Notch1 regulates the JNK signaling pathway and increases apoptosis in hepatocellular carcinoma

Abstract

Notch1-induced pathways are involved in cell growth, apoptosis, motility, and invasion in many cancers. In the present study, the expression of Notch1 and NICD1 was detected in hepatocellular carcinoma (HCC) tissues using in-vitro assays. And then, we explored cell biology and signaling pathways using Notch1 siRNA or plasmids. Here, the expression of Notch1 and NICD1 was significantly decreased in HCC tissues. In-vitro, Notch1 plasmids inhibited cell proliferation, migration and invasion, but enhanced apoptosis of HepG2 and Hep3B cells. Conversely, si-Notch1 enhanced cell proliferation, migration and invasion, but inhibited apoptosis of HepG2 and Hep3B cells. Mechanically, Notch1 decreased the expression of cyclin D1, MMP-9 and Bcl-2, but increased the expression of p-JNK, Bax and cleaved caspase 3 in HepG2 and Hep3B cells. Besides, si-JNK or JNK inhibitor SP600125 affected the activation of Notch1 signaling pathway, and prevents cell apoptosis. In conclusion, Notch1 regulates the JNK signaling pathway and increases apoptosis in HCC. Because patients with HCC have a poor prognosis, Notch1 pathway may provide a novel treatment strategy.

Keywords: HCC; JNK; Notch1.

Figure 1. The expression of Notch1 and NICD1 in liver cancer tissues and cells

(A) Low expression of Notch1 mRNA in tumor tissues than in normal tissues (*p<0.001). (B) 5 representative tissues were subjected to the western blot. T=tumor tissues, N=normal tissues (*p<0.001). (C) the plasmids and siRNA were used to establish HepG2 and Hep3B cell lines that expressed lower and higher levels of Notch1 and NICD1, respectively (*p<0.001). The vector and control siRNA-transfected cell line was designated as the control group. (D) The plasmids and siRNA were used to establish Hep3B cell lines that expressed lower and higher levels of Notch1 and NICD1, respectively (*p<0.001). The vector and control siRNA-transfected cell line was designated as the control group. * denotes significance at p<0.01 relative to control by student t-test.

Figure 2. Notch1 affects the proliferation of liver cancer cells

The HepG2 (A) and Hep3B (B) cells that were transfected with Notch1 plasmids or siRNA displayed significant growth changes compared with the cells that were transfected with the control after 24 h (*p<0.001). * denotes significance at p<0.01 relative to control by student t-test.

Figure 3. Notch1 affects apoptosis of HepG2 and Hep3B cells

(A) Apoptotic effects of plasmids and si-Notch1 on HepG2 and Hep3B cells were determined by TUNEL assay (green channel) at 90 minutes after treatment. DAPI (blue channel) is used to locate the nuclei of the cells. (*p<0.001). (B) HepG2 and Hep3B cells were transfected with Notch1 plasmids, Notch1 siRNA or the relative controls as indicated for 48 h, and then cells were stained with Annexin V-FITC/PI, and analyzed by flow cytometry as described in methods. The statistic data were presented as mean ± SEM from three independent experiments. Representative images for cell apoptosis stained with Annexin V-FITC/PI. (C) Representative micrographs (upper) and quantification (lower) of BrdU incorporation by different-treated cells. * denotes significance at p<0.01 relative to control by student t-test.

Figure 4. Cisplatin facilitates Notch1-induced cell apoptosis of HepG2 and Hep3B cells

We treated cells with apoptosis inducing agent cisplatin (0, 10, 20, 40μg/ml), and then cells were subjected to TUNEL assay (A), apoptosis assay (B) and BrdU incorporation assay (C). The statistic data were presented as mean ± SEM from three independent experiments. * denotes significance at p<0.01 relative to control by student t-test.

Figure 5. Notch1 affects proliferation and apoptosis-related gene expression

(A) Western blot analysis showed the expression of cleaved caspase-3, cyclin D1, MMP9, Bcl-2 and Bax of HepG2 cells was changed in the plasmids and si-Notch1 group than those in the control groups (*p<0.001). The statistic data were presented as mean ± SEM from three independent experiments. (B) Western blot analysis showed the expression of cleaved caspase-3, cyclin D1, MMP9, Bcl-2 and Bax of Hep3B cells was changed in the plasmids and si-Notch1 group than those in the control groups (*p<0.001). * denotes significance at p<0.01 relative to control by student t-test.

Figure 6. Notch1 affects the migration and invasion ability of liver cancer cells

A change in HepG2 and Hep3B cell migration (A, C) and invasion (B, D) was observed in the Notch1 plasmids or siRNA-transfected liver cancer cells (*p<0.001). * denotes significance at p<0.01 relative to control by student t-test.

Figure 7. Notch1 regulates JNK phosphorylation in liver cancer cells

(A) After transfection of the HepG2 and Hep3B cell lines, we tested the changes of total JNK levels and JNK phosphorylation. The knockdown of Notch1 significantly reduced the phosphorylation of JNK in both HepG2 and Hep3B cell lines, which is consistent with the general understanding that JNK is activated in transformed cells. However, the overexpression of Notch1 obviously enhanced the phosphorylation of JNK in both HepG2 and Hep3B cell lines (*p<0.001). (B, C) We transfected the si-JNK or si-control into HepG2 and Hep3B cells with Notch1 plasmids, and then the expression of p-JNK and t-JNK, and cell apoptosis were tested. The statistic data were presented as mean ± SEM from three independent experiments. * denotes significance at p<0.01 relative to control by student t-test. (D, E) We treated HepG2 and Hep3B cells with JNK inhibitor SP600125, and then the expression of p-JNK and t-JNK, and cell apoptosis were tested. The statistic data were presented as mean ± SEM from three independent experiments. * denotes significance at p<0.01 relative to control by student t-test.