Peiminine inhibits colorectal cancer cell proliferation by inducing apoptosis and autophagy and modulating key metabolic pathways

Abstract

Peiminine, a compound extracted from the bulbs of Fritillaria thunbergii and traditionally used as a medication in China and other Asian countries, was reported to inhibit colorectal cancer cell proliferation and tumor growth by inducing autophagic cell death. However, its mechanism of anticancer action is not well understood, especially at the metabolic level, which was thought to primarily account for peiminine's efficacy against cancer. Using an established metabolomic profiling platform combining ultra-performance liquid chromatography/tandem mass spectrometry with gas chromatography/mass spectrometry, we identified metabolic alterations in colorectal cancer cell line HCT-116 after peiminine treatment. Among the identified 236 metabolites, the levels of 57 of them were significantly (p < 0.05) different between peiminine-treated and -untreated cells in which 45 metabolites were increased and the other 12 metabolites were decreased. Several of the affected metabolites, including glucose, glutamine, oleate (18:1n9), and lignocerate (24:0), may be involved in regulation of the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway and in the oxidative stress response upon peiminine exposure. Peiminine predominantly modulated the pathways responsible for metabolism of amino acids, carbohydrates, and lipids. Collectively, these results provide new insights into the mechanisms by which peiminine modulates metabolic pathways to inhibit colorectal cancer cell growth, supporting further exploration of peiminine as a potential new strategy for treating colorectal cancer.

Keywords: cancer therapy; colorectal cancer; metabolomics; natural product; peiminine.

Figure 1. Peiminine inhibits growth and induces apoptosis in HCT-116 cells

(A) Molecular structure and formula of peiminine. (B) Results of viability assay showing dose-dependent decrease in HCT-116 cell viability after peiminine treatment at 0, 50, 100, 200 and 400 μM concentrations. (C) Double staining of peiminine-treated and untreated HCT-116 cells with propidium iodide and annexin V and vehicle control (DMSO) for 24 hours for flow cytometry. (D) Percentage of dead and early-apoptotic cells in three independent experiments.* p ≤ 0.05. DMSO, dimethyl sulfoxide. (E) Confocal microscopy images of the Hela cells treated with DMSO(0), 50, 100 and 200 μM peiminine for 24 h. Green coloring indicates the presence of stably expressed LC3II. Red coloring indicates the presence of lysosomes (magnification, ×630). (F) Dose-dependent increases in LC3B-II/LC3B-I ratio with peiminine treatment at 0, 50, 100, 200 and 400 μM concentrations in HCT-116, HeLa, and MEF cells. Three independent immunoblot assays were performed and gray scale images of each assay were scanned and presented as a bar chart in which the vertical axis is the mean gray scale ± SD. * indicates statistical differences at p < 0.05 and ** indicates statistical differences at p < 0.01.

Figure 2. Heat map representation of 236 metabolites between treated and untreated group in clustering analysis

Each line represents a metabolite. The deeper the red color, the higher its content in the cell lines; similarly, the deeper the green color, the lower its content in the cell lines. Three metabolites mentioned in the text are shown in greater detail by boxplot. The p values were calculated between the peiminine-treated and untreated groups. List of 236 metabolites is provided in Supplementary Table 1.

Figure 3. Metabolites found at significantly different levels in treated and untreated HCT-116 cells

(A) Plot of the scores from the PLS-DA model of the treated and untreated groups. (B) Loading plot from the PLS-DA model of the treated and untreated groups. The boxes represent the tested samples while metabolites are displayed as triangles. Metabolites playing key roles for separation are marked with red triangle.

Figure 4. Metabolic pathways involving metabolites that were significantly increased or decreased in peiminine-treated HCT-116 cells

Metabolites undetected in both the treated and untreated groups are shown in the black boxes. Metabolites in red or blue exhibited increased or decreased levels, respectively, in the peiminine-treated group. GPC: glycerophosphocholine; GPE: glycerophosphoethanolamine; GPI: glycerophosphoinositol; GPS: glycerophosphatidylserine; SAH: S-adenosylhomocysteine; GSH: glutathione, reduced; GSSG: glutathione, oxidized; DHA: docosahexaenoate; DPA: docosapentaenoate; EPA: eicosapentaenoate.