Licochalcone-A induces intrinsic and extrinsic apoptosis via ERK1/2 and p38 phosphorylation-mediated TRAIL expression in head and neck squamous carcinoma FaDu cells

Abstract

We investigated Licochalcone-A (Lico-A)-induced apoptosis and the pathway underlying its activity in a pharyngeal squamous carcinoma FaDu cell line. Lico-A purified from root of Glycyrrhiza inflata had cytotoxic effects, significantly increasing cell death in FaDu cells. Using a cell viability assay, we determined that the IC50 value of Lico-A in FaDu cells was approximately 100 µM. Chromatin condensation was observed in FaDu cells treated with Lico-A for 24 h. Consistent with this finding, the number of apoptotic cells increased in a time-dependent manner when FaDu cells were treated with Lico-A. TRAIL was significantly up-regulated in Lico-A-treated FaDu cells in a dose-dependent manner. Apoptotic factors such as caspases and PARP were subsequently activated in a caspase-dependent manner. In addition, levels of pro-apoptotic factors increased significantly in response to Lico-A treatment, while levels of anti-apoptotic factors decreased. Lico-A-induced TRAIL expression was mediated in part by a MAPK signaling pathway involving ERK1/2 and p38. In xenograft mouse model, Lico-A treatment effectively suppressed the growth of FaDu cell xenografts by activating caspase-3, without affecting the body weight of mice. Taken together, these data suggest that Lico-A has potential chemopreventive effects and should therefore be developed as a chemotherapeutic agent for pharyngeal squamous carcinoma.

Keywords: Apoptosis; Chemoprevention; FaDu cells; Licochalcone-A; Pharyngeal squamous carcinoma.

Conflict of interest statement

Conflict of Interest

The authors declare that there are no conflicts of interest.

Copyright © 2014 Elsevier Ltd. All rights reserved.

Figures

Fig. 1

Licochalcone-A (Lico-A) selectively induces apoptotic cell death in FaDu cells, but not in human normal oral keratinocytes (hNOKs). (A) Lico-A has cytotoxic effects in FaDu cells, but not in hNOKs. hNOK and FaDu cells were treated with different doses of Lico-A (0, 25, 50, 100, and 125 µM) for 24 h. Under the indicated treatment conditions, cell viability was measured using an MTT assay. The data represent the results of three independent experiments and are expressed as the mean ± standard deviation (SD; *p

Fig. 2

Lico-A-induced FaDu cell death is mediated by both intrinsic and extrinsic apoptotic pathways. FaDu cells were treated with 50 and 100 µM Lico-A for 24 h. After treatment, both total protein and conditioned media were harvested FaDu cells and then western blots were performed. (A) Lico-A increases TRAIL expression in both cell lysate and conditioned media as a dose-dependent manner. (B) Lico-A induces the extrinsic apoptotic signaling pathway. (C) Lico-A induces the intrinsic apoptotic signaling pathway. (D) Lico-A induces FaDu cell death by activating caspase-3 and poly (ADP ribose) polymerase (PARP).

Fig. 3

Lico-A-induced apoptosis in FaDu cells is dependent on caspase activation. (A) Lico-A induces the caspase-3/-7 activation in FaDu cells. A caspase-3/-7 intracellular activity assay was performed using PhiPhiLux-caspase-3/-7 substrate. The stained FaDu cells were observed and imaged using an inverted phase-contrast microscope. (B) Z-VAD-FMK, a pan-caspase inhibitor, inhibited Lico-A-induced activation of caspase-3/-7 and PARP in FaDu cells. FaDu cells were stimulated with Lico-A in presence or absence in Z-VAD-FMK for 24 h. After treatment, total protein was extracted and western blotting was performed to assess the activation of caspase-3/-7 and PARP. (C) The inhibition of caspase activation suppresses Lico-A-induced cytotoxicity in FaDu cells. FaDu cells were stimulated with Lico-A in the presence or absence in Z-VAD-FMK (pan-caspase inhibitor), Z-IETD-FMK (caspase-8 inhibitor), or Z-LEHD-FMK (caspase-9 inhibitor) for 24 h. Cell cytotoxicity was measured by MTT assay.

Fig. 4

Lico-A-induced FaDu cell apoptosis is regulated by TRAIL expression induced by ERK1/2 and p38 MAPK phosphorylation. (A) Lico-A induced the phosphorylation of both ERK1/2 and p38 MAPK, but not JNK MAPK in FaDu cells. FaDu cells were stimulated with 100 µM Lico-A for the indicated treatment times. After treatment, total protein was extracted and western blotting was performed to detect the MAPK activation. (B) Lico-A induces TRAIL expression via ERK1/2 and p38 MAPK signaling pathways in FaDu cells. FaDu cells were treated with 100 µM Lico-A in the presence or absence of 25 µM PD98059 (ERK1/2 inhibitor) and 5 µM SB203580 (p38 inhibitor) for 24 h. After treatment, total protein was extracted and western blotting was performed to assess the changes in TRAIL, caspase-8, caspase-9, caspase-3, and PARP expression. (C) Inhibition of the ERK1/2 and p38 MAPK signaling pathways suppresses the Lico-A-induced FaDu cell apoptosis. FaDu cells were treated with 100 µM Lico-A in the presence or absence of 25 µM PD98059 and 5 µM SB203580 for 24 h. After treatment, cell cytotoxicity was measured by MTT assay.

Fig. 5

Lico-A inhibits the volume of FaDu tumors in a xenografted animal model. (A) Lico-A significantly reduces tumor volume in a FaDu xenograft animal model. Lico-A (10 mg/kg) and vehicle were intravenously injected into experimental and control groups, respectively, three times per week for 8 consecutive weeks. Tumor sizes were measured using a caliper prior to intravenous injections. (B) Lico-A (10 mg/kg) has no effect on animal body weight. (C) Lico-A (10 mg/kg) induces the activation of caspase-3 in tumor tissue collected from FaDu cell xenografted animals. After the treatment period, tumor tissues were dissected from xenografted animals perfused and fixed using saline and 4% paraformaldehyde, respectively. Dissected tumor tissues were post-fixed using 4% paraformaldehyde and were embedded in paraffin for sectioning. Immunohistochemistry was performed using caspase-3 antibody as described in the Materials & Methods. Sections were counterstained using hematoxylin and eosin. Immunoreactive cells are indicated by white arrows.

Fig. 6

Apoptotic signaling pathway induced by Licochalcone-A in head and neck squamous carcinoma FaDu cells.