Kava root extracts hinder prostate cancer development and tumorigenesis by involvement of dual inhibition of MAO-A and LSD1

Aim: Here, we aim to evaluate the chemopreventive efficacy of kava root extracts (KRE) in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice and investigate potential molecular targets of kavalactones, the main components of kava. Methods: TRAMP mice were administrated with KRE formulated food for different periods of time, and then the incidences of high-grade prostatic intraepithelial neoplasia (HG-PIN) and adenocarcinomas and tumor burdens were compared between vehicle control and KRE food fed groups. In addition, the inhibitory effect of the KRE and kavalactones on monoamine oxidase A (MAO-A) and lysine-specific demethylase 1 (LSD1) enzyme activities were examined by commercially available inhibitor screening kits. Histone H3 lysine 9 dimethylation was also evaluated in prostate cancer cells and tumor tissues using Western blotting analysis. Results: Dietary feeding of 0.3% and 0.6% KRE to TRAMP mice from ages of 6 weeks to 12 weeks inhibited HG-PIN by 43.5% and 59.7%, respectively, and prostate adenocarcinoma by 53.5% and 66.4%, respectively. In addition, 0.6% KRE fed TRAMP mice from ages of 6 weeks to 24 weeks exhibited a significant reduction of genitourinary weight (a surrogate of tumor burden) by 54.5% and reduced body weight gain. Furthermore, the KRE and kavalactones showed a significant inhibition of LSD1 and MAO-A enzyme activities. Conclusion: Our results suggest that consumption of kava products through diet can delay prostate cancer development and progression and that kavalactones may be a new structure model for developing a potent dual inhibitor of LSD1 and MAO-A.


INTRODUCTION
Prostate cancer has a projected incidence of ~248,530 new cases diagnosed, accounting for ~34,130 deaths in 2021 in the United States, ranking the second leading cause of cancer death in men [1] . The majority of men diagnosed with prostate cancer (about 4 in 5 men) die from other reasons rather than prostate cancer. More than 3.1 million men are alive with a diagnosis of prostate cancer in the United States [1] . In addition, over one-third of prostate cancer is slowly growing or progressing over several decades. This group of patients without cancer-related symptoms do not warrant aggressive, immediate treatment [2] . Instead, they are closely monitored for their indolent status using prostate-specific antigen (PSA) kinetics, periodic biopsies for histologic progression, and possibly surveillance magnetic resonance imaging (MRI) until further treatment is needed as judged by physicians. This emergent strategy for the management of indolent prostate cancer is called active surveillance [2] . Men on active surveillance have high anxiety, depression and doubts about the possible progression of the disease and are highly motivated to prevention approaches using dietary or pharmacological means or positive lifestyle changes [3] . Therefore, men on active surveillance have been thought to be an ideal population for chemo/dietary prevention methods to further reduce disease progression and anxiety during the period of active surveillance. Currently very few chemopreventive agents are effective for their testing in chemoprevention studies in this population.

Kava (Piper methysticum Forst) is a continually regrowing shrub native to the South Pacific
Islands [4] . Traditionally, kava root extracts (KRE) have been prepared with coconut milk and water as a drink for a social gathering in the Pacific Islands for thousands of years [4] . The kava drink improves sleep quality and produces distinct muscle relaxation without drug addiction [4] . Several clinical studies have shown commercially available KRE to reduce anxiety and sold as an anxiolytic agent for several decades [5][6][7] . Kava drinking was also linked to lower cancer risk by an epidemiological study [8] . In addition, KRE has demonstrated potent anti-carcinogenic activity in several cancers, including bladder cancer, lung cancer, and others [9][10][11][12] . Intriguingly, compared to other areas of the world, incidences of prostate cancer in kava drinking countries, such as Fiji, are very low. However, when Fijian men moved to Australia, their prostate cancer incidence raised by 5.1-fold [13,14] .
These results have led us to investigate whether kava consumption can affect prostate cancer development and progression. We have previously shown that oral administration of KRE through dietary supplementation effectively reduced the growth of patient-derived xenograft (PDX) tumors and down-regulated the protein levels of androgen receptor (AR) and the expression of AR target genes PSA and transmembrane protease, serine 2 (TMPRESS2) [9] . Given the above-described properties of KRE, KRE would have the promise to be further studied as a chemopreventive agent for reducing progression risk and anxiety in prostate cancer patients under active surveillance.
To evaluate the chemopreventive effect of KRE, transgenic adenocarcinoma of the mouse prostate (TRAMP) mice were administrated with vehicle control or KRE formulated food in different stages of tumor development to test its ability to prevent prostate cancer development [i.e., occurrence of high-grade prostatic intraepithelial neoplasia (HG-PIN) and prostate adenocarcinomas] or delay progression. In addition, the effects of the KRE and its main components of kavalactones, including kawain, 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.

