T24, MCF7 and A549 cells were purchased from the China Center for Type Culture Collection (Wuhan, China). The T24 cells were cultured in RPMI 1640 medium (Gibco Life Technologies, Carlsbad, CA, USA) and both the MCF7 and A549 cells were cultured in Dulbecco's modified Eagle's medium (Gibco Life Technologies) containing 10% fetal bovine serum (FBS; Tianjin Hao Yang Biological Manufacture Co., Ltd., Tianjin, China) at 37°C with 5% CO₂. The media contained 100 U/ml penicillin (Sigma-Aldrich, St. Louis, MO, USA) and 100 µg/ml streptomycin (Sigma-Aldrich). The cells were passaged every 3 days and were diluted every day prior to each experiment.

The LC was purchased from Shanghai Lichen Trading Co., Ltd. (Shanghai, China). The MTT (Beyotime Institute of Biotechnology, Haimen, China) assay was used to evaluate viability of cells, which is based on the conversion of MTT to formazan crystals by mitochondrial dehydrogenases (20). Cells were seeded onto 96-well plates (8×10⁴ cells/ml) and incubated overnight in 100 µl of the culture medium. The cells were treated with a range of concentrations of LC (0, 25, 30, 35, 40 and 45 µg/ml) for 24 h. Following incubation, 20 µl MTT (5 mg/ml) was added to each well, which were then incubated for 4 h prior to removal of the supernatant. A total of 150 µl dimethyl sulfoxide (DMSO; Sigma-Aldrich) was added to each well. Absorbance at 570 nm was measured using a fluorescence plate reader (3001; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The data were expressed as the percentage cell viability compared with the control (DMSO). The inhibition rate was quantified using the following formula:

To investigate whether LC induces apoptosis in T24 cells, the cells were plated in a 4-well chamber slide at 2×10⁴ cells/slide, and treated with increasing concentrations of LC (0, 25, 30, 35, 40 and 45 µg/ml) for 24 h to examine the apoptosis of T24 cells. The cells were fixed in formaldehyde (40 g/l; Sigma-Aldrich) in phosphate-buffered saline (PBS) for 20 min followed by Hoechst 33258 (10 mg/l; Sigma-Aldrich) staining for 30 min in the dark at 37°C. Cell nuclei were then analyzed under a computer-assisted microscope (459330; Carl Zeiss AG, Oberkochen, Germany) by fluorescence microscopy. Apoptotic cells were characterized by chromatin condensation and multiple chromatin fragments (21).

Apoptotic rates were determined by staining cells with annexin V fluorescein isothiocyanate (FITC) and propidium iodide (PI) labeling (22). The Annexin V/PI Apoptosis kit was purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). Cells (1.5×10⁵ cells/ml) were incubated with LC for 24 h following which they were washed twice with ice-cold PBS, and 5 µl annexin V-FITC and 5 µl PI (1 mg/ml) were added to stain the cells. The cell staining was analyzed using the FACStar Flow Cytometer (BD Biosciences, San Jose, CA, USA). Viable cells were regarded to be negative for PI and annexin V-FITC, apoptotic cells were positive for annexin V-FITC and negative for PI, whereas late apoptotic dead cells displayed clear annexin V-FITC and PI labeling. Non-viable cells, which underwent necrosis, were positive for PI however were negative for annexin V-FITC.

Total RNA was extracted using TRIzol (Sangon Biotech Co., Ltd., Shanghai, China) according to the manufacturer's instructions. RNA quality was assessed using the A260/A280 ratio with a Nanodrop Spectrophotometer (ND-2000; Thermo Fisher Scientific, Inc., Pittsburgh, PA, USA) and 1.5% agarose gel electrophoresis (Biodee Biotechnology Co., Ltd., Beijing, China). Following extraction, 3 µl total RNA was reverse-transcribed to cDNA using a RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific) in a 20 µl reaction volume. The reaction conditions of reverse transcription PCR were established using 12.5 µl 2X Taq PCR MasterMix (Tiangen Biotech Co., Ltd., Beijing, China), 3 µl cDNA template and 0.5 µl of each primer synthesized by Sangon Biotech. Thermocycling conditions were as follows: Pre-denaturation at 94°C for 3 min, 30 cycles of denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec and extension at 72°C, and a final extension at 72°C for 10 min. The RT-qPCR products were quantified using a Bio-Rad gel imaging system (Bio-Rad Laboratories, Inc.) with GelPro analysis software 4.0 (Media Cybernetics, Rockville, MD, USA). The primer sequences are presented in Table I.

