Cell lines and reagents
Prostatic cell lines LNCaP and VCaP were purchased from the Cell Bank of the Chinese Academy of Sciences (China). LNCaP cells were maintained in complete Roswell Park Memorial Institute 1640 culture medium (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA) at 37 °C. VCaP cells were maintained in Dulbecco’s modified Eagle’s medium (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA) at 37 °C. NF-kB inhibitor pyrrolidinedithiocarbamate ammonium (PA), c-Myc inhibitor Mycro 3 were purchased from MCM (USA). Docetaxel (Doc), Abiraterone (Abi) and doxorubicin (Dox) were purchased from Sigma (USA). Other chemical reagents or materials were of high-performance liquid chromatography grade and obtained from Biyuntian (China).
Tumor tissues collection
Human prostatic tumor tissue samples were obtained from the Tongji Hospital. The tumor tissues were collected in 10% formalin after surgical operation. Patients were divided into the chemo-sensitive (C-S) and chemo-resistant (C-R) groups according to follow-up visit. The protocols were approved by the ethics board of the Tongji Hospital. Written informed consent was obtained from patients before clinical experiments, and all protocols were designed in accordance with the Declaration of Helsinki.
Cell apoptosis analysis
The cytotoxicity of LNCaP and VCaP cells to agents was determined by the FITC-Annexin V/ PE-PI apoptosis detection kit (Becton and Dickinson Co., USA). Briefly, agents treated tumor cells were resuspended and stained with Annexin V/PI staining solution for 15 min at room temperature. Then cells apoptosis was analyzed on a C6 flow cytometer (Becton and Dickinson Co., USA). Each experiment was repeated for three independent times.
The targeted genes expression of LNCaP and VCaP cells were examined using quantitative real-time PCR. 1μg cDNA was used as template for amplification with SYBRÔ Green Real-Time PCR master mixes (Thermo Fisher Scientific, MA, USA). The primer pairs were used as follow: human ABCB1 forward primer 5′-TTGGCTGATGTTTGTGGGAAG-3′, and reverse primer 5′-CCAAAAATGAGTAGCACGCCT-3′; human ABCB2 forward primer 5′-TGCCCCGCATATTCTCCCT-3′, and reverse primer 5′-CACCTGCGTTTTCGCTCTTG-3′; human ABCG2 forward primer 5′-CAGGTGGAGGCAAATCTTCGT’, and reverse primer 5′-ACCCTGTTAATCCGTTCGTTTT-3′; human ABCC1 forward primer 5′-CTCTATCTCTCCCGACATGACC-3′, and reverse primer 5′-CTGAAGACTGAACTCCCTTCCT-3′; human ABCC3 forward primer 5′-TGGGGTGAAGTTTCGTACTGG-3′, and reverse primer 5′-CACGTTTGACTGAGTTGGTGATA-3′; human ABCC5 forward primer 5′-AGTCCTGGGTATAGAAGTGTGAG-3′, and reverse primer 5′-ATTCCAACGGTCGAGTTCTCC-3′; human ABCC6 forward primer 5′-AAGGAGGTACTAGGTGGGCTT-3′, and reverse primer 5′-CCAGTAGGACCCTTCGAGC-3′; human SLC16A1 forward primer 5′-AGGTCCAGTTGGATACACCCC-3′, and reverse primer 5′-GCATAAGAGAAGCCGATGGAAAT-3′; human SLC22A3 forward primer 5′-ATCGTCAGCGAGTTTGACCTT-3′, and reverse primer 5′-ACCTGTCTGCTGCATAGCCTA-3′; human SLC22A1 forward primer 5′-ACGGTGGCGATCATGTACC-3′, and reverse primer 5′-CCCATTCTTTTGAGCGATGTGG-3′; human SLCEA5 forward primer 5′-GAGCTGCTTATCCGCTTCTTC-3′, and reverse primer 5′-GGGGCGTACCACATGATCC-3′; human SLC7A8 forward primer 5′-AGGCTGGAACTTTCTGAATTACG-3′, and reverse primer 5′-ACATAAGCGACATTGGCAAAGA-3′; human SLC16AE forward primer 5′-CGTGGAGGCTTCTCTCACAG-3′, and reverse primer 5′-CGTAGGACAGCCCGTTTATCG-3′; human GAPDH forward primer 5′-GGAGCGAGATCCCTCCAAAAT-3′, and reverse primer 5′-GGCTGTTGTCATACTTCTCATGG-3′; the GAPDH was set as a control. Each experiment was performed in triple.
