Sodium butyrate and tributyrin induce in vivo growth inhibition and apoptosis in human prostate cancer - PubMed (original) (raw)

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

R Kuefer et al. Br J Cancer. 2004.

Abstract

Histone deacetylase inhibitors (HDACs) are known to exhibit antiproliferative effects on various carcinoma cells. In this study, the in vivo efficiency of two HDACs, sodium butyrate and tributyrin, on prostate cancer growth inhibition were investigated. To gain an insight into the possible underlying pathways, cell culture experiments were performed focusing on the expression of p21, Rb and c-myc. For in vivo testing, prostate cancer cell lines (PC3 and TSU-Pr1) were seeded on the chorioallantois membrane (CAM) and implanted in a xenograft model using nude mice. Standard Western blot analysis was performed for protein expression of p21, Rb and c-myc in HDAC-treated vs untreated prostate cancer cells. Both sodium butyrate and tributyrin had a considerable treatment effect on microtumours on the chicken egg at already very low concentrations of 0.1 mM. Tributyrin-treated tumours showed the strongest effect with 38% apoptotic nuclei in the prostate cancer cell line PC3. In the mouse model, there was almost no difference between sodium butyrate and tributyrin. In untreated animals the tumours were almost double the size 4 weeks after implantation. Tumours of the treatment groups had a significantly lower percentage of Ki-67-positive-stained nuclei. As demonstrated by Western blot analysis, these effects seem to be independent of p53 status and a pathway via p21-Rb-c-myc is possibly involved. In this study we have demonstrated a substantial in vivo treatment effect, which can be induced by the application of sodium butyrate or the orally applicable tributyrin in human prostate cancer. The given results may provide the rationale to apply these drugs in well-controlled clinical trials in patients being at high risk of recurrence after specific therapy or in patients with locally or distant advanced prostate cancer.

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Figures

Figure 1

Figure 1

Increasing concentrations of sodium butyrate were applied on the human prostate cancer cell line PC3 starting at an initial concentration of 0.5 m

M

up to 5.0 m

M

. Growth inhibition was correlated to absorbance determined with the XTT assay. The control was set to 100%. The percentage of viable cells is given as mean values with standard deviation of repeated experiments.

Figure 2

Figure 2

Prostate cancer microtumours growing on the CAM. (A) In the mock-treated control, only very few cells of the tumour, sitting on top of the CAM, show signs of apoptosis. (B) Strong labelling (red staining) of tumour cells by immunohistochemistry with an anti-human antibody to demonstrate the human origin of the evaluated cells (MNF 116 cytoceratin cocktail (DAKO, Germany). (C) Tributyrin-treated tumours growing on the CAM. TUNEL assay reveals dark brown staining as a correlate to apoptotic changes (magnification × 20). (D) Higher magnification of (C). Nuclei-bound staining by TUNEL assay is indicated by white arrows (× 800 magnification).

Figure 3

Figure 3

(A) Induction of apoptosis in LNCaP, PC3 and TSU-Pr1 human prostate cancer cells by increasing concentrations of sodium butyrate. The final in vivo concentration of i.v.-applied sodium butyrate varied from 0.1 to 5.0 m

M

. Normal saline injection served as a negative control. Apoptotic cells were assessed using the TUNEL assay. (B) Induction of apoptosis in the three cell lines LNCaP, PC3 and TSU-Pr1 with increasing concentrations of tributyrin.

Figure 4

Figure 4

(A) PC3 cells were transplanted in male nude mice. Tumour volume was measured once weekly and is given as an average of 12 tumours. The mice were treated with either normal saline, sodium butyrate or tributyrin by i.p. injections with an estimated plasma concentration of 10 m

M

for the compounds. (B) Tumour volume after implantation of TSU-Pr1 prostate cancer cells in male nude mice. Same control and treatment groups as given in (A) for PC-3 cells.

Figure 5

Figure 5

Immunohistochemistry was performed for protein expression of Ki-67. Control mouse tumours, sodium butyrate- and tributyrin-treated mouse tumours were stained with anti-human Ki-67 monoclonal antibody. Ki-67-positive cells are stained dark brown. Nuclear staining was evaluated with a computerised system. Mean percentages of positive nuclei are graphically given with 95% CI. Examples of tissue sections stained for Ki-67 for the control group, the sodium butyrate- and tributyrin-treated group are given.

Figure 6

Figure 6

Western blot analysis using specific antibodies for detection of p21, Rb and c-myc expression. The presented blots represent expression of the targets of sodium butyrate-treated PC3 human prostate cancer cells. Expression was evaluated at several time points after treatment (internal loading control not shown). P21 expression was induced by sodium butyrate with a maximum at 12 h post-treatment. Rb expression shifted to the hypophosphorylated form in a time-dependent pattern. Simultaneously, c-myc expression decreased. Molecular weights are given in kDa.

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