Hempseed Lignanamides Rich-Fraction: Chemical Investigation and Cytotoxicity towards U-87 Glioblastoma Cells

Abstract

The weak but noteworthy presence of (poly)phenols in hemp seeds has been long overshadowed by the essential polyunsaturated fatty acids and digestible proteins, considered responsible for their high nutritional benefits. Instead, lignanamides and their biosynthetic precursors, phenylamides, seem to display interesting and diverse biological activities only partially clarified in the last decades. Herein, negative mode HR-MS/MS techniques were applied to the chemical investigation of a (poly)phenol-rich fraction, obtained from hemp seeds after extraction/fractionation steps. This extract contained phenylpropanoid amides and their random oxidative coupling derivatives, lignanamides, which were the most abundant compounds and showed a high chemical diversity, deeply unraveled through high resolution tandem mass spectrometry (HR-MS/MS) tools. The effect of different doses of the lignanamides-rich extract (LnHS) on U-87 glioblastoma cell line and non-tumorigenic human fibroblasts was evaluated. Thus, cell proliferation, genomic DNA damage, colony forming and wound repair capabilities were assessed, as well as LnHS outcome on the expression levels of pro-inflammatory cytokines. LnHS significantly inhibited U-87 cancer cell proliferation, but not that of fibroblasts, and was able to reduce U-87 cell migration, inducing further DNA damage. No modification in cytokines' expression level was found. Data acquired suggested that LnHS acted in U-87 cells by inducing the apoptosis machinery and suppressing the autophagic cell death.

Keywords: Cannabis sativa L.; U-87 glioblastoma cells; cytotoxicity; hemp seeds; high resolution tandem mass spectrometry; lignanamides; phenylamides.

Figure 1

(A) and (B) TOF-MS and TOF-MS/MS spectra for compound 1; (C) proposed fragmentation pathway of the [M − H]⁻ ion; (D) UV-DAD spectrum. In C panel, the theoretical m/z value is reported below each structure.

Figure 2

TOF-MS/MS spectra of compound 5 (A) and of its 3d-derived (B). UV-DAD spectrum of the compound (C). Proposed fragmentation pathway of the [M − H]⁻ ion for compound 5 (D) and the 3d-derived (E); theoretical m/z value is reported below each structure.

Figure 3

TOF-MS/MS spectra of compounds 15 and 22 (A and C, respectively). UV-DAD spectra of the compounds (B and D). In grey panel, the structure of cannabisin B is reported, without emphasizing stereochemical features.

Figure 4

TOF-MS/MS spectra of compounds (A) 19 and (B) 20, tentatively identified as cannabisin H isomers. The proposed fragmentation pathway of their [M − H]⁻ ion was reported (C); the theoretical m/z value is reported below each structure.

Figure 5

TOF-MS/MS spectra of compounds (A) 23, (B) 24, (C) 25, and (D) 35.

Figure 6

Cell viability of U-87 and human HF cell (A and B, respectively) was assessed by MTT assay after 24, 48 and 72 h of exposure. Data from LDH release assay at 24, 48 and 72 h exposure times were in panels C (U-87 cells) and D (HF cells). Values are the mean ± SE of two independent experiments performed in triplicate. *p < 0.05 vs. untreated cells. (E) Representative images from colony forming efficiency of U-87 cells grown in presence of LnHS or vehicle control for ten days; the experiment was performed in duplicate.

Figure 7

Representative images of U-87 and HF cells treated with different LnHS doses and subjected to the comet assay (panels A and B respectively). ctrl: untreated cells; green arrows indicate comets with a tail. Experiments were performed in duplicate.

Figure 8

U-87 cells underwent a scraped wound and were then treated with different LnHS doses (0.5, 2.5, 5, 25 and 50 μg/mL). Cells were photographed immediately following the scratch (0 h), after 12, 24, 36, 48, 72 and 96 h. Untreated cells (ctrl), were used as a control. Representative figures are shown from one of two independent experiments.

Figure 9

Levels of Bcl-2, Beclin-1, p-AKT, E-cadherin and ULK-1 in U-87 and HF cell types were detected using western blotting with respective antibodies. Graphical representation of pixel quantization of Bcl-2, Beclin-1, p-AKT, E-cadherin, and ULK-1 normalized to GAPDH (panels A, C). The intensities of signals were expressed as arbitrary units. *p < 0.05 vs. untreated cells (ctrl). Representative western blotting image of Bcl-2, Beclin-1, p-AKT, E-cadherin, ULK-1 and GAPDH are in panels B (U-87 cell type), and D (HF cell type).