Transcriptional Regulation of G2/M Regulatory Proteins and Perturbation of G2/M Cell Cycle Transition by a Traditional Chinese Medicine Recipe

Transcriptional regulation of G2/M regulatory proteins and perturbation of G2/M Cell Cycle transition by a traditional Chinese medicine recipe

Su Su Thae Hnit1,2, Mu Yao1,2, Chanlu Xie1,2, Ling Bi3, Shenyi Jin3, Lijing Jiao3, Ling Xu3, Lina Long4,5, Hong Nie4,5, Yu Jin6, Linda Rogers7,8, Natalka Suchowerska7,8, Matthew Wong9,10, Tao Liu9,10, Paul De Souza11, Zhong Li12# and Qihan Dong1,2,13#

1Chinese Medicine Anti-Cancer Evaluation Program, Greg Brown Laboratory, Central Clinical School and Charles Perkins Centre, The University of Sydney, Sydney, Australia 2Department of Endocrinology, Royal Prince Alfred Hospital, Sydney, Australia 3Department of Oncology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China 4International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development of Chinese Ministry of Education (MOE), School of Pharmacy, Jinan University, China 5Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou, China 6School of Pharmacy, East China University of Science and Technology, China 7VectorLAB, Department of Radiation Oncology, Chris O’Brien Lifehouse, Sydney, Australia 8School of Physics, The University of Sydney, Sydney, Australia 9Children’s Cancer Institute Australia for Medical Research, Sydney, NSW, Australia 10Centre for Childhood Cancer Research, UNSW Medicine, Sydney, Australia 11School of Medicine, Western Sydney University, Australia 12Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, China 13School of Science and Health, Western Sydney University, Australia

#Corresponding author: Qihan Dong; School of Science and Health, The University of Western Sydney, Penrith South, NSW 2751, Australia. Phone: +61 2 46203633; Fax: +61 2 46203025; Email: [email protected] Zhong Lee: Beijing University of Traditional Chinese Medicine, Beijing, 201203, China. Phone: 86-15000775800; Email: [email protected]

Key words: G2 to M transition, prostate cancer, traditional chinese medicine, Hedyotis diffusa, Scutellaria barbata

Running Title: Impeding of G2 to M transition in prostate cancer cells by HS 1

Abstract Ethnopharmacological Relevance: Hedyotis diffusa (H) and Scutellaria barbata (S) are ancient anti-cancer Chinese herb medicines. When combined, known as HS, it is one of the most commonly prescribed Chinese Medicine for cancer patients today in China. Aim of the study: The prevention of disease progression is a dominant concern for the growing number of men with prostate cancer. The purpose of this work is to evaluate the action and mode of action of Chinese Medicine recipe HS in inhibiting prostate cancer progression in preclinical models. Methods: Effects of HS were analyzed in prostate cancer cell lines by evaluating proliferation, cell cycle profile, DNA damage and key regulators responsible for G2 to M phase transition. The transcriptional activities of these regulators were determined by RT-PCR and ChIP. The efficacy of HS in vitro was validated in an animal model. Results: HS treatment was observed to reduce DNA content and accumulated prostate cancer cells at the G2/M phase. Immunolabeling for phospho-Histone H3 in association with nocodazole to capture mitotic cells confirmed that HS impeded G2 to M transition. After excluding DNA damage-induced G2 arrest, it was revealed that HS reduced expression of Cyclin B1, CDK1, PLK1 and Aurora A at both protein and mRNA levels, with concomitant reduction of H3K4 tri-methylation at their promoter-regions. Animals that received oral administration of HS with a dosage relevant to clinical application showed reduced tumor volume and weight with a reduction of Cyclin B1, CDK1, PLK1 and Aurora A protein levels. Conclusions: HS acts by impeding the G2 to M transition of prostate cancer cells. It is likely that the mode of action is transcriptionally suppressing proteins governing mitotic entry, without eliciting significant DNA damage.

