The T24 and RT4 human BC cell lines were obtained from the Chinese Academy of Science (Shanghai, China). The BC cells were cultured in Dulbecco's modified Eagle's medium (DMEM; cat. no. MGC803; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% FBS and 100 U/ml penicillin/strep tomycin (HyClone™; GE Healthcare Life Sciences, Logan, UT, USA) at 37°C in a humidified atmosphere containing 5% CO₂. Curcumin (Sigma-Aldrich; EMD Millipore, Billerica, MA, USA) was dissolved in dimethylsulfoxide (DMSO) and stored at −20°C. 3–4,5-dimethyl-2- thiazolyl-2, 5-diphenyl-2-H-tetrazolium bromide (MTT; CAS no. 57360-69-7) was obtained from Sigma-Aldrich; EMD Millipore. Lipofectamine 2000 was purchased from Invitrogen; Thermo Fisher Scientific, Inc. Primary antibodies targeting Trop2 (#90540), p27 (#2552), and cyclin E1 (#4129), and monoclonal anti-β-actin (#3700) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Secondary antibodies (#A-11031 and #A-11034) were obtained from Thermo Fisher Scientific, Inc.

The T24 and RT4 cells were plated in 96-well plates (5×10³ cells/well) and cultured for ~24 h. The cells were treated with curcumin (10, 15, 20 and 25 µM) at 37°C for 48 and 72 h. The experimental control group (0.1% DMSO) was set up as the zero group. Each group contained at least five wells. Cell growth abilities were assessed using MTT assays following the manufacturer's protocols. The MTT solution (10 µl; 0.5 mg/ml) was added to each well and incubated for 4 h. The supernatant was then removed, and 100 µl DMSO was added to dissolve the formazan product. Cell viability was determined by detecting the absorbance at 590 nm.

The BC cells (2×10⁵ cells/well) were seeded in 6-well plates. A series of concentrations of curcumin were added and the cells were cultured for 2 days. Cell apoptotic death was assessed with an Annexin V/FITC Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA). The curcumin-treated BC cells were harvested and stained with propidium iodide (PI) and annexin V-FITC, and were measured using a FACS calibur flow cytometer (BD Biosciences).

The T24 and RT4 BC cells were seeded in 6-well plates and treated with curcumin for 468. The cells were collected by trypsinization and washed with PBS and then suspended with pre-cooling alcohol (70%). Following fixing overnight, the cells were precipitated by centrifugation for 5 min at 200 × g and pelleted cells were washed three times with 10 ml PBS. The cells were then incubated with RNase and PI. Cell cycle distributions were detected using a flow cytometer (BD Biosciences).

The BC cells were plated into a 6-well plate and incubated until the cells grew to ~90% confluence. The confluent monolayer was injured with a sterile 100-µl tip and a rectangular wound was created. Cell debris was carefully removed, and the cells were cultured for ~16 h. The cells migrated into the wound were visualized using an inverted microscope. The wound size was scored by measuring the lesion border and comparing with the size of the initial wound.

Transwell chambers (8-µm pore size, Corning Incorporated, Corning, NY, USA) were used to assess the invasive ability of the BC cells. Following culture for 24 h at 37°C, the cells were suspended in the FBS-free DMEM. The cells (5×10⁴ cells; 200 µl) were placed in the Matrigel (BD Biosciences) pre-coated upper chamber, following which 600 µl of complete DMEM was added to the lower chamber. Following incubation for 468, the remaining cells were removed with cotton wool. Cells invading through the filters were stained with Calcein-AM for 10 min, followed by rinsing the filters with water. The stained cells were observed under a light microscope.

The BC cells were transfected with pcDNA3.1-Trop2- or Trop2-targeting siRNAs with Lipofectamine™ 2000 transfection reagent, following the manufacturer's protocol. The siRNA sequences (sense, 5′-ACA CTT GGA GGT TTT GGC CAC TGA CTG ACT CCA AGT GTC TGC TGC TCAA-3′; antisense, 3′-CCT GTT GAG CAG CAG ACT TGG AGG TCA GTC AGT CAG TGG CCA AAA CCT CCA AGT GTC TGC TGC TCA AC-5′) targeting human Trop2 were purchased from GenePharma (Shanghai, China).

