Two human ESCC cell lines (ECA-109 and TE-1) were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 37°C in a 5% CO₂ atmosphere. Normal human esophageal epithelial cells were obtained from ScienCell Research Laboratories (San Diego, CA, USA) and maintained in Epithelial Cell Medium-2 (ScienCell Research Laboratories) containing 10% FBS.

Plumbagin (>95% in purity) was purchased from Sigma-Aldrich (Merck KGaA) and dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA) to yield a stock solution of 20 mM. ECA-109 and TE-1 cells were exposed to different concentrations of plumbagin (1-16 μM) (12) for 48 h and examined for viability, proliferation, cell cycle distribution and apoptosis. If not stated otherwise, 8 μM of plumbagin was used in in vitro experiments.

Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In brief, following plumbagin treatment, cells were incubated with 0.5 mg/ml MTT (Sigma-Aldrich; Merck KGaA) at 37°C for 4 h, followed by the addition of DMSO. The absorbance of the MTT formazan reduction product was measured at 570 nm.

Cell proliferation was measured using 5-bromo-2′-deoxyuridine (BrdU) incorporation assay (13). In brief, cells were labeled with BrdU (Roche Applied Science, Penzberg, Germany; 10 μM) during the last 4 h of plumbagin treatment. For quantification of BrdU incorporation, cells were incubated with anti-BrdU conjugated to peroxidase (Roche Applied Science), followed by addition of 3,3′,5,5′-tetramethylbenzidine substrate solution (Roche Applied Science). The absorbance was measured at 450 nm using a microplate reader.

For analysis of cell cycle distribution, cells were fixed in ice-cold 70% ethanol and resuspended in staining solution containing DNase-free RNase A (0.2 mg/ml) and propidium iodide (PI; 20 μg/ml), both from Sigma-Aldrich (Merck KGaA). Following incubation for 15 min in the dark, cellular DNA content was analyzed by flow cytometry. For measurement of apoptosis, cells were fixed and stained with fluorescein isothiocyanate (FITC)-conjugated Annexin V and PI using the Annexin V-FITC Apoptosis Detection kit (R&D systems, Minneapolis, MN, USA). Stained cells were subjected to flow cytometric analysis using FlowJo software (version 10.0.4; Tree Star, Inc., Ashland, OR, USA).

Cells were lysed in ice-cold radio-immunoprecipitation assay (RIPA) buffer (Sigma-Aldrich; Merck KGaA) containing a mixture of protease inhibitors (Pierce; Thermo Fisher Scientific, Inc.). Protein concentrations were measured using the BCA assay kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of protein (40 μg) were separated by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. Following blocking with 5% fat-free milk, membranes were incubated with primary antibodies overnight at 4°C prior to incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:2,000; cat. no. sc-2005; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or HRP-conjugated goat anti-rabbit IgG (1:2,000; cat. no. sc-2030; Santa Cruz Biotechnology, Inc.) for 1 h. The primary antibodies recognizing tumor protein p53 (cat. no. ab131442; 1:500), cyclin-dependent kinase inhibitor 1A (also known as p21; cat. no. ab109520; 1:500), cyclin D1 (cat. no. ab134175; 1:500), cyclin-dependent kinase (CDK) 4 (cat. no. ab108357; 1:300), induced myeloid leukemia cell differentiation protein Mcl-1 (Mcl-1; cat. no. ab32087; 1:500), and β-actin (cat. no. ab8227; 1:2,000) were purchased from Abcam (Cambridge, UK). The antibodies against phospho-STAT3 (Tyr705; cat. no. 9131; 1:300), STAT3 (cat. no. 9132; 1:500), cleaved caspase-3 (cat. no. 9661; 1:300), and cleaved poly(ADP-ribose) polymerase (PARP; cat. no. 5625; 1:300) were from Cell Signaling Technology, Inc. (Danvers, MA, USA). Protein signals were visualized using enhanced chemiluminescence reagent (Amersham; GE Healthcare, Chicago, IL, USA) and quantified using Quantity One software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

A STAT3-responsive firefly luciferase plasmid (pSTAT3-Luc) was purchased from Panomics (Redwood City, CA, USA). A Renilla luciferase-expressing plasmid (pRL-TK) was purchased from Promega Corporation (Madison, WI, USA). A construct expressing constitutively active STAT3 mutant (pSTAT3-C) and empty vector were obtained from Addgene (Cambridge, MA, USA).

For over-expression of constitutively active STAT3, ESCC cells were seeded 12 h prior to transfection. At 80% confluence, they were transfected with pSTAT3-C or empty vector using Lipofectamine 2000 following the manufacturer's instructions (Invitrogen; Thermo Fisher Scientific, Inc.). At 24 h after transfection, cells were treated with plumbagin and subjected to proliferation and apoptosis assays. For the luciferase reporter assay, ESCC cells were co-transfected with pSTAT3-Luc (0.5 μg) and pRL-TK (20 ng) 24 h prior to plumbagin treatment. Following incubation for an additional 48 h, cells were lysed and luciferase activities were measured using a luciferase assay kit (Promega Corporation). The firefly luciferase activity was normalized to that of Renilla luciferase.

