Cucurbitacin D induces cell cycle arrest and apoptosis by inhibiting STAT3 and NF-κB signaling in doxorubicin-resistant human breast carcinoma (MCF7/ADR) cells

Abstract

Breast cancer is the most common cancer for women and is a major cause of mortality in women. Doxorubicin is a generally used chemotherapy drug for breast cancer. However, multidrug resistance of breast cancer interferes with the chemotherapy. We examined whether cucurbitacin D affects doxorubicin resistance of MCF7/ADR breast cancer cells. Cell viability was measured by MTT assay. Levels of p-STAT3, p-NF-κB, IκB, and caspases were measured by Western blot analysis. Nuclear staining of Stat3 and NF-κB was measured by immunocytochemistry. STAT3 and NF-κB transcriptional activity was detected by STAT3 and NF-κB luciferase reporter gene assays. Analysis of cell cycle arrest was performed by flow cytometry. Induction of apoptosis by cucurbitacin D was measured by Annexin V-FITC/propidium iodide assay. More than 90% of MCF7/ADR cells lived upon treatment with doxorubicin for 24 h. However, upon treatment with cucurbitacin D, cell death was more than 60%. Co-administration of cucurbitacin D and doxorubicin induced apoptosis, and G2/M cell cycle arrest, and inhibited upregulated Stat3 by doxorubicin on MCF7/ADR cells. Additionally, cucurbitacin D led to an increase in the IκBα level in the cytosol and a decrease in the p-NF-κB level in the nucleus. Finally, cucurbitacin D inhibited translocation of Stat3 and NF-κB and decreased transcriptional activity in the nucleus. Cucurbitacin D decreases cell proliferation and induces apoptosis by inhibiting Stat3 and NF-κB signaling in doxorubicin-resistant breast cancer cells. Cucurbitacin D could be used as a useful compound to treat adriamycin-resistant patients.

Keywords: Breast cancer; Cucurbitacin D; Doxorubicin; MCF7 cell; MCF7/ADR cell; Multidrug resistance.

Fig. 1

Effect of doxorubicin, cucurbitacin D, and doxorubicin with cucurbitacin D on MCF7 and MCF7/ADR cell viability. MCF7 and MCF7/ADR cells were treated with different concentrations of doxorubicin, cucurbitacin D, cucurbitacin D (0.5, 2 μg/mL), and doxorubicin (1 μM) for 24, 48, and 72 h. Cell viability was then measured using the MTT assay. Each value represents the mean ± SD. All data are p < 0.0001 by Student t-test

Fig. 2

Cucurbitacin D suppresses p-Stat3 expression in MCF7/ADR Cells. Constitutive activation of Stat3 was detected in MCF7/ADR cells. p-STAT3 was strongly expressed in MCF7/ADR cells. a Doxorubicin increases constitutive STAT3 phosphorylation in a time-dependent manner in MCF7 cells. b Cucurbitacin D inhibits p-Stat3 expression in MCF7/ADR cells. c Whole-cell lysates were analyzed by Western blot with anti-pStat3, anti-Stat3, and anti-tubulin antibodies

Fig. 3

Cucurbitacin D inhibits NF-κB signaling in MCF7/ADR cells. MCF7/ADR cells were treated with cucurbitacin D (0.5, 2 μg/mL) in the presence and absence of doxorubicin (1 μM). Nuclear and cytosolic extracts of cultured cells were then prepared and analyzed by Western blot to measure NF-κB levels

Fig. 4

Cucurbitacin D inhibits nuclear translocation of Stat3 and NF-κB in MCF7/ADR cells. MCF7/ADR cells were treated with cucurbitacin D (0.1 μg/mL) in the presence and absence of doxorubicin (1 μM), and then submitted to immunocytochemistry for the detection of nuclear NF-κB (a) and Stat3 (b)

Fig. 5

Cucurbitacin D inhibits Stat3 and NF-κB transcription in MCF7 cells. MCF7 cells were transfected with the indicated siRNA or plasmid, and then treated with each drug for 24 h. MCF7 cells were then treated with cucurbitacin D (0.5 μg/mL). Afterward, lysates were analyzed using the dual-luciferase reporter assay. Each value represents the mean ± SD. All data are p < 0.0001 by Student t-test

Fig. 6

Cucurbitacin D induces G2/M cell cycle arrest and apoptosis in MCF7/ADR cells. MCF7/ADR cells were treated with cucurbitacin D (0.5 μg/mL) in the presence and absence of doxorubicin (1 μM) for 24 h. Cell cycle distribution was analyzed using a FACS flow cytometer (a). MCF7/ADR cells were treated with the indicated drugs for 24 h and subjected to Annexin V/PI assay. b Effect of cucurbitacin D on the expression of cleaved caspase-3, cleaved caspase-8, and cleaved PARP. MCF7/ADR cells were treated with cucurbitacin D (0.5 μg/mL) in the presence and absence of doxorubicin (1 μM) for 24 h. The cell lysates were subjected to Western blot analysis using specific antibodies (c)

Fig. 6

Cucurbitacin D induces G2/M cell cycle arrest and apoptosis in MCF7/ADR cells. MCF7/ADR cells were treated with cucurbitacin D (0.5 μg/mL) in the presence and absence of doxorubicin (1 μM) for 24 h. Cell cycle distribution was analyzed using a FACS flow cytometer (a). MCF7/ADR cells were treated with the indicated drugs for 24 h and subjected to Annexin V/PI assay. b Effect of cucurbitacin D on the expression of cleaved caspase-3, cleaved caspase-8, and cleaved PARP. MCF7/ADR cells were treated with cucurbitacin D (0.5 μg/mL) in the presence and absence of doxorubicin (1 μM) for 24 h. The cell lysates were subjected to Western blot analysis using specific antibodies (c)