Brain-derived neurotrophic factor involved epigenetic repression of UGT2B7 in colorectal carcinoma: A mechanism to alter morphine glucuronidation in tumor

Uridine diphosphate-glucuronosyltransferase (UGT) 2B7, as one of significant drug enzymes, is responsible on the glucuronidation of abundant endobiotics or xenobiotics. We here report that it is markedly repressed in the tumor tissues of colorectal carcinoma (CRC) patients. Accordingly, morphine in CRC cells will stimulate the expression of its main metabolic enzyme, UGT2B7 during tolerance generation by activating the positive signals in histone 3, especially for trimethylated lysine 27 (H3K4Me3) and acetylated lysine 4 (H3K27Ac). Further study reveals that brain-derived neutrophilic factor (BDNF), a secretory neurotrophin, enriched in CRC can interact and inhibit UGT2B7 by primarily blocking the positive signals of H3K4Me3 as well as activating H3K27Ac on the promoter region of UGT2B7. Meanwhile, BDNF repression attributes to the sensitizations of main core factors in poly-comb repressive complex (PRC) 1 rather than PRC2 as the reason of the depression of SUZ12 in the later complex. Besides that, the productions of two main morphine glucuronides are both increased in the BDNF deficient or TSA and BIX-01294 treated morphine tolerance-like HCT-116 cells. On the same condition, active metabolite, morphine-6-glucuronide (M6G) was accumulated more than inactive M3G. Our findings imply that enzymatic activity enhancement and substrate regioselective catalysis alteration of UGT2B7 may release morphine tolerance under the cure of tumor-induced pain.


Cell cultivation and compounds incubation
All the human cell lines were cultured at an atmosphere of 5% CO 2 and 95% air at 37°C and passaged by 0.25% EDTA mixed trypsin. HCT-116, HCT-15, HT-29 cell lines were cultivated by McCoy's 5A; NCM460 (human normal colorectal cell line), LoVo, SW480, SW620, Caco-2 and HEK293 were all cultivated by Dulbecco's Modified Eagle's Medium (DMEM). Each medium was added 10% fetal bovine serum (FBS), 1% penicillin, streptomycin. After the cells growing to 70% convergence degree, the drugs or chemical compounds can be added. In brief, before they were diluted with DMSO and added into DMEM medium, all the chemical compounds were incubated for a period of indicated time. Next, we changed the old medium to fresh one containing different concentrations of compounds every 24h. Same volumes of DMSO (less than 0.1%) were treated the cells as control.

DNA plasmids and siRNA transfection
HCT-116 cell line was primary seeded in 6-well plates at a density of 0.5×10 6 per well and pre-cultured to 60% confluence. Then, 2μg DNA plasmid mixed with P3000 reagent or 400nM siRNA were treated in nonserum McCoy's 5A medium combining with the reagent of Lipofectamine 3000 and incubated 5min at room temperature. After that, all the mixture solutions were subjected to the cells. Experiencing 5.5 hours' incubation, the serum-free medium was changed by complete medium including different chemical compounds and culturing for another 48h (transfer the medium again at 24h). Then all the cells can be harvested for further experiment.

Luciferase assay
HEK293 cell line was first seeded in 24-well plate at a density of 0.5×10 5 /well and pre-cultured to 60% confluence. In each hole of plate, we transfected 500ng expression plasmid, 250ng report gene plasmid and 50ng pRL-TK SV40 enhancer plasmid based on the methods we referred to previously [28]. Then we added different chemical compounds in complete medium after 5.5 hours` incubation, and repeated this operation every 24h. After 48 hours` cultivation, the cells were harvested and subjected to the luciferase assay using chemiluminescence apparatus (Promega GloMax 96) combing with dual-luciferase reporter mode in its system according to the manuscript protocol. Relative light unit (RLU) value in each sample was normalized to the ratio of firefly luciferase activity from the promoter of UGT2B7 to renilla luciferase.

Chromatin immunoprecipitation (ChIP) assay
ChIP assay was carried out as the procedures provided by the manufacturer of kit. The breakage of UGT2B7 genome by Bioruptor UCD200 ultrasonic cell disruptor (Belgium) was primary decided as sizes 400-800bp through agarose gel electrophoresis (AGE). Then all the samples were all repeated this ultrasound method again for next immunoprecipitation and purification assays. Realtime-PCR was applied to do a further analysis. The enrichment of each histone modification signal was indicated as percentage of input. Pulling down the protein IgG was used as negative control to normalize other signals` alteration.

Ethics
All the human adjacent normal and CRC tumor tissues were collected from the Specimen Bank of Zhejiang Cancer Hospital (Hangzhou, China) and strictly conducted in accordance with the protocols approved by the Institutional Review Board of Zhejiang Cancer Hospital. The paperwork was according to the documentation of 'The Detailed Rules and Regulations of Medical Animal Experiments Administration and Implementation' (Document No. 1998-55, Ministry of Public Health, China).

