Paxilline derived from an endophytic fungus of Baphicacanthus cusia alleviates hepatocellular carcinoma through autophagy-mediated apoptosis

Abstract

Hepatocellular carcinoma (HCC) is a malignancy for which no effective drugs are available. Paxilline is derived from an endophytic fungus of the leaves of Baphicacanthus cusia (Nees) Bremek. In a previous study, we found that paxilline inhibited the proliferation of HepG2 cells; however, its mechanism remains unclear. In this study, ESI⁺ and NMR were used to characterize the paxilline structure. Network pharmacology analysis was performed with databases and software to obtain the core targets and signaling pathways associated with the anti-HCC effects of paxilline. Molecular docking was performed to validate the preliminary affinity of paxilline for the core targets. For further in vitro experiments, a CCK8 assay was performed to detect cell viability, a wound healing assay was performed to detect cell migration, an Annexin V-FITC assay was performed to detect the cell cycle and apoptosis rate in HepG2 cells, RT-qPCR analysis was performed to detect the expression of cell cycle-related genes and autophagy-related genes, Immunofluorescence staining was performed to detect the expression of LC3B, and Western blotting was performed to detect the expression of apoptosis-related proteins and autophagy-related proteins. As a result, we obtained a white powder, which was identified as paxilline. Network analysis and molecular docking results revealed that apoptosis-related and autophagy-relatted protein were key targets (mTOR and PI3K) for paxilline anti-HCC. Further examination revealed that paxilline promoted HepG2 cell apoptosis, inhibited HepG2 cell migration, and arrested HepG2 cell in the S phage. RT-qPCR analysis revealed that paxilline markedly downregulated the mRNA expression of Cyclin D1, CDK4, LC3B, mTOR, Parkin, and PINK1. Immunofluorescence staining demonstrated a significant upregulation of LC3B protein expression following paxilline treatment. Further validation by Western blotting showed that paxilline significantly increased the expression of LC3B II/I, bax, cleaved-PARP, and cleaved-caspase 3, while significantly decreased the expression of bcl-2. Additionally, a significant promotion of cellular apoptosis and expression of apoptotic proteins when treatment with chloroquine (CQ)/rapamycin (Rapa). Meanwhile, when combined with paxilline, it was found that paxilline may have a synergistic effects with Rapa, jointly promoting cellular apoptosis and the expression of proapoptotic proteins. In conclusion, these findings reveled paxilline alleviates HCC through autophagy-mediated apoptosis.

Keywords: Apoptosis; Autophagy; Hepatocellular carcinoma; Network pharmacology; Paxilline.

Fig. 1

Network analysis of paxilline for the treatment of HCC. (A) Flow chart of network pharmacology. (B)Venn diagram of paxilline and HCC targets. (C) PPI diagram of the paxilline anti-HCC agent. The more pink and larger the nodes, the greater the contribution of the target to the network.

Fig. 2

GO, KEGG, and MCODE analyses. (A) GO analysis of the core targets. (B) KEGG analysis of the core targets. The greener and greater the number of targets, the greater the significance of the pathways. (C–F) The core targets were analyzed using the MCODE plugin and clustered according to apoptosis, energy metabolism, extracellular matrix remodeling, and cell cycle clustering. (G) Targets from cluster 1-enriched signaling pathways.

Fig. 3

Molecular docking results showed that paxilline acts on apoptosis-related or autophagy-related targets. (A) Docking cartoon between paxilline and core targets. Molecular docking simulations were carried out using AutoDock Tools (version 1.5.7), with subsequent visualization performed in PyMOL (version 2.5.0). (B) The affinity between paxilline and core targets was determined. The redder and larger the dots, the greater the absolute value of the affinity.

Fig. 4

Screening of the effects of various concentrations of paxilline on HepG2 cells for growth and movement. (A) Images of HepG2 cells after treatment with different concentrations of paxilline (0, 10, 20, 40, and 80 μg/mL); scale bar = 100 μm; arrows indicate cell morphology. (B) The viability of HepG2 cells after treatment with different concentrations of paxilline (0, 10, 20, 40, and 80 μg/mL) was determined by the CCK8 assay. (C) HepG2 cell movement was evaluated by conducting wound-healing assays at 0, 12, and 24 h after paxilline treatment (10, 20, and 40 μg/mL). *P < 0.05 and **P < 0.01 compared to the control.

Fig. 5

Paxilline arrested HepG2 cells in the S phase. (A) Cell cycle distribution of paxilline-treated HepG2 cells after 48 h was determined by flow cytometry. (B, C) RT-qPCR of paxilline-treated HepG2 cells for 24 h at the mRNA level of cell cycle-related genes. *P < 0.05 and **P < 0.01 compared to the control.

Fig. 6

Paxilline promoted apoptosis in HepG2 cells. (A, B) Effects of treatment with different concentrations of paxilline (10, 20, and 40 μg/mL) for 24 h on the percentage of apoptotic HepG2 cells. (C) Effects of treatment with different concentrations of paxilline (10, 20, and 40 μg/mL) for 12 h on the levels of apoptosis-related proteins. *P < 0.05 and **P < 0.01 compared to the control.

Fig. 7

Paxilline regulates autophagy in HepG2 cells. (A–D) The mRNA levels of autophagy-related genes in HepG2 cells exposed to different concentrations of paxilline (10, 20, and 40 μg/mL) for 24 h, as determined by RT-qPCR. (E, F) Paxilline inhibited the expression of LC3B in HepG2 cells for 24 h, as shown by immunocytochemistry; scale bar = 20 μm. G Protein levels of LC3B in HepG2 cells exposed to different concentrations of paxilline (10, 20, and 40 μg/mL) for 24 h, as determined by Western blotting analysis. *P < 0.05 and **P < 0.01 compared to the control.

Fig. 8

Paxilline promoted apoptosis by mediating autophagy in HepG2 cells. (A, B) Annexin V-FITC analyses of the apoptotic rates of HepG2 cells exposed to paxilline (40 μg/mL) with or without CQ (10 μM)/Rapa (100 nM) for 24 h. (C) The expression of LC3B, Bax, Bcl-2, cleaved-caspase 3, and cleaved-PARP in HepG2 cells exposed to paxilline (40 μg/mL) with or without CQ (10 μM)/Rapa (100 nM) for 24 h. *P < 0.05 and **P < 0.01 compared to the control; ^#P < 0.05 and ^##P < 0.01 compared to the CQ group; ^{^}P < 0.05 and ^{^^}P < 0.01 compared to the Rapa group.