Antitumor Activity of Pulvomycin via Targeting Activated-STAT3 Signaling in Docetaxel-Resistant Triple-Negative Breast Cancer Cells

Abstract

Although docetaxel-based regimens are common and effective for early-stage triple-negative breast cancer (TNBC) treatment, acquired drug resistance frequently occurs. Therefore, a novel therapeutic strategy for docetaxel-resistant TNBC is urgently required. Signal transducer and activator of transcription 3 (STAT3) plays a pivotal role in the tumorigenesis and metastasis of numerous cancers, and STAT3 signaling is aberrantly activated in TNBC cells. In this study, a docetaxel-resistant TNBC cell line (MDA-MB-231-DTR) was established, and mechanisms for the antitumor activity of pulvomycin, a novel STAT3 inhibitor isolated from marine-derived actinomycete, were investigated. Levels of activated STAT3 (p-STAT3 (Y705)) increased in docetaxel-resistant cells, and knockdown of STAT3 recovered the sensitivity to docetaxel in MDA-MB-231-DTR cells. Pulvomycin effectively inhibited the proliferation of both cell lines. In addition, pulvomycin suppressed the activation of STAT3 and subsequently induced G₀/G₁ cell cycle arrest and apoptosis. Pulvomycin also significantly inhibited the invasion and migration of MDA-MB-231-DTR cells through the modulation of epithelial-mesenchymal transition markers. In an MDA-MB-231-DTR-bearing xenograft mouse model, the combination of pulvomycin and docetaxel effectively inhibited tumor growth through STAT3 regulation. Thus, our findings demonstrate that the combination of docetaxel and STAT3 inhibitors is an effective strategy for overcoming docetaxel resistance in TNBC.

Keywords: docetaxel; metastasis; pulvomycin; resistance; signal transducer and activator of transcription 3; triple-negative breast cancer.

Figure 1

Relationship between signal transducer and activator of transcription 3 (STAT3) expression and overall survival (OS) in breast cancer, and the effect of pulvomycin on p-STAT3 in MDA-MB-231 cells. (A) The Kaplan–Meier survival curve for breast cancer-associated OS with STAT3 expression. (B) The Kaplan–Meier survival curve for TNBC-associated OS with STAT3 expression. (C) Levels of p-STAT3 expression in several TNBC cell lines were analyzed using Western blot analysis. β-Actin was used as an internal control. (D) Chemical structure of pulvomycin (E) MDA-MB-231 cells were treated with the indicated concentrations of pulvomycin for 24 h, and levels of p-STAT3 (Y705) and STAT3 expression were determined using Western blot analysis. β-Actin was used as an internal control.

Figure 2

Signal transducer and activator of transcription 3 (STAT3)-dependent effects of docetaxel on the viability of human triple-negative breast cancer (TNBC) cells. (A) Cells were treated with the indicated concentrations of pulvomycin for 24 h, and levels of p-STAT3 (Y705) and STAT3 expression were determined using Western blot analysis. β-Actin was used as an internal control. (B) Western blot analysis of STAT3 expression in MDA-MB-231 cells 48 h after transfection with the control vector or STAT3 plasmid. β-Actin was used as an internal control. Relative intensities of indicated proteins were semi-quantitatively analyzed using NIH’s ImageJ v1.52a software. (C) The effect of docetaxel on the viability of MDA-MB-231 cells with the overexpression of STAT3. Cells transfected with either control vector or STAT3 plasmid were treated with various concentrations of docetaxel for 72 h, and cell viability was measured using the MTT assay. Data are presented as the mean ± SD (n = 3). * p < 0.05 and ** p < 0.01 compared with the vector-transfected control. (D) MDA-MB-231-DTR cells were transfected with scrambled siRNA or STAT3 siRNA for 48 h, and levels of STAT3 expression were determined using Western blot analysis. β-Actin was used as an internal control. Relative intensities of indicated proteins were semi-quantitatively analyzed using NIH’s ImageJ v1.52a software. (E) The effect of docetaxel on the viability of MDA-MB-231-DTR cells with the knockdown of STAT3. Cells transfected with either scrambled siRNA or STAT3 siRNA #2 were treated with various concentrations of docetaxel for 72 h, and cell viability was measured using the MTT assay. Data are presented as the mean ± SD (n = 3). * p < 0.05 and ** p < 0.01 compared with the vector-transfected control. (F) Cell viability was measured after combined pulvomycin and docetaxel treatment for 72 h in MDA-MB-231-DTR cells. Based on cell viability results, CI values were calculated to demonstrate the effect of the drug combination on MDA-MB-231-DTR cells.

