Preferential targeting of cancer stem cells in the radiosensitizing effect of ABT-737 on HNSCC

Abstract

Head and neck squamous cell carcinomas (HNSCC) are common human malignancies with poor clinical outcomes. The 5-year survival rates for patients with advanced stage HNSCC have not changed appreciably in the past few decades, underscoring a dire need for improved therapeutic options. HNSCC is frequently characterized by overexpression of anti-apoptotic Bcl-2 family members. Increased levels of these anti-apoptotic proteins have been associated with radio- and chemoresistance and poor clinical outcome. The aim of this study was to evaluate combined effects of radiation and ABT-737, a BH3-mimetic molecule, in HNSCC. Although ABT-737, as a single agent, was largely ineffective at promoting HNSCC cell death, we found that combining ABT-737 and radiation induced strong synergistic apoptosis in HNSCC cell lines and delayed tumoral growth in vivo. Moreover, we demonstrated for the first time that ABT-737, alone or in combination with radiation, can efficiently eliminate cancer stem cells (CSCs). Altogether, our results indicate that therapy targeting anti-apoptotic Bcl-2 family members could be a highly effective potential adjuvant to radiotherapy capable of targeting CSCs in HNSCC and therefore overcoming cancer recurrence and metastasis.

Keywords: ABT-737; Bcl-2 family; cancer stem cells; head and neck squamous cell carcinoma; radiation.

Figure 1. Study of the sensitivity of four human HNSCC cell lines to ABT-737

(A) Correlation between the sensitivity to ABT-737 (IC50) and the radiosensitivity of cell lines (SF2). (B) Expression of Bcl-2 family proteins of the four cell lines and the CSC sub-population (SP⁺/CD44^high/ALDH^high) was performed by Western blotting experiments. The correlation between sensitivity to ABT-737 (IC50) and (C) Bak expression, or (D) Bcl-X_L expression were normalized to the expression of α-tubulin.

Figure 2. Radio-sensitization of HNSCC cell lines

(A) Four HNSCC cell lines were treated with 0.1% DMSO (Control: Ctr) or 10 μM ABT-737 (ABT-737) 20 h after a 10 Gy irradiation. Cell death quantification was measured by the percentage of cells in the sub-G1 phase after 24 h, 72 h and 120 h after treatment +/– irradiation. (B) The clonogenic assay was performed on the most radioresistant cell line, SQ20B. The survival fraction at 2 Gy (SF2) was 0.81 (+/– 0.15) for control cells, and decrease to a value of 0.60 (+/– 0.16) after treatment of cells with 10 μM ABT-737. *p < 0.05; **p < 0.01.

Figure 3. Treatment with ABT-737 before X-ray exposure triggers radiation-induced intrinsic apoptosis in SQ20B cell line and intra-mitochondrial oxidative stress

SQ20B cells were treated with 0.1% DMSO (Control: Ctr) or 10 μM ABT-737, 20 h before a 10 Gy irradiation. After 24 h, 48 h, 72 h and 120 h, (A) cells were fixed and the percentage of TUNEL-positive cells were measured by flow cytometry analysis or (B) the percentage of cells having a caspase activity was measured on alive cells by flow cytometry analysis. (C) A Western blot analysis was performed to determine the specific activation of the procaspases-3 by cleavage. (D) The mitochondrial ROS production was validated with a positive (Antimycin A treated cells) control by fluorescence microscopy. Scale bar, 5 μm. (E) Specific mitochondrial ROS production was investigated by flow cytometry analysis using the MitoSOX labeling. (F) The loss of mitochondrial outer membrane potential (ΔΨm) was measured through a JC-1 staining on living cells. *p < 0.05; **p < 0.01; ***p < 0,001.

Figure 4. ABT-737 combined with irradiation alters expression levels of Bcl-2 family members

Representative Western blot analysis of Bcl-2 family members were performed 24 h after irradiation. Tubulin is shown as a loading control. Blots are representative of three independent experiments.

Figure 5. Treatment with ABT-737 increases X-ray-induced apoptosis of the SP+/CD44^high/ALDH^high cells sub-population

A SP⁺/CD44^high/ALDH^high sub-population sorted from the SQ20B cell line was treated with DMSO 0.1% (Control: Ctr) or 10 μM ABT-737 20 h before a 10 Gy irradiation. 24 h, 48 h, 72 h and 120 h after irradiation, (A) the percentage of sub-G1 cells and (B) the loss of mitochondrial outer membrane potential (ΔΨm) were measured by flow cytometry analysis. (C) The expression of Bcl-2 family proteins in the SP⁺/CD44^high/ALDH^high sub-population was performed by Western blot analysis, 7 hours and 24 hours after irradiation, respectively. Tubulin is shown as a loading control. Blots are representative of three independent experiments ***p < 0.001.

Figure 6. Treatment with ABT-737 delays tumoral growth in vivo

Mice were treated intraperitoneally with 0.2% DMSO (Control: Ctr) or with ABT-737 (20 mg/kg/day) 1 hour before irradiation of tumor xenografts (2 Gy/day; Ctr + 10 Gy and ABT-737 + 10 Gy) and this during 5 consecutive days. Tumor volume was monitored up to 40 days. *p < 0.05.