RESUMO
BACKGROUND: In this study, we have investigated the chemotherapeutic potential of a purple violet pigment (PVP), which was isolated from a previously undescribed Antarctic Janthinobacterium sp. (Ant5-2), against murine UV-induced 2237 fibrosarcoma and B16F10 melanoma cells. METHODS: The 2237, B16F10, C50, and NIH3T3 cells were treated with PVP at different doses and for different times, and their proliferation and viability were detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle arrest induced by PVP in 2237 fibrosarcoma cells was assessed by flow cytometry and expression analysis of cell cycle regulatory proteins were done by Western blot. Apoptosis induced by PVP in 2237 cells was observed by annexin-V/propidium iodide double staining flow cytometry assay and fluorescence microscopy. To further determine the molecular mechanism of apoptosis induced by PVP, the changes in expression of Bcl-2, Bax and cytochrome c were detected by Western blot. The loss of mitochondrial membrane potential in PVP treated 2237 cells was assessed by staining with JC-1 dye following flow cytometry. Caspase-3, Caspase-9 and PARP cleavage were analyzed by Western blot and Caspase-3 and -9 activities were measured by colorimetric assays. RESULTS: In vitro treatment of murine 2237 cells with the PVP resulted in decreased cell viability (13-79%) in a time (24-72 h) and dose (0.1-1 µM)-dependent manner. The PVP-induced growth inhibition in 2237 cells was associated with both G0/G1 and G2/M phase arrest accompanied with decrease in the expression of cyclin dependent kinases (Cdks) and simultaneous increase in the expression of cyclin dependent kinase inhibitors (Cdki) - Cip1/p21 and Kip1/p27. Further, we observed a significant increase in the apoptosis of the 2237 fibrosarcoma cells which was associated with an increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic proteins Bcl-2, disruption of mitochondrial membrane potential, cytochrome c release, activation of caspase-3, caspase-9 and poly-ADP-ribose-polymerase (PARP) cleavage. CONCLUSIONS: We describe the anti-cancer mechanism of the PVP for the first time from an Antarctic bacterium and suggest that the PVP could be used as a potent chemotherapeutic agent against nonmelanoma skin cancers.
