The human prostate cancer cell lines PC3 (cell identification number: 20160628-01) and LNCaP (cell identification number: 20180528-01) were obtained from Beijing Beina Chuanglian Biotechnology Institute; XHP (approval number: z11020073, batch number: 17043278), Beijing Tong Ren Tang, Beijing, China; DMEM F12 medium, GIBCO, Grand Island, NY, USA; fetal bovine serum (FBS), GIBCO; CCK-8, GIBCO; 3-MA (IM0190), Beijing Solarbio Technology Co., Ltd.; VEGFR (Ab 133273), Abcam, Cambridge, UK; αSMA (Ab7817) Abcam; PI3K (60225-1-ig), Proteintech, Chicago, IL, USA; Akt (Ab185633), Abcam; mTOR (Ab32028), Abcam; p-PI3K (ab182651), Abcam; p-Akt (ab192623), Abcam; p-mTOR (Ab137133), Abcam; cleaved caspase3 (ab214430), Abcam; caspase9 (ab202068), Abcam; PCNA (10205-2-AP), Proteintech; and β-actin (66009-1-Ig), Proteintech.

XHP 3 g was dissolved in 6 ml double distilled water for 24 h. The mixture was dissolved with the ultrasonic dissolver for 2 min. Then, the solubilized suspension was centrifuged at 6,000 rpm for 10 min. Both the supernatant and sediment were collected. The collected sediment was further dissolved in 6 ml DMSO with an ultrasound device, followed by 6,000 rpm centrifugation for 10 min. The XHP solution with the final concentration of 500 mg/ml was prepared by mixing the supernatant from the first and second centrifugation. The prepared solution was stored at 4°C for future use. In the in vitro assay, the solution was diluted to 5, 2.5, 1.25, and 0.625 mg/ml (crude dose) with the serum-free medium, respectively. According to the instructions, 5 mg 3-MA was dissolved in 3.2523 ml DMSO to the final concentration of 10 mM. [Woo Y, Jung YJ. Angiotensin II receptor blockers induce autophagy in prostate cancer cells. Oncol Lett. 2017 May; 13(5):3579-3585].

PC3 and LNCaP cells (5,000 cells/well) were seeded in 96-well plates and treated with different concentrations of XHP (0.625, 1.25, 2.5, and 5 mg/ml) for 6, 12, 24, or 48 h, then 10 μl of Cell Counting Kit-8 (CCK-8) solution was added to each well and the cells were cultured at 37°C for 2 h. The cell viability was confirmed by reading the absorbance at 450 nm in a microplate reader (TECAN, Switzerland).

PC3 cells were seeded in the T25 culture flask and incubated with or without XHP (1.25 mg/ml) at 37°C. After incubation, cells were collected and fixed with 70% ethanol at 4°C for overnight, followed by staining with 50 μg/ml propidium iodide (Sigma, St. Louis, MO, USA) for 45 min in the dark. A Canto II flow cytometer (Beckman, A00-1-1102) was utilized for cell cycle analysis.

PC3 cells were seeded in the T25 culture flask and incubated with or without XHP (1.25 mg/ml) at 37°C for 24 h. Then, cells were collected and fixed with precooled 4% paraformaldehyde. After washing with PBS, the fixed cells were digested with 0.25% trypsin (excluding EDTA), and centrifuged at 2000 rpm for 5 min. After a further PBS washing step, the collected cells were mixed with 500 μl binding buffer. Then, 5 μl propidium iodide and Annexin V-APC (Nanjing Kgi, KGA1030-1007) were added and the mixture was placed in the dark at room temperature for 5–15 min. A Canto II flow cytometer (Beckman, A00-1-1102) was utilized for apoptosis analysis.

Serum-free medium 300 μl was mixed with 50 μl matrix gel. 5 × 10⁵ PC3 cell suspension 100 μl was placed into the upper chamber of the Transwell chamber; meanwhile, 500 μl fetal bovine serum was added into the lower chamber of the Transwell chamber. After being cultured for 24 h, the chamber was fixed with 4% paraformaldehyde at 4 C. Finally, 0.1% crystal violet (G1061, Solarbio) was added and incubated for 30 min at room temperature. After being cleaned with PBS twice, the cells on the upper surface of the chamber were gently wiped with cotton balls and observed and counted under a microscope (ZEISS, Oberkochen, Germany). The experiment was repeated thrice.