1 Introduction Prostate cancer is a heterogeneous disease with manifestations ranging from asymptomatic to widespread metastasis. Active surveillance, rather than immediate aggressive treatment, has emerged as a viable option for men with early stage, localised prostate cancer (Tosoian et al. 2016). However, after 5 years of surveillance, 24– 40% of men progress and need surgical or medical treatment (Carter 2016). Currently, there is neither a biomarker with sufficient sensitivity and specificity to predict the progression of prostate cancer nor is there any clinical modality that can suppress progression of prostate cancer. Therefore, the active surveillance protocol requires regular biopsy of the prostate gland. Understandably, many men under active surveillance have developed anxiety issues and reported a reduction in quality of life (Klotz 2013). Recent reports indicate that the proportion of men on active surveillance has increased from 6.7% between 1990 and 2009 to 40.4% between 2010 and 2013 (Tosoian et al. 2016). Moreover, men with metastatic prostate cancer have few treatment options once androgen deprivation therapy fails, with or without newer androgen-axis active drugs such as abiraterone and enzalutamide; a considerable proportion is not suitable for chemotherapy. Considering prostate cancer is predicted to remain one of the top cancer diagnoses by 2030 (Rahib et al. 2014), there is a critical need to develop well-tolerated therapies that can maintain disease control and reduce rates of disease progression, regardless of whether men are on active surveillance protocols or have established metastatic disease. The Chinese herb medicine Hedyotis diffusa (Chinese name: Bai Hua She She Cao) and Scutellaria barbata (Chinese name: Ban Zhi Lian) has been prescribed for hundreds of years and remain the most prescribed core treatment of Chinese Medicine for cancer patients today in China (Chao et al. 2014; Yeh et al. 2014). In Taiwan, more than 20,000 prescriptions of either Hedyotis diffusa or Scutellaria barbata were issued for breast cancer patients in 2009 alone (Yeh et al. 2014). A promising efficacy and favorable toxicity profile of Scutellaria barbata has been reported in clinical trials on patients with advanced breast cancer (Fong et al. 2008; Perez et al. 2010; Rugo et al. 2007). For prostate cancer, only a few studies report preclinical evaluation of Scutellaria barbata (Marconett et al. 2010; Shoemaker et al. 2005; Wong et al. 2009). While an anti-proliferating action has been described, a clear mechanism of action of Hedyotis diffusa and Scutellaria barbata has not been established. Here we present evidences that Hedyotis diffusa and Scutellaria barbata can impede mitotic entry of prostate cancer cells into the cell cycle and transcriptionally suppress proteins governing G2 to M transition without eliciting DNA damage.

2 Materials and Methods 2.1 Cell lines The prostate cancer cell line derived from a metastasis to a lymph node, LNCaP (CRL- 1740; American Type Culture Collection) and the prostate cancer cell line derived from a metastasis to bone, PC-3 (CRL-1435; American Type Culture Collection), were grown in RPMI 1640 supplemented with 10% v/v fetal bovine serum (FBS; AusGeneX, Brisbane, QLD, Australia), penicillin (100 U/mL) and streptomycin (100 µg/mL;). The cells were cultured at a temperature of 37°C and in 5% CO2 /95% air. 2.2 Preparation of Hedyotis Diffusa and Scutellaria Barbata (HS) Hedyotis Diffusa (H) and Scutellaria Barbata (S) were certificated based on authentication by the Chinese Pharmacopoeia. In a weight ratio of 1:1, H and S were decocted twice with 20 times volume of water for 30 min each time. The two filtrated supernatants were combined and water was added to 160 mL, to which ethanol was added until its volume proportion reached 70%. After thorough mixing and standing for 12 hours, the supernatant was collected and the alcohol was removed through rotary evaporation at 50°C under reduced pressure, and then freeze-dried into powder. Three batches of HS preparation were used in this study. The quality control of each batch was confirmed by HPLC analysis that was shown to be stable and good repeatability (Figure S3). The HPLC analysis was performed on a Unitary C18 column (150×2.1 mm i.d, 5 µm, Acchrom, China). The mobile phase A was 0.1% formic acid aqueous solution, and the mobile phase B was 0.1% formic acid acetonitrile. The gradient condition was 0-10 min, 10% -15% B; 10-15 min, 15% -19% B; 15-25 min, 19% B; 25-35 min, 19% -30% B. The flow rate was 0.2 mL/min and the detective wavelength was 334 nm. For in vitro experiments, the HS powder was dissolved in culture medium. The culture medium without HS was used as control. 2.3 SYBR Green assay Cells were pre-seeded in 96-well plates and cultured in 200 µL of complete culture medium for two days as previously described (Xi et al. 2016; Yao et al. 2015). The medium was then replaced with fresh medium containing different concentrations of HS (0.25 to 1 mg/mL) and the cell culture plates were incubated for a further 3 days. In parallel, the cells as baseline control, ie time zero, were stored at -80 °C until use. After treatment, the medium was gently aspirated from each well and stored at -80 °C. 2.4 Immunocytochemistry of BrdU incorporation Cells were treated with HS for 3 days in T75 flasks, and BrdU (B9285, Sigma-Aldrich) at a final concentration of 10 µM was added 6 hr prior to cell harvesting. The cells were trypsinized and fixed in 10% formalin solution, followed by embedding in paraffin blocks. BrdU was performed as previously described (Xi et al. 2016; Yao et al. 2015). The images were acquired from each immunostained section using a microscope (BX51, Olympus) equipped with CellSens software and quantified using ImageJ Fiji (version 1.51g). The positivity of the immunolabeling was calculated as DAB density/(DAB plus haematoxylin density), and converted into percentages by designating vehicle control as 100%. 2.5 Colonogenic assay Cells in T25 flasks were treated with HS for 3 days and then trypsinized and counted. LNCaP (5000 cells) and PC-3 (1000 cells) were seeded into new T25 flasks with drug free fresh complete medium and cultured for another 10 days without changing medium to generate single cell-derived colonies. After removal of the medium from the flasks, the colonies were briefly fixed with 1% formalin, rinsed with PBS and dried in the air prior to being stained with methylene blue for 30 min. The stained colonies were rinsed with water and dried again in the same manner. The flasks were then scanned by an instrument (Colcount, Oxford Optronix) and any colony with more than 50 cells was recorded. The number, size and density of colonies were integrated into a colony index representing a measure of cancer cell survival and viability. 2.6 Measurement of culture medium for pH and osmolality The pH and osmolality of the culture medium containing HS with a dose range (0-2 mg/mL) were determined by using a bench top pH meter (Seven Compact ™, Mettler Toledo) and Fiske® 210 Micro-Sample Osmometer (Advanced Instrument).

