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Page 4 Li et al. J Transl Genet Genom 2021;5:163-72 https://dx.doi.org/10.20517/jtgg.2021.22

was measured by a fluorescence spectrophotometer using the excitation value of 530 nm and the emission
value of 585 nm.

Western blot analysis
Protein lysates preparation and Western blotting analysis were performed according to our previously
[22]
published paper . Tubulin levels were used as a loading control.

Immunohistochemistry
Antigen retrieval method, titration of anti-human Ki67 (1:100) antibody, and immunohistochemistry
staining of prostate tissues from control and the KRE containing food fed TRMAP mice were carried out by
following our published papers [9,16] .

Molecular docking studies [23]
First, kawain and methysticin molecules were generated by Pymol software and converted to the pdbqt
format using Open Babel. Then, the autodock 4 software from the Scripps Research Institute was used to
dock kawain and methysticin with LSD1 protein (PDB code: pdb 2hkO) or MAO-A (pdb 2bxs) from
MGLTools. Finally, Pymol was used to image the conformations of the molecule and protein interaction.

Statistical analysis
Analysis of variance or Student’s t-test followed by the Bonferroni t-test for multiple comparisons was used
to compared means of organ and body weights and food consumptions between vehicle control and KRE
treatments over time. The Mann-Whitney U and Kolmogorov-Smirnov test was used for GU weight
comparison among different treatment groups. The comparisons of the percentages of mice with different
2
pathologic stages or with palpable tumors among different treatment groups were tested by using the χ or
Fisher exact test.

RESULTS
Dietary feeding of the KRE for 6 weeks inhibits mouse HG-PIN and prostate adenocarcinoma in
TRAMP mice
To examine whether the KRE inhibits HG-PIN and prostate adenocarcinoma occurrence, TRAMP mice
were given vehicle control, 0.3% KRE, or 0.6% KRE containing food from 6 weeks of age to 12 weeks of age
[Figure 1A]. Figure 1B and C show that dietary feeding of 0.3% and 0.6% KRE inhibited HG-PIN by 43.5%
and 59.7%, respectively, and prostate adenocarcinomas by 53.5% and 66.4%, respectively (P < 0.05; Fisher
exact test). These results suggest that the KRE has a cancer-preventive activity for early-stage prostate
cancer.

Dietary feeding of the KRE for 18 weeks reduced tumor burden in TRAMP mice
To determine whether the KRE can affect tumor burdens at late stage of prostate cancer, TRAMP mice at 6
weeks of age were administrated with vehicle control, 0.3% or 0.6% KRE containing food for 18 weeks. The
mean GU weight of the mice fed with 0.6% KRE containing food was significantly reduced when compared
to the control group (Figure 2A; 1.98 g ± 2.07 g vs. 3.63 g ± 4.28 g; P < 0.05; Mann-Whitney U and
Kolmogorov-Smirnov test). The percentages of large tumors (GU weight > 0.9 g) were also decreased from
86.4% in the control group to 52.2% and 43.5% in 0.3% and 0.6% KRE food groups, respectively [Figure 2B].
In addition, administration of 0.6 % KRE containing food to TRAMP mice led to a decrease in food
consumption, and both 0.3% and 0.6% KRE food suppressed the body weight gain over time
[Figure 2C and D], as well as increased liver weight (data not shown) compared to control food.