In this study we show that
diindolylmethane (DIM) induces autophagy in
ovarian cancer cells by regulating endoplasmic reticulum (ER) stress and AMPK. Treatment of SKOV-3, OVCAR-3 and TOV-21G
ovarian cancer cells with varying concentrations of DIM for 24 hours resulted in a concentration dependent induction of autophagy as measured by flowcytometry. Electron microscopy confirmed the presence of autophagosomes in DIM treated cells. Western blot analysis showed that DIM treatment increased the expression of LC3B, a hall mark of autophagy as well as p62 and Atg 12
proteins that are accumulated during autophagy. Autophagy inhibitors bafilomycin or
chloroquine inhibited DIM induced autophagy. Furthermore, DIM treatment significantly increased the expression of ER stress regulators such as
Grp78, IRE1 and GADD153.
Cycloheximide or ER stress inhibitor
mithramycin not only blocked ER
stress proteins that were activated by DIM but also autophagy. Silencing
Grp78 or GADD 153 significantly blocked the expression of LC3B and p62 indicating that autophagy in our model is mediated by ER stress. Knocking out LC3B inhibited DIM induced autophagy. DIM treatment increased the cytosolic
calcium levels which lead to the activation of AMPK in our model. Chelating cytosolic
calcium with BAPT-AM abrogated not only the phosphorylation of AMPK but also prevented DIM induced autophagy. Inhibiting AMPK by a chemical inhibitor or
siRNA blocked the induction of LC3B or p62, indicating that DIM mediated autophagy requires activation of AMPK.
Oral administration of DIM significantly suppressed SKOV-3
tumor xenografts in nude mice. Activation of ER stress and autophagy were observed in the
tumors of DIM treated mice. Taken together, these results suggest that induction of autophagy by DIM in
ovarian cancer cells was associated with ER stress and AMPK activation.