2.7 Flow cytometry assay Propidium iodide (PI) staining was used to analyze cell cycle phase distribution. LNCaP (1.5×105 cells) and PC-3 (1.2×105 cells) cells were seeded in T25 flasks for 2 days prior to being treated with HS for 3 days. Cells were harvested, fixed in 70% cold ethanol and stained in PBS containing RNase A (R6513, Sigma-Aldrich) at 10 µg/mL and PI (P4170, Sigma-Aldrich) at 20 µg /mL at 37 °C in the dark for 60 min. The cells were then washed and re- suspended in PBS. The cellular DNA content in individual cells was first assessed using a flow cytometer (FACS Calibur, BD Biosciences) and then analyzed by using FlowJo software (version 8.1.1). To quantify the proportion of cells at mitotic phase, the fixed cells were first permeabilized with 0.1% saponin in PBS with 1% BSA for 10 min at room temperature. After being rinsed with PBS, the cells were incubated with an antibody against phospho- Histone H3 (3377, Cell Signaling Technology) in PBS with 1% BSA at 4°C overnight. On the following day, the cells were sequentially labeled with a secondary antibody conjugated with AlexaFluor488 (A11008, Life Technology) and PI staining. Thereafter, the cells were washed and re-suspended in PBS prior to the data acquisition and analysis as described above. 2.8 Immunoblotting PC-3 and LNCaP cells were treated in T25 flasks and cell lysates were prepared in a lysis buffer as described previously [23]. Primary antibodies against Cyclin B1 (SC-752), GAPDH (SC-137179) and α-tubulin (SC-5286) were purchased from Santa Cruz Biotechnology; Histone H3 (9715), phospho-Histone H3 Ser10 (9701), CDK1 (9116), PLK1 (4513) and Aurora A (12100) were obtained from Cell Signaling Technology. 2.9 Radiation treatment LNCaP and PC-3 cells were seeded in T25 flasks (Sarsdedt Australia Pty Ltd, Technology Park, South Australia) for 48 hr. Immediately before radiation, the culture medium was topped up to 20 mL and the cells were irradiated under full scatter conditions, using a 6 MV photon beam produced by a Varian Novalis TX linear accelerator as previously described (Mackonis et al. 2012). The cells were irradiated to a dose of 2 Gy at a dose rate of 6 Gy/minute. For all experiments, unexposed controls were prepared in the same way. The cells were harvested by trypsinization and fixation with 10% buffered formalin solution after 3 hr of radiation treatment. The cells were then embedded in paraffin blocks for immunofluorescence. 2.10 Immunofluorescence LNCaP and PC-3 cells were treated, harvested and processed as described in Radiation treatment. The paraffin blocks containing the cells were cut into sections, rehydrated and subjected to antigen retrieval. After blocking with 10% BSA in Tris-buffered saline containing TWEEN-20 (TBST), the sections were incubated with primary antibody against to phospho-Histone H2AX Ser139 (ab184520, Abcam) or 53BP1 (4937, Cell Signalling Technology) at 4oC overnight. After being rinsed in TBST, the sections were labeled with a secondary antibody conjugated with AlexaFluor488 (A11029, Life Technology) for phospho-Histone H2AX Ser139 and another secondary antibody conjugated with AlexaFluor594 (A-11037, Life Technology) for 53BP1. The labelled sections were then counterstained for nuclei with 4′, 6-Diamidino2- phenylindole dihydrochloride (D8417, Sigma-Aldrich) and cover-slipped. The images of phospho-H2AX or 53BP1 labeling and corresponding nuclear staining of each sample were obtained from the labelled sections using a microscope (Olympus, BX53) equipped with CellSens software. The acquired images of phospho-H2AX or 53BP1 labeling and corresponding nuclear staining were superimposed and saved in the format of portable network graphics. 2.11 RT-PCR LNCaP and PC-3 cells were treated and harvested by aspirating the medium followed by washing with cold PBS. mRNA from the samples was extracted using Purelink RNA Mini Kit (12183018A, Life Technologies). cDNA was generated, using the iScript™ cDNA Synthesis Kit (BioRad). The total cDNA was mixed with SYBR Green containing SensiMixTM master mix (QT650-05, Bioline) and subjected to a thermocycle at 95 ºC for 10 min followed by 45 cycles of PCR at 95 ºC for 15 s, at 60 ºC for 15 s and at 72 ºC for 15 s. The primer sequences are listed in the supplementary information (Table S1). 2.12 Chromatin immunoprecipitation The chromatin immunoprecipitation (ChIP) was carried out as previously described (Bi et al. 2018). Briefly, the

