in all cancer tissues. It is noteworthy that the levels of Notch1 expression in different types of colon cancers were associated positively with the pathologic grades and metastatic status of colon cancers. It is also worth noting that, although moderate to high levels of cytoplasmic Notch1 expression were detected in the majority of grade 2 and 3 adenocarcinomas and mucinous adenocarcinomas, high levels of Notch1 expression were observed predominately in the nuclei of signet ring cell carcinomas. Upon activation, Notch usually is cleaved, and NICD is translocated into the nucleus of cells for transcriptional activation of the downstream genes. The cytoplasmic accumulation of Notch1 in adenocarcinomas and mucinous adenocarcinomas may reflect cytoplasmic signal activation of Notch1 for regulating the growth of adenocarcinomas and mucinous adenocarcinomas. Although the mechanism underlying the cytoplasmic distribution of Notch1 is unclear, Notch1 activation can induce the expression of Hes3 and sonic hedgehog (Shh) by activating cytoplasmic serine/threonine kinase Akt, signal transducer and activator of transcription 3 (STAT3), and mammalian target of rapamycin (mTOR), promoting the survival of neural stem cells.25 Therefore, the differential distribution of Notch1 in colon cancers suggests that Notch1 may be engaged by different ligands that regulate the growth of different types of colon cancers through different pathways. We are interested in further investigating the regulatory role of Notch1-related signaling in the development and growth of different types of human colon cancers.

To determine the role of Notch1-related signaling in the growth of colon cancers, we established Notch1-knockdown and Notch1-overexpressing, stable SW480 and HT-29 cell lines by inducing siRNAs that targeted Notch1-specific mRNA or the NICD transgene. We observed that knockdown of Notch1 expression significantly inhibited cell proliferation and colony formation, induced cell cycle arrest in G0/G1 phase but promoted the apoptosis of SW480-siNotch1 and HT-29–siNotch1 cells in vitro. In parallel, the induction of Notch1 overexpression promoted cell proliferation, colony formation, cell cycling but inhibited spontaneous apoptosis of SW480-NICD and HT-29–NICD cells in vitro. We also observed that, whereas the knockdown of Notch1 expression retarded the development and growth of implanted colon cancers, the induction of Notch1 overexpression accelerated the development and growth of implanted colon cancers in vivo. These 2 lines of evidence demonstrate that Notch1-related signaling is a critical regulator of the development and growth of colon cancers. Our data are in agreement with previous findings in other types of tumors,26-29 suggesting that Notch1-related signaling may have a similarly regulatory effect on the growth of different types of human malignancies.

Recently, several studies have suggested the existence of CSCs in colon cancers,17-20 and it is believed that those CSCs are responsible for tumor initiation, progression, metastasis, and resistance to therapy.21-24, 30 Indeed, it has been reported that Notch-related signaling regulates the proliferation of CSCs in human lung, breast, medulloblastoma, and pancreas cancers.21, 24, 31, 32 We characterized the role of Notch1 in the proliferation of colon CSCs by using tumorsphere formation assays and observed that the knockdown of Notch1 expression significantly mitigated the formation of tumorspheres, whereas the induction of Notch1 overexpression remarkably increased the number of tumorspheres formed in an in vitro culture condition that allowed the proliferation of only CSCs and progenitor cells.11-13 Hence, Notch1-related signaling positively regulated the proliferation of CSCs and progenitor cells in colon cancers. Therefore, it is possible that Notch1-related signaling may regulate the growth of human colon cancers by promoting the proliferation of both CSCs and cancer cells. We did not purify CSCs for tumorsphere formation in vitro, because there are no reliable surface markers for identifying colon CSCs.17-20 We are interested in further examining whether the proposed surface markers, such as cluster of differentiation 44 (CD44) (a cell surface glycoprotein), CD24 (a cell adhesion molecule), CD133 (a transmembrane glycoprotein), epithelial-specific antigen (ESA), CD166 (a protein encoded by the activated leukocyte cell adhesion molecule gene), and aldehyde dehydrogenase 1 (ALDH1), could be used to identify colon CSCs and to investigate the role of Notch1-related signaling in the proliferation and differentiation of colon CSCs.

During the preparation of this article, a new report indicated that treatment with γ-secretase inhibitors (GSI) and oxaliplatin significantly increased the apoptosis of HCT-116 cells,33 suggesting that modulation of Notch-related signaling may effectively control the progression of colon cancers. However, γ-secretase can cleave many proteins, including Notch, E-cadherin, N-cadherin, and CD44,34 and the inhibition of its activity may result in severe side effects. Indeed, clinical trials of γ-secretase have been hindered because of widespread toxicity.35 We used siRNA technology to successfully implement the knockdown of Notch1 expression and significantly inhibited the proliferation, colony formation, and tumorsphere formation of colon cancer cells in vitro and the growth of colon cancers in vivo. Because of its high specificity, potentially, the siRNA-mediated knockdown of Notch1 expression may be a novel and safe strategy for intervention in human colon cancers.

In summary, our data indicate that Notch1 is overexpressed in colon cancer cells, and levels of Notch1 expression are associated with the pathologic grade and metastasis of colon cancer. Furthermore, Notch1-related signaling may positively regulate the growth of human colon cancers by promoting the proliferation and survival of CSCs and colon cancer cells. Conceivably, the modulation of Notch1-related signaling by the siRNA-mediated knockdown of Notch1 expression may be a promising therapy for patients with colon cancer.

CONFLICT OF INTEREST DISCLOSURES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONFLICT OF INTEREST DISCLOSURES
  7. REFERENCES

Supported by Grants 30571951 and 30725043 from the National Natural Science Foundation of China.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONFLICT OF INTEREST DISCLOSURES
  7. REFERENCES