Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer. OSCC cells are highly invasive, a characteristic that involves epithelial-mesenchymal transition (EMT); the conversion of immotile epithelial cells into motile mesenchymal cells. EMT is involved in the progression of various types of cancer by promoting tumour cell scattering and conferring to these cells cancer stem cell (CSC)-like characteristics, such as self-renewal. Hepatocyte growth factor (HGF) signalling plays an important role in EMT induction and, therefore, contributes to cell invasion and metastasis in cancer. Due to its potential chemopreventative and anti-tumour activities, curcumin has attracted much interest and has been shown to act as a potent EMT inhibitor in various types of cancer. However, at present, the potential effects of curcumin on HGF-induced EMT in OSCC have not been investigated. Here, we demonstrated that HGF signalling could induce EMT in the HSC4 and Ca9-22 OSCC cell lines via the HGF receptor c-Met and downstream activation of the pro-survival ERK pathway. Notably, curcumin inhibited HGF-induced EMT and cell motility in HSC-4 and Ca9-22 cells via c-Met blockade. Therefore, these findings establish curcumin as a candidate drug for OSCC treatment. Furthermore, curcumin was able to effectively inhibit the HGF-induced increase in the levels of vimentin by downregulating the expression of phosphorylated c-Met, an ERK. In conclusion, the results of the present study demonstrated that curcumin was able to reverse HGF-induced EMT, possibly by inhibiting c-Met expression in oral cancer cells, providing a strong basis for the development of novel approaches for the treatment of oral cancer.
Despite progress in the treatment of cancer through surgery, radiotherapy, and chemotherapy, the incidence of cancer remains high, which is largely due to the aging and growth of the world population. This high incidence of cancer is also associated with an increase in cancer-causing behaviours, particularly smoking, in economically developing countries (
Initially identified during embryogenesis, epithelial-mesenchymal transition (EMT) has been described as an essential process related to cell differentiation and morphogenesis (
Hepatocyte growth factor (HGF) and its receptor c-Met have been implicated in EMT in numerous types of cancer (
The human tongue-derived OSCC cell line HSC-4 and Ca9-22 was purchased from the RIKEN BioResource Center (Ibaraki, Japan). HSC-4 and Ca9-22 cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% (v/v) foetal bovine serum (FBS) in a humidified incubator at 37°C and 5% CO2. DMEM and FBS were purchased from Gibco (Life Technologies, Tokyo, Japan). HGF and curcumin were purchased from Sigma-Aldrich. All antibodies used in this study were commercially available and included antibodies against c-Met and phosphorylated-cMet (phospho-c-Met, Tyr1234/1235) (Cell Signaling Technology), α-tubulin (Sigma-Aldrich), E-cadherin and vimentin (Merck Millipore), and ERK and phospho-Tyr204-ERK (Tyr204) (both from Santa Cruz Biotechnology).
Cell migration was determined using a scratch wound healing assay, as described previously (
The gelatinolytic activity of HSC-4 cells was examined by gelatin zymography. Proteins from serum-free conditioned medium were diluted in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and separated using a 12.5% (w/v) polyacrylamide gel containing 0.1% (w/v) gelatin. For each sample, the amount of material loaded onto the gel was corrected for the number of cells in culture and corresponded to the proteins secreted by ten cells. Electrophoresis was carried out for 2 h at 60 mA and 4°C, and the gel was incubated overnight in 50 mM Tris-HCl buffer (pH 7.5) containing 10 mM CaCl2 and 1X Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). Finally, the gel was stained using 0.25% Coomassie Blue in a methanol:acetic acid:water (50:10:40) solution and destained using the same solution without dye. Negative staining (white bands against a dark background) indicated proteolysis and, therefore, gelatinolytic activity.
Following treatment, HSC-4 and Ca9-22 cells were collected, washed with PBS, and lysed using RIPA buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% (v/v) Nonidet P-40, 0.5% (w/v) deoxycholic acid, 0.1% (w/v) SDS, 5 mM ethylenediaminetetraacetic acid, 1X Halt Protease Inhibitor Cocktail, and 1X Halt Protein Phosphatase Inhibitor (Thermo Fisher Scientific, Inc.). The protein concentration of the cell lysates was determined using a BCA Protein Assay kit (Thermo Fisher Scientific, Inc.), and equal amounts of protein were subjected to SDS-PAGE. The separated proteins were transferred onto a PVDF membrane (GE Healthcare), which was then blocked for 1 h at room temperature with 5% (w/v) bovine serum albumin in Tris-buffered saline (TBS)/Tween-20 (TBS-T) to prevent non-specific binding. The membrane was incubated overnight at 4°C with antibodies diluted in TBS-T, washed, and incubated with HRP-conjugated secondary antibodies diluted in TBS-T. Finally, antibody-antigen complexes were detected using the ECL Plus Western Blotting Detection Reagent (GE Healthcare).
All data are presented as the mean ± standard deviation from three independent experiments unless stated otherwise. The statistical significance of a difference between two groups was evaluated using unpaired Student's t-test. P<0.05 was considered to indicate a statistically significant difference.
