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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.

SUBMITTER: Park MR

PROVIDER: S-EPMC4522946 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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

Park Mi-Ra MR Kim Su-Gwan SG Cho In-A IA Oh Dahye D Kang Kyeong-Rok KR Lee Sook-Young SY Moon Sung-Min SM Cho Seung Sik SS Yoon Goo G Kim Chun Sung CS Oh Ji-Su JS You Jae-Seek JS Kim Do Kyung DK Seo Yo-Seob YS Im Hee-Jeong HJ Kim Jae-Sung JS

Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 20150105

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 ...[more]

PMID: 25572524

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Project description:Epithelial cell adhesion molecule EpCAM is a single transmembrane protein, which is involved in numerous cellular processes including cell adhesion, proliferation, maintenance of stemness of embryonic cells and progenitors, migration, and invasion. Activation of signal transduction by EpCAM is warranted by regulated intramembrane proteolysis and nuclear translocation of the intracellular domain EpICD. Here, we describe matrix metalloproteinase 7 (MMP-7) as a target gene of EpCAM signalling via EpICD nuclear translocation. EpCAM and MMP-7 expression pattern and levels positively correlated in vitro and in vivo, and were strongly elevated in primary carcinomas of the head and neck area. Hence, MMP-7 is a novel target of EpCAM signalling. FaDu hypopharynx carcinoma cells were transiently transfected with a control siRNA or an EpCAM-specific siRNA. After two days, expression of EpCAM was assessed upon flow cytometry with antibodies, which specifically bind within the EGF-repeat in the extracellular domain of EpCAM (Balzar, et al., 1999). Compared to control siRNA, EpCAM siRNA induced a mean 43% reduction of EpCAM expression at the cell surface. Reduction of EpCAM expression upon siRNA treatment was confirmed by RT-PCR and qPCR. EpCAM down-regulation resulted in diminished cell proliferation assessed upon cell counting. The observed 43% decrease in cell surface expression of EpCAM translated in cell numbers diminished by 35%. This is a confirmation of earlier findings on the reduced proliferation of breast and colon cancer cell lines following siRNA-mediated knock-down of EpCAM (Maetzel, et al., 2009, Munz, et al., 2004, Osta, et al., 2004). Equal amounts of total RNA (RIN score = 10) from control- and EpCAM-siRNA-treated FaDu cells were next subjected to a single, explorative cDNA microarray analysis with the aim to identify potential target genes of the signal transduction cascade of EpCAM. All genes, which were up- or down-regulated by at least two-fold (>2-fold; 716 genes up-regulated, 552 genes down-regulated), were then subjected to a Panther classification software analysis (http://www.pantherdb.org/). Special emphasis was put on the regulation of genes associated with signalling pathways. Angiogenesis, Alzheimer's disease-presenilin pathway, Wnt signalling pathway, inflammation and cadherin signalling pathways were most strongly affected by EpCAM knock-down. mRNA levels of selected genes differently regulated following partial knock-down of EpCAM included the co-regulated genes for secreted frizzled-related protein 5, protocadherin 10, dickkopf homolog 2, MMP24, SPARC, Myst3, ErbB4, MMP12, WISP2, MMP7, Nanos1, WNT9A, and the counter-regulated genes for MMP11, fibronectin 1, DACT3, WNT2B, WNT5A, APC2, Spondin 1, and cateninα2. All genes except for protocadherin 10, Myst3, ErbB4, and WNT2B were validated in RT-PCR assays. Since several matrix metalloproteases were regulated after EpCAM knock-down and EpCAM is known to correlate with invasion, we chose this protein family for in-depth analysis. Matrix metalloprotease 7 (MMP-7) is a soluble factor, which, unlike other family members, is preferentially expressed in epithelial cells (Wilson, et al., 1995). Since EpCAM also displays highest expression in epithelia, we chose to proceed with the analysis of MMP7. SiRNA-mediated knock-down of EpCAM resulted in a comparable 50% down-regulation of MMP7 mRNA levels in RT-PCR and qPCR.