4 assay was performed according to the protocol of ChIP assay kit (17-295, Merck) together with ChIP grade rabbit anti-H3K4me3 (ab8580, Abcam) and isotype IgG (SC-2027, Santa Cruz Biotechnology) as control. Immunoprecipitated DNA was quantified by real-time PCR. The primer sequences are listed in the supplementary information (Table S2). ChIP assays were repeated three times independently and calculated as fold enrichment relative to the control IgG and normalized with respect to input DNA. 2.13 Xenograft model of human prostate cancer in mice To establish a xenograft model of human prostate cancer, 1 x 106 of PC-3 cells in 0.2 mL of PBS was subcutaneously inoculated into the left flank of 8 week-old male nude mice (SLAC Laboratory Animal, Shanghai). The PC-3 xenograft was monitored by calibre measurement and tumor volume was calculated as volume = (length × width2)/2. The human daily dose of HS is 30 g in total (H: 15 g and S: 15 g). As the ratio of HS herb to dried powder was 21:1 (w/w), the human daily dose is equivalent to 1.428 g powder. According to the conversion coefficient of 0.0026 for 20 g mice, 3.7 mg dried powder dissolved in 0.2 mL of normal saline (or 0.9% NaCl) and was given by gavage for each mouse per day as “low dose”, and 7.4 mg as “high dose”. For the control group, 0.2 mL 0.9% normal saline was given by gavage. The mice were anesthetized 29 days after cancer cell implantation, and their tumors were excised, weighted and then fixed in 10% formalin for immunohistochemistry. The study was approved by Yueyang Hospital of Integrated Traditional Chinese and Western Medicine (Institute Animal Ethics Approval Number: SYXK2018-0040) and University of Sydney Animal Ethics Committee (2014/622). 2.14 Immunohistochemistry Tumors were resected immediately after euthanasia and fixed in 10% buffered formalin solution. The samples were then dehydrated and embedded in paraffin blocks. Sections of 5 µm thickness were cut and rehydrated. After antigen retrieval and blocking, the sections were respectively labeled with primary antibodies against Cyclin B1 (SC-752, Santa Cruz Biotechnology), CDK1 (1161-1, Epitomics), PLK1 (4513, Cell Signalling Technology) and Aurora A (14475, Cell Signalling Technology) at 4oC overnight. In the following days, the sections were sequentially labeled as described previously (Xi et al. 2016; Yao et al. 2015).The imaging and quantification of immuno-labeled proteins were conducted as described in Immunocytochemistry of BrdU incorporation. 2.15 Statistical Analysis Statistical software v12.0 (NCSS LLC) and Prism version 7.0b (GraphPad) were used for analysis. One-Way ANOVA was implemented to determine the difference between individual groups of data. Fisher’s LSD Multiple- Comparison Test was used to determine whether the difference between individual groups (P<0.05) was considered significant.

