Targeting Notch1 signaling pathway positively affects the sensitivity of osteosarcoma to cisplatin by regulating the expression and/or activity of Caspase family

Abstract

Background: The introduction of cisplatin has improved the long-term survival rate in osteosarcoma patients. However, some patients are intrinsically resistant to cisplatin. This study reported that the activation of Notch1 is positively correlated with cisplatin sensitivity, evidenced by both clinical and in vitro data.

Results: In this study, a total 8 osteosarcoma specimens were enrolled and divided into two groups according to their cancer chemotherapeutic drugs sensitivity examination results. The relationship between Notch1 expression and cisplatin sensitivity of osteosarcoma patients was detected by immunohistochemistry and semi-quantitative analysis. Subsequently, two typical osteosarcoma cell lines, Saos-2 and MG63, were selected to study the changes of cisplatin sensitivity by up-regulating (NICD1 plasmid transfeciton) or decreasing (gamma-secretase complex inhibitor DAPT) the activation state of Notch1 signaling pathway. Our results showed a significant correlation between the expression of Notch1 and cisplatin sensitivity in patient specimens. In vitro, Saos-2 with higher expression of Notch1 had significantly better cisplatin sensitivity than MG63 whose Notch1 level was relatively lower. By targeting regulation in vitro, the cisplatin sensitivity of Saos-2 and MG63 had significantly increased after the activation of Notch1 signaling pathway, and vice versa. Further mechanism investigation revealed that activation/inhibition of Notch1 sensitized/desensitized cisplatin-induced apoptosis, which probably depended on the changes in the activity of Caspase family, including Caspase 3, Caspase 8 and Caspase 9 in these cells.

Conclusions: Our data clearly demonstrated that Notch1 is critical for cisplatin sensitivity in osteosarcoma. It can be used as a molecular marker and regulator for cisplatin sensitivity in osteosarcoma patients.

Figure 1

Immunohistochemistry showed the expression of Notch1 and HES1 in cisplatin sensitive group and insensitive group. The histopathology revealed the irregular nuclei, some spindle cells, and even destroyed bone (A and B, Column 1). Immunohistochemical examination of Notch1 and HES1 were carried out in different patients. There is significant more expression of Notch1 and HES1 in cisplatin sensitive patients than cisplatin insensitive patients in gross appearance (A and B, Column 2 and 3) and semi-quantitative scatter plot below (C and D). Column 4 in A and B was the negative control with the secondary antibody alone without primary antibody. (**P < 0.01).

Figure 2

The expression of Notch1 in MG63 and Saos-2 cell lines and their sensitivity to cisplatin. Real-time PCR revealed that Saos-2 cells had approximately 7 fold higher Notch1 expression than MG63 cells (A). We further carried out Western Blot and immunocytochemistry to determine the protein expression in MG63 and Saos-2. The level of Notch1 in Saos-2 is significantly higher than MG63 (error bar means s.d.) (B and C). Both two cell lines showed dose and time dependent response to cisplatin (D). Saos-2 performed significantly higher sensitivity to cisplatin (E). (*P < 0.05, **P < 0.01).

Figure 3

Activation of Notch1 signaling pathway increased the sensitivity of MG63 and Saos-2 to cisplatin. Real-time PCR indicated that HES1 mRNA in MG63 and Saos-2 had significantly more expression after NICD-1 plasmid transfection (A and B). The sensitivity of MG63 and Saos-2 to cisplatin has significantly increased after the activation of Notch1 signaling pathway (C and D). (*P < 0.05; **P < 0.01).

Figure 4

Inhibition of Notch1 signaling pathway decreased the sensitivity of MG63 and Saos-2 to cisplatin. Real-time PCR indicated that HES1 mRNA in MG63 and Saos-2 had significantly less expression after DAPT treatment (A and B). The sensitivity of MG63 and Saos-2 to cisplatin has significantly decreased after the inhibition of Notch1 signaling pathway (C and D). (*P < 0.05; **P < 0.01).

Figure 5

Notch1 signaling pathway regulated cisplatin sensitivity of MG63 and Saos-2 via changing the apoptosis effect. After overexpression of NICD-1, cisplatin could induce more apoptosis in MG63 and Saos2 cells than their control group (A and B). When Notch1 signaling pathway was inhibited by DAPT, cisplatin induced fewer apoptosis cells in both osteosarcoma cell lines (C and D). (*P < 0.05; **P < 0.01).

Figure 6

Inhibition of Notch1 signaling pathway decreased the expression and/or activity of Caspase3, Caspase8 and Caspase9. In the two cell lines, realtime PCR indicated that the gene expression of Caspase3, Caspase8 and Caspase9 were reduced to some extent after a simple DAPT treatment for 24 hours. Further reduction was observed with cisplatin subsequent treatment for 12 hours (A, B, C and D). Simultaneously, western blot was carried out to detect the protein expression of Pro-Casp3, Cleaved-Casp3, Casp8, Pro-Casp9 and Cleaved-Casp9. The expression of these several proteins significantly decreased after DAPT treatment in MG63 and Saos-2 cells. Further, such difference between two groups could be obviously magnified after cisplatin treatment (E, F, G and H). (*P < 0.05; **P < 0.01).