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. 2011 May 1;128(9):2038-49.
doi: 10.1002/ijc.25562.

Interleukin-8 is associated with proliferation, migration, angiogenesis and chemosensitivity in vitro and in vivo in colon cancer cell line models

Yan Ning  1 , Philipp C Manegold, Young Kwon Hong, Wu Zhang, Alexandra Pohl, Georg Lurje, Thomas Winder, Dongyun Yang, Melissa J LaBonte, Peter M Wilson, Robert D Ladner, Heinz-Josef Lenz
Affiliations

Interleukin-8 is associated with proliferation, migration, angiogenesis and chemosensitivity in vitro and in vivo in colon cancer cell line models

Yan Ning et al. Int J Cancer. .

Abstract

Interleukin-8 (IL-8), a chemokine with a defining CXC amino acid motif, is known to possess tumorigenic and proangiogenic properties. Overexpression of IL-8 has been detected in many human tumors, including colorectal cancer (CRC), and is associated with poor prognosis. The goal of our study was to determine the role of IL-8 overexpression in CRC cells in vitro and in vivo. We stably transfected the IL-8 cDNA into two human colon cancer cell lines, HCT116 and Caco2, and selected IL-8-secreting transfectants. Real-time RT-PCR confirmed that IL-8 mRNA was overexpressed in IL-8 transfectants with 45- to 85-fold higher than parental cells. The IL-8-transfected clones secreted 19- to 28-fold more IL-8 protein than control and parental cells as detected by ELISA. The IL-8 transfectants demonstrated increased cellular proliferation, cell migration and invasion based on functional assays. Growth inhibition studies showed that IL-8 overexpression lead to a significant resistance to oxaliplatin (p < 0.0001). Inhibition of IL-8 overexpression with small interfering RNA reversed the observed increases in tumorigenic functions and oxaliplatin resistance, suggesting that IL-8 not only provides a proliferative advantage but also promotes the metastatic potential of colon cancer cells. Using a tumor xenograft model, IL-8-expressing cells formed significantly larger tumors than the control cells with increased microvessel density. Together, these findings indicate that overexpression of IL-8 promotes tumor growth, metastasis, chemoresistance and angiogenesis, implying IL-8 to be an important therapeutic target in CRC.

