A ruthenium anticancer compound interacts with histones and impacts differently on epigenetic and death pathways compared to cisplatin

Abstract

Ruthenium complexes are considered as potential replacements for platinum compounds in oncotherapy. Their clinical development is handicapped by a lack of consensus on their mode of action. In this study, we identify three histones (H3.1, H2A, H2B) as possible targets for an anticancer redox organoruthenium compound (RDC11). Using purified histones, we confirmed an interaction between the ruthenium complex and histones that impacted on histone complex formation. A comparative study of the ruthenium complex versus cisplatin showed differential epigenetic modifications on histone H3 that correlated with differential expression of histone deacetylase (HDAC) genes. We then characterized the impact of these epigenetic modifications on signaling pathways employing a transcriptomic approach. Clustering analyses showed gene expression signatures specific for cisplatin (42%) and for the ruthenium complex (30%). Signaling pathway analyses pointed to specificities distinguishing the ruthenium complex from cisplatin. For instance, cisplatin triggered preferentially p53 and folate biosynthesis while the ruthenium complex induced endoplasmic reticulum stress and trans-sulfuration pathways. To further understand the role of HDACs in these regulations, we used suberanilohydroxamic acid (SAHA) and showed that it synergized with cisplatin cytotoxicity while antagonizing the ruthenium complex activity. This study provides critical information for the characterization of signaling pathways differentiating both compounds, in particular, by the identification of a non-DNA direct target for an organoruthenium complex.

Keywords: ER stress; cisplatin; epigenetics; p53; ruthenium.

Figure 1

(A) Schematic representation of cisplatin and RDC11. (B) Survival curve of cancer cells AGS treated with cisplatin or RDC11. Cells were treated for 48 hours in 96-well plates and their survival was evaluated by MTT assay (n = 8). (C) Table indicating the number of peptides of histones and histone related proteins present in the RDC11 affinity chromatography and mass spectrometric analysis of cell extracts. Repeats are the number of experiment with presence of the peptides out of three experiments done. (D) Migration of histone H3-RDC11 complex on non-denaturing SDS-Page. 100 ng of purified histone H3 was incubated with increased concentrations of RDC11 (R 0.05 to 0.25 μM) for 1 hour. M is the molecular marker. Image is a silver stained gel of the complex showing the monomeric, dimeric and trimeric forms. (E) Proteins were extracted with the indicated buffer (NP40 or sample buffer) from AGS cells treated with RDC11 at the IC50 and the IC75 for 6 hours. Western blot analysis revealed histone H3 and actin protein levels.

Figure 2

(A) Proteins were extracted from AGS treated with RDC11 (R6, R24) or cisplatin (C6, C24) for the indicated time (6 h, R6 and C6; 24 h, R24 and C24) using sample buffer. Western blot analysis revealed histone H3 acetyl lysine 7 (H3AK7), histone H3 (H3) and actin expression. Actin blot are shown in panel C. Quantifications of H3AK7/H3 are indicated below as measured by Pixi imager. (B) Curves are fold induction versus the control (Ct) for selected histone-modifying enzymes in RDC11 and cisplatin conditions. mRNA levels were assayed in AGS gastric cancer cells by RT-qPCR. Curves are means of fold induction versus the control (Ct) with SD (n = 3). *: p < 0.01. (C) Proteins were extracted from AGS treated with RDC11 (R6, R24) or cisplatin (C6, C24) for the indicated time. Western blot analysis revealed EZH2, HDAC4, and actin expression. (D) mRNA levels of HDAC4 were assayed by RT-qPCR in fragments of human colon cancer xenografted in nude mice and after the treatment with cisplatin or RDC11. Graphs are means of fold induction versus the control (Ct) with SD (n = 5). *:p < 0.01.

