4-oxo-N-(4-hydroxyphenyl)retinamide: two independent ways to kill cancer cells

Abstract

Background: The retinoid 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) is a polar metabolite of fenretinide (4-HPR) very effective in killing cancer cells of different histotypes, able to inhibit 4-HPR-resistant cell growth and to act synergistically in combination with the parent drug. Unlike 4-HPR and other retinoids, 4-oxo-4-HPR inhibits tubulin polymerization, leading to multipolar spindle formation and mitotic arrest. Here we investigated whether 4-oxo-4-HPR, like 4-HPR, triggered cell death also via reactive oxygen species (ROS) generation and whether its antimicrotubule activity was related to a ROS-dependent mechanism in ovarian (A2780), breast (T47D), cervical (HeLa) and neuroblastoma (SK-N-BE) cancer cell lines.

Methodology/principal findings: We provided evidence that 4-oxo-4-HPR, besides acting as an antimicrotubule agent, induced apoptosis through a signaling cascade starting from ROS generation and involving endoplasmic reticulum (ER) stress response, Jun N-terminal Kinase (JNK) activation, and upregulation of the proapoptotic PLAcental Bone morphogenetic protein (PLAB). Through time-course analysis and inhibition of the ROS-related signaling pathway (upstream by vitamin C and downstream by PLAB silencing), we demonstrated that the antimitotic activity of 4-oxo-4-HPR was independent from the oxidative stress induced by the retinoid. In fact, ROS generation occurred earlier than mitotic arrest (within 30 minutes and 2 hours, respectively) and abrogation of the ROS-related signaling pathway did not prevent the 4-oxo-4-HPR-induced mitotic arrest.

Conclusions/significance: These data indicate that 4-oxo-4-HPR anticancer activity is due to at least two independent mechanisms and provide an explanation of the ability of 4-oxo-4-HPR to be more potent than the parent drug and to be effective also in 4-HPR-resistant cell lines. In addition, the double mechanism of action could allow 4-oxo-4-HPR to efficiently target tumour and to eventually counteract the development of drug resistance.

Figure 1. Effects of vitamin C treatment on 4-oxo-4-HPR-induced ROS generation and apoptosis.

(A) Analysis of ROS production in A2780 cells treated for 4 hours with 5 µM 4-oxo-4-HPR, with or without 100 µM vitamin C. The analysis was performed by flow cytometry after addition of the redox-sensitive dye CM-H₂DCFDA. The graph shows representative flow cytometry fluorescence profiles in different conditions of treatment (one representative experiment of three). A shift to the right from control indicates increased ROS levels. (B) A2780 cells were treated for 24 hours with 5 µM 4-oxo-4-HPR, with or without 100 µM vitamin C and apoptosis, evaluated as DNA fragmentation, was measured by an ELISA assay. Data are means of three independent experiments; vertical bars are standard deviations. Asterisk indicates significant difference (P<0.05).

Figure 2. Effects of 4-oxo-4-HPR and vitamin C treatments on ER stress marker expression and JNK phosphorylation.

(A) A2780 cells treated for 24 hours with 5 µM 4-oxo-4-HPR, with or without 100 µM vitamin C, were subjected to RT-PCR assay to analyze the splicing of the 25 bp intron from XBP-1 transcript and western blot analysis for the expression of GRP-78/Bip, peIF2α, eIF2α. For reverse transcription–PCR, β-actin was amplified as internal control. For western blot analysis, as a control for loading, the blot was incubated with actin antibody. (B) Cells treated as in (A) were subjected to western blot analysis for the expression of pJNK and JNK.

Figure 3. Role of PLAB upmodulation in 4-oxo-4-HPR-induced apoptosis.

(A) Western blot analysis for PLAB expression in A2780 cells treated for 24 hours with 5 µM 4-oxo-4-HPR, with or without 100 µM vitamin C. As a control for loading, the blot was incubated with actin antibody. (B) Western blot analysis for PLAB expression in A2780 cells stably transfected with a plasmid containing a PLAB siRNA or a scrambled nonsilencing siRNA following addition of 5 µM 4-oxo-4-HPR for 24 hours. As a control for loading, the blot was incubated with actin antibody. (C) Detection of 4-oxo-4-HPR-induced apoptosis in A2780 stably transfected with a plasmid containing a PLAB siRNA (black columns) or a scrambled nonsilencing siRNA (grey columns). Transfected cells were treated for 24 hours with 5 µM 4-oxo-4-HPR and apoptosis, evaluated as DNA fragmentation, was measured by an ELISA assay. Data are means of four independent experiments; vertical bars are standard deviations. Asterisk indicates significant difference (P<0.01).

