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Page 8 Li et al. J Transl Genet Genom 2021;5:163-72 https://dx.doi.org/10.20517/jtgg.2021.22

Figure 5. Dietary feeding of KRE enhances H3K9 dimethylation and inhibits cell proliferation in vivo. (A, B) Western blot analysis of
H3K9 dimethylation in mouse dorsolateral prostates of TRAMP mice and prostate cancer PDX tumors, respectively, in vehicle control
and 0.6% KRE treatment groups. (C) Ki67 immunohistochemistry staining was shown in the prostates of TRAMP mice that was treated
with vehicle control vs. 0.6% KRE containing food for 18 weeks. 200× magnifications of images are presented. (D) The percentage of
positive stained cells was calculated after 20 fields from ten mouse prostates were counted in each group. KRE: Kava root extract;
H3K9: histone H3 lysine 9; TRAMP: transgenic adenocarcinoma of the mouse prostate; PDX: patient-derived xenograft; LSD1: lysine-
specific demethylase 1.

LSD1 was reported to interact with the AR and serves as a co-factor to regulate the transcriptional
expression of AR target genes [25,26] . Our previous studies demonstrated that kavalactones inhibited mRNA
expression of PSA and TMPRESS2, two key AR target genes, in LNCaP cells and tumor tissues of prostate
cancer PDX model . Whether the inhibitory effect of kawain and methysticin on AR signaling is
[9]
dependent, at least in part, on LSD1 expression warrants further studies. These studies will facilitate further
understanding of molecular mechanisms by which these kavalactones regulate AR transcription.

Previous studies by our group and others have demonstrated that kava chalcones (flavokawain A) and a
kavalactone-rich kava fraction B (free of flavokawains), respectively, are effective in preventing the
occurrences of HG-PIN and early prostate adenocarcinoma and reducing tumor burdens in the TRAMP
model [10,16] . In addition, we have shown that administration of flavokawain A containing food to TRAMP
mice resulted in the eradication of distant organ metastasis , whereas a kavalactone-rich kava fraction B
[16]
was shown to significantly decrease the incidences of neuroendocrine carcinoma in TRAMP mice by
absolute 42.9% . The KRE used here contains both kavalactones and flavokawains, including 2.7% kawain,
[10]
1.4% methysticin, 1.75% 5,6-dehydrokawain, 3.08% Yangonin, 0.33% flavokawain B, and 0.21% flavokawain
A . The KRE may be able to target different stages and heterogenity of prostate cancer. Consistent with the
[9]
previous studies, the KRE has similar potency as a kavalactone-rich kava fraction B for its chemopreventive
effect in prostate cancer in the TRAMP model. However, further studies are still needed to investigate
whether there are significant synergistic or antagonistic effects between flavokawains and kavalactones on
prostate cancer.

Dietary feeding of the KRE used here resulted in decreased body weight gain and liver enlargement (data
[10]
not shown). This result is also consistent with the report by Tang et al. that the kavalactone-rich kava
fraction B (free of flavokawains) suppressed body weight gain and induced liver enlargement