Crebanine, an aporphine alkaloid, induces cancer cell apoptosis through PI3K-Akt pathway in glioblastoma multiforme

Abstract

Glioblastoma multiforme (GBM) is one of the most prevalent and lethal primary central nervous system malignancies. GBM is notorious for its high rates of recurrence and therapy resistance and the PI3K/Akt pathway plays a pivotal role in its malignant behavior. Crebanine (CB), an alkaloid capable of penetrating the blood-brain barrier (BBB), has been shown to have inhibitory effects on proinflammatory molecules and multiple cancer cell lines via pathways such as PI3K/Akt. This study aims to investigate the efficacy and mechanisms of CB treatment on GBM. It is the first study to elucidate the anti-tumor role of CB in GBM, providing new possibilities for GBM therapy. Through a series of experiments, we demonstrate the significant anti-survival, anti-clonogenicity, and proapoptotic effects of CB treatment on GBM cell lines. Next-generation sequencing (NGS) is also conducted and provides a complete list of significant changes in gene expression after treatment, including genes related to apoptosis, the cell cycle, FoxO, and autophagy. The subsequent protein expressions of the upregulation of apoptosis and downregulation of PI3K/Akt are further proved. The clinical applicability of CB to GBM treatment could be high for its BBB-penetrating feature, significant induction of apoptosis, and blockage of the PI3K/Akt pathway. Future research is needed using in vivo experiments and other therapeutic pathways shown in NGS for further clinical or in vivo studies.

Keywords: aporphine alkaloid; brain tumor; crebanine; glioblastoma multiforme; natural product.

FIGURE 1

CB treatment–induced cell death in GBM cells. (A) Reduced viability of GBM cells was shown after CB treatment in the MTT assay. (B) The reduction in colony formation of GBM cells was shown after CB treatment in the colony formation assay. (C,D) Increased proportions of the sub-G1 group in GBM cells were shown after CB treatment in the cell cycle analysis. Data are shown as mean ± S.D. (*p < 0.05; **p < 0.01; ***p < 0.001).

FIGURE 2

CB caused apoptosis in GBM cells. (A,C) Annexin V/PI double staining demonstrated the proportions of early apoptosis, late apoptosis, and necrosis in CB-treated GBM cells. The results are also shown in bar charts. (B,D) Hoechst 33342 staining demonstrated morphological changes in apoptotic GBM cells after CB treatment. The results, as represented in the bar charts, show increased proportions of apoptotic GBM cells after CB treatment. Data are shown as mean ± S.D. (*p < 0.05; **p < 0.01; ***p < 0.001).

FIGURE 3

The 24-h CB-treated GBM cells were tested by NGS with the GO database. These genes are categorized on the basis of their functions. (A) The categories that changed significantly in count are shown on the chart and sequenced in the order of their p values. (B) The categories that changed significantly in ratio are shown on the chart and sequenced in the order of their ratios. Their p values are shown in color. (C) The heat map shows in color the p values for changes in gene expression. The categories for these genes are also shown.

FIGURE 4

The 24-h CB-treated GBM cells were tested by NGS with the KEGG database. These genes are categorized on the basis of their functions. (A) The categories that changed significantly in count are shown on the chart and sequenced in order of their p values. (B) The categories that changed significantly in ratio are shown on the chart and sequenced in the order of ratio. Their p values are shown in color. (C) The heat map shows in color the p values of the changes in gene expression.

FIGURE 5

Western blotting revealed the expression of apoptotic proteins of the PI3K/Akt signaling pathway and caspase cascade in CB-treated GBM cells.