title: Differential gene expression of chemokines in KRAS and BRAF mutated colorectal cell lines: role of cytokines.
abstract: OBJECTIVE
  To study KRAS/BRAF mutations in colorectal-cancer (CRC) that influences the efficacy of treatment. To develop strategies for overcoming combination of treatment.
  
  
  METHODS
  Five colonic cell-lines were investigated: DLD-1 with KRAS (G13D) mutation, HT 29 and Colo 205 with BRAF (V600E) mutation as well as the wild type (Wt) cell-lines Caco2 and Colo-320. DLD-1 (KRAS), HT-29 (BRAF) and Caco2 (Wt) cell lines were treated with cytokines (TNFα 50 ng, IL-1β 1 ng and IFNγ 50 ng) and harvested at different time points (1-24 h). KRAS inhibition was performed by the siRNA-approach. Two colorectal cancer cells DLD-1 and Caco2 were used for KRAS inhibition. About 70% confluency were confirmed before transfection with small interferring RNA (siRNA) oligonucleotides. All the synthetic siRNA sequences were designed in our laboratory. Total RNA and protein was isolated from the cells for RT-PCR and Western blotting. Densitometry of the Western blotting was analyzed with the Image J software (NIH). Results are shown as mean ± SD.
  
  
  RESULTS
  RT-PCR analysis in non-stimulated cells showed a low basal expression of TNFα and IL-1β in the DLD-1 KRAS-mutated cell-line, compared to Caco2 wild type. No detection was found for IL-6 and IFNγ in any of the studied cell lines. In contrast, pro-angiogenic chemokines (CXCL1, CXCL8) showed a high constitutive expression in the mutated cell-lines DLD-1 (KRAS), HT-29 and Colo205 (BRAF), compared to wild type (Caco2). The anti-angiogenic chemokine (CXCL10) showed a high basal expression in wild-type, compared to mutated cell-lines. KRAS down-regulation by siRNA showed a significant decrease in CXCL1 and CXCL10 gene expression in the DLD-1 (KRAS) cell-line in comparison to wild type (Caco2) at 72 h after KRAS silencing. In contrast, the specific KRAS inhibition resulted in an up-regulation of CXCL1 and CXCL10. The results of our study show a higher expression of pro-angiogenic chemokines at basal level in mutated cell-lines, which was further increased by cytokine treatment.
  
  
  CONCLUSIONS
  To summarize, basal chemokine gene expression for pro-angiogenic chemokines was high in mutated as compared to wild type cell-lines. This reflects the likely existence of a different microenvironment in tumours consistent of wild type or mutated cells. This may help to rationalize the choice of molecular targets for suitable therapeutic investigation in clinical studies.
authors.affiliation: Sajjad Khan, Silke Cameron, Martina Blaschke, Federico Moriconi, Naila Naz, Ahmad Amanzada, Giuliano Ramadori, Ihtzaz Ahmed Malik, Clinic of Gastroenterology and Endocrinology, University Medical Center, Robert-Koch-Str. 40, 37075 Goettingen, Germany.
authors.forename: Sajjad
authors.initials: S
authors.lastname: Khan
authors.affiliation: Sajjad Khan, Silke Cameron, Martina Blaschke, Federico Moriconi, Naila Naz, Ahmad Amanzada, Giuliano Ramadori, Ihtzaz Ahmed Malik, Clinic of Gastroenterology and Endocrinology, University Medical Center, Robert-Koch-Str. 40, 37075 Goettingen, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Cameron
authors.affiliation: Sajjad Khan, Silke Cameron, Martina Blaschke, Federico Moriconi, Naila Naz, Ahmad Amanzada, Giuliano Ramadori, Ihtzaz Ahmed Malik, Clinic of Gastroenterology and Endocrinology, University Medical Center, Robert-Koch-Str. 40, 37075 Goettingen, Germany.
authors.forename: Martina
authors.initials: M
authors.lastname: Blaschke
authors.affiliation: Sajjad Khan, Silke Cameron, Martina Blaschke, Federico Moriconi, Naila Naz, Ahmad Amanzada, Giuliano Ramadori, Ihtzaz Ahmed Malik, Clinic of Gastroenterology and Endocrinology, University Medical Center, Robert-Koch-Str. 40, 37075 Goettingen, Germany.
authors.forename: Federico
authors.initials: F
authors.lastname: Moriconi
authors.affiliation: Sajjad Khan, Silke Cameron, Martina Blaschke, Federico Moriconi, Naila Naz, Ahmad Amanzada, Giuliano Ramadori, Ihtzaz Ahmed Malik, Clinic of Gastroenterology and Endocrinology, University Medical Center, Robert-Koch-Str. 40, 37075 Goettingen, Germany.
authors.forename: Naila
authors.initials: N
authors.lastname: Naz
authors.affiliation: Sajjad Khan, Silke Cameron, Martina Blaschke, Federico Moriconi, Naila Naz, Ahmad Amanzada, Giuliano Ramadori, Ihtzaz Ahmed Malik, Clinic of Gastroenterology and Endocrinology, University Medical Center, Robert-Koch-Str. 40, 37075 Goettingen, Germany.
authors.forename: Ahmad
authors.initials: A
authors.lastname: Amanzada
authors.affiliation: Sajjad Khan, Silke Cameron, Martina Blaschke, Federico Moriconi, Naila Naz, Ahmad Amanzada, Giuliano Ramadori, Ihtzaz Ahmed Malik, Clinic of Gastroenterology and Endocrinology, University Medical Center, Robert-Koch-Str. 40, 37075 Goettingen, Germany.
authors.forename: Giuliano
authors.initials: G
authors.lastname: Ramadori
authors.affiliation: Sajjad Khan, Silke Cameron, Martina Blaschke, Federico Moriconi, Naila Naz, Ahmad Amanzada, Giuliano Ramadori, Ihtzaz Ahmed Malik, Clinic of Gastroenterology and Endocrinology, University Medical Center, Robert-Koch-Str. 40, 37075 Goettingen, Germany.
authors.forename: Ihtzaz Ahmed
authors.initials: IA
authors.lastname: Malik
mesh_terms: D000230:Adenocarcinoma / Q000378:metabolism*; D018938:Caco-2 Cells; D003110:Colonic Neoplasms / Q000378:metabolism*; D016207:Cytokines / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D019073:HT29 Cells; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D011518:Proto-Oncogene Proteins / Q000235:genetics; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins / Q000235:genetics
chemical_list: D016207:Cytokines; D020672:I-kappa B Proteins; C117307:KRAS protein, human; C000606204:NFKBIA protein, human; D011518:Proto-Oncogene Proteins; D000072000:NF-KappaB Inhibitor alpha; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins
keywords: BRAF; CXCL1 (GROα); CXCL10 (IP-10); CXCL8 (IL-8); Colorectal-cancer; Interferon-γ; Interleukin-1β; KRAS; Tumor necrosis factor-α; siRNA