The GTA+ve fraction showed a 40% reduction in cell viability at a dose of 80 ug/ml (selleck chemicals llc Figure 3A) while GTA-ve treatment had no effect. Treatment up to 48 hrs using 80 ug/ml showed the same 40% reduction Selleck Tideglusib as early as 12 hrs, which dropped further to 70% by 48 hrs (Figure 3B). No effect on cell proliferation was observed with the GTA-ve fraction or vehicle (DMSO). Evidence of apoptotic activity was determined by the detection of poly(ADP-ribose) polymerase (PARP) cleavage products through Western blot (Figure 3C). A number of PARP cleavage products including the hallmark 89 and 24 kDa fragments,
as well as others (Figure 3C), were induced following 48 hrs treatment with GTA+ve fraction, but not with GTA-ve treatment, suggesting a possible pro-apoptotic function of GTAs. Figure 3 Proliferation of SW620 cells treated with GTA+ve and GTA-ve extracts. (A) SW620 cells were incubated with increasing concentrations of GTA+ve and GTA-ve extracts for 24 hours and proliferation assayed by MTT. (B) The 80 ug/ml concentration
of GTA+ve and GTA-ve extracts was then used to treat cells for up to 48 hours and the effect on cell proliferation assayed by MTT. Data are PI3K inhibitor expressed as percent of vehicle or 0 hrs ± 1S.D. (C) Representative Western blot analysis of caspase-mediated PARP Etomidate cleavage fragments resulting from treatment with GTA+ve and -ve extracts. Experiments were repeated at least three times. We repeated the studies in MCF7 cells, which upon treatment with GTA+ve fraction showed gross cellular changes visible through phase-contrast microscopy including the appearance of apoptosomes and enlarged nuclei that were not observed with vehicle or GTA-ve treatments (Figures 4A, B and 4C). GTA+ve treatment in MCF-7 cells also resulted in the exclusive induction of the 24
kDa PARP cleavage product relative to vehicle or GTA-ve treatment (Figure 4D), further suggesting a pro-apoptotic activity of GTA-containing extracts. Figure 4 Treatment of MCF7 cells with GTA+ve and GTA-ve extracts. MCF7 cells were incubated with vehicle (A), 80 ug/ml GTA+ve extract (B), and 80 ug/ml GTA-ve extract (C) and cells photographed using phase-contrast light microscopy (200×). (D) Western analysis of PARP cleavage products; ns, non-specific. GTA+ve extracts inhibit pro-inflammatory markers The structural resemblance of GTAs to the inflammation-resolving protectins and resolvins prompted us to investigate the effect of GTA+ve extract on pro-inflammatory markers.