Deguelin Induces Both Apoptosis and Autophagy in Cultured Head and Neck Squamous Cell Carcinoma Cells

<div><p>Head and neck squamous cell carcinoma (HNSCC) represents more than 5% of all cancers diagnosed annually in United States and around the world. Despite advances in the management of patients with this disease, the survival has not been significantly improved, and the search for potential alternative therapies is encouraging. Here we demonstrate that deguelin administration causes a significant HNSCC cell death. Deguelin induces both cell apoptosis and autophagy by modulating multiple signaling pathways in cultured HNSCC cells. Deguelin inhibits Akt signaling, and down-regulates survivin and cyclin-dependent kinase 4 (Cdk4) expressions, by disrupting their association with heat shock protein-90 (Hsp-90). Deguelin induces ceramide production through de novo synthase pathway to promote HNSCC cell death. Importantly, increased ceramide level activates AMP-activated protein kinase (AMPK), which then directly phosphorylates Ulk1 and eventually leads to cell autophagy. We found that a low dose of deguelin sensitized HNSCC cells to 5-FU. Finally, using a nude mice Hep-2 xenograft model, we also showed a significant anti-tumor ability of deguelin in vivo. Together, we suggest that deguelin may represent a novel and effective chemo-agent against HNSCC.</p> </div

Deguelin’s <i>in vivo</i> anti-tumor effects in a Hep-2 xenograft model.

<p>Female BALB/c nude mice, 8–10 weeks of age, weighing 22 to 34 g, were acclimatized for 1 week before being injected s.c. with 3.5×10⁶ Hep-2 cells that had been re-suspended in 100 µL of medium. After 5 days when established tumors around 0.3 cm³ in diameter were detected, mice were randomized and divide in two groups. Ten mice per group were orally treated with deguelin (4 mg/kg) on days 1, 3, and 5 of each week for 3 weeks. Control animals received equal volume of saline solution. Tumor size was measured weekly using method mentioned above (A). Mice survival rate were also measured in (B). <i>p<0.05</i> vs. vehicle control group. Statistical significance was analyzed by ANOVA.</p

AMPK-dependent autophagy pathway contributes to deguelin-induced Hep-2 cell death.

<p>Hep-2 cells were pretreated with Z-VAD-fmk (50 µM) or 3-MA (2 mM) for 1 hr before deguelin exposure for 72 hours, cell viability was measured by MTT assay (A). Hep-2 cells were exposed to 100 µM of deguelin for indicated time points, followed by western blot detecting LC3B, tubulin, phospho- and total- level of Ulk1 (B). The association between AMPKα1 and Ulk1 after deguelin exposure (100 µM, 12 h) was determined by IP assay (C–D). Hep-2 cells were transfected with scramble or AMPKα1 siRNA (100 nM) for 48 hours. Western blot was then utilized to test AMPKα expression in transfected cells. Successfully AMPK knocking-down cells and their parental cells were treated with deguelin for indicated time points. AMPK, LC3B, tubulin, phospho- and total- level of Ulk1 were tested by western blots (E), LC3B and phospho-Ulk1 levels were quantified (F). Hep-2 cells were pretreated with 3-MA (2 mM) for 1 hr before deguelin (100 µM) exposure for 72 hours, cell apoptosis was measured by enzyme-linked immunosorbent cell apoptosis assay (G) and hoechst nuclear staining (H). (I) The proposed signaling pathway in this study: deguelin induces both cell apoptosis and autophagy by modulating multiple signaling pathways in cultured HNSCC cells: deguelin inhibits Akt signaling, and down-regulates survivin and cyclin-dependent kinase 4 (Cdk4) expressions, by disrupting their association with Hsp-90. Deguelin induces ceramide production through <i>de novo</i> synthase pathway to promote HNSCC cell death. Ceramide activates AMPK, which directly phosphorylates Ulk1 to promote an early cell autophagy. Cell autophagy is pro-apoptotic in our system. The mean of at least three independent experiments performed in triplicate is shown. Statistical significance was analyzed by ANOVA.</p

Deguelin down-regulates Akt signaling.

<p>Hep-2 cells were exposed to deguelin at indicated concentration (10, 25, 50 and 100 µM) for 24 hours, total- and phospho- levels of Akt as well as phosphorylation level of S6K were measured by Western blot using indicated antibodies (A).The association between Akt and Hsp 90 and the association between Akt and ubiquitin after deguelin treatment (Deg, 100 µM, 12 hours) were measured by immunoprecipitation (IP) assay (B-C). Effect of MG-132 (1 µM, 12 hour pretreatment) on deguelin-induced Akt degradation was shown in (D–E). The mean of at least three independent experiments performed in triplicate is shown. Statistical significance was analyzed by ANOVA.</p

Deguelin induces cellular ceramide synthesis.

