Chemoprevention of Urothelial Cell Carcinoma Tumorigenesis by Dietary Flavokawain A in UPII-Mutant Ha-ras Transgenic Mice

Abstract

Non-muscle-invasive bladder cancer (NMIBC) has one of the highest recurrence rates among all solid cancers and the highest lifetime treatment cost per patient. Therefore, the development of chemoprevention strategies for reducing the occurrence and recurrence of NMIBC as well as its burdens on the healthcare system is valuable. Our aim was to determine whether flavokawain A (FKA), a kava chalcone isolated from the kava plant, can target the in vivo activated Ha-ras pathway for prevention and treatment of NMIBC. UPII-mutant Ha-ras transgenic mice that develop papillary urothelial cell carcinoma were fed orally with vehicle control or FKA-formulated food for 6 months starting at 6 weeks of age. Seventy-nine percent (15/19) of male mice fed with 6 g FKA per kilogram (kg) of food survived beyond the 6 months of treatment, while 31.6% (6/19) of control food-fed male mice survived the 6-month treatment period (p = 0.02). The mean bladder weights in FKA vs. control food-fed mice were 0.216 ± 0.033 vs. 0.342 ± 0.039 g in male mice (p = 0.0413) and 0.043 ± 0.004 vs. 0.073 ± 0.004 g in female mice (p < 0.0001); FKA reduced bladder weight by 37% and 41%, respectively. The tumor burdens, determined by the wet bladder weight, in these mice were inversely related to plasma FKA concentrations. In addition to decreased bladder weight, FKA treatment significantly reduced the incidences of hydronephrosis and hematuria. FKA-treated mice exhibited more well-differentiated tumors in the bladder and ureter. Immunohistochemical analysis of FKA-treated tumors compared to those in the control group revealed fewer Ki-67- and survivin-positive cells and an increased number of p27- and TUNEL-positive cells, indicating that FKA inhibits proliferation and induces apoptosis. Overall, the results suggest that FKA can target the in vivo activated Ha-ras pathway for the prevention and treatment of NMIBC.

Keywords: UPII-mutant Ha-ras transgenic mice; flavokawain A; urothelial cell carcinoma.

Figure 1

The chemopreventive efficacy of dietary FKA on the survival of UPII-mutant Ha-ras urothelial-tumor-bearing mice. (A,B) Male and female UPII-mutant Ha-ras mice were fed daily with control diet and diet containing 0.6% FKA starting at 6 weeks of age until their death or up to 6 months of age. (C,D) The mean body weights were recorded and compared every month between male and female mice fed control diet and 0.6% FKA diet.

Figure 2

The chemopreventive efficacy of dietary FKA on urothelial tumorigenesis of homozygous UPII-mutant Ha-ras transgenic mice. (A) Bladder weights of wild-type and UPII-mutant Ha-ras transgenic mice fed with control diet or diet containing 0.6% FKA for 6 months. Mean bladder weight ± SD. (B,C) Macroscopic examination revealed enlarged bladders, ureters, and kidneys in control diet groups compared to FKA diet groups in both male and female mice.

Figure 3

The chemopreventive efficacy of dietary FKA on pathological progression of urothelial carcinogenesis in male UPII-mutant Ha-ras transgenic mice. (A) Macroscopic examination of bladders, ureters, and kidneys after male UPII-mutant Ha-ras transgenic mice were fed with control diet or diet containing 0.6% FKA for 6 months. Magnification: 200×. (B) Percentages of papillary hyperplasia, nodular hyperplasia, papilloma, and papillary carcinoma after male UPII-mutant Ha-ras transgenic mice were fed with control diet or diet containing 0.6% FKA for 6 months.

Figure 4

The chemopreventive effect of dietary FKA on hematuria and hydronephrosis. (A,B) Photographs of dipsticks after loading with urine from male and female homozygous UPII-mutant Ha-ras transgenic mice which were fed with control diet or diet containing 0.6% FKA for 4 months (left panel), and percentage of mice with hematuria in control and FKA diet groups (right panel). (C,D) Macroscopic examination showing enlarged kidneys in control-diet-fed mice compared to 0.6% FKA-diet-fed mice (left panel), and percentage of mice with hydronephrosis in control and FKA diet groups (right panel).

Figure 5

Plasma FKA concentrations are inversely related to tumor burdens. (A) Chromatographic separation of FKA was achieved using an ACQUITY UPLC (Waters, UK) with a reversed-phase ACQUITY UPLCTM BEP C18 column (1.7 μm, 2.1 × 50 mm, Waters). FKA was monitored as a precursor ion with an m/z value of 315 and a fragment ion with a value at 181.14. Correlation analysis between FKA plasma concentrations and bladder weights in male (A) and female (B) UPII-mutant Ha-ras transgenic mice. The food consumption of male (C) and female (D) mice was weighted and changed every three days.

Figure 6

The in vivo mechanisms of dietary FKA in chemoprevention of urothelial carcinogenesis. Bladder tumor sections of 6 randomly selected individual mice from mice fed control or FKA diet for IHC staining of proliferation markers (Ki-67 and p27/Kip1) and apoptotic markers (TUNEL and survivin) expression. (A) Representative DAB-stained tissue sections from control- and 0.6% FKA-fed groups showing brown-colored positive cells are depicted at ×200 magnification. (B) Positive-staining cells were counted in 12 fields in each group. The percentage of positive cells was calculated and presented as mean ± SD in histography; Student’s t-test, all p-values < 0.01. Ki-67, TUNEL, and survivin showed nuclear staining (red arrows), whereas p27 staining was in the cytoplasm (yellow arrows).