Targeting Inhibition of Notch1 Signaling Pathway on the Study of Human Gastric Cancer Stem Cells with Chemosensitization

Abstract

Background: Gastric cancer is the second most frequent cause of cancer death worldwide, although much geographical variation in incidence exists. Prevention and personalized treatment are regarded as the best options to reduce gastric cancer mortality rates (Hartgrink et al., 2009). Numerous studies have suggested that Notch1 and its ligands are overexpressed in gastric cancer, and its knockdown can inhibit the proliferation and survival of gastric cancer cells.

Objective: To investigate the effect of Notch1 on the stemness and drug sensitivity of human gastric cancer SGC-7901 cells.

Methods: Highly expressed Notch1 intracellular domain (NICD1) and Notch1-shRNA lentiviral expression vector were used to infect human gastric cancer SGC-7901 cells cultured in vitro, and western blot and immunofluorescence staining were used to identify highly expressed NICD and Notch1 silenced cells. The percentage of CD133+ cells was analyzed by flow cytometry, the expression of nestin and CFAP by immunofluorescence staining, the formation rate of tumor cell spheres and the tumorigenicity of SCID mice in vivo, and the regulation of cell stemness by Notch1. The sensitivity of each group of cells to the chemotherapeutic drugs teniposide (VM-26) and carmustine (BCNU) was also detected by the MTT method.

Results: The stemness phenotype of tumor cells with the increased NICD expression was enhanced, such as an increased proportion of CD133+ cells, enhanced nestin expression, decreased GFAP expression, increased tumor cell sphere formation rate and tumorigenic rate of SCID mice implantation, and decreased sensitivity to VM-26 and BCNU. In contrast, the stemness phenotype of tumor cells with downregulated Notch1 gene expression was significantly suppressed, while the sensitivity to VM-26 and BCNU was increased.

Conclusion: High Notch1 expression increased the stemness of SGC-7901 cells and decreased the sensitivity of SGC-7901 cells to chemotherapeutic drugs.

Figure 1

The protein expression of Notch1 in each group detected by western blot. Mean ± SD. n = 3. ^∗∗P < 0.01 vs. pLVX; ^##P < 0.01 vs pLKO-Notch1-NC the protein expression of Notch1 in each group was detected by western blot.

Figure 2

Identification of NICD expression in each group of cells by immunofluorescence staining.

Figure 3

Percentage of CD133 + cells in each group of cells. Mean ± SD. n = 3. ^∗∗P < 0.01 vs. pLVX; ^##P < 0.01 vs. pLKO-Notch1-NC.

Figure 4

The expression of nestin (red) in each group detected by immunofluorescence staining.

Figure 5

The expression of GFAP (red) in each group detected by immunofluorescence staining.

Figure 6

The formation rate of tumor spheres in each group cultured with stem cell medium. Mean ± SD. n = 3. ^∗P < 0.05 vs. pLVX; ^#P < 0.05 vs pLKO-Notch1-NC. The rate of formation of tumor spheres in each group cultured with stem cell medium.

Figure 7

Overexpression of NICD enhanced the formation of xenograft tumor in SCID mice.

Figure 8

The effects of chemotherapeutics on the survival rate of the cells with different treatments. Mean ± SD. n = 3. ^∗∗P < 0.01 vs. pLVX; ^##P < 0.01 vs. pLKO-Notch1-NC.