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. 2019 Jul;15(4):1001-1009.
doi: 10.5114/aoms.2018.78742. Epub 2018 Oct 3.

Genistein-triggered anticancer activity against liver cancer cell line HepG2 involves ROS generation, mitochondrial apoptosis, G2/M cell cycle arrest and inhibition of cell migration

Qian Zhang  1 , Juan Bao  2 , Jiehua Yang  1
Affiliations

Genistein-triggered anticancer activity against liver cancer cell line HepG2 involves ROS generation, mitochondrial apoptosis, G2/M cell cycle arrest and inhibition of cell migration

Qian Zhang et al. Arch Med Sci. 2019 Jul.

Abstract

Introduction: Liver cancer is one of the most common malignancies across the globe and one of the major causes of cancer-related mortality. With limited available treatment options, there is an urgent need to look for new available options. Genistein is an important plant flavonoid and has been shown to possess tremendous pharmacological potential. The objective of the present study was therefore to evaluate the anticancer effect of the genistein.

Material and methods: The antiproliferative activity and IC50 of genistein were determined by MTT assay. Reactive oxygen species (ROS) and cycle distribution were investigated by flow cytometry. Apoptosis was detected by DAPI and annexin V/IP staining. Cell migration was investigated by wound healing assay. Protein expression was estimated by western blotting.

Results: MTT assay revealed that genistein reduced the cell viability of HepG2 cancer cells in a dose-dependent manner. Genistein also reduced the colony forming potential of the HepG2 cell concentration dependently. The IC50 of genistein was found to be 25 μM. Genistein caused G2/M cell cycle arrest and G2/M cells increased from 4.2% in the control to 56.4% at 100 μM concentration. Genistein prompted generation of significant (p < 0.01) amounts of ROS, ultimately favouring cell death. Genistein also triggered apoptosis which was associated with upregulation of cytosolic cytochrome c, Bax, cleaved caspase 3 and 9 expression and downregulation of Bcl-2 expression in HepG2 cells.

Conclusions: We propose that genistein exhibits significant anticancer activity against liver cancer and therefore may prove beneficial in the management of liver cancer.

Keywords: HepG2; anticancer; genistein; liver cancer.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Assessment of cell viability at indicated doses of genistein at indicated time intervals Results are the mean of three biological replicates and are expressed as mean ± SD. The values were considered statistically significant at *p < 0.01, **p < 0.001 and ***p < 0.0001.
Figure 2
Figure 2
Colony formation assay of HepG2 cells: A – inhibition of colony formation, B – percentage of colony inhibition at indicated doses of genistein Results are the mean of three biological replicates and are expressed as mean ± SD. The values were considered statistically significant at *p < 0.01, **p < 0.001 and ***p < 0.0001.
Figure 3
Figure 3
Cycle distribution of HepG2 cells at indicated doses of genistein. Results are representatives of three biological replicates
Figure 4
Figure 4
Determination of ROS at indicated doses of genistein at indicated time intervals. A – Flow cytometric analysis, B – Quantification Results are the mean of three biological replicates and are expressed as mean ± SD. The values were considered statistically significant at *p < 0.01, **p < 0.001 and ***p < 0.0001.
Figure 5
Figure 5
Detection of apoptosis by DAPI staining at indicated doses of genistein. Results are representatives of three biological replicates
Figure 6
Figure 6
Estimation of apoptotic HepG2 populations at indicated doses by annexin V/IP using flow cytometric analysis. The images are representatives of three biological replicates
Figure 7
Figure 7
The effect of indicated doses of genistein on the expression of caspase-dependent mitochondrial apoptosis pathway proteins in HepG2 cells. Representative images of cytochrome c, Bax, Bcl-2, PARP, cleaved caspase 9 and cleaved caspase 3 protein expression detected by western blot. β-actin was used as a control. All experiments were carried out in three biological replicates
Figure 8
Figure 8
Effect of genistein at IC50 concentration on cell migration as shown by the wound healing assay. All experiments were carried out in triplicate
See this image and copyright information in PMC

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