Antitumor Effect of Pseudolaric Acid B Involving Regulation of Notch1/Akt Signaling Response in Human Hepatoma Cell In Vitro

Abstract

Background. Liver cancer, particularly hepatocellular carcinoma (HCC), is the fourth leading cause of cancer-related death worldwide. Sorafenib is a crucial drug for the treatment of advanced HCC, but it is difficult to meet the challenge of increasing clinical demands due to its severe side effects and drug resistance. Hence, development of novel antitumor drugs is urged. Previous studies showed that pseudolaric acid B (PAB) could reduce the expression of protein kinase B (PKB/Akt), a downstream effector of Notch signaling, facilitating cell apoptosis in HCC. The disruption of Notch signaling was verified to exacerbate malignant progression and drug resistance, however, the antitumor effect of PAB on Notch signaling in HCC remains unclear. Thus, this study aims to investigate the anti-HCC effect of PAB in association with the regulation of Notch1/Akt signaling. Methods. CCK-8 assay and transwell assay were used to examine the cell proliferation and invasion in Huh7 cells after treatment with PAB and a Notch inhibitor DAPT. Moreover, the cell cycle of Huh7 cells after treatment with PAB was analyzed using flow cytometry. Finally, the changes of Notch1, Jagged1, Hes1, and Akt expression at the protein and mRNA level in Notch1/Akt signaling in Huh7 cells after treatment with PAB and DAPT were analyzed using immunofluorescence assay and real-time qPCR. Results. The proliferation rate of Huh7 cells exposed to PAB of 0.5, 1, 2, 4, 8, 10, 20, 40, 80, 100, and 200 μmol/L revealed a time-and dose-dependent decrease in vitro, showing cell cycle arrest at G2/M phase $\left(P<0.05\right)$ . Furthermore, compared with the untreated group, at the concentration of 40 μmol/L, the proliferation rate and invasion rate of Huh7 cells in PAB, DAPT, and PAB-DAPT combination (PAB + DAPT) group were significantly decreased $\left(P<0.05\right)$ , but the PAB + DAPT showed no synergistic antiproliferation and anti-invasion effect in comparison with PAB treatment alone $\left(P>0.05\right)$ . In addition, compared with the untreated group, PAB and DAPT alone significantly downregulated the expression of Notch1, Jagged1, Hes1, Akt mRNA, or/and protein in Huh7 cells $\left(P<0.05\right)$ , but there was no significant difference in synergistic downregulated effect between the PAB + DAPT group and the PAB group $\left(P>0.05\right)$ . Conclusion. PAB can suppress proliferation and invasion of HCC cells through downregulating the expression of Notch1/Akt signaling protein and mRNA, and may be a potential novel antitumor drug candidate for the clinical treatment of HCC in the future.