A novel sulindac derivative lacking cyclooxygenase-inhibitory activities suppresses carcinogenesis in the transgenic adenocarcinoma of mouse prostate model

Abstract

Nonsteroidal anti-inflammatory drugs including sulindac are well documented to be highly effective for cancer chemoprevention. However, their cyclooxygenase (COX)-inhibitory activities cause severe gastrointestinal, renal, and cardiovascular toxicities, limiting their chronic use. Recent studies suggest that COX-independent mechanisms may be responsible for the chemopreventive benefits of nonsteroidal anti-inflammatory drugs and support the potential for the development of a novel generation of sulindac derivatives lacking COX inhibition for cancer chemoprevention. A prototypic sulindac derivative with a N,N-dimethylammonium substitution called sulindac sulfide amide (SSA) was recently identified to be devoid of COX-inhibitory activity yet displays much more potent tumor cell growth-inhibitory activity in vitro compared with sulindac sulfide. In this study, we investigated the androgen receptor (AR) signaling pathway as a potential target for its COX-independent antineoplastic mechanism and evaluated its chemopreventive efficacy against prostate carcinogenesis using the transgenic adenocarcinoma of mouse prostate model. The results showed that SSA significantly suppressed the growth of human and mouse prostate cancer cells expressing AR in strong association with G(1) arrest, and decreased AR level and AR-dependent transactivation. Dietary SSA consumption dramatically attenuated prostatic growth and suppressed AR-dependent glandular epithelial lesion progression through repressing cell proliferation in the transgenic adenocarcinoma of mouse prostate mice, whereas it did not significantly affect neuroendocrine carcinoma growth. Overall, the results suggest that SSA may be a chemopreventive candidate against prostate glandular epithelial carcinogenesis.

Fig. 1

A, the chemical structure of sulindac sulfide and its N, N dimethylaminoethylamide derivative SSA. B, the growth inhibitory response curves of prostate cancer cells exposed to SSA for 48 h by crystal violet staining. Each point represents Mean ± SEM, n=3. C, cell cycle distribution of LNCaP, LNCaP C4-2 and TRAMP-C2 cells after 24h of SSA treatment. D, Suppression of cyclin D₁ and cyclin E₁ abundance after 24h of SSA treatment and, induction of cleaved-PARP and apoptotic nucleosomal fragmentation (Death ELISA) after 6 h or 24 h of SSA treatment in LNCaP cells. Flow cytometry experiments and ELISA were done in triplicate flasks. Statistical significance from control *P<0.05, **P<0.01.

Fig. 2

SSA suppresses AR expression and antagonizes AR signaling in human prostate cancer cells. A, Down regulation by 24 h of SSA treatment of both AR mRNA (top panel, real-time PCR) and AR protein (bottom panel, western-blot) levels in LNCaP C4-2 cells. The mRNA levels were normalized to those of the vehicle controls. B, Suppression by 24 h of SSA treatment of PSA mRNA level (real-time PCR) in LNCaP C4-2 cells. The mRNA levels were normalized to those of the vehicle controls. C, dose-dependent inhibition of PSA-luc reporter in LNCaP cells by SSA treatment for 24h. The luciferase activities were normalized to protein concentration, and then were converted into the relative folds to the mibolerone-stimulated control. Mibolerone is a stable analogue of dihydrotestosterone. Decursin is an inhibitor of AR signaling used as a positive control (44). PSA-luc is a luciferase reporter driven by a six-kb human PSA promoter. The assays of real-time PCR and luciferase reporter were done in triplicate. Statistical significance from control *P<0.05, **P<0.01.

Fig. 3

Effect of dietary SSA on the body weight and genital urinary (GU) tract weights in TRAMP and wild type mice. WT−, wild type mice without SSA supplement (n=8); WT+, wild type mice with 2000ppm SSA (n=9); TRAMP−, TRAMP mice without SSA supplement (n=20); TRAMP+, TRAMP mice with 2000ppm SSA (n=18). A, Growth curves of mice based on the actual (left panel) and normalized body weight to initial weight at enrollment (right panel). BW: body weight. Normalized BW: body weight was normalized against the initial enrollment body weight (as 100%). B, C and D, the actual (left panels) and relative weights of the GU tracts, DLP lobes and VP lobes, respectively. GUTW: GU tract weight. DLPW: DLP lobe weight. VPW: VP lobe weight. GUTW/BW, DLPW/BW and VPW/BW: the relative weights of GU tract, DLP lobe and VP lobe, which were normalized against the endpoint body weight, respectively, and shown as %. ANOVA test was used to determine the significance among the groups. Statistical significance from TRAMP(−) group, ***p <0.001.

Fig. 4

Histopathological evaluation of the effects of dietary SSA consumption on the TRAMP epithelial lesions and NE-like carcinomas. A, Typical AR-dependent glandular epithelial lesions in the DLP and VP lobes of the TRAMP vs. histology of the wild type mice (magnification, 200x). IHC staining: E-cad, E-cadherin; Syn, synaptophysin; AR, androgen receptor. B, typical AR-negative NE-carcinomas. All the NE-like lesions were poorly differentiated (PD) lesions, synaptophysin-positive (arrow), but absent of E-cadherin and AR. The trapped glands (*) were E-cadherin- and AR-positive (arrow head), but absent of synaptophysin. C, Distribution patterns of the AR-dependent glandular epithelial lesions among the specific groups. WT−, wild type mice without SSA supplement (n=8); WT+, wild type mice with 2000ppm SSA (n=9); TRAMP−, TRAMP mice without SSA supplement (n=17); TRAMP+, TRAMP mice with 2000ppm SSA (n=17). N: normal; LP: low grade PIN (prostatic intraepithelial neoplasia), HP: high grade PIN, WD: well-differentiated carcinoma, MD: moderate-differentiated carcinoma. Mean ± SEM. Statistical significance from TRAMP(−) group was calculated by 2-tailed t-test. D, Summary of the AR-independent NE-carcinomas and metastases. PD, poorly differentiated carcinoma.

Fig. 5

Western blot analyses of effects of SSA on molecular biomarkers in TRAMP. DLP and NE-Ca: DLP lobes and NE-like carcinomas. WT: wild type mice, TRAMP+: SSA supplemented TRAMP mice. Syn: synaptophysin, E-cad: E-cadherin

Fig. 6

Detection of SSA and its back conversion product sulindac sulfide in (A) serum, mean ± SEM, N = 5 and (B) in anterior prostate gland, each bar represents an individual mouse. * indicates below limit of detection.