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Figures

Figure 1
Figure 1. Detection of IL-8 in stably transfected colon cancer cells and growth rate of IL-8-expressing clones and parental cells
A, Real time RT-PCR determined the level of IL-8, CXCR1 and CXCR2 mRNA in parental colon cancer cell lines (HCT116 and Caco2) and IL-8 transfected cell lines (HCT116-E2 and Caco2-IIIe). Normalized transcript levels were calculated as 2−ΔΔCT ×100. Data shown are expressed in arbitrary units (a.u.). B, ELISA measured IL-8 production from IL-8-transfected, vector-transfected and parental colon cancer cells. The results were normalized to cell numbers. C, In Vitro proliferation assay. HCT116 and Caco2 cells (5×103 cells/well) were treated with rhIL-8 (2.5, 5, 10, 20, 40 ng/ml) for 72 h, then fixed with 60% cold methanol, cells were stained with crystal violet, then solubilized by adding 10% SDS, and read absorbance at A570nm on plate reader. D, (i.) HCT116 cells with or w/o treatment rhIL-8 (10 ng/ml) and HCT116-E2 cells and (ii.) Caco2-vector cells with or w/o treatment rhIL-8 (10 ng/ml), Caco2-IIIe in a 12-well culture plate (5×103 cells/well) were incubated with media for 1, 2, 4, 6, 7 and 8 d. Then fixed, stained and solubilized as mentioned as above. In A, B, C and D, the results are representative of a minimum of three independent experiments. Data shown represent the mean ±standard deviation (*, P< 0.05, **, P<0.005.).
Figure 2
Figure 2. Overexpressed IL-8 is involved in cell migration and invasion in colon cancer cells
A, Migration assay. (i.) HCT116 and HCT116-E2 cells (2×105 cells/well) in a 24-well Boyden chambers with feeder tray were incubated with media for 48 h. Cell migration was determined by staining with crystal violet as mentioned above. Cells were photographed. Dark staining were migratory cells (original magnification, ×200). (ii.) Migration assay was quantitated. Columns shows fold change over the number of HCT116 control cells that migrated (**, P<0.005). B, Invasion assay. (i.) HCT116 and HCT116-E2 cells (2×105 cells/well) were plated on porous cell culture inserts which coated with Matrigel and allowed to invade for 48 h. Dark staining were invasive cells (original magnification, ×200). (ii.) Invasion assay was quantitated. Columns shows fold change over the number of HCT116 control cells that invaded (**, P<0.005). C, Q-PCR analysis of IL-8 expression knockdown in HCT116 and HCT116-E2 cells. All siRNA experiments were done with the following cells: untransfected cells, mock-transfected cells (control siRNA), siRNA-transfected cells (siRNA #2261). D, Quantitation of migration and invasion after siRNA transfection. HCT116, HCT116-E2, control-transfected HCT116-E2, and siRNA transfected HCT116-E2 cells were incubated on Boyden chambers or Matrigel for 48 h as described in Materials and Methods. Columns shows fold change over the number of HCT116 control cells that migrated or invaded. The results are representative of a minimum of three independent experiments. Data shown represent the mean ± standard deviation (**, P<0.005).
Figure 3
Figure 3. Overexpressed IL-8 increases NF-κB, Akt and MAPK activities in colon cancer cells
A, HCT116, HCT116-E2 and Caco2 transfectants were transiently transfected with pGL4.32 [luc2P/NF-κB-RE/Hygro] vector and cultured in media for 48 h. Promoter activity was measured by luciferase assay and normalized against Renilla activity (*, P<0.05). B and C, Cell lysates of each cell line were polyacrylamide gel electrophoresis and immunoblotted with anti-phospho-NF-κB-p65, Akt (60 kDa), and p44/42 MAP kinase or anti-total NF-κB-p65, Akt and p44/42 MAP kinase antibodies. In B and C, β-tubulin as a loading control.
Figure 4
Figure 4. Growth inhibition assay and clonogenicity analysis of IL-8-transfected cells treated with oxaliplatin
A, Growth inhibition assay. HCT116, HCT116-E2, HCT116 treated with rhIL-8 and HCT116-E2 transfected with IL-8 siRNA cells were measured for growth inhibition using CellTiter 96 AQueous One solution. After treatment for 72 h with oxaliplatin, IC50 were calculated using GraphPad Prism software. Data are presented as the mean ± standard deviation of three experiments carried out in triplicate. B, Clonogenicity assay. All cells mentioned above were treated with oxaliplatin at different concentration 0.5, 1, 1.5, 2.5, and 5 μM for 72 h. Colonies were fixed with 60% methanol after 3 weeks incubation with drug-free medium, then colonies were staining with crystal violet, (i.), Colonies were quantitated, (ii.), Wells were scanned. Results are representative of three independent experiments. Data shown represent the mean ± standard deviation.
Figure 5
Figure 5. Examination of IL-8 level and detection of microvessel density in xenograft of colon cancer cells
A, Examination of tumor growth in xenograft of colon cancer cells. HCT116, HCT116-E2, Caco2 and Caco2-IIIe subcutaneously injected into nude mice. The graphs indicate the mean tumor growth rates ±SD of three animals per experimental condition. P < 0.05 was obtained by ANOVA. B, Sera IL-8 productions were measured by ELISA, and the results were normalized to whole blood volume. C and D (bottom panel), Immunohistochemistry analysis of IL-8 expression and localization of microvessel in tumor tissue slides. In HCT116 and HCT116-E2 tumor specimens, rabbit anti-human IL-8 or rat anti-mouse CD31 antibodies were added to tissue sections. All sections imaged by Zeiss Axiovision software program. The images are presented at 200× magnification. D (top panel), Blood vessel density was quantified by counting the total number of CD31-positive vessels across the whole section of tumors. For these analyses, six different tumor samples were evaluated per experimental condition. Data shown represent the mean ± standard deviation (*, P<0.05; **, P<0.005).
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