Synergistic antitumor effect of brusatol combined with cisplatin on colorectal cancer cells

Abstract

Colorectal cancer (CRC) is a common and life‑threatening type of malignant cancer, which is associated with a high mortality rate. Cisplatin (CDDP) is a commonly used chemotherapy drug with significant side effects. Brusatol (BR) is one of the principal chemical compounds isolated from the Chinese herb Bruceae Fructus, which has been reported to markedly inhibit the proliferation of numerous cancer cell lines. The present study aimed to investigate the possible synergistic anticancer effects of CDDP combined with BR on CT‑26 cells, and to evaluate the underlying mechanisms of action. The growth inhibitory effects of BR, CDDP, and BR and CDDP cotreatment on CT‑26 cells were assessed by MTT assay. Cell apoptosis were determined by flow cytometry and western blot analysis. The results indicated that compared with single‑agent treatment, cotreatment of CT‑26 cells with CDDP and BR synergistically inhibited cell proliferation and increased cellular apoptosis. Furthermore, treatment of CT‑26 cells with CDDP and BR resulted in a marked increase in the release of cytosolic cytochrome c, decreased expression of procaspase‑3 and procaspase‑9, and upregulation of the B‑cell lymphoma 2 (Bcl‑2)‑associated X protein/Bcl‑2 ratio compared with treatment with BR or CDDP alone. These results strongly suggested that the combination of CDDP and BR was able to produce a synergistic antitumor effect in CRC cells, thus providing a solid foundation for further development of this combination regimen into an effective therapeutic method for CRC.

Figure 1

Chemical structure of brusatol.

Figure 2

Inhibitory rate of CT-26 cells following treatment with various concentrations of BR (0.75–15 μg/ml) and CDDP (0.75–15 μg/ml) either alone or in combination. Data are presented as the means ± standard deviation of three independent experiments. BR, brusatol; CDDP cisplatin.

Figure 3

Fa-CI plot for brusatol and cisplatin cotreatment on CT-26 cells. CI, combination index.

Figure 4

Cellular apoptosis observed with Hoechst 33342 staining (magnification, ×200). CT-26 cells were treated with BR (0.27 μg/ml), CDDP (1.44 μg/ml), or their combination for 48 h. Chromatin condensation, nuclear fragmentation and apoptotic bodies are indicated by small arrows. (A) Control group; (B) BR (0.27 μg/ml)-treated group; (C) CDDP (1.44 μg/ml)-treated group; and (D) BR (0.27 μg/ml) + CDDP (1.44 μg/ml)-treated group. BR, brusatol; CDDP, cisplatin.

Figure 5

Apoptosis of CT-26 cells mediated by BR and CDDP, alone or in combination. (A) Apoptosis was measured by flow cytometry after PI/Annexin V-FITC staining. Q1, PI⁺ (cells undergoing necrosis); Q2, Annexin V-FITC⁺ PI⁺ (cells in the late period of apoptosis and undergoing secondary necrosis); Q3, Annexin V-FITC⁺ PI⁻ (cells in the early period of apoptosis); Q4, Annexin V-FITC⁻ PI⁻ (living cells). Total apoptotic rate was calculated as Q2 + Q3. (B) Apototic rates were calculated. The proportion of early and late apoptotic cells stained with Annexin V and PI is presented for each group. Data are presented as the means ± standard deviation of three independent experiments. **P<0.01 compared with the Con group; ^##P<0.01 compared with the BR and CDDP monotherapy groups. BR, brusatol; CDDP, cisplatin; Con, control; FITC, fluorescein isothiocyanate; PI, propidium iodide

Figure 6

Protein expression levels of procaspase-3, procaspase-9 and cyt c in CT-26 cells treated with BR and CDDP, alone or in combination. (A) Total cell extracts were prepared and subjected to western blot analysis to monitor the protein expression levels of procaspase-3, procaspase-9 and cyt c in CT-26 cells. β-actin was used as the protein loading control. (B-D) Protein expression levels (relative to β-actin) of (B) cyt c, (C) procaspase-3 and (D) procaspase-9 were determined. All data are presented as the the means ± standard deviation of at least three independent experiments. *P<0.05 and **P<0.01 compared with the control group; ^#P<0.05 and ^##P<0.01 compared with the BR or CDDP monotherapy groups. BR, brusatol; CDDP, cisplatin; cyt c, cytochrome c.

Figure 7

Protein expression levels of Bax, Bcl-2 and Bax/Bcl-2 ratio in CT-26 cells treated with BR and CDDP, alone or in combination. (A) Total cell extracts were prepared and subjected to western blot analysis to monitor the protein expression levels of Bax and Bcl-2 in CT-26 cells. β-actin was used as the protein loading control. (B) Bax/Bcl-2 ratio, and protein expression levels (relative to β-actin) of (C) Bcl-2 and (D) Bax were determined. All data are presented as the means ± standard deviation of at least three independent experiments. *P<0.05 and **P<0.01 compared with the control group; ^##P<0.01 compared with the BR or CDDP monotherapy groups; NS, no statistical significance, P>0.05. Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; BR, brusatol; CDDP, cisplatin