Avenanthramides decrease breast cancer viability in vitro
To determine if AVN-A, B, and C have chemotherapeutic potential we first utilized an MTT cell viability assay. The effect of AVN-A, AVN-B and AVN-C on breast cancer cell viability is shown in Figs. 2, 3 and 4, respectively. Viability was assessed at, 50, 100, 200, and 400 µM treatments for AVN-A, B, and C at 48, 72, and 96 h timepoints. AVN-A was the first to show significance at the 400 µM concentration after 48 h (Fig. 2a). AVN-B and C didn’t show significance at the same concentration until the 96-h timepoint (Figs. 3c, 4c). Significant differences were noted in all three AVN treatments after 96 h with AVN-A remaining significant only at the highest dose, while AVN-B showed significance at both 200, and 400 µM and AVN-C showed significance as low as 50 µM after 96 h. Although AVN-C showed significance at a later time point than A or B, its observed decrease in viability was twice that of its counterparts. It is clear that AVN-C has a dramatic effect on breast cancer cell viability, while AVN-A and B display modest results, albeit sooner. Because of the observed differences in both timing and degree of decreased viability from the different treatments we hypothesized that different mechanisms may be at play and expected to see varying results from the treatments in further testing. Due to the fact that significance was observed in all three treatments only at 400 µM after 96 h, all following studies were conducted at this time and concentration unless noted otherwise.
Avenanthramide-C causes DNA fragmentation
We hypothesized that AVN may be decreasing cell viability by either inhibiting the cell cycle or activating apoptosis in the tumorigenic cells. As a result, we determined the percentage of cells in each stage of the cell cycle after 96 h of treatment with AVN at 400 µM (Fig. 5). PI staining revealed no significant difference between the vehicle and AVN-A and B for any stage of the cell cycle. Vehicle control showed 9.26% cells sub G1, 69.53% cells in the G1 stage, 9.23% cells in the S stage, and 11.97% cells in the G2 stage. AVN-A treatment resulted in 15.79% cells sub G1 (p = 0.42), 62.85% cells in the G1 stage (p = 0.25), 10.96% cells in the S stage (p = 0.57), and 10.4% cells in the G2 stage (p = 0.48). AVN-B treatment caused 14.2% cells sub G1 (p = 0.46), 62.49% cells in the G1 stage (p = 0.14), 12.75% cells in the S stage (p = 0.19), and 10.56% cells in the G2 stage (p = 0.60). The cells seemed to be replicating normally despite treatment and a decrease in viability. AVN-C however showed a massive accumulation of cells in a sub G1 population (71.03% of cells, p = 1.14E−5) resulting in significant changes to the G1 (20.69% of cells, p = 2.18E−6), and G2 (2.44% of cells, p = 2.52E−4) stages and a moderate but insignificant change to the S stage (5.85% of cells, p = 0.20).
The increase in the sub G1 population shows that AVN-C is causing DNA fragmentation; this is indicative of an apoptotic mechanism. It is of note that the AVN-C dependent viability decrease (75.6%) is nearly identical to the percent of cells found in the Sub G1 population (71%). We theorize that AVN-C works by activating apoptosis in this cell line. With no changes in cell cycle distribution for AVN-A and B their mechanism of action remained unclear. We observed an increase in sub G1 cells for both AVN-A and B that was accompanied by a decrease of cells in the G1 phase of equal magnitude, but these changes were not significant.
Avenanthramide-C activates apoptosis in breast cancer cells
To confirm that AVN-C was activating apoptosis we conducted two experiments. First, we co-stained treated cells with PI and annexin V and second, we analyzed the caspase activity present in each sample. In both experiments we compared our treatments to a DMSO vehicle dose given at the maximum AVN treatment concentration and a treatment with a 0.75 µM staurosporine (STS), a known apoptosis activator.
When staining with annexin V and PI we observed a shift from viable cells found in quadrant (Q) 4 to apoptotic and dead cells quadrants (Q3 and Q2 respectively) (Fig. 6); this occurred in all treatment groups and at both 48 and 96-h time points. After 48 h of treatment AVN-C and STS showed 35% (p = 0.04) and 39% (p = 0.0076) annexin V positive (Q3 + Q4) cells respectively, while AVN-A and B had no significant changes in annexin positive cells (Fig. 6a). At the same time, the treatment with the most cells in Q2 was AVN-A at 0.02%. However, when compared to vehicle (0.46% of cells in Q2) all trials indicated significance. When doubling the treatment time to 96 h AVN-C again had the most profound effect, leaving just over 2% of observed cells in Q4. In addition, nearly 98% of cells stained positive for the presence of annexin V (Figs. 6b, c). Of the 98% annexin positive cells, a final 27% of cells were undergoing apoptosis while 71% were dead. Taken together, these time points indicate that AVN-C induces apoptosis beginning before 48 h, and by 96 h viability has been reduced to 29 while 93% of cells still alive are on their way to an apoptotic death. At 400 µM AVN-C and 96 h we found a greater number of dead cells than after treatment with STS, indicating that AVN-C is killing cells more effectively than our positive control. All AVN-C and STS treated quadrants were significantly different than vehicle. AVN-A and B also showed marked departure from the vehicle by 96 h, in both a decrease in viable Q4 cells and an increase in apoptotic Q3 cells. Due to variance in the replicate experiments however, the p values indicated no significant difference in Q4 and Q3 as determined by a two tailed T test (0.054 and 0.08 respectively for AVN-A, and 0.092 in both quadrants for AVN-B).
To further confirm the apoptotic effects of AVN-C we analyzed the activity of caspases 3/7. We treated cells with each AVN and STS, but analyzed the samples to detect the activity of caspase 3/7, late stage apoptosis caspases (Fig. 7). As with the annexin V/PI co-staining, this assay allows the user to set up quadrants again detailing if cells are alive (Q4), dead (Q2), or undergoing apoptosis (Q3). For AVN-C, the results were nearly identical to those observed when staining with annexin V and PI at the same time point: 91% of cells showed caspase fluorescence and 8% of cells were found to be viable. Significance was observed in all three quadrants (p values 4.99E−8, 0.016, and 6.6E−8 for Q4, 3, and 2 respectively).