1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes induce autophagic cell death in estrogen receptor negative breast cancer

The effects of the C-DIMs on apoptotic, necrotic and autophagic cell death were determined using caspase inhibitors, measurement of lactate dehydrogenase release, and several markers of autophagy including Beclin and light chain associated protein 3 expression (LC3).The C-DIM compounds did not induce apoptosis and only DIM-C-pPhCF3 exhibited necrotic effects. However, treatment of MDA-MB-231 and MDA-MB-453 cells with C-DIMs resulted in accumulation of LC3-II compared to LC3-I protein, a characteristic marker of autophagy, and transient transfection of green fluorescent protein-LC3 also revealed that treatment with C-DIMs induced a redistribution of LC3 to autophagosomes after C-DIM treatment. In addition, the autofluorescent drug monodansylcadaverine (MDC), a specific autophagolysosome marker, accumulated in vacuoles after C-DIM treatment, and western blot analysis of lysates from cells treated with C-DIMs showed that the Beclin 1/Bcl-2 protein ratio increased.The results suggest that C-DIM compounds may represent a new mechanism-based agent for treating drug-resistant ER-negative breast tumors through induction of autophagy.Studies in this laboratory have investigated the mechanisms of cell death induced by a new series of anticancer agents that are derivatives of phytochemicals expressed in crucifers. Indole-3-carbinol is a phytochemical found as a conjugate in cruciferous vegetables, and both indole-3-carbinol and one of its major metabolites, 3,3'-diindolylmethane (DIM), exhibit a broad range of anticancer and antitumorigenic activities against multiple tumor types [1-6]. Epidemiology studies have correlated consumption of cruciferous vegetables with decreased risk for certain types of cancer [7-11], and indole-3-carbinol and DIM may contribute to cancer chemoprevention associated with these vegetables. The mechanisms of growth inhibition induced by DIM have been well-studied and include G0/G1 cell cycle arrest, induction of ER stress, induction of apoptosis, ac