In vitro effects of imatinib mesylate on radiosensitivity and chemosensitivity of breast cancer cells

Abstract

Background: Breast cancer treatment is based on a combination of adjuvant chemotherapy followed by radiotherapy effecting intracellular signal transduction. With the tyrosine kinase inhibitors new targeted drugs are available. Imatinib mesylate is a selective inhibitor of bcr-abl, PRGFR alpha, beta and c-kit. The purpose of this study was to determine whether Imatinib has an influence on the effectiveness of radiotherapy in breast cancer cell lines and if a combination of imatinib with standard chemotherapy could lead to increased cytoreduction.

Methods: Colony-forming tests of MCF 7 and MDA MB 231 were used to study differences in cell proliferation under incubation with imatinib and radiation. Changes in expression and phosphorylation of target receptors were detected using western blot. Cell proliferation, migration and apoptosis assays were performed combining imatinib with doxorubicin.

Results: The combination of imatinib and radiotherapy showed a significantly stronger inhibition of cell proliferation compared to single radiotherapy. Differences in PDGFR expression could not be detected, but receptor phosphorylation was significantly inhibited when treated with imatinib. Combination of imatinib with standard chemotherapy lead to an additive effect on cell growth inhibition compared to single treatment.

Conclusions: Imatinib treatment combined with radiotherapy leads in breast cancer cell lines to a significant benefit which might be influenced through inhibition of PDGFR phosphorylation. Combining imatinib with chemotherapy enhances cytoreductive effects. Further in vivo studies are needed to evaluate the benefit of Imatinib in combination with radiotherapy and chemotherapy on the treatment of breast cancer.

Figure 1

Imatinib inhibits PDGF BB dependent cell proliferation in breast cancer cell lines. Cells were incubated with increasing concentrations of PDGF BB and imatinib (IC 50). Cell growth was measured by MTT-assay.

Figure 2

Imatinib inhibits PDGF BB dependent cell migration in breast cancer cells. Migration assays were performed in breast cancer cell lines. Cells were incubated with either imatinib, PDGF BB or their combination for up to 72 hours. Cell migration was measured and student's t-test was performed. * indicates p-values < 0.05. Representative images of cell migration after 48 and 72 hours of incubation are displayed.

Figure 3

Combination of imatinib with doxorubicin leads to enhanced cell growth inhibition. Cells were incubated with doxorubicin or imatinib alone or in combination. Cell proliferation was assessed after six days of incubation. Combination indices were calculated using Calcusyn software. * = p-value 0.05 determined by student's t-test.

Figure 4

Effect of imatinib and doxorubicin on apoptosis of breast cancer cells. Cells were incubated with imatinib, doxorubicin alone or their combination for 24 and 48 hours. Apoptotic fraction was estimated by TUNEL assay and apoptotic cells were counted using fluorescence microscopy. For each sample, a total of five times 200-500 cells were scored and apoptotic fractions are expressed as percentage of total cells counted. Apoptotic cells show a green nuclear staining compared to vital cells.

Figure 5

Combination of imatinib with irradiation reduces breast cancer cell growth in vitro. Cells were incubated with imatinib alone, received irradiation of 2 Gy per day for 5 days or the combination of both. Colony-forming tests were performed and the surviving fraction was calculated dividing the number of colonies of treated cells by the number of colonies of non radiated control cells. Representative colonies of MCF 7 cells are shown.

Figure 6

PDGFR β expression is not effected by irradiation and imatinib. Breast cancer cell lines underwent irradiation, incubation with imatinib or the combination of both. Total protein was extracted and western blot was carried out using antibodies against PDGFR β.

Figure 7

Imatinib causes PDGFR β inactivation in combination with irradiation. Cell lines were treated with imatinib alone or in combination with irradiation. Total protein was extracted and western blot analysis was carried out using antibodies against phospho-PDGFR β. Equal loading was confirmed by β-Actin detection.