The primary antibodies used in western blot analysis were as follows: Rabbit anti-cyclin D1 (catalog no. 2922), rabbit anti-cyclin-dependent kinase (CDK)2 (catalog no. 2546), rabbit anti-CDK4 (catalog no. 12790), mouse anti-cytochrome c (catalog no. 12963), rabbit anti-phospho-PI3K (catalog no. 4228), rabbit anti-PI3K (catalog no. 4257), mouse anti-phospho-Akt (catalog no. 12694), and mouse anti-Akt (catalog no. 2920) (all from Cell Signaling Technology, Inc., Danvers, MA, USA), mouse anti-cyclin-dependent kinase inhibitor 1 (p21; catalog no. sc-136020), rabbit anti-cyclin-dependent kinase inhibitor 1B (p27; catalog no. sc-528), mouse anti-β-actin (catalog no. sc-81178), mouse anti-B-cell lymphoma 2-like protein 4 (Bax; catalog no. sc-20067) (all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and rabbit anti-Heat shock protein 60 (catalog no. ab46798; Abcam, Cambridge, UK). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc.

The human bladder cancer T24 and 5637 cell lines and the immortalized normal human uroepithelial SV-HUC-1 cell line were purchased from the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). They were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 µg/ml) (all from Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in a humidified 5% CO₂ atmosphere. Cells were treated with genipin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), which was dissolved in 0.1% dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA). DMSO-treated cells were used as a vehicle control.

A constitutively active Akt construct (Myr-Akt plasmid) and empty vector were purchased from AddGene (Cambridge, MA, USA). Cells were transfected with 0.3 µg Myr-Akt or vector using Lipofectamine 2000^® (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. At 24 h post-transfection, cells were exposed to genipin for additional 48 h prior to cell viability and apoptosis analysis. For each condition, three replicates were performed.

Cells were plated in 96-well plates at a density of 2×10⁴ cells/well and incubated with different concentrations (10, 30, 60, 100, and 300 µM) of genipin for 48 h. DMSO-treated cells were used as a control. Each assay was performed in quadruplicate. Cell viability was measured using the MTT assay. Briefly, cells were added with MTT (0.5 mg/ml; Sigma-Aldrich; Merck KGaA) and allowed to incubate for 4 h at 37°C. Following removal of MTT, the resulting formazan was dissolved in DMSO. Absorbance was measured at 570 nm using a microplate reader (xMark; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

T24 and 5637 cells (5×10³ cells/well) were seeded in triplicate onto 6-well plates and treated with 30 or 60 µM of genipin or DMSO (vehicle control) on the following day. DMEM containing genipin was renewed every 3 days. Following 10 days of culture, cells were stained with 0.5% crystal violet (Sigma-Aldrich; Merck KGaA). Colonies (>50 cells) were counted in 10 independent microscopic fields (magnification, ×40) using an inverted microscope.

A total of 12 athymic male BALB/c (nu/nu) mice, 4 to 6 weeks old and weighing 16–20 g, were obtained from the Animal Center of the Chinese Academy of Medical Science (Beijing, China). The mice were housed 4 per cage at 22°C and 80% humidity in a 12-h light/dark cycle with access to food and water ad libitum. Mice were injected subcutaneously in the left flank with 4×10⁶ T24 cells. When xenograft tumors reached a diameter of ~5 mm, the mice were divided into 3 groups (n=4 for each group) and DMSO (vehicle control) or genipin (20 and 50 mg/kg) was administered intraperitoneally three times/week for 4 weeks. The tumors were measured every week with microcalipers. Tumor volume was calculated using the following equation: Tumor volume (mm³) = 1/2 × (tumor length) × (tumor width)². Mice were sacrificed 5 weeks after cell injection and tumors were resected and weighted. Animal experiments were approved by the Institutional Animal Care and Use Committee of Chinese Academy of Medical Science. Animal care and treatment were performed in accordance with the international guidelines for laboratory animals use (https://www.apa.org/science/leadership/care/guidelines.aspx).

Cell cycle distribution was determined by flow cytometry following staining with propidium iodide (PI). Briefly, subsequent to 30 or 60 µg of genipin or DMSO (vehicle control) for 48 h at 37°C, T24 and 5637 cells were collected and fixed in 70% ethanol overnight at 4°C. The cells were resuspended in staining solution containing 0.05 mg/ml PI and 7 U/ml RNase A (Sigma-Aldrich; Merck KGaA) for 30 min at 37°C. Stained cells were analyzed by a flow cytometer (FACSCanto II; BD Biosciences, San Jose, CA, USA) with FlowJo software version 7.6.1 (TreeStar, Inc., Ashland, OR, USA). Cell apoptosis was examined using the Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit (Beyotime Institute of Biotechnology, Haimen, China), as per the manufacturer's instructions. Following incubation with Annexin V-FITC and PI, T24 and 5637 cells were analyzed by flow cytometry using FlowJo software version 7.6.1. Each assay was performed in triplicate.

For preparation of the whole cellular lysates, cells were suspended in radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Inc.) containing protease inhibitors on ice for 15 min. For extraction of mitochondrial and cytosolic fractions, a commercial mitochondria/cytosol fractionation kit (Beyotime Biotechnology, Inc.) was used. Briefly, cells were suspended in ice-cold cytosolic separation buffer containing protease inhibitors for 15 min and centrifuged at 800 × g for 5 min at 4°C. The supernatant was centrifuged at 12,000 × g for 30 min at 4°C to obtain the cytosolic fraction. The pellet was resuspended in mitochondrial separation buffer for 30 min and then centrifuged at 10,000 × g for 10 min at 4°C to obtain the mitochondrial fraction. Total protein concentrations were determined using a Pierce Bicinchoninic Acid Protein Assay kit (Thermo Fisher Scientific, Inc.).

