1H-NMR metabolomics reveals a multitarget action of Crithmum maritimum ethyl acetate extract in inhibiting hepatocellular carcinoma cell growth

Abstract

Hepatocellular carcinoma (HCC) is nowadays the sixth cause of tumour-related deceases worldwide, estimated to become the third in Western countries by 2030. New drugs for HCC treatment still have many adverse effects. Several lines of evidence indicate that plant metabolites offer concrete opportunities for developing new therapeutic strategies for many diseases, including cancer. We previously reported that ethyl acetate extract of a spontaneous edible plant harvested in Apulia, Crithmum maritimum, significantly inhibited cell growth in HCC cells. By ¹H-NMR spectroscopy, here we show that Crithmum maritimum ethyl acetate extract counteracts the Warburg effect, by reducing intracellular lactate, inhibits protein anabolism, by decreasing amino acid level, and affects membrane biosynthesis by lowering choline and phosphocholine. Also, we observed an effect on lipid homeostasis, with a reduction in triglycerides, cholesterol, monounsaturated fatty acids (MUFA), and diunsaturated fatty acids (DUFA), and an increase in polyunsaturated fatty acids (PUFA). Taken together, these data demonstrate that Crithmum maritimum-induced cytostasis is exerted through a multi-effect action, targeting key metabolic processes in HCC cells. Overall, our findings highlight the role of Crithmum maritimum as a promising tool for the prevention and the improvement of the therapeutic options for HCC and other types of tumours.

Figure 1

(a) Cartoon outline of the experimental workflow (b) Effect of the different Crithmum maritimum extracts on cell proliferation in three different HCC cell lines (Huh7, HepG2, HLE) as determined by crystal violet staining. Cells were treated for 72 h with the four different Crithmum maritimum extracts at 0.5 μM. **** p < 0.0001, *** p < 0.001 as determined by 2-way ANOVA analysis followed by Dunnett’s multiple comparisons tests. (c) Cartoon showing the proposed multi-target effect exerted by Crithmum maritimum ethyl acetate extract on HCC cells. DMSO Vehicle treatment, CM Crithmum maritimum treatment, [Hex] Hexane, [EA] Ethyl Acetate, [MetOH] Methanol, [EtOH] Ethanol. Results are expressed as the mean ± s.e.m. of at least three independent biological replicates, each conducted in triplicate.

Figure 2

¹H-NMR typical spectrum (600 MHz, CD₃OD:CDCl₃) of Crithmum maritimum ethyl acetate extract sample in (a) high, (b) middle, and (c) low-frequency regions, showing the main metabolite classes associated with principal peaks.

Figure 3

OPLS-DA scores plots (left panel) and corresponding coefficient loading plots (right panel) derived from the ¹H-NMR spectra of HepG2 (a) and Huh7 (b) cell extracts aqueous fraction obtained from different groups. DMSO Vehicle treatment, CM Crithmum maritimum treatment.

Figure 4

(a) Quantitative comparison of metabolites found in the aqueous cell extract. Mean and relative standard error mean refers to the relative integrals of metabolites, determined from cell extract 1D ¹H-NMR spectra of each group (CM and DMSO treatments). Letters in parentheses indicate peak multiplicities (s, singlet; d, doublet, dd, doublet of doublet; m, multiplet). DMA dimethylamine, PC O-phosho-choline, GPC sn-glycero-3-phoshocholine. *A p-value threshold of 0.05 was obtained from t-test analysis. (b,c) Box-and-Whisker plots illustrating the trend of significant metabolites grouped according to CM: Crithmum maritimum treatment; DMSO: Vehicle treatment, derived from the ¹H NMR spectra of (b) HepG2 and (c) Huh7 aqueous cell extracts. DMSO Vehicle treatment, CM Crithmum maritimum treatment.

Figure 5

OPLS-DA scores plots (left panel) and corresponding coefficient loading plots (right panel) derived from the ¹H-NMR spectra of HepG2 (a) and Huh7 (b) cell culture media samples obtained from different groups. DMSO Vehicle treatment, CM Crithmum maritimum treatment.

Figure 6

(a) Quantitative comparison of metabolites found in cell culture media. Mean and relative standard error mean refers to the relative integrals of metabolites, determined from cell culture media 1D ¹H-NMR spectra of each group (CM and DMSO treatments). Letters in parentheses indicate peak multiplicities (s, singlet; d, doublet, dd, doublet of doublet; q, quartet, m, multiplet). * A p-value threshold of 0.05 was obtained from t-test analysis. (b,c) Box-and-Whisker plots illustrating the significant metabolites trend grouped according to CM Crithmum maritimum treatment, DMSO Vehicle treatment, derived from the ¹H-NMR spectra of (a) HepG2 and (b) Huh7 cell culture media.

Figure 7

OPLS-DA scores plots (left panel) and corresponding coefficient loading plots (right panel) derived from the ¹H-NMR spectra of (a) HepG2 and (b) Huh7 cell lipid extracts obtained from different groups. DMSO Vehicle treatment, CM Crithmum maritimum treatment.

Figure 8

(a) Quantitative comparison of metabolites found in cell lipid extracts. Mean and relative standard error mean refers to the relative integrals of metabolites, determined from lipid cell extract 1D ¹H-NMR spectra of each group (CM and DMSO treatments). Letters in parentheses indicate the peak multiplicities (s singlet, d doublet, dd doublet of doublet, q quartet, m multiplet). *A p-value threshold of 0.05 was obtained from t-test analysis. (b,c) Box-and-Whisker plots illustrating the significant metabolites trend grouped according to CM Crithmum maritimum treatment, DMSO DMSO treatment, derived from the ¹H-NMR spectra of (a) HepG2 and (b) Huh7 cell lipid extracts.