Background

Apigenin, one of the most common flavonoids, is widely distributed in many fruits and vegetables, including parsley, onions, orange, tea, chamomile, wheat sprouts and in some seasonings [1]. Apigenin has potential uses in cancer prevention and therapy, and it suppresses cell growth against many human cancer cell lines, including breast, colon, skin, thyroid, leukemia, and prostate cancer cells [2–5]. Unlike other structurally related flavonoids, apigenin is non-mutagenic [6]. Although previous reports have shown the inhibitory effect of apigenin on other human cancer cells, there are few reports indicating the inhibitory effect on human bladder cancer cells.

Bladder cancer is the most common malignant tumor of the urinary tract. Worldwide, bladder cancer is the seventh most common cancer. An average of 386,300 new cases of urinary bladder cancer are diagnosed worldwide every year, accounting for 150,200 deaths [7]. In recent decades, bladder cancer was shown of a rising overall incidence [8]. In most cases of nonmuscle invasive bladder cancer, tumors are treated initially with TURBT (Transurethral Resection of Bladder Tumor). A careful cystoscopic examination of the entire urethra and all bladder surfaces precedes resection. Intravesical therapy can also be employed in an expectant way as opposed to an induction course alone to provide long-term immunostimulation or chemotoxicity and thereby prevent disease recurrence [9, 10]. Our earlier studies have shown that EGCG [Cell apoptosis assay

The extent of apoptosis was evaluated by annexin V-FITC and flow cytometry. Cells were grown at a density of 1 × 106 cells in six-well culture dishes and were treated with different concentrations of apigenin (0–80 μM in DMSO) for 24 h. Following treatment, the cells were harvested, washed twice with pre-chilled PBS, and resuspended in 1× binding buffer at a concentration of 1 × 106 cells/ml. One hundred microliters of such solution was mixed with 5 μL annexin V-FITC and 5 μL propidium iodide for 15 min, and then 400 μL 1× binding buffer was added. Analysis was carried out using a FC500 flow cytometer with CXP software (Beckman Coulter, Fullerton, CA, USA) within 1 h. The percentage of apoptotic cells was assessed by CXP software.

Cell cycle assay

Cells were plated in six-well culture dishes at concentrations determined to yield 60–70% confluence within 24 h. Cells were then treated with different concentrations of apigenin (0–160 μM in DMSO). After 24 h, cells were washed twice with PBS then centrifuged. The pellet was fixed with 70% ethanol for 1 h at 4°C. The cells were washed with PBS and resuspended with propidium iodide solution (0.05 mg/mL) containing RNase, incubated at room temperature in the dark for 30 min. DNA content was then analyzed using the FC500 flow cytometer.

Western blot analysis

Cell were harvested at 24 h following apigenin treatment, washed, and lysed with lysis buffer (10 mmol/L Tris-HCl, 0.25 mol/ L sucrose, 5 mmol/L EDTA, 50 mmol/L NaCl, 30 mmol/L sodium pyrophosphate, 50 mmol/L NaF, 1 mmol/LNa3VO4, 1 mmol/L PMSF, and 2% cocktail [pH 7.5]). Protein concentration in the resulting lysate was determined using the bicinchoninic acid protein assay. Appropriate amounts of protein (20–30 μg) were separated by electrophoresis in 10–12% Tris-glycine polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked then incubated overnight with the appropriate primary antibody at dilutions specified by the manufacturer. They were next washed and incubated with the corresponding HRP-conjugated secondary antibody at 1:1000 dilution in Tris-buffered saline-Tween 20 (10 mM Tris-Cl [pH 7.4], 150 mM NaCl, 0.1% Tween-20). Bound secondary antibody was detected using an enhanced chemiluminescence system (Pierce Biotechnology).

Statistical analysis

Statistical significance was compared between various treatment groups and controls using ANOVA. Data were considered statistically significant when P-values were <0.05.