A Combined Phytochemistry and Network Pharmacology Approach to Reveal the Potential Antitumor Effective Substances and Mechanism of Phellinus igniarius

Abstract

Phellinus igniarius (P. igniarius) is a medicinal fungus that is widely used in East Asia for the adjuvant treatment of cancer. To elucidate the antitumor effective substances and mechanism of P. igniarius, we designed an approach incorporating cytotoxicity screening, phytochemical analysis, network pharmacology construction, and cellular and molecular experiments. The dichloromethane extract of P. igniarius (DCMPI) was identified as the active portion in HT-29 cells. Nineteen constituents were identified, and 5 were quantified by UPLC-ESI-Q/TOF-MS. Eight ingredients were obtained in the network pharmacology study. In total, 473 putative targets associated with DCMPI and 350 putative targets related to colon cancer were derived from online databases and target prediction tools. Protein-protein interaction networks of drug and disease putative targets were constructed, and 84 candidate targets were identified based on topological features. Pathway enrichment analysis showed that the candidate targets were mostly related to reactive oxygen species (ROS) metabolic processes and intrinsic apoptotic pathways. Then, a cellular experiment was used to validate the drug-target mechanisms predicted by the system pharmacology analysis. Experimental results showed that DCMPI increased intracellular ROS levels and induced HT-29 cell apoptosis. Molecular biology experiments indicated that DCMPI not only increased Bax and Bad protein expression and promoted PARP and caspase-3/9 cleavage but also down-regulated Bcl-2 and Bcl-xl protein levels to induce apoptosis in HT-29 cells. In conclusion, our study provides knowledge on the chemical composition and antitumor mechanism of P. igniarius, which may be exploited as a promising therapeutic option for colon cancer.

Keywords: Phellinus igniarius; antitumor; effective substances; mitochondrial apoptosis pathway; network pharmacology; phytochemistry.

FIGURE 1

(A) Cytotoxic effects of DCMPI on the A549, SGC-7901, HT-29, SMMC7721, BGC-823 and HepG2 cell lines as determined by the MTT assay for 72 h. (B) Microscopic observation of HT-29 cells after administrating DCMPI for 72 h (a): untreated; (b): treated with 12.5 μg/mL DCMPI; (c): treated with 25 μg/mL DCMPI; (d): treated with 50 μg/mL DCMPI).

FIGURE 2

(A) UPLC-ESI-Q/TOF-MS BPI chromatogram of DCMPI. (B) Mass fragment spectrum of Phelligrin A in the negative ion mode. (C) Possible fragmentation mechanistic pathway of Phelligrin A. (D) Typical mixed standard substance chromatograms of protocatechuic aldehyde, osmundacetone, eriodictyol, naringenin, and sakuranetin.

FIGURE 3

GO analysis of putative DCMPI targets and compound-target network construction. (A) Chemical structures of the eight main ingredients. (B) GO analysis of BP, cell component, and MF terms was performed on putative DCMPI targets, and the top 20 terms with P < 0.05 are shown. (C) The compound-target network was constructed by linking the 8 main compounds (red dots) and their potential targets (green dots).

FIGURE 4

Identification of colon cancer-related targets using pre-existing microarray data. Two hundred DEGs identified by the limma package were highly related to colon cancer. P < 0.05 and FC > 2 were considered cut-off values.

FIGURE 5

Candidate target identification and ClueGO pathway analysis. (A) Core CPPI network of DCMPI targets. (B) Large hubs of the DCMPI CPPI network extracted from (A) whose degrees were more than twice the median degree of all nodes in the network. (C) PPI network of the main DCMPI targets extracted from (B) constructed by calculating 6 topological features. (D,E) A functionally grouped network of enriched categories was generated for the target genes. GO terms are represented as nodes, and the node size represents the term’s enrichment significance. Functionally related groups partially overlap. Only the most significant term in the group is labeled. Representative enriched pathway (P < 0.05) interactions among the main DCMPI targets.

FIGURE 6

DCMPI induced apoptosis in HT-29 colon cancer cells. (A) Nuclear morphological changes were observed in DCMPI-exposed HT-29 cells by DAPI staining. (B) The percentage of apoptotic cells induced by DCMPI treatment was detected by Annexin V/PI double-staining analysis. (C) The effect of DCMPI on the mitochondrial membrane potential, which was determined using a JC-1 staining assay and flow cytometry. (D) The effect of DCMPI on the intracellular ROS, which was detected using DCFH-DA probes and flow cytometry. ^∗∗P < 0.01. The data are presented as the mean ± SD from at least three independent experiments.

FIGURE 7

Western blot analysis of apoptosis-related protein expression. (A) Bad, Bax, Bcl-xl, Bcl-2, PARP, caspase-3, caspase-9, and caspase-8 protein expression in HT-29 cells after DCMPI treatment (30 and 60 μg/mL) for 24 h. (B) Densitometric analysis of Bad, Bax, Bcl-xl, Bcl-2, PARP, caspase-3, caspase-9, and caspase-8 expression. ^∗∗P < 0.01. The data are presented as the mean ± SD from at least three independent experiments.

FIGURE 8

The effect of Z-VAD-FMK (50 μM) pretreatment and DCMPI (60 μg/mL) treatment on the cleavage of PARP and caspase-3/9 in HT-29 cells. (A) PARP, caspase-3, caspase-9, and caspase-8 protein expression in HT-29 cells after treatment for 24 h. (B) Densitometric analysis of PARP, caspase-3, caspase-9, and caspase-8 expression. ^∗∗P < 0.01. The data are presented as the mean ± SD from at least three independent experiments.

FIGURE 9

Molecular mechanisms underlying the antitumor activity of DCMPI. DCMPI increased intracellular ROS levels and induced HT-29 cell apoptosis mainly mediated via the mitochondrial apoptosis pathway.