Introduction:For advanced hepatocellular carcinoma (HCC), resistance to conservative treatments remains a challenge. In previous studies, the therapeutic effectiveness and DNA damage responses of boric acid-mediated boron neutron capture therapy (BA-BNCT) in HCC have been demonstrated in animal models and HCC cell line. On the other hand, numerous studies have shown that high linear energy transfer (LET) radiation can overcome tumor resistance. Since BNCT yields a mixture of high and low LET radiation, we aimed to explore whether and how BA-BNCT could eliminate radioresistant HCC cells. Methods: Radioresistant human HCC (HepG2-R) cells were established from HepG2 cells via intermittent irradiation. HepG2 and HepG2-R cells were then irradiated with either γ-ray or neutron radiation of BA-BNCT. Colony formation assays were used to assess cell survival and the relative biological effectiveness (RBE). The expression of phosphorylated H2AX (γH2AX) was also examined by immunocytochemistry and Western blot assays to evaluate the extent of DNA double-strand breaks (DSBs). Finally, the expression levels of DNA damage response-associated proteins were determined, followed by cell cycle analysis and caspase-3 activity analysis. Results: Our data demonstrated that under the same dose by γ-ray, BNCT effectively eliminated radioresistant HCC by increasing the number of DNA DSBs (p < 0.05) and impeding their repair (p < 0.05), which verified the high RBE of BNCT. We also found that BNCT resulted in delayed homologous recombination (HR) and inhibited the nonhomologous end-joining (NHEJ) pathway during DNA repair. Markedly, BNCT increased cell arrest (p < 0.05) in the G 2 /M phase by altering G 2 checkpoint signaling and increased PUMA-mediated apoptosis (p < 0.05). Conclusion: Our data suggest that DNA damage and repair responses could affect the anticancer efficiency of BNCT in radioresistant HepG2-R cells, which highlights the potential of BNCT as a viable treatment option for recurrent HCC.

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