Chinese Herbal Mixture, Tien-Hsien Liquid, Induces G2/M Cycle Arrest and Radiosensitivity in MCF-7 Human Breast Cancer Cells through Mechanisms Involving DNMT1 and Rad51 Downregulation

Abstract

The Chinese herbal mixture, Tien-Hsien Liquid (THL), has been proven to suppress the growth and invasiveness of cancer cells and is currently regarded as a complementary medicine for the treatment of cancer. Our previous study using acute promyelocytic leukemia cells uncovered its effect on the downregulation of DNA methyltransferase 1 (DNMT1) which is often overexpressed in cancer cells resulting in the repression of tumor suppressors via hypermethylation. Herein, we explored the effects of THL in MCF-7 breast cancer cells that also demonstrate elevated DNMT1. The results show that THL dose-dependently downregulated DNMT1 accompanied by the induction of tumor suppressors such as p21 and p15. THL arrested cell cycle in G2/M phase and decreased the protein levels of cyclin A, cyclin B1, phospho-pRb, and AKT. DNMT1 inhibition was previously reported to exert a radiosensitizing effect in cancer cells through the repression of DNA repair. We found that THL enhanced radiation-induced clonogenic cell death in MCF-7 cells and decreased the level of DNA double-strand break repair protein, Rad51. Our observations may be the result of DNMT1 downregulation. Due to the fact that DNMT1 inhibition is now a mainstream strategy for anticancer therapy, further clinical trials of THL to confirm its clinical efficacy are warranted.

Figure 1

THL inhibited the growth of MCF-7, MDA-MB-231, and BT-474 breast cancer cells. Cells were treated with various concentrations of THL (0.75–6 mg/mL) or PBS only as a control for 72 h and cell viability was determined by the SRB assay. Data (mean ± SE in triplicate) are expressed as a percentage compared to the PBS-treated control.

Figure 2

THL arrested the cell cycle of MCF-7 cells in G2/M phase. MCF-7 cells were treated with increasing concentrations of THL (0.75–6 mg/mL) or PBS only, for 72 h, and the cell cycle distribution was analyzed by flow cytometry. THL increased the percentage of G2/M phase in a dose-dependent manner. The percentage of cells in different phases of the cell cycle is shown in Table 1.

Figure 3

THL decreased G2/M-related cyclins A and B1 in MCF-7 cells. (a) THL decreased cyclin A and cyclin B1 protein levels in a dose-dependent manner but did not significantly change the Cdc2 (CDK1) protein level. Cells were treated with PBS or THL (0.75, 1.5, 3, and 6 mg/mL) for 72 h and then harvested. Cell lysates were then prepared for Western blot analysis. (b) The blots were normalized to actin or tubulin, and the fold change in protein expression levels in comparison to the PBS-treated control is depicted in this panel.

Figure 4

THL downregulated DNMT1 and DNMT3A accompanied by the induction of cyclin-dependent kinase inhibitors (p21 and p15) and the reduction of pRb phosphorylation in MCF-7 cells. THL also diminished the DNMT1 protein level in BT-474 cells. (a) (A) THL dose-dependently decreased the protein levels of DNMT1 and DNMT3A in MCF-7 cells. (B) THL dose-dependently increased the protein level of p21. (C) THL dose-dependently increased the protein level of p15. (D) THL dose-dependently decreased the phosphorylation (inactivation) of Rb protein (pRb) in MCF-7 cells. (E) THL dose-dependently diminished the protein level of DNMT1 in BT-474 cells. Cells were treated with PBS (control) or THL for 72 h and then harvested. Cell lysates were then prepared for Western blot analysis. (b) The blots were normalized to tubulin or GAPDH, and the fold change protein level expression is reported in comparison to PBS-treated control.

Figure 5

THL inhibited AKT and ERK signaling pathways in MCF-7 cells. (a) (A) THL decreased both the phosphorylated (p-AKT) and total AKT proteins in a dose-dependent manner. (B) THL decreased phosphorylated ERK protein (p-ERK) at doses above 1.5 mg/mL but did not affect the total ERK protein level. Cells were treated with PBS or THL for 72 h and then harvested. Cell lysates were then prepared for Western blot analysis. (b) The blots were normalized to actin or tubulin, and the fold change protein level expression is shown in comparison to PBS-treated control.

Figure 6

THL sensitizes MCF-7 cells to radiation. (a) Cells were pretreated with THL for 24 h and then irradiated with 4–24 Gy of ionizing radiation as indicated. Colony formation of the irradiated cells was assayed after 8 days of incubation. (b) Representative picture of colony formed by the cells described in (a). Data (mean ± SE in triplicate) are expressed as a percentage of the PBS control. ^∗ p < 0.05, compared to the respective THL free group treated with the same dose of radiation.

Figure 7

THL decreased DNA double-strand break repair protein Rad51 in MCF-7 cells but increased the level of the protein in primary HS68 fibroblast cells. (a) (A) THL dose-dependently decreased the homologous recombination DNA repair protein Rad51 in MCF-7 cells. (B) Pretreatment with THL dose-dependently decreased the Rad51 protein level in irradiated MCF-7 cells but did not reduce the nonhomologous end joining DNA repair proteins Ku70 and Ku86. (C) In contrast to its effect on MCF-7 cells, THL increased the Rad51 protein level in HS68 human foreskin fibroblast cells. In (A) and (C), cells were treated with PBS or THL for 72 h and then harvested. In (B), cells were pretreated with THL for 24 h and then irradiated with 6 Gy of ionizing radiation. Cells were harvested 24 h after irradiation, after which cell lysates were prepared for Western blot analysis. (b) The blots were normalized to actin or tubulin, and the fold change protein level expression is reported in comparison to PBS-treated control.