The BC cells were inoculated into the culture plate at a concentration of 1×10⁴ cells/cm². The cells were then transfected with pcDNA3.1-Trop2- or Trop2-targeting siRNAs or treated with curcumin for a designated time course. The pretreated BC cells were precipitated and lysed in lysis buffer supplemented with protease inhibitor cocktail and PMSF (Roche Diagnostics, Basel, Switzerland). A BCA protein assay was used to quantify protein concentrations. Protein samples (30 µg) were separated by 10% SDS-PAGE. The separated proteins were then transferred onto a nitrocellulose membrane and were blocked with 5% skimmed milk at room temperature for 1 h. The primary antibodies (Trop2, 1:1,500; p27, 1:1,000; cyclin E1, 1:2,000; and anti-β-actin, 1:3,000) were added and incubated at 4°C overnight. After washing with TBS-Tween-20, a suitable secondary antibody (anti-mouse, 1:2,500; anti-rabbit, 1:2,500) was added and incubated at room temperature for ~1 h. The target proteins were developed onto a film by an ECL imaging system (Pierce; Thermo Fisher Scientific, Inc.). The protein expression levels were semi-quantitated via densitometry using ImageJ software version 1.51 (National Institutes of Health, Bethesda, MD, USA; https://imagej.nih.gov/ij/).

Data are presented as the mean ± standard deviation of the mean following analysis with GraphPad Prism 4.0 (GraphPad Software, Inc., La Jolla, CA, USA). Statistical significance values were evaluated through one-way analysis of variance with a Dunnett's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Curcumin has been reported to suppress proliferation in various types of cancer. The present study aimed to determine whether curcumin inhibits T24 and RT4 BC cell growth. The BC cells were treated with curcumin for 48 and 72 h, respectively. An MTT assay was performed and the resulting data showed that curcumin suppressed BC cell proliferation in a time- and dose-dependent manner (Fig. 1A). These data supported the hypothesis that curcumin exerts its antitumor growth property in BC cells.

Subsequently, the present study detected whether curcumin affects apoptotic death of BC cells. Annexin V-FITC/PI apoptosis analysis was performed in T24 and RT4 BC cells following curcumin treatment. As shown in Fig. 1B and C, curcumin induced apoptotic death of T24 and RT4 cells in a dose-dependent manner. For example, treatment with 10 and 15 µM curcumin caused increased apoptotic death of RT4 cells from 3.64% in the control group to 19.95 and 26.36% (Fig. 1B and C), respectively. It was found that curcumin mainly induced late apoptosis, increasing from 1.83% in the control group to 8.32 and 13.42% in T24 cells treated with 10 and 15 µM curcumin, respectively (Fig. 1B). A similar induction of late apoptosis was induced by curcumin in RT4 cells (Fig. 1B). These results demonstrated that curcumin promoted significant apoptotic death and may facilitate inhibiting the proliferation of BC cells.

Further detection of the cell cycle distribution of bladder cancer cells was performed following curcumin treatment. Flow cytometry was performed following PI staining. The findings revealed that curcumin treatment caused a significant increase in the numbers of cells in the G2/M phase in a dose-dependent manner, compared with control cells (Fig. 2A and B). For example, treatment with 10 and 15 µM curcumin increased the G2/M cell populations to 14.95 and 23.21% in RT4 cells, from 9.02% in the control (Fig. 2A and B). A similar cell cycle arrest pattern was observed in T24 cells (Fig. 2A and B). These findings confirmed that curcumin induced G2/M cell cycle arrest in the BC cells.

In order to detect the effects of curcumin on cell motility, wound-healing assays were performed in T24 and RT4 cells, respectively. As shown in Fig. 3A, curcumin treatment significantly inhibited cell migration in the two BC cell lines. These results revealed that curcumin treatment notably inhibited cell migratory activity in the T24 and RT4 cancer cells.

The present study also measured whether curcumin inhibits cell invasive ability using Transwell chambers. It was found that the number of cells invaded through the pores of Matrigel-coated filters was reduced in the two curcumin-treated BC cell lines in a dose-dependent manner (Fig. 3B). It is important to note that 15 mM curcumin had a cytotoxic effect with ~20% growth inhibition in the T24 cells and RT4 cells at 468. (Fig. 1A). However, treatment with 15 mM curcumin for 24 h did not cause any cell growth inhibition in the cell lines (data not shown). The invasion and migration were measured in the two cell lines following treatment with 15 mM curcumin for 20 h. Therefore, the effects on migration and invasion by curcumin treatment were not due to cell viability inhibition. The findings demonstrated that curcumin treatment significantly inhibited the cell invasion potential of the T24 and RT4 BC cells.

