Dipeptide γ-secretase inhibitor treatment enhances the anti-tumor effects of cisplatin against gastric cancer by suppressing cancer stem cell properties

Abstract

The γ-secretase inhibitor blocks Notch activity by preventing its cleavage at the cell surface. In the present study, the effect of the γ-secretase inhibitor on the viability of gastric cancer cells when administered in combination with cisplatin was investigated, with particular focus on CD44^highLgr-5^high cancer cells. The four gastric cancer cell lines, MKN45, MKN74, SC-6-JCK and SH-10-TC, were used for the experiments. In the MTT assay, treatment with 25 µM dipeptide γ-secretase inhibitor (DAPT) alone did not affect cell proliferation in any of the four cell lines. Gastric cancer cells subjected to combination treatment with DAPT and cisplatin exhibited decreased viability when compared with those treated with cisplatin alone. Flow cytometry was performed to evaluate the expression of cluster of differentiation (CD)-44 and leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr-5), two cancer stem cell markers in gastric cancers. Treatment with cisplatin alone significantly increased the proportion of CD44^highLgr-5^high cells. However, the addition of DAPT to cisplatin reduced the CD44^highLgr-5^high fraction, suggesting that DAPT reduced the number of gastric cancer cells. In conclusion, the present study demonstrated the synergistic effects of DAPT in combination with cisplatin by decreasing the survival of gastric cancer cells. In addition, combination treatment with DAPT reduced the number of CD44^highLgr-5^high cells, which are thought to exhibit cancer stem cell properties. These results highlight the therapeutic potential of DAPT in gastric cancer treatment.

Keywords: Notch pathway; cancer stem cell; gastric cancer; γ-secretase inhibitor.

Figure 1.

Expression of NICD and the downstream protein HES1 in MKN45 cells. MKN45 cells were treated with 50 µM DAPT for 24, 48 and 72 h. Then, cells were lysed and evaluated via western blot analysis using antibodies targeting NICD and HES1. Antibodies against β-actin were used to verify the equal loading of cellular proteins. Representative blots of >3 independent experiments are presented. NICD, intracellular domain of the Notch receptor; HES1, Hes family basic helix-loop-helix transcription factor 1; DAPT, dipeptide γ-secretase inhibitor.

Figure 2.

Effect of CDDP in (A) MKN45 and (B) MKN74 cells. Cells were treated with 0.1, 0.5 or 1.0 µg/m cisplatin for 72 h. Then, cells were lysed and cell proliferation was measured via the modified MTT assay. *P<0.05, as indicated. Data are presented as the mean ± standard deviation of three independent experiments. CDDP, cisplatin.

Figure 3.

Expression of NICD and the downstream protein HES1 in (A) MKN45 and (B) MKN74 cells. Cells were treated with CDDP (0.5 µg/ml) and/or DAPT (25 or 50 µM) for 72 h. Then, cells were lysed and analyzed via immunoblot analysis using antibodies targeting NICD and HES1. Antibodies against β-actin were used to verify equal loading of cellular proteins. Representative blots of >3 independent experiments are presented. NICD, intracellular domain of the Notch receptor; HES1, Hes family basic helix-loop-helix transcription factor 1; DAPT, dipeptide γ-secretase inhibitor; CDDP, cisplatin.

Figure 4.

Proliferation in the four gastric cancer cell lines, (A) MKN45, (B) MKN74, (C) SC-10-TC and (D) SH-6-JCK upon combined treatment with DAPT and CDDP. Cells were treated with CDDP (0.5 µg/ml) and/or DAPT (25 or 50 µM) for 72 h. Then, cells were lysed and analyzed via MTT assay. *P<0.05 and **P<0.01, as indicated. Data are presented as the mean ± standard error of the mean of >3 independent experiments performed in triplicate. N.S., not significant; DAPT, dipeptide γ-secretase inhibitor; CDDP, cisplatin.

Figure 5.

Expression of CD44 and Lgr-5 in (A) MKN45, (B) MKN74, (C) SH-10-TC and (D) SC-6-JCK cells upon combined treatment with DAPT and CDDP. Cells were cultured with or without CDDP and DAPT for 72 h. Then, cells were analyzed via flow cytometry using anti-CD44 and anti-Lgr-5 antibodies. Representative plots from >3 independent experiments are presented. CD44, cluster of differentiation 44; Lgr-5, leucine-rich repeat-containing G-protein coupled receptor 5; DAPT, dipeptide γ-secretase inhibitor; CDDP, cisplatin.

Figure 6.

Statistical analysis of CD44 and Lgr-5 expression in (A and B) MKN45, (C and D) MKN74, (E and F) SH-10-TC and (G and H) SC-6-JCK cells. Cells were cultured without cisplatin or DAPT (untreated controls), with cisplatin alone, or with DAPT and cisplatin for 72 h. Then, cells were analyzed via flow cytometry using anti-CD44 and anti-Lgr-5 antibodies. Three samples were used to calculate the mean ± standard deviation. *P<0.05 and **P<0.01, as indicated. N.S., not significant; CD44, cluster of differentiation 44; Lgr-5, leucine-rich repeat-containing G-protein coupled receptor 5; DAPT, dipeptide γ-secretase inhibitor.