The prostate cancer cell lines, PC-3, LNCaP and 22RV1, were purchased from the Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences, Shanghai, China). C4-2 cells were a gift from Dr Guo-wen Lin (Fudan University Shanghai Cancer Center, Shanghai, China). The normal human prostate epithelial cell line, RWPE-1, was purchased from the American Type Culture Collection (Manassas, VA, USA). PC-3, LNCaP, C4-2 and 22RV1 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (all from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). RWPE-1 cells were cultured in keratinocyte serum-free medium (K-SFM; cat. no. 17005-042; Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 0.05 mg/ml bovine pituitary extract (BPE), 5 ng/ml epidermal growth factor (EGF; both from K-SFM medium kit; cat. no. 17005-042; Invitrogen; Thermo Fisher Scientific, Inc), according to the manufacturer's protocols, and 1% of penicillin-streptomycin (Gibco). All media was changed every 2–3 days, and the cells were passaged at 80–90% confluency. The prostate cancer tissue array was purchased from Biomax USA (US Biomax, Inc., Derwood, MD, USA).

Cell Counting kit-8 (CCK-8) assays were performed by plating 5×10³ LNCaP cells per well in a 96 well plate (Thermo Fisher Scientific, Inc.). The following day, cells were treated with 0, 10, 100 nM, and 1, 5, 10 µM of R-(a)-methylhistamine (RAMH; cat. no. B5010; Apexbio, Houston, TX, USA) and 0, 10, 100 nM and 1, 5, 10 µM ciproxifan (CPX; cat. no. S2813; Selleck, Houston, TX, USA). The cells were exposed to the drugs for 1–2 days prior to the addition of CCK-8 reagent (cat. no. 40203ES60; Yeasen; Shanghai Yi San Biotechnology Co., Ltd., Shanghai, China) for 2 h, according to the manufacturer's protocol, and the absorbance of each well was detected using a microplate reader (Thermo Fisher Scientific, Inc) at a wavelength of 450 nm.

Transient knockdown was performed using siRNA targeting H3R (Ruibobio, Guangzhou, Guangdong, China) and non-targeting siRNA as a negative control. The siRNA sequences were as follows: si-H3R, 5′-GCTATGCCGAGTTCTTCTACA-3′, and negative control, 5′-TTCTCCGAACGTGTCACGT-3′. In brief, LNCaP cells (5×10⁴ in 24-well plate) were transfected with siRNA (both 20 pmol in 500 µl/well) with Lipofectamine 2000 (1 µl in 500 µl/well, Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The LNCaP cells used for transplant were transfected with si-H3R-packaged lentivirus (MOI=20) and selected using puromycin (2 µg/ml for 10 days) (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) according to the manufacturer's protocol.

The upper chamber of a 24-well plate (Corning Incorporated, Corning, NY, USA) was pre-coated with 50 µl Matrigel (BD Biosciences, Franklin Lake, NJ, USA) for the invasion assay. The following steps were the same for the two assays. A total of 1×10⁵ LNCaP cells were suspended in 200 µl serum-free RPMI-1640 medium and seeded into the upper chamber of a 24-well plate. A total of 900 µl RPMI-1640 medium supplemented with 20% FBS was added to lower chamber of each well. After 24 h, the cells were fixed using 4% paraformaldehyde for 30 min at room temperature (Mairel; Shanghai Miner Chemical Technology Co., Ltd., Shanghai, China) and stained with 0.1% crystal violet (Mairel; Shanghai Miner Chemical Technology Co., Ltd) for 30 min at room temperature. Cells on the upper surface of the Transwell insert were removed with a cotton swab and cells on the lower surface were counted under a light microscope (Olympus Corporation, Tokyo, Japan) in three fields of view (magnification, ×100).

Measurement of in vitro apoptosis was performed by Annexin V-FITC/propidium iodide (PI) staining Apoptosis Detection kit (Yeasen; Shanghai Yi San Biotechnology Co., Ltd.), according to the manufacturer's instructions, using a flow cytometer (Beckman Coulter, Inc., Brea, CA, USA).

Total RNA was extracted using a Takara RNeasy Mini kit (cat. no. 9767; Takara Bio, Inc., Otsu, Japan), according to manufacturer's instructions. RNA was transcribed into cDNA using a PrimeScript RT reagent kit (cat. no. RR036A; Takara Bio, Inc.). A Takara qPCR kit (cat. no. RR420Q; Takara Bio, Inc.) was used to assess H3R gene expression using an ABI 7500 real-time system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 30 sec at 95°C, 5 sec at 95°C, 34 sec at 60°C and 40 cycles of 15 sec at 95°C, 60 sec at 60°C, and 15 sec at 95°C. Relative gene expression was normalized to that of GAPDH. Gene expression was quantified using the 2^−ΔΔCq method (15). The primer sequences used were as follows: H3R forward, 5′-GCCACTGCTATGCCGAGTT-3′ and reverse, 5′-TGCGCCTCTGGATGTTCAG-3′; GAPDH forward, 5′-TTTACCTTCCAGCAGCCCTA-3′ and reverse, 5′-GACAGAGTCCCAGATGAGCA-3′.

