Modulation of CXCL-8 expression in human melanoma cells regulates tumor growth, angiogenesis, invasion, and metastasis

Abstract

CXCL-8, a chemokine secreted by melanoma and stromal cells, serves as a growth and angiogenic factor for melanoma progression. This study evaluated how modulation of CXCL-8 levels in melanoma cell lines with different tumorigenic and metastatic potentials affected multiple tumor phenotypes. A375P cells (CXCL-8 low expressor) were stably transfected with a CXCL-8 mammalian expression vector to overexpress CXCL-8, whereas A375SM cells (CXCL-8 high expressor) were transfected with a CXCL-8 antisense expression vector to suppress CXCL-8 expression. Subsequent cell proliferation, migration, invasion, and soft-agar colony formation were analyzed, and in vivo tumor growth and metastasis were evaluated using mouse xenograft models. Our data demonstrate that overexpression of CXCL-8 significantly enhanced primary tumor growth and lung metastasis, accompanied by increased microvessel density in vivo, as compared with vector control-transfected cells. We also observed increased clonogenic ability, growth, and invasive potential of CXCL-8 overexpressing cells in vitro. Knockdown of CXCL-8 using an antisense vector resulted in increased cell death and reduced tumor growth relative to control. Taken together, these data confirm that CXCL-8 expression plays a critical role in regulating multiple cellular phenotypes associated with melanoma growth and metastasis.

Keywords: Angiogenesis; CXCL-8; melanoma; metastasis; proliferation.

Figure 1

CXCL-8 overexpression and knockdown in A375P and A375SM melanoma cells. (A) Northern blot analysis to confirm CXCL-8 mRNA expression in A375P-control, A375P-CXCL-8, A375SM-control, and A375SM-anti-CXCL-8 cells. GAPDH was used as control. (B) CXCL-8 protein secretion in CXCL-8 overexpressing and knockdown cells as determined using ELISA and presented as mean CXCL-8 levels in pg/μg of total protein ± SEM.

Figure 2

Modulation of cell proliferation following altered CXCL-8 expression in melanoma cells. (A) and (B) In vitro cell proliferation of A375P-control, A375P-CXCL-8, A375SM-control, and A375SM-anti-CXCL-8 cells was determined at 72 h by MTT assay. (C) A375P cells were treated with media alone or media containing different concentrations of CXCL-8. (D) A375SM cells were incubated with media alone, media containing control IgG or neutralizing anti-CXCL-8 antibody. Cell proliferation was determined using MTT assay and presented as mean absorbance OD_570nm ± SEM. *Significantly different from controls (P < 0.05).

Figure 3

Modulation of CXCL-8 expression regulates cell motility and invasion. A375P-control, A375P-CXCL-8, A375SM-control, and A375SM-anti-CXCL-8 cells were seeded on noncoated (cell motility, A and B) or Matrigel-coated (cell invasion, C and D) membranes for migration assays. Migrated (A and B) and invaded (C and D) cells were counted and presented as average number of cells/field ± SEM (a representative of three experiments done in triplicate). *Significantly different from controls (P < 0.05).

Figure 4

Modulation of CXCL-8 expression alters melanoma growth in vivo. Melanoma cells (A375P-control, A375P-CXCL-8, A375SM-control, and A375SM-anti-CXCL-8) were injected s.c. and tumor volume was monitored. (A) Growth of A375P-control and A375P-CXCL-8 tumors. (B) Growth of A375SM-control and A375SM-anti-CXCL-8 tumors. (C) and (D) Immunohistochemical staining for CXCL-8 in A375P-control, A375P-CXCL-8, A375SM-control, and A375SM-anti-CXCL-8 tumors to show differential levels of CXCL-8. *Significantly different from controls (P < 0.05).

Figure 5

Melanoma CXCL-8 expression modulates tumor neovascularization. A375P-control, A375P-CXCL-8, A375SM-control, and A375SM-anti-CXCL-8 tumors were immunostained for microvessel density. (A) A375P-CXCL-8 tumors show higher immunostaining as compared with A375P-control tumors. (B) A decrease in microvessel density in A375SM-anti-CXCL-8 tumors as compared with A375SM-control tumors. The representative pictures are shown at 200×. Quantitation of microvessel density in tumors with different CXCL-8 levels was made using a 5 × 5 reticle grid at 400× magnifications and presented as average number of microvessels ± SEM. *Significantly different from controls (P < 0.05).

Figure 6

Altered melanoma cell proliferation in vivo in melanoma tumors expressing different levels of CXCL-8. PCNA immunohistochemical staining was performed, and the number of proliferating cells in A375P-control and A375P-CXCL-8 tumors (A) and in A375SM-control and A375SM-anti-CXCL-8 tumors (B) was counted and presented as average number of cells/field ± SEM (right panel). *Significantly different from controls (P < 0.05).

Figure 7

Frequency of apoptotic melanoma cells in vivo in melanoma tumors expressing different levels of CXCL-8. TUNEL staining was performed in the tumor section. The number of apoptotic cells in A375P-control and A375P-CXCL-8 tumors (A), and A375SM-control and A375SM-anti-CXCL-8 tumors (B) was counted and presented as average number of cells/field ± SEM (right panel). *Significantly different from controls (P < 0.05).

Figure 8

Serum CXCL-8 level correlates with tumor burden and spontaneous lung metastasis. Serum from tumor-bearing animals was collected at the time of primary tumor removal, and CXCL-8 levels were determined using ELISA. The values are mean CXCL-8 levels (in pg/mL) ± SEM. *Significantly different from controls (P < 0.05).