Specific Targeting of Antiapoptotic Bcl-2 Proteins as a Radiosensitizing Approach in Solid Tumors

Abstract

Avoidance of therapy-induced apoptosis is a hallmark of acquired resistance towards radiotherapy. Thus, breaking resistance still challenges modern cancer therapy. The Bcl-2 protein family is known for its regulatory role in apoptosis signaling, making Bcl-2, Mcl-1 and Bcl-x_L promising targets. This study evaluates the effects of highly specific inhibitors for Bcl-x_L (WEHI-539), Bcl-2 (ABT-199) and Mcl-1 (S63845) as radiosensitizers. Covering a broad spectrum of solid tumors, Non-Small-Cell Lung Cancer (NSCLC), Head and Neck Squamous Cell Carcinoma (HNSCC) and synovial sarcoma cell lines were exposed to fractionated radiation as standard therapy with or without Bcl-2 protein inhibition. Protein expression was detected by Western blot and cell death was assessed by flow cytometry measuring apoptosis. In contrast to NSCLC, a high level of Bcl-x_L and its upregulation during radiotherapy indicated radioresistance in HNSCC and synovial sarcoma. Radioresistant cell lines across all entities benefited synergistically from combined therapy with Bcl-x_L inhibition and fractionated radiation. In NSCLC cell lines, Mcl-1 inhibition significantly augmented radiotherapy independent of the expression level. Our data suggest that among antiapoptotic Bcl-2 proteins, targeting Bcl-x_L may break resistance to radiation in HNSCC, synovial sarcoma and NSCLC in vitro. In NSCLC, Mcl-1 might be a promising target that needs further investigation.

Keywords: Bcl-2; Bcl-xL; Mc-1; cell death; head and neck squamous cell carcinoma; non-small-cell lung cancer; radiotherapy; synovial sarcoma; therapy resistance.

Figure 1

Effects of fractionated radiation on NSCLC, HNSCC and synovial sarcoma cell lines. (A) FACS analysis of cell death induction during fractionated radiation. Experiments were performed in triplicate and cell death was measured as mean ± SD. The depicted FACS analyses are representative of at least three independent experiments. (B) Representative Western blot analysis of protein development during fractionated radiation and basal protein expression of the Bcl-2-family with Tubulin serving as loading control. Below, a densitometric analysis was performed by norming the expression bands to their respective loading control and using for each entity one cell line as reference. Relative expression was compared between three independent experiments and measured as mean ± SD.

Figure 2

Correlation of relative Bcl-x_L expression with radiation induced cell death. (A) The coefficient of determination (R²) was calculated by plotting the results of a densitometric analysis of shown Western blots against radiation-induced cell death at delta 4 × 4 Gy. Representative Western blots; Tubulin/Actin served as loading control. HNSCC and synovial sarcoma cell lines were grouped together due to their similar reaction to radiation with Bcl-x_L upregulation. (B) Graphical depiction via heatmap of inhibitor-induced cell death. FACS analysis of cell death induction during treatment with BH3 mimetics in different concentrations. Experiments were performed in triplicate and cell death was measured as mean ± SD. Findings are depicted graphically in a heat map for overview purposes. Results are representative of at least three independent experiments.

Figure 3

Set up for in vitro experiments and radiosensitization results of NSCLC cell lines (A) Cell lines undergoing a fractioned radiation schedule during molecular inhibition with BH3 mimetics. (B) Combination of irradiation and molecular inhibition in NSCLC cell lines and FACS analysis of induced cell death. Experiments were performed in triplicate and cell death was measured as mean ± SD. The depicted FACS analyses are representative of at least three independent experiments. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 4

Radiosensitization results of HNSCC and synovial sarcoma cell lines Combination of irradiation and molecular inhibition in (A) HNSCC and (B) synovial sarcoma cell lines measured by FACS analysis. Experiments were performed in triplicate and cell death was measured as mean ± SD. The depicted FACS analyses are representative of at least three independent experiments. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 5

Graphical overview of NSCLC, HNSCC and synovial sarcoma cell lines undergoing combined fractioned radiation and BH3-inhibition as depicted in Figure 3B and Figure 4. The label synergistic was defined as the combinatory effect surpassing the additive effect of irradiation and molecular inhibition.