LNCaP and VCaP cells were lysed using radioimmunoprecipitation assay buffer (Beyotime, China). Protease inhibitors (Beyotime, China) was added into the lyse buffer to avoid protein degradation. Samples (20 μg) were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Then samples were transferred onto nitrocellulose membranes. After that, samples were incubated with following primary antibodies: anti-AhR (1:500, Abcam, UK), anti-P-gp (1:1000, Abcam, UK), anti-β-actin (1:1000, Abcam, UK) and anti-c-Myc (1800, Abcam, UK). chemiluminescence kit (Beyotime, China) were used for protein detection. Each experiment was repeated for three independent times.
For TDO2 and c-Myc overexpression, cDNAs for human TDO2 and c-Myc were synthesized by Ruibo Inc., China, The TDO2 or c-Myc cDNAs were inserted into pLVX-EF1α-IRES-Puro lentiviral vector (Takara, Japan) for stable overexpression in LNCaP and VCaP cells. The overexpression of TDO2 or c-Myc was examined by western blotting.
Immunohistochemical and immunofluorescence
Pathological sections of prostatic tissues were retrieved using microwave antigen retrieval (Thermo, USA). Then samples were blocked by 5% Bovine Serum Albumin, followed by incubating with anti-TDO2 antibody (1:200, abcam, UK), anti-Kyn antibody (1:300, abcam, UK), anti-AhR antibody (1:200, abcam, UK), anti- NF-κB (1:200, abcam, UK) and anti-c-Myc antibody (1:500, abcam, UK) for 4 °C overnight. For immunofluorescence staining, the sections were then incubated with secondary antibodies (1:1000; abcam, UK), and the nucleus was stained with DAPI. Images were captured using a FV1000 laser scanning confocal microscope (Leica, Germany). For immunohistochemical staining, the sections were then stained using the ABC HRP Kit (Thermo, USA) and counterstaining with hematoxylin. Immunohistochemical images were captured using microscope (Leica, Germany). The intensity of proteins in sections were analyzed by Image-Pro Plus 2.0 software.
The human Kyn Elisa kit was purchased from Keshun (China). The human Trp Elisa kit was purchased from BIOMAT (USA). For Kyn or Trp analysis, 105 tumor cells were cultured in 2 ml medium at 37 °C. After 0 and 48 h, the supernatant was collected for Kyn or Trp concentration analysis according to the guidance of Kit. Each experiment was performed in triple independently.
Female NOD-SCID mice (6 ~ 8 weeks) were purchased from Huafukang Co. (China) and raised in SPF room. All experiments and protocols were approved and monitored by the Animal Care and Use Committee of Tongji Hospital. For tumor suppressive effects, LNCaP cells (2 × 106 cells in 50 μl of PBS) were subcutaneously injected into NOD-SCID mice. After two weeks, the mice were treated with PBS, Doc (5 mg/kg), Abi (10 mg/kg), Mycro-3 (5 mg/kg) or combination every three days. The tumor sizes (n = 6 per group) and survival times (n = 6 per group) of the mice were recorded every day. The tumor volume was calculated according the formula: length × width2 × 0.5.
The survival of patients from TCGA database was downloaded from https://www.cbioportal.org/. Each experiment was performed in triplicate independently. Data are expressed as the mean ± standard deviation. Differences among groups were determined suing variance or Student’s t-test analysis by GraphPad Software (USA) and SPSS software (USA). Survival times were analyzed by a log-rank test. * means p < 0.05, ** means p < 0.01, and n.s means no significant difference.