Synergistic cytotoxicity of histone deacetylase and poly-ADP ribose polymerase inhibitors and decitabine in pancreatic cancer cells: Implications for novel therapy

Abstract

Histone deacetylase inhibitors (HDACi) can modulate the acetylation status of proteins, influencing the genomic instability exhibited by cancer cells. Poly (ADP ribose) polymerase (PARP) inhibitors (PARPi) have a direct effect on protein poly (ADP-ribosyl)ation, which is important for DNA repair. Decitabine is a nucleoside cytidine analogue, which when phosphorylated gets incorporated into the growing DNA strand, inhibiting methylation and inducing DNA damage by inactivating and trapping DNA methyltransferase on the DNA, thereby activating transcriptionally silenced DNA loci. We explored various combinations of HDACi and PARPi +/- decitabine (hypomethylating agent) in pancreatic cancer cell lines BxPC-3 and PL45 (wild-type BRCA1 and BRCA2) and Capan-1 (mutated BRCA2). The combination of HDACi (panobinostat or vorinostat) with PARPi (talazoparib or olaparib) resulted in synergistic cytotoxicity in all cell lines tested. The addition of decitabine further increased the synergistic cytotoxicity noted with HDACi and PARPi, triggering apoptosis (evidenced by increased cleavage of caspase 3 and PARP1). The 3-drug combination treatments (vorinostat, talazoparib, and decitabine; vorinostat, olaparib, and decitabine; panobinostat, talazoparib, and decitabine; panobinostat, olaparib, and decitabine) induced more DNA damage (increased phosphorylation of histone 2AX) than the individual drugs and impaired the DNA repair pathways (decreased levels of ATM, BRCA1, and ATRX proteins). The 3-drug combinations also altered the epigenetic regulation of gene expression (NuRD complex subunits, reduced levels). This is the first study to demonstrate synergistic interactions between the aforementioned agents in pancreatic cancer cell lines and provides preclinical data to design individualized therapeutic approaches with the potential to improve pancreatic cancer treatment outcomes.

Keywords: HDAC inhibitors; PARP inhibitors; decitabine; pancreatic cancer; synergistic cytotoxicity.

Figure 1. Dose-response curves of various drugs in three pancreatic cancer cell lines.

(A–F) Cells were seeded in 96-well plates overnight and exposed to drugs for 3 days as described in the Materials and Methods. Rate of cell proliferation was determined relative to control by MTT assay. Model-adjusted means are shown with 95% confidence intervals for the non-zero doses modeled, and solid points indicate a significant difference from the first non-zero dose (Supplementary Table 1). Each cell line of each drug was modeled independently.

Figure 2. Synergistic cytotoxicity of HDACi and PARPi.

Cells were seeded in 96-well plates overnight and exposed to different concentrations of individual drugs or to the two-drug combinations at a constant concentration ratio, and cell proliferation was analyzed after 3 days. The relationships between the calculated combination indexes (CI, Y-axis) and fractions affected (Fa, X-axis) are shown. CI <1.0 indicates synergism. The graphs are representative of two independent experiments. Abbreviations: Ola: Olaparib; Pano: panobinostat; SAHA: vorinostat; TLZ: talazoparib.

Figure 3. Colony formation assay.

Cells were seeded in 6-well plates overnight and exposed to individual drug or drug-combinations for 1–2 weeks and stained as described in the Materials and Methods (A). Colony formation is presented relative to control (B). Model-adjusted means are shown with 95% confidence intervals, and solid points indicate a significant synergistic difference from all the individual drugs (see Supplementary Table 3). Each cell line of each drug was modeled independently. Abbreviations: DAC/D: decitabine; Ola/O: olaparib; Pano/P: panobinostat; SAHA: vorinostat; TLZ/T: talazoparib.

Figure 4. Drug-mediated inhibition of cell proliferation and PARylation, and effects on survival and apoptosis protein markers.

Cells were seeded in T25 flasks overnight and exposed to individual drugs or drug combinations for 3 days, harvested, and analyzed for cell proliferation by MTT assay (A) and Western blotting (B). Model-adjusted means are shown with 95% confidence intervals, and solid points indicate a significant synergistic difference from all the individual drugs (Supplementary Table 2). Each cell line of each drug was modeled independently. Abbreviations: Casp: caspase; DAC/D: decitabine; Ola/O: olaparib; Pano/P: panobinostat; SAHA: vorinostat; TLZ/T: talazoparib.

Figure 5. Effects of drugs on the levels of various proteins involved in DNA repair/DNA damage response.

Cells were exposed to the indicated drug concentrations for 3 days prior to analysis by Western blotting. Abbreviations: NuRD: nucleosome remodeling and deacetylase; DAC: decitabine; DDR: DNA damage response; HR: Homologous recombination; NHEJ: Non Homologous End Joining; NuRD: The Nucleosome Remodeling and Deacetylase (NuRD); Ola: olaparib; Pano: panobinostat; SAHA: vorinostat; TLZ: talazoparib.