RAD51 inhibitor B02 exhibits radio-sensitizing properties in BNCT

B02, a RAD51 inhibitor, disrupts the interaction with damaged DNA, reducing RAD51 foci formation and weakening homologous recombination repair (HRR) [26]. It’s also utilized in liver cancer studies [27]. In this study, the IC50 of B02 in eliminating HepG2 and HepG2R cells was 20.99 μM and 20.71 μM, respectively (Fig. 1A). There was no significant difference in survival rates between the cell lines. The IC10 concentration was approximately 10 μM, significantly inhibiting RAD51 foci formation [28]. Therefore, 10 μM of B02 was used for subsequent experiments.

Fig. 1

Combined BNCT and B02 treatment effectively eliminates both HepG2 and HepG2R. A The resulting IC50 values of B02 were 20.99 μM for HepG2 cells and 20.71 μM for HepG2R cells. B The colony formation assay assessed cell viability after B02, 1 Gy BNCT, and their combination. C Colonies were stained using crystal violet, and the surviving fraction was quantified. (*p < 0.05, ***p < 0.001, mean ± SD)

The colony formation assay assessed the cell surviving fraction following the combined treatment with BA-BNCT and B02 (Fig. 1B). The surviving fraction of HepG2 cells treated with B02 alone was approximately 0.57 ± 0.02 compared to the untreated group. The BA-BNCT monotherapy group had a surviving fraction of 0.14 ± 0.05, and the combined treatment group had a surviving fraction of 0.03 ± 0.01. For the radioresistant HepG2R cells, the surviving fraction after adding B02 was approximately 0.48 ± 0.05. The BA-BNCT monotherapy group had a surviving fraction of 0.17 ± 0.02, and the combined treatment group had a surviving fraction of 0.05 ± 0.02 (Fig. 1C).

The results showed that after 1 Gy BA-BNCT irradiation, the group treated with 10 μM B02 more effectively eliminated HepG2 and HepG2R cells than BNCT monotherapy. This finding prompted further research into its superior sensitizer enhancement ratio (SER) [29]. Using the formula in Supplementary Table 2A, the SER was 1.30 for HepG2 and 1.25 for HepG2R, indicating BA-BNCT with B02 is superior. Furthermore, to assess B02’s radiation-sensitizing effect in BA-BNCT, we calculated the relative effect ratio (RER). An RER above 1 indicates sensitization [30]. The RER for HepG2 cells was 5.53, and for HepG2R cells was 3.81 (Supplementary Table 2B), showing B02's strong sensitizing effect, especially in HepG2 cells.

The combined treatment of BNCT and B02 can effectively enhance the elimination of liver cancer cells. B02 inhibits RAD51, reducing HRR. It significantly lowers cell survival rates post-BA-BNCT irradiation, with superior SER and RER values, particularly in HepG2 cells, demonstrating a robust sensitizing effect.

B02 exacerbates the DNA damage response following BNCT

The HRR pathway is crucial in DNA damage repair in cancer radiation biology. Western blot results showed the relative expression level of RAD51 in HepG2 cells was significantly increased by 0.18 ± 0.02- and 0.55 ± 0.12-fold compared to untreated control at 10 and 24 h after BNCT irradiation, respectively (Fig. 2A). Interestingly, HepG2R cells exhibited earlier RAD51 upregulation at 4 h, which was significantly increased by 0.38 ± 0.10-, 0.37 ± 0.06-, and 0.69 ± 0.08-fold compared to untreated control at 4, 10, and 24 h, respectively, indicating potential radioresistance (Fig. 2B).

