Cisplatin enhances radiation-induced apoptosis in peripheral lymphocytes of cancer patients: in vivo and in vitro findings

Abstract

Abstract Background Concurrent cisplatin and radiotherapy frequently cause severe acute hematologic toxicity. This study investigated whether lymphocyte apoptosis, quantified via the γ-H2AX pan-nuclear immunostaining assay, can serve as a reliable biomarker for the acute hematologic side effects of combined chemoradiation. Methods Blood samples were collected from cancer patients and healthy controls. Lymphocyte apoptosis was evaluated using the γ-H2AX pan-nuclear assay following in vitro and in vivo exposure to cisplatin and ionizing radiation (IR). In PBMCs, γ-H2AX results strongly correlated with conventional caspase-3 ( r = 0.9736) and annexin V ( r = 0.9780) apoptosis assays. Additionally, in vitro experiments assessed how phytohemagglutinin-L (PHA-L)-induced lymphocyte proliferation influences the combined effects of cisplatin and IR on apoptosis, alongside the regulatory role of the ATM protein. Results In vitro, cisplatin increased lymphocyte apoptosis asymptotically, plateauing at 10 µg/ml (11.6 ± 5.0%). Combined cisplatin and IR treatment increased apoptosis in a strictly additive manner (radiation effect: p < 0.0001), with no significant synergistic interaction effect between the two modalities ( p = 0.22). The apoptotic effect level of in vivo cisplatin administration did not differ significantly from the maximum effect observed following a 1-hour in vitro exposure to high cisplatin concentrations. In vivo, the additive effect of cisplatin on IR-induced apoptosis peaked up to 3 days post-administration before returning to baseline by day 6. Consistent with these findings, cisplatin and radiation induced a significant, additive increase in apoptosis within PHA-L-stimulated lymphocytes. Inhibition of ATM has no effect on apoptosis following treatment with cisplatin and IR. Conclusions The γ-H2AX pan-nuclear assay provides a valid, reliable method for quantifying radiation- and chemotherapy-induced lymphocyte damage. The time-dependent, additive toxicity observed indicates a critical clinical window (up to 3 days post-cisplatin) for maximum hematologic damage, allowing for optimization of treatment schedules. Clinical trial number Not applicable.