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Li et al. J Transl Genet Genom 2021;5:163-72 https://dx.doi.org/10.20517/jtgg.2021.22 Page 3

dihydrokawain, yangonin, and methysticin, on monoamine oxidase A (MAO-A) and lysine-specific
demethylase 1 (LSD1) enzyme activities were examined. Our results have shown that the KRE significantly
reduced the occurrences of HG-PIN and prostate adenocarcinomas and slowed tumor growth in the
TRAMP transgenic mice, and inhibited both MAO-A and LSD1 activities in prostate cancer cells.

METHODS
Study materials
Authenticated LNCaP and C4-2B cell lines without mycoplasma contamination were used as described in
[15]
details in our previous publication . The KRE (150 mg/mL kavalactones in 50% ethanol) was purchased
from Gaia Herbs (Brevard, NC). Main kavalactones, including kawain, 5’,6’-dehydrokawain, yangonin, and
methysticin, were isolated and purified by LKT Laboratories, Inc. (St. Paul, MN) from the KRE. Antibodies
against MAO-A and tubulin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Di-methylated
histone H3 lysine 4 (H3K4) and H3K9 and Ki67 antibodies were purchased from Abcam (Cambridge, MA).
MAO-A and LSD1 activity assay kits were from Cayman Chemical Company (Ann Arbor, Michigan).

Prevention and intervention protocols in TRAMP mice
A cohort of male hemizygous TRAMP mice was obtained by breeding female hemizygous C57BL/TGN
TRAMP mice with male FVB/N mice and genotyped by a polymerase chain reaction (PCR) method as
described in details in our previous publication . The 0.3% (3 g/kg food) or 0.6% (6 g/kg food) KRE
[16]
containing rodent food was formulated into AIN-93M rodent food by Dyets, Inc. through customer
services. University of California, Irvine approved protocol (#2007-2740) was followed for animal care and
treatments. For the prevention protocol, 0.3% or 0.6% KRE containing food or vehicle control food was
given to TRAMP mice from ages of 6 weeks to 12 weeks. For the intervention protocol, 0.3% or 0.6% KRE
containing food or vehicle control food was administrated to 6 weeks old TRAMP mice until they were at
the age of 24 weeks old.

The body weight and food consumption were recorded weekly until the end of the experiments. Organ
weights, including liver, spleen, kidney, etc. and genitourinary (GU) weights were also measured at the end
of the experiments and fixed in formalin for standard H&E slide preparation and examination . PIN
[16]
[17]
lesions and prostate adenocarcinoma were evaluated to Dr. Cardiff’s description and our previous
publication .
[16]
LSD1 inhibition assay [18]
LSD1 inhibitor screening kit was purchased from Cayman Chemicals with the catalog number 700120.
Human recombinant LSD1 enzymes are incubated with a selected concentration of compounds or with
DMSO for 10 min. Then a premix of reaction buffers solution that contains fluorescence substrate and
horseradish peroxide (HRP) was added to the LSD1 solution. Finally, methylated peptides, which are the
first 21 amino acids of the N-terminal tail of methylated H3K4, were added to the LSD1-buffer solution to
begin the enzymatic processing. This process took place at 37 ℃ for 60 min. In the process of
demethylation, hydrogen peroxide (H O ) is formed as a byproduct. HRP then can use H O to convert the
2
2
2
2
non-fluorescence substrate resazurin into the fluorescence substrate resorufin, which was measured by a
fluorescence spectrophotometer using the excitation value of 530 nm and the emission value of 585 nm.
MAO-A inhibition assay [19-21]
MAO-A activity assay kit was purchased from Cayman Chemicals. The measurement of MAO-A activity in
vehicle control (0.1% DMSO) or kavalactones (50 μM) or the KRE (5 μg/mL) treated LNCaP and C4-2B
cells were carried out by following the kit instruction. HRP used the H O byproduct in the MAO-A
2
2
reaction to convert the non-fluorescence substrate resazurin into the fluorescence substrate resorufin, which