We showed that low doses of DIM (1 µM) stimulate BRCA1 expression and protect cells against oxidative stress, in part, through BRCA1. Protection against oxidative stress by DIM (and I3C) is consistent with the previous finding that BRCA1 protects against oxidative stress (25
). The BRCA1 protection was attributed, in part, to stimulation of an antioxidant response via NRF2, a redox-sensitive transcription factor that stimulates expression of phase II detoxifying enzymes and antioxidant genes through the ARE (33
). We identified several mechanisms by which DIM protects against oxidative stress, including the BRCA1-dependent stimulation of NRF2 activity. DIM stimulated several NRF2-regulated promoters, including NQO1 (an oxidoreductase), GST-α1 (a glutathione S-transferase), and x-CT (cystine/glutamate transporter). NRF2 appears to be a target for some dietary agents proposed for cancer prevention (39
), although this was not demonstrated previously for DIM or I3C.
Interestingly, DIM stimulated BRCA1 phosphorylation on S1387 and S1524 before any increase in BRCA1 protein levels. DIM-induced phosphorylation of S1387 was observed after 10 min and showed a time course suggestive of a signaling event. In cell lines lacking functional BRCA1, S1387 and S1524 were required for protection against oxidative stress by exogenous BRCA1. While the significance of these phosphorylations is not clear, prior studies suggest that these sites are phos-phorylated by ATM and/or ATR in response to ionizing or UV radiation (40
). ATM was required for DIM-induced phosphorylation of BRCA1 on S1387, suggesting that DIM signals through ATM.
In U87MG cells (which do not express BRCA1), DIM conferred additional protection against H2O2 beyond that afforded by exogenous BRCA1; but BRCA1 with an S1387A mutation failed to support DIM-mediated protection. In contrast, HCC1937 cells, which express mutant BRCA1, showed no additional protection from DIM beyond that due to exogenous BRCA1. It is possible that the endogenous mutant BRCA1 interferes with the transfected BRCA1 proteins, that the relevant phosphorylations cannot be induced or enhanced by DIM in this cell type, or that the protection by exogenous BRCA1 is already maximal and cannot increase further.
While the mechanism by which DIM induces BRCA1 phosphorylation is unclear, DIM and I3C can each activate an endoplasmic reticulum stress response (unfolded protein response) that is BRCA1-dependent; and this response is required for I3C stimulation of BRCA1 expression (15
). We speculate that DIM induces low grade stress signaling that directly or indirectly leads to rapid phosphorylation of BRCA1. One possibility is that DIM interacts with one or more stress-responsive proteins and the signal is transmitted to BRCA1. While DIM and/or I3C can bind to several proteins (eg
., aryl hydrocarbon receptor, estrogen receptor, and androgen receptor) (17
), relatively little is known about how DIM interacts with cell proteins or other macromolecules.
Another interesting finding was that H2
caused little or no apoptosis. Reactive oxygen species, including H2
, can cause different combinations of autophagy, apoptosis, and/or necrosis in a cell line and stimulus dependent manner (42
). Autophagy is a process by which cells degrade macromolecules and organelles that can lead to cell survival (eg
., under austere conditions in tumors) or cell death in different contexts. In our assays, H2
-induced autophagy was cytotoxic and DIM/BRCA1 inhibition of autophagy was cytoprotective, as evidenced by the following: 1) inhibition of autophagy by knockdown of beclin 1 (45
) increased cell survival; 2) DIM and wtBRCA1 inhibited H2
-induced autophagy and at the same time protected cells against cytotoxicity; and 3) we detected little or no H2
-induced apoptosis. Since the effects of oxidative stress are variable in terms of autophagy vs apoptosis, we cannot say whether DIM/BRCA1 inhibition of autophagy would be cytoprotective in a different context (eg
., starvation rather than oxidative stress).
caused a large increase in the percentage of cells undergoing autophagy that was inhibited by DIM or wtBRCA1. Consistent with this finding, H2
caused conversion of LC3-I to LC3-II, a marker of autophagy (37
); and the conversion of LC3-I to LC3-II was attenuated by DIM or wtBRCA1. Consistent with the idea that the protective effect of DIM is mediated by BRCA1, wtBRCA1 blocked the H2
-induced up-regulation of beclin 1. These findings link DIM and BRCA1 to regulation of autophagy.
Low doses of DIM also stimulated BRCA1 signaling and expression and protected against oxidative stress in normal or non-tumor-derived human mammary epithelial cells. These findings are important because normal cell types are presumably the main target for cancer prevention. Since we did not test whether H2O2 causes and DIM blocks autophagy in these cell types, we cannot say whether the observed protection reflects autophagy inhibition.
Our findings have implications for understanding chemoprevention and its limitations. Since DIM’s protective effects occurred at physiologic concentrations, it is reasonable to speculate that DIM blocks carcinogenesis, in part, by enabling normal cells to mount a more effective antioxidant response. This antioxidant response may include an increased ability of cells to repair oxidative DNA damage, since BRCA1 contributes to various DNA repair processes (46
). Tumor cells often exhibit oxidative stress due to impaired antioxidant defenses. Thus, the presence of pre-existing cancer cells that are protected by antioxidants is a possible explanation for the mixed results obtained in clinical studies using antioxidants to prevent cancer (47
). Here, the ability of DIM to promote survival of oxidatively stressed tumor cells could limit its activity as a chemoprevention agent.
Autophagy is regarded as a double-edged sword. On the one hand, autophagy is a tumor suppressor mechanism that can serve as a second type of programmed cell death replacing apoptosis. On the other hand, it can be utilized as a survival mechanism by tumor cells in nutrient limiting conditions (50
). As noted above, we do not know whether DIM inhibition of autophagy will extend to other cell types and other causes of autophagy (eg
., starvation). Nor do we know the extent to which autophagy inhibition by DIM is the consequence of a reduced stimulus for autophagy due to an increased cellular antioxidant response. Thus, at present, it is difficult to assess the contribution of autophagy as a target for DIM-mediated chemoprevention.