Oridonin inhibits the migration and epithelial-to-mesenchymal transition of small cell lung cancer cells by suppressing FAK-ERK1/2 signalling pathway

Abstract

Small cell lung cancer (SCLC) is a severe malignant with high morbidity; however, few effective and secure therapeutic strategy is used in current clinical practice. Oridonin is a small molecule from the traditional Chinese herb Rabdosia rubescens. This study mainly aimed to investigate the role of oridonin on inhibiting the process of H1688, a kind of small cell lung cancer cells from human. Oridonin could suppress H1688 cell proliferation and induce their apoptosis in a high dosage treatment (20 μmol/L). Meanwhile, cell migration was suppressed by oridonin (5 and 10 μmol/L) that did not affect cell proliferation and apoptosis. The expression level of E-cadherin was significantly increased, and the expression of vimentin, snail and slug was reduced after administration of oridonin. These expression changes were associated with the suppressed integrin β1, phosphorylation of focal adhesion kinase (FAK) and ERK1/2. In addition, oridonin (5 and 10 mg/kg) inhibited tumour growth in a nude mouse model; however, HE staining revealed a certain degree of cytotoxicity in hepatic tissue after treatment oridonin (10 mg/kg). Furthermore, the concentration of alanine aminotransferase (ALP) was significantly increased and lactate dehydrogenase (LDH) was reduced after oridonin treatment (10 mg/kg). Immunohistochemical analysis further revealed that oridonin increased E-cadherin expression and reduced vimentin and phospho-FAK levels in vivo. These findings indicated that oridonin can inhibit the migration and epithelial-to-mesenchymal transition (EMT) of SCLC cells by suppressing the FAK-ERK1/2 signalling pathway. Thus, oridonin may be a new drug candidate to offer an effect of anti-SCLC with relative safety.

Keywords: focal adhesion kinase; migration; oridonin; small cell lung cancer.

Figure 1

Effect of oridonin on the viability of H1688, BEAS‐2B and HBE cells. A, Chemical structure of oridonin. B, H1688, (C) BEAS‐2B and (D) HBE cells were treated with oridonin (0, 2.5, 5, 10, 20 and 40 μmol/L) for 24 and 48 h and assessed by MTT assay. The data represent the means ± SD of three independent experiments; *P < .05, **P < .01, *** P < .001 compared with the control

Figure 2

The effect of oridonin on the apoptosis of H1688 cells was determined with Hoechst 33258 and Annexin V‐TITC/PI staining. A, H1688 cells were treated with oridonin (0, 5, 10 and 20 μmol/L) for 24 h, and morphology changes in cell nuclei were observed by Hoechst 33258 staining. A marked increase in apoptotic cells exhibiting a heterogeneous intensity, chromatin condensation and fragmentation appeared after 24‐h treatment with 20 μmol/L oridonin. Scale bars = 50 μm. The pooled data from nine sections for each group are summarized in (B). C, Annexin V‐TITC/PI staining measured by flow cytometry revealed a significant increase in the apoptotic cell ratio in the 20 μmol/L oridonin treatment group. D, Quantification of the results of flow cytometry. ***P < .001; ns, not significant

Figure 3

The migration of H1688 cells was reduced by oridonin treatment via suppression of the EMT process. A, Cell migration in H1688 cells treated with oridonin (5 and 10 μmol/L) and control H1688 cells was determined by wound‐healing assay. Representative sections indicating a significant decrease in migration index after 24 h of oridonin treatment (left); scale bars = 100 μm. Analysis of data representing three independent experiments (right). B, Cell migration was determined by transwell assay after oridonin (0, 5 and 10 μmol/L) treatment for 24 h. Cells that travelled through the transwell membrane in each group were stained with crystal violet and obviously decreased after 24 h of oridonin (5 and 10 μmol/L) treatment (left); scale bars = 100 μm. Analysis of data representing three independent experiments (right). C, Western blotting was applied and showed that the protein expression of E‐cadherin was significantly increased after 24 h of oridonin (5 and 10 μmol/L) treatment, whereas the expression of vimentin, snail and slug was decreased in the oridonin‐treated groups. D, Relative mRNA expression of E‐cadherin, vimentin, snail and slug in H1688 cells was analysed by quantitative real‐time PCR and normalized to GAPDH mRNA expression. The data represent three independent experiments. E, Immunofluorescent staining was performed and showed that the expression of E‐cadherin (red) was increased and the expression of vimentin (green) was decreased in H1688 cells treated with oridonin (10 μmol/L) for 24 h. * P < .05, ** P < .01, *** P < .001

