Notch 2 signaling contributes to cell growth, anti-apoptosis and metastasis in laryngeal squamous cell carcinoma

Abstract

Notch signaling is important during the development of a variety of human tumors. Depending on the context, Notch signaling can be either oncogenic or anti‑proliferative, and therefore, its effects in cancer are unpredictable. The aim of the present study was to identify the importance of Notch 2 in the cell growth and metastasis of laryngeal squamous cell carcinoma (LSCC). The current study performed quantum dots‑based immunofluorescence histochemistry to determine expression of Notch 2 in 72 LSCC samples without lymph node metastasis, 23 LSCC samples with lymph node metastasis and 31 samples from vocal cord polyps. It was observed that Notch 2 was upregulated in LSCC tissue compared with normal vocal cord polyps. This upregulation was further enhanced in LSCC tissues with lymph node metastasis compared with LSCC tissues without lymph node metastasis. Following knockdown of NOTCH2 expression in LSCC cells, the in vitro tumorigenicity of Hep‑2 cells was inhibited, with growth, migration, invasion and proliferation reduced, and apoptosis induced. Additionally, following downregulation of Notch 2 protein expression, the protein expression levels of phospho‑mitogen‑activated protein kinase 1 (p‑ERK), v‑myc avian myelocytomatosis viral oncogene homolog and B‑cell CLL/lymphoma 2 (Bcl2) were also downregulated, whereas, Bcl2‑associated X protein expression was upregulated. There were no changes detected in the protein expression levels of total‑ERK, phospho‑v‑akt murine thymoma viral oncogene homolog 1 (p‑Akt) and total‑Akt. The results of the present study suggest that Notch 2 is important for the cell growth, anti‑apoptosis and metastasis of LSCC. Therefore, Notch 2 inhibitors may have therapeutic potential for the treatment of patients with LSCC via the inhibition of cancer cell growth and metastasis.

Figure 1

Notch 2 expression level is upregulated in LSCC samples. Representative images of quantum dots-based immunofluorescence histochemistry for Notch 2 expression in tissue from (A) LSCC with lymph node metastasis, (B) LSCC without lymph node metastasis and (C) vocal cord polyps. Magnification, ×100. White arrows indicate the Notch 2 expression. LSCC, laryngeal squamous cell carcinoma.

Figure 2

NOTCH2 was knocked down using shRNA. The expression of NOTCH2 mRNA and Notch 2 protein in Hep-2 cells 48 h after shRNA transfection measured by (A) reverse transcription-quantitative polymerase chain reaction, and (B) western blot with densitometric analysis, respectively. Notch 2-shRNA samples demonstrate a significantly reduced level of Notch 2. The data represent the results of three independent experiments. ^*P<0.05 vs. non-transfected samples, ^#P<0.05 vs. negative-shRNA samples. No significant difference was observed between the non-transfected and the negative-shRNA samples (P>0.05). shRNA, short hairpin RNA.

Figure 3

Effects of Notch 2 on cell morphology and viability. Notch 2-shRNA cells exhibited a higher rate of apoptosis compared with non-transfected or negative-shRNA cells. (A) Photographs of Hep-2 cell morphology captured 72 h after shRNA transfection. (B) Proliferation of Hep-2 cells was detected by Cell Counting kit-8 reagent following shRNA transfection. (C) Flow cytometry data of apoptotic Hep-2 cells and (D) the percentage of apoptotic Hep-2 cells 72 h after shRNA transfection. The data represent the results of three independent experiments. ^*P<0.05 vs. non-transfected cells, ^#P<0.05 vs. negative shRNA. shRNA, short hairpin RNA.

Figure 4

Knockdown of NOTCH2 inhibits the migration and invasion of Hep-2 cells. (A) NOTCH2 shRNA transfected Hep-2 cells exhibited inhibited cellular motility compared with the control cells. (B) The number of cells that migrated through uncoated filters (no Matrigel) represents the migratory ability of Hep-2 cells. (C) Representative images of the Transwell assay without Matrigel (upper panel) or pre-coated with Matrigel (lower panel) following shRNA transfection. (D) The number of cells that were able to pass through filters pre-coated with Matrigel represents the invasive ability of Hep-2 cells. The cell counts are presented as the mean ± standard deviation of ≥5 randomly selected low-power fields (×200) from three independent experiments. ^*P<0.05 vs. non-transfected samples, ^#P<0.05 vs. negative-shRNA samples. shRNA, short hairpin RNA.

Figure 5

Knockdown of NOTCH2 affects the expression of Notch 2 signaling pathway target genes in Hep-2 cells. (A) Western blot analysis demonstrating that the expression levels of p-ERK, c-Myc and Bcl-2 were downregulated, and the expression of Bax was upregulated. No differences were observed in the expression of t-ERK, p-Akt and t-Akt. (B) Quantification of the protein bands by densitometry. The results of three independent experiments are presented. All the histograms present the GAPDH-normalized mean ± standard deviation of the band density from the three experiments. ^*P<0.05 vs. non-transfected cells, ^#P<0.05 vs. negative shRNA. shRNA, short hairpin RNA; p, phospho; t, total; ERK, mitogen-activated protein kinase; AKT, v-akt murine thymoma viral oncogene homolog 1; c-Myc, v-myc avian myelocytomatosis viral oncogene homolog; Bax, BCL2-associated X protein; Bcl2, B-cell CLL/lymphoma 2.