CCL21/CCR7 promotes G2/M phase progression via the ERK pathway in human non-small cell lung cancer cells

Abstract

C-C chemokine receptor 7 (CCR7) contributes to the survival of certain cancer cell lines, but its role in the proliferation of human non-small cell lung cancer (NSCLC) cells remains vague. Proliferation assays performed on A549 and H460 NSCLC cells using Cell Counting Kit-8 indicated that activation of CCR7 by its specific ligand, exogenous chemokine ligand 21 (CCL21), was associated with a significant linear increase in cell proliferation with duration of exposure to CCL21. The CCL21/CCR7 interaction significantly increased the fraction of cells in the G(2)/M phase of the cell cycle as measured by flow cytometry. In contrast, CCL21/CCR7 had no significant influence on the G(0)/G(1) and S phases. Western blot and real-time PCR indicated that CCL21/CCR7 significantly upregulated expression of cyclin A, cyclin B1, and cyclin-dependent kinase 1 (CDK1), which are related to the G(2)/M phase transition. The expression of cyclin D1 and cyclin E, which are related to the G(0)/G(1) and G(1)/S transitions, was not altered. The CCL21/CCR7 interaction significantly enhanced phosphorylation of extracellular signal-regulated kinase (P-ERK) but not Akt, as measured by Western blot. LY294002, a selective inhibitor of PI3K that prevents activation of the downstream Akt, did not weaken the effect of CCL21/CCR7 on P-ERK. Coimmunoprecipitation further confirmed that there was an interaction between P-ERK and cyclin A, cyclin B1, or CDK1, particularly in the presence of CCL21. CCR7 small interfering RNA or PD98059, a selective inhibitor of MEK that disrupts the activation of downstream ERK, significantly abolished the effects of exogenous CCL21. These results suggest that CCL21/CCR7 contributes to the time-dependent proliferation of human NSCLC cells by upregulating cyclin A, cyclin B1, and CDK1 potentially via the ERK pathway.

Figure 1. Efficiency of CCR7 siRNA in A549 or H460 cells.

A549 (A) and H460 (B) cells were transfected with control siRNA or CCR7 siRNA (siCCR7). After transfection, the expression of CCR7 protein (a) and mRNA (b) was evaluated using Western blot (a) and RT-PCR (b) and compared to untransfected A549 or H460 cells. Each bar represents the mean ± SD of three independent experiments. *p<0.05, compared with control cells.

Figure 2. Effect of CCL21 at various concentrations on proliferation of A549 and H460 cells.

A549 (A) and H460 (B) cells were treated with CCL21 (50, 100 or 200 ng/mL) for 24, 48, or 72 h, and cell vitality was estimated using the CCK-8 assay. Each bar represents the mean ± SD of three independent experiments. **p<0.01, compared with cells treated with CCL21 (50 ng/mL).

Figure 3. Effect of CCL21/CCR7 on proliferation of A549 and H460 cells.

A549 (A) and H460 (B) cells were treated with CCL21 (100 ng/mL) for 24, 48, or 72 h, and cell vitality was estimated using the CCK-8 assay. Each bar represents the mean ± SD of three independent experiments. **p<0.01, compared with control cells.

Figure 4. Effect of CCL21/CCR7 on the expression of cyclins and cyclin-dependent kinases in A549 and H460 cells.

A549 (A) and H460 (B) cells were treated with CCL21 (100 ng/mL) for 24 h, and the protein (a) and mRNA (b) levels of cyclins A, B1, D1, and E and of CDK1 were estimated using Western blot (a) and real-time PCR (b). Each bar represents the mean ± SD of three independent experiments. *p<0.05 or **p<0.01, compared with control cells.

Figure 5. Effect of CCL21/CCR7 on the expression of ERK, JNK, p38, and Akt in A549 and H460 cells.

A549 (A) and H460 (B) cells were treated with CCL21 (100 ng/mL) for 12, 24, or 48 h, and normal and phosphorylated (P-) expression levels were estimated by Western blot. Each bar represents the mean ± SD of three independent experiments. *p<0.05 or **p<0.01, compared with control cells.

Figure 6. Effects of CCL21/CCR7 on the expression of P-ERK after inhibiting activation of Akt in A549 and H460 cells.

A549 (A) and H460 (B) cells were treated with CCL21 (100 ng/mL) for 24 h after LY294002, a selective inhibitor of PI3K that consequently prevents activation of the downstream Akt, was applied for 1 h. The expression of P-ERK was estimated using Western blot. Each bar represents the mean ± SD of three independent experiments. **p<0.01, compared with control cells.

Figure 7. Effect of CCL21/CCR7 on proliferation of A549 and H460 cells after inhibiting ERK activation.

A549 (A) and H460 (B) cells were treated with CCL21 (100 ng/mL) for 24, 48, or 72 h after PD98059, a selective inhibitor of MEK that disrupts activation of downstream ERK, was applied for 1 h. Cell vitality was estimated using the CCK-8 assay. Each bar represents the mean ± SD of three independent experiments. *p<0.05 or **p<0.01, compared with control cells.

Figure 8. Effect of CCL21/CCR7 on the expression of P-ERK, cyclin A, cyclin B1, and CDK1 after inhibiting the activation of ERK.

A549 (A) and H460 (B) cells were treated with CCL21 (100 ng/mL) for 24 h after PD98059, a selective inhibitor of MEK that disrupts activation of downstream ERK, was applied for 1 h. The expression levels of these components were estimated using Western blot. Each bar represents the mean ± SD of three independent experiments. *p<0.05 or **p<0.01, compared with control cells.

Figure 9. The interaction between P-ERK and cyclin A, cyclin B1, or CDK1 in the absence or presence of CCL21 or PD98059.

A549 (A) and H460 (B) cells in the absence or presence of CCL21 (100 ng/mL) for 24 h were subjected to immunoprecipitation with antibodies against P-ERK or IgG, followed by Western blotting for cyclin A, cyclin B1, and CDK1 (a). Reciprocal immunoprecipitation with antibodies against cyclin A, cyclin B1, CDK1, or IgG were analyzed by Western blotting for P-ERK (b). A549 and H460 cells in the absence or presence of PD98059 for 1 h were subjected to immunoprecipitation with P-ERK or IgG antibodies followed by Western blotting for cyclin A, cyclin B1, and CDK1 (c).