Acrolein preferentially damages nucleolus eliciting ribosomal stress and apoptosis in human cancer cells

Abstract

Acrolein (Acr) is a potent cytotoxic and DNA damaging agent which is ubiquitous in the environment and abundant in tobacco smoke. Acr is also an active cytotoxic metabolite of the anti-cancer drugs cyclophosphamide and ifosfamide. The mechanisms via which Acr exerts its anti-cancer activity and cytotoxicity are not clear. In this study, we found that Acr induces cytotoxicity and cell death in human cancer cells with different activities of p53. Acr preferentially binds nucleolar ribosomal DNA (rDNA) to form Acr-deoxyguanosine adducts, and induces oxidative damage to both rDNA and ribosomal RNA (rRNA). Acr triggers ribosomal stress responses, inhibits rRNA synthesis, reduces RNA polymerase I binding to the promoter of rRNA gene, disrupts nucleolar integrity, and impairs ribosome biogenesis and polysome formation. Acr causes an increase in MDM2 levels and phosphorylation of MDM2 in A549 and HeLa cells which are p53 active and p53 inactive, respectively. It enhances the binding of ribosomal protein RPL11 to MDM2 and reduces the binding of p53 and E2F-1 to MDM2 resulting in stabilization/activation of p53 in A549 cells and degradation of E2F-1 in A549 and HeLa cells. We propose that Acr induces ribosomal stress which leads to activation of MDM2 and RPL11-MDM2 binding, consequently, activates p53 and enhances E2F-1 degradation, and that taken together these two processes induce apoptosis and cell death.

Keywords: DNA damages; RPL11-MDM2-p53; acrolein; rDNA/ rRNA; ribosomal stress/ nucleolar stress.

Figure 1. Acrolein induces the same cytotoxic effect in A549 and HeLa cells

(A) Exponentially growing A549 and HeLa cells were treated with different concentrations of Acr (0–250 μM) for 24 h and the cell survival was determined by MTT assay (left panel) and LDH assay (right panel) as described in Materials and Methods. (B) Flow cytometric DNA profiles after propidium iodide (PI) staining of HeLa and A549 cells treated with Acr (75 μM, 0–24 h). The percentage of cell population in each cell cycle phase represents the mean of three different experiments. (C) Flow cytometry analysis of apoptosis/ necrosis by Annexin V and PI staining in Acr-treated A549 and HeLa cells (0–100 μM, 24 h).

Figure 2. Acrolein induces Acr-dG adducts and 8-oxo-dG adducts in nucleoli

HeLa cells were treated with Acr (75 μM, 3 h), Act D (20 ng/ml, 3 h) or H₂O₂ (1 mM, 3 h), fixed, stained with (A) anti-Acr-dG and (B) anti-8-oxo-dG antibody followed by goat anti-mouse FITC-conjugated secondary antibody and then examined by microscopy. Nucleolin (NCL) was used to stain nucleoli (A and B). The specificity of Acr-dG or 8-oxo-dG antibody was also confirmed by pre-incubating these antibodies with a 15 to 20-fold excess of soluble Acr-dG and 8-oxo-dG adducts. (C) DNase and RNase treatment in Acr-treated HeLa cells was described in Materials and Methods. Scale bar: 10 mm. Note: RNAse digests RNA containing oxidative DNA damage recognized by 8-oxo-dG antibody but not DNA (DAPl stained) indicating that Acr-induces 8-oxo-G adducts in RNA.

Figure 3. Acrolein decreases nucleolar RNA polymerase I (Pol I) and UBF translocation and induces nucleolar disintegration

(A and B) Immunofluorescence staining of RNA Pol I and UBF in Acr- and Act D-treated HeLa cells. Cells were treated with Acr (75 μM, 3 h) or Act. D (20 ng/ml, 3 h), fixed, stained with RNA Pol I and UBF antibody followed by goat anti-rabbit Rhodamine-conjugated secondary antibody and then examined by microscopy. B23 was used to stain nucleoli. The magnification of Act D-treated cells in white dashed rectangular box was shown in the lowest row. Scale bar: 40 mm. (C) Visualization of nucleolar structure in HeLa cells treated with Acr (75 μM) or Act D (20 ng/ml) for 3 h using immunofluorescence staining of B23 and NCL antibody. Nuclei were counter-stained with DAPI. Scale bar: 10 mm. Quantifications of nucleolar Pol I and UBF are showed in (D). Histograms show the values (mean ± s.d.) of three independent experiments. *P value < 0.05, **P value < 0.01. Student's t-test was used to calculate significance between control and treatment.

Figure 4. Acrolein interrupts the synthesis of rRNA through inhibition of transcriptional activation of RNA Pol I and UBF

(A) Acr inhibits RNA synthesis in nucleoli. HeLa cells were treated with Acr (75 μM) or Act D (20 ng/ml) for 3 h, labeled newly synthesized RNA with 5-fluorouridine (5-FU) for 15 min, and 5-FU labeld RNA's were labeled with specific FITC-conjugated monoclonal antibodies. Nucleolin (NCL) was used to stain nucleoli and DAPI counter-stained nuclei. Scale bar: 10 mm. Quantifications of nucleolar 5-FU staining is shown in (B). Histograms show the values (mean ± s.d.) of three independent experiments. *P value < 0.05, **P value < 0.01. Student's t-test was used to calculate significance between control and treatment. (C) ChIP assay for RNA Pol I and UBF binding at the promoter region and the transcribed region of 45S rRNA in HeLa cells treated with Acr (75 μM, 3 h). (D) Evaluation of 45S and 18S rRNA expression in HeLa cells treated with Acr (0–100 μM, 3 h) using real-time RT-PCR analysis.