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 [16] . The 0.3% (3 g/kg food) or 0.6% (6 g/kg food) KRE 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 [16] . PIN lesions and prostate adenocarcinoma were evaluated to Dr. Cardiff's description [17] 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 Nterminal tail of methylated H3K4, were added to the LSD1-buffer solution to begin the enzymatic processing. This process took place at 37 °C for 60 min. In the process of demethylation, hydrogen peroxide (H 2 O 2 ) is formed as a byproduct. HRP then can use H 2 O 2 to convert the 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 2 O 2 byproduct in the MAO-A reaction to convert the 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.

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

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 pathologic stages or with palpable tumors among different treatment groups were tested by using the χ 2 or Fisher exact test.

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.

Kavalactones and KRE inhibit MAO-A activities in prostate cancer cell lines LNCaP and C4-2B cells
Several studies reported that MAO-A was associated with aggressive prostate cancer and promoted prostate tumor growth and metastasis [20,21,24] . In addition, MAO-A is a key target for the treatment of neuropsychiatric and neurodegenerative disorders [19] . We therefore have examined whether the KRE and its active components kavalacotones can inhibit cellular MAO-A activity in LNCaP and C4-2B cells. First, we used the autodock program to dock kavalactones with MAO-A protein (PDB code: pdb 2bxs). Kawain and methysticin have predicted IC 50 values of 3.85 μM and 880.78 nM, respectively. Pymol program was used to observe the superimposed binding of kawain and methysticin to the flavin adenosine dinucleotide (FAD)-binding and the active site of MAO-A and the kavalactones were predicted to form hydrogen bonds [ Figure 3A and B].
In addition, the cellular MAO activities were examined by MAO-GLO assay after LNCaP and C4-2B cells were treated with KRE and kavalactones for 24 h. Figure 3C and D show that kavalactones and KRE significantly inhibited cellular MAO activities by about 25% to 51% (P < 0.05). These results suggest that kavalactones and KRE are weak inhibitors of MAO activity in prostate cancer cells.

Kavalactones inhibit LSD1 activities in prostate cancer cells and dietary feeding of KRE is associated with reduced LSD1 activities in prostate tumors
LSD1 is an epigenetic enzyme responsible for demethylation of H3K4 and H3K9 for reprogramming gene expression profile and suggested to have a critical role in the progression of castration-resistant prostate cancer via interacting with AR [25,26] . Given the catalytic activity of both MAO-A and LSD1 resides in the amine oxidase domain and is dependent on its co-factor FAD [18] , we therefore examined whether kavalactones could also affect LSD1 activity in prostate cancer. Molecular docking analysis revealed that kawain and methysticin superimpose FAD and binds to the FAD-binding domain of LSD1 and interacts with important amino acids, methionine 332 and arginine 316, respectively [ Figure 4A and B].
In vitro LSD1 inhibitory activity assay shows that kawain and methysticin have the most significant inhibition against LSD1 enzyme activity among the tested kavalactones [ Figure  4C]. In addition, protein expression levels of dimethylated H3K9 were observed to be significantly increased in KRE, methysticin, 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.
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 [9] . Whether the inhibitory effect of kawain and methysticin on AR signaling is 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 [16] , whereas a kavalactone-rich kava fraction B was shown to significantly decrease the incidences of neuroendocrine carcinoma in TRAMP mice by absolute 42.9% [10] . The KRE used here contains both kavalactones and flavokawains, including 2.7% kawain, 1.4% methysticin, 1.75% 5,6-dehydrokawain, 3.08% Yangonin, 0.33% flavokawain B, and 0.21% flavokawain A [9] . The KRE may be able to target different stages and heterogenity of prostate cancer. Consistent with the 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 not shown). This result is also consistent with the report by Tang et al. [10] that the kavalactone-rich kava fraction B (free of flavokawains) suppressed body weight gain and induced liver enlargement (hepatomegaly) without noticeable changes in liver function. In contrast, the changes in body weight gain and organ weight after long-term dietary feeding of flavokawain A in TRAMP mice have not been observed in our previous studies [16] . Therefore, it is unlikely that flavokawain A is responsible for the reported "kava hepatotoxicity". Flavokawain A could be a safer product for cancer prevention than the KRE and the kavalactone-rich kava fraction B.
In summary, the KRE product containing both kavalactones and flavokawains reduced the incidence of HG-PIN and early-stage prostate adenocarcinoma and inhibited tumorigenesis. The KRE and kavalactone weakly inhibited both MAO-A and LSD1 activities in prostate cancer, leading to their inhibitory effect on AR signaling and cell proliferation. This study has provided a new knowledge basis for developing more effective dual inhibitors of MAO-A and LSD1 enzymes for prostate cancer prevention and treatment.