Effect of Helix aspersa extract on TNFα, NF-κB and some tumor suppressor genes in breast cancer cell line Hs578T

Abstract

Background: The garden snail, Helix aspersa, is a big land snail widely found in the Mediterranean countries. It is one of the most consumed species and widely used in zootherapy.

Objective: The present study was carried out to investigate for the first time the first time the antitumor activity of an aqueous extract from Helix aspersa.

Materials and methods: The effect of H. aspersa extract was studied on a triple negative breast cancer cell line Hs578T. Firstly, the morphological changes and the mode of cell death induced by the extract have been evaluated by microscopy and acridine orange/ethidium bromide staining. The effect of the extract at dilution 0.1% and 1% was then tested on some genes, regulators of cell death and proliferation like tumor necrosis factor α (TNFα), NF- κB, and the tumor suppressor genes P53 and PTEN.

Results: Data demonstrate that the extract induces necrosis in tumor cells. It enhances significantly the expression of TNFα; mRNA levels were 20 and 10 times more important in treated cells compared to nontreated cells. NF-κB and PTEN were inhibited with the dilution 1% after 8 and 24 hours of treatment. P53 expression was further inhibited but only with the highest dose, after 4, 8, and 24 hours.

Conclusion: Our results show that H. aspersa extract has an antitumor activity against Hs578T cells; it is a potent stimulator for TNFα and a good inhibitor for NF-κB. Abbreviations used: AO: acridine orange; Bcl-2: B cell lymphoma 2. cDNA: complementary DNA; ELISA: enzyme linked immunosorbent assay; EB: ethidium bromide; IC50: the half maximal inhibitory concentration; mRNA: messenger RNA. MAPK: mitogen-activated protein kinase; NF-κB: nuclearfactorkappa B; PBS: phosphate buffered saline. PI3K: phospho-inositol 3 kinase; PTEN: phosphatase and tensin homolog; ROS: reactive oxygen species. RT-PCR: reverse transcription polymerase chain reaction; TNFα: tumor necrosis factor alpha. TNFR1: TNF receptor-1; TP53: tumor protein 53.

Keywords: Breast cancer; Helix aspersa; NF-Kβ; TNFα; necrosis.

Figure 1

Microscope fluorescence image of Hs578T cells treated with H. aspersa extract and stained by AO/EB observed at 100X magnification: (A) Control. (B) Cells treated by the dilution 0.1%. (C) Cells treated by the dilution 1%. Live cell showed by a green arrow, apoptotic cell showed by a yellow arrow, necrotic cell showed by a red arrow.

Figure 2

Effect of H. aspersa extract on the expression of TNFα mRNA in breast cancer cell line Hs578T (*significant p < 0.05, *** significant p < 0.001, compared to control)

Figure 3

Effect of H. aspersa extract on TNFα concentration in culture supernatant of breast cancer cell line Hs578T (* significant p < 0.05, *** significant p < 0.001, compared to control)

Figure 4

Effect of H. aspersa extract on the expression of NF-κB mRNA in breast cancer cell line Hs578T (**significant p < 0.01, compared to control).

Figure 5

Effect of H. aspersa extract on the expression of PTEN mRNA in breast cancer cell line Hs578T (*significant p < 0.05, compared to control)

Figure 6

Effect of H. aspersa extract on the expression of p53 mRNA in breast cancer cell line Hs578T (*significant p < 0.05, ** significant p < 0.01, compared to control).