Antagonistic effects of sodium butyrate and N-(4-hydroxyphenyl)-retinamide on prostate cancer

Abstract

Butyrates and retinoids are promising antineoplastic agents. Here we analyzed effects of sodium butyrate and N-(4-hydroxyphenyl)-retinamide (4-HPR) on prostate cancer cells as monotherapy or in combination in vitro and in vivo. Sodium butyrate and 4-HPR induced concentration-dependent growth inhibition in prostate cancer cells in vitro. The isobologram analysis revealed that sodium butyrate and 4-HPR administered together antagonize effects of each other. For the in vivo studies, a water-soluble complex (4-HPR with a cyclodextrin) was created. A single dose of sodium butyrate and 4-HPR showed a peak level in chicken plasma within 30 minutes. Both compounds induced inhibition of proliferation and apoptosis in xenografts of the chicken chorioallantoic membrane. Analysis of the cytotoxic effects of the drugs used in combination demonstrated an antagonistic effect on inhibition of proliferation and on induction of apoptosis. Prolonged jun N-terminal kinase phosphorylation induced by sodium butyrate and 4-HPR was strongly attenuated when both compounds were used in combination. Both compounds induced inhibition of NF-kappaB. This effect was strongly antagonized in LNCaP cells when the compounds were used in combination. These results indicate that combinational therapies have to be carefully investigated due to potential antagonistic effects in the clinical setting despite promising results of a monotherapy.

Figure 1

Inhibitory effect of sodium butyrate and 4-HPR as monotherapy or in combination on proliferation of LNCaP cells. Cells were treated in vitro for 72 hours with increasing concentrations of sodium butyrate (A), 4-HPR (B), or 1 to 2 mM sodium butyrate and increasing concentrations of 4-HPR (C). Cell proliferation was analyzed by a modified formazan assay (XTT). The number of viable cells in the control group was set to 100%. Results are presented as mean ± SEM of three experiments. (D) Detection of the antagonistic effects of sodium butyrate and 4-HPR on proliferation of LNCaP by using the isobologram method. The IC₅₀ of 4-HPR in the presence of 2 mM sodium butyrate is 0.32 µM and in the presence of 1 mM sodium butyrate is 0.69 µM. The values lie above the line connecting two points representing the IC₅₀ of 4-HPR and sodium butyrate used alone indicating the antagonistic effects of drugs.

Figure 2

Inhibitory effect of sodium butyrate and 4-HPR as monotherapy or in combination on proliferation of PC-3 cells. Cells were treated in vitro for 72 hours with increasing concentrations of sodium butyrate (A), 4-HPR (B), or 1 to 2 mM sodium butyrate and increasing concentrations of 4-HPR (C). Cell proliferation was analyzed by a modified formazan assay (XTT). The number of viable cells in the control group was set to 100%. Results are presented as mean ± SEM of three experiments. (D) Detection of the antagonistic effects of sodium butyrate and 4-HPR on proliferation of PC-3 by using the isobologram method. The IC₅₀ of 4-HPR in the presence of 2 mM sodium butyrate is 1.43 µM and in the presence of 1 mM sodium butyrate is 2.1 µM. The values lie above the line connecting two points representing the IC₅₀ of 4-HPR and sodium butyrate used alone indicating the antagonistic effects of drugs.

Figure 3

Analysis of plasma pharmacokinetics of sodium butyrate and water-soluble 4-HPR/CD complex. (A) Top: structure of the (2-hydroxypropyl)-β-cyclodextrin (CD) molecule consisting of seven glucopyranosides. Middle: structure of 4-HPR. Bottom: graphical presentation of 4-HPR within the CD molecule. (B) Principle of CAM assay. For xenotransplantation of tumor cells onto CAM, cells were seeded in 20 µl medium/Matrigel (1:1) in a 6-mm silicone ring placed on CAM of fertilized chicken. Next day after seeding, drugs or the cyclodextrin vehicle were put onto the second ring. (C) For analysis of the plasma-level kinetics, 5 µmol of sodium butyrate in 20 µl was applied topically on the CAM. Blood was collected from chorioallantoic veins at different time points, anticoagulated with 5 mM EDTA, and used for plasma preparation. For each time point, blood from three eggs was analyzed. (D) Pharmacokinetic analysis of plasma levels of 4-HPR was performed essentially as for sodium butyrate; 0.6 µmol of 4-HPR in 15 µl was applied topically on the CAM.

Figure 4

Inhibitory effect of sodium butyrate and 4-HPR as monotherapy or in combination on proliferation of LNCaP cells in vivo in CAM assay. Histological and TUNEL staining of representative center tissue sections of the LNCaP xenografts. Six days after fertilization, 1 x 10⁶ cells were grafted onto the CAM of chicken eggs. Next day, the CAMs were topically treated with either sodium butyrate (20 µmol/20 µl), 4-HPR/CD (0.6 µmol/15 µl), or both substances three times a day for 4 days. Sections were stained with hematoxylin-eosin (HE), human cytokeratin, and Ki-67 proliferation antigen and photomicrographed at 50x magnification. Apoptosis was analyzed by TUNEL technique, counterstained with hematoxylin, and photomicrographed at 100x magnification. Data shown are representative of 4 to 6 eggs each.

Figure 5

Inhibitory effect of sodium butyrate and 4-HPR as monotherapy or in combination on proliferation of PC-3 cells in vivo in CAM assay. (A) Histological and TUNEL staining of representative center tissue sections of the PC-3 xenografts. Six days after fertilization, 1 x 10⁶ cells were grafted onto the CAMs of chicken eggs. Next day, the CAMs were topically treated either with sodium butyrate (20 µmol/20 µl), 4-HPR/CD (0.6 µmol/15 µl), or both substances three times a day for 4 days. Sections were stained with hematoxylin-eosin (HE), human cytokeratin, and Ki-67 proliferation antigen and photomicrographed at 50x magnification. Apoptosis was analyzed by TUNEL technique, counterstained with hematoxylin, and photomicrographed at 100x magnification. Data shown are representative of 4 to 6 eggs each. (B) Histomorphometric analysis of the proliferation antigen Ki-67. For histomorphometry we used digitalized color photomicrographs of serial 5-µm sections 100 µm apart from each other (n = 4–6 eggs in each group). All treatment groups expressed significantly less Ki-67 antigen than the cyclodextrin control group. Results aremean ± SEM of 4 to 6 eggs in each group. *P < .05, **P < .01 compared with control. (C) Histomorphometric analysis of apoptosis. Results presented as percent of terminal deoxynucleotidyl transferase (TdT)-positive cells. **P < .01.

Figure 6

Effects of sodium butyrate and 4-HPR on NF-κB and MAPK activation in LNCaP and PC-3. Western blotting analysis of phosphorylated IκBα, p38, ERK1/2, and JNK MAPK in the cells treated for 3 hours either with sodium butyrate (2 mM), with 4-HPR (1 µM LNCaP, 3 µM PC-3), or both. Actin served as a loading control. One of three independent experiments is shown.