<p>Hep-2 cells were exposed to 100 µM of deguelin with or without fumonisin B1 (20 µM) for indicated time points, intracellular ceramide level was analyzed using methods mentioned above (A). Hep-2 cells were treated with fumonisin B1 (20 µM) or C6-ceramide (10 µg/ml), followed by deguelin (100 µM) exposure, MTT assay was used to test cell viability after 72 hours (B), Hoechst staining (C) and enzyme-linked immunosorbent cell apoptosis assay (D) were utilized to test Hep-2 cell apoptosis after 48 hours. The mean of at least three independent experiments performed in triplicate is shown. Statistical significance was analyzed by ANOVA.</p

Low dose of deguelin sensitizes Hep-2 cells to 5-FU.

<p>Hep-2 cells were exposure with the indicated concentration of 5-FU in the presence or absence of deguelin (10 µM), MTT assay was used to test cell viability after 72 hours (A), Hoechst 33342 nuclear staining (B) and enzyme-linked immunosorbent cell apoptosis assay (C) were used to test cell apoptosis after 48 hours. The mean of at least three independent experiments performed in triplicate is shown. Statistical significance was analyzed by ANOVA.</p

Deguelin down-regulates survivin and Cdk4.

<p>Hep-2 cells were exposed to deguelin at indicated concentration (10, 25, 50 and 100 µM) for 24 hours, expression levels of survivin, Cdk4 and tubulin were measured by western blot (A).The association between survivin, Cdk4 and Hsp 90 as well as the association between survivin, Cdk4 and ubiquitin after deguelin treatment (Deg, 100 µM, 12 hours) were detected by immunoprecipitation (IP) assay (B–C). Effect of MG-132 (1 µM, 12 hour pretreatment) on deguelin-induced Cdk4 and survivin degradation was shown in (D). Western blots was performed to test the expression of Akt, phos-Akt, survivin, Cdk4 and tubulin after deguelin treatment in A253 (E) and SCC-9 cells (F). The mean of at least three independent experiments performed in triplicate is shown. Statistical significance was analyzed by ANOVA.</p

Cytotoxic effects of deguelin in cultured HNSCC cells.

<p>Hep-2 cells were exposed to deguelin at a various concentration (10, 25, 50, 100 and 200 µM) for 72 hours (A) or exposed to 100 µM of deguelin for different time points (12, 24, 48 and 72 hours) (B), cell viability was then measured by MTT assay. (C) Three different cell lines A253, SCC-9 and PANC-1 were exposed to 100 µM of deguelin (Deg) for 72 hours; cell viability was measured by MTT assay. Hep-2 cells were treated with indicated concentration of deguelin, hoechst nuclear staining and enzyme-linked immunosorbent cell apoptosis assay were utilized to analyze cell apoptosis (D–E), caspase-3 activity was also measured (F), apoptosis related proteins including cleaved caspase-3, cleaved caspase-9, Bcl-Xl were detected by Western blots (G), Bcl-Xl expression level was quantified using image J software (H). The mean of at least three independent experiments performed in triplicate is shown. Statistical significance was analyzed by ANOVA. *<i>p</i><0.01 vs Control.</p

Deguelin activates AMPK signaling.

<p>Hep-2 cells were exposed to 100 µM of deguelin for indicated time points, followed by western blot detecting phospho- and total- level of LKB1-AMPK-ACC using specific antibodies (A). Hep-2 cells were transfected with scramble or AMPKα1RNAi (100 nM each) for 48 hours. Western blot was utilized to test AMPKα1 expression after transfection. Successfully AMPK knockdown cells and their parental cells were treated with deguelin (100 µM) in the presence or absence of AICAR (1 mM), phospho- and total- level of AMPKα1 were analyzed in (B–C), MTT assay was then used to test cell viability after 48 hours (D). Hep-2 cells were pretreated with fumonisin B1 (20 µM), C6-ceramide (10 µg/ml) or NAC (500 µM) for 1 hr before deguelin exposure for indicated time points, phospho- and total level of AMPKα1 as well as tubulin were tested by western blots (E–F). The mean of at least three independent experiments performed in triplicate is shown. Statistical significance was analyzed by ANOVA.</p