Selective cytotoxicity of goniothalamin against hepatoblastoma HepG2 cells

Abstract

Liver cancer has become one of the major types of cancer with high mortality and liver cancer is not responsive to the current cytotoxic agents used in chemotherapy. The purpose of this study was to examine the in vitro cytotoxicity of goniothalamin on human hepatoblastoma HepG2 cells and normal liver Chang cells. The cytotoxicity of goniothalamin against HepG2 and liver Chang cell was tested using MTT cell viability assay, LDH leakage assay, cell cycle flow cytometry PI analysis, BrdU proliferation ELISA assay and trypan blue dye exclusion assay. Goniothalamin selectively inhibited HepG2 cells [IC₅₀ = 4.6 (±0.23) µM in the MTT assay; IC₅₀ = 5.20 (±0.01) µM for LDH assay at 72 hours], with less sensitivity in Chang cells [IC₅₀ = 35.0 (±0.09) µM for MTT assay; IC₅₀ = 32.5 (±0.04) µM for LDH assay at 72 hours]. In the trypan blue dye exclusion assay, the Viability Indexes were 52 ± 1.73% for HepG2 cells and 62 ± 4.36% for Chang cells at IC₅₀ after 72 hours. Cytotoxicity of goniothalamin was related to inhibition of DNA synthesis, as revealed by the reduction of BrdU incorporation. At 72 hours, the lowest concentration of goniothalamin (2.3 µL) retained 97.6% of normal liver Chang cells proliferation while it reduced HepG2 cell proliferation to 19.8% as compared to control. Besides, goniothalamin caused accumulation of hypodiploid apoptosis and different degree of G2/M arrested as shown in cell cycle analysis by flow cytometry. Goniothalamin selectively killed liver cancer cell through suppression of proliferation and induction of apoptosis. These results suggest that goniothalamin shows potential cytotoxicity against hepatoblastoma HepG2 cells.

Figure 1

Structure of goniothalamin.

Figure 2

Effects of goniothalamin treatment in HepG2 (A) and Chang cells (B) and doxorubicin treatment in HepG2 (C) and Chang cells (D). Twenty-four hours after seeding of cells in 96 well plates, goniothalamin and doxorubicin were added to the final concentrations shown in the figure. At 24 h (●), 48 h (■) and 72 h (♦) of treatment, effects of goniothalamin and doxorubicin against the viability of treated cells were evaluated through mitochondrial activity using the MTT assay.

Figure 2

Effects of goniothalamin treatment in HepG2 (A) and Chang cells (B) and doxorubicin treatment in HepG2 (C) and Chang cells (D). Twenty-four hours after seeding of cells in 96 well plates, goniothalamin and doxorubicin were added to the final concentrations shown in the figure. At 24 h (●), 48 h (■) and 72 h (♦) of treatment, effects of goniothalamin and doxorubicin against the viability of treated cells were evaluated through mitochondrial activity using the MTT assay.

Figure 3

LDH leakage in HepG2 (A) and Chang cells (B) treated with goniothalamin. Cells (1 × 10⁴) with different concentrations of goniothalamin were incubated for the indicated times. The cytotoxicity was expressed as percentage LDH release as compared to the maximum release of LDH from Triton-X100-treated cells.

Figure 3

LDH leakage in HepG2 (A) and Chang cells (B) treated with goniothalamin. Cells (1 × 10⁴) with different concentrations of goniothalamin were incubated for the indicated times. The cytotoxicity was expressed as percentage LDH release as compared to the maximum release of LDH from Triton-X100-treated cells.

Figure 4

Effects of goniothalamin and doxorubicin on the proliferation of HepG2 (A) and Chang (B) cells in vitro. Goniothalamin inhibits cell proliferation in a time- and dose-dependent manner. After treatment with 2.3 µM (IC₅₀ based on MTT assay results) and 150 µM of goniothalamin and IC₅₀ of doxorubicin (as positive control) for 24, 48 and 72 hours, cellular proliferation of HepG2 and Chang cells was assayed using BrdU incorporation ELISA.

Figure 4

Effects of goniothalamin and doxorubicin on the proliferation of HepG2 (A) and Chang (B) cells in vitro. Goniothalamin inhibits cell proliferation in a time- and dose-dependent manner. After treatment with 2.3 µM (IC₅₀ based on MTT assay results) and 150 µM of goniothalamin and IC₅₀ of doxorubicin (as positive control) for 24, 48 and 72 hours, cellular proliferation of HepG2 and Chang cells was assayed using BrdU incorporation ELISA.

Figure 5

HepG2 (A) Chang (B) cells were treated with goniothalamin (GTN) and at the indicated concentration and doxorubicin IC₅₀ (Doxo) for 24, 48 and 72 hours. The viable cell number was assessed by cell counting in a trypan blue assay; Control values were set as 100%.

Figure 5

HepG2 (A) Chang (B) cells were treated with goniothalamin (GTN) and at the indicated concentration and doxorubicin IC₅₀ (Doxo) for 24, 48 and 72 hours. The viable cell number was assessed by cell counting in a trypan blue assay; Control values were set as 100%.