Fig 1.
Effects of cantharidin on cell viability in SAS, CAL-27, and SCC-4 human tongue carcinoma cells and primary normal oral epithelial cells.
Cells were treated with cantharidin (1 to 50 μM) for 24 h. The cell viability was subsequently analyzed by MTT assay. Data are presented as mean ± SEM of three independent experiments (n = 6). *P < 0.05 versus control group (Con).
Fig 2.
Effects of cantharidin on protein expressions of caspases in SAS human tongue carcinoma cells.
Cells were treated with cantharidin (10 μM) for 14 to 24 h. (A) The protein expressions of pro-caspase-9, cleaved form of caspase-9, pro-caspase-7, cleaved form of caspase-7, pro-caspase-3, cleaved form of caspase-3 were determined by Western blotting. The protein expression of α-tubulin was as an internal control. In B-C, the protein expressions were quantified by densitometry and analyzed by ImageQant TL 7.0 software. Data are presented as mean ± SEM of three independent experiments (n = 6). *P < 0.05 versus control group for pro-caspases (Con). #P < 0.05 versus control group for cleaved form caspases.
Fig 3.
Effects of cantharidin on mitochondrial transmembrane potential (MMP) and protein expressions of cytochrome c and AIF in SAS human tongue carcinoma cells.
(A) Cells were treated with cantharidin (1–30 μM) for 24 h. The MMP was analyzed by flow cytometry with a fluorescent dye DiOC6. (B) Cells were treated with cantharidin (10 μM) for 18 or 24 h. The cytosolic fraction was then subjected to Western blot analysis for cytochrome c and AIF. The protein expression of α-tubulin was as an internal control. The protein expressions were quantified by densitometry and analyzed by ImageQant TL 7.0 software. Data are presented as mean ± SEM of three independent experiments (n = 6). *P < 0.05 versus control group (Con).
Fig 4.
Effects of cantharidin on protein expressions of Bax, Bid, Bak, and Bcl-2 in SAS human tongue carcinoma cells.
(A) Cells were treated with cantharidin (10 μM) for 1 to 4 h. The protein expressions were analyzed by Western blot analysis. The protein expression of α-tubulin was as an internal control. In B, the protein expressions were quantified by densitometry and analyzed by ImageQant TL 7.0 software. Data are presented as mean ± SEM of three independent experiments (n = 6). *P < 0.05 versus control group.
Fig 5.
Effects of cantharidin on protein expressions of phospho-eIF-2α, CHOP, Grp78, Grp94, and procaspase-12 in SAS human tongue carcinoma cells.
Cells were treated with cantharidin (10 μM) for 6 to 24 h. (A) The protein expressions of phospho-eIF-2α, CHOP, Grp78, Grp94 and procaspase-12 were analyzed by Western blotting. The protein expression of α-tubulin was as an internal control. In B, the protein expressions were quantified by densitometry and analyzed by ImageQant TL 7.0 software. Data are presented as mean ± SEM of three independent experiments (n = 6). *P < 0.05 versus control group.
Fig 6.
Effects of cantharidin on protein expressions of JNK, ERK, and p38 and their phosphorylation in SAS human tongue carcinoma cells.
Cells were treated with cantharidin (10 μM) for 1 to 4 h. (A) The protein expression of JNK, ERK, and p38 and their phosphorylation were analyzed by Western blotting. In B, the protein expressions were quantified by densitometry and analyzed by ImageQant TL 7.0 software. Data are presented as mean ± SEM of three independent experiments (n = 6). *P < 0.05 versus control group for phospho-JNK.
Fig 7.
Transfection of shRNA-JNK inhibited cantharidin-induced JNK phosphorylation in SAS human tongue carcinoma cells.
(A) Cells were transfected with sh-control (siRNA-con) or shRNA-JNK for 48 h, and the JNK-1 mRNA expression was detected by qPCR analysis. Data are presented as mean ± SEM of three independent experiments. *P < 0.05 versus sh-control group. (B) Cells were transfected with shRNA-JNK for 48 h, and then treated with cantharidin (10 μM) for 1 h. The JNK3/1 protein expression and phosphorylation were analyzed by Western blotting. The protein expression of α-tubulin was as an internal control. The protein expressions were quantified by densitometry and analyzed by ImageQant TL 7.0 software. Data are presented as mean ± SEM of three independent experiments. *P < 0.05 versus shRNA-control group. P < 0.05 versus shRNA-control with cantharidin group. (C) Cells were pretreatment with shRNA-JNK for 48 h, and then added cantharidin (10 μM) for 24 h. The cell viability was analyzed by MTT assay. Data are presented as mean ± SEM of three independent experiments (n = 6). *P < 0.05 versus sh-control group. #P < 0.05 versus shRNA-control combined with cantharidin group.
Fig 8.
Transfection of shRNA-JNK inhibited cantharidin-induced MMP depolarization and apoptosis in SAS human tongue carcinoma cells.
Cells were pretreatment with sh-control or shRNA-JNK for 48 h, and then added cantharidin (10 μM) for 24 h. Both MMP and apoptosis were analyzed by flow cytometry with the staining of DiOC6 and annexin V/PI, respectively. All data are presented as mean ± SEM of three independent experiments (n = 6). *P < 0.05 versus sh-control group. #P < 0.05 versus sh-control combined with cantharidin group.
Fig 9.
Transfection of shRNA-JNK reversed the effects of cantharidin on protein expressions of Bcl-2, Bax, phospho-eIF-2α, CHOP, and cleaved caspase-3 in SAS human tongue carcinoma cells.
Cells were transfected with sh-control (siRNA-con) or shRNA-JNK for 48 h, and then treated with cantharidin (10 μM) for 4 h (A) or 24 h (B). The protein expressions of Bcl-2 and Bax (A) and phospho-eIF-2α, CHOP, and cleaved caspase-3 (B) were analyzed by Western blottingh. The protein expression of α-tubulin was as an internal control. The protein expressions were quantified by densitometry and analyzed by ImageQant TL 7.0 software. Data are presented as mean ± SEM of three independent experiments (n = 6). *P < 0.05 versus sh-control group. #P < 0.05 versus sh-control combined with cantharidin group.
Fig 10.
The schematic representation of proposed mechanisms of cantharidin on oral squamous cell carcinoma cells.
Cantharidin induced cell apoptosis via the JNK-regulated mitochondrial and ER stress signaling pathways.