Role of JNK activation in paclitaxel-induced apoptosis in human head and neck squamous cell carcinoma

Abstract

It has been reported that paclitaxel activates cell cycle arrest and increases caspase protein expression to induce apoptosis in head and neck squamous cell carcinoma (HNSCC) cell lines. However, the potential signaling pathway regulating this apoptotic phenomenon remains unclear. The present study used OEC-M1 cells to investigate the underlying molecular mechanism of paclitaxel-induced apoptosis. Following treatment with paclitaxel, cell viability was assessed via the MTT assay. Necrosis, apoptosis, cell cycle and mitochondrial membrane potential (∆Ψm) were analyzed via flow cytometric analyses, respectively. Western blot analysis was performed to detect the expression levels of proteins associated with the MAPK and caspase signaling pathways. The results demonstrated that low-dose paclitaxel (50 nM) induced apoptosis but not necrosis in HNSCC cells. In addition, paclitaxel activated the c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase or p38 mitogen-activated protein kinase. The paclitaxel-activated JNK contributed to paclitaxel-induced apoptosis, activation of caspase-3, -6, -7, -8 and -9, and reduction of ∆Ψm. In addition, caspase-8 and -9 inhibitors, respectively, significantly decreased paclitaxel-induced apoptosis. Notably, Bid was truncated following treatment with paclitaxel. Taken together, the results of the present study suggest that paclitaxel-activated JNK is required for caspase activation and loss of ∆Ψm, which results in apoptosis of HNSCC cells. These results may provide mechanistic basis for designing more effective paclitaxel-combining regimens to treat HNSCC.

Keywords: HNSCC; JNK; apoptosis; caspase; paclitaxel.

Figure 1.

Low-dose paclitaxel (50 nM) induces apoptosis but not necrosis in OEC-M1 cells. Cells were treated without or with different concentrations of paclitaxel (5, 50, 250, 500 nM and 1 µM) for 48 h, and the percentage of (A) cell viability and (B) necrotic cell death were determined via the MTT assay and FACScan analysis following Annexin V and PI staining, respectively. (C) Cells were treated with or without 50 nM paclitaxel for 12, 24, 36 and 48 h. Following PI staining, cell cycle events were measured via FACScan analysis. Fractions of subG₁ phase (apoptosis) were quantified using CellQuest software. *P<0.05 vs. control. PI, propidium iodide.

Figure 2.

Activation of the MAPK pathway in low-dose paclitaxel-treated OEC-M1 cells. Cells were treated with control or 50 nM paclitaxel at different time points. (A) Western blot analysis was performed to detect the protein expression levels of JNK, ERK and p38 MAPK pathways in OEC-M1 cells. Activation of (B) JNK, (C) ERK and (D) p38 were quantified using Quantity One image analysis system. OEC-M1 cells were treated with control or 50 nM paclitaxel, without or with 10 µM SP, 5 µM PD and 5 µM SB, respectively, and (E) the expression levels of cytokeratin 18 were determined via ELISA. *P<0.05 vs. control; ^#P<0.05 vs. paclitaxel alone treatment. MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; C, control, P, paclitaxel; SP, JNK inhibitor-SP600125; PD, ERK inhibitor-PD184352; SB, p38 inhibitor-SB202190.

Figure 3.

JNK inhibitor (SP600125) inhibits paclitaxel-induced apoptosis and caspases effectors in OEC-M1 cells. JNK inhibitor, SP600125, (0.1, 1, 10 and 20 µM) inhibited paclitaxel-induced phosphorylation of (A) JNK and cleaved caspase-3, and (B) cleaved caspase-6 and cleaved caspase-7. Cells (6×10⁵) were subsequently treated with 50 nM paclitaxel, with or without different concentrations of SP600125 (10 and 20 µM) for 24 h and fixed in 70% alcohol. (C) Following PI staining, cell cycle events were measured via FACScan analysis. Fractions of (D) subG₁ phase (apoptosis), (E) G₀/G₁ phase and (F) G₂/M phase were quantified using CellQuest software. In (C) red and blue dotted lines were plotted to illustrate the changes of subG₁ (left to red line), G₀/G₁ (between red and blue lines) and G₂/M phases (right to blue line) in the different treatment groups. *P<0.05 vs. control; ^#P<0.05 vs. paclitaxel alone treatment. JNK, c-Jun N-terminal kinase; PI, propidium iodide.

Figure 4.

JNK inhibition prevents paclitaxel-induced activation of caspase-8 and −9 initiators in OEC-M1 cells. Cells were treated with 50 nM paclitaxel, with or without different concentrations of SP600125 (0.1, 1, 10 and 20 µM) for 24 h. Cleavage of initiator (A) caspase-8 and (B) caspase-9 were detected via western blotting and quantified, respectively. *P<0.05 vs. control; ^#P<0.05 vs. paclitaxel alone treatment. JNK, c-Jun N-terminal kinase.

Figure 5.

Caspase inhibitors partially rescue paclitaxel-induced apoptosis in OEC-M1 cells. (A) Cells were treated with 50 nM paclitaxel with either caspase-8 (Z-IETD-FMK) and/or caspase-9 (Z-LEHD-FMK) inhibitor (100 µM) for 24 h, and cell cycle events were measured via FACScan analysis following PI staining. Fractions of (B) subG₁ phase, (C) G₀/G₁ phase and (D) G₂/M phase were quantified using CellQuest software. (E) Combined usage of these two caspase inhibitors, with or without treatment with 50 nM paclitaxel for 24 h, and cell cycle events were measured via FACScan analysis following PI staining. (F) Fractions of subG₁ phase were quantified using CellQuest software. In (A) and (E) red and blue dotted lines were plotted to illustrate the changes of subG₁ (left to red line), G₀/G₁ (between red and blue lines) and G₂/M phases (right to blue line) in the different treatment groups. *P<0.05 vs. control; ^#P<0.05 vs. paclitaxel alone treatment. PI, propidium iodide.

Figure 6.

Inactivation of JNK inhibits late-phase (48 h) ∆Ψm loss in OEC-M1 cells. (A) Paclitaxel induced time-dependent ∆Ψm loss in OEC-M1 cells. (B) Inactivation of JNK with specific JNK inhibitor (SP600125, 10 µM) only significantly rescued the late-phase (48 h) ∆Ψm loss. (C) Bid truncation occurred at 36 and 48 h following treatment with paclitaxel. *P<0.05 vs. control; ^#P<0.05 vs. paclitaxel alone treatment. JNK, c-Jun N-terminal kinase; ∆Ψm, mitochondrial membrane potential; C, control, P, paclitaxel.

Figure 7.

Potential signaling pathways of paclitaxel-induced apoptosis in OEC-M1 cells. Paclitaxel-induced JNK activation plays an important role in promoting loss of ∆Ψm and activation of pro-apoptotic proteins, resulting in apoptosis of OEC-M1 cells. JNK, c-Jun N-terminal kinase; ∆Ψm, mitochondrial membrane potential.