Fig. 2

B02 suppresses the HRR pathway and enhances γH2AX expression during BNCT treatment. A, B The Western blot assay assessed HR-related protein, RAD51, post-BNCT in HepG2 and HepG2R cells, quantified by using ImageJ and normalized by GAPDH. The relative expression of the untreated control was set to 1.00. C The representative images depict γH2AX foci (DNA double-strand breaks marker) through immunocytochemistry (ICC). The green fluorescence corresponds to γH2AX, while the blue fluorescence corresponds to nuclei. (*p < 0.05, **p < 0.01, #p < 0.05, mean ± SD)

In the combination treatment of BNCT and B02 group, RAD51 expression in HepG2 was significantly decreased by 0.27 ± 0.07-, 0.26 ± 0.04-, and 0.58 ± 0.17-fold compared to BNCT treatment alone at 4, 10, and 24 h post-BNCT, respectively (Fig. 2A). On the other hand, the RAD51 expression of HepG2R was significantly decreased by 0.35 ± 0.11-, 0.57 ± 0.08-, and 0.61 ± 0.14-fold, respectively (Fig. 2B). Since inhibition of DNA repair processes exacerbates DNA damage, γH2AX was used as a marker to quantify the extent of DNA double-strand breaks [31]. γH2AX expression was increased in HepG2 and HepG2R cells at 10 and 24 h following BA-BNCT and B02 treatment, with a notable rise at 10 h (Fig. 2C). The combination treatment group had higher γH2AX expression than the monotherapy groups.

In short, B02 sensitizes cells under BA-BNCT, inhibiting the HRR pathway, suggesting a promising therapeutic approach to enhance BA-BNCT effectiveness in hepatic malignancies.

The combined treatment induces G 0/G 1 arrest in liver cancer cells

To investigate the subsequent cellular response, we assessed changes in cell cycle distribution after combination treatment. Figures 3A, B showed increased G2/M phase population in HepG2 and HepG2R cells at 4 and 10 h after 1 Gy BA-BNCT, indicating DNA repair before mitosis. Since B02 attenuates DNA HRR after BA-BNCT, we examined its effect on cell cycle progression. Figures 3C, E showed HepG2 and HepG2R cell distribution in the G0/G1, S, and G2/M phases. Figures 3D, F provided statistical analysis, focusing on the G0/G1 and G2/M phases at 4- and 10-h post-treatment.

Fig. 3

Combined treatment of BNCT and B02 leads to G0/G1 arrest in HepG2 and HepG2R. A Cell cycle distribution was assessed at 4 and 10 h following BNCT treatment, B02 treatment, and their combination in HepG2 cells. B Cell cycle distribution was analyzed at 4 and 10 h post-BNCT, B02, and combined treatments in HepG2R cells. C The DNA histogram for the cell cycle assay of HepG2 cells was generated using staining with 7-Amino-Actinomycin D (7-ADD). D Quantitative analysis of cell cycle assay for G0/G1 phase and G2/M phase in HepG2 cells. E The DNA histogram for the cell cycle assay of HepG2R cells F Quantitative analysis of cell cycle assay for G0/G1 phase and G2/M phase in HepG2R cells. (*p < 0.05, mean ± SD)

The addition of B02 reduced the G2/M phase arrest induced by BA-BNCT, which is evident in both non-irradiated and post-treatment groups. BA-BNCT and B02 combined treatment caused cells to accumulate in the G0/G1 phase, especially 10 h post-irradiation (Figs. 3C, E). In HepG2 cells, G2/M phase cells decreased by 13.79 ± 3.89% while G0/G1 phase cells increased by 13.05 ± 1.12% (Fig. 3D). In HepG2R cells, G2/M phase cells decreased by 18.26 ± 3.65% while G0/G1 phase cells increased by 14.79 ± 2.16% (Fig. 3F). B02 addition alters cancer cell responses, affecting cell division post-BNCT irradiation and influencing cellular fate.

B02 inhibits the autophagic flux induced by BA-BNCT

Studies show autophagy restores cellular homeostasis by degrading damaged DNA [32]. We investigated whether BA-BNCT treatment induces autophagy. LC3B marks early autophagy stages, forming autophagosomes [33]. p62 transports damaged materials to lysosomes for degradation, with decreased p62 indicating increased autophagy [34]. Figure 4A showed a Western blot analysis of LC3B and p62 in HepG2 and HepG2R cells post-BNCT, indicating increased LC3BII/LC3BI ratio and decreased p62, suggesting autophagy induction.

