Kavalactone Kawain Impedes Urothelial Tumorigenesis in UPII-Mutant Ha-Ras Mice via Inhibition of mTOR Signaling and Alteration of Cancer Metabolism

Abstract

UPII-mutant Ha-ras transgenic mice develop urothelial hyperplasia and low-grade papillary carcinoma, which mimics human non-muscle invasive bladder cancer (NMIBC). We investigated the effects and mechanisms of kawain, a main kavalactone in the kava plant, on oncogenic Ha-ras-driven urothelial carcinoma in these mice. The mice were fed at six weeks of age with vehicle control or kawain (6 g/kg) formulated food for approximately five months. Seventy-eight percent of the mice or more fed with kawain food survived more than six months of age, whereas only 32% control food-fed male mice survived, (p = 0.0082). The mean wet bladder weights (a surrogate for tumor burden) of UPII-mutant Ha-ras transgenic mice with kawain diet was decreased by approximately 56% compared to those fed with the control diet (p = 0.035). The kawain diet also significantly reduced the occurrence of hydronephrosis and hematuria in UPII-mutant Ha-ras transgenic mice. Histological examination and immunohistochemistry analysis revealed that vehicle control-treated mice displayed more urothelial carcinoma and Ki67-positive cells in the bladder compared to kawain treated mice. Global metabolic profiling of bladder tumor samples from mice fed with kawain food showed significantly more enrichment of serotonin and less abundance of xylulose, prostaglandin A2, D2 and E2 compared to those from control diet-fed mice, suggesting decreased shunting of glucose to the pentose phosphate pathway (PPP) and reduced inflammation. In addition, kawain selectively inhibited the growth of human bladder cancer cell lines with a significant suppression of 4E-BP1 expression and rpS6 phosphorylation. These observations indicate a potential impact of kawain consumption on bladder cancer prevention by rewiring the metabolic programs of the tumor cells.

The Potent Anti-Tumor Effects of Rhodiola Drinking Are Associated with the Inhibition of the mTOR Pathway and Modification of Tumor Metabolism in the UPII-Mutant Ha-Ras Model

“…Wild-type or nontransgenic types were utilized in safety studies or as controls. Mouse breeding, Southern blotting, and genotyping were performed as described in our previous publications [ 13 , 14 ]. Homozygous UPII-mutant Ha-ras mice have a 1:1 ratio of the mutant human Ha-ras gene to the endogenous UPII gene, and express high levels of Ras protein in bladder tissues as shown in Figure 1 A.…”

“…Histological lesions of each urinary bladder were evaluated by a pathologist (B.W.) blinded to the experimental groups and categorized into simple hyperplasia, papillary hyperplasia, nodular hyperplasia, papilloma, and papillary UCC, as detailed in our previous publications [ 13 , 14 , 16 ].…”

“…The urine of each mouse, at four months of age, was collected through bladder massage and tested with Chemstrip 4MD urinalysis test strips (Roche Diagnostics, Laval, QC, Canada). The ranges of glucose, protein, blood/hemoglobin, specific gravity, pH, Leukocytes, nitrite, ketones, urobilinogen, and bilirubin concentrations were determined semi-quantitatively as described previously [ 13 , 14 , 16 ].…”

“…Urothelial cells and tissues were scratched from freshly removed bladders of UPII-mutant Ha-ras mice and protein lysates were prepared through homogenization as previously described [ 13 , 14 , 16 ]. Amounts of 10 to 50 μg protein lysates were used for Western blotting analysis of the protein expression of TSC2, phospho-mTOR, 4E-BP1, S6, Skp2, and β-tubulin (as a loading control) using a previously established protocol [ 10 ].…”

Editorial: Biofunctional Molecule Exploratory Research on Application in Food and Health

“…On the other hand, Liu et al have shown that Kawain, a main kavalactone in the kava plant, exhibits chemopreventive effects against UPII-mutant Ha-Ras transgenic mice [ 6 ]. The research findings showed that Kawain enhanced survival and delayed the progression from hyperplasia to papillary carcinoma in non-muscle invasive urothelial tumor-bearing mice, as well as reducing urothelial cell carcinoma-induced hydronephrosis and hematuria.…”