The daily dosage of XHP for the adults is 6 g/d, which equals to 78 mg/kg for the mouse. The calculation is based on the ratio of body surface area between human and mouse. A 5% DMSO XHP extract suspension was prepared for animal study.

A subcutaneous transplantation mice model was established to further verify the anti-proliferative effect of XHP on prostate cancer. The study protocols were in accordance with the European Community guidelines on the use and care of laboratory animals and were approved by the Ethics Committee of the Central South University. The mice model of subcutaneous transplantation was established by PC cells injection. In general, 1×10⁷ PC3 cells were subcutaneously injected into the middle and rear armpit of the BALB/C male nude mice. As the average volume of subcutaneous transplantation reached to 200 mm³, the mice were randomized into the two groups, including the controls and the XHP treatment group (n = 6). The volume was calculated as (length × width × height) × π/6 (Kim and Kim, 2008). In the test group, mice received the XHP for 78 mg powder/kg per day for 2 weeks, whereas controls received the equal volume of normal saline. After treatment, tumor tissues were collected from the sacrificed mice. The tumor body weight inhibition rate (%) was calculated as [(average tumor volume in control group − average tumor volume in administration group)/average tumor volume in control group] × 100%. (Liu et al., 2015).

Tumor tissues were dissected into slides, fixed with 4% formaldehyde, and observed under an optical microscope. Paraffin slices were prepared as well, and the VEGFR and αSMA protein expression in the subcutaneous transplantation of prostate cancer tissues and the corresponding tissues were assessed with a Immunofluorescence 3′-diaminobenzidine (DAB) kit using the anti-VEGFR (Ab 133273; Abcam) and anti-αSMA (Ab7817; Abcam) monoclonal antibodies, respectively.

The protein concentration of the PC3 cells and tumor tissues was confirmed by the BCA protein assay (ComWin Biotech, Beijing, China). The proteins were separated by 12% SDS-PAGE and transferred to the polyvinylidene fluoride (PVDF) membrane. After blocking with the 5% skim milk, the membranes were incubated with the primary antibodies (anti-PI3K, anti-Akt, anti-mTOR, anti-p-PI3K, anti-p-PI3K, anti-p-Akt, anti-p-mTOR, anti-cleave-Caspase-3, anti-Caspase-9, and anti-PCAN antibody at 1:1000) at 4°C overnight. After washing with TBST, the membranes were incubated with the secondary antibodies (HRP conjugated-goat anti-mouse IgG, SA00001-1; Proteintech) for 2 h at room temperature. Immunoreactivity was determined by an advanced ECL kit (AWB0005b; Abiowell) and visualized using a chemiluminescence imaging system (Chemiscope 6,100; Clinx Science Instruments).

Total RNA was extracted with TRIzol^®Reagent (TIANGEN BIOTECH, Beijing, China) and reverse-transcribed with NovoScript^® Plus all-in-one first-strand cDNA Synthesis SuperMix (gDNA Purge) Reverse Transcriptase (Novoprotein, Shanghai, China) according to the manufacturer instructions. The primers were synthesized by the Sangon Biotech (Shanghai), and were listed in Table 1.

SPSS 22.0 (IBM, Armonk, NY, USA) was used for statistical analysis, and the data was expressed as x ¯ ± s . The GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA) was employed for plotting. t-Test was utilized to compare the difference between two groups of the independent sample which conformed to normal distribution. Kruskal–Wallis test was employed to compare the difference between two groups of the independent sample which was inconsistent with normal distribution. One-way ANOVA was used to compare the difference among multiple groups. The LSD test was utilized for pairwise comparison among multiple groups. All tests were two-sided tests, and p ≤ 0.05 or p ≤ 0.01 was considered statistically significant.

The bioactive components of XHP were determined as those screened with oral bioavailability (OB) ≥30% and drug likeness (DL) ≥0.18 as the limiting conditions on the TCMSP and BATMAN-TCM databases. Twenty-seven bioactive components (ellagic acid, pelargonidin, quercetin, etc.) of XHP were obtained by the software of Cytoscape 3.7.1, and 206 targets (without duplication) of the active components were acquired to construct the active component target interaction network. The bioactive ingredients were represented by the orange nodes and the potential targets of bioactive ingredients were represented by the green nodes. Degree indicated the number of routes connecting nodes in the network. The active ingredients with high degree value play an important role in the pharmacological function of XHP. The top three MOL 000098 (quercetin) degree was 77; MOL000358 [β-sitosterol, the degree of sitosterol was 16; MOL 000449 (stigmasterol) degree was 13] (Figure 1A).