Accumulating data have characterized Trop2 as a tumor driver in various types of cancer, and pharmacological inhibition of Trop2 may be a promising approach to treating BC. Therefore, the present study further examined whether curcumin treatment modulates the expression of Trop2 in BC cells. Following treatment with curcumin for 468, western blot analysis revealed that Trop2 was markedly reduced in the T24 and RT4 BC cells (Fig. 4A and B). It was also detected that the protein levels of p27 and cyclin E1, two typical cell cycle regulators, were modulated following curcumin treatment (Fig. 4A and B). p27, the cell cycle inhibitor, was significantly induced by curcumin treatment. cyclin E1, which is crucial in promoting cell cycle progression and contributes to tumorigenesis, was markedly suppressed in the presence of curcumin in BC cells. Taken together, these results suggested that curcumin exercised antitumor function in BC cells at least partially by downregulating the expression of Trop2. In addition, curcumin caused cell-cycle arrest, which may be attributed to the modulated expression of p27 and cyclin E1.

In order to further identify whether Trop2 was involved in the cytotoxic effects of curcumin, the present study induced the overexpression of Trop2 in BC cells via Trop2 cDNA transfection in the presence of curcumin. An empty vector was transfected as a control. The subsequent MTT assays demonstrated that the overexpression of Trop2 triggered BC cell proliferation (Fig. 5A). In addition, the cell growth inhibition induced by curcumin was partly abrogated under Trop2 overexpression (Fig. 5A).

The cellular invasive and migratory properties were measured to determine whether Trop2 regulates T24 and RT4 cell mobility. Using a wound-healing assay, an increase in the migration of BC cells was found under Trop2 overexpression (Fig. 5B). Consistently, the overexpression of Trop2 eliminated curcumin-induced suppression of cell mobility (Fig. 5B). The Transwell analysis demonstrated that the overexpression of Trop2 markedly increased the number of invasive BC cells (Fig. 5C). The overexpression of Trop2 reversed the curcumin-induced inhibition of cell invasion (Fig. 5C). Furthermore, the effect of the overexpression of Trop2 on apoptosis was detected. The resulting data demonstrated that the overexpression of Trop2 markedly reduced the apoptotic cell percentage in BC cells and abrogated curcumin-induced apoptotic cell death (Fig. 6A and B).

The protein levels of p27 and cyclin E1 were measured in BC cells following Trop2 cDNA transfection. It was observed that the overexpression of Trop2 significantly suppressed protein levels of p27 in the T24 and RT4 cells (Fig. 7A and B; P<0.05). When the overexpression of Trop2 was combined with curcumin treatment, the induced expression of p27 was partially abrogated (Fig. 7A and B; P<0.05). Conversely, the protein level of cyclin E1 was significantly induced in the two BC cell lines with the overexpression of Trop2 (Fig. 7B; p<0.05). The curcumin-induced suppression of cyclin E1 was partially abrogated when combined with Trop2 cDNA transfection. Taken together, these findings supported the hypothesis that curcumin exhibits anti-tumor activity in BC cells partially via regulating the expression of Trop2 and its downstream targets p27 and cyclin E1.

In addition, Trop2 siRNA was used to knock down Trop2 in the BC cells and its effects on cell proliferation, mobility, apoptosis, and the expression of p27 and cyclin E1 were examined. Following Trop2 siRNA transfection, the MTT assay results showed that T24 and RT4 cell proliferation was inhibited (Fig. 8A). When Trop2 was downregulated in the presence of curcumin, the suppression of cell proliferation was significantly enhanced compared with that in the cells treated with either curcumin or siRNA transfection alone (Fig. 8A; P<0.05). The downregulation of Trop2 combined with curcumin treatment also suppressed cell invasion and migration (Fig. 8B and C; P<0.05). The sensitivity of BC cells to apoptosis from curcumin was markedly enhanced in the Trop2-silenced cells (Fig. 9A and B). Furthermore, the downregulation of Trop2 altered the levels of p27 and cyclin E1 in the T24 and RT4 cells (Fig. 10A and B). Taken together, these findings demonstrated that Trop2 was associated with the curcumin-induced suppression of cell proliferation, increase of apoptosis, and inhibition of invasive and migration abilities in BC cells. Curcumin treatment together with the downregulation of Trop2 enhanced the antineoplastic property of curcumin in BC cells.