Reverse transcription-PCR (RT-PCR) and quantitative realtime-PCR
Total RNA was extracted from human tissues or cells and purified by kit. Then RNA was reverse transcribed to cDNA via PrimeScript RT Master Mix (Takara). After each sample was mixed with SYBR Premix EX Taq (Takara), the quantitative realtime PCR was performed to determine each target gene expression via specific primers (Supplementary Table 3). The relative expression of transcripts was quantified and normalized to the expression of housekeeping gene of PPIA for tissues and GAPDH for cells.

Co-immunoprecipitation (Co-IP) and western blotting assay
The Co-IP assay was performed following the manual of the kit. In brief, we extracted the total proteins from the cell lysates containing RIPA and 1×PMSF reagents, then added 0.7mL cell lysate solution with specific antibodies and Protein A/G Plus Agarose (kit, Beyotime) to rotate overnight. After eluting by Co-IP buffer several times and mixing with protein loading buffer supplied in the kit, all the samples were subjected to SDS-PAGE. For western blotting assay, 10-50μg protein samples measured by BCA assay in different tests were subjected for running the electrophoresis. PVDF membranes were used to transfer the proteins from gels for 2h, 200mA. Soon after that, the blots were merged in blocking solution containing 5% non-fat milk in TBST buffer (100mM Tris-HCl, pH7.4, 150mM NaCl and 0.1% Tween 20) for 1h at room temperature. Following, primary antibodies including UGT2B7(1:1000), BDNF (1:2000), SUZ12 (1:2000), GAPDH (1:2000), μ-opioid receptor (MOR, 1:500) were mixed with TBST and incubated to the blots overnight at 4°C. After washing three times to remove the primary antibody, the HRP-conjugated goat anti-rabbit or anti-mouse IgG (H+L) (1:2000) was used as secondary antibody for detection by ECL system. Target proteins were visualized to expose the membranes through G-box chemiluminescence imaging (Syngene). GAPDH was paralleled as an internal reference to control the expression of different target proteins.

Immunohistochemistry (IHC) assay
Normal or tumor tissues of CRC patients were fixed in 4% paraformaldehyde for 12h then preserved in 30% cold sucrose mixed with PBS solution overnight. Each section of tissue (100μm) were cut by freezing microtome and immerged in 0.3% H 2 O 2 -PBS solution for eliminating the endogenous peroxidase. Soon after that, they were incubated in 10% goat serum (Santa Cruz, Los Angeles, CA, USA) in PBS, and primary UGT2B7 rabbit polyclonal antibodies (diluted with 1:1000) overnight at 4°C before HRP-conjugated secondary goat anti-rabbit polyclonal antibody (1:2000) incubated for 1h. Following, they were treated with a peroxidase substrate solution, 3, 3'-diaminobenzidine tetra hydrochloride (DAB; Vector Laboratories) before counterstaining reagent, hematoxylin (Sigma, Sigma-Aldrich, Shanghai, China) were applied. The prepared slides were observed with a 400× of magnification under an Olympus BX41 microscope to measure the expression of UGT2B7. Negative control group without primary antibody was carried out with the same procedure as described above in the preliminary experiments.

Cell immunofluorescence assay
Cells were cultured on the glass slides of 6-wells and transfected with siRNAs or DNA plasmids as previously referred. Then, they were fixed by stationary liquid at 4°C overnight. Before thrice cleanings by PBST (1%Triton X-100 mixed with PBS, pH7.4), the glass slides were immerged into 5% non-fat milk in TBST buffer (100mM Tris-HCl, pH7.4, 150mM NaCl and 0.1% Tween 20) for 1h at room temperature. Following by incubating with BDNF or SUZ12 primary rabbit polyclonal antibody (1:100) TBST buffer for 4°C overnight and thrice PBST washes, Alexa Fluor 488 goat anti-rabbit IgG (H+L) antibody was then used to incubate the slides for 1h at room temperature. Finally, each target gene expression was examined under fluorescence microscope at 400× magnification. Nuclei were visualized and located by staining the dye of 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, Life Company).

Glucuronidation assay
Cells were primarily treated with 1μM morphine for 48h to induce cell tolerance status then transfected with siRNAs or treated with different chemical compounds at 80% confluence. We lysed the cells and extracted the total proteins. According to the protocols published previously [41], all the proteins were quantified by BCA assay and extracted 0.1mg/ml to incubate with 500μM morphine. 2μl HClO 4 (70%, v/v) was used to terminate the reaction. After that, acetonitrile was added to precipitate the protein in the samples at a ratio of 2:1 of solvent. After centrifuging at 13,000g for 10 min, the supernatant was separated. M6G-d3 (internal standard) was added into the supernatant with a final concentration of 10ng/ml in each sample. Next, the collected liquid was evaporated and dried in the centrifugal thickener (LABCONCO Company) for HPLC-MS/MS determination.

HPLC-MS/MS determination
The HPLC-MS/MS method for determining the concentration ratios of M3G and M6G was established previously [21]. Based on that, we acquired the data of morphine glucuronidation and made further estimation to the enzymatic activity and substrate regioselectivity of UGT2B7.