Sixty-nine nodes (40 targets, 27 active ingredients, 1 disease, and 1 drug) and 147 edges were obtained in the active ingredient prostate cancer target interaction network diagram by the Cytoscape 3.7.1 software. The red, light blue, green, and light red nodes represented the XHP, prostate cancer, active ingredients of XHP, and the common target of active ingredients of XHP and prostate cancer, respectively (Figure 1B).

Four hundred eighty-five therapeutic targets of prostate cancer were obtained by setting the correlation score ≥17.0 and deleting the repeated targets on gene cards and OMIM database; 485 targets for prostate cancer and 85 targets (including repetition) for drug active ingredients were screened out by R language software and Perl language program. A total of 40 targets were input into the Venny 2.1 software. The Venny diagram obtained was shown in Figure 1C. The string data platform was employed to input 40 common targets, and the PPI was constructed in the mode of “high confidence: 0.7” (Figure 1D). The top 30 protein targets with high frequency were CCND1, EGFR, ESR1, and MYC, which can be used as the potential targets of XHP in the treatment of prostate cancer.

R language software was used to analyze the go function of the above common targets. The bubble diagram and lollipop diagram are shown in Figures 1E,F, respectively. The biological processes were mainly related to ubiquitination of protein bodies, heterodimerization activity of proteins, and activity of transcription factors. XHP may regulate multiple complex biological processes to treat prostate cancer. The signal pathways with more than five enriched targets are listed in Table 2. Forty common targets are mainly distributed in PI3K-Akt, MAPK, AGE-RAGE, HIF-1, estrogens, and other signaling pathways, which suggests that XHP can treat prostate cancer by acting on multiple signaling pathways. The potential target of XHP in the treatment of prostate cancer based on the PI3K-Akt signaling pathway is shown in Figure 2.

According to the results of the network pharmacology, the potential mechanism of XHP to treat prostate cancer was via PI3K/Akt/mTOR signaling pathway. Next, the ultraperformance liquid chromatography coupled with quadruple time-of-ight mass spectrometry assay was used to control the quality of XHP. Finally, the experiments in vitro and in vivo were employed to verify the mechanism of XHP to treat prostate cancer (Figure 3).

Qualitative analysis of components XHP was performed by the LC-MS/MS method. Chromatograms of total ion in ESI positive and negative-ion mode for XHP are shown in Figures 4A,B. We preliminarily identified 37 compounds in this extract. They were D-9-AnthrylaAlanine,Trimethylolpropane triacrylate, Subaphyllin, 1,3,4-Oxadiazol-2(3H)-one, Octylacetate, Curzerenone, Ethyl2-acetamido-2-deoxyhexopyranoside, rel-1S, 2S-epoxy-3R-methoxy-4R-furanogermacr-10(15)-en-6-one, palmitic acid, 1,3,4-Oxadiazol-2(3H)-one, Taurocholic acid, Ricinoleic acid, Abietic acid, Ethylenediamine Diaceturate, Rhodomollein III, Myrrhone, glycocholic acid, Decyl acetate, Lauric Acid Isobutyl Ester, Myrrhone, 9,10-Dihydroxystearic acid, taurodeoxycholic acid, Cholic acid, Glycochenodeoxycholic acid, 1-Octanol, Oleic acid, Abietic acid, Fritillebic acid, Broussonin B, [2-(2-phenylethyl)phenyl]methanol, Eicosanetetraenoic acid, curzerenone, Myrrhone, 11-Oxo-β-boswellic acid, Diacetoxytetrahydroxytaxadiene, Anacardic acid B, and Acetyl-11-keto-β-boswellic acid, as shown in Table 3.

In order to explore the inhibitory effect of XHP on castration-resistant prostate cancer and hormone-sensitive prostate cancer, different concentrations of XHP [5, 2.5, 1.25, and 0.625 mg/ml (crude drug)] were used to treat PC3 cells (castration-resistant prostate cancer) and LNCaP cells (hormone-sensitive prostate cancer) for 6, 12, 24, and 48 h, respectively. CCK8 experiments were used to verify the inhibitory effect of XHP on different prostate cancer cells. As shown in Figures 5A,B, the concentration and time of XHP to reach IC₅₀ for different types of prostate cancer cells are different. In this study, the concentration IC50 of XHP on prostate cancer PC3 cells was 3250ug/ml at 48 h. The 24 h IC₅₀ concentration of XHP on prostate cancer LNCaP cells was 1100 µg/ml. The results showed that XHP had a well inhibitory effect on hormone-sensitive prostate cancer cells in a short period time, and the enhancement time also had an ideal inhibitory effect on PC3 cells. As shown in Figures 6A-C, the XHP exhibited cytotoxicity against PC3 and LNCaP cells in manners of dose and time-dependence.