The activity of caspase-3 was assessed using the Caspase-3 Colorimetric Assay kit (R&D Systems, Inc., Minneapolis, MN, USA), which is based on the spectrophotometric detection of the color reporter molecule p-nitroanaline (pNA) following cleavage from the labeled substrate DEVD-pNA (caspase-3) as an index. Cells were incubated with the designated concentrations of LC (0, 25, 35 and 45 µg/ml). The cells were washed with PBS and suspended in 5 volumes lysis buffer (20 mmol/l HEPES, pH 7.9, 20% glycerol, 200 mmol/l KCl, 0.5 mmol/l EDTA, 0.5% NP40, 0.5 mmol/l DTT and 1% protease inhibitor cocktail; Sigma-Aldrich). The lysates were collected and stored at −20°C until use. The protein concentration was determined by the Bradford method as per the manufacturer's instructions of the Caspase-3 Colorimetric Assay kit. Supernatant samples, containing 100 µg total protein, were added to 96-well plates with the DEVD-pNA and LEHD-pNA at 37°C for 1–2 h to determine caspase-3 activity. The optical density of each well was measured at 405 nm using a fluorescence plate reader (3001; Bio-Rad Laboratories, Inc). Each plate contained three wells of a given experimental condition and three control wells. The activity of caspase-3 was expressed in arbitrary absorbance units (absorbance at a wavelength of 405 nm).

Cells at a density of 1.5×10⁵ cells/ml were incubated with LC for 24 h. The soluble lysates (15 µl per lane) were subjected to 10% SDS-PAGE, then were transferred onto the nitrocellulose membranes (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) and blocked with 5% non-fat milk in Tris-buffered saline with Tween-20 (TBST; Biodee Biotechnology Co., Ltd) for 2 h at room temperature. Membranes were incubated with the respective primary antibody [anti-caspase-3 antibody (1:2,000; cat no. sc-65496), anti-poly(adenosine diphosphate-ribose) polymerase (PARP) antibody (1:2,000; cat no. sc-56196) or anti-β-actin antibody (1:2,000, cat no. sc-47778), all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA] at 4°C overnight and then incubated with horseradish peroxidase-conjugated bovine anti-mouse immunoglobulin G (1:10,000; cat no. sc-2371; Santa Cruz Biotechnology, Inc.) as the secondary antibody for 1 h at room temperature. Western blots were developed using enhanced chemiluminescence (Thermo Fisher Scientific) and were exposed on Kodak radiographic film (Kodak, Rochester, NY, USA).

The data were presented as the mean ± standard deviation from a minimum of three independent experiments and evaluated through analysis of variance followed by Student's t-test. P<0.05 was considered to indicate a statistically significant difference. The analyses were performed using the Origin software, version 8.0 (OriginLab, Northampton, MA, USA).

Breast, lung and bladder cancer are frequently malignant, thus have a clear effect on health due to high incidence and recurrence rates (23,24). The current study examined the proliferation inhibition of LC (0, 25, 30, 35, 40 and 45 µg/ml) against T24 (bladder cancer), MCF7 (breast cancer) and A549 (lung cancer) cells. Subsequent to treatment with LC (45 µg/ml) for 24 h, the rates of proliferation inhibition of T24, MCF7 and A549 cells were 68, 47 and 40% respectively (Fig. 2). As T24 cells were observed to exhibit a greater sensitivity to LC than MCF7 and A549 cells, T24 cells were selected for use in the subsequent experiments.

Morphological assessment with Hoechst staining verified the fact that LC induces T24 cell apoptosis, with LC-treated cells exhibiting typical morphological features of apoptosis, such as nuclear condensation and fragmentation (Fig. 3A). Annexin V-FITC-PI double-staining was used to detect phosphatidyl serine externalization, a hallmark of early apoptosis, to indicate whether LC-induced apoptosis occurred (25). Treatment of T24 cells with LC (0, 25, 35 and 45 µg/ml) for 24 h led to a significant increase in the percentage of apoptotic cells, from 0.6% in control cells to 30, 64 and 74% respectively (Fig. 3B). In addition, measurement of a key factor in apoptosis, caspase-3 activity, provided further support for the LC-induced apoptotic response (Fig. 3C).

As Bcl-2 family members serve a critical role in inducing caspase-3 activation, regulating apoptosis and irreversible cellular damage, they are suggested to be important in the determination of cell fate (25). LC-induced caspase-3 activation and apoptosis were observed in the present study. To investigate whether Bcl-2 family members are involved in the apoptosis of T24 cells induced by LC, the expression levels of Bcl-2 family members (Bax, Bim, Bcl-w, Bcl-2 and Bcl-XL) in T24 cells treated with LC were analyzed (Fig. 4A). Compared with the control group, exposure of T24 cells to LC (25, 35 and 45 µg/ml) resulted in a concentration-dependent reduction in the mRNA level of Bcl-2, Bcl-w and Bcl-XL, with a concomitant increase observed in the levels of Bax and Bim. Based on the importance of Bcl-2 family members in inducing apoptosis, and the alterations in the levels of Bcl-2 mRNA observed in the present study, an inhibitor of the Bcl-2 family (ABT-737) was used to confirm the role of Bcl-2 family members in LC-treated T24 cell apoptosis. As presented in Figure 4B, the Bcl-2 family inhibitor ABT-737 effectively blocked LC-induced apoptosis associated proteins (pro-caspase-3 and cleaved PARP).