Lepidium sativum Secondary Metabolites (Essential Oils): In Vitro and In Silico Studies on Human Hepatocellular Carcinoma Cell Lines

Abstract

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and the greatest cause of cancer-related death in the world. Garden cress (Lepidium sativum) seeds have been proven to possess extraordinary antioxidant, anti-inflammatory, hypothermic, and analgesic properties. In this study, in vitro cytotoxic efficiency evaluation of L. sativum fractions was performed against two hepatocellular carcinoma cell lines (HuH-7 and HEPG-2), and the expression of some apoptotic genes was explored. In addition, the chemical composition of a potent extract of L. sativum was analyzed using gas chromatography coupled with mass spectrometry. Then, molecular docking analysis was implemented to identify the potential targets of the L. sativum components' most potent extract. Overall, the n-hexane extract was the most potent against the two HCC cell lines. Moreover, these cytotoxicity levels were supported by the significant downregulation of EGFR and BCL2 gene expression levels and the upregulation of SMAD3, BAX, and P53 expression levels in both HuH-7 and HEPG2 cell lines. Regarding L. sativum's chemical composition, GC-MS analysis of the n-hexane extract led to the identification of thirty compounds, including, mainly, hydrocarbons and terpenoids, as well as other volatile compounds. Furthermore, the binding affinities and interactions of the n-hexane fraction's major metabolites were predicted against EGFR and BCL2 molecular targets using the molecular docking technique. These findings reveal the potential use of L. Sativum in the management of HCC.

Keywords: GC–MS analysis; Lepidium sativum; apoptosis; garden cress; hepatocellular carcinoma; molecular docking; qPCR.

Figure 1

Effect of different concentrations of methylene chloride, butanol, methanol, ethyl acetate, and n-hexane extracts on the cellular proliferation of HEPG2 (A), HuH-7 (B), and THLE 2 (C) cell lines following 48 h of treatment. Values are expressed as the mean ± SD of 3 separate experiments performed in quadruplicate. Statistical analysis was carried out utilizing analysis of variance (ANOVA) followed by post-hoc test, and Dunnett t-test was used for comparison with the control group. All tests were two-tailed. A p-value < 0.05 was considered significant (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001).

Figure 2

Gene expression levels after treating hepatocellular carcinoma cells with L. sativum n-hexane extract. (A): Expression of BCL2, BAX, P53, TGF-β, SMAD3, and EGFR in HEPG2 cells. Blue: untreated control, red: hexane extract. (B): Expression of BCL2, BAX, P53, TGF-β, SMAD3, and EGFR in HuH-7 cells. Blue: untreated control, red: hexane extract. Statistical analysis was performed using analysis of variance (ANOVA) followed by post-hoc test, and Dunnett t-test was used for comparison with the control group. All tests were two-tailed. A p-value < 0.05 was considered significant (** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001).

Figure 3

3D representations of the predicted (in grey) and crystal (in cyan) binding modes of (a) erlotinib (AQ4) with the active conformation of EGFR (PDB code: 1M17), (b) erlotinib with the inactive conformation of EGFR (PDB code: 4HJO), and (c) DRO with BCL2 (PDB code: 2W3L).

Figure 4

2D representations of the binding modes of potent n-hexane extract major metabolites inside the active site of inactive EGFR (PDB code: 4HJO). Interactions: conventional hydrogen bond (green), carbon–hydrogen bond (pale green), pi–sigma and pi–pi (violet), pi–sulfur (yellow), alkyl and pi–alkyl (pale violet).

Figure 5

2D representations of the binding modes of potent n-hexane extract major metabolites inside the active site of inactive EGFR (PDB code: 4HJO). Interactions: conventional hydrogen bond (green), carbon–hydrogen bond (pale green), pi–sigma and pi–pi (violet), pi–sulfur (yellow), alkyl and pi–alkyl (pale violet).