Figure 4. Effects of 4-oxo-4-HPR treatment at different time points on ROS generation and cell cycle distribution.

(A) Analysis of ROS production in A2780 cells treated with 5 µM 4-oxo-4-HPR for 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 16 hours, and 24 hours. The analysis was performed by flow cytometry after addition of the redox-sensitive dye CM-H₂DCFDA. The graphs show representative flow cytometry fluorescence profiles in different conditions of treatment (one representative experiment of three). A shift to the right from control indicates increased ROS levels. (B) Flow cytometric analysis of propidium iodide-stained A2780 cells treated with vehicle or 5 µM 4-oxo-4-HPR for 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 16 hours, and 24 hours. Numbers in the figure indicate the percentage of cells in the phase of cell cycle, according to the analysis performed with ModFit LT software. One experiment representative of three is shown.

Figure 5. Effects of 4-oxo-4-HPR and vitamin C treatments on cell cycle distribution and spindle assembly.

Flow cytometric analysis of propidium iodide-stained A2780 cells treated for 24 hours with 5 µM 4-oxo-4-HPR with or without 100 µM vitamin C. Numbers in the figure indicate the percentage of cells in the phase of cell cycle, according to the analysis performed with ModFit LT software. One experiment representative of three is shown. On the right is depicted a representative mitotic cell image for each treatment, obtained by immunostaining with α-tubulin antibody (green) and nuclear staining with Hoechst 33342 (blue). Scale bar  = 5 µm.

Figure 6. Effects of 4-oxo-4-HPR and vitamin C treatments on ROS generation and PLAB expression.

(A) Citotoxic effect of 4-oxo-4-HPR on T47D (□), HeLa (▵) and SK-N-BE (○) cell growth was estimated by using the sulforhodamine B assay. The antiproliferative activity of 4-oxo-4-HPR in each cell line was tested in three independent experiments with four replicate wells for each analysis; vertical bars are standard deviations. (B) Analysis of ROS production in T47D, HeLa and SK-N-BE cells treated for 4 hours with 5 µM 4-oxo-4-HPR, with or without 100 µM vitamin C. The analysis was performed by flow cytometry after addition of the redox-sensitive dye CM-H₂DCFDA. The graphs show representative flow cytometry fluorescence profiles in different conditions of treatment (one representative experiment of three). A shift to the right from control indicates increased ROS levels. (C) Western blot analyses for PLAB expression in T47D, HeLa and SK-N-BE cells treated with 5 µM 4-oxo-4-HPR for 24 hours, with or without 100 µM vitamin C. As a control for loading, the blot was incubated with actin antibody.

Figure 7. Effects of vitamin C treatment on 4-oxo-4-HPR antimitotic activities in T47D, HeLa and SK-N-BE cells.

Flow cytometric analysis of propidium iodide-stained T47D (A), HeLa (B) and SK-N-BE (C) cells treated for 24 hours with 5 µM 4-oxo-4-HPR with or without 100 µM vitamin C. Numbers in the figure indicate the percentage of cells in the phase of cell cycle, according to the analysis performed with ModFit LT software. One experiment representative of three is shown. On the bottom of each histogram, immunofluorescence analysis with α-tubulin antibody (green) of cells treated in the same way. Nuclear morphology was visualized by staining with Hoechst 33342 (blue). Scale bar  = 10 µm.

Figure 8. Scheme showing proposed cascade of events involved in 4-oxo-4-HPR-induced growth inhibitory effect.

4-oxo-4-HPR induces cell death through two independent mechanisms of action: 1) a ROS-related signaling cascade involving ER stress response, JNK activation and upregulation of the proapoptotic protein PLAB; and 2) antimicrotubule activities consisting in inhibition of tubulin polymerization, mitotic arrest and formation of multipolar spindles.