3 Results 3.1 HS suppresses proliferation in prostate cancer cells We firstly determined the anti-proliferative effect of HS on prostate cancer cells using the SYBR Green assay. In a 3 day course of treatment, HS significantly inhibited DNA synthesis in both LNCaP and PC-3 cells manifested by reduction in net gain of DNA content in a dose-dependent manner (Figure 1a). At the highest dose tested (1 mg/mL), HS appeared to cause cytotoxicity to LNCaP cells, as the DNA content fell below the initial input (i.e. baseline). In contrast, there was no noticeable cytotoxicity to PC-3 cells at any tested dose. A dose range between 0-0.75 mg/mL and 0-1 mg/mL was therefore applied to LNCaP and PC-3 cells, respectively, in the following experiments to focus on cytostatic action of HS at its relevant dose range. The dose range did not cause any alteration in either pH or osmolality of culture medium (Figure S1a and Figure S1b). Consistent with SYBR Green DNA analysis, treatment with HS also reduced BrdU incorporation (Figure 1b, Figure S1c). In order to determine the long-term effect of HS, LNCaP and PC-3 cells were treated with a dose range of HS for 3 days followed by an additional 10 days culture without HS at a density appropriate for formation of discernible colonies. HS significantly decreased the colony formation in both cell lines (Figure 1c, Figure S1d).

3.2 HS impedes G2 to M transition in prostate cancer cells Cell cycle phase analysis revealed that HS significantly increased the proportion of LNCaP and PC3 cells in G2/M phase at a dose of 0.75 mg/mL, and at 0.75-1 mg/mL, respectively (Figure 2a) after 3 days treatment. These data demonstrate that HS may inhibit prostate cancer cell proliferation by arresting cells at the G2/M phase. As cells at 5

G2 and M phase possess a similar amount of DNA content and cannot be differentiated from each other by staining of DNA with propidium iodide alone, a mitotic marker, phospho-histone H3 at ser10, was utilized together with propidium iodide to differentiate G2 from M phase. After 3 days treatment, there was no increase of cells in M phase, indicating the treated cells accumulated in G2 phase (Figure 2b). To confirm this finding, cells treated with HS were also harvested for immunoblotting. While there was no noticeable alteration in total histone H3, the intensity of phospho-histone H3 at ser10 was diminished by HS in a dose-dependent manner (Figure 2e and Figure S2a). To further verify that HS impedes G2 to M transition, cells were subjected to nocodazole to capture the mitotic cells in the presence or absence of HS. Compared to nocodazole alone, there was no significant change in the total percentage of cells in G2 plus M phase in the presence of HS (Figure 2c). However, the proportion of cells in G2 phase was significantly increased, indicating that fewer cells entered mitotic phase in the presence of HS (Figure 2d). Taken together, these data demonstrate that HS impedes G2 to M transition in prostate cancer cells. 3.3 HS did not elicit significant DNA damage Since a delay in G2 to M transition is often associated with DNA damage (Raleigh et al. 2000), we investigated whether the HS could elicit DNA damage in treated cells. To this end, cells were treated with HS and analyzed for phospho-H2AX and p53-binding protein 1 foci as indicators of DNA damage. In parallel, cells were exposed to ionizing radiation at 2 Gy to be used as a positive control for DNA damage. While the irradiated cells displayed conspicuous foci of both phospho-H2AX and p53-binding protein 1, HS treatment did not initiate apparent changes in both prostate cancer cell lines (Figure 3a, 3b and 3c).

