EGFR inhibitors in combination with cyclopamine as chemotherapeutic strategy for treating breast cancer

PurposeTo determine whether combination therapy targeting sonic hedgehog (HH) and epidermal growth factor receptor (EGFR) pathways can inhibit breast cancer growth.MethodsCell viability was determined by MTT assay, apoptosis assessed using flow cytometry and endogenous expression of Gli-1, SHH and SMO determined by real time RT-PCR.ResultsHigher endogenous expression of the hedgehog signaling genes Gli-1, SHH and SMO was observed in MCF-7 cells compared to MDA-MB-231 cells. In contrast, MDA-MB-231 cells have higher endogenous expression of EGFR. Among the two EGFR inhibitors studied, afatinib was more potent than gefitinib regardless of cell line and exposure time and cyclopamine more potently inhibited breast cancer cells than vismodegib. As determined using combination index analysis and three-dimensional response surface methodology, simultaneous combination of cyclopamine and afatinib or gefitinib was synergistic at selected concentrations and mixing ratios. Regarding exposure schedules, only sequential treatment of afatinib followed by cyclopamine exhibited a synergistic effect (CI = 0.24) in MDA-MB-231 cells. Also, combination of cyclopamine and afatinib or gefitinib was more potent in inducing apoptosis compared to monotherapy in MDA-MB-231 cells. Additionally, combination of cyclopamine and gefitinib more effectively downregulated Gli-1 expression in MDA-MB-231 breast cancer cells compared to monotherapy.ConclusionsCombination of cyclopamine and afatinib or gefitinib leads to a supra-additive inhibition of MDA-MB-231 and MCF-7 cell proliferation and was found to be dose, time, cell line and schedule dependent. Also, combination therapy more effectively induced apoptosis, inhibited cell migration and downregulated Gli-1 expression compared to monotherapy in breast cancer cells.

Three-dimensional graphs representing anticancer interaction between gefitinib and cyclopamine in human breast cancer cell lines. Cells were treated with gefitinib and cyclopamine at concentrations ranging from 0 to 100 μM for 24 and 72 h and cell viability determined using MTT assay. MCF-7 cells treated for (A) 24 h and (B) 72 h. MDA-MB-231 cells treated for (C) 24 h and (D) 72 h. Portions of plot above zero on the difference axis indicate combinations that are synergistic while areas below zero are antagonistic and those equal to zero are additive.Figure optionsDownload as PowerPoint slide