To assess the effect of curcumin on oral cancer cells, we first examined HGF-induced cell motility of HSC-4 cells using an invasion assay (
To further assess the effect of curcumin on HGF-induced cell motility, we also examined cell migration using a wound healing assay (
The concomitant down-regulation of the epithelial marker E-cadherin and up-regulation of the mesenchymal marker vimentin is recognised as a hallmark of cells undergoing EMT (
As mentioned previously, HGF signalling plays an important role in EMT induction. A crucial step is the homodimerisation and autophosphorylation of the c-Met receptor tyrosine kinase upon HGF binding (
The role of HGF signalling in the induction of EMT is mediated via the downstream activation of the AKT and ERK effector pathways (
Matrix metalloproteinases (MMPs) are major regulators of the extracellular matrix and are known to play important roles in tumour invasion and metastasis (
EMT is a highly conserved cellular program that allows polarised, immotile epithelial cells to convert into motile mesenchymal cells. Importantly, EMT plays physiological roles during embryonic development, but also pathological roles in cancer. HGF signalling plays an important role in EMT induction and involves homodimerisation and autophosphorylation of its receptor c-Met, which induces the transcription of downstream target genes. Moreover, the activation of c-Met has been shown to promote invasion and metastasis, as well as angiogenesis and tumorigenesis. The functional diversity of HGF signalling has attracted much interest in the clinical setting due to its potential prognostic and therapeutic value (
In the present study, we demonstrated that HGF signalling could induce EMT in the HSC-4 OSCC cell line and promote both cell migration and invasion. Importantly, EMT in HSC-4 and Ca9-22 cells involved down- and up-regulation of E-cadherin and vimentin expression, respectively. Grotegut
MMPs are known to play important roles in tumour invasion (
In a previous study, Davies
The HSC-4 and Ca9-22 human OSCC cell line was used as an experimental model in this study. HSC-4 and Ca9-22 cells are negative for cancer stemness (
Moreover, Siddappa
In conclusion, this study demonstrated that curcumin could inhibit HGF-induced EMT and cell motility in an OSCC cell line via c-Met blockade and inhibition of the ERK effector pathway. Importantly, these findings suggested that curcumin was a potent drug targeting invasive oral cancer cells. Therefore, we believe that our data provide a strong theoretical and experimental basis for the development of novel approaches and drugs for the treatment of oral cancer.
Not applicable.
The present study was supported by a KAKENHI grant (grant no. JP18K09736) from The Japan Society for the Promotion of Science.
The datasets used and/or analysed during the present study are available from the corresponding author on reasonable request.
YO and MN contributed to the conception and design of the experiments. YO, TS and HY performed the experiments. LZ, HH and HK contributed to the study conception and design. All authors read and approved the final version of the manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
oral squamous cell carcinoma
epithelial-mesenchymal transition
cancer stem cells
hepatocyte growth factor
matrix metalloproteinases
Cur inhibits HGF-induced cell invasion. Control and HGF-induced HSC-4 oral cancer cells with and without cur pre-treatment were cultured in Matrigel chambers, and the number of invading cells was scored after 48 h. (A) Representative bright-field microscopy images showing invading cells in the Matrigel chambers. Magnification, ×20. (B) Histogram showing relative cell migration (%) of invading cells after 48 h of culture, compared with HGF-induced cells. Data are presented as the mean ± SD. n=3. *P<0.05 vs. HSC-4. Cur, curcumin; HGF, hepatocyte growth factor.
Cur inhibits HGF-induced cell migration. Control and HGF-induced HSC-4 oral cancer cells with and without cur pre-treatment were cultured in monolayers, and cell migration was measured by a scratch wound healing assay. (A) Representative brightfield microscopy images show wound healing at 0 and 12 h. Magnification, ×10. (B) Histogram showing relative cell migration (%), compared with control cells. Data are presented as the mean ± SD. n=3. ***P<0.001 vs. HSC-4. Cur, curcumin; HGF, hepatocyte growth factor.
Cur inhibits epithelial-mesenchymal transition in HGF-induced cells. Representative western blot showing the levels of E-cadherin and vimentin in whole cell lysates from control and HGF-induced HSC4 (left) and Ca9-22 oral cancer cells (right) with and without cur pre-treatment. α-tubulin was used as a loading control. Cur, curcumin; HGF, hepatocyte growth factor.
Cur inhibits c-Met phosphorylation in HGF-induced cells. Representative western blot showing the levels of c-Met and p-c-Met in whole cell lysates from control and HGF-induced HSC4 (left)and Ca9-22 oral cancer cells (right)with and without cur pre-treatment. α-tubulin was used as a loading control. Cur, curcumin; HGF, hepatocyte growth factor; p, phosphorylated.
Cur-mediated c-Met blockade results in the downregulation of ERK phosphorylation. Representative western blot showing the levels of ERK and phospho-ERK in whole cell lysates from control and HGF-induced HSC4 oral cancer cells with and without cur pre-treatment. α-tubulin was used as a loading control. Cur, curcumin; HGF, hepatocyte growth factor.
Cur represses the production of gelatinolytic activity. Representative gelatin zymography showing the levels of the two principal gelatinolytic activities (pro-MMP2 and pro-MMP9) in conditioned culture medium from control and HGF-induced HSC4 oral cancer cells with and without cur pre-treatment. Cur, curcumin; HGF, hepatocyte growth factor; MMP, matrix metalloproteinase.