The Annexin V-APC staining assays, the Canto II flow cytometer, and the trans-well invasion assays were performed to analyze the apoptotic, cell cycle, and invasiveness features of PC3 cells treated with XHP for 24 h. As shown in Figure 6D, the apoptosis rate of the XHP group was significantly increased, compared with the control group; the p value (0.004) was calculated by Kruskal-Wallis test (n = 3) (p < 0.01). As shown in Figure 6E, XHP could block cell cycle from G1 phase to S phase, and the difference was statistically significant compared with the control group; all the p values (0.000) of different groups were calculated as by t-test (n = 3) (p < 0.01). As shown in Figure 6F, XHP could restrain the invasiveness of PC3 cells and the difference was statistically significant compared with the control group; the p value (0.004) was calculated by Kruskal–Wallis test (n = 3) (p < 0.01).

According to the above cell phenotype experiments, XHP can significantly promote cell apoptosis, block cell cycle, and inhibit cell invasion. In order to further verify the mechanism of XHP to treat the prostate cancer, the molecular mechanism experiments were adopted. The PC3 cells were treated with 1.25 mg/ml and XHP 5 mg/ml of XHP in 24 h, respectively; the blank serum group and 3-MA group were adopted as the control groups. As shown in Figures 6G,H, the HDXHP extracting solution restrained the p-PI3K, p-Akt, p-mTOR, PCAN protein, PI3K, Akt, and mTOR mRNA of PC3 cells significantly, compared to the control, LDXHP, and 3-MA groups (one-way ANOVA test and t-test) (p < 0.05). This study suggests that XHP can inhibit the activity of prostate cancer cells and affect the molecular mechanism of intercellular communication.

In order to assess whether XHP inhibited the tumor growth in vivo, xenograft mouse models were established by subcutaneous transplantation injection of PC3 cells into BALB/C male nude mice. As shown in Figures 7A–C, daily treatment of XHP at the concentration of (78 mg powder/kg/day) for 2 weeks could significantly inhibit the tumor growth in subcutaneous transplantation of prostate cancer PC3 cells (p = 0.03) (t-test). There was no significant change in body weight between the control group and XHP group (p > 0.05) (t-test). The results showed that XHP had satisfactory inhibitory effect on prostate cancer in vivo.

In order to observe the effect of XHP on the pathological tissue of subcutaneous transplantation of prostate cancer, the HE staining microscope was used to evaluate the changes of pathological tissue. The results showed that the tumor cells in the control group had irregular morphology, large and highly stained nuclei, more mitotic images, less cytoplasm, imbalance of nucleo-plasma ratio, unclear cell membrane, less stromal components, and hematopoietic processes between the beam and cable. As shown in Figure 7D, XHP could suppress the staining of tumor nucleus and the distribution of blood vessels in the prostate cancer stroma.

In order to evaluate the effects of XHP on the microenvironment of blood vessels and stroma in the subcutaneous transplantation of prostate cancer, the subcutaneous transplantation tissues were taken for immunofluorescence to observe vascular-related indicators VEGFR and αSMA. As shown in Figure 7E, XHP could inhibit the VEGFR level and increased αSMA level of subcutaneous transplantation of PC3 cells.

In order to further verify the mechanism of XHP to treat the prostate cancer, the vivo experiments were adopted. The PC3 subcutaneous transplantation mice were treated with XHP at 78 mg/kg per day for 2 weeks. As shown in Figures 7F,G, XHP could activate the cleave caspase-3 and caspase-9 protein and mRNA of the PC3 prostate cancer subcutaneous transplantation significantly, compared to the control group, p = 0.002. Moreover, as shown in Figures 7H,I, XHP could suppress the Akt (p = 0.024), p-PI3K(p = 0.000), p-Akt (p = 0.000), and p-mTOR (p = 0.000) protein of the PC3 prostate cancer subcutaneous transplantation significantly (p < 0.05) (t-test). These results confirm that XHP can promote apoptosis of prostate cancer cells and is related to the PI3K/Akt/mTOR signaling pathway.