Cantharidin Induced Oral Squamous Cell Carcinoma Cell Apoptosis via the JNK-Regulated Mitochondria and Endoplasmic Reticulum Stress-Related Signaling Pathways

<div><p>Oral cancer is a subtype of head and neck cancer which represents 2.65% of all human malignancies. Most of oral cancer is histopathologically diagnosed as oral squamous cell carcinoma (OSCC). OSCC is characterized by a high degree of local invasion and a high rate of metastasis to the cervical lymph nodes. How to prevention and treatment of OSCC is important and imperative. Here, we investigated the therapeutic effect and molecular mechanism of cantharidin, an active compound isolated from blister beetles, on OSCC <i>in vitro</i>. Results showed that cantharidin significantly decreased cell viability in human tongue squamous carcinoma-derived SAS, CAL-27, and SCC-4 cell lines. The further mechanistic studies were carried out in SAS cells. Cantharidin also significantly increased apoptosis-related signals, including caspase-9, caspase-7 and caspase-3 proteins. Besides, cantharidin decreased mitochondrial transmembrane potential (MMP) and induced cytochrome c and apoptosis inducing factor (AIF) release. Cantharidin also increased Bax, Bid, and Bak protein expressions and decreased Bcl-2 protein expression. Cantharidin could also increase the endoplasmic reticulum (ER) stress signals, including the expressions of phosphorylated eIF-2α and CHOP, but not Grp78 and Grp94. Furthermore, cantharidin reduced pro-caspase-12 protein expression. In signals of mitogen-activated protein kinases, cantharidin increased the phosphorylation of JNK, but not ERK and p38. Transfection of shRNA-JNK to OSCC cells effectively reversed the cantharidin-induced cell apoptotic signals, including the mitochondrial and ER stress-related signaling molecules. Taken together, these findings suggest that cantharidin induces apoptosis in OSCC cells via the JNK-regulated mitochondria and ER stress-related signaling pathways.</p></div

The schematic representation of proposed mechanisms of cantharidin on oral squamous cell carcinoma cells.

<p>Cantharidin induced cell apoptosis via the JNK-regulated mitochondrial and ER stress signaling pathways.</p

Effects of cantharidin on protein expressions of caspases in SAS human tongue carcinoma cells.

<p>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). *<i>P</i> < 0.05 versus control group for pro-caspases (Con). #<i>P</i> < 0.05 versus control group for cleaved form caspases.</p

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.

<p>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). *<i>P</i> < 0.05 versus sh-control group. #<i>P</i> < 0.05 versus sh-control combined with cantharidin group.</p

Transfection of shRNA-JNK inhibited cantharidin-induced JNK phosphorylation in SAS human tongue carcinoma cells.

<p>(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. *<i>P</i> < 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. *<i>P</i> < 0.05 versus shRNA-control group. <i>P</i> < 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). *<i>P</i> < 0.05 versus sh-control group. #<i>P</i> < 0.05 versus shRNA-control combined with cantharidin group.</p

Effects of cantharidin on cell viability in SAS, CAL-27, and SCC-4 human tongue carcinoma cells and primary normal oral epithelial cells.

<p>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). *<i>P</i> < 0.05 versus control group (Con).</p

Effects of cantharidin on mitochondrial transmembrane potential (MMP) and protein expressions of cytochrome c and AIF in SAS human tongue carcinoma cells.

<p>(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). *<i>P</i> < 0.05 versus control group (Con).</p

Effects of cantharidin on protein expressions of phospho-eIF-2α, CHOP, Grp78, Grp94, and procaspase-12 in SAS human tongue carcinoma cells.

<p>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). *<i>P</i> < 0.05 versus control group.</p

Transfection of shRNA-JNK inhibited cantharidin-induced MMP depolarization and apoptosis in SAS human tongue carcinoma cells.

<p>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). *<i>P</i> < 0.05 versus sh-control group. #<i>P</i> < 0.05 versus sh-control combined with cantharidin group.</p

Effects of cantharidin on protein expressions of JNK, ERK, and p38 and their phosphorylation in SAS human tongue carcinoma cells.

<p>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). *<i>P</i> < 0.05 versus control group for phospho-JNK.</p