Figure 2.

Proteasome inhibitors sensitize HC cells to radiation

(A) Two-dimensional colony formation assays in FaDu cells treated with PIs (BTZ: 5 nM, PT33: 10 nM) for 12 h and subsequently with a single dose of 0–4 Gy. (Left) Representative images. (Right) Survival curves and relative SER estimated survival rates of 50%, 30%, and 10%, shown as means ± SDs from 3 independent experiments (1-way ANOVA, DMSO as a vehicle control). (B and D) The combination index (CI) of XR (indicated doses) and PIs (indicated concentrations) on FaDu cells based on the results of cell viability assays using CCK-8 in (B) FaDu and (D) Detroit 562 cells. (C and E) Conditional 3D spheroid formation assays for FaDu cells treated with PIs (DMSO as vehicle control) plus XR (NR as control) starting on day 4 as in Figure S3A. (C) Representative images. (E) Numbers of spheres (diameter ≥75 μm) from 3 independent experiments, shown as means ± SDs (1-way ANOVA). Q was calculated as follows: Q = I_AB/[I_A + I_B (1 − I_A)], where I_A, I_B, and I_AB indicate the inhibitory rates of PIs, XR, and PIs + XR, respectively. Q < 0.85, 0.85 ≤ Q ≤ 1.15, and Q > 1.15 indicate antagonistic, additive, and synergistic effects, respectively. (F) Annexin V/PI double-staining assays with flow cytometry to clarify the percentage of apoptotic cells in FaDu and Detroit 562 cells at 72 h after XR treatment. Cells were pretreated with PIs (BTZ: 5 nM, PT33: 10 nM; DMSO as vehicle control) for 12 h before XR. The statistical data are presented as the means ± SDs of at least 3 independent experiments (1-way ANOVA). (G) Western blot analysis of the levels of PARP (pro-/cleaved) and caspase 3 (pro-/cleaved) in FaDu and Detroit 562 cells at 72 h after XR treatment, PIs (BTZ: 5 nM, PT33: 10 nM; DMSO as vehicle control) were pretreated for 12 h before XR. (H and I) The xenograft tumor model in nude mice showed that PIs enhanced the sensitivity of HC to radiotherapy in vivo. FaDu xenografts formed in the right flank of BALB/c nude mice were treated alternately with PIs (BTZ 0.5 mg/kg × 7 or PT33 1 mg/kg × 7; normal saline [NS] was used as negative control; purple arrows) and X-rays (2-Gy every other day × 7; orange arrows). (H) Xenograft tumors were dissected and weighed in the NS, BTZ, and PT33 groups (n = 8 tumors/group). (I) The tumor growth curves were drawn by measuring the tumor volume in different groups, shown as means ± SDs (1-way ANOVA). Q value in (F) and (H) indicates the combination effect of XR treatment and PIs (see the legend for E). All statistical data: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; N.S., not significant.