Figure 3

Effects of pulvomycin on cell cycle regulation. (A,B) MDA-MB-231-DTR cells were treated with the indicated concentrations of pulvomycin for 24 h and fixed with 70% ethanol for 24 h. Cell cycle distribution was determined in cells incubated with RNase A and PI for 30 min using flow cytometry. Data are presented as the mean ± SD (n = 3). * p < 0.05 and ** p < 0.01 compared with the control. (C) MDA-MB-231-DTR cells were treated with the indicated concentrations of pulvomycin for 24 h, and levels of cyclin E, CDK2, cyclin D1, CDK4, and CDK6 expression were determined using Western blot analysis. β-Actin was used as an internal control.

Figure 4

Effects of pulvomycin on apoptotic cell death. (A,B) MDA-MB-231-DTR cells were treated with the indicated concentrations of pulvomycin for 48 h and fixed with 70% ethanol for 24 h. After incubation with RNase A and PI for 30 min, cell cycle distribution was determined using flow cytometry. Data are presented as the mean ± SD (n = 3). ** p < 0.01 and *** p < 0.001 compared with the control. (C,D) MDA-MB-231-DTR cells were treated with the indicated concentrations of pulvomycin for 48 h and stained with Annexin V-fluorescein isothiocyanate (V-FITC) and PI. Annexin V/PI-positive cells were analyzed using flow cytometry to evaluate the apoptotic cell population. Data are presented as the mean ± SD (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the control. (E) MDA-MB-231-DTR cells were treated with the indicated concentrations of pulvomycin for 48 h, and levels of survivin, cleaved caspase-9, cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase (PARP) (D214) expression were determined using Western blot analysis. β-Actin was used as an internal control.

Figure 5

The effects of pulvomycin on epithelial-mesenchymal transition (EMT)-mediated cell invasion and migration. (A) The cells were treated with the indicated concentrations of pulvomycin for 24 h, and levels of p-STAT3 (Y705), STAT3, E-cadherin, N-cadherin, and vimentin expression were determined using Western blot analysis. β-Actin was used as an internal control. (B) The cells were pretreated with pulvomycin at the indicated concentration for 24 h, reseeded into the upper chambers of Transwell inserts, and incubated for 24 h. Cells that invaded the lower chambers were fixed, stained, imaged (left), and quantified using NIH’s ImageJ v1.52a software (right). Data are presented as the mean ± SD (n = 3). *** p < 0.001 compared with the control. (C) Cell monolayers were mechanically scratched and treated with pulvomycin for 24 h. Representative light microscopy images of wound closure are shown (left). Wound areas were quantified using NIH’s ImageJ v1.52a software (right). Data are presented as the mean ± SD (n = 3). ** p < 0.01 and *** p < 0.001 compared with the control.

Figure 6

The combined administration of pulvomycin and docetaxel can overcome docetaxel resistance in an in vivo xenograft model. (A) Tumor volumes of the MDA-MB-231-DTR xenograft mouse model intraperitoneally administered vehicle, docetaxel, pulvomycin, or a combination of docetaxel (10 mg/kg body weight) and pulvomycin (10 mg/kg body weight) three times per week for 24 days were measured every 3–4 days with an electronic caliper. * p < 0.05, ** p < 0.01, and *** p < 0.001 were compared with the vehicle-administered control group. (B) Mouse body weights were measured every 3–4 days to assess general toxicity. (C) Small portions of tumor tissue from each group were homogenized in complete lysis buffer (Active Motif). Levels of p-STAT3 (Y705) and STAT3 expression were determined using Western blot analysis. β-Actin was used as an internal control. (D) Small portions of tumor tissue from each group were homogenized in complete lysis buffer (Active Motif). Levels of Ki-67, p-STAT3 (Y705), and STAT3 expression were determined using Western blot analysis. β-Actin was used as an internal control.