Project description:Epithelial cell adhesion molecule EpCAM is a single transmembrane protein, which is involved in numerous cellular processes including cell adhesion, proliferation, maintenance of stemness of embryonic cells and progenitors, migration, and invasion. Activation of signal transduction by EpCAM is warranted by regulated intramembrane proteolysis and nuclear translocation of the intracellular domain EpICD. Here, we describe matrix metalloproteinase 7 (MMP-7) as a target gene of EpCAM signalling via EpICD nuclear translocation. EpCAM and MMP-7 expression pattern and levels positively correlated in vitro and in vivo, and were strongly elevated in primary carcinomas of the head and neck area. Hence, MMP-7 is a novel target of EpCAM signalling. FaDu hypopharynx carcinoma cells were transiently transfected with a control siRNA or an EpCAM-specific siRNA. After two days, expression of EpCAM was assessed upon flow cytometry with antibodies, which specifically bind within the EGF-repeat in the extracellular domain of EpCAM (Balzar, et al., 1999). Compared to control siRNA, EpCAM siRNA induced a mean 43% reduction of EpCAM expression at the cell surface. Reduction of EpCAM expression upon siRNA treatment was confirmed by RT-PCR and qPCR. EpCAM down-regulation resulted in diminished cell proliferation assessed upon cell counting. The observed 43% decrease in cell surface expression of EpCAM translated in cell numbers diminished by 35%. This is a confirmation of earlier findings on the reduced proliferation of breast and colon cancer cell lines following siRNA-mediated knock-down of EpCAM (Maetzel, et al., 2009, Munz, et al., 2004, Osta, et al., 2004). Equal amounts of total RNA (RIN score = 10) from control- and EpCAM-siRNA-treated FaDu cells were next subjected to a single, explorative cDNA microarray analysis with the aim to identify potential target genes of the signal transduction cascade of EpCAM. All genes, which were up- or down-regulated by at least two-fold (>2-fold; 716 genes up-regulated, 552 genes down-regulated), were then subjected to a Panther classification software analysis (http://www.pantherdb.org/). Special emphasis was put on the regulation of genes associated with signalling pathways. Angiogenesis, Alzheimer's disease-presenilin pathway, Wnt signalling pathway, inflammation and cadherin signalling pathways were most strongly affected by EpCAM knock-down. mRNA levels of selected genes differently regulated following partial knock-down of EpCAM included the co-regulated genes for secreted frizzled-related protein 5, protocadherin 10, dickkopf homolog 2, MMP24, SPARC, Myst3, ErbB4, MMP12, WISP2, MMP7, Nanos1, WNT9A, and the counter-regulated genes for MMP11, fibronectin 1, DACT3, WNT2B, WNT5A, APC2, Spondin 1, and cateninM-NM-12. All genes except for protocadherin 10, Myst3, ErbB4, and WNT2B were validated in RT-PCR assays. Since several matrix metalloproteases were regulated after EpCAM knock-down and EpCAM is known to correlate with invasion, we chose this protein family for in-depth analysis. Matrix metalloprotease 7 (MMP-7) is a soluble factor, which, unlike other family members, is preferentially expressed in epithelial cells (Wilson, et al., 1995). Since EpCAM also displays highest expression in epithelia, we chose to proceed with the analysis of MMP7. SiRNA-mediated knock-down of EpCAM resulted in a comparable 50% down-regulation of MMP7 mRNA levels in RT-PCR and qPCR. Comparison of FaDu cells expressing EpCam vs FaDu cells with downregulated EpCam

Project description:Human papillomavirus (HPV) is causative for a new and increasing form of head and neck squamous cell carcinomas (HNSCCs). Although localised HPV-positive cancers have a favourable response to radio-chemotherapy (RT/CT), the impact of HPV in advanced or metastatic HNSCC remains to be defined and targeted therapeutics need to be tested for cancers resistant to RT/CT. To this end, we investigated the sensitivity of HPV-positive and -negative HNSCC cell lines to TRAIL (tumour necrosis factor-related apoptosis-inducing ligand), which induces tumour cell-specific apoptosis in various cancer types. A clear correlation was observed between HPV positivity and resistance to TRAIL compared with HPV-negative head and neck cancer cell lines. All TRAIL-resistant HPV-positive cell lines tested were sensitised to TRAIL-induced cell death by treatment with bortezomib, a clinically approved proteasome inhibitor. Bortezomib-mediated sensitisation to TRAIL was associated with enhanced activation of caspase-8, -9 and -3, elevated membrane expression levels of TRAIL-R2, cytochrome c release and G2/M arrest. Knockdown of caspase-8 significantly blocked cell death induced by the combination therapy, whereas the BH3-only protein Bid was not required for induction of apoptosis. XIAP depletion increased the sensitivity of both HPV-positive and -negative cells to TRAIL alone or in combination with bortezomib. In contrast, restoration of p53 following E6 knockdown in HPV-positive cells had no effect on their sensitivity to either single or combination therapy, suggesting a p53-independent pathway for the observed response. In summary, bortezomib-mediated proteasome inhibition sensitises previously resistant HPV-positive HNSCC cells to TRAIL-induced cell death through a mechanism involving both the extrinsic and intrinsic pathways of apoptosis. The cooperative effect of these two targeted anticancer agents therefore represents a promising treatment strategy for RT/CT-resistant HPV-associated head and neck cancers.

Dataset Information

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

Publications

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

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