Redox modulation of oxidatively-induced DNA damage by ascorbate enhances both in vitro and ex-vivo DNA damage formation and cell death in melanoma cells.

Elevated genomic instability in cancer cells suggests a possible model-scenario for their selective killing via the therapeutic delivery of well-defined levels of further DNA damage. To examine this scenario, this study investigated the potential for redox modulation of oxidatively-induced DNA damage by ascorbate in malignant melanoma (MM) cancer cells, to selectively enhance both DNA damage and MM cell killing. DNA damage was assessed by Comet and ɣH2AX assays, intracellular oxidising species by dichlorofluorescein fluorescence, a key antioxidant enzymatic defence by assessment of catalase activity and cell survival was determined by clonogenic assay. Comet revealed that MM cells had higher endogenous DNA damage levels than normal keratinocytes (HaCaT cells); this correlated MM cells having higher intracellular oxidising species and lower catalase activity, and ranked with MM cell melanin pigmentation. Comet also showed MM cells more sensitive towards the DNA damaging effects of exogenous H2O2, and that ascorbate further enhanced this H2O2-induced damage in MM cells; again, with MM cell sensitivity to induced damage ranking with degree of cell pigmentation. Furthermore, cell survival data indicated that ascorbate enhanced H2O2-induced clonogenic cell death selectively in MM cells whilst protecting HaCaT cells. Finally, we show that ascorbate serves to enhance the oxidising effects of the MM therapeutic drug Elesclomol in both established MM cells in vitro and primary cell cultures ex vivo. Together, these results suggest that ascorbate selectively enhances DNA damage and cell-killing in MM cells. This raises the option of incorporating ascorbate into clinical oxidative therapies to treat MM.

Investigations of the novel checkpoint kinase 1 inhibitor SRA737 in non-small cell lung cancer and colorectal cancer cells of differing tumour protein 53 gene status.

Aim: In response to DNA damage the serine/threonine-specific protein kinase checkpoint kinase 1 (CHK1) is activated allowing cells to enter S phase (S) and G2 phase (G2) cell-cycle arrest. CHK1 inhibitors are expected to prevent cells from entering such arrest, thereby enhancing DNA damage-induced cytotoxicity. In contrast, normal cells with intact ataxia-telangiectasia mutated (ATM), CHK2 and tumour suppressor protein 53 (P53) signalling are still able to enter cell-cycle arrest using the functioning G1/S checkpoint, thereby being rescued from enhanced cytotoxicity. The main objective of this work is to investigate the in vitro effects of the novel CHK1 inhibitor SRA737 on pairs of non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) cell lines, all with genetic aberrations rendering them susceptible to replication stress but of differing tumour protein 53 (TP53) gene status, focusing on DNA damage induction and the subsequent effects on cell proliferation and viability. Methods: NSCLC cell lines H23 [TP53 mutant (MUT)] and A549 [TP53 wild-type (WT)] and CRC cell lines HT29 (TP53 MUT) and HCT116 (TP53 WT) were incubated with differing micromolar concentrations of SRA737 for 24 h and then analysed using alkaline comet and phosphorylated H2A.X variant histone (γH2AX)-foci assays to assess mostly DNA single strand break and double strand break damage, respectively. Cell-counting/trypan blue staining was also performed to assess cell proliferation/viability. Results: Clear concentration-dependent increases in comet formation and γH2AX-foci/cell were noted for the TP53 MUT cells with no or lower increases being noted in the corresponding TP53 WT cells. Also, greater anti-proliferative and cell killing effects were noted in the TP53 MUT cells than in the TP53 WT cells. Conclusions: This study's data suggests that P53 status/functioning is a key factor in determining the sensitivity of NSCLC and CRC cancer cells towards CHK1 inhibition, even in circumstances conducive to high replicative stress.

FLASH irradiation induces lower levels of DNA damage ex vivo, an effect modulated by oxygen tension, dose, and dose rate.

OBJECTIVE: FLASH irradiation reportedly produces less normal tissue toxicity, while maintaining tumour response. To investigate oxygen's role in the 'FLASH effect', we assessed DNA damage levels following irradiation at different oxygen tensions, doses and dose rates. METHODS: Samples of whole blood were irradiated (20 Gy) at various oxygen tensions (0.25-21%) with 6 MeV electrons at dose rates of either 2 kGy/s (FLASH) or 0.1 Gy/s (CONV), and subsequently with various doses (0-40 Gy) and intermediate dose rates (0.3-1000 Gy/s). DNA damage of peripheral blood lymphocytes (PBL) were assessed by the alkaline comet assay. RESULTS: Following 20 Gy irradiation, lower levels of DNA damage were induced for FLASH, the difference being significant at 0.25% (p < 0.05) and 0.5% O2 (p < 0.01). The differential in DNA damage at 0.5% O2 was found to increase with total dose and dose rate, becoming significant for doses ≥20 Gy and dose rates ≥30 Gy/s. CONCLUSION: This study shows, using the alkaline comet assay, that lower levels of DNA damage are induced following FLASH irradiation, an effect that is modulated by the oxygen tension, and increases with the total dose and dose rate of irradiation, indicating that an oxygen related mechanism, e.g. transient radiation-induced oxygen depletion, may contribute to the tissue sparing effect of FLASH irradiation. ADVANCES IN KNOWLEDGE: This paper is first to directly show that FLASH-induced DNA damage is modulated by oxygen tension, total dose and dose rate, with FLASH inducing significantly lower levels of DNA damage for doses ≥20 Gy and dose rates ≥30 Gy/s, at 0.5% O2.