3,3'-diindolylmethane and paclitaxel act synergistically to promote apoptosis in HER2/Neu human breast cancer cells.
BACKGROUND
HER2/neu positive breast tumors are difficult to treat. About 25 to 30% of invasive breast tumors overexpress the HER2/neu oncogene. These tumors are aggressive and become resistant to chemotherapeutic drugs. 3'3'-diindolylmethane (DIM), the active metabolite of indole-3-carbinol, a naturally occurring compound found in cruciferous vegetables, has been found to have anti-cancer properties in both humans and animals. DIM has been shown to induce cell cycle arrest and apoptosis in animal breast cancer models. Because HER2/neu overexpression confers resistance to paclitaxel, and DIM has anti-tumor effects, we hypothesized that DIM will enhance the cytotoxic effects of paclitaxel, a common taxane drug, on human Her2/neu breast cancer cells by potentiating its effect on cell cycle and stimulating apoptosis.


METHODS
The MDA-MB-435eB1 human Her2/neu breast cancer cells were treated with varying concentrations of DIM and paclitaxel. The cells were analyzed at different time points (24, 48, and 72 h). Proliferation was measured by a commercial cell proliferation assay (Promega Procheck Assay). Cell-cycle analysis and apoptosis were determined by flow cytometry. Western blot analysis was performed on to determine the effect of DIM and/or paclitaxel on the proteins involved in apoptosis, and epidermal growth factor-induced activation of HER2/neu and ERK1/2 signaling proteins.


RESULTS
Both DIM and paclitaxel exhibited time and concentration dependent inhibition of cell proliferation. TUNEL assay indicated that the combination also increased the number of apoptotic cells more than either agent alone. The presence of cleaved poly (ADP-Ribose) polymerase (PARP) significantly increased in the combination treatment, whereas Bcl-2 is decreased. DIM alone decreased the activation of the Her2/neu receptor; the combination decreased the activation of ERK1/ERK2.


CONCLUSIONS
DIM in combination with paclitaxel synergistically inhibits growth of Her2/neu human breast cancer cells through G2M phase cell-cycle arrest and induction of apoptosis/necrosis. The Her2/neu receptor and its downstream signaling protein ERK1/2 appear to be involved in DIM's affect on cell growth and differentiation, whereas apoptosis appears to be mediated through the mitochondrial pathway (Bcl-2/PARP). It appears DIM, a naturally occurring, nontoxic compound, may be a beneficial addition to a traditional (taxane-based) chemotherapy regimen.
Department of Surgery, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA. kmcguire76@hotmail.com
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McGuire
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Ngoubilly
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Neavyn
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Lanza-Jacoby
D016588:Anticarcinogenic Agents / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms / Q000737:chemistry / Q000473:pathology*; D002455:Cell Division / Q000187:drug effects; D004357:Drug Synergism; D066246:ErbB Receptors / Q000378:metabolism; D005260:Female; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D017239:Paclitaxel / Q000494:pharmacology*; D010766:Phosphorylation; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis*
D016588:Anticarcinogenic Agents; D000970:Antineoplastic Agents; D000972:Antineoplastic Agents, Phytogenic; D066246:ErbB Receptors; D007211:Indoles; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D017239:Paclitaxel; D018719:Erb-b2 Receptor Tyrosine Kinases; C016392:3,3'-diindolylmethane