3.4 Transcriptional suppression of regulatory proteins governing G2 to M transition by HS We then measured protein and mRNA levels of the regulators for G2 to M transition, namely CDK1, CyclinB1, Polo-like kinase 1 (PLK 1) and Aurora A. HS reduced all these regulators at protein (Figure 4a and Figure S2b) and mRNA (Figure 4b) levels. To address if HS reduced the stability of these mRNAs, cells were pre-treated with or without HS for 2 days followed by treatment with Actinomycin D for different durations to inhibit the synthesis of mRNAs. Subsequent RT-PCR showed that HS did not accelerate the degradation of the concerned mRNAs (Figure 4c). To gain an insight into the transcriptional regulation of these G2 to M transition regulators, chromatin immunoprecipitation was conducted to measure the degree of K4 tri-methylation on histone H3 (H3K4me3) (Schneider et al. 2004) near the promoter regions of all the regulator genes. Clearly, HS reduced the degree of this epigenetic modification (Figure 4d), indicating that HS diminishes gene expression of the regulators governing G2 to M transition at the transcriptional level in prostate cancer cells. 3.5 Efficacy of HS in xenograft model of human prostate cancer To validate the HS action in vivo, HS was introduced to a PC-3 xenograft model of human prostate cancer. Two doses of HS dissolved in saline were given orally. The low dose (3.7 mg/20g mouse) is equivalent to the human daily dose, and the high dose (7.4 mg/20 g mouse) is twice the human daily dose. While both doses of HS suppressed tumor growth compared to the control, high doses of HS did not have a further inhibitory effect (Figure 5a,c). At the end of treatment, the tumor weight was reduced by 46% and 44%, respectively, in the low and high dose arms (Figure 5b), while tumor volume was reduced by 38% and 49%, respectively. (Figure 5c,d). Both low and high doses were well tolerated in the animals and no significant change in body weight was noted compared with control (Figure 5e). Immunohistochemical staining also showed that all four proteins governing G2 to M transition were significantly reduced in the HS treated xenografts in comparison to the control (Figure 6).

4 Discussion Cancer is inevitably related to an anomaly in the regulation of the cell cycle. In prostate cancer, the percentage of Ki-67 positive cancer cells is low in low risk disease (Berges et al. 1995), but increases in high risk (Keshari et al. 2011) and advanced prostate cancer (Khatami et al. 2009), suggesting that an increase in the index of proliferation contributes to prostate cancer progression. Notably, only through proliferation are cancer cells with genetic and epigenetic alterations able to be selected and accumulate. The increased proliferative rate in tumours can further increase the chance of DNA replication errors. Therefore, we propose that a method of impeding cell cycle progression can prevent or delay progression of prostate cancer in patients; in order to be acceptable to patients, ideally that treatment would also have low toxicity to encourage long-term use.

Hedyotis diffusa and Scutellaria barbata, either alone or in combination, are among the best known traditional Chinese Medicines to treat cancer in Eastern Asia. As the traditional treatment of cancer is not organ specific, Hedyotis diffusa and Scutellaria barbata have been used to treat various types of cancer. Three studies on the preclinical activity of Scutellaria barbata have been conducted in prostate cancer (Marconett et al. 2010; Shoemaker et al. 2005; Wong et al. 2009) but there have been none involving Hedyotis diffusa. Scutellaria barbata has been shown to arrest LNCaP cells in the G2/M phase with concomitant decreases in cyclin B1 and CDK1 mRNA and protein levels (Marconett et al. 2010). In mice with transgenic adenocarcinoma (TRAMP), oral administration of Scutellaria barbata delayed the onset of palpable tumor and increased the percentage of tumor- free mice (Wong et al. 2009). Our study is consistent with the reported anti-proliferative action of Scutellaria barbata but we have further established the action and mode of action of the combination of H and S, as they are commonly used together as a core treatment for cancer (Yeh et al. 2014). HS in combination possesses anti-proliferative properties in both LNCaP and PC-3 prostate cancer cell lines as shown by assays on DNA content, BrdU incorporation and colony formation in our study. We have provided evidence that the previously reported G2/M phase arrest by Scutellaria barbata is likely due to impedance of cell cycle transition from G2 to M phase. As a delay in G2 to M transition is frequently associated with DNA damage (Raleigh et al. 2000), we also considered the possibility that HS causes DNA damage as a primary mechanism of impairment of G2 to M transition, but found no significant change in the number of phospho-H2AX and p53- binding protein 1 foci compared to control. Our attention then turned to the evaluation of other key regulators that govern the G2 to M transition.