Figure 3. Hierarchical clustering

Clustering of expression of 4540 probe-set significantly regulated in at least one treated group compared to control. Rows represent gene expression and columns biological samples. Gene expression levels are represented as scaled expression values (row Z-score from −3 to +3). Blue: low expression, Red: high expression, white: moderate expression. 17 different clusters are detected, indicated by their number and different color next the probe-set tree. C6: Cisplatin exposure for 6 hrs; C24: Cisplatin exposure for 24 hrs; R6: Ruthenium exposure for 6 hrs; R24: Ruthenium exposure for 24 hrs.

Figure 4. Signaling pathways and mechanisms regulated by cisplatin or RDC11

(A) Venn diagram. Significantly deregulated probe sets between control and exposed conditions are compared to highlight genes shared or specific to each group. C6: Cisplatin exposure for 6 hrs; C24: Cisplatin exposure for 24 hrs; R6: Ruthenium exposure for 6 hrs; R24: Ruthenium exposure for 24 hrs. (B, C, D) Graphs represents number of genes in the indicated pathways that are regulated by RDC11 at 6 hours or 24 hours (B) or by RDC11 and cisplatin at 24 hours (C, D). Microarray data were analyzed using AltAnalysis and R bioinformatics tools to identify in KEGG, Gene Ontology, miRNA, transcription factors databanks, the signaling pathways and mechanisms corresponding to the mis-regulated genes.

Figure 5. mRNA levels of gdf15 (A), fas (B), plk3 (C) and bak1 (D) were assayed in cancer cells by RT-qPCR

Curves are means of fold induction versus the control (Ct) with SD (n = 3). *:p < 0.01. (E) Proteins were extracted from cells treated with RDC11 (R6, R24) or cisplatin (C6, C24) for the indicated time. Western blot analysis revealed p53 and actin expression. (F) Survival curve of AGS cells treated with RDC11 and pifithrin-α (10 μM). Cells were treated for 48 hours in 96-well plates and their survival was evaluated by MTT assay (n = 8).

Figure 6. mRNA levels of ddit3 (A), atf4 (B), chac1 (C) and dnajb2 (D) were assayed in cancer cells by RT-qPCR

Curves are means of fold induction versus the control (Ct) with SD (n = 3). *:p < 0.01. (E) Proteins were extracted from HCT116 treated with RDC11 (R6, R24) or cisplatin (C6, C24) for the indicated time. Western blot analysis revealed XBP1s and actin expression. (F) Survival curve of AGS cells treated with RDC11 and salubrinal (10 μM). Cells were treated for 48 hours in 96-well plates and their survival was evaluated by MTT assay (n = 8). Bars are means of fold induction versus the control (Ct) with SD (n = 3). *:p < 0.01.

Figure 7. Survival curve of AGS cells treated with SAHA (A) and RDC11 (B)

Cells were treated for 48 hours in 96-well plates and their survival was evaluated by MTT assay (n = 8). Bars are means of fold induction versus the control (Ct) with SD (n = 3). *p < 0.01. Isobologram analyses and combinatory index representations of combinatory treatments between cisplatin with SAHA (C) and RDC11 with SAHA (D). Doses at IC₇₅, IC₆₀, IC₅₀, IC₃₀, IC₂₅ were combined and the results were analyzed with the algorithm of Chu and al. using the CompuSyn software. (E, G) Proteins were extracted from cancer cells treated with RDC11 (R24) or SAHA (S24) or RDC11 and SAHA (R = S) after 24 hours of treatment. Western blot analysis revealed p53 (E) and XBP1s (G) and actin expression. Quantifications are indicated below as measured by Pixi imager. (F, H). mRNA levels of p53 (A), noxa (B), ddit3 (C) and chac1 (D) were assayed in cancer cells by RT-qPCR. Graphs are means of fold induction versus the control (Ct) with SD (n = 3). *p < 0.01. (E) Proteins were extracted from cells treated with RDC11 (R24) or SAHA (S24) or RDC11 and SAHA (R+S) for the 24 hours.