For xenograft studies, a total of 10 male nude mice (aged 4-6 weeks) were used, which were purchased from the Experimental Animal Center of Zhengzhou University (Zhengzhou, China). Animals were housed in a laminar air flow hood under a 12/12 h light/dark cycle at 25°C and 50% humidity with free access to food and water. ECA-109 and TE-1 cells (2×10⁶ cells/mouse) were subcutaneously injected into the flanks of mice. When xenograft tumors reached 100 mm³, the tumor-bearing mice (n=5 for each group) were intraperitoneally administered with plumbagin or vehicle (0.05% DMSO) at a dose of 2 mg/kg/day 3 times per week for 4 weeks (14). Tumor growth was monitored by measuring tumor volume. All animals were sacrificed after 30 days of treatment. Tumors were removed and processed for immunohistochemical staining using anti-Ki-67 (cat. no. ab15580; 1:500; Abcam) and anti-phospho-STAT3 (1:100) antibodies (15). Over 1,000 cells were counted from 5 random microscopic fields, and the % of immunoreactive cells was calculated. All experiments involving animals were approved by the Experimental Animal Ethics Committee of Shanghai Jiao Tong University School of Medicine (Shanghai, China; Approval number: 2016-01-032).

Data are presented as the mean ± standard deviation. Statistical differences were determined using the Student's t-test for comparison of two groups or one-way analysis of variance followed by Tukey's post-hoc test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

To assess the cytotoxicity of plumbagin, two ESCC cell lines were exposed to different concentrations of plumbagin for 48 h. As determined by MTT assay (Fig. 1B), plumbagin treatment resulted in a concentration-dependent reduction in cell viability. The IC₅₀ values for plumbagin in ECA-109 and TE-1 cells were 6.2±0.5 and 2.4±0.6 μM, respectively. BrdU incorporation assay was preformed to validate the antiproliferative activity of plumbagin. The results demonstrated that treatment with 8 μM plumbagin for 48 h decreased the proliferation of ECA-109 and TE-1 cells by 45.5 and 62.6%, respectively (Fig. 1C). However, plumbagin, up to the concentration of 16 μM, had no significant impact on the viability of normal esophageal epithelial cells following treatment for 48 h (Fig. 1D).

Flow cytometric analysis was performed to determine the effect of plumbagin on cell cycle progression and apoptosis. As presented in Fig. 2A, treatment of ECA-109 cells with plumbagin for 48 h increased the proportion of cells in the G₀/G₁ phase of the cell cycle from 50 to 62% (P=0.001) and decreased the proportion of cells in the S phase from 29 to 18% (P=0.002) relative to control. In addition, plumbagin treatment resulted in a 4.7-fold (P=0.004) increase in the % of Annexin V-positive apoptotic cells, compared with vehicle-treated cells (Fig. 2B). Similar findings were observed in TE-1 cells following treatment with plumbagin (Fig. 2). Western blot analysis of several key genes involved in cell cycle progression and apoptosis confirmed that plumbagin treatment resulted in a marked increase in p53 and p21 and decrease in cyclin D1, CDK4 and Mcl-1 expression (Fig. 3A). In addition, cleavage of caspase-3 and PARP was markedly enhanced in plumbagin-treated ECA-109 and TE-1 cells compared with vehicle-treated cells (Fig. 3A).

Western blot analysis demonstrated that plumbagin treatment led to a significant decrease in the levels of phosphorylated STAT3, but not total STAT3 protein (Fig. 3B). Consistently, plumbagin-treated ESCC cells displayed a 3- to 6-fold reduction of STAT3 reporter gene activity, compared with control cells (Fig. 3C). These data indicated that plumbagin had the capacity to interfere with STAT3 activation.

Next, the hypothesis that the plumbagin-mediated cytotoxicity in ESCC cells was directly associated with inactivation of STAT3 signaling was examined. To this end, rescue experiments were performed with a constitutively active STAT3 variant. The results demonstrated that ectopic expression of constitutively active STAT3 (Fig. 4A) significantly attenuated the plumbagin-induced growth suppression (Fig. 4B) and apoptosis (Fig. 4C) in ESCC cells.

To determine the in vivo anticancer effect of plumbagin, ECA-109 and TE-1 cells were injected into nude mice, allowed to form xenograft tumors, and treated with plumbagin or vehicle in vivo. After 30 days, plumbagin-treated ECA-109 and TE-1 xenograft tumors displayed 72 and 54% reduction in tumor volume, respectively, relative to vehicle-treated tumors (Fig. 5A). Immunohistochemistry analysis of tumor specimens confirmed that plumbagin-treated tumors presented a significant reduction in the % of Ki-67-positive cells (P=0.011; Fig. 5B). Additionally, the levels of phosphorylated STAT3 were significantly lower in plumbagin-treated tumors compared with vehicle-treated tumors (P=0.001; Fig. 5C).