Antioxidant, Anti-Inflammatory and Anti-Proliferative Properties of Stachys circinata on HepG2 and MCF7 Cells

Abstract

According to the WHO, the overall age-standardized cancer rate keeps declining, and the number of cases diagnosed each year increases, remaining among the leading causes of death in 91 out of 172 recorded countries. In this context, novel cancer prediction and therapeutic protocols are compulsory. The effect of a Stachys circinata L'Hér dichloromethane extract (ScDME) on cell redox homeostasis and tumor proliferation was investigated. HepG2 cell feedback mechanisms to oxidative stress exposure were evaluated by determining catalase (CAT) and reduced glutathione (GSH), following the supply with ScDME (0.0-5.7 µg/µL). Cytotoxicity of ScDME against the human umbilical vein endothelial cell (HUVEC) and two human cancer cell lines (breast: MCF7; liver: HepG2) was evaluated by the MTT assay. H₂O₂-stressed HepG2 cells supplied with the S. circinata extracts exhibited significantly increased CAT and GSH activity as compared to unsupplied ones. The anti-inflammatory activity of the extracts was evaluated by real time-qPCR on IL-1, IL-6 and TNF-α expression. As a result, this research points out that S. circinata dichloromethane extract owns anti-inflammatory and anti-proliferative properties against MCF7 and HepG2 cells and activates CAT and GSH of the HepG2 cells' antioxidant enzyme system.

Keywords: anti-proliferative activity; bioactive molecules; cell proliferation; cellular mechanisms; gene expression; natural extract.

Figure 1

Dose-dependent cytotoxic activity of S. circinata extract on HUVEC, HepG2 and MCF7 cell lines. Cells were treated with increasing doses of the extract. “Ctrl” represents the cells cultured in the growing medium alone. Each cell type was incubated with the extracts for 24 h at 37 °C and subjected to MTT assays to measure % cell viability. The data were obtained from three independent assays using three wells for each assay.

Figure 2

Effect of S. circinata extracts on cellular catalase activity. Data are expressed as mean ± standard error of the mean (SEM) (n = 3; *** p ≤ 0.001).

Figure 3

Effect of S. circinata extracts on cellular GSH levels. Data are expressed as mean ± SEM (n = 3; *** p ≤ 0.001).

Figure 4

Effect of S. circinata extracts on cellular ROS levels on HUVEC, HepG2 and MCF7 cell lines. Data are expressed as mean ± SEM (n = 3; ** p ≤ 0.01, *** p ≤ 0.001).

Figure 5

Effect of S. circinata extracts on IL-1 expression levels. The mRNA levels for each gene were normalized to β-Actin and expressed as fold of change (2^−∆∆Ct) of the mRNA levels observed in undifferentiated control cells defined as 1 (mean ± SD; n = 6). Data are expressed as mean ± SD referred to the control (*** p ≤ 0.001).

Figure 6

Effect of S. circinata extracts on IL-6 expression levels. The mRNA levels for each gene were normalized to β-Actin and expressed as fold of change (2^−∆∆Ct) of the mRNA levels observed in undifferentiated control cells defined as 1 (mean ± SD; n = 6). Data are expressed as mean ± SD referred to the control (*** p ≤ 0.001).

Figure 7

Effect of S. circinata extracts on TNF-α expression levels. The mRNA levels for each gene were normalized to β-Actin and expressed as fold of change (2^−∆∆Ct) of the mRNA levels observed in undifferentiated control cells defined as 1 (mean ± SD; n = 6). Data are expressed as mean ± SD referred to the control (*** p ≤ 0.001).