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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 7

Figure 4. Kavalactones inhibits LSD1 enzyme activity. (A, B) Autodocktools program was used to dock kawain and methysticin with
LSD1 protein, and Pymol program was used to observe the superimposed binding of kawain and methysticin to LSD1, respectively. 2D
molecular structures of kawain and methysticin are shown to interact with key amino acids within the predicted binding site. (C) In vitro
LSD1 enzyme activities were analyzed using LSD1 inhibitor screening kit. (D) Western blotting analysis of LSD1 expression and H3K9
dimethylation. LSD1: Lysine-specific demethylase 1.

5’,6’-dehydrokawain, or kawain treated LNCaP cells without affecting LSD1 expression [Figure 4D]. Our
previous studies have also reported that KRE, methysticin and kawain inhibited the gene transcription of
PSA and TMPRSS2, two AR target genes. These results together suggest that KRE, methysticin, and kawain
reduce LSD1 enzyme activity in cells leading to inhibition of AR signaling.

KRE containing food increased H3K9 dimethylation and reduced Ki67 expression in tumor tissues
Similar to the in vitro findings as described above, Western blotting analysis confirmed that KRE containing
food increased H3K9 dimethylation without obvious effects on LSD1 expression in prostate tissues of the
TRAMP mice [Figure 5A] and tumor tissues of a prostate cancer PDX model from our previous studies
[Figure 5B]. In addition, immunohistochemistry analysis revealed that the number of Ki67-positive cells in
the prostate of KRE-fed TRAMP mice was 26.3% ± 4.7% compared with 49.1% ± 10.2% in those from the
vehicle control treatment (P < 0.05; Figure 5C and D). This finding suggests that KRE feeding inhibited
LSD1 activity, leading to the anti-proliferative effects of KRE on prostate tumor tissues of mouse models
and therefore delaying the progression of prostate cancer.

DISCUSSION
Although kavalactones have been suggested to be responsible at least in part for both the anxiolytic effect
and the anti-prostate cancer effect, the molecular targets of kavalactones remain understudied. In this study,
our results have shown that kawain and methysticin are weeks but cell-active inhibitors for both MAO-A
and LSD1 enzymes. Therefore, our study has provided the first structure base for further developing more
potent dual inhibitors of MAO-A and LSD1 through chemical modification of the kavalactone skeleton.
Given both MAO-A and LSD1 have been demonstrated to be critically involved in prostate cancer
progression. Dual inhibition of MAO-A and LSD1 may have significant translation values in prostate cancer
prevention and treatment.