Total protein was extracted with radioimmunoprecipitation assay (RIPA) buffer (Beyotime Institute of Biotechnology, Haimen, China) adding 1 mM protease inhibitor cocktail (Beyotime Institute of Biotechnology) at 4°C between 30 min and 1 h then concentrate under 12,000 rpm at 4°C for 15 min. Total protein concentration was determined using a bicinchoninic acid (BCA) kit (cat. no. P0010; Beyotime Institute of Biotechnology, Haimen, China). The protein (30 µg per lane) samples were resolved by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with 5% non-fat milk for 1 h at room temperature. The membranes were probed with the following primary antibodies overnight at 4°C: H3R (dilution, 1:5,000; cat. no. ab124732; Abcam, Cambridge, UK), BAX (BCL-2 associated X, apoptosis regulator; dilution, 1:1,000; cat. no. D2E11), BCL2 (dilution, 1:1,000; cat. no. D17C4; Cell Signaling Technology, Inc., Danvers, MA, USA), AR (dilution, 1:500; cat. no. sc-7305; Santa Cruz Biotechnology, Dallas, TX, USA), GAPDH (dilution, 1:5,000; cat. no. 70-Mab5465-040; Multi Sciences; Hangzhou Lianke Biotechnology Co., Ltd., Hangzhou, China). The membranes were then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody: Anti-rabbit IgG (dilution, 1:1,000; cat. no. 7074) or anti-mouse IgG (dilution, 1:1,000; cat. no. 7076; both Cell Signaling Technology, Inc.) at room temperature for 1 h. The two secondary antibodies were diluted at 1:1,000 and were purchased from Cell Signaling Technology, Inc. The blots were visualized using ECL-Plus reagent (cat. no. WBKLS0010; Merck Millipore; Merck KGaA, Darmstadt, Germany). Images were acquired with the ImageQuant LAS 4000mini (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) and analyzed with ImageJ 2.1.4.7 software (National Institutes of Health, Bethesda, MD, USA). The results were expressed as the target protein/GAPDH ratio and subsequently normalized to the values measured in the control groups.

Prior to immunohistochemistry, the tissue array and the xenograft tumor slides were placed in a washing solution of 3% hydrogen peroxide (H₂O₂) and 60% methanol phosphate buffered saline (PBS) (PH 7.4) for 30 min and subsequently treated with 0.01 mol/l sodium citrate buffer for 95°C in a microwave oven for 15 min for antigen retrieval. The tissue array and the xenograft tumor slides were subsequently blocked in 5% normal goat serum and 5% bovine serum albumin (Yeasen; Shanghai Yi San Biotechnology Co., Ltd) in PBS for 15 min at 37°C. Prior to each step, the tissue array and the xenograft tumor slides were rinsed three times in PBS buffer. The array was incubated with a H3R primary antibody (dilution, 1:100; cat. no. ab124732; Abcam) and the xenograft tumor slides with Ki67 (dilution, 1:400; cat. no. 9449; Cell Signaling Technology, Inc.) and TUNEL (dilution, 1:500; cat. no. ab206386; Abcam) each for 12 h at 4°C. Subsequent to rinsing with PBS, the sections were incubated with a secondary horseradish peroxidase-conjugated antibody anti-rabbit IgG (dilution, 1:200; cat. no. 7074; Cell Signaling Technology, Inc) for 1 h at room temperature. 0.05% diaminobenzidine (Beyotime Institute of Biotechnology) exposure was performed using chromogen at room temperature about 5 min. Finally, the array and the xenograft tumor slide were counterstained with 0.025% hematoxylin (Beyotime Institute of Biotechnology) at room temperature for ~30 sec.

All animal procedures were performed according to the National Animal Experimentation guidelines and were approved by the ethics committee of the Shanghai Jiao Tong University (Shanghai, China). Male 4–8 week-old nude mice weighing 20–25 g were used in the following experiments. A total of 8 mice were divided equally and randomly into 2 groups. All mice were bred in aseptic conditions at a constant humidity (~40–70%) between 20 and 26°C with standard 12 h light-dark cycles and free access to food and water. For tumor xenograft studies, 5×10⁶ LNCaP cells were suspended in 0.1 ml serum-free media containing 50% Matrigel, and injected subcutaneously into the oxter of the mice, which were then observed every three days. Once the tumors reached 1 cm in diameter, the mice were sacrificed and the tumor volumes were calculated using the following formula: 0.5 × length (mm) × width² (mm²). The tumors were fixed in 10% formalin at 4°C between 24 and 48 h, and embedded in paraffin for incubation with the aforementioned primary antibodies, Ki67 and TUNEL.