Whole and fractionated cell lysates (50 µg total protein per lane) were mixed with loading buffer and subjected to 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% fat-free milk in TBS containing 0.05% Tween-20 for 1 h at room temperature and incubated with primary antibodies overnight at 4°C. All primary antibodies were diluted to 1:500. Subsequent to washing three times (10 min for each time), the membranes were incubated with HRP-conjugated secondary antibodies (dilution at 1:3,000) for 1 h. Membranes were developed with enhanced chemiluminescence reagents (Amersham Biosciences; GE Healthcare, Chicago, IL, USA). Densitometric analysis of protein signals was conducted with Quantity One software version 4.6.2 (Bio-Rad Laboratories). Each assay was performed in triplicate.

Measurement of Δψm was performed using the fluorescent probe JC-1, as previously described (17). At low Δψm, JC-1 exists primarily in a monomeric form and produces green fluorescence. At high Δψm, JC-1 aggregates in intact mitochondria and yields red fluorescence. In brief, T24 and 5637 cells were incubated for 30 min at 37°C with 2 µM JC-1 (Molecular Probes, Inc.; Thermo Fisher Scientific, Inc.). Cells were collected and analyzed by the FACSCanto II flow cytometer using FlowJo software version 7.6.1. Results are expressed as the red/green fluorescence intensity ratio; whose reduction indicates loss of Δψm.

Data are presented as the mean ± standard deviation, and were analyzed using one-way analysis of variance followed by pair-wise comparisons with Tukey's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

Genipin treatment for 48 h resulted in a concentration-dependent inhibition of the viability of T24 and 5637 bladder cancer cells, compared with the vehicle control (Fig. 1A). In contrast, ≤60 µM genipin did not exhibit a significant effect on the viability of non-malignant uroepithelial SV-HUC-1 cells. These results suggested that a low concentration of genipin was selectively cytotoxic to bladder cancer cells. In the following experiments, genipin was used at a final concentration of 30 or 60 µM unless indicated otherwise.

Next, the effect of genipin on clonogenic growth of bladder cancer cells was determined. As demonstrated in Fig. 1B, exposure to genipin significantly decreased the colony formation in T24 and 5637 cells by 2–3-fold. The in vivo antitumor effect of genipin on the growth of T24 xenograft tumors was also examined in nude mice. Compared to the DMSO-treated group, genipin treatment significantly inhibited the growth of T24 xenograft tumors (P<0.05; Fig. 1C). Genipin at the dose of 50 mg/kg caused significantly greater tumor suppression compared with 20 mg/kg genipin. At the end of the experiment, the tumor weight was reduced by >60% in the genipin (50 mg/kg)-treated group (Fig. 1D). These results confirmed the growth suppressive effects of genipin in bladder cancer.

Flow cytometric analysis revealed that genipin-treated T24 and 5,637 cells exhibited a marked increase in the percentage of cells in the G0/G1 phase and a concomitant reduction in the percentage of S phase cells (Fig. 2A). The percentage of cells in the G2/M phase was comparable between genipin-treated and DMSO-treated cells. Western blot analysis demonstrated that genipin treatment significantly reduced the expression levels of cyclin D1, CDK2 and CDK4 in T24 and 5,637 cells (Fig. 2B). By contrast, the levels of the CDK inhibitors (p21 and p27) were significantly raised in response to genipin exposure.

Annexin V/PI staining analysis demonstrated that genipin treatment resulted in a significant increase in the percentage of Annexin V-positive apoptotic cells, compared with DMSO treatment (P<0.05; Fig. 3A). Subsequent to treatment with 60 µM genipin, the percentage of apoptotic cells increased from 4.5±0.9 to 34.6±2.1% in T24 cells and from 3.9±0.8 to 28.5±2.4% in 5637 cells. To test the involvement of the mitochondrial pathway in genipin-mediated apoptosis, Δψm, Bax translocation into mitochondria and the release of mitochondrial cytochrome c were measured. As demonstrated in Fig. 3B, there was 70–80% loss in Δψm in the cells treated with 60 µM genipin, compared with DMSO-treated controls (P<0.05). Additionally, genipin-treated cells exhibited increased Bax and decreased cytochrome c expression in the mitochondrial fractions, compared with control cells (P<0.05; Fig. 3C). Additionally, there was decreased Bax and increased cytochrome c expression in the cytoplasmic fractions of genipin-treated cells (Fig. 3C). These results suggest that genipin-induced apoptosis involves mitochondrial damage and cytochrome c release.

Finally, the potential involvement of PI3K/Akt signaling in the anticancer activity of genipin was examined. Western blot analysis suggested that genipin treatment significantly (P<0.05) reduced the phosphorylation levels of PI3K and Akt in T24 and 5637 cells, compared to DMSO-treated cells (Fig. 4A). Of note, overexpression of constitutively active Akt significantly (P<0.05) increased the viability (Fig. 4B) and inhibited apoptotic response (Fig. 4C) in genipin-treated T24 and 5,637 cells.