The CDK1-Cyclin B1 complex is known as a master driver of mitosis (Gavet et al. 2010). The complex is activated by Cdc25 phosphatases that remove inhibitory phosphorylations at Thr14 and Tyr15 on CDK1 catalyzed by Wee1 and Myt1 (Lindqvist et al. 2009). A rise in PLK1 activity in late G2 precedes activation of the CDK1-Cyclin B1 complex and results in activating phosphorylation of Cdc25C1 at Ser75 (Gheghiani et al. 2017) and Ser198 (Toyoshima‐Morimoto et al. 2002). In turn, PLK1 is activated through phosphorylation at Thr210 by Aurora A (Seki et al. 2008). Aurora A and PLK1 are reported to be over-expressed in prostate cancer and its precursor lesion (Buschhorn et al. 2005; Weichert et al. 2004). CDK1 and Cyclin B1 are known to be elevated in cancer of breast (Kawamoto et al. 1997), colon (Wang et al. 1997), lung (Soria et al. 2000), esophagus (Murakami et al. 1999) and mouth (Kushner et al. 1999). Using the Sawyers–370 dataset from R2 microarray analysis and visualization platform (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi), we noticed that the mRNA levels of Cyclin B1, CDK1, PLK1 and Aurora A are significantly higher (p<0.001) in prostate cancer (n=300) than in benign prostatic hyperplasia (n=58). In addition, the same dataset also shows that mRNA levels of each of these proteins are significantly higher (p<0.001) in prostate cancer with metastasis (n=38) than in primary localized prostate cancer (n=262). Together, these data support the notion that there is an association between prostate cancer progression and the expression of these four genes. Our study revealed that HS reduced expression of these four genes at both protein and mRNA levels. After excluding the possibility that HS decreases the stability of the mRNA of these four genes, we demonstrate that the transcriptional activities of these genes are diminished as shown by the reduction of H3K4 tri-methylation near their promoter-regions. Importantly, the reduction of these four G2-M regulators is confirmed in animals implanted with PC-3 xenograft following oral administration of HS at a dose relevant to clinical use. Concomitantly, we observed a reduction of the xenograft volume by 38-49% and weight by 44-46% over a one-month study period. Although there is some indication that a higher dose could be more effective in reducing the protein levels of CDK1, PLK1 and Aurora A (Figure 6), there was no difference in efficacy between the two doses of HS on tumor volume and weight in vivo. The low dose (3.7 mg/20 g mouse) is equivalent to the human daily dose, and the high dose (7.4 mg/20 g mouse) is equivalent to twice the human daily dose. Further studies with a broader dose range may be needed to verify our observation.

5 Conclusion In conclusion, a combination of the Traditional Chinese Medicines Hedyotis diffusa and Scutellaria barbata transcriptionally represses the regulators that are essential for mitotic transition of prostate cancer cells without 7 provoking apparent DNA damage. Considering the G2 phase is twice as long as the M phase in cell cycle progression and that very few agents are available to impede transition at G2-M phases of the cell cycle, the potential of using HS to prevent the progression of prostate cancer in men with prostate cancer warrants further clinical evaluation.

Conflict of Interest The authors declare no competing interests.

Funding This study was supported by Sydney Medical School Foundation grant (QD), University of Western Sydney Partnership Grant (QD), Shanghai Municipal Health Commission: ZY(2018-2020)-CCCX-2004-09; Science and Technology Commission Shanghai Municipality: No.16401970700; Shanghai Municipal Education Commission, “Gao Yuan Gao Feng” Team; Shanghai Municipal Health Commission: ZYKC201601020; Shanghai Sailing Program: No. 19YF1450000.

Authorship S.H., M.Y., C.X., L.B., S.J., L.J., L.Y., Y. J., L.R., M.W. conducted experiments, analyzed data and wrote the manuscript. L.X., N.H., N.S., T.L., P.D.S., A.B., supervised research, interpreted data and wrote the manuscript. Z.L., Q.D. designed the study.

Acknowledgements The authors acknowledge the support received from Dr Shirley Nakhla from Live Cell Analysis Facility, Bosch Institute for flow cytometric analysis; Ms Sanaz Maleki from Histopathology Facility for technical support on the immunofluorescence, and a generous donation of PuraPharm Corporation to the Chinese Medicine Anti-Cancer Evaluation Program (QD) in Central Clinical School of the University of Sydney.

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Figure legend

Figure 1. HS inhibited the DNA synthesis and colony formation in prostate cancer cells. (a) LNCaP and PC-3 cells were treated with HS for 3 days and harvested for SYBR Green assay. Expressed as the mean ± SD of three independent experiments. Means without a common letter differ, p<0.05. (b) Cells were treated with HS for 3 days and BrdU was added in 6 hours prior to harvest for immunocytochemistry. (c) Cells were treated with or without HS for 3 days followed by replating for 10 days in the absence of the HS. The colony consisting of more than 50 cells was then stained and quantified.

