Sodium butyrate and tributyrin induce in vivo growth inhibition and apoptosis in human prostate cancer

16 Nov.,2022


what is tributyrin

Cell culture and reagents

The LNCaP, PC3 and Tsu-Pr1 cell lines were obtained from American Type Culture Collection (Rockwell, MD, USA). All cell lines were cultured and maintained in 5% CO2 at 37°C in RPMI 1640 (Life Technologies, Eggenstein, Germany), supplemented with 10% heat-inactivated foetal calf serum, 2 mM L-glutamine, 100 U ml−1 penicillin G and 100 U ml−1 streptomycin (Life Technologies, Eggenstein, Germany). Sodium butyrate and tributyrin were obtained from Sigma-Aldrich Chemie GmbH (Deisenhofen, Germany). A stock solution of tributyrin was prepared using 100% ethanol. Sodium butyrate was dissolved in sterile water.

Growth inhibition in cell culture

For determining cell proliferation, the viable cell numbers were counted using the Cell Proliferation Kit II (Roche Diagnostics GmbH, Mannheim, Germany) based on the XTT assay (Roehm et al, 1991). In brief, cells were grown in microtitre plates to 80% confluency. Cells were incubated with the given final concentration of the butyrates for 72 h. After incubation, 50 μl of XTT labelling mixture was added to each well. The microtitre plate was incubated for another 4 h before measuring absorbance at 492 nm (reference wavelength 690 nm) of the samples using a microtitre plate reader (SLT spectra, Tecan GmbH, Crailsheim, Germany).

Chorioallantois membrane

Preparation and procedure of grafting

Fertilised chicken eggs were incubated at 37.8°C and 60% relative humidity. On day 4 of incubation, implantation was prepared. Standard microbiology testing was used to rule out subclinical infections. The procedure of preparing the CAM was carried out as described previously (Kunzi-Rapp et al, 2001). Briefly, a part of the CAM was exposed by peeling a round aperture of 2 cm diameter. The resulting window was covered with a self-adhesive stripe and incubation was continued. At day 7 of incubation, a silicone ring with a thickness of 0.5 mm and an inner diameter of 6 mm was placed onto the membrane. The cells were seeded into the ring at a concentration of 5 × 105 cells in 25 μl serum-free RPMI 1640. Tumour growth and viability of the embryo were controlled daily by stereo microscopy.

In vivo apoptosis induction

Tumours were established as described 3 days after the inoculation of tumours on the CAM, sodium butyrate or tributyrin was administered intravenously (i.v.) into the CAM vessels using a 0.3 × 13 mm needle in a total volume of 100 μl. The final in vivo concentration was calculated for a total blood volume of 2 cm3 and varied from 0.1 to 5.0 mM for tributyrin and sodium butyrate. At 48 h after application, the tumour, together with the CAM, was sampled for immunohistochemistry and apoptosis detection.

Quantitative assessment of apoptosis in implanted tumours

The detection of apoptotic cells was performed as described previously (Maier et al, 2000). Tumours were sampled 5 days after seeding and embedded in paraffin. Sections of 3 μm were mounted on poly-L-lysine-coated slides. After deparaffinising and rehydration, slides were washed twice in PBS and treated with 0.1% H2O2 in PBS for 15 min. After repeated washing in dH2O and TdT buffer (30 mM Tris, pH 7.2, 140 mM sodium cacodylate, 1 mM cobalt chloride), slides were incubated with TdT–Biotin–dUTP mix (100 μl TdT buffer, 30 U TdT, 0.5 μl Biotin–dUTP; Boehringer Mannheim, Indianapolis, IN, USA) for 1 h at 37°C in a humid chamber. The reaction was stopped and unspecific binding was blocked by incubation in 2% BSA for 10 min at room temperature. Slides were incubated with a secondary antibody (ABC Vectastain, Vector Laboratories, Inc., Burlingame, CA, USA) for 30 min in a humid chamber at room temperature. Slides were washed and stained with 3-amino-9-ethylcarbazol (AEC). After washing in dH2O, slides were counterstained with haematoxylin, dehydrated and mounted. For determination of apoptosis, 500 cells were evaluated in representative fields at × 400 magnification, and apoptotic cells were calculated in percent of the total number of counted cells.

