Do protons and X-rays induce cell-killing in human peripheral blood lymphocytes by different mechanisms?

Abstract

PurposeSignificant progress has been made in the technological and physical aspects of dose delivery and distribution in proton therapy. However, mode of cell killing induced by protons is less understood in comparison with X-rays. The purpose of this study is to see if there is any difference in the mode of cell-killing, induced by protons and X-rays in an ex vivo human peripheral blood lymphocyte (HPBL) model.Materials and methodsHPBL were irradiated with 60 MeV proton beam or 250-kVp X-rays in the dose range of 0.3–4.0 Gy. Frequency of apoptotic and necrotic cells was determined by the Fluorescein (FITC)-Annexin V labelling procedure, 1 and 4 h after irradiation. Chip-based DNA Ladder Assay was used to confirm radiation-induced apoptosis and necrosis. Chip-based DNA Ladder Assay was used to confirm radiation-induced apoptosis.ResultsEx vivo irradiation of HPBL with proton beams of 60 MeV or 250 kVp X-rays resulted in apoptotic as well as necrotic modes of cell-killing, which were evident at both 1 and 4 h after irradiation in the whole dose and time range. Generally, our results indicated that protons cause relatively higher yields of cell death that appears to be necrosis compared to X-rays. The analysis also demonstrates that radiation type and dose play a critical role in mode of cell-killing.ConclusionObtained results suggest that X-rays and protons induce cell-killing by different modes. Such differences in cell-killing modes may have implications on the potential of a given therapeutic modality to cause immune modulation via programmed cell death (X-rays) or necrotic cell death (proton therapy). These studies point towards exploring for gene expression biomarkers related necrosis or apoptosis to predict immune response after proton therapy.

Comparison of time and dose dependent gene expression and affected pathways in primary human fibroblasts after exposure to ionizing radiation

“…However, predicted downstream effects from gene expression in mouse blood cells tend to differ from available human samples. In particular, cell death of lymphocytes was predicted to be inactivated in mice, whereas lymphocytes in human samples are known to activate processes of cell death after radiation exposure (Miszczyk et al 2018 ). This was also observed in human samples in our comparison analysis (Web Figure 11 ).…”

Radiation, inflammation and the immune response in cancer

“…As an example, irradiation of human peripheral blood lymphocytes induces threefold more cytotoxicity with proton radiation (dose rate [DR] 4.5 Gy/min), compared to X-ray irradiation (DR 9 Gy/min) across a dose range of 0.3–4 Gy (Miszczyk et al 2018). Similar results were obtained with patient-derived glioma stem cells, where doses of 0–10 Gy proton irradiation (DR 2 Gy/min) induced more single and DSB, and ~ 25% greater apoptosis when compared to X-ray radiation (DR 2 Gy/min).…”

“…It is interesting to note that the mechanism of cell death is influenced by radiation type as much as the above biological factors (Miszczyk et al 2018). This is likely in part due to the difference in LET/RBE (Table 1) and therein the number of DNA strand breaks between the two radiation types.…”

“…Necrotic cell death was observed at doses of 0.3–4 Gy from both proton and X-ray radiation (Miszczyk et al 2018). The radiation type influenced the ratio of apoptotic:necrotic cell death with 25:75% observed for proton therapy, and 40:60% observed for X-ray therapy at 4-h post-irradiation.…”

“…Of all the cell death mechanisms, necrosis is the most morphologically distinct, with profound cellular damage including clumping and degradation of DNA, swelling and rupture of the plasma membrane, organelle degradation, mitochondrial swelling, increased vacuolation and the release of DAMPs (Gamrekelashvili et al 2015 ). Necrotic cell death was observed at doses of 0.3–4 Gy from both proton and X-ray radiation (Miszczyk et al 2018 ). The radiation type influenced the ratio of apoptotic:necrotic cell death with 25:75% observed for proton therapy, and 40:60% observed for X-ray therapy at 4-h post-irradiation.…”