An antagonist of retinoic acid receptors more effectively inhibits growth of human prostate cancer cells than normal prostate epithelium

Abstract

Screening of synthetic retinoids for activity against prostate carcinoma cell lines has identified antagonists of retinoic acid receptors (RARs) as potent growth inhibitors (Hammond et al, 2001, Br J Cancer 85, 453-462). Here we report that 5 days of exposure to a high-affinity pan-RAR antagonist (AGN194310) abolished growth of prostate carcinoma cells from 14 out of 14 patients, with half-maximal inhibition between 200 and 800 nM. It had similar effects (at approximately 250 nM) on the prostate carcinoma lines LNCaP, DU-145 and PC-3. AGN194310 inhibited the growth of normal prostate epithelium cells less potently, by 50% at approximately 1 microM. The growth of tumour cells was also inhibited more than that of normal cells when RARbeta together with RARgamma, but not RARalpha alone, were antagonised. Treatment of LNCaP cells with AGN194310 arrested them in G1 of cell cycle within 12 h, with an accompanying rise in the level of p21(waf1). The cells underwent apoptosis within 3 days, as indicated by mitochondrial depolarisation, Annexin V binding and DNA fragmentation. Apoptosis was caspase-independent: caspases were neither cleaved nor activated, and DNA fragmentation was unaffected by the pan-caspase inhibitor Z-VAD-FMK. The ability of AGN 194310 to induce apoptosis of prostate cancer cells and its differential effect on malignant and normal prostate epithelial cells suggests that this compound may be useful in the treatment of prostate cancer.

Figure 1

AGN194310 potently inhibits the growth of patients' prostate carcinoma cells. Activity of AGN194310 against carcinoma cells from core biopsies of 14 patients with prostatic carcinoma was measured by seeding cells into wells of a microtitre plate, treating with agent immediately and at day 2, and measuring cellular ATP levels at day 5. The top panel shows the titration curves obtained for each of the patients' cells. The IC₅₀ values shown in the table are means±s.e. of data obtained from three separate experiments.

Figure 2

Pan-RAR antagonist AGN194310 affects the growth of prostate carcinoma cells more than that of normal prostate epithelium. The top panel shows the activity of the pan-RAR antagonist AGN194310 against LNCaP cells (closed circles), PC-3 cells (closed triangles), DU-145 (squares) a patient's primary carcinoma cells (open triangles) and normal prostate epithelium (open circles). The lower panels show the activities of antagonists of RARβγ (AGN194431) and RARα (AGN196996) against LNCaP cells (closed circles) and normal prostate epithelium (open circles). Activities were measured by seeding cells into wells of a microtitre plate, treating with agents immediately and at day 2, and measuring cellular ATP levels at day 5. Data are means±s.e. of values obtained from six (A), four (B) and three (C) experiments. The P-values obtained when the IC₅₀ value for AGN194310 against normal prostate epithelium was compared with IC₅₀ values against LNCaP, PC-3 and DU-145 cells are 0.008, 0.001 and 0.009, respectively. Comparison of the IC₅₀ values for AGN194431 against normal prostate epithelium and LNCaP cells gave a P-value of 0.1, and a P-value of 0.4 was obtained for this comparison for AGN196996.

Figure 3

AGN194310 causes LNCaP cells to arrest in G1 and later undergo apoptosis. (A) Kinetics of changes to the cell cycle status of LNCaP cells treated with 1 μM AGN194310 (open circles) as compared to the status of control (untreated) cultures (closed circles). Cell cycle status was measured after staining cells with propidium iodide, and representative profiles are shown. (B) Time course for the induction of mitochondrial membrane depolarisation (open circles) and the appearance of DNA-strand breaks (TUNEL assay, open triangles) when LNCaP cells were treated with 1 μM AGN194310. The right panel shows that AGN194310-induction of DNA-strand breaks was largely unaffected by treating LNCaP cells with the pan-caspase inhibitor (at 50 μM) for 1 h before adding 1 μM AGN194310 for 3 days. By contrast, Z-VAD-FMK blocked AGN193198-induced apoptosis. Data are means±s.e. of values obtained from three time course experiments, and five experiments using the pan-caspase inhibitor. ^*Denotes P-values <0.05.

Figure 4

AGN194310 increases p21^waf1 protein levels in LNCaP cells. Blots of cell extracts were immunostained with antibodies to p21^waf1 and p27^kip1. The figure shows that p21, but not p27, increased in level when LNCaP cells were treated with AGN194310. To control for the detection of p27, LNCaP cells were treated with the RRM AGN193198.

Figure 5

LNCaP cells undergoing AGN194310-induced apoptosis bind Annexin V. LNCaP cells were treated with 1 μM AGN194310, or vehicle control (DMSO) and stained with Annexin V and propidium iodide. Fluorescence microscopy at day 3 (A) reveals early apoptotic cells (Annexin V +ve; green), as well as late apoptotic/necrotic cells (Annexin V +ve/propidium iodide +ve; yellow-red) in AGN194310-treated cultures. (B) Representative FACS cytograms of stained cells. Viable cells (Annexin V and propidium iodide −ve) are in the lower left-hand quadrant. Early apoptotic cells (Annexin V +ve/propidium iodide −ve) are in the lower right-hand quadrant. Terminal apoptotic/necrotic cells (Annexin V +ve/propidium iodide +ve) are in the upper right-hand quadrant. (C) Results of the flow cytometry analyses are summarised. Data are means±s.e. of values from at least three experiments. ^**Denotes P-values <0.001.

Figure 6

Caspases are neither cleaved nor activated in AGN194310-treated LNCaP cells. (A) that caspases-3, -8 and -9 were not activated in LNCaP cells treated with 1 μM AGN194310. To control for detection of activated enzymes, Jurkat T cells were treated for 6 h with 4 μM AGN193198 (bottom panel). Enzyme activities were measured using peptide substrates. (B) Caspases-3, -8 and -9 were not cleaved in AGN194310 (1 μM)-treated LNCaP cells, as revealed by immunostaining blots of cell extracts. To control for detection of cleaved (active) forms of caspases, Jurkat T cells were treated with 4 μM AGN193198 (right immunoblots).