GFP-expressing prostate cancer cells demonstrate attenuated tumorigenicity through regulation of energy metabolism, translational activity, and cytoskeletal dynamics: a proteomic study

Abstract Background Green Fluorescent Protein is widely used as a cellular marker tool, but its potential influence on cells has been questioned. Although the potential off-target effects of GFP on tumor cells have been studied to some extent, the findings at the molecular level are insufficient to explain the effect of GFP expression on the tumorigenic capacity of cancer cells. Here, we aimed to investigate the effect of GFP expression on the tumorigenicity of PC3 prostate cancer cells. Methods and Results Using GFP-expressing and wild-type PC3 cells, xenograft models were generated in athymic BALB/C mice. To identify differentially expressed proteins, the change in the proteome of cells was investigated by label‐free quantification with nano‐high performance liquid chromatography to tandem mass spectrometry (nHPLC‐MS/MS). The proteins that showed significantly altered expression levels were evaluated using the bioinformatics tools. The results showed that GFP-expressing cells, in contrast to the wild-type cells, failed to develop tumor. Label-free quantification revealed a total of 216 differentially regulated proteins, of which 98 were upregulated and 117 were downregulated. Conclusion Upon GFP expression, the immune system, translational machinery, energy metabolism, elements of cytoskeletal and VEGF signaling pathway were regulated. Alterations in these cellular events caused attenuated tumorigenicity. Therefore, ultimate care should be taken into account to prevent reporting deceitful mechanisms generated from studies utilizing GFP.