Fig. 4

The combined treatment of B02 and BNCT effectively inhibits autophagic flux. A The Western blot revealed autophagy-related protein levels after 4, 10, and 24 h of BNCT in HepG2 and HepG2R cells. B The Western blot analysis revealed autophagy-related protein levels at 4, 10, and 24 h post-BNCT and combined treatment in HepG2 and HepG2R cells. C LC3BII/LC3BI protein ratio was quantified using ImageJ. D p62 protein expression was quantified using ImageJ. GAPDH was used as a loading control. The relative expression of the untreated control was set to 1.00. (*p < 0.05, **p < 0.01, mean ± SD)

Autophagic flux involves autophagosome formation, lysosome fusion, and degradation, marked by increased LC3BII/LC3BI ratio and decreased p62 levels as substances decompose [35]. To investigate the impact of HRR inhibitor B02 on autophagy induced by BA-BNCT, we assessed the response at 4, 10, and 24 h post-irradiation (Fig. 4B). In the combination treatment group, the LC3BII/LC3BI ratio was increased in HepG2 by 0.50 ± 0.10-, 0.67 ± 0.12-, and 0.65 ± 0.13-fold, and in HepG2R was increased by 1.01 ± 0.20-, 0.35 ± 0.10-, and 0.49 ± 0.16-fold compare to BNCT monotherapy, respectively (Fig. 4C). This suggests that B02 may enhance autophagosome formation, potentially facilitating the encapsulation of damaged DNA for degradation.

The quantification of p62 revealed a cumulative increase after adding B02. At 4, 10, and 24 h, p62 levels in HepG2 was increased by 0.81 ± 0.13-, 0.75 ± 0.08-, and 0.34 ± 0.16-fold, and in HepG2R by 0.88 ± 0.16-, 0.71 ± 0.24-, and 0.20 ± 0.20-fold, respectively (Fig. 4D). This suggests the autophagosome may not be fusing with the lysosome for degradation.

The apoptotic pathway is upregulated by the addition of B02

The inhibition of autophagic flux prevents the efficient degradation of autophagosomes containing damaged DNA, increasing cellular stress [36]. We speculate that BA-BNCT and B02 treatment causes autophagosome buildup, elevating stress and triggering apoptosis. We assessed Caspase-3 levels to quantify apoptosis post-treatment.

We monitored Caspase-3 levels at 24 h after BA-BNCT irradiation. Compared to the untreated group (Fig. 5A, red line), the B02-treated group without BA-BNCT (Fig. 5A, yellow line) showed a slight increase in Caspase-3. In HepG2 and HepG2R, Caspase-3 increased by 0.61 ± 0.08- and 0.74 ± 0.09-fold, respectively (Fig. 5B). BA-BNCT monotherapy (Fig. 5A, green line) caused a noticeable increase: 2.32 ± 0.20-fold in HepG2 and 0.85 ± 0.14-fold in HepG2R (Fig. 5B).

Fig. 5

The B02 induces more intensive apoptosis caused by BNCT treatment. A The histogram of caspase-3 activity for the apoptosis assay was obtained through PE staining. B Quantitative assessment of caspase-3 relative fluorescence intensity. (*p < 0.05, **p < 0.01, mean ± SD)

After 24 h of BA-BNCT combined with B02 treatment (Fig. 5A, blue line), a substantial increase in Caspase-3 can be observed. The data indicated that in the combined treatment group, HepG2 and HepG2R increased by 3.8 ± 0.34- and 1.7 ± 0.25-fold compared to the untreated group, respectively (Fig. 5B). Both HepG2 and the radioresistant HepG2R exhibited enhanced apoptosis when treated with B02 and BA-BNCT.