Studies on the mode of action of synthetic diindolylmethane derivatives against triple negative breast cancer cells.

Abstract

Diindolylmethane (DIM) is a metabolic product of indole-3-carbinol (I3C), the major phytochemicals present in cruciferous vegetables, which can modulate multiple signalling pathways in cancer. The present study deals with the mechanism of action of two synthetic biaryl conjugates of DIM in triple negative breast cancer cells. Out of 12 DIM derivatives tested, two compounds, DIM-1 and DIM-4, exhibit cytotoxicity with GI50 values of 9.83 ± 0.2195 μM and 8.726 ± 0.5234 μM, respectively, in 2D culture. In 3D culture, DIM-1 and DIM-4 show GI50 values of 24.000 ± 0.7240 μM and 19.230 ± 0.3754 μM, respectively. The non-toxic nature of the compounds was also established by the toxicity studies using the zebrafish model system. The two compounds induced apoptosis and anoikis in the cancer cells, which was confirmed by morphological analysis, nuclear fragmentation, membrane integrity assay, caspase activity measurements and modulation of pro/anti-apoptotic proteins. The compounds inhibited cell migration and MMP-2 and MMP-9 activities indicating their anti-metastatic property. They also reduced the expression of active Ras, phosphorylated forms of PI3K, Akt and mTOR. Immunofluorescence studies revealed the reduced expression of EGFR and pEGFR in treated cells. To conclude, DIM-1 and DIM-4 induced anti-breast cancer effects by blocking EGF receptor and subsequently inhibiting Ras-mediated PI3K-Akt-mTOR signalling pathway.