Sulforaphane suppresses oral cancer cell migration by regulating cathepsin S expression

Abstract

Sulforaphane has been demonstrated to exert numerous biological effects, such as neuroprotective, anti-inflammatory, and anticancer effects. However, the detailed effects of sulforaphane on human oral cancer cell migration and the underlying mechanisms remain unclear. In this study, we observed that sulforaphane attenuated SCC-9 and SCC-14 cell motility and invasiveness by reducing cathepsin S expression. Moreover, sulforaphane increased microtubule-associated protein 1 light chain 3 (LC3) conversion, and the knockdown of LC3 by siRNA increased cell migration ability. Regarding the mechanism, sulforaphane inhibited the cell motility of oral cancer cells through the extracellular signal-regulated kinase (ERK) pathway, which in turn reversed cell motility. In conclusion, sulforaphane suppress cathepsin S expression by inducing autophage through ERK signaling pathway. Thus, cathepsin S and LC3 may be new targets for oral cancer treatment.

Keywords: LC3; cathepsin S; migration; oral cancer; sulforaphane.

Figure 1. Effect of sulforaphane on cell viability and cell motility of SCC-9 and SCC-14 cells

SCC-9 (A) and SCC-14 (B) cells were treated with different concentrations (0, 2.5, 5 and 10 μM) of sulforaphane for 24 h and 48 h before being subjected to an MTT assay for cell viability. The values represented the means ± SD of at least three independent experiments. ^*p < 0.05, compared with the vehicle group. The motility of SCC-9 (C) and SCC-14 (D) cells were assessed by in vitro wound closure assay with different concentration of sulforaphane (0, 2.5, 5 and 10 μM) at different time points. A quantitative assessment of cell number in the denuded zone is the mean ± SD (n = 3). ^*p < 0.05, compared with the vehicle group.

Figure 2. Effect of sulforaphane on cell migration and invasion of SCC-9 and SCC-14 cells

The cell migration (A–B) and invasion (C–D) were measured using a Boyden chamber for 24 h and 48 h with polycarbonate filters. The number of cells which invaded the underside of the porous polycarbonate was counted for assessing the migration and invasion abilities of SCC-9 (A, C) and SCC-14 (B, D) cells. The values represented the means ± SD of at least 3 independent experiments. ^*p < 0.05, compared with the vehicle group.

Figure 3. Effect of sulforaphane on the protein expression of cathepsin S of SCC-9 and SCC-14 cells

(A) The cell protein lysate was collected and detected the protein expression by Human Proteinases Array. (B) The expressions of cathepsin S protein on the treatments of SCC-9 and SCC-14 cells were assessed by western blot. The values represented the means ± SD of at least 3 independent experiments. (C–D) SCC-9 and SCC-14 cells transfected with the siRNA of cathepsin S for 48 h and analyzed by western blot and Boyden chamber migration assay. (E–F). The cells were treated with cathepsin S inhibitor Z-FL-COCHO (20 μM) for 24 h then analyzed by western blot and Boyden chamber migration assay. The values represented the mean ± S.D. from 3 determinations per condition repeated 3 times. ^*p < 0.05, compared with the vehicle group.

Figure 4. Effect of sulforaphane on the LC3 expression of SCC-9 and SCC-14 cells

The expressions of LC3 protein on the treatments of (A) SCC-9 and (B) SCC-14 cells were assessed by western blot. The values represented the means ± SD of at least three independent experiments. ^*p < 0.05, compared with the vehicle group. (C). GFP-LC3 dots were observed after the cells transfected with the plasmid and sulforaphane treatment.

Figure 5. Effect of LC3 siRNA on the cell migration of SCC-9 and SCC-14 cells

(A) SCC-9 and (B) SCC-14 cells transfected with the siRNA of LC3 for 48 h and analyzed by Boyden chamber migration assay. The values represented the mean ± S.D. from 3 determinations per condition repeated 3 times. ^*p < 0.05, compared with the vehicle group. The (C) SCC-9 and (D) SCC-14 cells were treated with cathepsin S siRNA and cathepsin S inhibitor Z-FL-COCHO (20 μM) for 24 h then analyzed by western blot.

Figure 6. Effect of sulforaphane on the mTOR/AKT and MAPKs pathway

(A). After a 24 h culture in various concentrations of sulforaphane (0, 2.5, 5 and 10 μM) for 24 h, the lysates of SCC-9 cells were subjected to SDS-PAGE followed by western blots with anti-mTOR, anti-AKT, anti-Beclin 1, anti-ERK, anti-JNK and anti-p38 (total and phosphorylated) antibodies. (B–C). SCC-9 cells were pretreated with or without U0126 (10 μM) for 1 h and then incubated in the presence or absence of combined sulforaphane (10 μM) for 23 h. The weastern blot assay was used for measurement of LC3 expression. Analysis of (C) cell migration of SCC-9 cells were assessed as described in Methods Section. (D) SCC-9 cells were pretreated with or without ERK siRNA (80 pmole) for 24 h and then incubated in the presence or absence of combined sulforaphane (10 μM) for 24 h. The values represented the means ± SD of at least 3 independent experiments. ^*p < 0.05, compared with the vehicle group. ^#p < 0.05, compared with the sulforaphane treated group.