In vitro anti-proliferative effect of capecitabine (Xeloda) combined with mocetinostat (MGCD0103) in 4T1 breast cancer cell line by immunoblotting

Abstract

Objectives: Mouse breast cancer cell line 4T1 can accurately mimic the response to immune receptors and targeting therapeutic agents. Combined therapy has emerged as an important strategy with reduced side effects and maximum therapeutic effect. Mocetinostat (MGCD0103) is one of the members of Class I Histone Deacetylase Inhibitors (HDACi) and its mechanism of action has not been defined, yet. Capecitabine (Xeloda) is an antimetabolite and currently is widely utilized to treat a wide range of solid tumors. The aim of this study was to investigate the effects of the capecitabine, mocetinostat and their combined application on the 4T1 cell line.

Materials and methods: The effects of combined administration of mocetinostat and capecitabine on 4T1 cells were investigated by cell viability and migration assays, apoptosis analysis, and Western blotting technique.

Results: The concentrations of drugs that give a half-maximal response (IC₅₀) were detected for capecitabine (1700 µM), mocetinostat (3,125 µM), and 50 µM Capecitabine+1,5 µM Mocetinostat for 48 hr. In capecitabine+mocetinostat combine group, we observed that cell migration decreased, DNA fragmentation increased compared to the control group. capecitabine + mocetinostat group induced apoptosis by decreasing Bcl-2, PI3K, Akt, c-myc protein levels, while increasing Bax, Caspase-3, PTEN, cleaved-PARP, Caspase-7, Caspase-9, p53, cleaved-Cas-9 protein levels in 4T1 cells.

Conclusion: Capecitabine and mocetinostat played a toxic role through inducing apoptosis on 4T1 cancer cells in a time- and concentration-dependent manner. These results showed that combined therapy with low concentrations were detected to be more effective than that with high-concentration alone drug treatment.

Keywords: Apoptosis; Breast neoplasms; Capecitabine; Drug synergism; Histone deacetylase - inhibitors.

Figure 1

Effect of capecitabine, mocetinostat, and capecitabine+ mocetinostat on 4T1 breast cancer cell viability for 48 hr. The effect of drugs on the survival rate of 4T1 cells was quantified by the MTT method. The data are shown as the mean SD of three separate experiments, with error bars denoting SD. *P< 0.05, **P< 0.01, ***P< 0.001 when compared with untreated cells

Figure 2

Time-dependent morphological image of 4T1 cells treated with mocetinostat capecitabine and combination of two drugs (Nikon Eclipse TS100)

Figure 3

Time dependency of the viability of 4T1 breast cancer cells after capecitabine, mocetinostat, and capecitabine+mocetinostat treatment

Figure 4

DNA laddering was performed in the control and treatment groups in an agarose gel to support the induction of apoptosis by capecitabine, mocetinostat, and capecitabine+mocetinostat for 48 hr

Figure 5

Wound healing in treated groups of 4T1 cells indicates reduction of cellular migration. (1) Control (without any treatment), (2) 1700 μm capecitabine, (3) 3,125 μm mocetinostat, and (4) 50 μm capecitabine, 1,5 μm mocetinostat. Combined treatment of capecitabine and mocetinostat reduced cellular density more than concentrations of mocetinostat and capecitabine applied alone

Figure 6

Capecitabine, mocetinostat, capecitabine+mocetinostat induces apoptosis by altering the protein expression of Bcl-2, Hdac I, Akt, Bax, caspase 3, Pten, cleaved-PARP, Caspase-7, Caspase-9, p53, PI3K, c-myc, and Hdac III in 4T1 cells. Bands were quantified by densitometry and normalized to the model control values. A statistical analysis of proteins adjusted to β-actin was performed. The data are shown as the mean standard deviation of three separate experiments. * P<0.05, ** P<0.01, *** P<0.001, respectively