Abstract
Herceptin failure is a major clinical problem in breast cancer. A subset of breast cancer patients with high HER-2/neu levels eventually experience metastatic disease progression when treated with Herceptin as a single agent. Mechanistic details of development of this aggressive disease are not clear. Therefore, there is a dire need to better understand the mechanisms by which drug resistance develops and to design new combined treatments that benefit patients with aggressive breast cancer and have minimal toxicity. We hypothesized that 3, 3′-diindolylmethane (DIM), a non-toxic agent can be combined with Herceptin to treat breast cancers with high levels of HER-2/neu. Here, we evaluated the effects of Herceptin alone and in combination with DIM on cell viability, apoptosis and clonogenic assays in SKBR3 (HER-2/neu-expressing) and MDA-MB-468 (HER-2/neu negative) breast cancer cells. We found that DIM could enhance the effectiveness of Herceptin by significantly reducing cell viability, which was associated with apoptosis-induction and significant inhibition of colony formation, compared with single agent treatment. These results were consistent with the down-regulation of Akt and NF-kB p65. Mechanistic investigations revealed a significant upregulation of miR-200 and reduction of FoxM1 expression in DIM and Herceptin-treated breast cancer cells. We, therefore, transfected cells with pre-miR-200 or silenced FoxM1 in these cells for understanding the molecular mechanism involved. These results provide experimental evidence, for the first time, that DIM plus Herceptin therapy could be translated to the clinic as a therapeutic modality to improve treatment outcome of patients with breast cancer, particularly for the patients whose tumors express high levels of HER-2/neu.
Highlights
Herceptin (Trastuzumab), a humanized monoclonal antibody targeting HER-2/neu, has shown some benefit as a treatment for patients with HER-2/neu-expressing breast cancer, but clinical studies show that patients with high levels of HER-2/neu who are treated with a single dose of Herceptin progress to metastatic disease within one year [1,2,3,4,5]
We showed that DIM could reduce the concentration of Taxotere needed to induce an inhibitory effect on breast cancer cells [13,14,17,19], which raises the possibility that combination of DIM with Herceptin might help overcome the shortcomings and increased higher efficacy of Herceptin
Cell Growth Inhibition by DIM and Herceptin Treatment We tested several doses of DIM and Herceptin at different time points and found that treatment of SKBR3 and MDA-MB-468 breast cancer cells with 15 mM DIM for 72 h caused 40–60% growth inhibition which is irrespective of HER-2/ neu status
Summary
Herceptin (Trastuzumab), a humanized monoclonal antibody targeting HER-2/neu, has shown some benefit as a treatment for patients with HER-2/neu-expressing breast cancer, but clinical studies show that patients with high levels of HER-2/neu who are treated with a single dose of Herceptin progress to metastatic disease within one year [1,2,3,4,5]. The potential mechanisms underlying Herceptin failure are found in altered EGFR receptors, increased Akt activity and IGF-IR signaling, reduced p27kip and PTEN level in breast cancer cell [3,6]. These signaling pathways have been reported to be modulated by a natural nontoxic agent, 3, 3-diindolylmethane (DIM) [7,8,9] which raises the possibility that combination of DIM with Herceptin might help to enhance the antitumor activity of Herceptin against HER-2/neuexpressing breast cancer. We believe that combination of DIM and Herceptin is more potent than individual compounds, which could maximize the effect of Herceptin-based therapies and target multiple signaling pathways
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