DNA Damage Repair in Glioblastoma: A Novel Approach to Combat Drug Resistance

Abstract

Due to the lack of effective therapeutic approach, glioblastoma (GBM) remains one of the most malignant brain tumour. By in vitro investigations on primary GBM stem cells, we highlighted one of the underlying mechanisms of drug resistance to alkylating agents, the DNA damage responses. Here, flow cytometric analysis and viability and repopulation assays were used to assess the long-term cytotoxic effect induced by the administration of a fourth-generation platinum prodrug, the (OC-6-44)-acetatodiamminedichlorido(2-(2-propynyl)octanoato) platinum(IV) named Pt(IV)Ac-POA, in comparison to the most widely used Cisplatin. The immunofluorescence studies revealed changing pathways involved in the DNA damage response mechanisms in response to the two chemotherapies, suggesting in particular the role of Poly (ADP-Ribose) polymerases in the onset of resistance to Cisplatin-induced cytotoxicity. Thus, this research provides a proof of concept for how the use of a prodrug which allows the co-administration of Cisplatin and an Histone DeACetylase inhibitors, could suppress DNA repair mechanisms, suggesting a novel effective approach in GBM treatment.

Keywords: DNA damage response; chemotherapy; drug resistance; glioblastoma.

FIGURE 1

Curve representing viability of human (A) BT 487 and (B) BT 517 spheroids, and (C) astrocytes obtained using MTT assay after standard acute exposure, that is, 48 h continuous treatment, to increasing Cisplatin (0–60 μM) or Pt(IV)Ac‐POA (0–15 μM) concentrations. The relative cell viability is expressed as a percentage relative to the untreated control cells. Data representing the mean value ± SEM, statistical significance as **** for p value < 0.0001. (D) Histograms representing the distributions of GSCs cells, BT 487 on the left and BT 517 on the right, in the cellular cycle phases after each treatment at 48 h and after 7 days from the wash out.

FIGURE 2

(A) Viability of GSCs cells, BT 487 on the left and BT 517 on the right, treated with Cisplatin 40 μM or Pt(IV)Ac‐POA 10 μM for 6 h (t1), 48 h (t2) and after 7 days of wash out, by Trypan blue exclusion test. Data representing the mean value ± SEM, statistical significance as: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.1, specifically *CTR versus Cisplatin, #CTR versus Pt(IV)Ac‐POA, §Cisplatin versus Pt(IV)Ac‐POA. (B) Histograms representing the number of spheres classified by size after 6 h (t1) and 48 h (t2) from the beginning of the treatments with Cisplatin 40 μM or Pt(IV)Ac‐POA 10 μM, after 7 days from the wash out (t3) and after 96 h from the recovery period (t4). Data, from BT 487 on the left and BT 517 on the right, representing the mean value ± SEM. (C) Representative table of BT 487 cells, showing the resulting spheres after 6 h (t1) and 48 h (t2) from the beginning of the treatments with Cisplatin 40 μM or Pt(IV)Ac‐POA 10 μM, after 7 days from the wash out (t3) and after 96 h from the recovery period (t4). Magnification 4×, bar of 200 μm.

FIGURE 3

Proliferation and DNA repair activity. Immunolabelling for PCNA and PARP1 (in green) and BAP1 (in red): In the control condition and differently treated GSCs, that is, after 48 h‐CT Cisplatin 40 μM or Pt(IV)Ac‐POA 10 μM. DNA was stained with Hoechst 33,258 (blue fluorescence). Magnification 25×, bar of 40 μm. The histograms show the mean fluorescence intensity value of the immunolabelling ± SEM for (A) PCNA, (B) PARP1, and (C) BAP1. Statistical significance calculated as follows: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.1.

FIGURE 4

Hypoxic conditions, cell death and inflammatory response. Immunolabelling for HIF1α and AIF (in red) and COX2 (in green): In the control condition and differently treated GSCs, that is, after 48 h‐CT Cisplatin 40 μM or Pt(IV)Ac‐POA 10 μM. DNA was stained with Hoechst 33,258 (blue fluorescence). Magnification 25×, bar of 40 μm. The histograms show the mean fluorescence intensity value of the immunolabelling ± SEM for (A) HIF1α, (B) AIF, and (C) COX2. Statistical significance calculated as follows: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.1.

FIGURE 5

Oxidative stress. Immunolabelling for SOD2 and GPx4 (in green): In the control condition and differently treated GSCs, that is, after 48 h‐CT Cisplatin 40 μM or Pt(IV)Ac‐POA 10 μM. DNA was stained with Hoechst 33,258 (blue fluorescence). Magnification 25×, bar of 40 μm. The histograms show the mean fluorescence intensity value of the immunolabelling ± SEM for (A) SOD2 and (B) GPx4. Statistical significance calculated as follows: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.1.