Growth of cells in nude mice and procedure of treatment

Evaluation of drug-induced effects in the mouse model was performed using fast growing, hormone-independent PC3 and TSU-PR1 cells. Cells were maintained in culture as described and found to be free of mycoplasma contamination. Cells were harvested from tissue culture flasks after reaching semiconfluence. Before injection, cells were washed and resuspended in Dulbecco's phosphate-buffered saline (PBS). In all, 105 cells were transplanted on both sides subcutaneously through 0.4 × 19 mm needles in projection of the scapulae of 7-week-old NMR/−nu/nu male mice with a mean weight of 30 g (Bomholtgard, Denmark). For each treatment protocol, six mice were used resulting in 12 tumours per setting. For intraperitoneal application, both sodium butyrate and tributyrin were dissolved in sterile saline. Treatment was started 24 h after implantation of the cells. During the first week, drug administration was performed on a daily basis for a calculated final plasma concentration of 10 mM. As the mice were estimated with a 2.4 ml blood volume, 24 mg of sodium butyrate and 7.9 mg of tributyrin were used (Egorin et al, 1999). From week 2 until the end of week 4, when the experiment was stopped, application was carried out every second day. As control, a mock-treated group with mice growing xenograft tumours and getting normal saline injections was observed. Tumour size was measured once a week and tumour volume was calculated according to the formula 1/2 × L × W × H in millimetres (Tomayko and Reynolds, 1989). The weight of all mice was measured weekly. All animal experiments were carried out with ethical committee approval (#631, Regierungspräsidium Tübingen, Germany) and met the standards as defined by the UKCCCR guidelines (Workman et al, 1998).

Immunohistochemistry and quantitative assessment of KI-67 protein expression

Detection of the nuclear protein Ki-67 has been chosen, as it is preferentially expressed during the active phases of the cell cycle, but not in the G0-phase. Ki-67 is an accepted means for the detection of proliferating cells in paraffin-embedded tissue sections (Cattoretti et al, 1992; Gerdes et al, 1992; Mucci et al, 2000), and its expression has been shown to be associated with poor outcome in prostate cancer patients (Borre et al, 1998). Standard avidin–biotin complex immunohistochemistry was used for staining the animal tumours. Pretreatment was performed by exposure for 10 min in sodium citrate buffer in a microwave oven. The slides were then incubated sequentially with primary antibody (1 : 100 dilution, monoclonal mouse anti-human-Ki-67 antibody, DAKO Diagnostika, Hamburg, Germany), biotinylated secondary antibody, avidin–biotin complex and chromogenic substrate 3,3′-diaminobenzidine. Slides were evaluated for adequacy using a standard bright field microscope. Digital images were acquired and protein expression was scored as positive or negative using the MDS 5.8 system from Applied Imaging Corporation (Santa Clara, CA, USA). For objective evaluation, eight representative areas were defined. In the case of KI-67 protein expression, the system was optimised for nuclear staining, calculating the percentage of stained nuclei. Data were transferred to a spreadsheet for subsequent analysis.

Western blotting

The expression of proteins was determined by Western blot analysis using specific primary antibodies for p21WAF1, Rb and c-myc (Oncogene, Cambridge, USA). Briefly, cells were lysed using RIPA solution (1% nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 μg ml−1 phenylmethylsulphonylfluoride, 30 μl ml−1 aprotinin, 50 μg ml−1 leupeptin, in PBS, pH 7.4; chemicals were purchased from Sigma, Steinheim, Germany) at 4°C for 10 min. Lysates were centrifuged and the protein concentration of the supernatant was determined using the Bradford assay. Laemmli sample buffer at a ratio of 1 : 2 was mixed to the sample. Following electrophoresis, the proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad, Munich, Germany). Blocking was carried out with freshly prepared TBST plus 10% nonfat milk (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween-20). After washing, the membrane was incubated for 1 h with primary antibodies diluted in TBST (100–1.500-fold). Appropriate horseradish peroxidase-conjugated secondary antibodies (Amersham, Buckinghamshire, UK) were incubated overnight. For detection, autoradiography using ECL was performed according to the manufacturer's instructions (Amersham, Buckinghamshire, UK).

Statistical analysis

Treatment effect was statistically evaluated using the mean score result (i.e. percentage of stained nuclei) for each treatment group (i.e., control, sodium butyrate, tributyrin or various concentrations). To test for significant differences the one-way ANOVA test was performed. To determine the differences between all pairs, post hoc analysis using the Scheffé method was applied. Values are presented in a graphical format using error bars with 95% confidence intervals (CIs). P-values <0.05 were considered statistically significant.