Grapefruit-Derived Micro and Nanovesicles Show Distinct Metabolome Profiles and Anticancer Activities in the A375 Human Melanoma Cell Line

Abstract

Fruit juice is one of the most easily accessible resources for the isolation of plant-derived vesicles. Here we found that micro- and nano-sized vesicles (MVs and NVs) from four Citrus species, C. sinensis, C. limon, C. paradisi and C. aurantium, specifically inhibit the proliferation of lung, skin and breast cancer cells, with no substantial effect on the growth of non-cancer cells. Cellular and molecular analyses demonstrate that grapefruit-derived vesicles cause cell cycle arrest at G2/M checkpoint associated with a reduced cyclins B1 and B2 expression levels and the upregulation of cell cycle inhibitor p21. Further data suggest the inhibition of Akt and ERK signalling, reduced intercellular cell adhesion molecule-1 and cathepsins expressions, and the presence of cleaved PARP-1, all associated with the observed changes at the cellular level. Gas chromatography-mass spectrometry-based metabolomics reveals distinct metabolite profiles for the juice and vesicle fractions. NVs exhibit a high relative amount of amino acids and organic acids whereas MVs and fruit juice are characterized by a high percentage of sugars and sugar derivatives. Grapefruit-derived NVs are in particular rich in alpha-hydroxy acids and leucine/isoleucine, myo-inositol and doconexent, while quininic acid was detected in MVs. Our findings reveal the metabolite signatures of grapefruit-derived vesicles and substantiate their potential use in new anticancer strategies.

Keywords: Akt signalling; ERK signalling; antitumor activity; citrus; gas chromatography-mass spectrometry; metabolome; nanovesicles; vesicles.

Figure 1

Citrus-derived nano and microvesicles reduce cancer cell proliferation. Cell growth was measured by MTT assay after 24 h and 48 h incubation with 25 μg/mL (expressed in protein content) of nano- and microvesicles isolated from the fruit juices of the following Citrus species: (A) C. limon, (B) C. paradisi, (C) C aurantium and (D) C. sinensis. The values were plotted as fold changes concerning related mock-treated cells (100% viable). Each point represents the mean ± SD of three independent experiments. Asterisks denote statistically significant values in comparison to non-tumour cells (HaCaT) assessed by t-test (* p ≤ 0.05).

Figure 2

Grapefruit-derived vesicles induce cell cycle arrest and affect the expression of oncogenes and cell-cycle regulatory genes in melanoma cells (A) Flow cytometry analysis of melanoma A375 cells untreated (NT) or treated with 25 μg/mL nanovesicles (NVs) or microvesicles (MVs) for 24 h. Cell distribution in the different cell cycle phases was determined by propidium iodide staining. Each point represents the mean ± SD of three independent experiments. (B) Western blot analyses were performed on cells treated for 24 h with 25 μg/mL of C. paradisi-derived nanovesicles or microvesicles. Levels of proteins involved in cell proliferation (pERK and pAkt), oncogenes (ICAM 1 and cathepsin) and apoptosis (Parp-1) were evaluated. Blots were stripped and subsequently re-probed with an antibody against GAPDH to check equal loading of protein extracts. (C) Relative expression levels of cell-cycle regulatory genes in 24 h NV- and MV-treated melanoma cells by qRT-PCR. GAPDH was used as a reference gene to normalize the expression levels. Data represent mean ± SD of three technical repeats from three biological replicates, expressed as relative fold changes compared to their basal level in untreated A375 cells. Statistically significant values in untreated A375 cells assessed by t-test (* p ≤ 0.05).

Figure 3

Metabolite profiles of (A) whole juice, (B) microvesicles and (C) nanovesicles isolated from grapefruit and analysed by GC-MS in full scan mode. Chromatograms are showing the most abundant GC-MS peaks and their tentative identifications.

Figure 4

Distribution of main metabolite classes identified in grapefruit juice, and microvesicle and nanovesicle samples. Nanovesicles exhibit a high relative amount of amino acids and organic acids, while microvesicles and fruit juice are characterized by a high percentage of carbohydrates and derivatives.