Oridonin inhibited epithelial-mesenchymal transition of laryngeal carcinoma by positively regulating LKB1/AMPK signaling

Abstract

Oridonin is the main bioactive component of Rabdosia rubescens, and its anticancer activity has been reported in a variety of cancers. However, the molecular mechanism of oridonin in laryngeal carcinoma remains unclear. In the present study, the cytotoxic effect of oridonin on laryngeal carcinoma Hep-2 and TU212 cell lines were initially detected by modified MTT assay. The results showed that oridonin had a dose-dependent anti-proliferative effect on laryngeal carcinoma Hep-2 and TU212 cells. Next, we found that oridonin significantly inhibited the migration and invasion of human laryngeal carcinoma Hep-2 and TU212 cell lines by wound healing assay and transwell assay. Subsequently, the results of quantitative real-time PCR assay and western blotting assay confirmed that oridonin upregulated the expression of E-cadherin while downregulated the expression of N-cadherin in a concentration-dependent manner at mRNA and protein levels. In addition, phosphorylation levels of liver kinase B1 (p-LKB1) and AMP-activated protein kinase (p-AMPK) were also elevated upon oridonin treatment. To further verify the role of LKB1/AMPK signaling pathway in laryngeal carcinoma, overexpression of LKB1 was constructed by plasmid transfection. The data exhibited that overexpression of LKB1 could further reinforce the increase of E-cadherin level and decrease of N-cadherin level mediated by oridonin. Additionally, AMPK inhibitor compound C could reverse anti-metastatic effect of oridonin on laryngeal carcinoma, and antagonise EMT expression. In contrast, AMPK activator AICAR presented the opposite effect. In conclusion, our study revealed that oridonin could remarkably reverse the epithelial-mesenchymal transition of laryngeal carcinoma by positively regulating LKB1/AMPK signaling pathway, which suggested that oridonin may be a potential candidate for the treatment of laryngeal carcinoma in the future.

Keywords: AMPK; EMT; LKB1; Laryngeal carcinoma; Oridonin.

Figure 1

Oridonin suppressed the proliferation of laryngeal carcinoma cells. (A) The chemical structure of oridonin. Hep-2 (B, D) and TU212 (C, E) cells with 90 percent density were exposed to different concentrations of oridonin for 24h (0, 1, 2.5, 5.0, 10.0, 15.0, 20.0, 30.0 μM). Then the viability of laryngeal carcinoma Hep-2 and TU212 cell lines was evaluated by MTT assay. The optical density and cell inhibitory rate were presented. Error bars indicated mean ± SD. Five independent experiments were performed.

Figure 2

Oridonin repressed metastatic phenotype of laryngeal carcinoma Hep-2 and TU212 cells. Wound healing assay was used for the detection of anti-migratory activity of oridonin on Hep-2 and TU212 cells. The width of scratches was assessed in negative control or oridonin (10 μM) group in Hep2 and TUT212 cells (2A, 2B). Using Transwell migration assay and Matrigel invasion assay, Hep-2 and TU212 cells were exposed to oridonin treatment (10 μM) for 24 h and the number of migrated or invaded cells per chamber was assessed. The experiments were performed in triplicate (****, P<0,0001).

Figure 3

Oridonin remarkably reversed EMT in laryngeal carcinoma cells. Quantitative real-time PCR was used to explore the mRNA level of E-cadherin and N-cadherin in Hep-2 and TU212 cell lines upon oridonin treatment (2.5, 5.0 and 10 μM) (A and B). Hep-2 and TU212 cells pre-treated with certain doses of oridonin (2.5, 5.0 and 10 μM) were subjected to western blotting for E-cadherin (E-Ca), N-cadherin (N-Ca) and β-actin (C and D). Representative protein bands from five experiments were shown. EMT, epithelial-mesenchymal transition.

Figure 4

Oridonin reversed EMT of laryngeal carcinoma by upregulating LKB1/AMPK signaling. Hep-2 and TU212 cells pre-treated with certain doses of oridonin (2.5, 5.0 and 10 μM) were subjected to western blotting for phosphorylated-LKB1, phosphorylated-AMPK and β-actin. Representative protein bands from five experiments were shown (A and B). (C and D) Cells overexpressing LKB1 by plasmid transfection were synergistically treated with oridonin to detect the change of EMT markers in Hep-2 and TU212 cells. Western blotting was used to analyze the expressions of phosphorylated-LKB1, phosphorylated-AMPK, E-cadherin (E-Ca), N-cadherin (N-Ca) and β-actin. EMT, epithelial-mesenchymal transition.

Figure 5

Inactivation of AMPK by Compound C (ComC) attenuated the anti-metastatic effect of oridonin on laryngeal carcinoma cells. (A and B) Transwell migration assay and Matrigel invasion assay were used to explore the metastatic phenotype of Hep-2 and TU212 cells with different treatments (oridonin with or without compound C). Five random fields were observed by microscopy. All the experiments were performed in triplicate. (C and D) Western blotting was performed to explore the expressions of phosphorylated-AMPK, E-cadherin (E-Ca), N-cadherin (N-Ca) and β-actin in Hep-2 and TU212 cells with different treatment (oridonin with or without compound C). Representative results from five independent experiments were shown.

Figure 6

Activation of AMPK by AICAR further weakened migration and invasion, and reversed EMT in oridonin-treated laryngeal carcinoma cells. (A and B) Using Transwell migration assay and Matrigel invasion assay, the migrated and invaded Hep-2 and TU212 cells were assessed upon different treatments (oridonin with or without AICAR). (C and D) Hep-2 and TU212 cells co-treated with oridonin and AICAR were immunoblotted for phosphorylated-AMPK, E-cadherin (E-Ca), N-cadherin (N-Ca) and β-actin. Representative results from five independent experiments were shown. EMT, epithelial-mesenchymal transition.

Figure 7

Oridonin prominently decreased laryngeal carcinoma tumorigenicity in a nude mouse xenograft model. (A) Representative picture of xenografts in control and oridonin groups were presented at the 18th day. 5 x10⁶ Hep-2 cells were subcutaneously injected into the right flank of nude mice. (B) The quantification analysis of tumor mass in control and oridonin groups were shown as mean ± SD (****P<0.0001). (C) Tumor volumes and (D) body weight of nude mice were detected every 3 days. The statistical results were presented as mean ± SD of five mice (***P<0.001, ****P<0.0001). (E) The protein levels of phosphorylated LKB1, phosphorylated AMPK, E-cadherin, N-cadherin and β-actin were detected by Western blot. The proteins were collected from the dissected tumor tissues of control and oridonin groups).