title: TNF-alpha and melphalan modulate a specific group of early expressed genes in a murine melanoma model.
abstract: BACKGROUND
  Cutaneous melanoma displays high morbidity and mortality rates. Isolated limb perfusion with melphalan (Mel) is used for the treatment of non-resectable, locally advanced extremity melanomas. When combined with tumor necrosis factor alpha (TNF-alpha) treatment, the complete response varies between 70% and 90%. The mechanisms underlying the effects of Mel and TNF-alpha are not completely understood. We evaluated the impact of systemic Mel and TNF-alpha administration on tumor growth, analyzed the morphological changes promoted by each treatment, and identified early expressed genes in response to Mel and TNF-alpha treatment, either alone or in combination, in a murine melanoma model.
  
  
  METHODS
  Six- to eight-week-old male mice were subcutaneously inoculated with B16F10 melanoma cells and then intravenously injected with TNF-alpha, melphalan or a combination of both drugs when the tumors reached 1.0 cm(2). Tumor growth was monitored every other day, and histological analysis was performed when the tumors reached 3.0 cm(2). Total RNA was extracted from the resected tumors and submitted to amplification, labeling and hybridization on an oligonucleotide microarray (Fox Chase Cancer Center). Tumor growth and histological parameters were compared using ANOVA. Survival curves were calculated using the Kaplan-Meier method. Two-way ANOVA was used to identify differentially expressed genes among the various treatments, and Dunn's test was used for pair-wise comparisons.
  
  
  RESULTS
  Systemic administration of Mel impaired tumor growth (p<0.001), improved animal survival (p<0.001), and decreased mitotic rate (p=0.049). Treatment with TNF-alpha alone had no impact, neither on tumor growth, nor on survival, but it increased necrosis (p<0.024) and decreased mitotic rates (p=0.001) in the tumors. Combined treatment with Mel and TNF-alpha had similar effects in tumor growth, survival, necrosis and mitotic rate as observed with individual treatments. Moreover, 118 genes were found differentially expressed by microarray analysis and 10% of them were validated by RT- real time PCR. In our model we found that the treatments regulate genes that play important roles in tumorigenesis such as cell adhesion (Pard3, Pecam1, Ilk, and Dlg5), proliferation (Tcfe3 and Polr1e), cell motility (Kifap3, Palld, and Arhgef6), apoptosis (Bcl2l11), and angiogenesis (Flt1 and Ptprj).
  
  
  CONCLUSIONS
  Our data reproduces, in mice, some of the features observed in melanoma patients treated with the combination of Mel and TNF-alpha. The identification of genes with altered expression by these drugs both individually and in combination might help in the understanding of their mechanism of action and, as a consequence, improved strategies that could impact their clinical application.
authors.affiliation: Laboratório de Inflamação e Câncer, Fundação Antônio Prudente, Hospital AC Camargo, São Paulo, Brazil. vladmirlima@yahoo.com.br
authors.forename: Vladmir Cláudio Cordeiro
authors.initials: VC
authors.lastname: de Lima
authors.forename: Alex Fiorini
authors.initials: AF
authors.lastname: de Carvalho
authors.forename: Mariana
authors.initials: M
authors.lastname: Morato-Marques
authors.forename: Vivian Lika
authors.initials: VL
authors.lastname: Hashimoto
authors.forename: Graziela Machado Gruner Turco
authors.initials: GM
authors.lastname: Spilborghs
authors.forename: Sarah Martins
authors.initials: SM
authors.lastname: Marques
authors.forename: Gilles
authors.initials: G
authors.lastname: Landman
authors.forename: Cesar
authors.initials: C
authors.lastname: Torres
authors.forename: Karina
authors.initials: K
authors.lastname: Braga Ribeiro
authors.forename: Helena
authors.initials: H
authors.lastname: Brentani
authors.forename: Luiz F L
authors.initials: LF
authors.lastname: Reis
authors.forename: Adriana Abalen Martins
authors.initials: AA
authors.lastname: Dias
mesh_terms: D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D002448:Cell Adhesion / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D020869:Gene Expression Profiling; D008297:Male; D008545:Melanoma / Q000188:drug therapy* / Q000235:genetics; D008558:Melphalan / Q000627:therapeutic use*; D051379:Mice; D008810:Mice, Inbred C57BL; D009368:Neoplasm Transplantation; D009389:Neovascularization, Pathologic / Q000235:genetics; D016896:Treatment Outcome; D014409:Tumor Necrosis Factor-alpha / Q000627:therapeutic use*
chemical_list: D018906:Antineoplastic Agents, Alkylating; D014409:Tumor Necrosis Factor-alpha; D008558:Melphalan