Figure 5. Acrolein interferes with ribosomal assembly and global protein synthesis

(A) Effect of Acr treatment (75 μM, 3 h) on the distribution of monosomes and polysomes in HeLa cells. The monsomes (80S) and polysomes (indicated by a bar) were separated by sucrose gradient centrifugation and each fraction was measured by absorption at 254 nm. (B) RNA extracts from different fractions were analyzed by gel electrophoresis. (C) Effect of Acr (0–100 μM), Act D (20 ng/ml) or cyclohexymide (CHX) (50 μM) for 3 or 24 h treatment on total protein synthesis. HeLa cells were treated with Acr (0–100 μM), Act D (20 ng/ml) or cyclohexymide (CHX) (50 μM) for 3 or 24 h, and then with puromycin analog o-propargyl-puromycin (OPP) for 30 min. OPP incorporation at the C-terminus of translating polypeptide chains, stops translation. These truncated C-terminal alkyne -labeled proteins were then subsequently detected via copper-catalyzed click chemistry using 5 FAM-Azide followed by flow cytometry. Histograms show the values (mean ± s.d.) of three independent experiments. (D) Gel electrophoresis of total rRNA in HeLa cells treated with Acr (0–100 μM, 3 h).

Figure 6. Acrolein stabilizes and activates p53 in p53-active A549 cells

(A) Representative western blot of total MDM2, p53, and the phosphorylated form of MDM2 (p-MDM2, Ser166) and p53 (p-p53, Ser15) expression in control and Acr (0–100 μM, 3 h)-treated A549 and HeLa cells. (B) Time course of total MDM2, p53, p-MDM2, and p-p53 expression in A549 and HeLa cells treated with Acr (75 μM, 0–24 h). Note: Acr treatment increases p-53 in a concentration and time dependent fashion In A549 cells but not in HeLa cells.

Figure 7. Acrolein increases proteasomal degradation of E2F-1 in p53-active A549 and p53-inactive HeLa cells

(A) Effect of Acr treatment (0–100 μM, 3 h) on E2F-1 expression in A549 and HeLa cells. Protein levels detected by Western blots are shown in the left and mRNA levels detected by real-time RT-PCR are shown at right. Values are mean ± s.d. of three experiments. (B) Time course of E2F-1 protein and mRNA expression in A549 and HeLa cells treated with Acr (75 μM, 0–24 h). Representative Western blot E2F-1 protein is shown on the left and real-time RT-PCR quantification of E2F-1 mRNA is shown on the right. Values are mean ± s.d. of three experiments. (C) Western blot showing E2F-1 protein expression in control and acrolein-treated HeLa cells (0–100 μM, 3 h) either pre-treated or not treated with MG-132 (10 μM, 2 h). (D) Time course analysis of E2F-1 protein expression in control and Acr-treated HeLa cells (75 μM, 0–24 h) either pre-treated or not treated with MG-132 (10 μM, 2 h).

Figure 8. Acrolein induces binding of RPL11 to MDM2, p53 stabilization, degradation of E2F-1 and Bcl2, and activates apoptotic enzymes caspase 9 and 3

(A) Effects of Acr treatment (75 μM, 6 h) on the binding of MDM2 with RPL11 p53 and E2F-1 were determined by using an immunoprecipitation method. Cell lysates of Acr-treated A549 and HeLa cells were immunoprecipitated with anti-MDM2 polyclonal antibodies (MDM2-IP), followed by immunoblotting with anti-MDM2, p53, E2F-1, and RPL11 antibodies. For each lysate, 20% of the quantity used for immunoprecipitation was loaded as input control (input). (B and C) Dose effect and time course of Acr effects on cleavage of PARP, and procaspase 9 and 3 (pro-Cas 9 to c-Cas 9; pro-Cas 3 to c-Cas 3), PUMA induction and degradation of Bcl-2 in A549 and HeLa cells. For time course analysis cells were treated with Acr (75 μM) and incubated for different time periods (C) and for dose effect cells were treated with different concentrations of Acr and incubated for 24 h (B). Symbols: p-Cas 9, procaspase 9, c-Cas 9, cleaved caspase 9; p-Cas 3, procaspase 3, c-Cas 3, cleaved caspase 3.

Figure 9. Acrolein induces apoptosis in A549 cells with knockdown of p53 via E2F1 degradation

A549 cells transfected with control siRNA and p53 siRNA were treated with different concentrations of Acr (0–100 μM, 3h), or treated with Acr (75 μM) for different time periods. Expression of (A and B) MDM2, p53, E2F-1, (C and D) PARP, caspase 9, caspase 3 (pro-Cas 9 to c-Cas 9; pro-Cas 3 to c-Cas 3), PUMA and Bcl-2 were detected in these cells as described in Materials and Methods. (E) Flow cytometric DNA profiles after propidium iodide (PI) staining of A549 cells with control siRNA and p53 siRNA treated with Acr (75 μM) for different time periods. The percentage of cell population in each cell cycle phase represents the mean of three different experiments. (F) Flow cytometry analysis of apoptosis/ necrosis by Annexin V and PI staining in Acr-treated A549 cells with control siRNA and p53 siRNA (0–100 μM, 24 h). Symbols: p-Cas 9, procaspase 9, c-Cas 9, cleaved caspase 9; p-Cas 3, procaspase 3, c-Cas 3, cleaved caspase 3.

Figure 10. Model of Acr-induced ribosomal stress responses in p53-active and -inactive cancer cells