BCL-2 inhibition impairs mitochondrial function and targets oral tongue squamous cell carcinoma

Abstract

Purpose: To understand the role of Bcl-2 overexpression in oral tongue squamous cell carcinoma (OTSCC) patients and investigate the efficacy of targeting Bcl-2 in OTSCC.

Methods: The expression level of Bcl-2 on normal tongue cells and OTSCC cells were measured by real-time PCR and western blotting. The functional roles of Bcl-2 were examined by MTS, flow cytometry and xenograft cancer mouse model. Mechanism studies were performed by analyzing mitochondrial functions in a panel of OTSCC cell lines.

Results: Bcl-2 is up-regulated at mRNA and protein levels in a panel of OTSCC cell lines compared to normal tongue epithelial cells (NTEC). Importantly, overexpression of Bcl-2 confers resistance of OTSCC cells to chemotherapeutic drug cisplatin treatment. Overexpression of Bcl-2 in NTEC significantly increased cell growth. In contrast, inhibition of Bcl-2 by genetic and pharmacological approaches inhibits proliferation and induces apoptosis in OTSCC cells. Mechanistically, Bcl-2 inhibitor ABT-199 impairs mitochondrial functions as shown by the decreased levels of mitochondrial membrane potential, mitochondrial respiration and ATP, and the increased levels of ROS in OTSCC cells. In addition, ABT-199 inhibits proliferation and induces apoptosis and mitochondrial dysfunctions in NTEC cells, but to a less extent than in OTSCC cells. We further show that ABT-199 augments the effects of cisplatin in eliminating OTSCC cells in in vitro tongue cancer cellular system and in vivo tongue cancer xenograft mouse model.

Conclusions: Inhibition of Bcl-2 effectively targets OTSCC cells through inhibiting proliferation and inducing apoptosis. Inhibition of Bcl-2 also augments the inhibitory effects of cisplatin in vitro and in vivo.

Keywords: ABT-199; Bcl-2; Mitochondria; Tongue squamous carcinoma.

Fig. 1

BCL-2 is up-regulated and confers resistance to cisplatin in OTSCC cells. a mRNA and b protein levels of BCL-2 in normal tongue epithelial cell (NTEC) and OTSCC cell lines. c Protein levels of Bcl-2 after Bcl-2 transfection (pEMD-Bcl2) in NTEC cells. d Overexpression of Bcl-2 promotes NTEC growth. Cells are electroporated with 1.5 μg pEMD, or pEMD-Bcl2 and cultured for 48 h prior to WB and monitoring growth. Different inhibitory effects of cisplatin on the proliferation (e) and survival (f) in SCC-25, SCC-9, CAL27 and Tca3188 cells. Cisplatin at 5 µM was used. Data were presented as mean ± SD. *p < 0.05

Fig. 2

BCL-2 inhibition inhibits proliferation and induces apoptosis of OTSCC cells. a Bcl-2 protein expression level on BCL-2 knockdown by siRNA in SCC-25, SCC-9, CAL27 and Tca3188 cells. b Proliferation is decreased and c apoptosis is increased in Bcl-2 depleted OTSCCs. Cells are electroporated with 100 nM siRNA control or siRNA BCL-2 and cultured for 48 h prior to WB, proliferation and apoptosis assays. ABT-199 inhibits proliferation (d) and induces apoptosis (e) of OTSCCs in a dose-dependent manner. Data were presented as mean ± SD. *p < 0.05

Fig. 3

The effects of BCL-2 inhibitor ABT-199 on NTEC cells. ABT-199 at 1 and 5 µM significantly inhibits proliferation (a) and induces apoptosis (b) of NTEC cells. ABT-199 at 0.5 µM has no significant inhibitory effects in NTEC cells. Data were presented as mean ± SD. *p < 0.05

Fig. 4

Bcl-2 inhibitor impairs mitochondrial functions in OTSCCs. a ABT-199 decreases mitochondrial membrane potential in SCC-25, SCC-9, CAL27 and Tca3188 cells. Cells were stained with JC-1 dye prior to flow cytometry. b ABT-199 dose-dependently inhibits mitochondrial respiration. OCR was measured using a Seahorse XF24 extracellular flux analyser in the absence of compound injection. ABT-199 increases ROS (c) and decreases ATP (d) levels in OTSCCs. Cells were treated with ABT-199 for 24 h prior to mitochondrial function assays. Data were presented as mean ± SD. *p < 0.05

Fig. 5

Bcl-2 inhibitor impairs mitochondrial functions in NTEC cells. ABT-199 at 1 and 5 µM decreases mitochondrial membrane potential (a), mitochondrial respiration (b), increases ROS (c) and decreases ATP (d) levels in NTEC cells. Data were presented as mean ± SD. *p < 0.05

Fig. 6

Bcl-2 inhibitor augments the inhibitory effects of cisplatin in tongue cancer cells in vitro. Combination of ABT-199 and cisplatin is superior in inducing apoptosis than single drug alone. The concentrations of ABT-199 and cisplatin used in combination studies are 0.5 and 1 µM, respectively. Data were presented as mean ± SEM. *p < 0.05, compared to single arm treatment

Fig. 7

Bcl-2 inhibitor augments the inhibitory effects of cisplatin in tongue cancer cells in vivo. a Combination of ABT-199 with cisplatin inhibits much more growth of tongue cancer xenograft mouse model than single drug alone. ABT-199 and cisplatin inhibits growth of tumor derived from SCC-9 cells as a single agent. Combination of ABT-199 with cisplatin completely arrests tumor growth throughout the duration of treatment. There are 10 mice in each group. Data were presented as mean ± SEM. *p < 0.05, compared to single arm treatment. b Representative photos of H&E staining. Increased cell death shown by cytoplasmic swelling, vacuolization or disintegration were observed in combination group compared to control or single treatment group. Scale bar presents 50 μM