Figure 4

The anti‐EMT effect of oridonin was mediated by inactivation of the FAK/ERK1/2 signalling pathway. A, Representative Western blots showing that integrin β1 p‐FAK and p‐ERK1/2 levels were reduced in the oridonin (5 and 10 μmol/L) treatment groups (left). Bar graphs represent quantitative differences in the expression of integrin β1 p‐FAK and p‐ERK1/2 (right). Data represent the means ± SD of four independent experiments. B, Representative Western blots showing that PF573228 (10 μmol/L), an inhibitor of FAK, reduced the expression of p‐FAK and p‐ERK1/2, which was similar to the effect of oridonin (10 μmol/L). In addition, the expression of p‐ERK1/2 was down‐regulated by PD98059 (left), and data represent the means ± SD of four independent experiments (right). C, The protein expression of E‐cadherin and vimentin was detected by Western blotting (left), and the data represent the means ± SD of four independent experiments (right). D, The protein expression of snail and slug was detected by western blotting, and the data represent the means ± SD of four independent experiments (right). E, Representative sections indicated that the migration index wound space was significantly decreased after 24 h of oridonin (10 μmol/L), PF573228 (10 μmol/L) or PD98059 (10 μmol/L) treatment (left); scale bars = 100 μm. Analysis of data representing three independent experiments (right). F, Cells that travelled through the transwell membrane in each group were stained with crystal violet and obviously decreased after 24 h of oridonin (10 μmol/L), PF573228 (10 μmol/L) and PD98059 (10 μmol/L) treatment (left); scale bars = 100 μm. Analysis of data representing three independent experiments (right). *P < .05; **P < .01; ***P < .001

Figure 5

Specific knockdown of FAK, ERK1 and ERK2 could reduce the migration of H1688 cells. A, The mRNA levels of FAK, ERK1 and ERK2 were significantly reduced after 48 h of siRNA‐mediated knockdown. B, The protein expression of p‐ERK1, p‐ERK2, p‐FAK, E‐cadherin and vimentin was detected by Western blotting (left), and the data represent the means ± SD of four independent experiments (right). C, Representative sections indicated that the migration index wound space was significantly decreased after siRNA‐mediated knockdown (left); scale bars = 100 μm. Analysis of data representing three independent experiments (right). D, Cells that travelled through the transwell membrane in each group were stained with crystal violet and obviously decreased after siRNA‐mediated knockdown (left); scale bars = 100 μm. Analysis of data representing three independent experiments (right). *P < .05; **P < .01; ***P < .001

Figure 6

The daily injection of oridonin could reduce tumour weight and volume and exhibited a high level of safety. A, Representative images of tumour from three groups. 1: control group, 2: oridonin (5 mg/kg) group and 3: oridonin (10 mg/kg) group. Scale bars = 1 cm. B, The tumour width (W) and length (L) were measured by a micrometre every other day. Tumour volumes were calculated as (L × W²)/2. C, The tumour weight was significantly reduced after treatment with oridonin (5 and 10 mg/kg). D, Body weight was not obviously altered after treatment with oridonin (5 and 10 mg/kg). E, The administration of oridonin (5 and 10 mg/kg) had no effect on the health of nude mice, as assessed by body weight. F, H&E staining to examine major organs in oridonin‐treated mice. There was no obvious pathological change in the lung, heart and kidney; however, the hepatic cord was narrowed following treatment with a high concentration of oridonin; scale bars = 100 μm. *P < .05; **P < .01; ***P < .001

Figure 7

Histochemical analysis was used to detect the effect of oridonin on the expression of E‐cadherin, vimentin and p‐FAK in vivo in a nude mouse xenograft model. Scale bars = 50 μm