Abstract
We aimed to explore the anticancer potential of violacein and its time, dose dependent mechanism of action in human MCF-7 breast cancer cells. We observed, violacein inhibit MCF-7 cells viability in a time and dose-dependent manner, IC50 value was 4.5 mu M in 24 h, 1.7 mu M in 48 h and 0.51 mu M in 72 h. Violacein triggered generation of intra cellular ROS even from the lower doses, significant ROS production was observed from 0.25, 0.45 mu M dose range and it is relative to higher doses. Further we fixed 0.45 mu M and 4.5 mu M as an experimental dose for relative dose dependent analysis. In nuclear staining, after 48 h 0.45 mu M dose showed characteristic apoptotic morphological changes such as, 59% of cells in apoptosis and 11% of cells in necrotic stage, also in 72h we found 68% in apoptosis and 12% in necrotic stage. However, 4.5 mu M (IC50) dose of violacein, 78% of cells became apoptotic and 21% in necrotic after 48 h; but in 72 h only 61% cells are in apoptotic, necrosis was increased to 38%. Violacein increased both mitochondrial and extra mitochondrial apoptotic pathway related gene expressions; it was confirmed by increased CYP1A, GPX, GSK313 and TNF-alpha gene. Further, 0.45 and 4.5 mu M of violacein increased apoptotic genes, such as Bax, p53, caspase 3, Fas, FADD and markedly reduced Bc1-2 and MDM2 expression levels to two fold when compared to control. In addition violacein upregulated poly ADP-ribose polymerase (PARP), CDKN1A and caspase-9 significantly (p < 0.05) when compared to control. Relative quantification of caspase-8 was differently expressed; there were no changes in 0.45 mu M, but in 4.5 mu M we found two fold increased caspase-8 expression. In conclusion, lower dose of violacein treatment induced apoptosis in human breast cancer MCF-7 cells through TNF-alpha and p53 dependent mitochondrial pathways. (C) 2015 Elsevier GmbH. All rights reserved.