B-Raf inhibition in conjunctival melanoma cell lines with PLX 4720.
OBJECTIVE
Mutations in the gene coding for the kinase B-Raf are associated with tumour growth in conjunctival melanoma. The purpose of this study is to explore effects of pharmacological B-Raf inhibition in conjunctival melanoma cell lines.


METHODS
The B-Raf genotypes were assessed by PCR and subsequent sequencing. Cytotoxicity, cell viability, proliferation, apoptosis rate and phosphorylation rate of ERK and Akt were analysed in three different conjunctival melanoma cell lines under the influence of the B-Raf inhibitor PLX 4720 at various concentrations.


RESULTS
The cell lines CRMM-1 and CM2005.1 showed the B-Raf V600E mutation, whereas CRMM-2 expressed a B-Raf wild type. CM2005.1 was highly sensitive to PLX 4720, showing a complete cytotoxic effect for >1 µM, as well as a significant concentration-dependent reduction of the proliferation rate and viability rate. Even though CRMM-1 also carries the B-Raf V600E mutation, it did not react as sensitive to PLX 4720 inhibition as CM2005.1, but showed a significant concentration-dependent reduction regarding proliferation and viability. PLX 4720 had only slight impact on CRMM-2 in high concentrations (10 µM) regarding cytotoxicity, proliferation and viability. Fluorescence-activated cell sorting analysis revealed that PLX 4720 acted predominantly antiproliferative and not via an induction of apoptosis. The phosphorylation rate of ERK was significantly reduced in CRMM-1 and CM2005.1, while it remained unchanged in CRMM-2. The phosphorylation rate of Akt was significantly elevated in CRMM-2.


CONCLUSIONS
Proliferation inhibition of conjunctival melanoma cells by PLX 4720 depends on their B-Raf genotype. Therefore, therapeutic application of B-Raf inhibitors should take into account the specific B-Raf genotype.
Augenklinik, Charité-Universitätsmedizin Berlin, Berlin, Germany Experimentelle Ophthalmologie, Charité Universitätsmedizin Berlin, Berlin, Germany.
Aline I
AI
Riechardt
Augenklinik, Charité-Universitätsmedizin Berlin, Berlin, Germany Experimentelle Ophthalmologie, Charité Universitätsmedizin Berlin, Berlin, Germany.
Anna-Karina B
AK
Maier
Hämatoonkologie, Charité-Universitätsmedizin Berlin, Berlin, Germany.
Anika
A
Nonnenmacher
Experimentelle Ophthalmologie, Charité Universitätsmedizin Berlin, Berlin, Germany.
Nadine
N
Reichhart
Hämatoonkologie, Charité-Universitätsmedizin Berlin, Berlin, Germany.
Ulrich
U
Keilholz
Experimentelle Ophthalmologie, Charité Universitätsmedizin Berlin, Berlin, Germany.
Norbert
N
Kociok
Experimentelle Ophthalmologie, Charité Universitätsmedizin Berlin, Berlin, Germany.
Olaf
O
Strauss
Augenklinik, Charité-Universitätsmedizin Berlin, Berlin, Germany Experimentelle Ophthalmologie, Charité Universitätsmedizin Berlin, Berlin, Germany.
Antonia M
AM
Joussen
Augenklinik, Charité-Universitätsmedizin Berlin, Berlin, Germany Experimentelle Ophthalmologie, Charité Universitätsmedizin Berlin, Berlin, Germany.
Enken
E
Gundlach
D017209:Apoptosis; D049109:Cell Proliferation; D003230:Conjunctival Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay; D005838:Genotype; D006801:Humans; D015151:Immunoblotting; D007211:Indoles / Q000494:pharmacology*; D008545:Melanoma / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D016133:Polymerase Chain Reaction; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors* / Q000235:genetics; D013449:Sulfonamides / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D007211:Indoles; C528407:PLX 4720; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf
Conjunctiva; Neoplasia