SPSS 22.0 (IBM Corp., Armonk, NY, USA) and Graph Pad Prism (version 5; Graph Pad Software, Inc., La Jolla, CA, USA) were used to analyze the data and all values are expressed as mean ± standard deviation. One-way analysis of variance followed by the LSD or Dunnett's T3 post-hoc tests was used for multiple comparisons. The Mann-Whitney U test was used to compare differences between H3R expression in prostate cancer and normal tissues. Fisher's exact test was used to assess differences in H3R expression in prostate cancer tissues with different Gleason scores. P<0.05 was considered to indicate a statistically significant differences.

Previous studies have demonstrated that H3R is overexpressed in various types of cancer, however, there are limited reports of the expression of H3R in prostate cancer. In the present study, the tissue array demonstrated positive immunoreactivity of H3R in the majority of cancer tissues but negative immunoreactivity in normal tissues, and the two tissue groups were significantly different (P<0.01) (Fig. 1A-F; Table I). It was demonstrated that the expression of H3R was positively associated with Gleason score (P<0.01; Table II). RT-qPCR analysis demonstrated that H3R mRNA is overexpressed in prostate cancer cells. In LNCaP, 22rv1 and C4-2 cells, the expression of H3R was >20-fold than that of normal prostatic epithelial cells (P<0.01) (Fig. 1G). Western blotting demonstrated the same result at the protein level. All cancer cell lines exhibited higher expression of H3R than normal prostatic epithelial cells (P<0.05). LNCaP cells exhibited the highest expression of H3R (Fig. 1H-I), and were therefore, selected for further experimentation. Thus, the tissue array, RT-qPCR analysis and western blotting all suggested that expression of H3R in prostate cancer is significantly higher than that in normal prostatic epithelial tissues.

In order to reveal the role of H3R in LNCaP cells, a specific H3R agonist (RAMH) and antagonist (CPX) were used. Pretreatment with RAMH promoted the proliferation of LNCaP cells in a dose-dependent manner. In contrast, pretreatment with CPX suppressed proliferation of LNCaP cells in a dose-dependent manner (Fig. 2A-C). In previous studies by our group, it was reported that histamine receptor antagonists may function without a histamine receptor. Therefore, an siRNA was designed to target H3R. Following si-H3R treatment of LNCaP cells (Fig. 2D-F) their proliferation declined by 20% more than that of si-NC LNCaP cells in one day (P<0.01) (Fig. 2G).

Knockdown of H3R expression decreased the migration (P<0.01) (Fig. 3A-C) and invasion (P<0.01) (Fig. 3D-F) of the LNCaP cells compared with si-NC-transfected cells.

Flow cytometric analysis of apoptosis revealed that knockdown of H3R lead to increased apoptosis than control si-NC cells (P<0.01) (Fig. 4E-F and H). Treatment with 10 µM H3R agonist decreased apoptosis whereas treatment with 10 µM H3R antagonist increased apoptosis. When used in conjunction, the effects of both drugs were weakened (Fig. 4A-D and H). These results indicate that knockdown of H3R lead to apoptosis of LNCaP cells.

BCL-2 signaling pathways have been reported to serve a key role in prostate cancer-cell apoptosis (16). The BCL-2 family contains BCL-2, BAX, BAK and BCL-XL. It was demonstrated that knockdown of H3R significantly decreased the level of BCL2 (anti-apoptosis gene) expression (P<0.01), and increased the level of BAX (apoptosis-associated gene) expression (P<0.05). It also confirmed in specific H3R agonist and antagonist assay (Fig. 4).

Numerous pathways have been demonstrated to be involved in the development and progression of prostate cancer, including AR, PI3K/AKT, MYC and PTEN (17–20). AR has been demonstrated to accelerate the proliferation of prostate cancer cells, therefore, in the present study, the expression of AR was investigated following H3R-knockdown. Western blotting revealed that the expression of AR was significantly reduced following knockdown of H3R compared with si-NC-transfected cells (P<0.05). This indicates that the expression of AR is associated with the expression of H3R (Fig. 4G and I). The H3R agonist and antagonist were used in parallel and CPX reduced the expression of AR while RAMH increased the expression of AR (Fig. 4).

To reveal the effect of H3R-knockdown in vivo, a xenograft model was established in nude mouse using si-H3R-transfected LNCaP cells (LNCaP siH3R) and negative control (NC) transfected LNCaP cells (LNCaP si-NC). The length and width of the tumor were measured twice a week. After 8 weeks, the tumors in the LNCaP si-NC group grew to ~1 cm while tumors in the LNCaP si-H3R group only grew to ~0.5 cm, and the difference was significantly different (P<0.05) (Fig. 5A-E). Furthermore, immunohistochemistry revealed that the expression of Ki67 (proliferation marker) was significantly in the LNCaP si-H3R group than the LNCaP si-NC group (P<0.01) (Fig. 5F-H). The expression of TUNEL (apoptosis marker) was significantly increased in the LNCaP si-H3R group compared with the LNCaP si-NC group (P<0.01) (Fig. 5I-K).