Systematic analyses of the cytotoxic effects of compound 11a, a putative synthetic agonist of photoreceptor-specific nuclear receptor (PNR), in cancer cell lines

Abstract

Photoreceptor cell-specific receptor (PNR/NR2E3) is an orphan nuclear receptor that plays a critical role in retinal development and photoreceptor maintenance. The disease-causing mutations in PNR have a pleiotropic effect resulting in varying retinal diseases. Recently, PNR has been implicated in control of cellular functions in cancer cells. PNR was reported to be a novel regulator of ERα expression in breast cancer cells, and high PNR expression correlates with favorable response to tamoxifen treatment. Moreover, PNR was shown to increase p53 stability in HeLa cells, implying that PNR may be a therapeutic target in this and other cancers that retain a wild type p53 gene. To facilitate further understanding of PNR functions in cancer, we characterized compound 11a, a synthetic, putative PNR agonist in several cell-based assays. Interestingly, we showed that 11a failed to activate PNR and its cytotoxicity was independent of PNR expression, excluding PNR as a mediator for 11a cytotoxicity. Systematic analyses of the cytotoxic effects of 11a in NCI-60 cell lines revealed a strong positive correlation of cytotoxicity with p53 status, i.e., p53 wild type cell lines were significantly more sensitive to 11a than p53 mutated or null cell lines. Furthermore, using HCT116 p53+/+ and p53-/- isogenic cell lines we revealed that the mechanism of 11a-induced cytotoxicity occurred through G1/S phase cell cycle arrest rather than apoptosis. In conclusion, we observed a correlation of 11a sensitivity with p53 status but not with PNR expression, suggesting that tumors expressing wild type p53 might be responsive to this compound.

Figure 1. The effect of 11a on PNR, TLX, COUP-TFI and COUP-TFII activation of the DR2-luciferase reporter.

(A) Chemical structure of 11a. HEK293T cells transfected with the indicated constructs were treated in triplicate with 0.1% DMSO, 15 nM, 30 nM, 60 nM, 120 nM or 150 nM 11a. Data are expressed as relative luciferase units normalized to the DMSO control ± SD. (B) Comparison between different nuclear receptors with increasing 11a concentrations. (C) Comparison between various doses of 11a with different nuclear receptors.

Figure 2. 11a cytotoxicity is independent of PNR overexpression in breast cancer cell lines.

(A) Breast cancer cells were infected with retroviruses expressing GFP or PNR. PNR expression was detected in the Western blot and Hsp90 was used as the loading control. (B) MCF7, (C) MDA-MB-231, (D) LM2 and (E) MDA-MB-468 breast cancer cells were treated with 11a concentrations ranging from 10^-8 to 10^-3 M for 72 hours, and 11a IC₅₀ values were obtained by MTT cell proliferation assays.

Figure 3. p53 wild type cells exhibit higher sensitivity towards 11a than p53 mutated or null cell lines.

11a GI₅₀ values (µM) are plotted against p53 WT and Mut/Null groups in the box chart. Minimum and maximum values, median values and mean values are shown. Significance testing was carried out by two-sided unpaired Wilcoxon rank sum test. *, p<0.05.

Figure 4. 11a induces minimal cell apoptosis in ovarian cancer cell lines and a breast cancer cell line.

SKOV3, A2780 and OVCAR3 ovarian cancer cells (A) and MCF7 breast cancer cells (C) were treated with the indicated doses of 11a or doxorubicin for 24 hours. Total cell lysates were probed for PARP cleavage using anti-PARP antibody in western blots. β-Actin was used as a loading control. The black arrows indicate positions of non-cleaved and cleaved PARP proteins. (B) and (D) After 24 hours treatment with 2 µM 11a, cells were collected and stained with Annexin V/PI and subjected to flow cytometry. 50 µM etoposide (B) or 1 µM staurosporine (STS) (D) served as positive controls for apoptosis. The statistical significance were shown as **p<0.01, ***p<0.001 compared with DMSO control.

Figure 5. Isogenic HCT116 p53+/+ and p53-/- cell lines show differential sensitivity towards 11a.

(A) IC₅₀ values of 11a in p53+/+ and p53-/- HCT116 cell lines. The cells were seeded in quadruplicate in the 384-well plates and treated with the indicated concentrations of 11a for 7 days. The growth inhibition was determined by CellTiter Glo luminescent cell viability assay. (B) IC₅₀ values of 11a were examined in the MTT cell viability assays after 72 hours incubation with 11a. (C) The two cell lines were treated with 0, 1, 10, 100 or 1000 nM 11a for 24 hours and subjected to Western blot using anti-PARP antibody to detect PARP cleavage. Hsp90 was used as a loading control. (D) The cells were treated with indicated concentrations of 11a in the presence or absence of 2 mM 3-aminobenzamide (3-AB) for 72 hours and then subjected to MTT assays. (E) After 24 hours treatment with 1 µM 11a, cells were collected and stained with Annexin V/PI and subjected to flow cytometry. 1 µM staurosporine (STS) served as a positive control for apoptosis. The statistical significance were shown as **p<0.01, ***p<0.001 compared with DMSO control. (F) The two cells were transfected with 1 µg GFP or PNR for 24 hours followed by treatment with DMSO or 1 µM 11a for another 24 hours. Western blot was performed to examine the PARP cleavage.

Figure 6. 11a induces G₁/S phase cell cycle arrest in a time dependent manner in HCT116 isogenic cell lines.

(A) Western blot against p53, Cyclin D1, p21 and Hsp90, which was used as the loading control (B) Histograms of the FACS analysis (C) Quantification of the FACS analysis and cell cycle distribution of the two cell lines treated with 0.1% DMSO or 50 nM 11a.

Figure 7. 11a induces G₁/S phase cell cycle arrest in a dose dependent manner in HCT116 isogenic cell lines.

(A) Western blot against p53, Cyclin D1, p21 and Hsp90 as the loading control. (B) Histograms of the DNA FACS analysis of the cultures from panel A. (C) Quantification of the FACS analysis and cell cycle distribution of the two cell lines treated with 0, 5, 10, 50, or 100 nM 11a in 0.1% DMSO for 12 hours.