Figure 2. HS increased cancer cells at G2/M phase and impeded the transition from G2 to M phase. (a) LNCaP and PC-3 cells were treated with HS for 3 days and harvested for PI-staining and flow cytometric analysis. (b) The cells were treated with HS for 3 days and labeled with both PI and antibody against phospho-Histone H3 followed by cell cycle analysis. (c & d) LNCaP and PC-3 cells were treated with nocodazole in the presence or absence of HS at 0.75 mg/mL in LNCaP or 1 mg/mL in PC-3 cells for 16 hours. Cells were then labeled with PI or both PI and phospho-Histone H3. (e) Representative images of immunoblotting in LNCaP and PC-3 cells treated with HS for 3 days. All data were expressed as the mean ± SD from three independent experiments. * p <0.01 compared to Control.

Figure 3. HS did not elicit significant DNA damage in prostate cancer cells. LNCaP and PC-3 cells were treated with or without 0.75 and 1 mg/mL of HS respectively for 3 days followed by immunofluorescence of phospho-H2AX (a) or 53BP1 (b) for detection of the DNA damage. Radiation treated cells were used as positive control for DNA damage. The number of DNA foci from both phospho-H2AX and 53BP1 immunofluorescence were quantified and presented as the mean ± SD from three different experiments (c). * p <0.05 compared to Control.

Figure 4. HS suppressed the transcription of the proteins which govern G2 to M phase transition. Cells were treated with HS for 3 days followed by immunoblotting (a) and RT-PCR (b). LNCaP and PC-3 cells were treated with or without HS at 0.75 and 1 mg/mL respectively for 48 h, and exposed to Actinomycin D (AD) at 0.4 µg/mL for 2, 4 and 6 hrs with 0 h time point of each cohort as baseline. Cells were harvested and analysed by RT-PCR

(c). PC-3 cells were treated with HS for 48 h and harvested for ChIP assay with anti-H3K4me3 antibody or rabbit IgG in PC-3 cells. The data were shown as the mean ± SD from three different experiments. * p<0.05 compared to Control.

Figure 5. HS inhibited the growth of PC-3 xenograft in vivo. The left flanks of male nude mice were injected with PC-3 cells subcutaneously. Mice received a daily gavage of HS, which begun one day after inoculation of PC- 3 cells and continued for 29 days. The measurable tumor volume over the study period (a) and tumor weight at day 29 (b) were presented as the mean ± S.D from 9 mice of each group. A scatter plot of tumor volume from each mouse at day 11, 17, 23 and 29 were presented. The bar indicated the mean and 1S.D (c). Photos of recovered tumors at day 29 (d). Animal body weight (e). *p<0.05 compared with the control.

Figure 6. The levels of proteins governing G2/M transition were reduced in xenografts treated with HS. The animals with PC-3 xenograft were treated with saline or HS for 29 days. The xenografts harvested at the end of the study were paraffin-fixed, sectioned and immune-stained using antibody against (a) Cyclin B1, (b) CDK1, (c) PLK1 and (d) Aurora A. (e) For quantification of protein levels, two images of the immuno-stained sections were acquired and quantified using ImageJ Fiji (version 1.51g). Expressed the mean ± SD of 9 xenografts from each group. *p<0.05 compared with the vehicle control. Supplementary Figure 1. Quantification of BrdU and colony formation assay. (a) Each immunostained section was photographed for three images and three independent experiments were performed. The mean of 9 sections ± SD. (b) The number of colonies of each dose were quantified and the mean numbers from three independent experiments were presented with ± SD. *p<0.05 vs. control. Complete culture media containing a dose range of HS from three independent experiments were tested for (c) pH and (d) osmolality and presented as mean ± SD. Supplementary Figure 2. Quantification of Histone H3, Cyclin B1, CDK1, PLK1 and Aurora A protein levels in HS treated cancer cells. Immunoblot images were quantified using ImageJ Fiji (version 1.51g) and the values plotted were mean ± SD from four independent experiments. *p<0.05 vs. control. Supplementary Figure 3. High-performance liquid chromatography (HPLC) analysis of HS. HPLC method was used to analyze the stability and reproducibility of HS. The comparison of three different batches of HS.