Topotecan inhibits cancer cell migration by down-regulation of chemokine CC motif receptor 7 and matrix metalloproteinases

Abstract

Aim: The aim of this study was to investigate the effect of topotecan (TPT) on cancer cell migration.

Methods: Growth inhibition of TPT was analyzed by MTT assay, and cancer cell migration was measured by transwell double chamber assay. To verify the effect of TPT on the chemokine receptors CXCR4 and CCR7, quantitative PCR, semi-quantitative PCR and Western blot analysis were performed. The secretion of MMP-2 and MMP-9 was detected by enzyme-linked immunosorbent assay (ELISA) and gelatin zymography. To evaluate possible contributions of CCR7 to MMP secretion, the overexpression vectors pcDNA3.1(+)-CCR7 and CCR7 siRNA were transiently transfected into MDA-MB-435 cells.

Results: TPT inhibited cancer cell migration in a dose-dependent manner. Additionally, TPT significantly decreased the expression of CCR7 in both MDA-MB-435 and MDA-MB-231 cells and moderately reduced the expression of CXCR4 in MDA-MB-435 cells. The secretion of MMPs (MMP-2, MMP-9) was also inhibited by TPT. Overexpression of CCR7 increased the secretion of MMP-2/9 and cancer cell migration, whereas knockdown of CCR7 reduced active MMP-2/9 production and migration of MDA-MB-435 cells.

Conclusion: TPT inhibited cancer cell migration by down-regulation of CCR7 and MMPs (MMP-2 and MMP-9).

Figure 1

Effect of TPT on cancer cell migration. The data shown are representatives of three independent experiments. (A) Evaluation of cell viability of MDA-MB-435 cells and MDA-MB-231 cells treated with different concentrations of TPT for 24 h. Cell survival was determined by MTT assay. (B) TPT decreased cancer cell migration in MDA-MB-435 cells and MDA-MB-231 cells. Cells were cultured with different doses of TPT for 24 h, and representative pictures of cancer cell migration are shown. Microscopic images of cells that migrated into the lower chamber. (C) Cell migration was quantified by counting migrated cells in five randomly selected fields 24 h after seeding. Control cells remained untreated. Data are presented as means±SD. ^bP<0.05, ^cP<0.01 vs control.

Figure 2

Effect of TPT on the expression of chemokine receptors in MDA-MB-435 and MDA-MB-231 cells. Cells were incubated with different concentrations of TPT for 24 h or cultured with 0.01 μmol/L TPT (MDA-MB-435) or 0.1 μmol/L TPT (MDA-MB-231) for different times. The Effect of TPT on the expression of chemokine receptors was analyzed by quantitative PCR, semi-quantitative PCR and Western blotting. Experiments were performed in triplicate and representative data are presented. (A) Gene expression of CXCR4 and CCR7 in MDA-MB-435 and MDA-MB-231 cells challenged with TPT. Total cellular RNA was extracted and subjected to RT-PCR using specific primers. (B) Relative gene expression of CXCR4 and CCR7 in MDA-MB-231 and MDA-MB-435 cells. Total RNA was tested for CXCR4 and CCR7 mRNA levels by qPCR. (C) CXCR4 and CCR7 proteins were detected by Western blotting. Protein samples were collected and separated by 12% SDS–PAGE and immunoblotted with the anti-CXCR4 and anti-CCR7 antibodies. ^bP<0.05, ^cP<0.01 vs control group.

Figure 3

Effect of TPT on the secretion of MMPs in MDA-MB-435 (A) and MDA-MB-231 cells (B). Cells were treated with increasing concentrations of TPT for 24 h, after which supernatants were collected and active MMP-2/9 secretion was analyzed by ELISA and gelatin zymography. Results are representatives of three independent experiments. Asterisks indicate significant difference (^bP<0.05, ^cP<0.01). Upper panel, ELISA; lower panel, gelatin zymography.

Figure 4

Role of CCR7 in mediating MMP secretion and cancer cell migration. The secretion of MMP-2 and MMP-9 was increased after plasmid pcDNA3.1⁺-CCR7 was transiently transfected into MDA-MB-435 cells (435/CCR7); 50 nmol/L CCR7 siRNA (siCCR7) reduced the production of both MMPs. Cells transfected with empty vector pcDNA3.1⁺ (435/EV) and untreated cells were used as controls. MMP production was measured by ELISA (A) and gelatin zymography (B). Overexpression of CCR7 promoted cell migration, whereas knockdown of CCR7 decreased cell migration in MDA-MB-435 cells. (C) Representative photos of cancer cell migration are shown. (D) Cell migration was quantified by counting migrated cells in five randomly selected fields 24 h after seeding. Data are presented as means±SD. ^bP<0.05, ^cP<0.01 vs control.

Figure S1

Knockdown of CCR7 by RNA interference. (A) Expression of CCR7 mRNA in MDA-MB-435 cells transfected with different concentrations of CCR7 siRNA. Relative gene expression was measured by quantitative PCR; (B) CCR7 protein was detected by Western blotting in MDA-MB-435 cells transfected with different concentrations of CCR7 siRNA. Data shown were representatives of three independent experiments. ^bP<0.05, ^cP<0.01 vs control.