Radiosensitizing Effect of Trabectedin on Human Soft Tissue Sarcoma Cells

Figure 1. Evaluation of the cytotoxic effect of trabectedin in STS cell lines. An MTS assay was used to determine cell viability of rhabdomyosarcoma RD (A), liposarcoma SW872 (B), leiomyosarcoma HS5.T (C), and fibrosarcoma HS 93.T (D) cells treated with trabectedin.

Figure 2. Evaluation of surviving fraction after irradiation with increasing doses in rhabdomyosarcoma RD (A), liposarcoma SW872 (B), leiomyosarcoma HS5.T (C), and fibrosarcoma HS 93.T (D) cell lines, respectively, the control, pretreated with trabectedin, were irradiated with 0–6 Gy. Surviving fraction is shown as the mean ± SEM. * p ≤ 0.05 vs. the control.

Figure 3. Trabectedin (trb) combined with radiation-induced synergistic effect in leiomyosarcoma (HS5.T) and liposarcoma (SW872) cells. (A) Leiomyosarcoma HS5.T. (B) Liposarcoma SW872. Observed combination: surviving fraction observed after irradiation (IR) with trabectedin (trb) treatment. Expected combination: surviving fraction calculated as the product of the surviving fractions observed after treatment with trabectedin or radiation alone. Data are shown as the means ± SEM. * p ≤ 0.05 vs. the expected surviving fraction. Ctrl: control.

Figure 4. Invasion assay in STS cell lines. The matrigel cell invasion assay was performed in each STS cell line after irradiation (IR) and/or in the presence of trabectedin (trb). Columns: capability of invasion reported in % (left) and at 24 h (middle), quantification of 595 nm with a plate reader (right) for rhabdomyosarcoma RD (A), liposarcoma SW872 (B), leiomyosarcoma HS5.T (C), and fibrosarcoma HS 93.T (D) * p ≤ 0.05 vs. control, # p ≤ 0.05 vs. trb, $ p ≤ 0.05 vs. IR.

Figure 5. Cell cycle progression in response to trabectedin. Representative flow cytometry analysis showing cell cycle progression in each STS cell line after treatment with trabectedin and/or 4 Gy. (A–D) Rhabdomyosarcoma RD, liposarcoma SW872, leiomyosarcoma HS5.T, and fibrosarcoma HS 93.T cells, respectively. Analysis was performed by quantifying DAPI nuclear staining using flow cytometry. DNA content and the number of events were analyzed 24 h after treatment. Relative percentage in the G0/G1, G2/M, and S cell phases are plotted after FACS analysis. (E) Relative percentage in the G0/G1, G2/M, and S cell phases are plotted after FACS analysis (* p < 0.05 vs. control, # p < 0.05 vs. trabectedin, $ p < 0.05 vs. irradiation).

Figure 6. Trabectedin significantly increased the mean immunofluorescence emitted by γ-H2AX foci in irradiated rhabdomyosarcoma RD (A), liposarcoma SW872 (B), and leiomyosarcoma HS5.T (C) cell lines. The mean γ-H2AX immunofluorescence is reported as a percentage of the control and shown as the mean ± SEM. * p ≤ 0.05 vs. control (ctrl); # p ≤ 0.05 vs. trabectedin (trb); $ p ≤ 0.05 vs. irradiation (IR).

Figure 7. Trabectedin affects protein expression in leiomyosarcoma HS5.T (A) and liposarcoma SW872 (B). Western blot analysis of the expression of ATM, RAD50, VEGF, Ang-2, and PDL-1 in leiomyosarcoma (HS5.T) and liposarcoma SW872 cells treated with the vehicle (control, ctrl), 4 Gy irradiation (IR) alone, or with trabectedin (Trb), respectively. GAPDH and STAT1 were used as loading controls. Data reported as the percentage versus the respective control (set at 100) are shown as the mean ± SEM. * p ≤ 0.05 vs. control, # p ≤ 0.05 vs. trabectedin, $ p ≤ 0.05 vs.IR.